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WO2015171741A1 - Compositions and methods for identification assesssment, prevention, and treatment of cancer using nfs1 biomarkers and modulators - Google Patents

Compositions and methods for identification assesssment, prevention, and treatment of cancer using nfs1 biomarkers and modulators Download PDF

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WO2015171741A1
WO2015171741A1 PCT/US2015/029439 US2015029439W WO2015171741A1 WO 2015171741 A1 WO2015171741 A1 WO 2015171741A1 US 2015029439 W US2015029439 W US 2015029439W WO 2015171741 A1 WO2015171741 A1 WO 2015171741A1
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cancer
biomarker
human
activity
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Mark Bittinger
Jessie M. ENGLISH
Kwok-Kin Wong
Sima ZACHAREK
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Dana Farber Cancer Institute Inc
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Dana Farber Cancer Institute Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • malignant neoplasms represent the second most frequent cause of death, worldwide surpassed only by heart diseases.
  • effective anti-cancer treatments exist for many malignancies, such treatments are directed against well-known targets that do not fully control such malignancies. Accordingly, there is a great need to identify new cancer-related targets and bioniarkers useful for the identification, assessment, prevention, and treatment of cancer.
  • the present invention is based, at least in part, on the disco very that the iron-sulfur cluster biosynthesis pathway plays a significant role in driving hyperproiiferative eel! growth and that modulating the pathway (e.g., inhibiting the function of one or more iron- sulfur cluster biosynthesis pathway members) can inhibit such hyperproiiferative cell growth.
  • modulating the pathway e.g., inhibiting the function of one or more iron- sulfur cluster biosynthesis pathway members
  • btomarkers related to the iron-sulfur cluster biosynthesis pathway have been identified that are useful for identifying and assessing modulation of such
  • a method of treating a subject afflicted with a cancer comprising administering to the subject an agent that inhibits the copy number, amount, and'or activity of at least one biomarker listed in Table i , thereby treating the subject afflicted with the cancer.
  • the agent is administered in a pharmaceutically acceptable formulation, in another embodiment, the agent directly binds the at least one biomarker listed in Table 1 .
  • the at least one biomarker listed in Table 1 is human NFS 1 or an ortholog thereof.
  • the method further comprises administering one or more additional anti-cancer agents, optionally comprising mitochondrial cefaclor therapy.
  • a method of inhibiting hyperproiiferaiive growth of a cancer cell or cells comprising contacting the cancer cell or cells with an agent that inhibits the copy number, amount, and/or activity of at least one biomarker listed in Table 1, thereby inhibiting hyperproiiferaiive growth of the cancer cell or ceils, is provided, in one embodiment, the step of contacting occurs in vivo., ex vivo, or in vitro.
  • the agent is administered in a pharmaceutically acceptable formulation.
  • the agent directly binds the at least: one biomarker listed in Table 1.
  • the at least one biomarker listed in Table I is human NFS! or an ortholog thereof
  • the method further comprises administering one or more additional anti-cancer agents, optionall comprising mitochondrial cofactor therapy.
  • method of determining whether a subject afflicted with a cancer or at risk for developing a cancer would benefit from iron-sulfur cluster (ISC) biosynthesis pathway inhibitor therapy, the method comprising: a) obtaining a biological sample from the subject; b) determining the copy number, amount, and/or activity of at least one biomarker listed in Table 1 in.
  • ISC iron-sulfur cluster
  • the method further comprises recommending, prescribing, or administering ISC biosynthesis pathway inhibitor therapy if the cancer is determined to benefit from ISC biosynthesi pathway inhibitor therapy.
  • the method further comprises recommending, prescribing, or administering anti-cancer therapy other than ISC biosynthesis pathway inhibitor therapy if the cancer is determined to not benefit from ISC biosynthesis pathway inhibitor therapy, in still another embodiment, the anti-cancer therapy is selected from the group consisting of targeted therapy, chemotherapy, radiation therapy, and/or hormonal therapy, in yet another embodiment;
  • the control sample is determined from a cancerous or non-cancerous sample from either the patient or a member of the same species to which the patient belongs. In another embodiment, the control sample comprise ceils.
  • the method further comprises determining responsiveness to ISC biosynthesis pathway inhibitor therapy measured by at least one criteria selected from the group consisting of clinical benefit rate, survival until mortality, pathological complete response, semiquantitative measures of pathologic response, clinical complete remission, clinical partial remission, clinical stable disease, recurrence-free survival, metastasis free survival, disease free survival, circulating tumor cell decrease, circulating marker response, and RECIST criteria.
  • a method of assessing the efficacy of an agent for treating a cancer in a subject comprising: a) detecting in a first subject sample and maintained in the presence of the agent the copy number, amount or activity of at least one biomarker listed in Table 1 ; b) detecting the copy number, amount, and/or activity of the at least one biomarker listed in Table 1 in a second subject sample and maintained in the absence of the test compound; and c) comparing the copy number, amount, and/or activity of the at least one biomarker listed in Table I from steps a) and b), wherein a significantly increased copy number, amount, and/or activity of the at least one biomarker listed in Table I in the first subject sample relative to the second subject sample, indicates that the agent treats the cancer in the subject, is provided.
  • a method of monitoring the progression of a cancer in a subject comprising; a) detecting in a subject sample at a first point in time the copy number, amount, and/or activity of at least one biomarker listed in Table 1 ; b) repeating step a) during at least one subsequent point in time after administration of a therapeutic agent; and c) comparing the copy number, amount, and/or acti vity detected in steps a) and b), wherein a significantly increased copy number, amount, and/or activity of the at least one biomarker listed in Table I in the first subject sample relative to at least one subsequent subject sample, indicate that the agent treats the cancer in the subject, is provided, in one embodiment, the subject has undergone treatment, completed treatment, and/or is in remission for the cancer in between the first point in time and the subsequent point in time, In another embodiment, the subject has undergone ISC biosynthesis pathway inhibitor therapy in between the first point in time and the subsequent point in time, in still another embodiment, the first and/or at least one
  • a cell-based method for identifying an agent which inhibits a cancer comprising: a) contacting a cell expressing at least one biomarker listed in Table 1 w ith a. test agent; and b) determining the effect of the test agent on the copy number, level of expression, or level of activity of the at least one biomarker listed in Table 1 to thereby identify an agent that inhibits the cancer, is provided, in one embodiment, the cells are isolated from an animal model of a cancer.
  • the cells are from a subject afflicted with a cancer, in sti!i another embodiment, the cells are unresponsive to ISC biosynthesis pathway inhibitor therapy, in yet another embodiment, the step of contacting occurs in viva, ex vivo, or in vitro, in another embodiment, the method further coinpn ' scs determining the ability of the test agent to bind to the at least one biomarker listed in Table 1 before or after determining the effect of the test agent on the copy number, level of expression, or level of activity of the at least one biomarker listed in Table 1 ,
  • the sample comprises ceils, cell lines, histological slides, paraffin embedded tissue, fresh frozen tissue, fresh tissue, biopsies, blood, plasma, serum, buccal scrape, saliva, cerebrospinal fluid, urine, stool, mucus, or bone marrow, obtained from the subject.
  • a cell-free method for identifying a compound which inhibits a cancer die method comprising: a) determining the effect of a test compound on the amount or activity of at least one biomarker listed in Table 1 contacted with a test compound; b) determining the amount or activity of the at least one biomarker listed in Table 1 maintained in the absence of the test compound; and c) comparing the amount and/or activity of the at least one biomarker Hsted in Table 1 from steps a) and b), wherein a significantly increased amount, and or activity of the at least one biomarker listed in Table I in step a) relative to step b), identifies a compound which inhibits the cancer, is provided.
  • the method further comprises determining the ability of the test compound to bind to the at least one biomarker l isted in Table 1 before or after determining the effect of the test compound on the amount or activity of the at least one biomarker .
  • the steps a) and b) are selected from the group consisting of a methylene blue assay, a ?-azido-4-tneihylcoumarin (A MC.) assay, an alanine assay, and a mass spectrometry assay.
  • the methylene blue assay compri es i) reacting the at least one biomarker listed in Table 1 in a buffer comprising a) cysteine, b) a pyridoxal phosphate cofactor, and c) optionall the test compound; ii) stopping the reaction by adding N,N-dimethyi-p-phenyienedi amine and iron chloride (FeCB) in hydrogen chloride (HQ) solution, and in) determining the production of methylene blue via absorhance of light having a wavelength of 670 tim.
  • the AxMC assay comprises i) reacting the at least one biomarker listed in Table 1 in a buffer
  • test compound comprising a) cysteine, b) a pyridoxal phosphate cofactor, e) glutathione as reducing agent, d) bovine serum albumin, e) 7-azido-4-raetaykoumarin, and f) optionally, the test compound; and ii) fiuorometricaily monitorin the reaction product, 7-amino-4- methylcoumarin.
  • the alanine assay comprises i) reacting the at least one biomarker listed in Table I in a buffer comprising a) cysteine, b) a pyridoxal phosphate cofactor, e) DTT as reducing agent, and d) optionally, the test compound; ii) performing a. secondary reaction to measure alanine production in a buffer containing a) NAD (nicotinamide adenine dinucleoride) and b) alanine dehydrogenase enzyme; and in) fiuorometricaiiy measuring the reaction product, NADH.
  • the mass spectrometry assay comprises i) reacting the at least one biomarker listed in Table 1 in a buffer comprising a) cysteine, b) a pyridoxal phosphate cofactor, and c) optionally the test compound; and ii) determining the production of alanine using mass spectrometry.
  • the copy number is assessed by mieroarray, quantitative PCR (qPCR), high-throughput sequencing, comparative genomic hybridization (CGH), or fluorescent in situ hybridization (FISH).
  • the amount of the at least one biomarker is assessed by detecting the presence in the samples of a polynucleotide molecule encoding the biomarker or a portion of said polynucleotide molecule.
  • polynucleotide molecule is a mRNA, cDNA, or functional variants or fragments thereof, in yet another embodiment, the step of detecting further comprises amplifying the
  • the amount of the at least one biomarker is assessed by annealing a nucleic acid probe with the sample of the polynucleotide encoding the one or more biomarkers or a portion of said polynucleotide molecule under stringent hybridization conditions, in still another embodiment, the amount of the at least one biomarker is assessed b detecting the presence a polypepti de of the at least one biomarker.
  • fits presence of a polypeptide is detected using a reagent which specifically binds with the polypeptide (e.g., a reagent selected from the group consisting of an antibody, an antibody deri vative, and an antibody fragment).
  • the acti ity of the at least one biomarker is assessed by determining the magnitude of modulation of at least one NFS! pharmacodynamic biomarker listed in Table 1.
  • the activity of the at least one biomarker is assessed by determining the magnitude of modulation of the activity or expression level of at least one downstream target of the at least one biomarker.
  • the ISC biosynthesis pathway inhibitor agent or test compound modulates a biomarker selected from the group consisting of human NFS I , human LY M4, human !SCU, human FXN, human NFUJ , human GLRX5, human BOLA3, human HSCB, human KSPA9, human
  • the ISC biosynthesis pathway inhibitor agent or test compound is an inhibitor selected from the group consisting of a small molecule, antisense nucleic acid, interfering RNA, shR A, siRNA , aptamer, ribozyme, dominant-negative protein binding partner, and combinations thereof.
  • the at least one biomarker is selected .from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more biomarkers.
  • the at least one biomarker is selected from the group of I SC biosynthesis pathway biomarkers listed in Table L
  • the ISC biosynthesis pathwa biomarkers listed in Table 1 are selected from the group consisting of human NFS ! ceremoni human LYRM4, human ISCU, human FXN, human NFU I , human GLRX5, human BOLA3, hitman HSCB, human HSPA9, human ISC A 1 , human ISCA2, human IBA57, human NUBPL, human SLC25A28, human FDXR, human FDX2, and orthoiogs of said biomarkers thereof.
  • the at least one biomarker is selected from the group of NFS 1 pharmacodynamic biomarkers listed in Table 1.
  • the NFS I pharmacodynamic biomarkers listed in Table 1 are selected from the group consisting of human aconitase, human succinate dehydrogenase, human ferritin, human transferrin- reeeptor, human HifZalpha, human PTGS2, and lipid reactive oxygen species ( OS).
  • the cancer is selected from the group consisting of paragangliomas, colorectal cancer, cervical cancer, lung adenocarcinoma, ovarian cancer, and myeloid cancer within a hypoxic tumor microenvvronment.
  • the subject is a mammal, such, as an animal model of cancer or a human.
  • Figure 1 shows a schematic diagram of the iron-sulfur cluster biogenesis pathway as adapted from Lira et al. (2013) Htm. Mol Genet. 22:4460-4473.
  • Figure 2 shows the results of NFS I amplification assessed across available TCGA (The Cancer Genome Atlas) datasets using the cBioPortal for Cancer Genomics (available on the World Wk!e Web at cbioportai.org pubh ' c-portal ).
  • the inset shows the correlation between copy-number alterations (x-axis, as determined by GISTIC) and mRNA expression (y-axis, by RNASeq) from a representative dataset (colorectal cancer). All histograms shown represent: amplifications.
  • Figure 3 shows the results of collective alterations of NFS! , LYR 4/ISD1 1 , ISCU, and FXN evaluated across available TCGA datasets using the cBioPortal for Cancer Genomics (available on the World Wide Web at cbioportal.org/public-portal/).
  • Top bar of histogram amplification; middle bar of histogram: deletion; bottom bar of histogram: imitation; gray: multiple alterations.
  • the tipper inset shows the distribution of alterations between NFS1 , LYRM4, ISCU, and FXN in ovarian cancers.
  • the lower insets show the correlation between copy-number alterations (x-axis, as determined by GISTIC) and mRNA expression (y-axis, by RNASeq) for NFS 1 (left) and LYRM4 (right) from a representative dataset.
  • Figure 4 shows the results of mutation analyses of solute carrier family 25 mitochondrial iron transporter, member 28 (SLC25A28), a mediator of iron uptake, assessed across TCGA samples using datasets from the cBioPortal for Cancer Genomics (available on the World Wide Web at cbioportai.org/pablic-portai/) and the Memorial Sloan-Ketterlng Cancer Center (MSKCC). All markers shown represent raissense mutations, except, for the seventh market " from the left located at the -terminus of the Mito_carr domain, which represents a frameshift deletion.
  • Figure 5 shows that inducible knockdown of NFS l using siiRNAs causes clonogenic growth inhibition in M N74 cells.
  • Figure 6 shows the results ofe ' DNA rescue experiments confirming on-target activity of NFS ! shRNAs.
  • Figure 7 shows the results of NFS .1 knockdown and resulting cell growth effects across a panel of cell lines.
  • Figure 8 shows the results of NFS I sh shut-off experiments confirming restoration of clonogenic growth following derepression of NFS 1 function.
  • Figure 9 shows the results of aconitase activity and SDH activity measured in lysates of mitochondria isolated from: GAL-NFS ⁇ cells harboring plasmid- ome copies of WT NFSl, fl/S'iLM/AA, or vector without insert, as indicated, and grown for 40 hours in glucose-containing medium. Enzymatic activities were measured and plotted relative to the non-iron-sulfur cluster protein, malate dehydrogenase. The figure is adapted from
  • Figure 10 shows thai transient knockdown of NFS ' ! in Heia ce!is shows alterations in mitochondrial structure and significantly decreased activity of iron-sulfur-dependent enzymes, including aconitase and SDH. The figure is adapted from Biederbick el al(2006) ⁇ Cell, Biol 26:5675-5687.
  • Figure 11 shows a schematic diagram illustrating an iron-dependent form of nou- apoptotic cell death known as ferroptosis. The. figure is adapted from. Dixon ei al (2012) Cell 149:4060- 1072.
  • Figure 12 includes 2 panels, identified as panels A aikf B, which show data adapted from Yang et al. (2014) Cell 156:317-331 indicating that upregulation of PTGS2 expressio occurs upon crastin and ⁇ 1S,3&)-RSL3 treatment (panel A) and further showing that PTGS2 expression is induced by PE (panel B).
  • the oncogenic RAS-seieciive lethal small molecule erasfin. triggers ferroptosis, which is dependent upon intracellular iron, but not other metals, and is morphologically, biochemically, and genetically distinct from apoptosis, necrosis, and autophagy.
  • Erastin like glutaraate, inhibits cysteine uptake by the cysteine/glutairiate antiporter (system xc--), creating; a void in the antioxidant defenses of the eel! and ultimately leading to iron-dependent, oxidative death.
  • Figure 13 shows that iRP ⁇ I represses HIF2a translation and acti vity. The figure is adapted from Zimmer el al. (2008) Mol Cell 32:838-848.
  • Figure 14 shows dat confirming that candidate biomarkers for iran-suifur cluster biosynthesis pathway modulation and iron-dependent cell death (ferroptosi ) correlate with NFSl inhibition.
  • Figure 15 includes 5 panels, identified as panels A, B, C, D, and E, which demonstrate modulation of biomarkers of ISC biosynthesis pathway modulation and iron- dependent cell-death (ferroptosis) associated with NFSl knockdown.
  • Panel A shows decrease in ferritin protein le vels and increases in TFRC protein le vels.
  • Panels B and C shows decreases in aconitase activity.
  • Panel F. shows decreases in succinate dehydrogenase activity.
  • Panel F confirms knockdown of NFS i protein levels in the samples analyzed in panels E and F.
  • Figure 16 shows that NFS 1 knockdown in the C2BBE1 colorectal cell hoe correlates with down-regulation of HlF2a protein.
  • the asterisk (*) indicates treatment for 24 hours with cobalt chloride, a hypoxia-mimieking agent.
  • Figure 17 includes 2 panels, identified as panels A and B, which demonstrate that NFS ' ! is essential in MKN74, an NPS-i amplified cell line.
  • Panel A shows that NFS I is amplified in the MKN74 gastric cell line.
  • Panel B shows the results of cDNA rescue of M N74 stable inducible NFS 1 shRNA lines with either wild type NFS! or a dominani- negative NFS! mutant.
  • Figure 18 shows that cDNA rescue with WT FSi similar to that described in Figure 17 restores the NFS 1 -sh5 ⁇ dependent effect oo aconiiase activity.
  • Figure 19 shows that WT NFSI t but »ot NFSl°* iA , rescues the NFSl -shS- dependetit inhibition of FTH1 protein levels and the up-regu!ation of Tfitc protein levels.
  • Figure 20 shows that the NFS 1 t hi catalytic mutant causes a decrease in aeonitase acti vity and ferritin levels comparable to that with NFS ! -sh l or NFS l-sh5.
  • Figure 21 shows representative sulfide-based (e.g., methylene blue assays or flurogenic sulfide probes, such as AzMC to AMC detection.) or alanine-based (e.g., alanine dehydrogenase activity) detection methods for analyzing NFS. I activity.
  • sulfide-based e.g., methylene blue assays or flurogenic sulfide probes, such as AzMC to AMC detection.
  • Figure 22 includes 2 panels, identified as panels A and B, which provide a representative methylene blue assay suitable for high-through put formats (panel A) and representative sulfide detection range analyses (panel B).
  • a value of > 0.5 is preferred for enzymatic assays and Z * was calculated as equaling 1 - [ 3 (SD of signal + SD of background) / (Mean of signal - Mean of background) ' ).
  • Figure 23 shows the loss of sulfide from solution in a methylene blue assay over time.
  • Figure 24 includes 3 panels, identified as panels A, B, and C, which provide a representative AzMC assay suitable for high-through put formats (panel A) and representative sulfide detection range analyses (panel B). V values were calculated as equaling .1 - 3 (SD of signal ⁇ * ⁇ SD of background) (Mean of signal - Mean of background)). Panel C shows the enzyme kinetics of IscC using an AzMC assay optimized for high-throughput analyses.
  • Figure 25 includes 3 panels, identified as panels A, .8, and C, which provide a representati ve alanine assay suitable for high-through put formats (panel A) and
  • Panel C shows the enzyme kinetics of IscC using an alanine assa optimized for high-throughput analyses.
  • Figure 26 shows exemplary reporter constructs useful for screening for NFS i inhibitors and/or inhibitors of the iron-sulfur cluster biosynthesis pathway.
  • the asterisks (*) represent the use of luciferase, GFP, and RFP containing destabilizing sequences from mouse ornithine decarboxylase at their C-terminus.
  • Iron-sulfur cluster biogenesis is necessary for the generation of iron-sulfur
  • iron-sulfur cluster biogenesis pathway members are biomarkers for the diagnosis, prognosis, and treatment of cancers.
  • a variety of cancers can be so analyzed and treated, such as those having overexpression of NFS1 and/or those having activating mutations in the HIF2a pathway.
  • an element means one element or more than one element.
  • altered amount refers to increased or decreased copy number (e.g. , gcrmline and/or somatic) of a biomarker nucleic acid, e.g., increased or decreased expression level in a cancer sample, as compared to the expression level or copy number of the biomarker nucleic acid in a control sample.
  • altered amount of a biomarker also includes an increased or decreased protein level of a biomarker protein in a sample, e.g., a cancer sample, as compared to the. corresponding protein level in a normal, control sample.
  • an altered amount of a biomarker protein may be determined by detecting posttraiislatKraai modificatioii such as methylatioii status of the marker, which may affect the expression or activity of the biomarker protein.
  • the amount of a biomarker in a subject is "significantly" higher or lower than the normal amount of the biomarker, if the amount of the biomarker is greater or less, respectively, than the normal level by an amount greater than the standard error of the assay employed to assess amount, and preferably at least 203 ⁇ 4, 30%, 40%, 503 ⁇ 4, 60%, 70%, 803 ⁇ 4, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or than that amount.
  • the amount of the biomarker in the subject can be considered "significantly" higher or lower than the normal amount if the amount is at least about two, and preferably at ieast about three, four, or five times, higher or lower, respectively, than the normal amount of the biomarker.
  • altered level of expression of a biomarker refers to an expression level or copy number of the biomarker in a test sample, e.g., a sample derived from a patient suffering from cancer, that is greater or less than the standard error of the assay employed to assess expression or copy number, and is preferably at least twice, and more preferably three, .four, five or ten or more times the expression level or copy number of the biomarker in a control sample (e.g. sample from a healthy subjects not having the associated disease) and preferably, the average expression level or copy number of the biomarker in several control samples.
  • a test sample e.g., a sample derived from a patient suffering from cancer
  • a control sample e.g. sample from a healthy subjects not having the associated disease
  • the altered level of expression is greater or less than the standard error of the assa employed to assess expression or copy number, and is preferably at least twice, and more preferably three, four, five or ten or more times the expression level or copy number of the biomarker in a control sample (e.g., sample from a healthy subjects not having the associated disease) and preferably, the average expression level or copy number of the biomarker in several control samples.
  • altered activity of a biomarker refers to an activity of the biomarker which is increased or decreased in a disease state, e.g., in a cancer sample, as compared to the activity of the biomarker in a normal, control sample.
  • Altered activity of the biomarker may be the result of for example, altered expression of the biomarker, altered protein level of the biomarker, altered structure of the biomarker, or, e.g., an altered interaction with other proteins involved in the same or different pathway as the biomarker or altered interactio with transcriptional activators or inhibitors.
  • altered structure of a biomarker refers to the presence of mutations or allelic variants within a biomarker nucleic acid or protein, e.g., mutations which affect expression or acti vity of the biomarker nucleic acid or protein, as compared to the normal or wild-type gene or protein.
  • mutations include, but are not limited to substitutions, deletions, or addition mutations, Mutations may be present in the coding or non-coding region of the biomarker nucleic acid.
  • antibody broadly encompass naturally-occurring forms of antibodies (e.g. I ' gG, igA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments and derivatives have at least an antigenic binding site.
  • Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody.
  • antibody as used herei also includes an "antigen-binding portion" of an antibody (or simply “antibody portion”).
  • antigen-binding portion refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g. , a biomarker polypeptide or fragment thereof). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full- length antibody. Examples of binding fragments encompassed within the term "antigen- binding portion” of an antibod include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (it) a Fi ' ab')?
  • CDR complementarity determining region
  • Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.
  • Any VH and VL sequences of specific seFv can be linked to human immunoglobulin constant region cD A or genomic sequences, in order to generate expression vectors encoding complete I ' gG polypeptides or oilier isotypes.
  • VH and VL can also be used in the generation of Fab, Fv or other fragments of immunoglobulins using either protein chemistry or recombinant DMA technology.
  • Other forms of single chain antibodies, such as diabodies are also encompassed.
  • Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see &g. r Hoiliger et a!. (1993) Proc. ⁇ Acad. Set. U.S.A. 90:6444-6448; Poljak et al. (1994) Structure 2: 1 121 - 1 123).
  • an antibody or an tigen-binding portion thereof may be part of larger immunoadbesion polypeptides, formed by co valent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides.
  • immunoadhesion polypeptides include use of the streptavidin core region to make a tctramcric scFv polypeptide (Kipriyanov et al. (1 95) Human Antibodies and Hyhrtdamas 6:93-101) and use of a cysteine residue, biomarker peptide and a C-terminal polyhistidmc tag to make bivalent and biotinylated scFv polypeptides (Kipriyanov et al.
  • Antibody portions such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such, as papain or pepsin digestion, respectively, of whole antibodies.
  • antibodies, antibody portions and immunoadbesion polypeptides can be obtained using standard recombinant DNA
  • Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof (e.g. humanized, chimeric, etc.). Antibodies may also be fully human. Preferably, antibodies of the invention bind specifically or substantially
  • monoclonal antibodies and “monoclonal antibody composition”, as used herein, refer to a population of antibody polypeptides that contain only one species of an antigen binding site capable of imniunoreaeting with a particular epitope of an an tigen, whereas the term “polyclonal antibodies” and “polyclonal antibody composition” refer to a population of antibody polypeptides that contain multiple species of antigen bi nding sites capable of interacting with a particular antigen.
  • a monoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it immunoreacts.
  • Antibodies may also be "humanized,” which is intended to include antibodies made by a non-human celi having variable and constant regions which have been altered to more closely resemble antibodies that would be made by a human ceil. For example, by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germlme immunoglobtslin sequences.
  • the humanized antibodies of the invention may include amino acid residues not encoded by human germlme immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or b somatic imitation in vivo), for example in the CDRs.
  • the term "humanized antibody”, as used herein, also includes antibodies in which CDR sequences derived from the germtine of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • the term "assigned score” refers to the numerical value designated for each of the biomarkers after being measured in a patient sample.
  • the assigned score correlates to the absence, presence or inferred amount of the biomarker in the sample.
  • the assigned score can be generated manually (e.g., by visual inspection) or with the aid of instrumentation for image acquisition and analysis.
  • the assigned score is determined by qualitative assessment, for example, detection of a fluorescent readout on a graded scale, or uantitative assessment:.
  • an "aggregate score” which refers to the combination of assigned scores from a plurality of measured biomarkers, i s determined, in one embodiment the aggregate score is a summa tion of assigned scores.
  • combination of assigned scores involves performing mathematical operations on the assigned scores before combining them into an aggregate score.
  • the aggregate score is also referred to herein as the predictive score.”
  • biomarker refers to a measurable entity of the present invention that has been determined to be predictive of anti-cancer therapy ( g., iron-sulfur cluster biosynthesis pathway inhibitory therapy) effects on a cancer.
  • Biomarkers can include, without limitation, nucleic acids (e.g. , genomic nucleic acids and or transcribed nucleic acids) and proteins, particularly those involved shown in Table 1.
  • nucleic acids e.g. , genomic nucleic acids and or transcribed nucleic acids
  • proteins particularly those involved shown in Table 1.
  • Many biomarkers listed in Table 1 are also useful as therapeutic targets, hi one embodiment, such targets are the iron-sulfur cluster biosynthesis pathway members shown in section A of Table I .
  • a “blocking” antibody or an antibody “antagonist” is one which inhibits or reduces at least one biological activity of the antigen(s) it binds, in certain embodiments, the blocking antibodies or antagonist antibodies or fragments thereof described herein substantially or completely inhibit a given biological acti vity of the antigen(s).
  • body fluid refers to fluid that are excreted or secreted from the bod as well as fluid that are normally not (e.g.
  • amniotic fluid aqueous humor, biie f blood and blood plasma, cerebrospinal fluid, cerumen and earwax, co per's fluid or pre-ejaculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, and vomit).
  • cancer or “tumor” or “hyperprolifeTath3 ⁇ 4' ? refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. In some embodiments, such cells exhibit such characteristics in past or in full due to the expression and activity of oncogenes, such as e- MY Cancer cells are often in the form of a tumor, but such cells may exist alone within an animal, or may be a non-htmorigenie cancer ceil, such as a leukemia cell. As used herein, the term "cancer” includes premalignant as well as malignant cancers.
  • Cancers include, but are not limited to, B cell cancer, e.g., multiple myeloma, Waldenstrom's macroglobulinemia, the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gamniopathy, and
  • immunoeytic amyloidosis melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, sali ary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematologic tissues, and the like.
  • types of cancers applicable to the methods encompassed by the present invention include human sarcomas and carcinomas, e.g., fibrosarcoma,
  • myxosarcoma myxosarcoma, liposarcoma, chondrosarcoma,, osteogenic sarcoma, chordoma,
  • angiosarcoma endotheliosareoma, lymphangiosarcotna, lymphaagioendomcitosarcoraa, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain iiimor, testicular cancer, lung carcinoma, small ceil lung carcinoma, bladder carcinoma, epithelial
  • cancers are epithlelia.l in nature and include but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer.
  • the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer- n still other embodiments, the epithelial cancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovarian carcinoma), or breast carcinoma.
  • the epithelial cancers may be characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear ceil, Brenner, or undifferentiated.
  • coding region refers to regions of a nucleotide sequence comprising eodons which are translated into amino acid residues
  • non-coding region refers to regions of a nucleotide sequence that are not translated into amino acids (e.g., 5' and 3' untranslated regions).
  • an adenine residue of a first nucleic acid region ts capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which, is antiparallel to the first region if the residue is thymine or uracil.
  • base pairing specific hydrogen bonds
  • a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine.
  • a first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparaliel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region.
  • the first region comprises a first portion and the second region comprises a second portion, whereby, when the first: and second portions are arranged in an antiparaliel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in die second portion. More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
  • control refers to any reference standard suitable to provide a comparison to the expression products in the test sample
  • the control comprises obtaining a "control sample” from which expression product levels are detected and compared to the expression product levels from the test sample.
  • a control sample may comprise any suitable sample, including but not limited to a sample from a control cancer patient (can be stored sample or previous sample measurement) with a known outcome; normal tissue or cells isolated from a subject, such as a normal patient or the cancer patient, cultured primary cells/tissues isolated from a subject such as a normal subject or the cancer patient, adjacent norma!
  • control may comprise a reference standard expression product level from any suitable source, including but not limited to housekeeping genes, an expression product level range from normal tissue (or other previously analy ed control sample), a previously determined expression product level range within a test sample from a group of patients, or a set of patients with a certain outcome (for example, survival for one, two, three, four years, etc.) or receiving a certain treatment (for example, standard of care cancer therapy).
  • a certain outcome for example, survival for one, two, three, four years, etc.
  • a certain treatment for example, standard of care cancer therapy
  • control samples and reference standard expression product levels can be used in combination as controls in the methods of the present invention.
  • the control may comprise normal or non-cancerous cell/tissue sample
  • the control may comprise an expression level for a set of patients, such as a set of cancer patients, or for a set of cancer patients receiving a certain treatment, or for a set of patients with one outcome versus another outcome.
  • the specific expression product leve! of each patient can be assigned to a percentile level of expression, or expressed as either higher or lower than the mean or average of the reference standard expression level
  • the control may comprise norma! cells, cells from patients treated with combination chemotherapy,, and cells from patients having benign cancer.
  • control may a!s comprise measured value for example, average ievel of expression of a particular gene in a population compared to the level of expression of a housekeeping gene in the same population.
  • a population may comprise normal subjects, cancer patients who have not undergone any treatment (i.e. , treatment naive), cancer patients undergoing standard of care therapy, or patients having benign cancer
  • the control comprises a ratio transformation of expression product levels, including but not limited to determining a ratio of expression product levels of two genes in the test sample and comparing it to any suitable ratio of the same two genes in a reference standard;
  • the con trol comprises a control sample which is of the same lineage and/or ty pe as the test sample.
  • the control may comprise expression product levels grouped as percentiles within or based on a set of patient samples, such as ail patients with cancer.
  • a control expression product level is established wherein higher or lower levels of expression product relative to, for instance, a particular percentile, are used as the basis for predicting outcome.
  • a control expression product level is established using expression product levels from cancer control patients with a known outcome, and the expression product levels from the test sample are compared to the control expression product level as the basts for predicting outcome.
  • the methods of the invention are not limited to use of a specific cut -point in comparing the level of expression product in the test sample to the control.
  • the "copy number" of a btomarker nucleic acid refers to the number of DM A sequences in a cell (e.g., germline and/or somatic) encoding a particular gene product. Generally, for a given gene, a mammal has two copies of each gene. The cop number can be increased, however, by gene amplification or duplication, or reduced by deletion. For example, germline copy number changes include changes at one or more genomic loci, wherein said one or more genomic loci are not accounted for by the number of copies in the normal complement of Gerrline copies in a control (e.g. , the normal copy number in germUne DNA for the same species as that from which the speci fic germilme DNA and corresponding copy number were determined).
  • Somatic copy number changes include changes at one or more genomic loci, wherein said one or more genomic loci are not accounted for by the number of copies in germline DN A of a control (3 ⁇ 4 ⁇ ., copy number in gerraline DNA for the same subject as that from which the somatic DNA and corresponding copy number were determined).
  • the ''normal copy number (e.g. , germline and/or somatic ) of a biomarker nucleic acid or "normal" level of expression of a biomarker nucleic acid, or protein is the activity/level of expression or copy number in a biological sample, e.g. , a sample
  • tissue containing tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bone marrow, from a subject, e.g., a. human, not afflicted with cancer, or from a corresponding non-cancerous tissue in the same subject who has cancer.
  • determining a suitable treatment regimen for the subject is taken to mean the determination of a treatment regimen U e, , a single therapy or a combination of different therapies that are used for the pre vention and/or treatment of the cancer in the subject) for a subject that is started, modified and/or ended based or essentially based or at least partially based on die results of the analysis according to die present invention.
  • One example is determining whether to provide targeted therapy against a cancer to provide antt-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy).
  • antt-cancer therapy e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy
  • Another example is starting an adjuvant therapy after surgery whose purpose is to decrease the risk of recurrence, another would be to modify die dosage of a particular chemotherapy.
  • the determination can, in addition to the results of the analysis according to the present invention, be based on personal characteristics of the subject to be treated. In most eases, the aciual determination of the suitable treatment
  • expression signature refers to a group of two or more coordinate!;-- expressed biomarkers.
  • the genes, proteins, and the like making up this signature may be expressed in a specific cell lineage, stage of differentiation, or during a particular biological response.
  • the biomarkers can reflect biological aspects of the tumors in which they are expressed, such as the cell of origin of the cancer, the nature of the non-malignant ceils in the biopsy, and the oncogenic mechanisms responsible for the cancer.
  • Expression data and gene expression levels can be stored on computer readable media, e.g., the computer readable medium used in conjunction with a rnkroarray or chip reading device.
  • a molecule is "fixed” or "affixed” to a substrate if it is eovalently or nort-eovalently 5 associated with the substrate such that the substrate cart be rinsed with a fluid (e.g. standard saline citrate, pH 7.4) without a substantial fraction of the molecule dissociating from the substrate.
  • a fluid e.g. standard saline citrate, pH 7.4
  • highly structured 5' untranslated region (' UTR) * " refers to the region of an mRNA directly upstream from the initiation codon, which J ) begins at the transcription it ) start site and ends one nucleotide (nt) before the initiation codes) (usually AUG) of the coding region and 2) contains a hairpin loop or other secondary structures.
  • Such secondary structures are usually predicted by modeling but there are experimental means to define them more quantitatively, such as fay measuring the resistance of the structure to nucleases which do not attack double stranded regions or performing physical techniques, such as
  • the highly structured 5' UTR renders the mRNA a -relatively poor substrate for translation.
  • mRNAs encoding proteins necessary for cell growth and survival typically contain a complex, highly structured 5 ' UTR in order to limit the availability of the protein.
  • 25 mRNA having a free energy of 30-70 keal/mol or less is able to inhibit translation
  • Non-limiting, representative examples of 0 RN As with a highly structured 5' UTR include transferrin, transferrin receptor, c-. YC, X- tirrked inhibitor of apoptosis protein (XIAP), and ornithine decarboxylase (ODCT ).
  • homologous refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When a nucleotide residue position in both region is occupied b the same nucleotide residue, then the regions are homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position of cadi region is occupied by the same residue. Homology between two regions is expressed in terms of the proportion of nucleotide residue positions of the two regions that are occupied by the same nucleotide residue.
  • a region having the nucleotide sequence 5 - ATTGCC-3* and a region having the nucleotide sequence S'-TATGGC-S' share 50% homology.
  • the first region comprises a first portion and the second region comprises a second portion, whereby, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residue positions of each of the portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.
  • cancer includes the decrease, limitation, or blockage, of, for example a particular action, function, or interaction.
  • cancer is ' nhibited” if at least one symptom of the cancer is alleviated, terminated, slowed, or prevented.
  • cancer is also "inhibited' '' if recurrence or metastasis of the cancer is reduced, slowed, delayed, or prevented.
  • interaction when referring to an interaction between two molecules, refers to the physical contact (e.g. , binding) of the molecules with one another. Generally, such an interaction results in an acti vity (which produces a biological effect) of one or both of said molecules.
  • iron-sulfur cluster biogenesis pathway refers to the full set, or relevant subsets thereof, of proteins required for generating iron-sulfur (Fe-S) clusters composed of iron and inorganic sulfur for itse as cofactors in generating Fe-S proteins (see, for example, Lill et al ' . (2012) Biochmt Biophys. Acta 1823: 1491 - 1508; Lill and MuSenhoff (2005) Trends Biochem. Set 30: 133- 141 ; Renault (2012) Dm. Mode! Meek 5: 155-164, Ye and Touault (2010) Biochem. 49:4945-4956, and Rouauh and Tong (2005) Nat. Rev. Mot. Cel! Biol.
  • Iroii-sulfur clusters are critical for the production of a subset of enzymes invol ved in critical cellular processes, such as oxidative phosphorylation, the citric acid cycle, heme biosynthesis, iron homeostasis, and DNA repair.
  • Figure 1 shows an exemplary schematic diagram of the pathway. Members of the pathway, including terminology, sequences, and function, are well known in the art.
  • NFSl refers to the nitrogen fixation i homolog cysteine desulfitrase member of the class- V family of yridoxal phosphate-dependent aminotransferase family and is alternatively known as "iscS," " PS, and "HUSSY-OB.' " NFSl , whose structure- function relationship is known, supplies inorganic sulfur to iron-sulfur clusters by removing the sulfur from cysteine thereby creating alanine in the process ( Fartian et ai (2014) Mol. Genet. G no . Med. 2:73-80; urihara el ai. (2003) Biochim. Biophys. Acta 1647:303-309; Cupp- Viekery et al.
  • the NFS1 gene uses alternate in- frame translation initiation sites to generate mitochondrial forms and eytopiasmie nuelear forms. Selection of the alternative initiation sites is determined by the cytosolic pH.
  • mitochondrial forms are itsed according to the present invention, in another embodiment, cytoplasmic/nuciear forms are used according to the present invention.
  • human NSF I transcript variant I (NMJ)21 .100.4) encodes the long human NSF I isoform .1 (NPJ ) 66923.3).
  • Human NSF I transcript variant 2 (NM 001 1 8989.1) lacks an in -frame exon in the 5 ' coding region compared to variant 1 , resulting in an isoform (NP 001 185 18.1 ) that is shorter compared to isoform 1.
  • Nucleic acid and polypeptide sequences of N FS orfhologs in species other than humans are also well known and include, for example, monkey NFS 1 (XMJ ) 0! 0976989.2, XPJ10.1097699.1, XMJKH 097983.2, and .XPJMM097983. l X dog NFS!
  • NFSl orfhologs Representative sequences of NFSl orfhologs are presented below in Table 1 .
  • Anti-NFS 1 agents including antibodies, nucleic acids, and the like are well-known in the art and include, for example, iron, L-alanine, L-cysteine, pyridoxal 5 '-phosphate and derivatives.
  • NFS I molecules can further be used to refer to arty combination of features described herein regarding NFS I molecules.
  • sequence composition percentage identify, sequence length, domain structure, functional acti vity, etc. can be used to describe an NFS 1 molecule of the present invention.
  • LYR 4" refers to the LYR motif containing 4 and is alternatively known as "homolog of yeast Isdl 1 " and "mitochondrial matrix Nfsl interacting protein,"
  • the LYRM4 gene encodes the ISDl I protein that forms a stable complex in vim with the human cysteine desulfitrase !SCS to generate the inorganic sulfur needed for iron- sulfur protein biogenesis (Shi et l. (2009) Hum. Mai, Genet .1 :3014-3025).
  • NM 020408.5 encodes the short human LYRM ' 4 isoform 1 (NP 065141 .3), Human LYR 4 transcript variant 2 (NMJ)Ol 164840.2) contains an alternate 3' terminal exon to create a different 3 5 coding region and 3' UTR compared to variant 1 and to thereby encode an isoform (NP 001 1583.12.1 ) having a distinct C-terminiis and a longer sequence than that of isoform 1 .
  • Human LYRM4 transcript variant 3 (NM 001 164841.2) includes an additional exon that results in an altemate 3' coding region and ' UTR compared to variant 1 to thereby encoded an isoform f NPJK) 1 158313.1) having a distinct C-terminus and a longer sequence than that of isoform 1 .
  • Each of the isoforms is functional.
  • Nucleic acid and polypeptide sequences of LYRM4 orthologs in species other than humans are also well known and include, for example, monkey LYRM4 (XM_00.1095995.2 and
  • XP . 00iO95995.2X dog LYRM4 (XM . 005640157.1 and XP .. 005640214.1 ), cow LYRM4 (NM .. 001076306.1 and NP .00! 069774.1), mouse LYR 4 (NM ... 201358.2 and
  • Anti- LYRM4 agents including antibodies, nucleic acids, and the like are well-known in the art and include, for example, dominant-negative binding proteins such as versions of NFS 1 without a catalytic domain, it is to be noted that the term can further be used to refer to any combination of features described herein regarding LYRM4 molecules. For example, any combination of sequence composition, percentage identify, sequence length, domain structure, functional activity, etc. can be used to describe an LYRM4 molecule of the present invention.
  • ISCU refers to the iron-sulfur cluster assembly enzyme and is alternatively known as “iSU2/ ; "NIFU,” and “NIFUN.”
  • the ISCU gene encodes two isomeric forms, ISCU I and ISCU2, of the iron-sulfur cluster scaffold protein and the structures of the proteins in complex with other iron-sulfur cluster assembly proteins is biown (see, for example, ajewska er a!, (2013) J, Biol Chem. 288:29134-29142).
  • ISCU l is used according to the present invention.
  • ISCU2 is used according to the present invention.
  • both ISCU l and ISCU2 are used n combination according to the present invention.
  • At least two splice variants encoding the two distinct human ISO.) isofomis exist and sequences are publicly available on the GenBank database maintained by the U.S. National Center for
  • human ISCU transcript variant 1 human ISCU transcript variant 1
  • ISCU 1 isoform MP 0551 1 .1
  • the ISCU i isoform is found, io the eytosol and nucleus.
  • Human ISCU transcript variant 2 (NM_213595.2) encodes the longer ISCU2 isoform (NP 998760.1 ), which is found in mitochondria.
  • Nucleic acid and polypeptide sequences of NFS orthologs is species other than humans are also well known and. include, for example, monkey ISCU (NM 001261474.1 and
  • ISCU orthoiogs Representative sequences of ISCU orthoiogs are presented below in Table 1.
  • Anti-ISCU agents including antibodies, nucleic acids, and the like are well-know in the art and include, for example, iro and derivati ves thereof. It is to be noted that the term can further be used to refer to any combination of features described herein, regarding ISCU molecules. For example, any combination of sequence composition, percentage identify, sequence length, domain structure, functional activity, etc. can be used to describe an ISCU molecule of the present invention.
  • FXN refers to frataxin and is alternatively known as “CyaY” and “FARR.”
  • the FXN gene encodes the mitochondrial frataxin protein that functions in regulating mitochondrial iron transport and respiration (Stemmler ei al. (2010) J. Biol. Chem. 285: 26737-26743; Gentry ei al. (20.13) Biochem. 52:6085-6096; Abruzzo m al (2013) BioMed R . Intl. 20 3, article ID 276808; and Pastore and Puccio (2013 ⁇ J.
  • human FXN transcript variant I (NMJiOO.144.4) encodes the long human FXN isoform I
  • Human FXN transcript variant 2 (NM. 181425.2) uses an alternate splice site in the 3' coding region compared to variant 1 resulting in a frameshift and encodes isoform 2 (NPJ$52090.l) that is shorter and has a distinct C-terminus compared to that of isoform .1.
  • the mature peptide is represented by residues 56-1 6 and the proprotcirt is represented by residues 42-1 6.
  • NM_00.1 161706.1 uses an alternate ex on in the 3' coding region compared to variant 1 that results irt a .frame-shift and encodes isoform 3 (NP_001 155178,1) that is shorter and has a distinct C-tertninus compared to that of isoform 1.
  • the mature peptide is represented by residues 56-171 and the proprotetn is represented by residues 42-171, Each of the isoforms is functional.
  • Nucleic acid and polypeptide sequences of FXN orthologs in species other than humans are also well known and include, for example, chimpanzee FXN
  • Anti-FXN agents including antibodies, nucleic acids, and the like are well-known irt the art and include, for example, iron and heme, it is to be noted that the term can further be used to refer to any combination of features described herein -regarding FXN molecules. For example, any combination of sequence composition, percentage identify, sequence length, domain structure, functional activity, etc. can be used to describe an FXN molecule of the present invention.
  • NFXJ l encodes a protein that is localized to mitochondria and plays a critical role in iron-sulfur cluster biogenesis (Li et ah (2013) Biachem, 52:4904-4913).
  • the encoded protein assembles and transfers 4Fe-4S clusters to target apoproteins including succinate dehydrogenase and lipoic acid synthase.
  • Nucleic acid and polypeptide sequences of FU 1 orthologs in species including humans are also well known and include, for example, human NFUl (NMJM 5700.3, NPJ>56S!
  • NMJM002755.2, NPJX)l002755.i mature peptide represented by residues 10-254, NMJ.IOl 002756.2, and NP . 001.002756, 1 , all of which isoforms are fotiettonal.i, cow NFUl (NM. 001 46566.2 and P 1 001040031 .1), mouse NFU 1 (NMJ)01170591 J , NP_001 1.64062.1 , NM_020045.3, and NP_064429.2), rat NFU l (NM . 001 106606.2 and NP 001100076,2), and chicken NFUl (NM 001006305.2 and NP . OOl 006305.2 ).
  • GL.RX5 encodes a mitochondrial protein, whose crystal structure- function relationship is known, that i involved in the biogenesis of iron-sulfur clusters and is required for normal homeostasis (Ye ei al. (2010) J, Clin. Invest 120; 1749- 17 1 and Johansson et al. (201 1) Biochem J. 433:303-31 1 ).
  • Nucleic acid and polypeptide sequences of GLRX5 orthologs in species including humans are also well known and include, for example, human GLRX5 (NM 016417.2 and NP .
  • BOLA3 encodes a protein, for which the structure-functi n relationship is known, that plays an essential, role in the production of iron-sulfur (Fc-S) clusters for the normal maturation of itpoate-containing 2-oxoacid dehydrogenases, and for the assembly of the mitochondrial respirators' chain complexes (Cameron et al (201 1 ) Am. J. Hum. Genet. 89:486-495; Zhou et al. (2008) Afo/. Ceil. Biochem. 317:61-68; and asai et al (2004) Protein Set 13:545-548), Two alternatively spliced transcript variants encoding different isoforms with distinct subcellular localization are known. Isoform 1 (NM_212552.2 and NP_9977.I 7.2) are mitochondrial, whereas isoform 2 (NM_0010 5505. 1 and
  • NP_00i030582, l are cytoplasmic.
  • Nucleic acid and polypeptide sequences of BOLA3 orthologs in species other than humans are also well known and include, for example, chimpanzee BOLA3 (XM J)01 153666.2, XP_00? 153666.1, XM_515554.2, and
  • HSCB also known as the HscB iron-sulfur cluster co-ehaperone homolog
  • HSCB also known as the HscB iron-sulfur cluster co-ehaperone homolog
  • Nucleic acid and polypeptide sequences of HSCB orthologs in species including humans are also well known and include, for example, human HSCB (NM 172002.3 and NP .. 741 9 .3 (mature peptide represented b residues 30-235)), chimpanzee HSCB
  • monkey HSCB NMJIOl 194228.1 and NPJXH 181157.1
  • dog HSCB XM.J34725.4 and XPJ34725.2
  • cow HSCB NMjOOl 102340.1 and NP ⁇ OO 109581.0.1
  • mouse HSCB KM J 53571.2 and P . 705799,2
  • rat HSCB NM 001 108340 J and P .001 101810.
  • chicken BSCB XM_003 42207.2 artel XPJ
  • HSPA9 also known as mortalm
  • the encoded protein for which the structure-function relationship is known, is primarily locaiized to the mitochondria but is also found in the endoplasmic reticulum, plasma membrane and cytoplasmic vesicles (Lao el al (2010) Protein. Expr, Purif. 72:75- B 1 and Craig and Marszalek (2002) Cell Mol Life Scl 59; 1 58-1 65) .
  • HSPA9 orthologs in species including humans include, for example, human HSPA9 (NM 004134.6 and MP 004125.3 (mature peptide represented by residues 47-679)), chimpanzee HSPA9 (XMJXM 171426.3 and XPJ)01171426.2), dog HSPA9 (XM 531923.4 and XPJ31923.2), cow HSPA9
  • NPJB4 11 .2) rat HSPA9 (NM 001 1006 .2 and N J ) 01 94.128.2)
  • chicken HSPA9 NM 00100 1 7.1 and NP . 001 06147.1 ⁇ .
  • ISCA 1 also known as iron-sulfur cluster assembly 1 , encodes a mitochondrial protein involved in the biogenesis and assembly of iron-sulfur clusters, which play a role in electron-transfer reactions.
  • the encoded protein for which the structure-func tion relationship is known, is primarily localized to the mitochondria but is also found in the endoplasmic reticulum, plasma membrane and cytoplasmic vesicles (Cozar-Casteliano el al. (2004) Biochim. Biop ys. Acta. 1700: 179-188; Lu et l. (2010) Blochem. J. 428: 1 25- 131; and Song ei al. (2009) J. Biol. Chem. 284:35297-35307).
  • Nucleic acid and polypeptide sequences of ISCA 1 orthologs in species including humans are also well known and include, for example, human ISCA 1 (NM_030940.3 and N1 12202.2 (mature peptide represented by residues 13-129», chimpanzee ISC 1 (NM_O01242612.1 and NP formulate 001229541.1), dog ISCA1 (XMJS44342.3 and XT 49435.2), cow ISCA1
  • ISCA2 also known as iron-sulfur cluster assembly 2
  • Isoform .1 M_ 1 4279.3 and NPJ? .1 255.2 (mature peptide represented by residues 9-154)) represents the longer isoform and isoform 2
  • NMJ Ol 272007.
  • i and NPjOOl 258936 J (mature peptide represented by residues 9-60)) is encoded by a nucleic acid that lacks an alternate coding exon compared to transcript variant. 1 resulting in a frameshift and a. shorter isoform having a distinct C-termtnus relative to isoform 1.
  • Each isoform is functional.
  • Nucleic acid and polypeptide sequences of ISCA2 orthologs in species other than humans are also well known and include, for example, chimpanzee ISCA2 (XM .. 001. 143075.3 and XP ... 001 143075.2), monkey ISCA2
  • IBA57 also known as MMDS 3, encodes a protein involved in iron-stiifur protein biosynthesis and normal heme biosynthesis (BoJar t al. (2013) Hum. MoL Genet. 22:2590- 2602; Sheftel etai (2012) MoL Bioi. Ceil, 23: 1157-1 166; and NHsson et ai. (2009) Ceil Mei boi 10: 1 19-130).
  • Nucleic acid and polypeptide sequences of ISCA1 ortlioiogs m species including humans are also well known and include, for example, human iSCA 1 (NM_00.1010867.2 and NP_0 1.010867.1 ; the mature peptide is represented by residues 40- 356 since residues 1-39 represent a transit, peptide), chimpanzee ⁇ 57 (XMJ514253.3 and X " P_514253.2), monkey 1BA57 (XM 001083460.2 and XP .
  • NP 001 102297.1 NP 001 102297.1
  • chicken 1BA57 NM. 001030958.2 and NP 001026129.2
  • NUBPL also known as IND l and HU ' LND l , encodes a member of the Mrp NBP35
  • the encoded protein is required for the assembly of the respirators' chain NA.DH dehydrogenase (complex I) onto an oligomeric enzymatic complex located in the inner mitochondrial membrane.
  • the respiratory complex 1 consists of 45 subunits and 8 iron-stilfur (Fe/S) clusters.
  • This protein is an Fe/S protein that plays a critical role in the assembly of .respiratory comple i, likely by transferring Fe/S into the Fe/S-contairang complex 1 subuniis (Sheftel et ai (2009) MoL Bioi Cell. 29:6059-6073; Calvo et ai. (2010) Nat. Genet.
  • Isoform i (NMJI25152.2 and N.PJ)79428.2 (mature peptide represented by residues 39-319)) represents the longest isoform, whereas isoform 2 (NMJK ) 120.1573.1 and NP ...
  • NUB PL orthologs in species other than humans are also well known and include, for example, monkey NUBPL (XM 001 108145,2 and
  • SLC2SA28 also known as solute carrier family 25 (mitochondrial iron transporter) member 28 and mitofe.rr.m2 and MRS3/4, encodes a mitochondrial iron transporter that mediates iron uptake and is required for heme synthesis of hemoproteins and Fe-S cluster assembl in non-etytliroid ceils.
  • the iron delivered into the mitochondria presumably as Fe(2+), is then delivered to ferroeheiatase to catalyze Fe(2+) incorporation into
  • Nucleic acid and polypeptide sequences of SLC25A28 orthologs in species including humans arc also well known and include, for example, human SLC25A2S (NM 031212,3 and NPJ 12489.3), monkey SLC25A28 ( M 001265757. 1 and NP_00! 252686.1), dog SLC25A28 (XM 846248.3 and XP .. 851341.2), cow SLC25A28 (NM.001205552.1 and NP .001192481.1 mouse SLC25A28 (NM . 1451 6.1 and NP . 6601 8.1 ), rat SLC25A28 (NMJKi l 109 15.1 and NPJIOI 102985.1), and chicken SLC25A28 (XM_421702.3 and XM_421702.3).
  • FD.X L also known as ferrodoxin reductase, encodes a mitochondrial flavoprotein that initiates electron transport for cytochromes P450 receiving electrons froni ADPR (Slit et al (2012) Biochim. Biophys. Acta 1 823:484-492; Lin et al ( 1990) Proa Natl. Acad. Set. U.S.A. 87:8516-8520; and Liu and Chen (2002) Oncogene 21:7195-7204)... Seven alternatively spliced human transcript variants encoding seven different isoforms are known (NM . 024417,3, NP 077728.2 (mature peptide represented by residues 33-4 1),
  • Nucleic acid and polypeptide sequences of FDXR orthologs in species other than humans are also well known and include, for example, chimpanzee FDXR
  • FDX2 also known as ferrodoxin 1 like, encodes a mitochondrial ferrodoxin required for iron-sulfur protein biogenesis and cellular iron homeostasis as F.D.X2 deficienc leads to increased cellular iron uptake, iron accumulation in mitochondria, and impaired Fe/S protein biogenesis (Sheftel et oi (2010) Prae. Nail. Acad Set. U.S.A.
  • Nucleic acid and polypeptide sequences of FDX2 orthologs in species including humans are well known and include, for example, human FDX2 (NM 001031734.2 and NP 001026904.1 (mature peptide represented by residues 53-183)), chimpanzee FDX2 (XM. 512366.4 and
  • XP_512366.3 monkey FDX2 (XMJ ) O i 105309.2 and XPJX ) l 105309.2), dog FDX2 (XMJ42073.4 and XPJ42073.1), cow FDX2 (NMJK) 1080226.2 and NPJ)0i 073695.1 ), mouse FDX2 (NMJK) 1 39824.2 and NPJK) 1034 13.1 ), and rat FDX2 (NM J ) 0.1 1 8002.1 and NP 001 101472.1 ),
  • an “'isolated protein” refers to a protein that is substantially free of other proteins, cellular material, separation medium, and culture medium: when isolated from cell or produced by recombinant DNA techniques, or chemical precitrsors or other chemicals when chemically synthesized.
  • An “isolated” or “purified” protein or biologically active portion thereof is substantiall free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody, polypeptide, peptide or fusion protein i deri ved, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of a biomarker polypeptide or fragment thereof in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • the language “substantially free of cellular material” includes preparations of a biomarker protein or fragment thereof, having less than about 30% (by dry weight) of non-biomarker protein (also referred to herein as a "contaminating protein”), more preferably less than about 20% of non-biomarker protein, stil! more preferably less than abo ut 10% of non-biomarker protein,, and most preferably less than about 5% non- biomarker protein.
  • polypeptide, peptide or fusion protein or fragment thereof e.g. , a biologically active fragment thereof
  • it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
  • kits is any manufacture (e.g. a package or container) comprising at least one reagent, e.g. a probe or small molecule, for specifically detec ting and/or affecting the expression of a marker of the invention.
  • the kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention.
  • the kit may comprise one or more reagents necessary to express a composition useful in the methods of the present invention, in certain embodiments, the kit may further comprise a reference standard, e.g., a nucleic acid encoding a protein that does not affect or regulate signaling pathways controlling cell growth, division, migration, survival or apoptosis.
  • control proteins including, but not limited to, common molecular tags ⁇ e.g., green fluorescent protein and heta-gaiaetosidase), proteins not classified in any of pathway encompassing cell growth, division, migration, survival or apoptosi by
  • Reagents in the kit may be provided in individual containers or as mixtures of two or more reagents in a single container.
  • instructional materials which describe the use of the compositions within the kit can be included.
  • neoadjuvant therapy refers to a treatment given before the primary treatment.
  • neoadjuvant fherapy can include chemotherapy, radiation therapy, and hormone therapy.
  • neoadjuvant therapy can allows patients with large breast cancer to undergo breast-conserving surgery.
  • NFS! pharmacodynamic biomarkers refers to biomarkers and related assays whose modulation is correlated with that of NFS 1 such that they can be used as surrogates, combinations, or other readouts associated with NFS] modulation.
  • Representative examples include, without limitation 1) decreased conversion of cysteine to alanine or methylene blue; 2) induction and/or promotion of mitochondrial dysfunction, such ⁇ ts a) a decrease in aconitase copy number, amount, and/or activity and/or b) a decrease in succinate dehydrogenase copy number, amount-, and/or activity; 3 ⁇ induction and/or promotion of iron regulatory protein dysfunction, such as a) a decrease in ferritin copy number, amount, and/or activity and/or b) an increase in transfcrria-rccep or copy number, amount, and/or activity and/or c) a decrease in Hif2alpha copy number, amount, and/or activity; and 4) induction and/or promotion of ferroptosis, such as a) an increase and/or accumulation of lipid reactive oxygen species (ROS) and/or b) art increase in PTGS2 (COX2) copy number, amount, and/or activity.
  • ROS lipid reactive
  • iron-sulfur proteins that function to catalyze the conversion of citrate to isoeitrate.
  • iron -responsive elements IREs
  • the protein binds to IRE, it resul ts in repression of translation of ferritin mRNA, and inhibition of degradation of the otherwise rapidly degraded transferrin receptor mRNA.
  • aconitascs there are two forms of aconitascs in mammalian cells, including a cytoplasmic aconitase encoded by Acol and a mitochondrial aconitase encoded by Aco2.
  • the term "aconiiase” encompasses the combination of nucleic acids and/or proteins of Acol and Aco2.
  • the term ""aconitase” encompasses the nucleic acids and/or proteins of Acol alone or of Aco2 alone.
  • Acol encodes a bifonctionai, cytosolic protein that functions as an essential enzyme in the TCA cycle and interacts with mRNA to control the levels of iron inside cells. When cellular iron levels are high, this protein binds to a 4Fe-4S cluster and functions as an acom ' fcse (Philpott et al. (1 94) Free. Nail Acad. Set. U.S.A. 91:7321-7325; Bra-ssolotto et al (1999) J. Biol. Chem. 274:21625-21630; aptain et al. (1991) Proc. Natl Acad. Set U.S.A.
  • Transcript variant 1 (NM 001278352. J ) represents the longer transcript and transcript variant 2 (NM 0021 7,2) differs from transcript variant 1 b having a different 5' UTR despite the fact that both transcript variants encode the same protein (NP_002 ' l88. ' l and NP 00 i 265281.1 ).
  • Nucleic acid and polypeptide se uences of Aco 1 orthologs in species other than humans are also well known and include, for example, chimpanzee Acol (XM ⁇ OOH 56102.3 and XPJ)0 I I 56102.1), monkey Acol (NMJXH25786S.J and NPJM ) 1244794.1 ), dog Acol (XM Stamm 538698.4 and XP 38698.2), cow Acol
  • NP 031412.2 rat Acol (NM.. 017321.1 and NP 059017.1), and chicken Acol
  • Aco2 encodes a Afunctional, mitochondrial protein that catalyzes the
  • Nucleic acid and polypeptide sequences of Aco2 ortho!ogs in species including humans are well known and include, for example, human Aco2 (NM 001098.2 and NP 001089.1 (mature peptide represented by residues 28-780)), monkey Aco2 (NMJ1 1261 164.2 and NP ⁇ OO 1248093.1), dog Aco2 (XMJJ44073.3 and XP 84 166.1), cow Aco2 (NM 173977.3 and NP 776402.1), mouse Aco2 (NMJ>80633.2 and NP J42364.1 ), rat Aeo2 (NM 024398.2 and NP 077374.2), chicken Aco2
  • reductase SQ
  • respiratory Complex 11 is a well-known enzyme complex that exists in. bound form on the inner mitochondrial membrane of mammalian mitochondria
  • SDH catalyzes the oxidation of succinate to marate with the reduction of ubiquinone to ubiquinol.
  • Mammalian and mitochondrial SDH are composed of four subunits: two hydrophilic and two hydrophobic. The first two subtmits. a t!avoprotein (SdhA) and an iron-sulfur protein ( SdhB), are hydrophilic.
  • SdhA contains a covalentiy attached flavin adenine dinucleotide (FAD) cofaeior and the succinate binding site and SdhB contains three iron-sulfur clusters: [2Fe-2S],
  • the second two subunits are hydrophobic membrane anchor subunits, SdhC and SdhD.
  • Human mitochondria contain two distinct isoform of SdhA (Fp subunits type I and type II). The subunits form a membrane-bound cytochrome b complex with six transmembrane helices containing one heme b group and a ubiquinone-btnding site.
  • phospholipid molecules Two phospholipid molecules, one cardiolipin and one phosphatidylethanolamine, are also found in the SdhC and SdhD subunits and serve to occupy the hydrophobic space below the heme.
  • UBQ inhibitors include carboxin and the noytaifiuoroacetone.
  • Succinate-analogue inhibitors include the synthetic
  • oxaloacetate is one of the most potent inhibitors of Complex 11.
  • assays for analyzing SDH activity are well known in the art and include, for example,
  • Feritin refers to a well-known intracellular protein that stores and releases iron and exists as a globular protein complex consisting of 12 or 24 protein subunits wherein the submits associate to form a spherical nanocage. Ferritin that is not combined with iron is referred to "apoferritin.” A "ferritin protein subunif is defined as one of the 12 or 24 polypeptide subunits that make up a ferritin protein.
  • the numbering system used herein for the identification of amino acids within, ferritin sobitriiis is based on the original sequence of horse spleen L ferritin (Swiss Protein Database Accession Number P027 ),
  • the horse spleen numbering system can be easily converted to a numbering system based on the human H sequence (Swiss Protein Database accession number P02794; the human L sequence accession number is P02792), which has four additional amino acids at the N- terminus.
  • the human H sequence numbering therefore adds 4 to the corresponding amino acid number in horse spleen ferritin.
  • LI 34 by horse spleen numbering corresponds to Li 38 by human li sequence numbering. Alignments of ferritin suburtit sequences can be found, e.g., in Theil, E. , in Handbook of Metalloproieins,
  • the subunits are both the fight (L) and the heavy (H) type with an apparent molecular weight of 19 kDa or 21 kDa respectively.
  • Some ferritin complexes in vertebrates are hete.tO-oitgom.ers of two highly related gene products with slightly different physiological properties. The ratio of the two homologous proteins in the comple depends on the relative expression levels of the two genes. Assays for analyzing ferritin present, amount, and acti vity are well known in the art as described above. 'Transferrin receptors" are carrier protein for transferrin.
  • the term “transferrin receptor” encompasses the combination of nucleic acids and/or proteins of TFR1 and TFR2. In other embodiments, the term “transferrin receptor” encompasses the nucleic acids and/or proteins ofTFR! alone or of TFR2 alone.
  • transcript variant .1 (NM 003234.2) represents the longer transcript and transcript variant 2 ( M_001 12 148.1) differs from transcript variant I by having a different 5" UTR despite the fact that both transcript variants encode the same protein ( PJ ) 03225.2 and NPJXM 121620.1 ; mature peptide represented by residues 101-760)).
  • Nucleic acid and polypeptide sequences of TFR 3 orthologs in species other than humans are also well known and include, for example, monkey TFR 1 ( M_001257303.1 and
  • NPJM 1244232.
  • i k dog TFR 1 (NM. 0010031 11.1 and NPJM) 10031 1 1.1
  • cow TFR 1 (NM 001206577.1 and NP ..001 193506.1 )
  • mouse TFR1 (NM_01 1638.4 and
  • NPJB5768.1 NM_20525 J and P_990S87.1.
  • TFR2 encodes a transferrin receptor thai is highly homologous to TFR! that mediate cellular uptake of tiansfemn-boitnd iron but whose expression is largely restricted to hepatocytes (Daniels ei al. (2006) Gin. Immunol. 121 : 144- 158 and Zhao ei al. (2013) Biochem. 52:3310-3319).
  • isoform 1 (NM_003227,3 and ⁇ )03218.2) represents the longer isofomi and isofbrm 2 (NM 001206855.1 and NP ..
  • TFR2 orthoiogs in species other than humans are also well known and include, for example, chimpanzee TFR2 (X 003318650.1 and
  • NPJ ⁇ 56614.3 rat TFR2 (NM jO t 105916.1 and NP OO10993S6.1 ), and zebrafish TFR2 (NMJM00 916.1 and PjOOl 09916.1 ⁇ .
  • i 4 Hif2a also known as endothelial PAS domain protein 1 , encodes a transcription factor invoi ved in the induction of genes regulated by oxygen, which is induced as oxygen levels fall (Mastrogiannaki et at (2013 ⁇ Blood 122:885-892 and Haase (2010) Am. J.
  • the encoded protein contains a basic-helix-loop- helix domain protein dimerizafioti domain as well as a domain found in proteins in signal transduction pathways which respond to oxygen levels.
  • Nucleic acid and polypeptide sequences of.H.iSa orthoiogs in species including humans are well known and include, for example, human Hi£2a (NM. 001430.4 and NP . 001421.2), chimpanzee Flifia
  • PTGS2 refers to a specific isozyme of the prostaglandin-endoperoxicle synthase (PTGS), also known as cyciooxyge.nase-2, which is the key enzyme in prostaglandin biosynthesis and acts as both a dioxygenase and as a peroxidase.
  • PTGS prostaglandin-endoperoxicle synthase
  • cyciooxyge.nase-2 the key enzyme in prostaglandin biosynthesis and acts as both a dioxygenase and as a peroxidase.
  • PTGS prostaglandin-endoperoxicle synthase
  • PTGS2 encodes the inducible isozymes and is regulated by specific stimulatory events, indicating that it is responsible for the prostanoid biosynthesis .tnvoived in inflammation, and mitogencsis (Percy et l. ( 1998) Analyst 123:41- 50), Nucleic acid and polypeptide sequences of PTGS2 orthoiogs in species including humans are well known and include, for example, human PTGS2 (NM_J)00963.3 and NP 000954.1 (signal peptide sequence represents residues 1-23), chimpanzee PTGS2 (XM . 524999.4 and XP 524999.3), monke PTGS2 (XM JIOI 107538.2 and
  • Lipid reactive oxygen species (ROS)' ' refer to lipids that can participate in reactions that give rise to free radicals to thereby cause oxidative damage. Lipids are prone 5 to oxidative damage since ROS species can act on unsaturated lipids to yield reactive
  • Oxidized lipids may be identified by presence of lipid peroxides.
  • Exemplary ROS include hydroxy! radicals (OH,), superoxide radical (02.-), it ) nitric oxide (NO.), thyi (RS,), peroxyi (R02.), and lipid peroxyi (LOO,).
  • Lipids can form lipid ROS when present in conditions of oxidative challenge or stress, wherein lipids are vulnerable to oxidative damage.
  • An oxidative challenge can involve the introduction of free radicals, ROS, or reacti ve nitrogen species, such as to RBC or lysed RBC, for example in an assay of antioxidant activity.
  • the oxidative challenge may be created by adding a free radicals, ROS, or reacti ve nitrogen species, such as to RBC or lysed RBC, for example in an assay of antioxidant activity.
  • the oxidative challenge may be created by adding a free radicals, ROS, or reacti ve nitrogen species, such as to RBC or
  • the "norma! level of expression, of a biomarker is the level of expression of the biomarker in cells of a subject, e.g., a human patient, not afflicted with a cancer.
  • expression level in a test sample that is greater than the standard error of the assay employed to assess expression, and is preferably at least twice, and more preferably 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, UK 10,5, 1 1, 12, 13, 14, 1 , 16, 17, 18, 19, 20 times or more higher than the expression activity or
  • a "significantly lower level of expression" of a biomarker refers to an expression level, in a test sample that is at least twice, and more preferably 2.1 , 2,2, 2,3, 2,4, 2,5, 2,6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6,5, 7, 7.5, 8, 8.5, 0 9, 9.5, 10, 10.5, .1 1 , 12, 13, 14, 15, 16, 1.7, 18, 19, 20 times or more lower than the
  • control sample e.g. , sample from a healthy subject not having the biomarker associated disease
  • average expression level preferably, the average expression level, of the biomarker in several control samples.
  • signaling levels can also be applied to an other measured parameter described herein, such as for expression, inhibition,
  • the term “predictive"' incl udes the use of a biomarker nucleic acid and/ or protein status, e.g., over- or under- activity, emergence, expression, growth, remission, recurrence or resistance of tumors before, during or after therapy, for determining the likelihood of response of a cancer to anti-cancer therapy, such as iron-sulfur cl uster biosynthesis pathway inhibitor treatment (e.g., NFS ! inhibitors).
  • Such predictive use of the biomarker may be confirmed by, e.g., (1 ) increased or decreased copy number ⁇ e.g., by FISH, FISH plus SKY, single-molecule sequencing, e.g.. as described in the ar at least at J.
  • BiotechnoL, 86:289- 30 i , or qPCR overexpression or underexpression of a biomarker nucleic acid (e.g., by ISH, Northern Blot, or qPCR), increased or decreased biomarker protein (e.g., by !HC) and/or biomarker target, or increased or decreased activity, e.g., in more than about 5%, 6%, 7%, 8%, 9%, 10%, I i%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more of assayed human cancers types or cancer samples; (2) its absolute or relatively modulated presence or absence in a biological sample, e.g., a sample containing tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool or bone marrow, .f om a subject, e.g.
  • a human, afflicted with cancer (3) its absolute or relatively modulated presence or absence in clinical subset of patients with cancer (e.g., those responding to a particular anti-cancer therapy (e.g. , iron-sulfur cluster biosynthesis pathway inhibitory therapy) or those developing resistance thereto).
  • a particular anti-cancer therapy e.g. , iron-sulfur cluster biosynthesis pathway inhibitory therapy
  • prevent refers to reducing the probability of developing a disease, disorder, or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder, or condition.
  • probe refers to any molecule which is capable of selectively binding to a specifically intended target molecule, for example, a nucleotide transcript or protein encoded by or corresponding to a biomarker nucleic acid. Probes can be either synthesized by one skilled in the art, or derived from appropriate biological preparations. For purposes of detection of the target molecule, probes may be specifically designed to be labeled, as described herein. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.
  • prognosis includes a prediction of the probable course and outcome of cancer or the likelihood of recovery from the disease, in some embodiments, the use of statistical algorithms provides a prognosis of cancer in an individual.
  • the prognosis can be surgery, development of a clinical subtype of cancer (e.g., solid tumors, such as lung cancer, melanoma, and renal cell carcinoma), development of one or more clinical factors, development of intestinal cancer, or recovery from the disease.
  • response to anti-cancer therapy relates to any response of the hyperproltferattve disorder (e.g., cancer) to an anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy), such as anti-NFS 1 inhibitor therapy), preferably to a change in tumor mass and/or volume after initiation of neoadjuvant or adjuvant chemotherapy.
  • anti-cancer therapy e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy
  • Hyperproliferative disorder response may he assessed , for example for efficacy or in a neoadjuvant or adjuvant situation, where the size of a tumor after systemic intervention can be compared to the initial size and dimensions a measured by CT, PET, mammogram:, ultrasound or palpation. Responses may also be assessed by caliper measurement or pathological e amination of the tumor after biopsy or surgical resection. Response may be recorded in a quantitative fashion like percentage change in tumor volume or in a qualitative fashion like "pathological complete response” ipCRk "clinical complete remission' ' (eCR), “clinical partial remission' (cPR), “clinical stable disease” (cSD), “clinical progressive disease” (cPD) or other qualitative criteria.
  • pathological complete response ipCRk "clinical complete remission' ' (eCR), “clinical partial remission' (cPR), “clinical stable disease” (cSD), “clinical progressive disease” (cPD) or other qualitative criteria.
  • Assessment of hyperproHferative disorder response may be done early after the onset of neoadjuvant or adjuvant therapy, e.g., after a few hours, days, weeks or preferabl after a few months.
  • a typical endpoint for response assessment is upon termination of neoadjuvant chemotherapy or upon surgical removal of residual tumor cells and/or the tumor bed. This is typically three months after initiation of
  • clinical efficacy of the therapeutic treatments described herein may be determined by measuring the clinical benefit rate (CBR).
  • CBR clinical benefit rate
  • the clinical benefit rate is measured by determining the sum of the percentage of patients who are in complete remission (CR), the number of patients who are in partial remission (PR) and the number of patients having stable disease (SD) at a time point at least 6 months out from the end of therapy.
  • the shorthand for this formula is CBR s R+PR+SD over 6 months.
  • the CBR for a particular cancer therapeutic regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more.
  • Additional criteria for evaluating the response to cancer therapies are related to "survival,” which includes all of the following: sitrvi va! until mortality, also known as overall survival (wherein said mortality may be either irrespective of cause or tumor related); "recurrence- free survival" (wherein the terra recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the terra disease shall include cancer and diseases associated therewith).
  • the length of said survival may be calculated by reference to a defined start point (e.g., time of diagnosis or start of treatment) and end point (e.g., death, recurrence or metastasis).
  • criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence, for example, in order to determine appropriate threshold values, a particular cancer therapeutic regimen can be administered to a population of subjects and the outcome can be correlated to biomarker measurements that were determined prior to administration of any cancer therapy.
  • the outcome measurement may be pathologic response to therapy gi en in the neoadjuvant setting.
  • outcome measures such as overall survival and disease- free survival can be monitored over a period of time for subjects following cancer therapy for whom biomarker measurement values are known.
  • the doses administered are standard doses known in the art for cancer therapeutic agents.
  • the period of time for which subjects are monitored can vary. For example, subjects may be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 51 or 60 months.
  • Biomarker measurement threshold values that con-elate to outcome of a cancer therapy can be determined using well-known methods in the art, such as those described in the Examples section.
  • resistance refers to an acquired or natural resistance of a cancer sample or mammal to a cancer therapy (i.e., being nonresponsive to or having reduced or limited response to the therapeutic treatment), such as having a reduced response to a therapeutic treatment by 25% or more, for example, 30%, 40%, 50%, 60%, 70%, 80%, or more, to 2- foid, 3-fold, 4-foSd, 5-fold, 10-fold, 15-fold, 20-fold or more.
  • the reduction in response can he measured by comparing with the same cancer sample or mammal before the resistance is acquired, or by comparing with a different cancer sample or a mammal who is known to have no resistance to the therapeutic treatment.
  • multidrug resistance A typical acquired resistance to chemotherapy is called "multidrug resistance.”
  • the multidrug resistance can be mediated by P-glycoprotein or can be mediated by other mechanisms, or it can occur when a .mammal is infected with a mute -drug-resistant microorganism or a combination of microorganisms.
  • the term “reverses resistance'” means that the use of a second agent in combination with a primary cancer therapy ( g., chemotherapeutic or radiation therapy) is able to produce a significant decrease in tumor volume at a level of statistical significance (e.g., p ⁇ 0.05) when compared to tumor volume of untreated tumor.
  • a primary cancer therapy e.g., chemotherapeutic or radiation therapy
  • the primary cancer therapy e.g. , chemotherapeutic or radiation therapy
  • response refers to an anti-cancer response, e.g. in the sense of reduction of tumor size or inhibiting tumor growth.
  • the terms can also refer to an improved prognosis, for example, as reflected by art increased time to recurrence, which is the period to first recurrence censoring for second primary cancer as a first event or death, without evidence of rec urrence, or an increased overall survival, which is the period from treatment to death from any cause.
  • beneficial endpoint attained when exposed to a stimulus.
  • a negati ve or detrimental symptom is minimized, mitigated or attenuated on exposure to a stimulus.
  • evaluating the likelihood that a tumor or subject will exhibit a favorable response is equivalent to evaluating the likelihood that the tumor or subject will not exhibit favorable response ⁇ i.e. , will exhibit a lack of response or be iion-responsi ve).
  • R A interfering agent is defined as any agent which interferes with or inhibits expression of a target biomarker gene by RNA interference (RNAi).
  • RNA interfering agents include, but are not limited to, nucleic acid molecules including RNA molecules which are homologous to the target biomarker gene of the invention, or a fragment thereof, short interfering R (siR A), and small molecules which interfere with or inhibit expression of a target biomarker nucleic acid by RNA interference (RNAi).
  • RNA interference is an evolutionally conserved process whereby the expression or introduction of RNA of a sequence that is identical or highly similar to a target biomarker nucleic acid results in the sequence specific degradation or specific post- transcriptiona! gene silencing (PIGS) of messenger RN (mRNA) transcribed from that targeted gene (see Cobnrn, G. and Cullen, B. (2002) J, ofVimlogy 76(18):9225), thereby inhibiting expression of the target biomarker nucleic acid, in one embodiment, the RNA is double stranded RNA fdsRNA). This process has been described in plants, invertebrates, and mammalian cells.
  • RNAi is initiated by the dsRNA-specifie endonac!ease Dicer, which promotes processive cleavage of long dsRNA into double-stranded fragments termed siRNAs.
  • siRNAs are incorporated into a protein complex that recognizes and cleaves target mRNAs.
  • RNAi can also be initiated by introducing nucleic acid molecules, e.g., synthetic siRNAs, shR As, or other RNA interfering agents, to inhibit or silence the expression of target faiomarker nucleic acids.
  • inhibiting target biomarker nucleic acid expression includes any decrease in expression or protein activity or level of the target biomarker nucleic acid or protein encoded by the target biomarker nucleic acid.
  • the decrease may be of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as compared to the expression of a target biomarker nucleic acid or the activity or level of the protein encoded by a target biomarker nucleic acid which has not been targeted b an RNA interfering agent.
  • sample used for detecting or determining the presence or le vel of at least one biomarker is typically whole blood, plasma, scrum, saliva, urine, stool (e.g., feces), tears, and any other bodily fluid (e.g., as described above under the definition of "body fluids"), or a tissue sample (e.g., biopsy) such as a small intestine, colon sample, or surgical resection tissue, in certain instances, the method of the present in vention further comprises obtaining the sample from the individual prior to detecting or determining the presence or level of at least one marker in the sample.
  • cancer therapy e.g., iron-sulfur cluste biosynthesis pathway inhibitor, chemotherapeutic, and/or radiation therapy.
  • normal cells are not affected to an extent that causes the normal cells to be unduly injured by the anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy).
  • An increased sensitivity or a reduced sensitivity to a therapeutic treatment is measured according to a known method in the art for the particular treatment and methods described herein below, including, but not limited to, cell proliferative assays (Tanigawa N, Kern D H, Kikasa Y, Morton D L, Cancer Res 1 82; 42: 2159-2164), ceil death assays (Weisenthal L M, Shoemaker R H, Marsden J A, Dill P L, Baker j A, Moran E M, Cancer Res 1984; 94: 161 - 173; Weisenthal L M, Lippman M E, Cancer Treat Rep 1985; 69: 615-632; Weisenthal L , in: Kaspers G J L, Pieters R, Twenfy an P R, Weisenthal L M, Veerman A J P, eds.
  • the sensitivity or resistance may also be measured in animal by
  • a composition or a method sensitizes response to a therapeutic treatment if the increase in treatment sensitivity or the reduction in resistance is 25% or more, for example, 30%, 40%, 50%, 60%, 70%, 80%, or more, to 2 -fold, 3-fold, 4-fold, 5- fcld, 10-fold, 15-fold, 20-fold or more, compared to treatment sensitivity or resistance n the absence of such composition or method.
  • sensitivity or resistance to a therapeutic treatment is routine in the art and within the skill of n ordinarily skilled clinician, ft is to be understood that any method described herein for enhancing the efficacy of a cancer therapy can be equally applied to methods for sensitizing hyperproh erative or otherwise cancerous cells (e.g., resistant cells) to the cancer therapy.
  • iron- sulfur cluster biosynthesis pathway inhibitor agents refers to the combined effect- of two or more iron- sulfur cluster biosynthesis pathway inhibitor agents can be greater than the sum of the separate effects of the anticancer agents alone,
  • RNA Short interfering RNA
  • small interfering RNA small interfering RNA
  • RNA is defined as an agent: which functions to inhibit expression of a target bioroarker nucleic acid, e.g. , by RNAi.
  • An siRNA may be chemically synthesized, ma be produced by in vitro transcription, or may be produced within a host cell.
  • siR A is a double stranded RNA (dsRNA) molecule of about 15 to about 40 nucleotides in length, preferably about 15 to about 28 nucleotides, more preferably about 1 to about 25
  • the siRN A is capable of promoti ng RN A interfere nce through degradation or specific post-transcriptional gene silencing (PTGS) of the target messenger RNA (raRNA).
  • PTGS post-transcriptional gene silencing
  • an siRNA is a small hairpin (also called stem loop) RNA (shRNA).
  • shRNAs are composed of a short (e.g., 19-25
  • shRNAs may be contained in plasmtds, retroviruses, and lentiviruses and expressed from, for example, the pel III U6 promoter, or another promoter (see, e.g., Stewart, ei ai. (2003) RNA Apr;9(4):493-501 incorporated fay reference herein).
  • RNA interfering agents e.g.. siRNA molecules
  • subject refers to any healthy animal, mammal or human, or any animal, mammal or human afflicted with a cancer, e.g., lung, ovarian, pancreatic, liver, breast, prostate, and colon carcinomas, as well as melanoma and multiple myeloma.
  • a cancer e.g., lung, ovarian, pancreatic, liver, breast, prostate, and colon carcinomas, as well as melanoma and multiple myeloma.
  • subject is interchangeable with "patient.”
  • survival includes all of the following: survi val until mortality, also known as overall survival (wherein said mortality may be either irrespective of cause or tumor related); "recurrence-free survival” (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith).
  • the length of said survival may be calculated by reference to a defined start point (e.g. time of diagnosis or start of treatment) and end point (e.g. death, recurrence or metastasis), in addition, criteria for efficacy of treatment ca t be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.
  • therapeutic effect refers to a local or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance.
  • the term thus means any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental development and conditions in an animal or human.
  • therapeutic iy- effeefive amount means that amount of such a substance that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment.
  • a therapeutically effective amount of a compound will depend on its therapeutic index, solubility, and the like.
  • certain compounds discovered by the methods of the present invention may be administered in a sufficient amount to proditce a reasonable benefit/risk ratio applicable to such treatment.
  • the tons "therapeutically-efJfective amount” and “effective amount” as used herein means that amount of a compound, material, or composition comprising a compound of the present invention which is effective for producing some desired therapeutic effect in at ieast a sub-population of ceils in an animal at a reasonable benefit/risk ratio applicable to any medical treatment.
  • Toxicity and therapeutic efficacy of subject compounds may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, £?.g. , for determining the L so and the EDsu. Compositions that exhibit large therapeutic indices are preferred.
  • the L3 ⁇ 4> (lethal dosage) can be measured and can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more reduced for the agent relative to no administration of the agent.
  • the ED3 ⁇ 4 > i.e.
  • the concentration which achieves a half-maximal inhibition of symptoms can be measured and can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increased for the agent relative to no administration of the agent.
  • the IC > « i.e., the concentration which achieves haif-maximaJ cytotoxic or cytostatic effect on cancer cells
  • the concentration which achieves haif-maximaJ cytotoxic or cytostatic effect on cancer cells can be measured and can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increased for the agent relative to no administration of the agent, in some embodiments, cancer ceil growth in an assay can he inhibited by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,, 65%, 70%, 75%,, 80%, 85%, 90%,, 95%, or even 100%.
  • At least about a 1.0% , 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 1.00% decrease in a solid malignancy can. be achieved.
  • a “transcribed polynucleotide” or “nucleotide transcript” is a polynucleotide (e.g. an raRNA, hnRNA, a cDNA, or an analog of such RNA or cDNA) which is complementary to or homologous with all or a portion of a mature mRNA made by transcription of a bio-marker nucleic acid and normal post-rranscriprional processing (e.g. splicing), if any, of the RNA transcript, and reverse transcription of the RNA transcript.
  • a polynucleotide e.g. an raRNA, hnRNA, a cDNA, or an analog of such RNA or cDNA
  • nucleotide triplet An important and well known feature of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one coding nucleotide triplet may be employed (illustrated above). Therefore, a number of different nucleotide sequences may code tor a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrmridine may be found in a given nucleotide sequence. Such methylations do not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.
  • the nucleotide sequence of a D A or R A encoding a biomarker nucleic acid can be used to derive the polypeptide amino acid sequence, using the genetic code to translate the DNA or RNA into an amino acid sequence.
  • corresponding nucleotide sequences that can encode the polypeptide can be deduced from the genetic code (which, because of its redundancy, will produce multiple nucleic acid sequences for any given amino acid sequence).
  • description and/or disclosure herein of a nucleotide sequence which encodes a polypeptide should be considered to also include description ancl or disclosure of the amino acid sequence encoded by the nucleotide sequence.
  • description and/or disclosure of a poly peptide amino acid sequence herein should be considered to also include description and/or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.
  • nucleic acid and amino acid sequence information for the loci and biomarkers of the present invention and related biomarkcrs are well known in the art and readily available on publicly available databases, such as the National Center for Biotechnology information (NCBl).
  • NCBl National Center for Biotechnology information
  • exemplary nucleic acid and amino acid sequences derived from publicly available sequence databases are provided below.
  • biomarkers described above are presented below in Table 1. It is to be noted that the terms described above can further be used to refer to any combination of features described herein regarding the biomarkers. For example, any combination of sequence composition, percentage identify, sequence length, domain structure, functional activity, etc. can be used to describe a biomarker of the present invention.
  • ROS lipid reactive oxygen species
  • RNA nucleic acid molecules e.g., thy mines replaced with uredines
  • nucleic acid moiecuies encoding orthologs of the encoded proteins
  • DNA or RNA nucleic acid sequences comprising a nucleic acid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with the nucleic acid sequence of any SEQ ID NO listed in Table I , or a portion thereof.
  • nucleic ac id molecules can have a function of the full-length nucleic acid as described further herein.
  • polypeptide moiecuies comprising an amino acid sequence having at least %, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with an amino acid sequence of any SEQ ID NO listed in Table 1 , or a portion thereof.
  • polypeptides can have a function of the full-length polypeptide as described further herein.
  • the subject for whom cancer treatment is administered or who is predicted likelihood of efficacy of an anti-cancer therapy is determined, is a mammal (e.g., mouse, rat, primate, non-human mammal, domestic animal such as dog, cat, cow, horse), and is preferably a human.
  • an anti-cancer therapy e.g. , iron-sulfur cluster biosynthesis pathway inhibitory therapy
  • the sub ject has not undergone treatment, such as chemotherapy, radiation therapy, targeted therapy, and/or anti-cancer therapy (e.g., iron-stilmr cluster biosynthesis pathway inhibitory therapy), in still another embodiment, the subject has undergone treatment, such as chemotherapy, radiation therapy, targeted therapy, and/or anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy).
  • treatment such as chemotherapy, radiation therapy, targeted therapy, and/or anti-cancer therapy
  • anti-cancer therapy e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy
  • the subject has had surgery to remove cancerous or precancerous tissue.
  • the cancerous tissue has not been removed, e.g. , the cancerou tissue may be located in an inoperable region of the body, such as in a tissue that is essential for life, or in a region where a surgical procedure would cause considerable risk of harm to the patient.
  • the methods of the invention can be used to determine the responsiveness to anticancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy) of many different cancers in subjects such as those described above, in one embodiment, the cancers are hematologic cancers, such as leukemia. In another embodiment, the cancers are solid tumors, such as lung cancer, melanoma, and/or renal ceil carcinoma.
  • anticancer therapy e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy
  • the cancers are hematologic cancers, such as leukemia.
  • the cancers are solid tumors, such as lung cancer, melanoma, and/or renal ceil carcinoma.
  • the cancer is an epithelial cancer such as, but not limited to, brain cancer (e.g., glioblastomas) bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer.
  • brain cancer e.g., glioblastomas
  • breast cancer e.g., cervical cancer, colon cancer
  • gynecologic cancers e.g., renal cancer, laryngeal cancer
  • lung cancer e.g., oral cancer, head and neck cancer
  • ovarian cancer e.g., pancreatic cancer, prostate cancer, or skin cancer.
  • biomarker presence, absence, amount, and/or activity measurements) in a sample from a subject is compared to a predetermined control (standard) sample.
  • the sample from the subject is typically from a diseased tissue, such as cancer ceils or tissues.
  • the coturoi sample can be from the same subject or from a different subject.
  • the control sample is typicall a normal non-diseased sample. However, in some embodiments, such as for staging of disease or for evaluating the efficacy of treatment, the control sample can be from a diseased tissue.
  • the control sample can be a combination of sample from several different subjects. n some embodiments, the biomarker amount and/or activity measurements) from a subject is compared to a pre-determined level.
  • This predetermined level is typically obtained from normal samples, such as the normal copy number, amount, or activity of a biomarker in the cell or tissue type of a member of the same species as from which the test sample was obtained or a non-diseased cell or tissue from the subject from which the test samples was obtained.
  • a "predetermined" biomarker amount and/or activity measurements may be a bioniarker amount and/or activity measurements) used to, by way of example only, evaluate a sub jec t that may be selected for treatment, evaluate a response to an anti-cancer therapy (e.g., iron- sulfur cluster biosynthesis pathway inhibitory therapy), and/or evaluate a response to a combination anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory- therapy plus immunoinhibitory inhibitor therapy).
  • a pre-determined biomarker amount and/or activit measurement 8 ma be determined in populations of patients with or without cancer.
  • the pre-determined biomarker amount and/or activity measurements can be a single number, equally applicable to every patient, or the pre-determined biomarker amount and/or activit measurements) can vary according to specific subpopitlations of patients. Age, weight, height, and other factors of a subject may affect the pre-determined biomarker amount and/or activity measurements) of the individual. Furthermore, the predetermined biomarker amount and/or activity can be determined for each subject individually, in one embodiment, the amounts determined and/or compared in a method described herein are based on absolute measurements. In another embodiment, the amounts determined and/or compared in a method described herein are based on relative measurements, such as ratio (e.g., biomarker expression normalized to the expression of a housekeeping gene, or gene expression at various time points).
  • ratio e.g., biomarker expression normalized to the expression of a housekeeping gene, or gene expression at various time points.
  • the pre-determined biomarker amount and/or activity measurement ' s) can be any suitable standard.
  • the pre-determined biomarker amount and/or activity measurements can be obtained from the same or a different human for whom a patient selection is being assessed, in one embodiment, the pre-determined biomarker amount and/or activity measurements) can ' be obtained from a previous assessment of the same patient In such a maimer, the progress of the selection of the patient can be monitored over time.
  • the control can be obtained from an assessment of another human or multiple humans, e.g.
  • selected groups of humans if the subject is a human, in such a manner, the extent of the selection of the human for whom selection is being assessed can be compared to suitable other humans, .g., other humans who are in a similar situation to the human of interest, such as those suffering from similar or the same conditions) and/or of the same ethnic group.
  • the change of biomarker amount and/or activity measurements.) from the predetermined level is about 0.5 fold, about 1 ,0 fold, about 1 .5 fold, about 2.0 fold, about 2.5 fold, about 3.0 fold, about 3,5 fold, about 4.0 fold, about 4.5 fold, or about 5.0 fold or greater.
  • the fold change is less than about 1 , less than about 5, less than about 10, less than about 20, less than about 30, less than about 40, or less than about 50. io.
  • the fold change in biomarker amount and/or activity' measurements) compared to a predetermined level is more than about 1 , more than about 5, more than about 10, more than about 20, snore than about 30, more than about 40, or more than about 50,
  • Body fluids refer to fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g.
  • amniotic fluid amniotic fluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, co per's fluid or pre-ejaculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomit).
  • the subject and/or control sample is selected from the group consisting of cells, cell lines, histological slides, paraffin embedded tissues, biopsies, whole blood, nipple aspirate, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bone marrow.
  • the sample is serum, plasma, or urine, in another embodiment, the sample is scrum,
  • the samples can be collected from individuals repeatedly over a longitudinal period of time (e.g., once or more on the order of days, weeks, months, annually, biannually, etc.). Obtaining numerous samples from an indi vidual over a period of time can be used to verify results from earlier detections and/or to identify an alieration in biological pattern as a result of, for example,, disease progression, drug treatment etc.
  • subject samples can be taken and monitored every month, every two mouths, or combinations of one, two, or three month intervals according to the invention, in addition, the biomarker amount and/or activity measurements of the subject obtained over time can be conveniently compared with each other, as well as with those of normal control during the monitoring period, thereby providing the subject's own values, as an internal, or personal, control for long-term monitoring.
  • Sample preparation and separation can involve any of the procedures, depending on the type of sample collected and/or analysis of biomarker measurements).
  • Such procedures include, by way of example only, concentration, dilution, adjustment of pH, removal of high abundance polypeptides ⁇ e.g., albumin, gamma globulin, and transferrin, etc.), addition of preservatives and calibrants, addition of protease inhibitors, addition of denaturants, desalting of samples, concentration of sample proteins, extraction and purification of lipids.
  • the sample preparation can also isolate molecules that are bound in non-eovalent complexes to oilier protein (e.g., carrier proteins).
  • oilier protein e.g., carrier proteins
  • This process may isolate those molecules bound to a specific carrier protein (e.g. , albumin), or use a more general process, such as the release of hound molecules from. all. carrier proteins via. protein denaturation, for example using an acid, followed by removal of the carrier proteins.
  • undetectable proteins from a sample can be achieved using high affinity reagents, high, molecular weight filters, uStraeentrifugatton and/or e!eeirodia!ysis.
  • High affinity reagents include antibodies or other reagents (e.g., aptamers) that selectively bind to high abundance proteins.
  • Sample preparation could also include ion exchange chromatography, metal ton affinity chromatography, gei filtration, hydrophobic chromatography, chromatofocusing, adsorption chromatography, isoelectric focusing and related techniques.
  • Molecular weight filters include membranes that separate molecules on the basis of size and molecular weight. Such filters may further employ re verse osmosis, nanofiltration, ultrafiltration and microfiltratton.
  • Ultracenrrifugation is a method for removing undesired polypeptides from a sample
  • Uliraeentrifugation is the eentrifisgation of a sample at about 15,000-60,000 rpm while monitoring with an optical system the sedimentation (or lack thereof) of particles.
  • Electrodialysis is a procedure which uses an eiectromembnme or semipermable membrane in a process in which ions are transported through semi-permeable membranes from one solution to another under the influence of a potential gradient. Since the membranes used in electrodialysis may have the ability to selectively transport ions having positive or negative charge, reject ions of the opposite charge, or to allow species to migrate through a semipermabie membrane based on size and charge, it renders electrodialysis useful for concentration, removal, or separation of electrolytes.
  • Separation and purification in the present invention may include any procedure known in the art, such as capillary electrophoresis (e.g., in capillary or on-chip) or
  • Electrophoresis is a method which can be used to separate ionic molecules under the influence of an electric field. Electrophoresis can be conducted in a gel, capillars', or in a mierocharmel on a chip.
  • gels used for electrophoresi include starch, aerylamide, polyethylene oxides, agarose, or combinations thereof.
  • a gel can be modified fay its cross-linking, addition of detergents, or denatnrants, immobilization of enzymes or antibodies (affinity
  • capillaries used for electrophoresis include capillaries that interface with an electrospray.
  • CE Capillary electrophoresis
  • CE can be further segmented into separation techniques such as capillary zone electrophoresi (CZE), capillary isoelectric focusing (CIEF), capillary isotachophoresis (clTP) and capillary eleetroeliromatography (CEC).
  • CZE capillary zone electrophoresi
  • CIEF capillary isoelectric focusing
  • clTP capillary isotachophoresis
  • CEC capillary eleetroeliromatography
  • An embodiment to couple CE techniques to electrospray ionization involves the use of volatile solutions, for example, aqueous mixtures containing a volatile acid and/or base and an organic such as an alcohol or aceiomtrile,
  • Capillary tsotachophoresis is a technique in which the analytes move through the capillary at a constant speed but are nevertheless separated by their respective mobilities.
  • Capillary zone electrophoresis also known as free-solution CE (PSCE)
  • PSCE free-solution CE
  • CEC Capillary isoelectric focusing
  • HPLC high, performance liquid chromatography
  • Separation and purification techniques used in die present- invention include any chromatography procedures known in the art. Chromatography can be based on the differential adsorption and elution of certain anaiytes or partitioning of analy es between mobile and stationary phases. Different examples of chromatography include, but not limited to, liquid chromatography (LC), gas chromatography (GC), high performance liquid chromatography (H.PLC), etc,
  • nucleic acid molecule is intended to include DNA moiecuies (e.g., cDNA or genomic DNA) and RNA molecules (eg., tn ' RNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is doubie- stranded DN A.
  • nucleic acid molecule is one which is separated from other nucleic acid moiecuies which are present in the natural source of the nucleic acid molecule.
  • an "isolated" nucleic acid molecule is free of sequences (preferably protein- encoding sequences) which naturally flank the nucleic acid ⁇ i.e. , sequences located at the 5 * and 3' ends of the nucleic acid) in the genomic DNA of the organism from: which the nucleic acid is derived.
  • the isolated nucleic acid molecule can contain less than about 5 kB, 4 kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the eel! from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized,
  • a biomarker nucleic acid molecule of the present invention can be isolated using standard molecular biolog techniques and the sequence information in the database records described herein. Using all or a portion of such nucleic acid sequences, nucleic acid molecules of the invention can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al. y edminister Molecular Cloning: A Laboratory Manual, 2nd d., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). A nucleic acid molecule of the invention can be amplified using cDNA, mR A, or genomic DNA as a template and appropriate oligoiiueieotide primers according to standard PGR amplification techniques.
  • oligonucleotide* corresponding to ail or a portion of a nucleic acid molecule of the invention can be prepared by standard synthetic techniques, e.g., using an automated DN A synthesizer.
  • nucleic acid molecule of the in vention can comprise only a portion of a nucleic acid sequence, wherein die fail length nucleic acid sequence comprises a marker of the invention or which encodes a polypeptide corresponding to a marker of the invention.
  • Such nucleic acid molecules can be used, for example, as a probe or primer.
  • the probe/primer typically is used as one or more substantially purified oligonucleotides.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 7, preferably about 15, more preferably about 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or 400 or more consecutive nucleotides of a biomarker nucleic acid sequence.
  • Probes based on the sequence of a biomarker nucleic acid molecule can be used to detect transcripts or genomic sequences corresponding to one or more markers of the invention.
  • the probe comprises a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • a biomarker nucleic acid molecules that differ, due to degeneracy of the genetic code, from the nucleotide sequence of nucleic acid molecules encoding a protein which corresponds to the biomarker, and thus encode the same protein, are also contemplated.
  • thai DNA sequence polymorphisms that lead to changes in the amino acid sequence can exist within a population (e.g., the human population). Such genetic polymorphisms can exist among individuals within a population due to natural allelic variation. An allele is one of a group of genes which occur alternatively at a given genetic locus, in addition, it will be appreciated that D A polymorphisms that affect R A expression levels can also exist that may affect the overall expression level of that gene (e.g., by affecting regulation or degradation).
  • allele which is used interchangeably herein with “allelic variant” refers to alternative forms of a gene or portions thereof Alleles occupy the same locus or position on homologous chromosomes.
  • the subject is said to be homozygous for the gene or aiiele.
  • the subject- is said to be heterozygous for the gene or aiiele.
  • biomarker alleles can differ from each other in a single nucleotide, or several nucleotides, and can include substitutions, deletions, and insertions of nucleotides.
  • An allele of a gene can also be a fbrin of a gene containing one or more mutations.
  • allelic variant of a polymorphic region of gene refers to an alternative form of a gene having one of several possible nucleotide sequences found in that region of the gene in the population.
  • allelic variant is meant to encompass functional allelic variants, non-functional allelic variants, SNPs, mutations and polymorphisms.
  • single nucleotide polymorphism refers to a polymorphic site occupied b a single nucleotide, which is the site of variation between allelic sequences.
  • the site is usually preceded by and followed by highly conserved sequences of the allele (e.g., sequences that vary in less than 1/100 or 1/1000 members of a population).
  • a S P usually arises due to substitution of one nucleotide for another at the polymorphic site.
  • SNPs can also arise from a deletion of a nucleotide or an insertion of a nucleotide relative to a reference allele.
  • the polymorphic si te is occupied by a base other than the reference base.
  • the altered aiiele can contain a "C” (cytidhie), "G” (guanine), or "A” (adenine) at the polymorphic site.
  • SNP's may occur in protein-coding nucleic acid sequences, in which, ease they may give rise to a defective or otherwise variant protein, or genetic disease. Such a SNP may alter the coding sequence of the gene and therefore specify another amino acid (a "missense” SNP) or a SNP may introduce a stop codon (a "nonsense" SNP).
  • SNP When a SNP does not alter the amino acid sequence of a protein, the SNP is called "silent.” SNP's may also occur in noncoding regions of the nucleotide sequence. This may result in defective protein expression, e.g., as a result of alternative spicing, or it may have no effect on the function of the protein.
  • the terms "gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide corresponding to a marker of the invention. Such natural allelic variations can typically result in 1 -5% variance in the nucleotide sequence of a gi ven gene. Alternati ve alleles can be identi fied by sequencing the gene of interest in a number of different individuals. This can be readily carried out by using hybridization probes to identify the same genetic locus in a variety of individuals. Any and all such nucleotide variations and resulting amino acid
  • polymorphisms or variations that are die resu lt of natural allelic variation and that do not alter the functional activity are intended to be within the scope of the in vention,
  • a biomarker nucleic acid molecule is at least 7, 15, 20, 25, 30, 40, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 550, 650, 700, 800, 900, 1.000, i 100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1 00, 2000, 2200, 2400, 2600, 2S00, 3000, 3500, 4000, 4500, or more nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule corresponding to a. marker of the invention or to a nucleic acid molecule encoding a protein corresponding to a marker of the invention.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% (65%, 70%, 75% 80%, preferably 85%) identical to each other typically remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in sections 6,3.1 -6.3,6 of Current Protocols in Molecular Biology, John Wiley & Sons, N,Y, (1989).
  • a preferred, non-limiting example of stringent hybridization conditions are hybridization i 6X sodium chloride/sodium citrate (SSC) at about 45 :' C, followed by one or more washes in 0.2X SSC, 0.1 % SDS at 50-65°C.
  • sequence changes can be introduced by mutation thereby leading to changes in the amino acid sequence of die encoded protein, without altering the biological activity of die protein encoded thereby.
  • sequence changes can be introduced by mutation thereby leading to changes in the amino acid sequence of die encoded protein, without altering the biological activity of die protein encoded thereby.
  • a "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
  • amino acid residues that are not conserved, or only semi -conserved among homologs of various species may be non-essential for activity and thus would be likely targets for alteration.
  • amino acid residues that are conserved among the homologs of various species e.g., murine and human
  • amino acid residues that are conserved among the homologs of various species may be essentia! for activity and thus would not be likely targets for alteration.
  • nucleic acid molecules encoding a polypeptide of the invention that contain changes in amino acid residues that are not essential for activity.
  • polypeptides differ in amino acid sequence from the natnaturey-oceitrriog proteins which correspond to the markers of the invention, yet retain biological activity-
  • a biomarker protein has an amino acid sequence that is at least about 40% identical, 50%, 60%, 70%, 75%, 80%, 83%, 85%, 87,5%, 90%, 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or identical to the amino acid sequence of a biomarker protein described herein.
  • An isolated nucleic acid molecule encoding a variant protein can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of nucleic acids of the invention, such that one or more amino acid residue substitutions, additions, or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis- Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidrae
  • acidic side chains e.g. , aspartic acid, glutamic acid
  • uncharged polar side chains e.g.
  • glycine asparagine, giuta.oii.oe, serine, threonine, tyrosine, cysteine
  • non-polar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidirte.
  • mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity.
  • the encoded protein can be expressed recombinantly and the activity of the protein can be determined,
  • die present invention further contemplates the use of anii- biomarker antisense nucleic acid molecules, i.e., molecules which are complementary to a sense nucleic acid of the invention, e.g., complementary to the coding strand of a double- stranded cDNA molecule corresponding to a marker of the invention or complementar to an ra ' A sequence corresponding to a marker of the invention.
  • an antisense nucleic acid molecule of the invention can hydrogen bond to (i.e. anneal with) a sense nucleic acid of the invention.
  • the antisense nucleic acid can be complementary to an entire coding strand, or to only a portion thereof, e.g., ail or part of the protein coding region (or open reading frame).
  • An antisense nucleic acid molecule can also be antisense to ail or part of a non-coding region of the coding strand of a nucleotide sequence encoding a polypeptide of the invention.
  • the non-coding regions (“5' and 3' untranslated regions") are the 5' and 3' sequences which flank " the coding region and are not translated into amino acids.
  • An antisense oligonucleotide can be, for example, about 5, 10 tension 15, 20, 25, 30, 35,
  • An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5- fkiorouracii, S-broroouraci!, 5-ehlorouraeil, 5-iodouracil, hypoxanthine, xanthine, 4- aeety!eytosine, 5- ⁇ carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, S-earboxyrnetlwlaminoniethxdwacil, dihydrouracii, beta-D- alactosyk ucosinc, inosine, 6-isopentenyladenme, 1 -niethyiguaiiirie, l-meth Sinosirie, 2,2-diniethylgnanine,
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been sub-cloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orien tation to a target nucleic acid of ref t, described further in the following subsection).
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic D A encoding a polypeptide corresponding to a selected marker of the invention to thereb inhibit expression of the marker, e.g. , by inhibiting transcription and/or
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site or infusion of the antisense nucleic acid into a blood- or bone marrow-associated body fluid.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by
  • the antisense nucleic acid molecules can a! so be deli vered to cells using the vectors described herein.
  • vector constructs in which the antisense nucleic acid molecule is placed under die control of a strong pol 11 or poi ill promoter are preferred.
  • An antisense nucleic acid molecule of the invention can be an a-anomcric nucleic acid molecule.
  • An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ot-unsts, the strands run parallel to each other (Gaultier el at, 1987, Nucleic Ackis Res. 15:6625-6641).
  • the antisense nucleic acid molecule can also comprise a 2 ⁇ -c ⁇ methylribonucleotide (inoue et al, 1987, Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (inoue et L imi. FEBSLeit. 215:327-330).
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes as described in Haselhoff and Gerlach, 1 88, Nature 334:585-59 i
  • a ribozyme having specificity for a nucleic acid molecule encoding a polypeptide corresponding to a marker of the invention can be designed based upon the nucleotide sequence of a cDN A corresponding to the marker.
  • a deri vative of a Tetr hymen L-1 1 ' VS RN A can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved (see Cecil el el U ,S. Patent No. 4,987,0? I ; and Cech el o!.
  • an mRNA encoding a polypeptide of the invention can be used to select a catalytic R A having a specific ribonuclease acti vity from a pool of RNA molecules (see, e.g., Bartel and Szostak, 1993, Science 261 :141 1-1418).
  • the present invention also encompasses nucleic acid molecules which form triple helical structures.
  • expression of a biomarker protein can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the gene encoding the polypeptide (e.g. , m promoter and/or enhancer) to form triple helical structures thai prevent transcription of the gene in target cells.
  • nucleotide sequences complementary to the regulatory region of the gene encoding the polypeptide e.g. , m promoter and/or enhancer
  • the nuc eic acid molecules of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acid molecules (see Hyrup et al, 1996, Bioorganic & Medicinal Chemistry 4(1 ): 5- 23).
  • peptide nucleic acids refer to nucleic acid, mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of PNAs has been shown to allow for specific hybridization to DN A and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard, solid phase peptide synthesis protocols as described in Hyrup et al. (1996),, supra; Perry-O' eefe et al. (1996) Proc. Na . Acad. Sci. USA 93: 14670-675.
  • PNAs can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.
  • PNAs can also be used, e.g. , m the analysis of single base pair mutations in a gene by, e.g. , PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., SI nucleases (Hyrup (1 96), supra; or as probes or primers for DMA sequence and .hybridization (Hyrup, 1 96, supra; Perry-O'Keefe et al., 1996, Proc. Natl. Acad. Sci. USA 93: 14670-675).
  • PNAs can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-D A chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras can be generated which can combine the advantageous properties of P A ami DNA, Such chimeras allow DNA recognition enzymes, e.g., RNASE H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
  • PNA-DNA chimeras cart be linked using linkers of appropriate lengths selected in terms of base stacki ng, number of bonds between the nucleobases, and orientation (Hyrup, 1 96, supra).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996), supra, and Finn et al ⁇ 1 96) Nucleic Acids Res. 24( 17):3357-63.
  • a DNA chain can be synthesized on a solid support using standard phosp oramulitc coupling chemistry and modified nucleoside analogs.
  • PN A monomers are then coupled in a step-wise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn el at, ⁇ 996, Nucleic Acids Res. 24( 57): 3357-63), Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterscr et al. , 1 75 , Bioarganic Med. Chem. Lett. 5: 1 1 19-1 1124).
  • the oligonucleotide can include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. S t. USA 86:6553-6556; Lemaitre et al, 1987, Proc. Natl Acad. Set. USA 84:648-652; PCT
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across the cell membrane see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. S t. USA 86:6553-6556; Lemaitre et al, 1987, Proc. Natl Acad. Set. USA 84:648-652; PCT
  • oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., rol et at, 1988, Bio/Techniques 6:958-976) or intercalating agents (see, e,g grid Zon, 1988, Pharm. Res. 5:539-549).
  • the oligonucleotide can be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
  • polypeptide corresponding to a marker can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques
  • polypeptides corresponding to a marker of the invention are produced by recombinant DNA techniques.
  • a polypeptide corresponding to a marker of the invention can be synthesized chemically using standard peptide synthesis techniques.
  • substantially free of cellular material includes preparation of protein in which the protein is separated from DCluiar components of the ceils from which it is isolated or recombmantly produced.
  • protein that is substantially free of cellular materia! includes preparations of protein having less than about 30%, 20%, .1 %, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein").
  • culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation.
  • i is preferably substantially free of chemical precursors or other chemicals, i.e. , it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly such preparations of the protein ha ve less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide of interest.
  • Biomarker polypeptides include polypeptides comprising amino acid sequences sufficiently identical to or deri ved from a biomarker protein amino acid sequence described herein, but which include fewer amino acids than the full length protein, and exhibit at least one activity of the corresponding full-length protein.
  • biologically active portions comprise a domain or motif with at least one activity of the corresponding protein.
  • a biologically active portion of a protein of the invention can be a polypeptide which is, for example, 1 , 25, 50, 100 or more amino acids in length .
  • other biologically active portions, in which other regions of the protein are deleted can be prepared b recombinant techniques and evaluated for one or more of the functional activities of the native form of a polypeptide of the invention.
  • Preferred polypeptides have an amino acid sequence of a biomarker protein encoded by a nucleic acid molecule described herein.
  • Other useful proteins are substantially identical (e.g., at least about 40%, preferably 50%, 60%, 70%, 75%, 80%, 83%, 85%, 88%, 90%, 1 , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) to one of these sequences and retain the functional activity of the protein of the corresponding naturally-occurring protein yet differ in amino acid sequence due to natural allelic variation or mutagenesis.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity ::: # of identical positions/total # of positions (e.g., overlapping positions) xi 00). In one embodiment the two sequences are the same length.
  • the determination of percent identity between two sequences can he accomplished using a mathematical algorithm.
  • a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of arlin and Altsehul ( 1.990) Pmc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altsehul (1.993) Pmc. Nad. Acad Sci. USA 90:5873-5877. Such an algorithm is
  • BLAST nucleotide searches can. be performed with the NBLAST program, score - 100, wordlength - 12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the inven tion.
  • BLAST protein searches can be performed with the XBLAST program, score ⁇ - : 50, wordlength ⁇ - 3 to obtain amino acid sequences homologous to a protei molecules of the invention.
  • Gapped BLAST can be utilized as described in Altsehul ei ai. ( 1997) Nucleic Acids Res. 25:3389-3402.
  • PSI-BIast can be used to perform an iterated search which defects distant relationships between molecules.
  • BLAST, Gapped BLAST, and PSI -Blast programs the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See the National Center for Biotechnology Information (NCBI) website at ncbi.nlm.nih.gov.
  • NCBI National Center for Biotechnology Information
  • Another preferred, non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, (1988) C mpui Appf Biosci, 4:1 1-7. Such an algorithm is incorporated into the ALIGN program, (version 2.0) which is part of the GCO sequence alignment software package.
  • a P AMI 20 weight residue table When utilizing the ALIGN program for comparing amino acid sequences, a P AMI 20 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm as described in Pearson and Lipraan (1 88) Proa Natl Acad Sei. USA 85:2444-2448, When using the FASTA algorithm for comparing nucleotide or amino acid sequences, a PA 120 weight residue table can, for example, be used with a A-tuple value of 2.
  • the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.
  • the invention also provides chimeric or fusion proteins corresponding to a
  • a "chimeric protein'" or “fusion protein” comprises all or part (preferably biologically active part) of a polypeptide corresponding to a marker of the invention operably linked to a heterologous polypeptide (i.e., a polypeptide other than the polypeptide corresponding to the marker).
  • a heterologous polypeptide i.e., a polypeptide other than the polypeptide corresponding to the marker.
  • operably linked is intended to indicate that the polypeptide of the invention and the heterologous polypeptide are fused in-frame to each other.
  • the heterologous polypeptide can be fused to the araino-terarinus or the carboxyi-teoiiinus of the -polypeptide of the invention.
  • One useful fusion protein is a GST fusion protein in which a polypeptide corresponding to a marker of the invention is f sed to the carboxyl terminus of GST sequences. Such fusion proteins can facilitate the purification of a recombinant polypeptide of the invention.
  • the fusion protein contains a heterologous signal sequence, immunoglobulin fusion protein, toxin, or other useful protein sequence.
  • Chimeric and fusion proteins of the invention can be produced by standard recombinant DNA techniques, in another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PGR amplification of gene
  • fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see, e.g., Ausubel et ai, supra).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence
  • expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • a nucle ic acid encoding a polypeptide of the invention can be cloned into such an expression vector such ihat the fusion moiety is linked in-frame to the polypeptide of the invention.
  • a signal sequence can be used to facilitate secretion and isolation of the secreted protein or other proteins of interest.
  • Signal sequences are typicall characterized b a core of hydrophobic amino acids which arc generally cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides contain processing sites that 5 allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway .
  • a nucleic acid sequence encodin a signal sequence can be operably linked in an expression vector to a it ) protein of interest, such as a protein which is ordinarily not secreted or is otherwise difficult to isolate.
  • the signal sequence directs secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed,, and the signal sequence is subsequently or concurrently cleaved.
  • the protein can then be readily purified from the extracellular medium by art recognized methods.
  • the signal sequence can be
  • the presen t invention also pertains to variants of the biornarker polypeptides described herein. Such variants have an altered amino acid sequence which can function as either agonists (mimetics) or as antagonists. Variants can he generated bv mutagenesis.
  • An agonist can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of the protein.
  • An antagonist of a protein can inhibit one or more of the activities of the naturally occurring form of the protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the protein of interest.
  • Treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein can have fewer side effects in a subject relative to treatment with the naturally occurring form of the protein.
  • Variants of a biornarker protein which function as ei ther agonists (mimetics) or as 0 antagonists can be identified by screening combinatorial libraries of mutants, e.g. ,
  • a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of variants can be produced by, for example, enzymatically iigatimg a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display).
  • methods which can be used to produce libraries of potential variants of the polypeptides of the invention from a
  • oligonucleotides are known in the art (see, e.g., Narang, 1983, Tetrahedron 39:3; Itakura ttf a!. , 19 , Af>n . Rev. Biochem. 53:323; liakura et oL, 1 84, Science 198: 1056; ike et ttl, 1983 Nucleic Acki Res. 1 1 :4?7).
  • libraries of f agments of the coding sequence of a polypeptide corresponding to a marker of the invention can be used to generate a variegated population of polypeptides for screening and subsequent selection of variants.
  • a library ofcodiiig sequence fragments can be generated by treating a double stranded PGR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/an tiseose pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligatmg the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes amino terminal and Internal f agments of various sizes of the protein of interest.
  • combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property typically include cloning the gene library into repiieabie expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions In which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected.
  • Recursive ensemble mutagenesis (REM) a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify variants of a protein of the invention. (Arkin and Yourvan, 1 92, Pr e.
  • biomarker nucleic acid and/or biomarker polypeptide molecules described herein can be facilitated by using standard recombinant techniques, in some embodiments, such techniques use vectors, preferably expression vectors, containing a nucleic acid encoding a biomarker polypeptide or a portion of such a polypeptide.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • vectors are capable of autonomous replication in a it ) host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g. , rton-episoroal
  • mammalian vectors are integrated into the genome of a host ceil upon introduction into the host cell, and thereby arc replicated along with the host genome.
  • certain vectors namely expression vectors, are capable of directing the expression of genes to which they
  • vectors e.g. , replication, defective retroviruses, adenoviruses and adeno-associated viruses
  • viral vectors e.g. , replication, defective retroviruses, adenoviruses and adeno-associated viruses
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell.
  • the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operabiy linked to the nucleic acid sequence to be expressed.
  • 25 linked is intended to mean that the nucleotide sequence of interest is linked to the
  • regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when tie vector is introduced into the host cell).
  • the term, "regulatory sequence” is intended to include promoters, enhancer's and other expression control elements (e.g., polyadenylatton signals). 0 Such .regulatory sequences are described, for example, in Goeddei, Me (hods in Enzym log :
  • Regulatory seq uences include those which, direct constitutive expression of a nucleotide sequence in many types of host ceil and those which direct expression of the nucleotide sequence only in certain host ceils (e.g. , tissue-specific regulatory sequences), it will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as die choice of die host cell to be transformed, the level of expression of protein desired, and the like.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.
  • the recombinant expression vectors for use in the invention can be designed tor expression of a polypeptide corresponding to a marker of the invention in prokaryotie (e.g., E. coli) or eukaryotic cells (e.g., insect cells (using baculovirus expression vectors ⁇ , yeast cells or mammalian ceils). Suitable host ceils are discussed further in Goeddel, supra.
  • prokaryotie e.g., E. coli
  • eukaryotic cells e.g., insect cells (using baculovirus expression vectors ⁇ , yeast cells or mammalian ceils). Suitable host ceils are discussed further in Goeddel, supra.
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein .
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the sorubiUiy of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a iigand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1 88, Gene 67:3.1 -40), pMAL (New England Biolabs, Beverly, MA) and pRlT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • GST glutathione S-transferase
  • Suitable inducible non-fusion E, coii expression vectors include p ' frc (Amann et al, 1 88, Gene 69:301 -3 i 5) and pET i Id ( Srudier et al , p. 60-89, In Gene Expression Technology: Methods in Emymo gy vol.185, Academic Press, San Diego, CA, 1 91).
  • Target biomarker nucleic acid expression from the pTrc vector relies on host A polymerase transcription from a hybrid trp-lac fusion promoter.
  • Target btoniarker nucleic- acid expression from the pET 11 d vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a co-expressed viral R.
  • a polymerase (T7 gni). This viral polymerase is supplied by host strains BL21 (DE3) or HMS.174 ⁇ DB3) from: a resident prophage harboring a T7 gnl gene under the transcriptional control of Ac lacliV 5
  • nucleic acid sequence of the nucieic acid to be inserted into an expression vector so that it ) the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al, 1992, Nucleic Acids Res. 20:21 1 .1 -2118).
  • Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the expression vector is a yeast expression vector.
  • vectors for expression in yeast S. cerevtsiae include p YepSec l (Batdari et ol,
  • pJRY88 Schotz et a!., 1987, Gem 54: 1 13-1 3
  • pYES2 Invitrogen Corporation, San Diego, CA
  • pPieZ Invitrogen Corp, San Diego, CA
  • the expression vector is a baculovirus expression vector.
  • Baculovirus vectors available for expression of proteins in cultured insect ceils include
  • a nucleic acid of the present invention is expressed in mammalian cell using a mammalian expression vec tor.
  • mammalian expression vectors include pCDM8 (Seed, 1987, Nature 329:840) and pMT2PC (Kaufman
  • the expression vector's control functions are often provided by viral regulatory elements.
  • viral regulatory elements For example, commonly- used promoters are derived from polyoma. Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic ceils see chapters 16 and 17 of Sambrook et a ' supra,
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g. , tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are used to express the nucleic acid.
  • tissue-specific promoters include the albumin promoter (liver-specific; Pi «kert ⁇ ?/ ' al., 1987, Genes Dev. 1 :268-277), iymphoid-speeifk promoters (Calame and Eaton, 1.988,, Adv. Immunol, 43:235- 275), in particular promoters of T cell receptors (Winoto and Baltimore, 1 89, EMBOJ.
  • an immunoglobulins (Banerji i al.., .1 83, Cell 33:729-740; Queen and Baltimore, .1 83, Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1 89, Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas- specific promoters (Edlimd el al, 1985, Science 230:912-916), and mammary gland- specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264, 166).
  • neuron-specific promoters e.g., the neurofilament promoter; Byrne and Ruddle, 1 89, Proc. Natl. Acad. Sci. USA 86:5473-5477
  • pancreas- specific promoters (Edlimd
  • DevelopmentaUy-regulated promoters are also encompassed, for example the murine hox promoters ( essel and Gruss, 1 90, Science 249:374-379 ⁇ and the a-£etoprote1 ⁇ 2 promoter (Camper and Tilghman, 1989, Genes Dev. 3:537-546).
  • the present invention further provides a recombinant expression vector comprising a DNA molecule cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operabiy linked to a regulator ⁇ '' sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to the raRNA encoding a polypeptide of the invention. Regulatory sequences operabiy linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of eel!
  • the antisense expression vector can be in the form of a recombinant piasmid, phagemid, or attenuated virus in which antisense nucleic acids are produced, under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which, the vector is introduced.
  • Another aspect of the present invention pertains to host cells into which a
  • a host cell can be any prokaryotic ⁇ e.g., E. call) or eukaryotic cell (e.g. , insect cells, yeast or mammalian ceils).
  • Vector D A can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techttics.
  • Suitable methods for transforming or transfecting host cells can be found in Sambrook, et l ⁇ supra), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g. , for resistance to antibiotics) is generally introduced into the host ceils along with the gene of interest
  • Preferred selectable markers include those which confer resistance to drugs, such as G 1 8, hygromycin and methotrexate. Ceils stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • Biomarker nucleic acids and/or biomarker polypeptides can be analyzed according to the methods described herein and techniques known to the skilled artisan to identify such genetic or expression alterations useful for the present invention including, but not limited to, 1 ) an alteration in the level of a biomarker transcript or polypeptide, 2) a deletion or addition of one or more nucleotides from a biomarker gene, 4 ⁇ a substitution of one or more n ucleotides of a biomarker gene , 5) aberrant modification of a biomarker gene, such as an expression regulatory region, and the like,
  • a biological samp!e is tested for die presence of copy number changes in genomic loci containing die genomic marker.
  • the increased copy number of at least one biomarker listed in Tabic i is predictive of better outcome of iron-sulfur cluster biosynthesis pathway inhibitory therapy.
  • a copy number of at least 3, 4, 5, 6 relief 7, 8, , or .10 of at least one biomarker listed in Table 1 is predictive of likely responsive to iron-sulfur cluster biosynthesis pathway inhibitory therapy.
  • Methods of evaluating the copy number of a. biomarker locus include, but are not limited to, hybridization-based assays.
  • Hybridization-based assays include, but are not limited to, traditional "direct: probe” methods, such as Southern blots, in situ hybridization (e.g., FISH and FISH plus SKY) methods, and "comparative probe” methods, such as comparative genomic hybridization (CGH), e.g., cDNA-based or oligonueleotide-based CGH.
  • CGH comparative genomic hybridization
  • the methods can be used in a wide variety of formats including, but not limited to, substrate (e.g. membrane or glass) bound methods or array-based approaches.
  • evaluating the biomarker gene copy number in a sample involves a Southern Blot.
  • Southern Blot the genomic DNA (typically fragmented, and separated on an deeirophoreiie gel) is hybridized to a probe specific for the target region. Comparison of the intensity of the hybridization signal from the probe for the target region with control probe signal from analysis of normal genomic DN A (e.g., a non-amplified portion of the same or related cell, tissue, organ, etc.) provides an estimate of the relative copy number of the target nucleic acid.
  • normal genomic DN A e.g., a non-amplified portion of the same or related cell, tissue, organ, etc.
  • a Northern blot may be utilized for evaluating the copy number of encoding nucleic acid in a sample. In a.
  • mRNA is hybridized to a probe specific for the target region. Comparison of the intensity of the hybridization signal from the probe for the target region with control probe signai from analysis of normal R A (e.g., a non-amplified portion of the same or related cell, tissue, organ, etc. ' ) provides an estimate of the relative copy number of the target nucleic acid. Alternati ely, other methods well known in the art to detect RNA can be used, such that higher or lower expression relative to an appropri te control ⁇ e.g., a non-amplified portion of the same or related cell tissue, organ, etc..) provides an estimate of the relative copy number of the target nucleic acid.
  • site hybridization comprises the following steps: (I) fixation of tissue or biological structure to be analyzed; (2) prehybridization treatment of the biological structure to increase accessibility of target DNA, and to reduce nonspecific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological structure or tissue: (4) post-hybridization washes to remove nucleic acid fragments not bound in the hy bridization and (5) detection of the hybridized nucleic acid fragments.
  • fixation of tissue or biological structure to be analyzed fixation of tissue or biological structure to be analyzed
  • prehybridization treatment of the biological structure to increase accessibility of target DNA, and to reduce nonspecific binding
  • hybridization of the mixture of nucleic acids to the nucleic acid in the biological structure or tissue (4) post-hybridization washes to remove nucleic acid fragments not bound in the hy bridization and (5) detection of the hybridized nucleic acid fragments.
  • post-hybridization washes to remove
  • ceils are fixed to a solid support, typically a glass slide, if a nucleic acid is to be probed, the cells are typically denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of labeled probes specific to the nucleic acid sequence encoding the protein.
  • the targets e.g., cells
  • the targets are then typically washed at a predetermined stringency or at an increasing stringency until an appropriate signal to noise ratio is obtained.
  • probes are typically labeled, e.g., with radioisotopes or fluorescent reporters, in one embodiment, probes are sufficiently long so as to specifically hybridize with the target nucleic acid(s) under stringent conditions. Probes generally range tn length from about 200 bases to about 1 00 bases, in some applications it is necessary to block the hybridization capacity of repetitive sequences. Thus, in some embodiments, tRNA, huma genomic DNA, or Cot-I DNA is used to block non-specific hybridization.
  • genomic DNA is isolated from normal reference cells, as well as from test cells (e.g., tumor cells) and amplified, if necessary.
  • the two nucleic acids arc differentially labeled and then hybridized in situ to metaphasc chromosomes of a reference ceil.
  • the repetitive sequences in both the reference and test DNAs are either removed or their hybridization capacity is reduced by some means, for example by prehybridization with appropriate blocking nucleic acids and/or including such blocking nucleic acid sequences for said repetitive sequences during said hybridization.
  • the bound, labeled DNA sequences are then rendered in a visual izable form, if necessary.
  • Chromosomal regions in the test cells which are at increased or decreased copy number can be identified by detecting regions where the ratio of signal from the two DNAs is altered. For example, those regions that have decreased in copy number in the test cells will show relatively lower signal from the test DNA than the reference compared to other regions of the genome. Regions that have been increased fa copy number in the test cells will show relatively higher signal from, the test DN A. Where there arc chromosomal deletions or multiplications, differences in the ratio of the signals from the two labels will be detected and the ratio will provide a measure of the copy number.
  • array CGH the immobilized chromosome element is repiaced with a collection of solid support bound target nucleic acids on an array, allowing for a large or complete percentage of the genome to be represented in the collection of solid support bound targets.
  • Target nucleic acids may comprise c-DNAs, genomic DNAs, oligonucleotides (e.g., to detect single nucleotide polymorphisms) and the like.
  • Array-based CGH may also be performed with single-color labeling (as opposed to labeling the control and the possible tumor sample with two different dyes and mixing them prior to hybridization, which will yield a ratio due to competitive hybridization of probes on the arrays).
  • amplification-based assays can be used to measure copy number, in such amplification-based assays, the nucleic acid sequences act as a template in an amplification reaction (e.g.. Polymerase Chain Reaction (PCR)).
  • PCR Polymerase Chain Reaction
  • the amount of amplification product will be proportional to the amount of template in the original sample.
  • Fluorogenic quantitative PCR may also be used in the methods of the invention, in fluorogenic quantitative PCR, quantitation is based on amount of fluorescence signals, e.g., Taq an and SY.8R green.
  • ligase chain reaction LCR.
  • LCR. ligase chain reaction
  • Genomics 4 560
  • Barringer et al. 1.990
  • Gene 89: 1.17 transcription amplification
  • transcription amplification Kwoh, et al. (1 89) Proc. Mat!. Acad. Set USA 86: 1 173
  • self-sustained sequence replication (Guateili, ef ai. (1990) Proc. Na Aca Sci. USA 87: 1874), dot PCR, and linker adapter PCR, etc.
  • Loss of heterozygosity (LOH) and major copy proportion (MCP) mapping Wang, Z.C., et al. (2004) Cancer Res 64(1);64-71 ; Seymour, A. B., ei ei (1994) Cancer Res 54, 2763-4; Hahn, S. A,, et at. (3995) Cancer Res 55, 4670-5; Kimuta, M., et al. (3996) Gems Chromosomes Cancer 17, 88-93; Li et al, (2008) MBC Bkmfbrm. 9, 204-219) may also be used to identify regions of amplification or deletion.
  • Biomarker expression may be assessed by an of a wide variety of well-known methods for detecting expression of a transcribed molecule or protein.
  • Non-limiting examples of such methods include immunological methods for detection of secreted, celi- surface, cytoplasmic, or nuciear proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods.
  • activity of a particular gene is characterized by a measure of gene transcript (e.g. mRNA), by a measure of the quantity of translated protein, or by a measure of gene product activity.
  • Biomarker expression can be monitored in a variety of ways, including by detecting mRNA levels, protein levels, or protein activity, any of which can be measured using standard, techniques. Detection can involve quantification of the level of gene expression (e.g., genomic DNA, cDNA, mRNA, protein, or enzyme activity), or, alternatively, cati. be a qualitative assessment of the level of gene expression, in particular in comparison with a control level. The type of level being detected will be clear from the context.
  • detecting or determining expression levels of a biomarker and. functionally similar homoiogs thereof, including a fragment or genetic alteration
  • RNA levels for the marker of interest comprises detecting or determining RNA levels for the marker of interest
  • one or more cells from the subject to be tested are obtained and RNA is isolated from the cells.
  • a sample of breast tissue cells is obtained from the subject.
  • RNA is obtained from a single cell.
  • a cell can be isolated from a tissue sample by laser capture microdissection (LCM).
  • LCM laser capture microdissection
  • a cell can be isolated from a tissue section, including stained tissue section, thereby assuring that the desired cell is isolated (see, e.g. , Bonner et al. (1 97) Science 278: 1481; Emmert-Buck et al ( 1996) Science 274:998; Fend et al. (1999) Am. J. Path. 154: 61. and Murakami et al. (2000) Kidney int. 58; 1346),
  • Murakami et al describes isolation of a cell from a previously imimmostained tissue section.
  • RNA can. be extracted Methods for establishing cultures of non-transformed cells, i.e., primary cell cultures, are k own in the art.
  • RNA in the tissue and cells may quickly become degraded. Accordingly, in a preferred embodiment, the tissue or cells obtained from a subject is snap frozen as soon as possible.
  • RNA can be extracted from: the tissue sample by a variety of methods, e.g., the guanidium thiocyanate lysis followed by CsCl centrifugation (Chirgwin et al., 1979, Biochemistry i 8:5294-5299).
  • R from single cells can be obtained as described in methods for preparing cD A libraries from single cells, such as those described in Dulac, C. (.1998) Curr. Top. Dev. Biol. 36 245 and Jena et al. (1996) J. Immunol. Methods 1 0:1 9, Care to avoid RNA degradation must be taken, e.g. , b inclusion of RNA sin.
  • RNA sample can then be enriched in particular species.
  • poly(A)-*- RNA is isolated from the RNA sample, in general, such purification takes ad vantage of the poiy-A. tails on mR A.
  • poly-T oligonucleotides may be immobilized within on a sol id support to serve as affinity ligands
  • Kits for this purpose are commercially available,, e.g., the MessageMaker kit (Life Technologies, Grand Island, NY).
  • the RNA population is enriched in marker sequences. Enrichment can be undertaken, e.g., by primer-specific c ' DNA synthesis, or multiple rounds 5 of linear amplification based on cDNA synthesis and template-directed in vitro
  • RNA enriched or not in particular species or sequences
  • an "amplification process” is designed to it ) strengthen, increase, or augment a molecule within the RNA.
  • RNA is mRNA
  • art amplification process suc as RT-PCR can be utilized to amplify the mRNA, such that a signal is detectable or detection is enhanced.
  • Such art amplification process is beneficial particularly when the biological, tissue, or tumor sample is of a small size or volume.
  • RNA sequence can be reverse transcribe mRNA into cDNA followed by polymerase chain reaction (RT-PCR); or, to use a single enzyme for both steps as described in. U.S. Pat. No. 5,322,770, or reverse transcribe mR A into cDNA followed by symmetric gap ligase chain reaction (RT-AGLCR) as described by R. L. Marshall ; e al, PCR
  • Northern analysis involves running a preparation of RNA on a denaturing agarose gel, and transferring it to a suitable support, such as activated cellulose, nitrocellulose or glass or nylon membranes. Radiolabeled cD A or RNA is then hybridized to the preparation,, washed and analyzed by autoradiography.
  • In situ hybridization visualization may also be employed, wherein a radioaetively labeled ami sense RNA probe is hybridized with a thin section of a biopsy sample, washed, cleaved with RNase and exposed to a sensitive emulsion for autoradiography.
  • the samples may be stained with hematoxylin to demonstrate the histological composition of the sample, and dark field imaging with a suitable light filter shows the developed emulsion.
  • Non-radioactive labels such as digoxigenin may also be used.
  • mRNA expression can be detected on a DNA array, chip or a microarray.
  • Labeled nucleic acids of a test sample obtained from subject may be hybridized to a solid surface comprising biomarker DNA. Positive hybridization signal is obtained with the sample containing biomarker transcripts.
  • raRNA is extracted from the biological sample to be tested, reverse transcribed, and fiuorescentiy-labeled cDNA probes are generated.
  • the microarravs capable of hybridizing to marker cDNA are then probed with the labeled cDNA probes, the slides scanned and fluorescence intensity measured. This intensity correlates with the hybridization intensity and expression levels.
  • probes that can be used in the methods described herein include cDNA, riboprobes, synthetic oligonucleotides and genomic probes.
  • the type of probe used will generally be dictated by the particular situation, such as riboprobes for in siiu hybridization, and cDNA for Northern blotting, for example.
  • the probe is directed to nucleotide regions unique to the RN A.
  • the probes may be as short as is required to differentially recognize marker mRNA transcripts, and may be as short as, for example, 15 bases: however, probes of at least ! 7 remind 18, 19 or 20 or more bases can be used.
  • the primers and probes hybridize specifically under stringent conditions to a DNA fragment having the nucleotide sequence corresponding to the marker.
  • stringent conditions means hybridization will occur only if there is at least 95% identify in nucleotide sequences, in another embodiment, hybridization under
  • stringent conditions occurs when there is at least 97% identity between the sequences.
  • the form of labeling of the probes may be any that is appropriate, such as the use of radioisotopes, for example, J ⁇ P and *,5 S. Labeling with radioisotopes may be achieved, whether the probe is synthesized chemically or biologically, by the use of sui tably labeled bases.
  • the biological sample contains polypeptide molecules from the test subject.
  • the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.
  • the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting marker polypeptide, mRNA, genomic DNA, or fragments thereof, such that the presence of the marker polypeptide, mRNA, genomic DNA, or fragments thereof, is detected in the biological sample, and comparing the presence of the marker polypeptide, mRNA, genomic DNA, or fragments thereof, in the control sample with the presence of the marker polypeptide, mRNA, genomic DNA, or fragments thereof in the test sample,
  • the activity or level of a biomarker protein can be detected and/or quantified by detecting or quantifying the expressed polypeptide.
  • the polypeptide can be detected and quantified by any of a number of means well known to those of skill m the art Aberrant levels of polypeptide expression of the polypeptides encoded by a biomarker nucleic acid and functionally similar homologs thereof, including a fragment or genetic alteration thereof (e.g., in regulatory or promoter regions thereof) are associated with the likelihood of response of a cancer to an anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy).
  • an anti-cancer therapy e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy
  • Any method known in the art for detecting polypeptides can be used, such methods include, but are not limited to, immunodiffusion, immunoeiectrophoresis, radioimmunoassay ( IA), enzyme-linked immunosorbent assays (ELlSAs),
  • immunoflttorescent assays Western blotting, binder-ltgand assays, immuaohistochcraical techniques, agglutination, complement assays, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography; and the like (e.g., Basic and Clinical Immunology, Sites and Terr, eds., Appleton and Lange, Norwaik, Conn, pp 217-262, 1 1 which is incorporated by reference).
  • binder-iigand immunoassay methods including reacting antibodies with an epitope or epitopes and competitively displacing a labeled polypeptide or derivative thereof.
  • ELISA and A procedures may be conducted such that a desired biomarker protein standard is labeled (with a radioisotope such as ' or " ⁇ S, or art assayable enzyme, such as horseradish peroxidase or alkaline phosphatase), and, together with the unlabelled sample, brought into contact with the corresponding antibody, whereon a second antibody is used to bind the first, and radioactivity or the immobilized enzyme assayed (competitive assay).
  • a radioisotope such as ' or " ⁇ S, or art assayable enzyme, such as horseradish peroxidase or alkaline phosphatase
  • the biomarker protein in the sample is allowed to react with the corresponding immobilized antibody, radioisotope- or enzyme-labeled anti-biomarkcr protcinantibody is allowed to react with die system, and radioactivity or the enzyme assayed (ELiSA-sandwieh assay).
  • radioactivity or the enzyme assayed ELiSA-sandwieh assay.
  • Other conventional methods may also be employed as suitable .
  • a “one-step' ' assay involves contacting antigen with immobilized antibody and, without washing, contacting the mixture with labeled antibody.
  • "two-step” assay involve washing before contacting, the mixture with labeled antibody.
  • Other conventional method may also be employed as suitable.
  • a method for measuring biomarker protein levels comprises the steps of: contacting a biological specimen with an antibody or variant (e.g., fragme t) thereof which selectively binds the biomarker protein, and detecting whether said antibody or variant thereof is bound to said sample and thereby measuring the levels of the biomarker protein.
  • an antibody or variant e.g., fragme t
  • Enzymatic and radiolabeling of biomarker protein and/or the antibodies may be effected by conventional means.
  • Such means will generally include covaiest linking of the enzyme to the antigen or the antibody in question, such as by g!utara!dehyde, specifically so as not to adversely affect the activity of the enzyme, by which, is meant that the enzyme must still be capable of interacting with its substrate, although it is not necessary for all of the enzyme to be active, provided that enough remains active to permit the assay to be effected.
  • some techniques for binding enzyme are non-specific (such as using formaldehyde), and will only y ield a proportion of active enzyme.
  • sol id-phase enzyme it is possible to immobilize the enzyme itself on a support, but if sol id-phase enzyme is required, then this is generally best achieved by binding to antibody and affixing the antibody to a support, models and systems for which are well-known in the art.
  • Simple polyethylene may provide a suitable support.
  • Enzymes employable for labeling are not particularly limited, but may be selected from the members of the oxidase group, for example. These catalyze production of hydrogen peroxide by reaction with their substrates, and glucose oxidase is often used for its good stability, ease of availabiiitv and cheapness, as well as the ready availabiiitv of its substrate (glucose). Activity of the oxidase may be assayed by measuring the concentration of hydrogen peroxide formed after reaction of the enzyme-labeled antibody with the substrate under controlled conditions well-known in the art
  • bioraarker protein may be detected according to a practitioner's preference based upon the present disclosure.
  • One such technique is Western blotting (Towbin et at., Proe. Nat, Acad. Sci, 76:4350 (1979)), wherein a suitably treated sample is run on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose filter, Aoti-biomarket protein antibodies (unlabeled) are then brought into contact with the support and assayed by a secondary immunological reagent, such as labeled protein A or anti-iniraitnoglobulin (suitable labels including i2' l. horseradish peroxidase and alkaline phosphatase). Chromatographic detection may also be used.
  • immunohistochemistry may be used to detect expression of btomarker protein, e.g., in a biopsy sample.
  • a suitable antibody is brought into contact with, for example, a thin layer of cells, washed, and then contacted with a second, labeled antibody.
  • Labeling ma be by fluorescent markers, enzymes, such as peroxidase, avidin, or radioiabeliing. The assay is scored visually, using microscopy.
  • Anti-biomarker protein antibodies may also be used for imaging purposes, for example, to detect: the presence of btomarker protein in cells and tissues of a subject.
  • Suitable labels include radioisotopes, iodine ( 5 I, !J ! i), carbon ( i C.I, sulphur ( > ⁇ . tritium ( * H), indium ( u 3 ⁇ 4n), and technetium ( ' v m7c), fluorescent labels, such as fluorescein and rhodamine, and biotin.
  • markers for this purpose may be any that do not substantially interfere with the antibody binding, but which allow externa! detection.
  • suitable markers may include those that may be detected by X-radiography, NMR or MR!.
  • suitable markers include any radioisotope that emits detectable radiation but that is not overtly harmful to the subject, such as barium or cesium, for example.
  • suitable markers for NMR and MR! generally include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by suitable labeling of nutrients for the relevant hybridoma, for example.
  • the size of the subject, and the imaging system used, will determine the quantity of imaging moiety needed to produce diagnostic images, in the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of technetium- 9.
  • the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain biomarker protein. The labeled antibody or antibody fragment can then be detected using known techniques.
  • Antibodies that may be used to detect biomarker protein include any antibody, whether natural or synthetic, full length or a fragment thereof, monoclonal or polyclonal, that bind sufficiently strongly and specifically to the biomarker protein to be detected.
  • An antibody may have a 3 ⁇ 4 of at most about 10* M. ⁇ M, 10 '8 M, ]( u, H) "!0 M, ⁇ ⁇ , or JO ' ⁇ M.
  • the phrase "specifically binds" refers to binding of, for example, an antibody to an epitope or antigen or antigenic determinant in such a manner that binding can be clispiaeed or competed with a second preparation of identical or similar epitope, antigen or antigenic determinant.
  • An antibody may bind preferentially to the biomarker protein relative to other proteins, such as related proteins.
  • Antibodies are commercially available or may be prepared according to methods known in the art.
  • Antibodies and derivatives thereof that may be used encompass polyclonal or monoclonal antibodies, chimeric, human, humanized, primatized (CDR -grafted), veneered or single-chain antibodies as well as functional fragments, i.e., biomarker protein binding fragments, of antibodies.
  • antibody fragments capable of binding to a biomarker protein or portions thereof including, but not limited to, Fv, Fab, Fab' and F(ab') 2 fragments can be used.
  • Such fragments can be produced b enzymatic cleavage or by recombinant techniques. For example, papain or pepsin cleavage can generate Fab or F(ah') 2 fragments, respectively.
  • proteases with the requisite substrate specificity can also be used to generate Fab or F(ab') 2 fragments.
  • Antibodies can also bo produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site.
  • a chimeric gene encoding a F(ab') 2 heavy chain portion can be designed to include DNA sequences encoding the €H, domain and hinge region of the heavy chain.
  • agents that specifically bind to a biomarker protein other than antibodies are used, such as peptides.
  • Peptides that specifically bind to a biomarker protein can be identified by any means known in the ait. For example, specific peptide binders of a biomarker protein can be screened for using peptide phage display libraries. d. Methods for Detection of Biomarker Structural Alteration
  • biomarker nucleic acid and/or biomarker polypeptide molecule can be used to identify the presence of a structural alteration in a biomarker nucleic acid and/or biomarker polypeptide molecule in order to, for example, identify sequences or agents that affect translation of iron-sulfur cluster biosynthesis-related genes.
  • detection of the alteration involves the use of a
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of ceils from a subject, isolating nucleic acid (#.g. 5 genomic, mR A or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a biomarker gene under conditions such that hybridization and amplification of the biomarker gene (if present) occ urs, and detecting the presence or absence of an amplification product, or defecting the size of the amplification product and comparing the length to a control sample, it is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
  • Alternative amplification methods include: self sustained sequence replication
  • mutations in a biomarker nucleic acid from a sample cell can be identified by alterations in restriction enzyme cleavage patterns.
  • sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonuc!eases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.
  • sequence specific ribozymes see, for example, U.S. Pat, No, 5,498,531 ) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • biomarker nucleic acid can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA harm to high density arrays containing hundreds or thousands of oligonucleotide probes (Crontn, M. T, et al. ( 1996) Hum. Mutat 7:244-255; Kozal M. J, et al. ( 1996) Nat. Med,. 2:753-759).
  • biomarker genetic mutations can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin et al (1 96) supra.
  • a first hybridization array of probes can be used, to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected .
  • Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
  • biomarker genetic mutations can be identified in a. variety of contex ts, including, for example, germline and somatic mutations.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence biomarker gene and detect mutations by comparing the sequence of the sample biomarker with the corresponding wild-type (control) sequence.
  • Examples of sequencing reactions include those based on techniques developed by Maxam and Gilbert ( 1977) Pro . Nad Acad. ScL USA 74:560 or Sanger (.1977) . Proc. Natl. Acad Set USA. 74:5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve (1995)
  • RNA/RNA or RNA/DNA heterodupiex.es Other methods for detecting mutations in a biomarker gene include methods in. which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heterodupiex.es (Myers et al ( 1985) Science 230: 1242).
  • the art technique of "mismatch cleavage" starts by providing heteroduplexes formed by hybridizing (labeled) R A or D A containing the wild-type biomarker sequence with potentially mutant RNA or DNA obtained from a tissue sample.
  • the double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to base pair mismatches between the control and sample strands.
  • RNA/DNA duplexes can be treated with RNase and DNA DNA hybrids treated with SI nuclease to enzymatically digest the mismatched regions.
  • either DMA/DMA or R A/D A ditpiexes can be treated with hydroxyiaraine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl. Acad. Set. " USA 85:4397 and Saleeba et al ( 1992) Methods
  • control DMA or KNA can be labeled for defection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DMA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in biomarker cD As obtained from samples of cells.
  • the mutY enzyme of colt cleaves A at G/A mismatches and the thymidine DNA glyeosylase from HeLa ceils cleaves T at G/T mismatches (Hsu et al. (1 94) Carcinogenesis 15: 657-1662).
  • a probe based on a biomarker sequence e.g.
  • a wild-type biomarker treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like (e.g., U.S. Pat. No. 5.459.039.) in other embodiments, alterations in eiectrophoretic mobility can be used to identify mutations in biomarker genes.
  • alterations in eiectrophoretic mobility can be used to identify mutations in biomarker genes.
  • single strand conformation polymorphism iSSCP may be used to detect differences in eiectrophoretic mobility between mutant and wild type nucleic acids (Orita ei al. (1989) Proe Natl. Acad. Set USA 86:2766; see also
  • the subject- method ' utilizes heteroduplcx analysis to separate doable stranded heteroduplcx molecules on the basis of changes in eiectrophoretic mobility (Keen et al. (19 1 ) Trends Genet. 7:5).
  • mutant or wild-type fragments i polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DOGE) (Myers et i. (1 85) Nature 313:495).
  • DOGE denaturing gradient gel electrophoresis
  • DNA will be modified to ensure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp o higb- melting GC-rich DNA by PGR,
  • a temperature gradient is used in place of a denaturing gradient to identify differences in the mobil ity of control and sample DNA (Rosenbaum and Reissner (1987) Bi phys. Chem.
  • oligonucleotide primers may be prepared, in which the known mutation is placed centrally and then hybridized to target DNA. under conditions which permit hybridization onl if a perfect match is found (Saiki et al. (1 86) Nature 324:163; Saiki et ah ( 1989) Proc Natl. Acad. Set USA 86:6230).
  • Such allele specific oligonucleotides arc hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hy bridizing membrane and hybridized with labeled target DNA.
  • Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differentia! hybridization) (Gibbs el al. ( 1989) Nucleic Ackk Res'. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibiech 1 1 :238).
  • amplification ma also be performed using Taq ligase for amplification (Barany (1991) Proc, Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
  • anti-cancer therapy e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy
  • a cancer e.g., cancer
  • anti-cancer therapy e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy
  • combinations of therapies e.g.
  • anti-PD- 1 and anti-immunoiub.ibitory- therapies can be administered to a desired subject or once a subject is indicated as being a likely responder to anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy), in another embodiment, such anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy) can be avoided once a subject is indicated as not being a likely responder to the anti-cancer therapy (e.g., iron- sulfur cluster biosynthesis pathway inhibitory therapy) and an alternative treatment regimen, such as targeted and/or untaxgeted anti-cancer therapies can be administered.
  • Combination therapies are also contemplated and can comprise, for example, one or more chemotherapeutie agents and radiation, one or more chemotherapeutie agents and
  • immunotherapy or one or more chemotherapeutie agents, radiation and chemotherapy, each combination of which can be with or without anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy).
  • anti-cancer therapy e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy
  • iron-sulfur cluster biosynthesis pathway and exemplary agents useful for inhibiting the iron-sulfur cluster biosynthesis pathway, or other hiomarkers described herein, have been described above.
  • targeted therapy refers to admini tration of agents that selectively interact with a. chose biomolccule to thereby treat cancer.
  • targeted thcrepy regarding the inhibition of immune checkpoint inhibitor is useful in combination with the methods of the present invention.
  • immune checkpoint inhibitor means a group of molecules on the cell surface of CD * and/or CD8-3 ⁇ 4- T ceils that fine-tune immune responses by down-modulating or inhibiting an anti-tumor immune response.
  • Immune checkpoint proteins are well known in the art and include, without limitation, CTLA-4, Pi 1, VISTA, 87-112, B7-H3, PD-Ll , ⁇ 7- ⁇ ⁇ 4, B7-H6, 2B ICOS, HVEM, PD-L2.
  • Inhibition of one or more immune checkpoint inhibitors can block or otherwise neutralize inhibitory signaling to thereb upregulate an immune response in order to more efficaciously treat cancer.
  • immunotherapy is one form of targeted therapy that may comprise, for example, the use of cancer vaccines and/or sensitized antigen presenting cells.
  • an oncolytic virus is a virus that is able to infect and lyse cancer cells, while leaving normal cells unharmed, making them potentially useful in cancer therapy. Replication of oncolytic viruses both facilitates tumor cell destruction and also produces dose amplification at the tumor site. They may also act as vectors for anticancer genes, allowing them to be
  • the mununotherapy can involve passi ve immunity for short-term protection of a host, achieved by the administration of pre-formed antibody directed against a cancer antigen or disease antigen ⁇ ?.#., administration of a monoclonal antibody, optionally linked to a chemotherapeutie agent or toxin, to a tumor antigen).
  • a monoclonal antibody optionally linked to a chemotherapeutie agent or toxin, to a tumor antigen.
  • anti-VEGF and raTOR inhibitors are .known to be effective in treating renal cell carcinoma.
  • Immunotherapy can also focus on using the cytotoxic lymphocyte-recognized epitopes of cancer ceil lines.
  • antisense polynucleotides can be used to selectively modulate biomolecules that are linked to the initiation, progression, and/or pathology of a rumor or cancer.
  • nn targeted therapy referes to admi nistration of agents that do not selectively interact with a chosen biomolccule yet treat cancer.
  • untargeted therapies include, without limitation, chemotherapy, gene therapy, and radiation therapy.
  • mitochondrial coiactor therapy is useful
  • vitamin E is known to block cell death via ferroptosis such that mitochondrial coiactor therapy can alleviate or improve any toxicity associated with ISC biosynthesis pathway inhibition.
  • Mitochondrial cofaetor therapies are well known in the art and include, for example, coenzyme Q10 (ubiquinone), riboflavin, thiamin, niacin, vitamin K (phylloqulnone and menadione), creatine, carnitine, and other antioxidants such as ascorbic acid and lipoic acid (see, for example, M.an-iage el. al (2003) ,/ Am. Diet. Assoc. 103:1029-1038 and Parik el til. (2009) Curr. Trea Options Neurol. 1 :4.14-430).
  • Chemotherapy includes the
  • chemotherapeutie agent may be, but is not limited to, those selected from among the following groups of compounds: platinum compounds, cytotoxic antibiotics, antimetabolities, an ti -mitotic agents, alkylating agents, arsenic compounds, D A topoisomerase inhibitors, taxanes, nucleoside analogues, plant alkaloids, and toxins; and synthetic derivatives thereof.
  • Exemplary compounds include, but are not limited to, alkylating agents: cispfatin, treosulfan, and trofosfamide; plant, alkaloids: vinblastine, paclitaxel, docetaxol; DNA topoisomerase inhibitors: teniposide, crisnatol, and mitomycin; anti-folates: methotrexate, mycophetiolic acid, and hydroxyurea; pyrimidine analogs: 5-fluorouracil, doxiiliiridine, and cytosine atabinoside; purine analogs:
  • compositions comprising one or more chemotherapeutie agents (e.g., FLAG, CHOP) may also be used.
  • FLAG comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF
  • CHOP comprises cyclophosphamide, vincristine, doxorubicin, and prednisone.
  • PARP t ⁇ g., PARP-l and/or PARP-2
  • inhibitors arc well known in the art (e.g., Olaparib, ABT-888, BS!-20! , BGP-15 (N-Gene Research Laboratories, inc.); lNO-1 0 ' i (Inoiek Pharmaceuticals Inc.); PJ34 (Soriano et al., 2001 ; Paeher et ai, 2002b): 3-aminoberizamide (Trevigen); 4-amirto- l ,8-o.aphthalimide; (Trevigen); 6(5H)-phetianthridinone (Trevigen); benzamide (U.S.
  • PARP Poly(ADP-ribose) polymerase i
  • radiation therap is used.
  • the radiation used in radiation therapy can be ionizing radiation.
  • Radiation therapy can also be gamma rays. X-rays, or proton beams.
  • Examples of radiation therapy include, but are not limited to, external-beam radiation therapy, interstitial implantation of radioisotopes (1-125, palladium, iridium), radioisotopes such as strontium-89 f thoracic radiation therapy, intraperitoneal P-32 radiatton therapy, and/or total abdominal and pelvic radiation therapy.
  • the radiation therapy can be administered as external beam radiation or teletherapy wherein the radiation is directed from a remote source.
  • the radiation treatment can aiso be administered as internal therapy or brachytherapy wherein a radioactive source is placed inside the body close to cancer cells or a tumor mass.
  • photodynamic therapy comprising the administration of photosensitizers, such as hematoporphyrin and its derivatives, Vertoporfrn (BPD- A), phthalocyanine,
  • photosensitizer Pc4 demethoxy- ' hypocrellin A; and 2BA-2-DMHA.
  • hormone therapy is used.
  • Hormonal therapeutic treatments can comprise, for example, ' hormonal agonists, ormonal antagonists (e.g- , flutanride, bicaiutamide, tamoxifen, raloxifene, leupro!icle acetate (LUPR.ON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, and steroids (e.g.
  • ATRA all-trans retinoic acid
  • hyperthermia a procedure in which body tissue is exposed to high temperatures (up to i OtV .) is used. Heat may help shrink tumors by damaging cells or depriving them of substances they need to live. Hyperthermia therapy cart be local, regional, arid whole-body hyperthermia, using external and internal heating devices.
  • Hyperthermia is almost always used with other forms of therapy (e.g., radiation therapy, chemotherapy, and biological therapy) to try to increase their effectiveness.
  • Local hyperthermia refers to heat that is applied to a very small area, such as a tumor.
  • the area may be heated externally with high-frequency waves aimed at a tumor from a device outside the body.
  • sterile probes may be used, including thin, heated wires or hollow tubes filled with warm water; implanted microwave antennae; and radiofrequency electrodes.
  • an organ or a !imb is heated.
  • Magnets and devices that produce high energy are placed over the region to be heated, in another approach, called perfusion, some of the patient's blood is removed- heated, and then pumped, (perfused) into the region that is to be heated internally.
  • Whole- body heating is used to treat metastatic cancer that has spread throughout the body. It can be accomplished using warm-water blankets, hot wax, inductive coils (like those in electric biatikets), or thermal chambers (similar to large incubators). Hyperthermia does not cause any marked, increase in radiation side effects or complications. Heat applied directly to the skin, however, can cause discomfort or e ven signi ficant local pain in about half the patients treated, it can also cause blisters, which generally heal rapidly .
  • photodynamic therapy also called PDT, photoradiation therapy, phototherapy, or photochemotherapy
  • PDT photoradiation therapy
  • phototherapy phototherapy
  • photochemotherapy photochemotherapy
  • the photosensitizin agent When the treated cancer cells are exposed to laser f ght, the photosensitizin agent absorbs the light and produces an acti e form of oxygen thai destroys the treated cancer ceils. Light exposure must be timed carefully so that it occurs when most of the photosensitizing agent has left healthy cells but is still present in the cancer cells.
  • the laser light used in PDT can be directed through a fiberoptic (a very thin glass strand).
  • the fiber-optic is placed close to the cancer to deliver the proper amount of light
  • the fiber-optic can be directed through a bronchoscope into the lungs for the treatment of lung cancer or through an endoscope into the esophagus for the treatment of esophageal cancer.
  • PDT causes minima] damage to healthy tissue.
  • the laser light currently in use cannot pass through more than about 3 centimeters of tissue (a l ittle more than one and an eighth inch)
  • PDT is mainly used to treat tumors on or just under the skin or on the lining of internal organs.
  • Photodynamic therapy makes the skin and eyes sensitive to light for 6 weeks or more after treatment. Patients are advised to avoid direct sunlight and bright indoor light for at least 6 weeks. If patients must go outdoors, they need to wear protective clothing, including sunglasses. Other temporary side effects of PDT are related to the treatment of specific areas and can include coughing, trouble swallowing, abdominal pain, and painful breathing or shortness of breath.
  • FD A U.S. Food and Drug Administration
  • FD A approved a photosensitizing agent called porftmer sodium, or PhotofriniK
  • laser therapy is used to harness high-intensity light to destroy cancer cells. This technique is often used to relieve symptoms of cancer such as bleeding or obstruction, cspecialiv when the cancer cannot be cured by other trcatnicnis. It may also be used to treat cancer by shrinking or destroying tumors.
  • laser stands for light amplification by stimulated emission of radiation.
  • CO Carbon dioxide
  • iaser-This type of laser can remove thin layers from the skin's surface without penetrating me deeper layers. This technique is particularly useful in treating tumors that have not spread deep into the skin and certain precancerous conditions.
  • the CO> laser is also able to cut the skin. The laser is used in this way to remove skin cancers.
  • Lasers sterilizes the surgery site, thus reducing the risk of infection.
  • Less operating time may be needed because the precision of the laser allows for a smaller incision.
  • Healing time is often shortened; since laser heat seals blood vessels, there is less bleeding, swelling, or scarring.
  • Laser surgery may be less complicated. For example, with fiber optics, laser light can be directed to parts of the body without making a large incision. More procedures may ⁇ be done on an outpatient basis. Lasers can be used in two ways to teat cancer; by
  • a photosensitizing agent is retained in cancer cells and can be stimulated by light to cause a reaction that kills cancer cells.
  • CO? and d:YAG lasers are used to shrink or destroy tumors. They may be used with endoscopes, tubes that allow physicians to see into certain areas of the body, such as the bladder. The light from some lasers cm be transmitted through a flexible endoscope fitted with fiber optics. This allows physicians to see and work in parts of the body that could not otherwise be reached except by surgery and therefore allows very precise aiming of the la ser beam.
  • Lasers also may be used with low-power microscopes, giving the doctor a clear view of the site being treated. Used with other instruments, laser systems can produce a c utting area as small a 200 microns in diameter— less than the width of a very fine thread. Lasers are used to treat many types of cancer. Laser surgery is a standard treatment for certain stages of glottis (vocal cord), cervical, skin, lung, vaginal, vulvar, and penile cancers. In addition to its use to destroy the cancer, laser surgery is also used to help relieve symptoms caused by cancer (palliative care). For example, lasers may be used to shrink or destroy a tumor that is blocking a patient's trachea (windpipe), making it easier to breathe.
  • windshieldpipe trachea
  • LOT Laser- induced interstitial thermotherapy
  • hyperthermia a cancer treatment
  • lasers are directed to interstitial areas (areas between organs) in the body. The laser light then raises the temperature of the tumor, which damages or destroys cancer cells.
  • the duration and/or dose of treatment with anti-cancer therapy may vary according to the particular iron- sulfur cluster biosynthesis pathway inhibitor agent or combination thereof.
  • An appropriate treatment time for particular cancer therapeutic agent will be appreciated by the skilled artisan.
  • the invention contemplates the continued assessment of optimal treatment schedules for each cancer therapeutic agent, where the phenotype of the cancer of the subject as determined b the methods of the invention is a factor in determining optimal treatment doses and schedules.
  • the D A constructs are delivered to ceils by transfection, i.e. f by delivery of ' " naked" DNA or in a complex with a colloidal dispersion system.
  • a colloidal system includes macromolecule complexes, nanocapsules, microspheres, beads, and lipkl-based systems including oil-in- water emulsions, micelles, mixed micelles, and liposomes.
  • the preferred colloidal system of this invention is a lipid-eoniplexed or Uposome-formulated DNA.
  • a plasmid containing a transgeoe bearing the desired D A constructs may first be experimentally optimized for expression (e.g. t inclusion of an intron in the 5' untranslated region and elimination of unnecessary sequences (Feigner, et al, Ann NY Acad Sci 126-139, 1995).
  • Formulation of DNA, e.g. with various lipid or liposome materials may then be effected using known methods and materials and delivered to the recipient mammal. See, e.g., Canonieo et ai.
  • the targeting of l iposomes can be classified based on anatomical and mechanistic factors.
  • Anatomical classification is based on the level of selectivity, for example, organ- specific, cell-specific, and organelie-speeifie.
  • Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes die natural tendency of liposomes to distribute io ceils of the reticuloendothelial s stem (RES) in organs, which contain sinusoidal capillaries.
  • RES reticuloendothelial s stem
  • Active targeting involves alteration of the liposome by coupling die liposome to a specific iigand such as a monoclonal antibody, sugar, glycoiipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
  • a specific iigand such as a monoclonal antibody, sugar, glycoiipid, or protein
  • the surface of die targeted deli very system may be modified in a variety of ways.
  • lipid groups can be incorporated into the lipid bi ayer of the liposome in order to maintain the targeting Iigand in stable association with the liposomal bilayer.
  • Various linking groups can be used for joining the lipid chains to the targeting iigand. Naked DNA or DNA associated with a delivery vehicle, e.g., liposomes, can be administered to several sites in a subject, (see below).
  • Nucleic acids can be delivered in an desired vector.
  • viral or non- viral vectors including adenovirus vectors, adeno-associatcd virus vectors, .retrovirus vectors, lenti virus vectors, and plasmid vectors.
  • exemplary types of viruses include HSV (herpes simplex, virus), AAV (adeno associated virus), HIV (human immunodeficiency virus), BIV (bovine immunodeficiency virus), and MLV (murine leukemia virus), Nucieic
  • - 1.20 - acids can be administered in any desired format that provides sufficiently efficietu delivers' levels, including in virus particles, in liposomes, in nanoparticles, and comp!exed to polymers.
  • the nucleic acids encoding a protein or nucleic acid of interest may be in a piasmi or viral vector, or other vector as is known in the art. Such vectors are well known and an can be selected for a pariiculat" application.
  • the gene delivery vehicle comprises a promoter and a characteristic promoter coding sequence.
  • Preferred promoters are tissue-specific promoters and promoters which are activated by cellular proliferation, such as the thymidine kinase and thymidilate synthase promoters.
  • promoters which are activatable by infection with a virus such as the a- and ⁇ -iuterferon promoters, and promoters which are activatable by a hormone, such as estrogen.
  • promoter which can be used include the Moloney virus LTR, the CMV promoter, and the mouse albumin promoter, A promoter may be constitutive or inducible.
  • naked polynucleotide molecules are used as gene delivery vehicles, as described in WO 90/1 1092 and U.S. Patent 5,580,859.
  • gene delivery vehicles can be either growth factor DNA or RNA and, in certain embodiments, are linked to killed adenovirus. Curie! et al. Hum. Gene. Ther. 3: 147- 154, 1992.
  • Other vehicles which can optionally be used include D A-hgand (Wit et al., J. Biol Chem.
  • a gene delivery vehicle can optionally comprise viral sequences such as a viral ori gin of replication or packaging signal
  • viral sequences can be selected from viruses such as asirovirus, eoronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornaviats, poxvirus, retrovirus, togavirus or adenovirus.
  • the growth factor gene delivery vehicle is a recombinant retroviral vector. Recombinant retroviruses and various uses thereof have been described in numerous references including, for example, Mann et al., Ceil 33: 153, 1983, Cane and Mulligan, Proc. Natl Acad. Sci.
  • Herpes vims e.g., Herpes Simplex Vims (U.S. Patent No. 5,631 ,236 by Woo et al, issued May 20, 1 97 and WO 00/08191 by Neurovex), vaccinia virus (Rklgcway (i 988) Ridge ay, "Mammalian expression vectors," In; Rodriguez R L, Denhardt I ) T, ed.
  • Vectors A survey of molecular cloning vectors and their uses.
  • RNA viruses include an aiphavirus, a poxivirus, n arena virus, a vaccinia virus, a polio virus, and the like. They offer several attractive features for various mammalian cells (Friedmann (1989) Science, 244: 1275- 1281;
  • target DNA in the genome can be manipulated using well- known methods in the art.
  • the target DNA in the genome can be manipulated by deletion, insertion, and/or mutation are retroviral insertion, artificial chromosome techniques, gene insertion, random insertion with tissue specific promoters, gene targeting, transposabie elements and/or any other method for introducing foreign DNA or producing modified DNA/modified nuclear D A.
  • Other modification techniques include deleting DNA sequences from a genome and/or altering nuclear DNA sequences. Nuclear DNA sequences, for example, may be altered by site-directed mutagenesis.
  • biomarker polypeptides, and fragments thereof can be administered to subjects, in sotne embodiments, fusion proteins can be constructed and administered which have enhanced biological properties.
  • biomarker polypeptides, and fragment thereof can be modified according to well-known
  • pharmacological methods in the art e.g., pegylation, glycosylation, oiigotnertzation, etc.
  • pegylation e.g., pegylation, glycosylation, oiigotnertzation, etc.
  • desirable biological activities such as increased bioavaiiabiiity aad decreased proteolytic degradation.
  • Clinical efficacy can be measured b any method .known in the art.
  • the response to an aoti-eaoeer therapy e.g. , iron-sulfur cluster biosynthesis pathway inhibitory therapy
  • the cancer e.g., a tumor
  • the therapy preferably to a change in tumor mass and/or volume after initiation of neoadjuvant or adjuvant chemotherapy.
  • Tumor response may be assessed in a neoadjuvant or adj van situation where the size of a tumor after systemic intervention can be compared to the initial size and dimensions as measured by CT, PET, mammogram, ultrasound or palpation and the DClitlarity of a tumor can be estimated histologically and compared to the cellularuy of a tumor biopsy taken before initiation of treatment.
  • Response may also be assessed by caliper measurement or pathological examination of the tumor after biopsy or surgical resection.
  • Response may be recorded in a quantitative fashion like percentage change in tumor volume or cellularity or using a semi-quantitative scoring system such as residual cancer burden (Syraroans ei i, J. Clin. Oncol.
  • a typical endpoint for response assessment is upon termination of neoadjuvant chemotherapy or upon surgical removal of residual tumor cells and/or the rumor bed.
  • clinical efficacy of the therapeutic treatments described herein may be determined by measuring the clinical benefit rate (CBR).
  • CBR clinical benefit rate
  • the clinical benefit rate is measured by determining the sum of the percentage of patients who are m complete remission (CR), the number of patients who are in partial remission (PR) and the number of patients having stable disease (SO) at a time point at least 6 months out from the cod of therapy.
  • the shorthand .for this formula is CBR-CR-i-PR- SD ov er 6 months.
  • the CBR for a particular tron-sulfur cluster biosynthesis pathway inhibitor therapeutic regimen is at least 25%, 30%, 35%visor 40%, 45%, 50% cur 55%, 60%, 65% font 70%, 75%. 80%, 85%, or more.
  • Additional criteria for evaluating the response to anti-cancer therapy are related to "survival;' which includes all of the following: sitrvivai until mortality, also known as overall survival
  • recurrence- free survival wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith).
  • the length of said survival may be calculated by reference to a defined start point (e.g., time of diagnosis or start of treatment) and end point (e.g., death, recurrence or metastasis).
  • criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.
  • a particular iron- sulfur cluster biosynthesis pathway inhibitor therapeutic regimen can be administered to a populatio of subjects and the outcome can be correlated to biomarker measurements that were determined prior to administration of any anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy).
  • the outcome measurement may be pathologic response to therapy given in the neoadjuvant setting.
  • outcome measures such as overall sitrvivai and disease-free survival can be monitored over a period of time for subjects following anti-cancer therapy (e.g.. iron-sulfur cluster biosynthesis pathway inhibitors' therapy) for whom biomarker measurement values are known.
  • the same doses of iron-su!fur cluster biosynthesis pathwa inhibitor agents are administered to each subject, in related embodiments, the doses administered are standard doses known in the ait for iron-sulfur cluster biosynthesis pathway inhibitor agents.
  • the period of time for which subjects are monitored can vary. For example, subjects ma be monitored for at least 2, 4, 6, 8, 10, 12 f 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months.
  • Biomarker measurement threshold values that correlate to outcome of an anti-cancer therapy e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy
  • compositions described herein can be used in a variety of diagnostic, prognostic, and therapeutic applications regarding hiomarkers described herein, such as those listed in 1 able .5 .
  • the assays provide a method for identifying whether a cancer is likely to respond to anti-cancer therapy (e.g. , iron-sulfur cluster biosynthesis pathway inhibitory therapy) and/or whether an agent cars inhibit the growth of or kill a cancer cell that is unlikely to respond to anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitor ⁇ - therapy).
  • anti-cancer therapy e.g. , iron-sulfur cluster biosynthesis pathway inhibitory therapy
  • agent cars inhibit the growth of or kill a cancer cell that is unlikely to respond to anti-cancer therapy
  • the invention in one embodiment, relates to assays for screening test agents which bind to, or modulate the biological activity of, at least one biomarker listed in Table 1.
  • a method for identifying such an agent entails determining the ability of the agent to modulate, e.g. inhibit, the at least one biomarker fisted in Table 1.
  • an assay is a ceil-free or cell-based assay, comprising
  • test agent contacting at ieast one biomarker listed in Table 1 , with a test agent, and determining the ability of the test agent to modulate (e.g. inhibit) the enzymatic activity of the biomarker, such as by measuring direct binding of substrates or by measuring indirect -parameters as described below.
  • an assay is a cell-free or cell-based assay, comprising contacting at least one biomarker listed in Table 1 , with a test agent, and determining the ability of the test agent to modulate the ability of the biomarker to regulate NFS I or other iron-sulfur cluster biosynthesis pathway member, such as by measuring direct binding of substrates or by measuring indirect parameters as described below.
  • biomarker protein for their respective target polypeptides or molecules can be coupled with a radioisotope or enzymatic label such that binding can be determined by detecting the labeled protein or molecule in a complex.
  • the targets can be labeled with % ⁇ S, C, or J H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • the targets can be enzymatieally labeled with, for example, horseradish peroxidase, alkaline phosphatase, or hiciferase, and the enzymatic label detected by determination of con version of an appropriate substrate to product.
  • Determining the interaction between biomarker and substrate can also be accomplished using standard binding or enzymatic analysis assays.
  • binding of a test agent to a target can be accompli shed in any vessel suitable for containing the reaetants.
  • vessels include microliter plates, test tubes, and micro-centrifuge tubes
  • immobilized forms of the antibodies of the present invention can also include antibodies bound to a solid phase like a porous, microporous (with an average pore diameter less than about one micron) or macroporous (with an average pore diameter of snore than about 10 microns) material, such as a membrane, cellulose, nitrocellulose, or glass fibers; a bead, such as that made of agarose or
  • polyacrylamide or latex or a surface of a dish, plate, or well, such as one made of polystyrene,
  • determining the ability of the agent to modulate the interactio between the biomarker and its natural binding partner can be accomplished by determining the ability of the test agen t to modulate the activity of a polypeptide or other product that functions downstream or upstream of its position within die tron-snlfur chtster biosynthesis pathway.
  • the present invention further pertains to novel agents identified by the above- described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model.
  • an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an antibody identified as described herein can be used in an animal model to determine the mechanism of ac tion of such an agent,
  • the present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylaetieally.
  • diagnostic assays for determining the presence, absence, amount, and/or activity level of a biomarker described herein, such as those listed in Table 1, in the context of a biological sample (e.g., blood, serum,, cells, or ti sue) to thereby determine whether an individual afflicted with a cancer is likely to respond to anticancer therapy (eg., iron-sulfur cluster biosynthesis pathway inhibitory therapy ⁇ , whether in an original or recurrent cancer.
  • anticancer therapy eg., iron-sulfur cluster biosynthesis pathway inhibitory therapy ⁇ , whether in an original or recurrent cancer.
  • Such assays can be used for prognostic or predictive purpose to thereby prophylactieaily treat an individual prior to the onset or after recurrence of a disorder characterized by or associated with bioraarker polypeptide, nucleic acid expression or activity.
  • biomarkers described herein, such as those listed in Table 1
  • Another aspect of the present invention pertains to monitoring the influence of agents (e.g., drugs, compounds, and small nucleic acid-based molecules) on the expression or acti vity of a bioraarker listed in Table 1.
  • the methods of the present invention implement a computer program and computer system.
  • a computer program can be used to perform the algorithms described herein
  • a computer system can also store and manipulate data generated by the methods of the present invention which comprises a pluralit of bioraarker signal changes/profiles which can be used by a computer sy stem in Implementing the methods of this invention.
  • a computer system receives biomarker expression data; (ii) stores the data; and (iii) compares the data in any number of ways described herein (e.g., analysis relative to appropriate controls) to determine the state of informative biomarkers from cancerous or pre-eaneerous tissue.
  • a computer system (i) compares the determined expression biomarker level to a threshold value; and (ii) outputs an indication of whether said biomarker level is significantly modulated (e.g., above or below) the threshold value, or a phenotype based on said indication.
  • such computer systems are also considered part of the present invention.
  • Numerous types of computer systems can be used to implement the analytic methods of this invention according to knowledge possessed by a skilled artisan in the bioinformatics and/or computer arts.
  • Several software components can be loaded into memory during operation of such a computer system..
  • the software components can comprise both software components that are standard in the art and components that are special to the present invention (e.g., dCHIP software described in Lin el « . (2004) ioinform tics 20, ⁇ 233- 1240; radial basis machine learning algorithms (RBM) known in the art).
  • dCHIP software described in Lin el « . (2004) ioinform tics 20, ⁇ 233- 1240
  • RBM radial basis machine learning algorithms
  • the eotnpister comprises a database for storage of biomarker data.
  • biomarker data can be accessed and used to perform comparisons of interest at a later point in time.
  • biomarker expression profiles of a sample derived from the non-cancerous tissue of a subject and/or profiles generated from population-based distributions of informative loci of interest in relevant populations of the same species can be stored and later compared to that of a sample derived from the cancerous tissue of the subject or tissue suspected effacing cancerous of the subject,
  • the present invention provides, in part, methods, systems, and code for accurately classifying whether a biological sample is associated with a cancer that is likely to respond to anti-cancer therapy (e,g., iron-sulfur cluster biosynthesis pathway inhibitory therapy).
  • anti-cancer therapy e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy
  • tie present invention is useful for classifying a sample (e.g., from a subject) as associated with or at risk for responding to or not responding to anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy) using a statistical algorithm and/or empirical data (e.g., the amount or activity of a biomarker listed in Table I ).
  • An exemplary method for detecting the amount or activity of a biomarker listed in Table 1 and thus useful for classifying whether a sample is likely or unlikely to respond to anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy) involves obtaining a biological sample from a test subject and contacting the biological sample with an agent, such as a protein-binding agent like an antibod or antigen-binding fragment thereof, or a nucleic acid-binding agent like an oligonucleotide, capable of defecting the amount or activity of the biornarker in the biological sample.
  • an agent such as a protein-binding agent like an antibod or antigen-binding fragment thereof, or a nucleic acid-binding agent like an oligonucleotide, capable of defecting the amount or activity of the biornarker in the biological sample.
  • the statistical algorithm is a single learning statistical classifier system.
  • a single learning statistical classifier system can be used to classify a sample as a based upon a prediction or probability value and the presence or level of the biomarker.
  • a single learning statistical classifier system typically classifies the sample as, for example, a likely anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy) responder or progressor sample with a sensitivity, specificity, positive predictive value, negative predictive value, and/or overall accuracy of at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • a likely anti-cancer therapy e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy
  • learning statistical classifier systems include a machine learning algorithmic technique capable of adapting to complex data sets (e.g., parte! of markers of interest) and making decisions based upon such data sets.
  • a single learning statistical classifier system such as a classification tree (e.g., random forest) is used.
  • a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more learning statistical classifier systems are used, preferably in tandem.
  • Examples of learning statistical classifier systems include, but are not limited to, those using inductive learning (e.g. ,
  • decision/classification trees such as random forests, classification and regression trees (C& T), boosted trees, etc.), Probably Approximately Correct (PAC) learning, conneetioi!tist learning (e.g., neural networks (NN), artificial neural networks (ANN), neuro fuzzy networks (NFN), network structures, pereeptrons such as multi-layer pereeptrons, multi-layer feed-forward networks, applications of neural networks, Bayesian learning in belief networks, etc,), reinforcement learning (e.g.,, passive learning in a known environment such, as naive learning, adaptive dynamic learning, and temporal difference learning, passive learning in an unknown environment, active learning in an unknown environment, learning action-value functions, applications of reinforcement learning, etc), and genetic algorithms and evolutionary programming.
  • PAC Approximately Correct
  • conneetioi!tist learning e.g., neural networks (NN), artificial neural networks (ANN), neuro fuzzy networks (NFN), network structures, pereeptrons such as multi-layer pereeptrons, multi-layer feed-forward networks, applications of
  • learning statistical classifier systems include support vector machines (e.g. , Kernel methods), multivariate adaptive regression splines (MARS), Levenberg-Marquardt algorithms,, Gauss-Newton algorithms. mixtures of Gaussians, gradient- descent algorithms, and learning vector quantization
  • the method of the present invention further comprises sending the sample classification results to a clinician, e.g., an oncologist
  • diagnosis of a subject is followed by administering to the individual a therapeutically effective amount of a defined treatment based upon the diagnosis.
  • the methods further involve obtaining a control biological sample (e.g. , biological sample from a subject who does not have a cancer or whose cancer is susceptible to anti-cancer therapy (eg., iron-sulfur cluster biosynthesis path way inhibitory therapy), a biological sample from the subject during remission, or a biological sample from the subject during treatment for developing a cancer progressing despite anticancer therapy (eg., iron-sulfur cluster biosynthesis pathway inhibitory therapy).
  • a control biological sample e.g. , biological sample from a subject who does not have a cancer or whose cancer is susceptible to anti-cancer therapy (eg., iron-sulfur cluster biosynthesis path way inhibitory therapy)
  • anti-cancer therapy e.g., iron-sulfur cluster biosynthesis path way inhibitory therapy
  • a biological sample from the subject during remission e.g., iron-sulfur cluster biosynthesis path way inhibitory therapy
  • a biological sample from the subject during remission e.g., iron-sul
  • the diagnostic methods described herein can ftjrthermore be utilized to identify subjects having or at risk of developing a cancer that is likely or unlikely to be responsive to am -cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy).
  • am -cancer therapy e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy.
  • the assays described herein, such as the preceding diagnostic assays or the following assays can. be utilized to identify 1 a subject having or at risk of developing a disorder associated with a misreguiatiion of the amount or acti vity of at least one biomarker described in Table .1rada such as in cancer.
  • the prognostic assays can be utilized to identify a. subject having or at risk for developing a disorder associated with a misregulatiori of the at least one biomarker described in Table 1 , such as in cancer.
  • the prognostic assays described herein can be used to determine whether a subject can be administered an agent (eg., an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, or other drag candidate) to treat a disease or disorder associated with the aberrant biomarker expression or activity,
  • an agent eg., an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, or other drag candidate
  • Another aspect of the invention pertains to methods of modulating the expression or activity of one or more biomarkers described herein ⁇ e.g., those listed in Table I and the Examples or fragments thereof,) for therapeutic purposes.
  • the biomarkers of the present invention have been demonstrated to correlate with cancers. Accordingly, the activity and/or expression of the biomarker, as well as the interaction between one or more biomarkers or a fragment thereof and its naiurai binding partners) or a fragments) thereof, can be modulated in order to treat cancers.
  • Modulatory methods of the invention involve contacting a cell with one or more biomarkers of the invention, including one or more biomarkers of the invention, including one or more biomarkers listed in Tabic i and the Examples or a fragment thereof or agent that modulates one or more of the acti vities of biomarker activity associated with the ceil.
  • An agent that modulates biomarker activity can be an agent as described herein, such as a nucleic acid or a polypeptide, a naturally-occurring binding partner of the biomarker, an antibody against the biomarker, a combination of antibodies against the biomarker and antibodies against other immune related targets, one or more biomarkers agonist or antagonist, a peptidomimetie of one or more biomarkers agonist or antagonist, one or snore biomarkers peptide mimetic, other small molecule, or small RNA directed against or a tnitnic of one or more biomarkers nucleic acid gene expression product.
  • an agent as described herein such as a nucleic acid or a polypeptide, a naturally-occurring binding partner of the biomarker, an antibody against the biomarker, a combination of antibodies against the biomarker and antibodies against other immune related targets, one or more biomarkers agonist or antagonist, a peptidomimetie of one or more biomarkers agonist or antagonist
  • An agent that modulates the expression of one or more biomarkers of the present invention including one or more biomarkers of the invention, including one or more biomarkers listed in Table 1 and the Examples or a fragment thereof is, e.g., an antisense nucleic acid molecule, Ai molecule, shRNA, mature raiRNA, pre-iniRNA, pri-roiRNA, miRNA*, anri-miRNA, or a miRNA binding site, or a variant thereof or other small RNA molecule, triplex oligonucleotide, ribozyme, or recombinant vector for expression of one or more biomarkers polypeptide.
  • an oligonucleotide complementary to the area around one or more biomarkers polypeptide translation initiation site can be synthesized.
  • One or more antisense oligonucleotides can be added to cell media, typically at 200 ,ug mi, or administered to a pa tient to prevent the synthesis of one or more biomarkers polypeptide.
  • the antisense oligonucleotide is taken up by cells and hybridizes to one or more biomarkers niRNA to prevent translation.
  • an oligonucleotide which binds double- stranded DNA to form a triplex construct to preve t DNA unwinding and transcription can be used. As a result of either, synthesis of biomarker polypeptide is blocked.
  • biomarker expression is modulated, preferably, such modulation occurs by a means other than by knocking out the biomarker gene.
  • Agents which modulate expression by virtue of the fact that they control the amount of biomarker in a cell, also modulate the total amount of biomarker activity in a eel! ,
  • the agent stimulates one or more activities of one or more biomarkers of the invention, including one or more biomarkers listed in Table I and the Examples or a fragment thereof.
  • stimulatory agents include active biomarker polypeptide or a fragment thereof and a nucleic acid molecule encoding the biomarker or a fragment thereof that has been introduced into the cell (e.g., cDNA, mRNA, shR As, siR As, small NAs, mature mi ' RNA, pre-tmRNA, pri-miRNA, rm ' R ' NA*, anti- miRNA, or a miRNA binding site, or a variant thereof, or other functionally equivalent molecule known to a skilled artisan), in another embodiment, the agent inhibits one or more biomarker activities. In one embodiment, the agent inhibits or enhances the
  • inhibitory agents include antisense nucleic acid molecules, anti-bionwker antibodies, biomarker inhibitors, and compounds identified in the screening assays described herein.
  • modulatory methods can be performed m vitro (e.g., by contacting the cell with the agent) or, alternatively, by contacting an agent with cells in vivo (e.g., by
  • the present invention provides methods of treating an individual afflicted with a condition or disorder that would benefit from tip- or down-modulation of one or more biomarkers of the present invention listed in Table 1 or 2 and the Examples or a fragment thereof, e.g., a disorder characterized by unwanted, insufficient, or aberrant expression or activity of the biomarker or fragments thereof.
  • the method involves administering an agent (e.g.. an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) biomarker expression or activity.
  • the method involves administering one or more biomarkers polypeptide or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted biomarker expression or activity.
  • Stimulation of biomarker activity is desirable in situations in which the biomarker is abnormally dow.nregulated and/or in which increased biomarker activity is likely to have a beneficial effect.
  • inhibition of biomarker activity is desirable in .vi ' Zwations in which, biomarker is abnormally upregulated and/or in which decreased biomarker activity is likely to have a beneficial effect.
  • these modulatory agents can also be administered in combination therapy with, e.g., chemotherapeutic agents, hormones, antiangiogens, radioiabe!led, compounds, or with surgery, cryotherapy, and/or radiotherapy.
  • chemotherapeutic agents e.g., hormones, antiangiogens, radioiabe!led, compounds, or with surgery, cryotherapy, and/or radiotherapy.
  • the preceding treatment methods can be administered in conjunction with other forms of conventional therapy ⁇ .g., standard-of-care treatments for cancer well known to the skilled artisan), either consecutively with, pre- or post-conventional therapy.
  • these modulatory agents can bo administered with a therapeutically effective dose of ehemotherapeutie agent.
  • these modulatory agents are administered in conjunction with chemotherapy to enhance the activity and efficacy of the chemotherapeutie agent.
  • the Physicians' Desk Reference discloses dosages of chemotherapeutie agents that have bee used in the treatment of various cancers.
  • the dosing regimen and dosages of these aforementioned chemotherapeutie drugs that are therapeutically effective wii! depend on the particular melanoma, being treated, the extent of the disease and other factors familiar to the physician of skill in the art and can be determined, by the physician.
  • the present invention provides pharmaceutically acceptable compositions which comprise a merapeutica.lly ⁇ effect.ive amount of an agent that modulates (e.g. , decreases) biomarker expression and/or activity, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents.
  • compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) imravagtnally or intrarectaSiy, for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound.
  • oral administration for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes
  • parenteral administration for example, by subcutaneous, intramus
  • iiierapeutically-effeetive amount' as used herein means that amount of an agent that modulates (e.g., inhibits) biomarker expression and/or activity which is effective for producing some desired therapeutic effect, e.g., cancer treatment, at a reasonable benefit/risk ratio.
  • phrases "pharmaceutically acceptable' '" is employed herein to refer to those agents, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • phrases "phanraceuticaliy-acceplabie carrier” as used herein means a
  • pharoiaceuticaliy-acceptable material, composition or vehicle such as a liquid or solid filler, diluent, exeipient, solvent or encapsulating material, involved in carrying or
  • phan3 ⁇ 4aceuticaily-accepiabie carriers include: (1 ) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium earboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) exctpients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil saftlower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11 ⁇ po
  • esters such as ethyl oSeaic and ethyl laurate
  • agar such as agar
  • buttering agents such as magnesium hydroxide arid aluminum hydroxide
  • pharmaceuticallyUy-acceptable salts refers to the relatively non-toxic, inorganic and organic acid addition salts of the agents that modulates (e.g., inhibits) biomarker expression and/or activity. These salts can be prepared in situ during the final isolation and purification of the respiration uncoupling agents, or by separately reacting a purified respiration uncoupling agent in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed.
  • Representative salts include the hydrohromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oieate, pahnitate, stearate, laurate, bcnz contest, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, ghieohepfonate, tactobionate, and laurylsulphonate salts and the like (See, for example, Berge et ah (1977) "Pharmaceutical Salts", . Ph rm. SC 66: 1 -19).
  • the agents useful in the methods of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically- acceptable salts with pharraaceutically-acceptable bases.
  • pharmaceutically- acceptable salts in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of agents that modulates (e.g., inhibits) biomarker expression.
  • These salts can likewise be prepared in situ during the final isolation and purification of the respiration uncoupling agents, or by separately reacting the purified respiration uncoupling agent in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a phamiaceutieaily-aeeeptable metal cation, with ammonia, or with a pharaiaceuticaliy-acceptable organic primary, secondary or tertiary amine.
  • a suitable base such as the hydroxide, carbonate or bicarbonate of a phamiaceutieaily-aeeeptable metal cation, with ammonia, or with a pharaiaceuticaliy-acceptable organic primary, secondary or tertiary amine.
  • Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like.
  • Organic amines useful for the formation of base addition salts include et ' hylamine, iethylamine, ethylene-diamine, ethanolaniine, diethanolamine, piperazine and the like (see, for example, Berge et tl., supra).
  • wetting agents such as sodium lauryi sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: ( I ) water soluble antioxidants, such as ascorbic acid, cysteine ' hydrochloride, sodium bisu!fate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gaiiate, alp ' ha-tocop ' herol, and die like: and (3) metal chelating agents, such as citric acid, ethylenediamine tetraaeetie acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine ' hydrochloride, sodium bisu!fate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyani
  • Formulations useful in the methods of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient, which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.
  • Methods of preparing these formulations or compositions include the step of bringing into association an agent that modulates (e.g., inhibits) biomarker expression and/or activity, with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association a respiration uncoupling agent with liquid earners, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragaeanth), powders, granules, or as solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a
  • respiration uncoupling agent as an active ingredient.
  • a compound may also be administered as a bolus, electuary or paste.
  • the active ingredient is mixed with one or more pharmaeeutically-acceptabie carriers, such as sodium citrate or dicalciura phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymeihylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginie acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin : (6) absorption accelerators, such as quaternary ammonium compounds; (?) wetting
  • pharmaeeutically-acceptabie carriers such as sodium citrate or dicalciura phosphate
  • fillers or extenders
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high, molecular weight polyethylene glycols and the like.
  • a tablet- may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative,
  • disintegrant for example, sodium starch glycol ate or cross-linked sodium earboxyrnethyi cellulose
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered peptide or peptido imetie moistened w th an inert liquid diluent.
  • Tablets, and other solid dosage forms may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well ' known in the p annaceutieal-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and-'or microspheres.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient ' s) only, or preferentially, in a certain portion of the gastrointestinal trac t, optionally, in a delayed manner. Examples of embedding
  • compositions which can be used include polymeric substances and waxes.
  • the acti ve ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described exc ipients .
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyi alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions, n addition to the active agent ma contain suspending agents as, for example, ethoxyiated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, raicfocrystalline ccliiilose, aluminum metabydroxidc, bentonite, agar-agar and iragaeanth, and mixtures thereof.
  • Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more respiration uncoupling agents with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, suppository wa or a salicylate, and which is solid at room
  • Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such earners as are known in the art to be appropriate.
  • Dosage forms for the topical or transdermal administration of an agent that modulates (e.g., inhibits) biomarker expression and/or activity include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active component may be mixed under sterile conditions with a plmrmaceuticall -acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to a respiration uncoupling agent, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacaoth, cellulose deri vatives, polyethylene glycols, silicones, bentomtcs, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacaoth, cellulose deri vatives, polyethylene glycols, silicones, bentomtcs, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to an agent that modulates (e.g., inhibits) biomarker expression and/or activity, excipients sitch as lactose, tale, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as
  • the agent that modulates (tig., inhibits.) biomarker expression and/or activity can be alternatively administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A nona ueous (e.g. , fluorocarbon propellant) suspension could be used. Sonic nebulizers are preferred because they minimize exposing the agent to shear, which cart result: in degradation of the compound.
  • an aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically acceptable carriers and stabilizers.
  • the carriers and stabilizers vary with the requirements of the particular compound, but typically incl ude nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols.
  • Aerosols generally are prepared from isotonic solutions.
  • Transdermal patches have the added advantage of providing controlled delivery of a respiration uncoupling agent to the body.
  • dosage forms can be made by dissolving or dispersing the agent in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the pcptidomimetic across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the
  • Ophthalmic formulations are also contemplated as being within the scope of this invention.
  • compositions of this invention suitable for parenteral administration comprise one or more respiration uncoupling agents in combination with one or more pharfflaeeuticaily-aceeptable sterile isotonic aqueous or nonaqueou solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents,
  • aqueous and nonaqueous carriers examples include water, etltanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof vegetable oils, such as oiive oil, and injectable organic esters, such as ethyl oleate.
  • etltanol examples include water, etltanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof vegetable oils, such as oiive oil, and injectable organic esters, such as ethyl oleate.
  • vegetable oils such as oiive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservati ves, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured b the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like, it may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions, in addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum fnonostearifte and gelatin.
  • adjuvants such as preservati ves, wetting agents, emulsifying agents and dispersing agents.
  • the absorption of the drug in order to prolong the effec of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form.
  • delayed absorption of a parenteraily-administered drug form is accomplished by dissolving or suspending the drug in an oil -vehicle.
  • injectable depot forms are made by forming mieroeneapsu!e matrices of an agent that modulates (e.g., inhibits) biomarker expression and/or activity, in biodegradable polymers such as polylaetide-polyglyeolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and polyi anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions, which are compatible with bod tissue.
  • respiration uncoupling agents of the present invention are administered as pharmaceuticals, to humans and animals, the can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier,
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this in ention may be determined by the methods of the present invention so as to obtain an amount of the active ingredient, which i effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No, 5,328,470) or by stereotactic injection (see e.g. , Chen et aL (1994) Proc. Natl. Acad. ScL USA 91 :3054 3057).
  • Tire pharmaceutical preparation of the gene therapy vector can include the gene therap vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery s stem:.
  • kits for detecting and or modulating biomarkers described herein may also include instructional materials disclosing or describing the use of the kit or an antibody of the disclosed invention in a method of the disclosed invention as provided herein.
  • a kit may also include additional components to facilitate the particular application for which the kit is designed.
  • a kit may additionally contain means of detecting the label (e.g., enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a sheep anti-mousc-H P, etc.) and reagents necessary for controls (e.g. , control biological samples or standards),
  • A. kit may additionally include buffers and other reagents recognized for use in a method of the disclosed invention. Non-limiting examples include agents to reduce non-specific binding, such as a carrier protein or a detergent.
  • Example 1 Materials and Methods for Examples 2-5
  • YD38 was obtained from the Korean Ceil Line Bank; ⁇ 95 was from the Japanese Collection of Research Bioresources Cell Bank; MK 1 and KE97 were from the Riken Cell Bank; SNIJ-L W138 and MDA-M468 were from the American Type Culture Center; MK 74 and YCC1 were provided by the P. Jaime Lab; and NHBE were purchased from Lonza (CC-2540). Cells were cultured in DMEM (YCC I), DMEM plus 10 mg/L insulin ( ⁇ 95), RFM1 (MDA-MB469, MK l s MKN74, SNU! , KE97, and. YD38), or EMEM (WB8), All media was supplemented with 10% FBS. NHBE were cultured in BEGM growth medium bullet kit (Lonza CC-3170). b. RNAi and cDNA rescue constructs
  • Negative controls included a non-targeting shRNA (N I), and two separate sbRNAs specific for luciferase (Luc2 and Luc3).
  • sb IFl l which disrupts mitotic spindie dynamics, served as a positive control for cell death. Pairs of oHgos were synthesized (Eurofitis MWG Operons) for each, target sequence, following the genera!
  • OUgo A is: ACCGG- sense target sequence- GTTAATATTCATAGC(ioop)-antisense seqiiersce-TTTT, and Oiigo B is; the reverse complement of Oligo A .
  • A!i shRNAs were cloned into the Cellecta vectors pRSI6-U6-(sh)- UbiC-TagGFP-2A-Puro (for constitutive shRNA expression) and RSlTi2-U6/TO-(sh ⁇ - CMV-TetR-TagRFP-Puro (for Dox-indncihle shRNA expression) at the Bbsl sites.
  • Overlap PGR was used to generate NFSI-shRNA-resistant cDNA constructs (see Table 4). Wild-type NFS I cDNA. was mutagenized as listed in Table 3 using the primer pairs listed in Table 4, generating silent mutations in NFS I that cause resistance to knockdown by NFS l-shL sh4, or sh6.
  • the sequence-confirmed NFS l-shRNA-resisian: mutant PG products were introduced into the pDONR223 Gateway donor vector, then transferred into a Gateway-adapted pLVX-neo entry vector (by Gateway Cloning, Life Technologies). NFS! -sh5 i an UTR -targeted shRNA and was rescued using wild-type
  • Bold nucleotides are tlie silent mutations introduced in the shRNA-resistant CDS Italicized nucleotides are Gateway adapter sequences Scheme for overlap PCR method used to generate shNF S i -resistant cDNA clones
  • 293T cells were transfected using Lipofeciamine (Life Technologies) and packaging plas ids carrying VSV ' g and delta 8.2, along with shRNA or cDNA expression vector to generate lentiviral particles. Viral supematants were collected and cleared by
  • Supematants containing cDNA virus were concentrated using Lenti-X Concentrator (Clontech), titered by infection in limiting dilution, and used at 8 to 10 uL per 500,000 cells to achieve an MO! of approximately 0.5. infections using shRNA lertttvirases were carried out using 40 or 50 uL of viral supernatant (using pRS16 or pRSlT12 constructs, respectively) per 100,000 cells, corresponding to an approximate MOi of 3. in generating stable inducible shRNA lines, ceils were selected using puromyc in ( 1 -3 ug ml, depending on cell line) for 5-7 days.
  • Clonogenic assays were performed for longer-term assessment of proliferative phenotypes. Three days following doxycycline (dox)-inducdon (of stable, inducible shRNA lines using 0.5 ug/mL dox) or infection (for constitutive shRNA expression), cells were trypsinized, counted (Countess Automated Cell Counter, Life Technologies), and re- plated n duplicate or triplicaie in 6-weii dishes at 750 to 3000 cells per well depending on cell line.
  • dox doxycycline
  • Cionogenic growth was monitored by R.FP (pRSiT12 vector) or GFP (pRSI6 vector) expression at 1 week intervals using a laser scanning cytometer to measure colony area and numbers of colonies per well (IsoCyte, imageXpress Ve os). Two to three weeks after plating ceils, cells were stained using crystal violet as an additional visualization of colony formation. For shorter-term proliferative assays, cells were also plated onto 96-weil plates using the Ceil Titer-Glo Luminescent Cell Viability Assay (Promega G757J ⁇ to measure cell viability on the day of plating and 6 days post-plating.
  • Aconitase activity was assessed three to eleven days post-infection or dox induction in one million ceils using the Aconitase Activity Coiorimetric Assay Kit (Btovision K716- 100). Succinate dehydrogenase activity was assessed using the Succinate Dehydrogenase Activity Coiorimetric Assa Kit (Biovisio.n K66 -100). e. Da aj Uysis
  • TCOA Cancer Genome Atlas
  • Ly sates were cleared by centrifugation and quantified using a BCA Protein Assay Kit (Pierce Biotechnology, Inc.). Lysates were resolved by SDS-PAGE (using Mini-Protean TGX gradient gels, Bio-Rad Laboratories), transferred to nitrocellulose (using the iBIot Gel Transfer Device and Dry Blotting System, Life Technologies) and blocked in 5% nonfat milk (Bbtting-Gra.de Blocker, Bio-Rad). Antibodies for S ! (mouse monoclonal antibody B-7, Santa Cruz Biotechnology, inc.), FTHl (rabbit monoclonal antibody D1D4, Ceil. Signaling
  • GAP ' DH rabbit monoclonal antibody 14CI0, Cell Signaling
  • Example 2 NFS! and other members of the iron-sulfur cluster biosynthesis pathway are biomarkers of cancer and targets for inhibiting cancer
  • NFS 1 is a pyrio3 ⁇ 4xaI-5 '-phosphate-dependent cysteine desuifurase that removes inorganic sulfur from cysteine, creating alanine as a byproduct. It is primarily localized to mitochondria and is critical for iron-sulfur cluster biosynthesis and thiomodit!cation of transfer RNAs (tRNAs).
  • the accessory protein iSDl i promotes efficient interaction between NFS ! and it substrate cysteine.
  • NFS! exists as a heterodimer with ISDl .1 and binds to ISCU and FX during iron-sulfur cluster biogenesis, FXN (Frataxin) is an iron- binding protein thought to provide iron for Iron-Sulfur Cluster (ISC) formation.
  • FXN also interacts with NFS i , and may facilitate cysteine binding to NFS 1 by exposing its substrate binding sites (Pandey et al (2013) J. Biol. Che , 288(52», ISCU serves as a scaffold on which ISCs assemb le.
  • a numbe r of proteins HSCB, RSPA9, G RPEL 1/2, GLRX5, BOLA3, ISCA.l/2, 1.BA57, UBPL
  • FU 1 may serve as another scaffolding protein for ISC formation (Li et a I. (2013) Biochem. 52).
  • a schematic diagram further illustrating the iron-sulfur cluster biogenesis pathway is shown in figure 1.
  • NFS 1 is frequently amplified in " various human cancers.
  • Figure 2 shows the results of NFS I amplification assessed across available TCGA (The Cancer Genome Atlas) datasets using the cBioPortal for Cancer Genomics (available on the World Wide Web at cbioportal .org/publk-portal - Colorectal md cervical cancers exhibit the highest levels of NFS 1 amplification.
  • 15% colorectal cancers of a total of 212 cases showed NFS 1 amplification.
  • Approximately 3% stomach cancers of a total of 219 cases also showed NFSl amplification.
  • NFS! amplification within a span of 1 1 genes was also present in recurrent focal amplification in all lung cancers.
  • FIG. 3 shows the results of collective alterations of NFS L LY M4/1SD1 1, ISCU, and FXN evaluated across available TCGA datasets using the cBioPortal for Cancer Genomics (available on the World Wide Web at
  • Figure 4 shows the results of mutation analyses of solute carrier family 25 mitochondrial iron transporter, member 28 (SLC25A28), a mediator of iron uptake, assessed across TCGA samples using datasets from the cBioPortal for Cancer Genomics (available on the World Wide Web at cbiopoitai.org/pubiic-portal/) and the Memorial Sloan-Kettering Cancer Center (MS CC).
  • AH markers shown represent missense
  • SLC25A28 Rl I 2C/M is a recurring mutation in colorectal and gastric cancers.
  • S.LC25A28 gatn-of-function mutations are believed to be particularly dependent on NFS 1.
  • NFS inhibits clonogenic cell growth.
  • inducible knockdown of NFS 1 using sliRNAs causes potent growth inhibition in cells of the gastric cancer cell Sine, K /4.
  • NFS l is amplified in the MKN /4 ceil line.
  • ceil lines that are dependent on NFSl overexpression are well known in the art and include, for example, the AGS and KE39 ceil lines. Such ceil lines, including MKN74, can grow as xenografts in SG mice.
  • MK.N74 stable cell lines carrying dox-inducible shRNAs for Kifl 1 (positive kill control), luciferase (Luc2 or Luc3: negative controls), or NFS 1 shRNAs (NFS1-L -4, - 5, -6) were induced to express shRNAs by addition of dox (0,5 lig/mL) io the culture media, or left un-induced (no dox). Three days post-dox-ind crion, cells were trypsinized.
  • Clonogeme growth was monitored by red fluorescent protein (RFP) expression over two weeks scanning the plates on a laser scanning cytometer (I soCyte, ImageXpress Velos), The average coiony area at days 7 or 14 post-plating from 2 replicate wells was normalized to the average of the negative controls ( Figure 5). Clonogenic growth was also visualized by crystal violet (CV) staining at day 14 ( Figure 5; bottom panel).
  • RFP red fluorescent protein
  • NFS! knockdown was also determined to cause differential effects on cell growth across a panel, of different cell lines.
  • a panel of eel! lines were infected with constitutive NFS1 shRNAs (sh4, sh5, sli6) « negati ve control shRN As (for Luc2, Luc3), and positive kill. control shRNA. (for Kifl 1 ).
  • Three days post- infection cells were trypsinized, counted, and re-plated in 96-well plates. Cell viability was measured on the day of re-plating and at day 6 post-re-pi ating by Cell-Titer® Glo, and. the data was growth rate normalized (see Example I). Cells collected on the day of re-plating were assessed for NFS! D by cj-RT- PC R and Figure 7 shows that NFSi knockdown yielded a range of NFS 1 -dependency with respect to modulation of cell growth.
  • Example 4 Pharmacodynamic markers related to NFSI act as biomarkers for NFSI activity and cell growth modulation and ceil-free and cell-based screening strategies for identifying inhibitors of NFSI and/or other biomarkers listed in Table 1
  • iron-sulfur dependent mitochondrial enzymes such as succinate dehydrogenase (SDH) and aconitase (Aco) act as pharmacodynamic biomarkers for NFSI function.
  • SDH succinate dehydrogenase
  • Aco aconitase
  • Figure 9 shows that SDH and Aco activity are coordinately modulated according to NFSI activity (Majewska et aL (2013) J. Biol. Chem. 288 ;2 134-29142) and Figure 10 further shows thai SDH and Aco activity are significantly decreased following NFSI knockdown.
  • ferroptosis a non-apoptotic, iron-dependent form of cell death known as ferroptosis ( Figure 1 1 ).
  • Erastin, 1 S3R-RSL3, & PE induce ferroptosis and PTGS2 (Cox2) is a known biomarker of ferroptosis, as wel l as bio marker of oxidative stress, which is a hallmark of ferroptosis ( Figure 12 and Yang et aL (2014.) Cell 156:317-331 )
  • the implication of modulation of NFS! and free iron levels are also belie ved to involve the change in expression of ferritin and transferrin receptors, whose translation is regulated by iron-response-proteins.
  • HlF2a is modulated via NFS I modulation.
  • HIF2a is an "undruggab!e" transcription factor in oncology and inflammation that is activated by mutations (either directly or indirectly) in a subset of cancers.
  • Thompson el ctl. (2014) Blood 1 :366-376 demonstrate that HlF2a regulates neutrophil longevity and modulates inflammation.
  • Figure 13 shows that iron-regulatory protein 1 (IRP.1 ) inhibits HIF2a translation ant! activity such that ! Pi activators would downregulate the HlF2a pathway. NFS! inhibition leads to depletion of Pe ⁇ S clusters.
  • IRP.1 iron-regulatory protein 1
  • HIP 2a gain-of-function mutations have been described in congenital polycythemia and number of cancers including paragangliomas and these mutations are believed to be driver imitations.
  • NFS ! inhibition not only affects fundamental iron metabolism in tumors, but also directly shuts down signaling from a mutated driver mutation.
  • Mutations in HIF2a or other mutations that activate F1IF levels, such as imitations in succinate dehydrogenase, are thus predictive biomarkers for NFS 1 therapy.
  • HlF2a has been demonstrated to be important for myeloid cell function, in particular neutrophils (Thompson el al. (2014) Blood .123:366-376), and it is expected that NFS! will modulate activity of myeloid cells within the hypoxic tumor niieroeiiviroBirient.
  • Tumor-associated myeloid cells have been demonstrated to inhibit the anti-tumor immune response such that inhibition of NFS 1 is expected to lead to increased apoptosis of tumor associated myeloid cell which can have therapeutic benefit.
  • FIG. 14 shows the results of M N74 stable ceil lines carrying dox-indueible shRNAs for ift 1 (positive kill control), !ueiferase (Lue2 or Luc3: negative controls), or NFS I.
  • shRNAs (NFS! -4, -5, ⁇ 6) that were cultured in the presence or absence of doxycyciine (0.5 ug/mL).
  • doxycycline for 7 clays were then either lysed in RIPA buffer, quantified, and equall loaded on a SDS-PAGE gel tor analysis by Western by staining for TfRC, NFS1 , FTL, or GA ' PDH (loading control); or trypsintzed, counted, lysed, and analyzed for aconitase activity.
  • Aconitase activity was normalized to the protein concentration of each lysate. After .1 1 days of NFS 1 knockdown, decreases in aconitase activity were maintained along with decreases in succinate dehydrogenase (SDH) activity as determined by the readout of succinate conversion to iuraarate arid as normalized against protein
  • HTF2a is an "undruggable" transcription factor that is active in a subset of cancers, regulates neutrophil longevity, and modulates inflammation (Thompson et l. (2014) Blood .16:366-376).
  • NFS. I knockdown was determined to be associated with down-regulation of HlF2a protein levels ( Figure 16), which also correlated with the down- regulation ofFTHl protein levels, with, the effects on both proteins likely due to the activation of iron-regulatory protein 1 (1R.P1).
  • NFS 1 -amplified colorectal line C2BBE 1 were established following infection and
  • candidate biomarkers for iron-sulfur cluster biosynthesis pathway modulation and iron-dependent cell death (ferroptosis) correlate with NFS 1 inhibition.
  • Example 5 cD A rescue of NFS1 confirms on -target activity of NFS1 sh NAs
  • Figure 17A confirms thai the KN74 gastric cell line harbors amplifications of NFS I, as detern nedby fluorescent in situ hybridization (FISH) analyses relative to C.EP20, a chromosome 20 centromere marker, and DAP! used to stain genomic DNA.
  • FISH fluorescent in situ hybridization
  • FIG. 17B shows the results of the cells wherein the cells were either rescued with wild-type NFS 1 (WT NFS 1 ⁇ or a mutant NFS1 that is eatalyikaUy dead due to a C381 A mutation (NFS l cm ).
  • NFS 1 cmA acts as a dominant negative mutant with growth inhibitory effects comparable to that of NFSl -shS, Specifically, the stable
  • shRN A lines for the negative control shRNAs NT. I , l. c2, or Luc3, or the NFS 1 shRNA
  • lentivirus generated using the pLVX-neo vector
  • WT NFS! negative control
  • NFSl a>Wi ⁇ negative control
  • selected lines were then treated with do ycycline to induce shRNA expression, and at day 3 post-doxycyeline, these 0 cells were trypsinized and replated to assess their clonogenic growth in duplicate in 6- well plates.
  • Figure ⁇ 7 ⁇ shows that impaired clonogenic growth mediated by NFSI-sh.5, a UTR- targeted shRNA: KD » 87% by q PCR, was restored by WT NFS 1 , but not by NFS l slA , The combination of FSl-shS andNFS ' l t '" >,>lA completely inhibited clonogenic growth.
  • Figure 18 shows that cDNA rescue with WT NFS! (in the same clonogenic growth experiment: described in Figure 17 ⁇ restored the S ! -shS-dependent effect on aconitase activity.
  • cell lysates were prepared from 100cm plates of the same sets of infected ceils described in Figure 17, and analyzed for aconitase activity ten days 5 after doxycyciine treatment. Aconitase activity was normalized to protein concentration of each lysate. Similar experiments with NFS !' " "" ⁇ caused insufficient number of cells for analyses since the mutant MP Si strongly inhibited ceil growth.
  • NFS 1 ⁇ > ' catalytic mutant causes a decrease in aconitase activity and ferritin levels comparable to that with FS l-s i or NFS i -sh5 ( Figure 20).
  • M 74 cells were infected at an approximate MOI of 3 at time of cell replating into
  • NFSl -shl NFSl -shl , NFSl -sh.5, or negative control shRNAs, shhie2 or shluc3), or constitutive cDNAs (from pLVX-neo vector: WT NFS I , C381 A catalytic mutant NFS 1 , or GFP (negative control).
  • Selection of infec ted cells was carried out using puromycin (for pRSI6 vectors) or neomycin (for pLVX-neo vectors), beginning 2 days after infection. Seven days
  • cells were either lysed for further analysis of aconitase activity, or lysed with cold RIPA buffer, and lysates were then quantified and loaded in equal protein quantities onto an SDS-PAGE gel.
  • Western analysis was carried out to detect NFS! , FTHl, and vtnctiltn (loading control) levels.
  • Aconitase activity was normalized to protein
  • iron-sulfur cluster biosynthesis pathwa members and pharmacodynamic markers related to same can be used in various screening assay to identify modulators (eg., inhibitors) of iron-sulfur cluster biosynthesis pathway members of interest,
  • biochemical screening for evaluating expression arid activity of a biomarker listed in Table I such as due to application of inhibitors of a biomarker listed in Table I (e.g., NFS I inhibitors), are presented (see, for example, Li et al. (2004) Am. J. Physiol. Cell Physiol. 28?:C1547-C1559; Tsai and Baroadeau (2010)
  • Protein for use in the assays can be purified from any number of natural or recombinant sources (see, for example, Majewska et I. (2013) J. Biol Chem. 288:29134-29142).
  • . call expressing a bicistronic expression vector encoding both NFS! and ISD11 can be used to readily purify a complex of NFS! and 1SD1 1 by, for example, using a tagged protein (see, for example, Marelja et al. (2008) J. Biol. Chem, 283:25178-25185).
  • the expression vector or post-translation modifications can be engineered to remove mitochondrial signal peptides and oilier domains that are not associated with protein function.
  • the activity of a biomarker of interest such as the effect of a test agent or compound on a purified protein, can be analyzed using direct or indirect enzymatic assays. in an indirect method, analysis of an enzymatic reaction product can he analyzed as a surrogate for directly measuring enzyme action.
  • sulfide-based detection methods can be used to analyze FSl readout ( Figure 21).
  • sulfide-based detection methods e.g., methylene blue assays or fluorogenic sulfide probes, such as AzMC to AMC detection
  • alanine-hased detection methods can be used to analyze FSl readout ( Figure 21).
  • a reaction mixture containing a buffer, purified NFS 1/ISDl 1 , cysteine, and PLP cofactor can be created in the presence or absence of a test agent or compound and any enzymatic reaction can be stopped with the addition of ,N-dimeihyl-p-phenylenediami «e and FeC3 ⁇ 4 in HQ solution such that monitoring of the production of methylene blue at an absorbauce of 670 am will indicate the extent of inhibition.
  • the methylene blue-cysteine desulfurase assay is a standard coloriraetrk assa that is well known in the art (see, for example, Pandey el al (201 1) ./. Biol. Chem.
  • figure 22A provides a representative methylene blue assay suitable for a high-throughput format, This was accomplished by making a reaction buffer having 100 niM Tris, pH 8.0, 200 ffl:M NaCl, 100 uM pyrodoxial phosphate, 100 uM DTT, and 100 ⁇ of L-Cysteine. This mixture was then added to wells of a clear 96-weSl plate. After, a range of concentrations of sodium sulfide could be added for use as a standard.
  • Figure 22B demonstrates a representative sulfide detection range, wherein reactions were run with DMPPDA sulfide probe and a standard curve of sodium sulfide concentration as substrate was used to generate methylene blue.
  • the same protocol that was described for Figure 22A was used.
  • a fiuorogenic probe can be used, such as ?-a2ido-4-methylcoitinarin (AzMC) (Thorson el at. (2013) Angewantite Chemie. Intl. Ed. 52:4641 -4544).
  • AzMC reacts with and detects sulfide as NFS 1 produces it, so sulfide loss is minimized relative to a methylene blue assa in which DMPPDA isn't added until the NFS ! reaction is complete (e.g., for at least 20 minutes).
  • AzMC assays have not heretofore been adapted to measure NFS I acti vity .
  • Figure 24 A provides a representative AzMC assay suitable for a high-throughput format. This was accomplished by making a reaction buffer having 100 mM Tris, pH 8.0, 200 mM NaCL 100 ⁇ pyrodoxial phosphate, 5 mM glutathione, 100 uM of L-cysteine and 0,5 mg/mL ofBSA. This mixture was then added to wells of a. black, clear-bottom 96-well plate. " The AzMC probe was then added to each well to a final concentration of .1 ⁇ . Then, a range of Sodium Sulfide
  • Figure 24B demonstrates a representative sulfide detection range, wherein reactions were run with AzMC sulfide probe and a standard curve of sodium sulfide concentration as subs trate was used to generate die fluorophore f AMC. The same protocol that was described for Figure 24A was used.
  • Figure 24C demonstrates enzyme kinetics of IscS using the AzMC assay optimized for high-throughput analyses.
  • the same reaction buffer described for Figure 24A was made, without L-cystcinc.
  • the reaction mixture was thai added to a welis of a black, clear-bottom 96- well plate.
  • the AzMC probe was added, sodium sulfide standards were made as described above, and L-cysteine was added to different wells at different concentrations.
  • the react ion was then carried out at 37 e C with different concentrations of L-cysteine and at several time points in order to find the initial velocities for each substrate concentration.
  • the final IscS concentration used was 250 n. .
  • a Michaelis-Mmten Graph and a Lineweaver-Burk Plot were made, and the K m and V stsiK were subsequently determined.
  • alanine assays cati. be used (Colin et ah (2013) J. Amer. Chem. Sac. 1.35:733-740; Tsai and Baroadeau (201.0) Biechem. 49: 132-9139; Anthony et al. (20! I ) P oS ONE 6:e20374).
  • AzMC assay described herein, alanine assays have not heretofore been adapted to measure NFS! activity.
  • Figure 25 A provides a representative alanine assay suitable for a high-throughput format.
  • NFS1 reaction buffer having 100 mM Tris, pH 8.0, 200 mM aCl, 100 ⁇ pyrodoxiai phosphate, 100 ⁇ ' TT, 100 ⁇ of L-cysteine. " This mix ture was then added to wells of a clear, UV- transparent 96- well plate. If protein was being tested, it would be added at this point and incubated for the appropriate amount of time.
  • the pH was increased to 10 by adding a reaction mixture of 100 mM sodium carbonate buffer with the same concentrations of NaCl, pyrodoxiai phosphate, and DTT as described.
  • NAD* As a co-substrate, 1 mM NAD* was added. A range of L-alanine concentrations were used as standards, and were added to the same reaction mixture described above. Alanine dehydrogenase was added to a final concentration of 0.03 units/raL to each well, and this reaction was run for 30 minutes at room temperature. The alanine dehydrogenase converts alanine and NAD ' to NADH, which fluoresced and could be measured at an excitation of 340 am artel an emission of 460 nm. The blank for this reaction would be the reaction mixture without L-alanine and Figure 25B demonstrates a representative NADH detection range, wherein reactions were run with alanine
  • AlaDH dehydrogenase
  • AlaDH reaction buffer and subsequently the AlaDH were added, and the reaction was incubated at room temperature for 30 minutes, after which the fluorescence was measured via a plate reader.
  • the "blanks'" for the experiment were wells that received the same reagents without any L-alanine.
  • Figure 25C demonstrates enzyme kinetics of IscS using the alanine assay optimized for high- throughput analyses.
  • the same NFS! reaction buffer described for Figure 25A was made without L-eysteine.
  • the reaction mixture was then added to a series of PC tubes. Then, L-aianine was added to the standards tubes, and L-cysteine was added to different tubes at different concentrations.
  • the IscS protein was added to a final concentration of 250 nM
  • the IscS reaction was then carried out at 37 * C with different concentrations of L-eysteine and at different time points, in order to find the initial velocities for each substrate concentration.
  • the reactions at each time point would be halted via heat inactivation at 9S°C, and after ail reactions were finished, the AlaDH reaction was run under the conditions described in Figure 25A. After measuring the fluorescence and calculating the alanine concentration, the initial velocities were found, a Michaeiis-Menten Graph and a Lineweaver-Burk Plot were made and the K m and Vj impart ;a were subsequently determined.
  • a direct enzymatic assay such as that using the same enzymatic reaction mixture described above, but actively monitoring the production of alanine via a mass-spectrometer (e.g., such as the Agilent RapklFire# platform), can be used to identif the extent- or inhibition.
  • Test agents or compounds that reduce the production of alanine for example, can be identified as NFS I inhibitors.
  • This assay could be configured as a high content screen (Weerapana et al. ⁇ 2010) Nature 468:790-795), Alternatively, stable cell lines expressing reporter gene (e.g.. lueiferase, GFP, etc.) with t « NA context that contains iron response elements that will increase mRNA stability and ultimately reporter
  • reporter gene e.g.. lueiferase, GFP, etc.
  • the mRNA context of transferrin receptor can be used as model, wherein iron response elements in 3' UT that increase stabilit of mRNA under low Fe-S levels.
  • the fact that IREs in 3' UT stabilize mRNA, while 20 IRE in 5' UT reduces translation, can be exploited by generating at least two reporters, one that will be upregulated and one that will be down regulated upon inhibition, such as NFS ! inhibition (Fe-S cluster depletion), and the ratio of at least two reporters can be monitored. This can be done with either fluorescent proteins or lueiferase proteins or a combination of both.
  • Figure 26 shows art exemplary schematic diagram illustrating
  • the first reporter can have a 5 ' UTR arrangement similar to ferritin mRNA that will have low protein levels upon NFS I inhibition (e.g., 5 "UTR of ferritm-firefJy luficerase-2A-GFP reporter).
  • the second reporter can have a 3' UTR arrangement similar to transferrin receptor mRNA that will have increased protein levels of the report with increases in NFS 1 inhibition (e.g., Renil!a luciferase-2A-RFP
  • 0 reporter-3' UTR of transferrin receptor Drug selection markers, such as puromycin, can also be used.
  • the readout would be the ratio of at least two reporters. For example, NFS ! inhibition will increase the ratio of Renilla-to-firefly lueiferase ratios and or RFP-to-GFP ratios.
  • simplified reporters with just FTL.iron response element (IRE)-iuciferase and a corresponding control can be used.
  • the engineered constructs can be expressed via any number of well -known vectors, including viruses, such as lentiviral vectors shown in Figure 25, plasmids, and the like. Such a strategy, in addition to allowing the use of the luciferase ratio to identify NFSl inhibitors, could also be adapted for live ceil imaging using the ratio of, for example, two fluorescent markers.
  • Example 7 Microbial pathogen growth inhibited using inhibitors of NFS! homologs
  • NFS 1 homologs in microbial pathogens are believed to have therapeutic potential as inhibitors of microbial pathogenic growth since the catalytic cysteine residue is always conserved.
  • Such NFS! homologs are essential for growth in a variety of bacterial/fungal species including helicohacier pylori via its NFSl homolog, NifS (Olson ei a!. (2000) Bioc em. 30: 16213-16219; mycobctcierium tuberculosis via its NFSl homolog, IscSMtb (Rybmker et al (2014) Biochem. J.
  • any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) on the World Wtde Web and/or the National Center for Biotechnology information (NCBi) on the World W ide Web.
  • TIGR The Institute for Genomic Research
  • NCBi National Center for Biotechnology information

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Abstract

The present invention is based, in part, on the identification, of novel mitochondrial iron-sulfur (Fe-S) cluster biosynthesis pathway biomarkers and modulators, and methods of use thereof, for identifying, assessing, preventing, and treating cancer.

Description

COMPOSITIONS AND METHODS FOR IDENTIFICATION,
ASSESSMENT, PREVENTION, AND TREATMENT OF CANCER USING
NFSl BIOMARKERS AND MODULATORS
Cross-Referenee to Related Applications
This application claims the benefit of U.S. Provisional Application No, 61 /989,037, filed on 6 May 20 ! 4; the entire contents of said application are incorporated herein in their entirety fay this reference.
Background of t he aygntiou
Despite advances in understanding the etiology of cancer and effective methods for treating cancer, malignant neoplasms represent the second most frequent cause of death, worldwide surpassed only by heart diseases. Although effective anti-cancer treatments exist for many malignancies, such treatments are directed against well-known targets that do not fully control such malignancies. Accordingly, there is a great need to identify new cancer-related targets and bioniarkers useful for the identification, assessment, prevention, and treatment of cancer.
Summary of the Invention
The present invention is based, at least in part, on the disco very that the iron-sulfur cluster biosynthesis pathway plays a significant role in driving hyperproiiferative eel! growth and that modulating the pathway (e.g., inhibiting the function of one or more iron- sulfur cluster biosynthesis pathway members) can inhibit such hyperproiiferative cell growth. In addition, btomarkers related to the iron-sulfur cluster biosynthesis pathway have been identified that are useful for identifying and assessing modulation of such
hyperproiiferative ceil growth.
i one aspect, a method of treating a subject afflicted with a cancer comprising administering to the subject an agent that inhibits the copy number, amount, and'or activity of at least one biomarker listed in Table i , thereby treating the subject afflicted with the cancer, is provided. In one embodiment, the agent is administered in a pharmaceutically acceptable formulation, in another embodiment, the agent directly binds the at least one biomarker listed in Table 1 . In still another embodiment, the at least one biomarker listed in Table 1 is human NFS 1 or an ortholog thereof. In yet another embodiment, the method further comprises administering one or more additional anti-cancer agents, optionally comprising mitochondrial cefaclor therapy.
in another aspect:, a method of inhibiting hyperproiiferaiive growth of a cancer cell or cells, the method comprising contacting the cancer cell or cells with an agent that inhibits the copy number, amount, and/or activity of at least one biomarker listed in Table 1, thereby inhibiting hyperproiiferaiive growth of the cancer cell or ceils, is provided, in one embodiment, the step of contacting occurs in vivo., ex vivo, or in vitro. In another embodiment, the agent is administered in a pharmaceutically acceptable formulation. In still another embodiment, the agent directly binds the at least: one biomarker listed in Table 1. in yet another embodiment, the at least one biomarker listed in Table I is human NFS! or an ortholog thereof, in another embodiment, the method further comprises administering one or more additional anti-cancer agents, optionall comprising mitochondrial cofactor therapy.
In still another aspect, method of determining whether a subject afflicted with a cancer or at risk for developing a cancer would benefit from iron-sulfur cluster (ISC) biosynthesis pathway inhibitor therapy, the method comprising: a) obtaining a biological sample from the subject; b) determining the copy number, amount, and/or activity of at least one biomarker listed in Table 1 in. a subject sample; c) determining the copy number, amount, and/or activity of the at least one biomarker in a control; and d) comparing the copy number, amount, and/or activity of the at least one biomarker detected in steps b) and c); wherein a significant increase in the copy number, amount, and/or activity of the at least one biomarker in the subject sample relative to the control copy number, amount, and/or activity of the at least one biomarker indicates that the subject afflicted with the cancer or at risk for developing the cancer would benefit from ISC biosynthesis pathway inhibitor therapy, is provided, in one embodiment, the method further comprises recommending, prescribing, or administering ISC biosynthesis pathway inhibitor therapy if the cancer is determined to benefit from ISC biosynthesi pathway inhibitor therapy. In another embodiment, the method further comprises recommending, prescribing, or administering anti-cancer therapy other than ISC biosynthesis pathway inhibitor therapy if the cancer is determined to not benefit from ISC biosynthesis pathway inhibitor therapy, in still another embodiment, the anti-cancer therapy is selected from the group consisting of targeted therapy, chemotherapy, radiation therapy, and/or hormonal therapy, in yet another embodiment;, the control sample is determined from a cancerous or non-cancerous sample from either the patient or a member of the same species to which the patient belongs. In another embodiment, the control sample comprise ceils. In still another embodiment, the method further comprises determining responsiveness to ISC biosynthesis pathway inhibitor therapy measured by at least one criteria selected from the group consisting of clinical benefit rate, survival until mortality, pathological complete response, semiquantitative measures of pathologic response, clinical complete remission, clinical partial remission, clinical stable disease, recurrence-free survival, metastasis free survival, disease free survival, circulating tumor cell decrease, circulating marker response, and RECIST criteria.
in yet another aspect, a method of assessing the efficacy of an agent for treating a cancer in a subject, comprising: a) detecting in a first subject sample and maintained in the presence of the agent the copy number, amount or activity of at least one biomarker listed in Table 1 ; b) detecting the copy number, amount, and/or activity of the at least one biomarker listed in Table 1 in a second subject sample and maintained in the absence of the test compound; and c) comparing the copy number, amount, and/or activity of the at least one biomarker listed in Table I from steps a) and b), wherein a significantly increased copy number, amount, and/or activity of the at least one biomarker listed in Table I in the first subject sample relative to the second subject sample, indicates that the agent treats the cancer in the subject, is provided.
in another aspect, a method of monitoring the progression of a cancer in a subject, comprising; a) detecting in a subject sample at a first point in time the copy number, amount, and/or activity of at least one biomarker listed in Table 1 ; b) repeating step a) during at least one subsequent point in time after administration of a therapeutic agent; and c) comparing the copy number, amount, and/or acti vity detected in steps a) and b), wherein a significantly increased copy number, amount, and/or activity of the at least one biomarker listed in Table I in the first subject sample relative to at least one subsequent subject sample, indicate that the agent treats the cancer in the subject, is provided, in one embodiment, the subject has undergone treatment, completed treatment, and/or is in remission for the cancer in between the first point in time and the subsequent point in time, In another embodiment, the subject has undergone ISC biosynthesis pathway inhibitor therapy in between the first point in time and the subsequent point in time, in still another embodiment, the first and/or at least one subsequent sample is selected from, the group consisting of ex vivo and in vivo samples. In yet another embodiment, the first and/or at least one subsequent sample is obtained from an animal model of the cancer, in another embodiment, the first and/or at least one subsequent sample is a portion of a single sample or pooled samples obtained from the subject
in still another aspect, a cell-based method for identifying an agent which inhibits a cancer, the method comprising: a) contacting a cell expressing at least one biomarker listed in Table 1 w ith a. test agent; and b) determining the effect of the test agent on the copy number, level of expression, or level of activity of the at least one biomarker listed in Table 1 to thereby identify an agent that inhibits the cancer, is provided, in one embodiment, the cells are isolated from an animal model of a cancer. In another embodiment, the cells are from a subject afflicted with a cancer, in sti!i another embodiment, the cells are unresponsive to ISC biosynthesis pathway inhibitor therapy, in yet another embodiment, the step of contacting occurs in viva, ex vivo, or in vitro, in another embodiment, the method further coinpn'scs determining the ability of the test agent to bind to the at least one biomarker listed in Table 1 before or after determining the effect of the test agent on the copy number, level of expression, or level of activity of the at least one biomarker listed in Table 1 , In another aspect, the sample comprises ceils, cell lines, histological slides, paraffin embedded tissue, fresh frozen tissue, fresh tissue, biopsies, blood, plasma, serum, buccal scrape, saliva, cerebrospinal fluid, urine, stool, mucus, or bone marrow, obtained from the subject.
in yet another aspect, a cell-free method for identifying a compound which inhibits a cancer, die method comprising: a) determining the effect of a test compound on the amount or activity of at least one biomarker listed in Table 1 contacted with a test compound; b) determining the amount or activity of the at least one biomarker listed in Table 1 maintained in the absence of the test compound; and c) comparing the amount and/or activity of the at least one biomarker Hsted in Table 1 from steps a) and b), wherein a significantly increased amount, and or activity of the at least one biomarker listed in Table I in step a) relative to step b), identifies a compound which inhibits the cancer, is provided. In one embodiment, the method further comprises determining the ability of the test compound to bind to the at least one biomarker l isted in Table 1 before or after determining the effect of the test compound on the amount or activity of the at least one biomarker . In another embodiment, the steps a) and b) are selected from the group consisting of a methylene blue assay, a ?-azido-4-tneihylcoumarin (A MC.) assay, an alanine assay, and a mass spectrometry assay. In still another embodiment, the methylene blue assay compri es i) reacting the at least one biomarker listed in Table 1 in a buffer comprising a) cysteine, b) a pyridoxal phosphate cofactor, and c) optionall the test compound; ii) stopping the reaction by adding N,N-dimethyi-p-phenyienedi amine and iron chloride (FeCB) in hydrogen chloride (HQ) solution, and in) determining the production of methylene blue via absorhance of light having a wavelength of 670 tim. in yet another embodiment, the AxMC assay comprises i) reacting the at least one biomarker listed in Table 1 in a buffer
comprising a) cysteine, b) a pyridoxal phosphate cofactor, e) glutathione as reducing agent, d) bovine serum albumin, e) 7-azido-4-raetaykoumarin, and f) optionally, the test compound; and ii) fiuorometricaily monitorin the reaction product, 7-amino-4- methylcoumarin. In another embodiment, the alanine assay comprises i) reacting the at least one biomarker listed in Table I in a buffer comprising a) cysteine, b) a pyridoxal phosphate cofactor, e) DTT as reducing agent, and d) optionally, the test compound; ii) performing a. secondary reaction to measure alanine production in a buffer containing a) NAD (nicotinamide adenine dinucleoride) and b) alanine dehydrogenase enzyme; and in) fiuorometricaiiy measuring the reaction product, NADH. in still another embodiment, the mass spectrometry assay comprises i) reacting the at least one biomarker listed in Table 1 in a buffer comprising a) cysteine, b) a pyridoxal phosphate cofactor, and c) optionally the test compound; and ii) determining the production of alanine using mass spectrometry.
Other embodiments of the present invention are applicable to any of the methods, compositions, assays, and the like presented herein. For example, in one embodiment, the copy number is assessed by mieroarray, quantitative PCR (qPCR), high-throughput sequencing, comparative genomic hybridization (CGH), or fluorescent in situ hybridization (FISH). In another embodiment, the amount of the at least one biomarker is assessed by detecting the presence in the samples of a polynucleotide molecule encoding the biomarker or a portion of said polynucleotide molecule. In still another embodiment, the
polynucleotide molecule is a mRNA, cDNA, or functional variants or fragments thereof, in yet another embodiment, the step of detecting further comprises amplifying the
poly nucleotide molecule, in another embodiment, the amount of the at least one biomarker is assessed by annealing a nucleic acid probe with the sample of the polynucleotide encoding the one or more biomarkers or a portion of said polynucleotide molecule under stringent hybridization conditions, in still another embodiment, the amount of the at least one biomarker is assessed b detecting the presence a polypepti de of the at least one biomarker. In yet another embodiment, fits presence of a polypeptide is detected using a reagent which specifically binds with the polypeptide (e.g., a reagent selected from the group consisting of an antibody, an antibody deri vative, and an antibody fragment). In another embodiment, the acti ity of the at least one biomarker is assessed by determining the magnitude of modulation of at least one NFS! pharmacodynamic biomarker listed in Table 1. In still another embodiment, the activity of the at least one biomarker is assessed by determining the magnitude of modulation of the activity or expression level of at least one downstream target of the at least one biomarker. In yet another embodiment, the ISC biosynthesis pathway inhibitor agent or test compound modulates a biomarker selected from the group consisting of human NFS I , human LY M4, human !SCU, human FXN, human NFUJ , human GLRX5, human BOLA3, human HSCB, human KSPA9, human
ISCA1 , human 1SCA2, human ί.ΒΑ.57. human NUBPL, human SLC2SA28, human PDXR, human FDX2, and orthoiog of said biomarkers thereof. In another embodiment;, the ISC biosynthesis pathway inhibitor agent or test compound is an inhibitor selected from the group consisting of a small molecule, antisense nucleic acid, interfering RNA, shR A, siRNA , aptamer, ribozyme, dominant-negative protein binding partner, and combinations thereof. In another embodiment, the at least one biomarker is selected .from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more biomarkers. in still another embodiment:, the at least one biomarker is selected from the group of I SC biosynthesis pathway biomarkers listed in Table L In yet another embodiment, the ISC biosynthesis pathwa biomarkers listed in Table 1 are selected from the group consisting of human NFS !„ human LYRM4, human ISCU, human FXN, human NFU I , human GLRX5, human BOLA3, hitman HSCB, human HSPA9, human ISC A 1 , human ISCA2, human IBA57, human NUBPL, human SLC25A28, human FDXR, human FDX2, and orthoiogs of said biomarkers thereof. In another embodiment, the at least one biomarker is selected from the group of NFS 1 pharmacodynamic biomarkers listed in Table 1. In stil l another embodiment, the NFS I pharmacodynamic biomarkers listed in Table 1 are selected from the group consisting of human aconitase, human succinate dehydrogenase, human ferritin, human transferrin- reeeptor, human HifZalpha, human PTGS2, and lipid reactive oxygen species ( OS). In yet another embodiment, the cancer is selected from the group consisting of paragangliomas, colorectal cancer, cervical cancer, lung adenocarcinoma, ovarian cancer, and myeloid cancer within a hypoxic tumor microenvvronment. In another embodiment, the subject is a mammal, such, as an animal model of cancer or a human. Brief Description of the Drawings
Figure 1 shows a schematic diagram of the iron-sulfur cluster biogenesis pathway as adapted from Lira et al. (2013) Htm. Mol Genet. 22:4460-4473.
Figure 2 shows the results of NFS I amplification assessed across available TCGA (The Cancer Genome Atlas) datasets using the cBioPortal for Cancer Genomics (available on the World Wk!e Web at cbioportai.org pubh'c-portal ). The inset shows the correlation between copy-number alterations (x-axis, as determined by GISTIC) and mRNA expression (y-axis, by RNASeq) from a representative dataset (colorectal cancer). All histograms shown represent: amplifications.
Figure 3 shows the results of collective alterations of NFS! , LYR 4/ISD1 1 , ISCU, and FXN evaluated across available TCGA datasets using the cBioPortal for Cancer Genomics (available on the World Wide Web at cbioportal.org/public-portal/). Top bar of histogram: amplification; middle bar of histogram: deletion; bottom bar of histogram: imitation; gray: multiple alterations. The tipper inset shows the distribution of alterations between NFS1 , LYRM4, ISCU, and FXN in ovarian cancers. The lower insets show the correlation between copy-number alterations (x-axis, as determined by GISTIC) and mRNA expression (y-axis, by RNASeq) for NFS 1 (left) and LYRM4 (right) from a representative dataset.
Figure 4 shows the results of mutation analyses of solute carrier family 25 mitochondrial iron transporter, member 28 (SLC25A28), a mediator of iron uptake, assessed across TCGA samples using datasets from the cBioPortal for Cancer Genomics (available on the World Wide Web at cbioportai.org/pablic-portai/) and the Memorial Sloan-Ketterlng Cancer Center (MSKCC). All markers shown represent raissense mutations, except, for the seventh market" from the left located at the -terminus of the Mito_carr domain, which represents a frameshift deletion.
Figure 5 shows that inducible knockdown of NFS l using siiRNAs causes clonogenic growth inhibition in M N74 cells.
Figure 6 shows the results ofe'DNA rescue experiments confirming on-target activity of NFS ! shRNAs.
Figure 7 shows the results of NFS .1 knockdown and resulting cell growth effects across a panel of cell lines.
Figure 8 shows the results of NFS I sh shut-off experiments confirming restoration of clonogenic growth following derepression of NFS 1 function. Figure 9 shows the results of aconitase activity and SDH activity measured in lysates of mitochondria isolated from: GAL-NFS ί cells harboring plasmid- ome copies of WT NFSl, fl/S'iLM/AA, or vector without insert, as indicated, and grown for 40 hours in glucose-containing medium. Enzymatic activities were measured and plotted relative to the non-iron-sulfur cluster protein, malate dehydrogenase. The figure is adapted from
Majewska et al. (2013) J. Biol. Chem. 288:2 134-29142.
Figure 10 shows thai transient knockdown of NFS'! in Heia ce!is shows alterations in mitochondrial structure and significantly decreased activity of iron-sulfur-dependent enzymes, including aconitase and SDH. The figure is adapted from Biederbick el al(2006) Μοί Cell, Biol 26:5675-5687.
Figure 11 shows a schematic diagram illustrating an iron-dependent form of nou- apoptotic cell death known as ferroptosis. The. figure is adapted from. Dixon ei al (2012) Cell 149:4060- 1072.
Figure 12 includes 2 panels, identified as panels A aikf B, which show data adapted from Yang et al. (2014) Cell 156:317-331 indicating that upregulation of PTGS2 expressio occurs upon crastin and {1S,3&)-RSL3 treatment (panel A) and further showing that PTGS2 expression is induced by PE (panel B). The oncogenic RAS-seieciive lethal small molecule erasfin. triggers ferroptosis, which is dependent upon intracellular iron, but not other metals, and is morphologically, biochemically, and genetically distinct from apoptosis, necrosis, and autophagy. Erastin, like glutaraate, inhibits cysteine uptake by the cysteine/glutairiate antiporter (system xc--), creating; a void in the antioxidant defenses of the eel! and ultimately leading to iron-dependent, oxidative death.
Figure 13 shows that iRP~ I represses HIF2a translation and acti vity. The figure is adapted from Zimmer el al. (2008) Mol Cell 32:838-848.
Figure 14 shows dat confirming that candidate biomarkers for iran-suifur cluster biosynthesis pathway modulation and iron-dependent cell death (ferroptosi ) correlate with NFSl inhibition.
Figure 15 includes 5 panels, identified as panels A, B, C, D, and E, which demonstrate modulation of biomarkers of ISC biosynthesis pathway modulation and iron- dependent cell-death (ferroptosis) associated with NFSl knockdown. Panel A shows decrease in ferritin protein le vels and increases in TFRC protein le vels. Panels B and C shows decreases in aconitase activity. Panel F. shows decreases in succinate dehydrogenase activity. Panel F confirms knockdown of NFS i protein levels in the samples analyzed in panels E and F.
Figure 16 shows that NFS 1 knockdown in the C2BBE1 colorectal cell hoe correlates with down-regulation of HlF2a protein. The asterisk (*) indicates treatment for 24 hours with cobalt chloride, a hypoxia-mimieking agent.
Figure 17 includes 2 panels, identified as panels A and B, which demonstrate that NFS '! is essential in MKN74, an NPS-i amplified cell line. Panel A shows that NFS I is amplified in the MKN74 gastric cell line. Panel B shows the results of cDNA rescue of M N74 stable inducible NFS 1 shRNA lines with either wild type NFS! or a dominani- negative NFS! mutant.
Figure 18 shows that cDNA rescue with WT FSi similar to that described in Figure 17 restores the NFS 1 -sh5~dependent effect oo aconiiase activity.
Figure 19 shows that WT NFSI t but »ot NFSl°*iA, rescues the NFSl -shS- dependetit inhibition of FTH1 protein levels and the up-regu!ation of Tfitc protein levels.
Figure 20 shows that the NFS 1 t hi catalytic mutant causes a decrease in aeonitase acti vity and ferritin levels comparable to that with NFS ! -sh l or NFS l-sh5.
Figure 21 shows representative sulfide-based (e.g., methylene blue assays or flurogenic sulfide probes, such as AzMC to AMC detection.) or alanine-based (e.g., alanine dehydrogenase activity) detection methods for analyzing NFS. I activity.
Figure 22 includes 2 panels, identified as panels A and B, which provide a representative methylene blue assay suitable for high-through put formats (panel A) and representative sulfide detection range analyses (panel B). A value of > 0.5 is preferred for enzymatic assays and Z* was calculated as equaling 1 - [ 3 (SD of signal + SD of background) / (Mean of signal - Mean of background)').
Figure 23 shows the loss of sulfide from solution in a methylene blue assay over time.
Figure 24 includes 3 panels, identified as panels A, B, and C, which provide a representative AzMC assay suitable for high-through put formats (panel A) and representative sulfide detection range analyses (panel B). V values were calculated as equaling .1 - 3 (SD of signal ·*· SD of background) (Mean of signal - Mean of background)). Panel C shows the enzyme kinetics of IscC using an AzMC assay optimized for high-throughput analyses. Figure 25 includes 3 panels, identified as panels A, .8, and C, which provide a representati ve alanine assay suitable for high-through put formats (panel A) and
representative sulfide detection range analyses (panel B). Z* values were calculated as equaling 1 - [ 3 (SD of signal ·÷· SD of background) / (Mean of signal - Mean of
background)]. Panel C shows the enzyme kinetics of IscC using an alanine assa optimized for high-throughput analyses.
Figure 26 shows exemplary reporter constructs useful for screening for NFS i inhibitors and/or inhibitors of the iron-sulfur cluster biosynthesis pathway. The asterisks (*) represent the use of luciferase, GFP, and RFP containing destabilizing sequences from mouse ornithine decarboxylase at their C-terminus.
Note that for every figure containing a histogram, the bars from left to right for each discreet measurement correspond to the figure boxes from top to bottom in the figure legend as indicated.
Detailed Description of the Invention
Iron-sulfur cluster biogenesis is necessary for the generation of iron-sulfur
containing proteins. It lias been determined herein that the presence, absence, amount (e.g., copy number or level of expression), and/or activity of iron-sulfur cluster biogenesis pathway members are biomarkers for the diagnosis, prognosis, and treatment of cancers, A variety of cancers can be so analyzed and treated, such as those having overexpression of NFS1 and/or those having activating mutations in the HIF2a pathway.
L Definition
The articles Y and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.
The term: "altered amount" or "altered level" refers to increased or decreased copy number (e.g. , gcrmline and/or somatic) of a biomarker nucleic acid, e.g., increased or decreased expression level in a cancer sample, as compared to the expression level or copy number of the biomarker nucleic acid in a control sample. The term "altered amount" of a biomarker also includes an increased or decreased protein level of a biomarker protein in a sample, e.g., a cancer sample, as compared to the. corresponding protein level in a normal, control sample. Furthermore, an altered amount of a biomarker protein may be determined by detecting posttraiislatKraai modificatioii such as methylatioii status of the marker, which may affect the expression or activity of the biomarker protein.
The amount of a biomarker in a subject is "significantly" higher or lower than the normal amount of the biomarker, if the amount of the biomarker is greater or less, respectively, than the normal level by an amount greater than the standard error of the assay employed to assess amount, and preferably at least 20¾, 30%, 40%, 50¾, 60%, 70%, 80¾, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or than that amount. Alternatively, the amount of the biomarker in the subject can be considered "significantly" higher or lower than the normal amount if the amount is at least about two, and preferably at ieast about three, four, or five times, higher or lower, respectively, than the normal amount of the biomarker.
The term "altered level of expression" of a biomarker refers to an expression level or copy number of the biomarker in a test sample, e.g., a sample derived from a patient suffering from cancer, that is greater or less than the standard error of the assay employed to assess expression or copy number, and is preferably at least twice, and more preferably three, .four, five or ten or more times the expression level or copy number of the biomarker in a control sample (e.g... sample from a healthy subjects not having the associated disease) and preferably, the average expression level or copy number of the biomarker in several control samples. The altered level of expression is greater or less than the standard error of the assa employed to assess expression or copy number, and is preferably at least twice, and more preferably three, four, five or ten or more times the expression level or copy number of the biomarker in a control sample (e.g., sample from a healthy subjects not having the associated disease) and preferably, the average expression level or copy number of the biomarker in several control samples.
The term "altered activity" of a biomarker refers to an activity of the biomarker which is increased or decreased in a disease state, e.g., in a cancer sample, as compared to the activity of the biomarker in a normal, control sample. Altered activity of the biomarker may be the result of for example, altered expression of the biomarker, altered protein level of the biomarker, altered structure of the biomarker, or, e.g., an altered interaction with other proteins involved in the same or different pathway as the biomarker or altered interactio with transcriptional activators or inhibitors.
The term "altered structure" of a biomarker refers to the presence of mutations or allelic variants within a biomarker nucleic acid or protein, e.g., mutations which affect expression or acti vity of the biomarker nucleic acid or protein, as compared to the normal or wild-type gene or protein. For example, mutations include, but are not limited to substitutions, deletions, or addition mutations, Mutations may be present in the coding or non-coding region of the biomarker nucleic acid.
Unless otherwise specified here within, the terms "antibody" and "antibodies" broadly encompass naturally-occurring forms of antibodies (e.g. I'gG, igA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments and derivatives have at least an antigenic binding site. Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody.
The term "antibody" as used herei also includes an "antigen-binding portion" of an antibody (or simply "antibody portion"). The term "antigen-binding portion", as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g. , a biomarker polypeptide or fragment thereof). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full- length antibody. Examples of binding fragments encompassed within the term "antigen- binding portion" of an antibod include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (it) a Fi'ab')? fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341 : 544-546), which consists of a VH domain; and (vi) an isolated
complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent polypeptides (known as single chai Fv (scFv); see e.g.. Bird et al (1 88) Science 242:423-426; and Huston ei al (1 88) Prac. Natl Acad Sci. USA 85:5879-5883; and Osbourn et al 1998, Nature Biotechnology 16: 778). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Any VH and VL sequences of specific seFv can be linked to human immunoglobulin constant region cD A or genomic sequences, in order to generate expression vectors encoding complete I'gG polypeptides or oilier isotypes. VH and VL can also be used in the generation of Fab, Fv or other fragments of immunoglobulins using either protein chemistry or recombinant DMA technology. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see &g.r Hoiliger et a!. (1993) Proc. Ναίί Acad. Set. U.S.A. 90:6444-6448; Poljak et al. (1994) Structure 2: 1 121 - 1 123).
Still further, an antibody or an tigen-binding portion thereof may be part of larger immunoadbesion polypeptides, formed by co valent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion polypeptides include use of the streptavidin core region to make a tctramcric scFv polypeptide (Kipriyanov et al. (1 95) Human Antibodies and Hyhrtdamas 6:93-101) and use of a cysteine residue, biomarker peptide and a C-terminal polyhistidmc tag to make bivalent and biotinylated scFv polypeptides (Kipriyanov et al. ( 1994) Mol. Immunol 31 : 1047-1058). Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such, as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadbesion polypeptides can be obtained using standard recombinant DNA
techniques, as described herein.
Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof (e.g. humanized, chimeric, etc.). Antibodies may also be fully human. Preferably, antibodies of the invention bind specifically or substantially
specifically to a biomarker polypeptide or fragment thereof The terms "monoclonal antibodies" and "monoclonal antibody composition", as used herein, refer to a population of antibody polypeptides that contain only one species of an antigen binding site capable of imniunoreaeting with a particular epitope of an an tigen, whereas the term "polyclonal antibodies" and "polyclonal antibody composition" refer to a population of antibody polypeptides that contain multiple species of antigen bi nding sites capable of interacting with a particular antigen. A monoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it immunoreacts.
Antibodies may also be "humanized," which is intended to include antibodies made by a non-human celi having variable and constant regions which have been altered to more closely resemble antibodies that would be made by a human ceil. For example, by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germlme immunoglobtslin sequences. The humanized antibodies of the invention may include amino acid residues not encoded by human germlme immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or b somatic imitation in vivo), for example in the CDRs. The term "humanized antibody", as used herein, also includes antibodies in which CDR sequences derived from the germtine of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
The term "assigned score" refers to the numerical value designated for each of the biomarkers after being measured in a patient sample. The assigned score correlates to the absence, presence or inferred amount of the biomarker in the sample. The assigned score can be generated manually (e.g., by visual inspection) or with the aid of instrumentation for image acquisition and analysis. In certain embodiments, the assigned score is determined by qualitative assessment, for example, detection of a fluorescent readout on a graded scale, or uantitative assessment:. In one embodiment, an "aggregate score," which refers to the combination of assigned scores from a plurality of measured biomarkers, i s determined, in one embodiment the aggregate score is a summa tion of assigned scores. In another embodiment, combination of assigned scores involves performing mathematical operations on the assigned scores before combining them into an aggregate score. In certain, embodiments, the aggregate score is also referred to herein as the predictive score."
The term "biomarker" refers to a measurable entity of the present invention that has been determined to be predictive of anti-cancer therapy ( g., iron-sulfur cluster biosynthesis pathway inhibitory therapy) effects on a cancer. Biomarkers can include, without limitation, nucleic acids (e.g. , genomic nucleic acids and or transcribed nucleic acids) and proteins, particularly those involved shown in Table 1. Many biomarkers listed in Table 1 are also useful as therapeutic targets, hi one embodiment, such targets are the iron-sulfur cluster biosynthesis pathway members shown in section A of Table I .
A "blocking" antibody or an antibody "antagonist" is one which inhibits or reduces at least one biological activity of the antigen(s) it binds, in certain embodiments, the blocking antibodies or antagonist antibodies or fragments thereof described herein substantially or completely inhibit a given biological acti vity of the antigen(s). The term "body fluid" refers to fluid that are excreted or secreted from the bod as well as fluid that are normally not (e.g. amniotic fluid, aqueous humor, biief blood and blood plasma, cerebrospinal fluid, cerumen and earwax, co per's fluid or pre-ejaculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, and vomit).
The terms "cancer" or "tumor" or "hyperprolifeTath¾'? refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. In some embodiments, such cells exhibit such characteristics in past or in full due to the expression and activity of oncogenes, such as e- MY Cancer cells are often in the form of a tumor, but such cells may exist alone within an animal, or may be a non-htmorigenie cancer ceil, such as a leukemia cell. As used herein, the term "cancer" includes premalignant as well as malignant cancers. Cancers include, but are not limited to, B cell cancer, e.g., multiple myeloma, Waldenstrom's macroglobulinemia, the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gamniopathy, and
immunoeytic amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, sali ary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematologic tissues, and the like. Other non-limiting examples of types of cancers applicable to the methods encompassed by the present invention include human sarcomas and carcinomas, e.g., fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma,, osteogenic sarcoma, chordoma,
angiosarcoma, endotheliosareoma, lymphangiosarcotna, lymphaagioendomcitosarcoraa, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain iiimor, testicular cancer, lung carcinoma, small ceil lung carcinoma, bladder carcinoma, epithelial carcinoma,, glioma, astrocytoma, medullobSastoma, craniopharyngioma, ependymoma, pineaSoma, hemangiobiastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukeinias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myelofa!astic, promyelocyte, myelomonocytie, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkirfs disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's roacrogiobu!inemia, and heavy chain disease. In some embodiments, cancers are epithlelia.l in nature and include but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer- n still other embodiments, the epithelial cancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovarian carcinoma), or breast carcinoma. The epithelial cancers may be characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear ceil, Brenner, or undifferentiated.
The term "coding region" refers to regions of a nucleotide sequence comprising eodons which are translated into amino acid residues, whereas the term "non-coding region" refers to regions of a nucleotide sequence that are not translated into amino acids (e.g., 5' and 3' untranslated regions).
The term "complementary" refers to the broad concept of sequence
complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region ts capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which, is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparaliel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first: and second portions are arranged in an antiparaliel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in die second portion. More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
The term "control" refers to any reference standard suitable to provide a comparison to the expression products in the test sample, in one embodiment, the control comprises obtaining a "control sample" from which expression product levels are detected and compared to the expression product levels from the test sample. Such a control sample may comprise any suitable sample, including but not limited to a sample from a control cancer patient (can be stored sample or previous sample measurement) with a known outcome; normal tissue or cells isolated from a subject, such as a normal patient or the cancer patient, cultured primary cells/tissues isolated from a subject such as a normal subject or the cancer patient, adjacent norma! cells/tissues obtained from the same organ or body location of the cancer patient, a tissue or cell sample isolated from a normal subject, or a primary cells/tissues obtained from a depository. In another preferred embodiment, the control may comprise a reference standard expression product level from any suitable source, including but not limited to housekeeping genes, an expression product level range from normal tissue (or other previously analy ed control sample), a previously determined expression product level range within a test sample from a group of patients, or a set of patients with a certain outcome (for example, survival for one, two, three, four years, etc.) or receiving a certain treatment (for example, standard of care cancer therapy). It will be understood by those of skill in the art that such control samples and reference standard expression product levels can be used in combination as controls in the methods of the present invention. In one embodiment, the control may comprise normal or non-cancerous cell/tissue sample, in another preferred embodiment, the control may comprise an expression level for a set of patients, such as a set of cancer patients, or for a set of cancer patients receiving a certain treatment, or for a set of patients with one outcome versus another outcome. In the former case, the specific expression product leve! of each patient can be assigned to a percentile level of expression, or expressed as either higher or lower than the mean or average of the reference standard expression level In another preferred embodiment, the control may comprise norma! cells, cells from patients treated with combination chemotherapy,, and cells from patients having benign cancer. In another embodiment, the control may a!s comprise measured value for example, average ievel of expression of a particular gene in a population compared to the level of expression of a housekeeping gene in the same population. Such a population may comprise normal subjects, cancer patients who have not undergone any treatment (i.e. , treatment naive), cancer patients undergoing standard of care therapy, or patients having benign cancer, in another preferred embodiment, the control comprises a ratio transformation of expression product levels, including but not limited to determining a ratio of expression product levels of two genes in the test sample and comparing it to any suitable ratio of the same two genes in a reference standard;
determining expression product levels of the tw or more genes in the test sample and determining a difference in expression product levels in any suitable control; and determining expression product levels of the two or more genes in the test sample, normalizing their expression to expression of housekeeping genes in the test sample, and comparing to any suitable control. In particularly preferred embodiments, the con trol comprises a control sample which is of the same lineage and/or ty pe as the test sample. In another embodiment, the control may comprise expression product levels grouped as percentiles within or based on a set of patient samples, such as ail patients with cancer. In one embodiment a control expression product level is established wherein higher or lower levels of expression product relative to, for instance, a particular percentile, are used as the basis for predicting outcome. In another preferred embodime t, a control expression product level is established using expression product levels from cancer control patients with a known outcome, and the expression product levels from the test sample are compared to the control expression product level as the basts for predicting outcome. As demonstrated by the data below, the methods of the invention are not limited to use of a specific cut -point in comparing the level of expression product in the test sample to the control.
The "copy number" of a btomarker nucleic acid refers to the number of DM A sequences in a cell (e.g., germline and/or somatic) encoding a particular gene product. Generally, for a given gene, a mammal has two copies of each gene. The cop number can be increased, however, by gene amplification or duplication, or reduced by deletion. For example, germline copy number changes include changes at one or more genomic loci, wherein said one or more genomic loci are not accounted for by the number of copies in the normal complement of gernrline copies in a control (e.g. , the normal copy number in germUne DNA for the same species as that from which the speci fic gernilme DNA and corresponding copy number were determined). Somatic copy number changes include changes at one or more genomic loci, wherein said one or more genomic loci are not accounted for by the number of copies in germline DN A of a control (¾·., copy number in gerraline DNA for the same subject as that from which the somatic DNA and corresponding copy number were determined).
The ''normal" copy number (e.g. , germline and/or somatic ) of a biomarker nucleic acid or "normal" level of expression of a biomarker nucleic acid, or protein is the activity/level of expression or copy number in a biological sample, e.g. , a sample
containing tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bone marrow, from a subject, e.g., a. human, not afflicted with cancer, or from a corresponding non-cancerous tissue in the same subject who has cancer.
The term "determining a suitable treatment regimen for the subject" is taken to mean the determination of a treatment regimen U e, , a single therapy or a combination of different therapies that are used for the pre vention and/or treatment of the cancer in the subject) for a subject that is started, modified and/or ended based or essentially based or at least partially based on die results of the analysis according to die present invention. One example is determining whether to provide targeted therapy against a cancer to provide antt-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy). Another example is starting an adjuvant therapy after surgery whose purpose is to decrease the risk of recurrence, another would be to modify die dosage of a particular chemotherapy. The determination can, in addition to the results of the analysis according to the present invention, be based on personal characteristics of the subject to be treated. In most eases, the aciual determination of the suitable treatment regimen for the subject will be performed by the attending physician or doctor.
The term "expression signature" or "signature" refers to a group of two or more coordinate!;-- expressed biomarkers. For example, the genes, proteins, and the like making up this signature may be expressed in a specific cell lineage, stage of differentiation, or during a particular biological response. The biomarkers can reflect biological aspects of the tumors in which they are expressed, such as the cell of origin of the cancer, the nature of the non-malignant ceils in the biopsy, and the oncogenic mechanisms responsible for the cancer. Expression data and gene expression levels can be stored on computer readable media, e.g., the computer readable medium used in conjunction with a rnkroarray or chip reading device. Such expression data can be manipulated to generate expression signatures, A molecule is "fixed" or "affixed" to a substrate if it is eovalently or nort-eovalently 5 associated with the substrate such that the substrate cart be rinsed with a fluid (e.g. standard saline citrate, pH 7.4) without a substantial fraction of the molecule dissociating from the substrate.
The term "highly structured 5' untranslated region (5' UTR)*" refers to the region of an mRNA directly upstream from the initiation codon, which J ) begins at the transcription it) start site and ends one nucleotide (nt) before the initiation codes) (usually AUG) of the coding region and 2) contains a hairpin loop or other secondary structures. Such secondary structures are usually predicted by modeling but there are experimental means to define them more quantitatively, such as fay measuring the resistance of the structure to nucleases which do not attack double stranded regions or performing physical techniques, such as
15 measuring the optical density at 260 nm as function of temperature. In one embodiment, the highly structured 5' UTR renders the mRNA a -relatively poor substrate for translation. mRNAs encoding proteins necessary for cell growth and survival typically contain a complex, highly structured 5' UTR in order to limit the availability of the protein.
Structured 5' UTJRs prevent CAP-dependent initiation of translation. Regulation of
20 translation by structured 5' UTRs typically occurs due to long 5' UTRs and stable
secondary structures and sequence segments which comprise a high, proportion of guanine and cytosine bases since, when present in the 5' UTR of an mRNA, very efficientl inhibit the CAP-dependent initiation of protein biosynthesis according to the ribosome scanning model. In vitro in vestigations have shown that a hairpin structure in the 5' UTR of an
25 mRNA having a free energy of 30-70 keal/mol or less is able to inhibit translation
effectively. Thus, it has been possible to show that mRNAs coding for a particular protein and having a 5' UTR exhibiting such a structure are translated only very weakly, whereas mRNAs coding for the same protein and having a shorter 5" UTR with a weaker structure are translated considerably more efficiently. Non-limiting, representative examples of 0 RN As with a highly structured 5' UTR include transferrin, transferrin receptor, c-. YC, X- tirrked inhibitor of apoptosis protein (XIAP), and ornithine decarboxylase (ODCT ).
The term "homologous" refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When a nucleotide residue position in both region is occupied b the same nucleotide residue, then the regions are homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position of cadi region is occupied by the same residue. Homology between two regions is expressed in terms of the proportion of nucleotide residue positions of the two regions that are occupied by the same nucleotide residue. By way of example, a region having the nucleotide sequence 5 - ATTGCC-3* and a region having the nucleotide sequence S'-TATGGC-S' share 50% homology. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residue positions of each of the portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.
The term "inhibit" includes the decrease, limitation, or blockage, of, for example a particular action, function, or interaction. In some embodiments, cancer is ' nhibited" if at least one symptom of the cancer is alleviated, terminated, slowed, or prevented. As used herein, cancer is also "inhibited''' if recurrence or metastasis of the cancer is reduced, slowed, delayed, or prevented.
The term "interaction", when referring to an interaction between two molecules, refers to the physical contact (e.g. , binding) of the molecules with one another. Generally, such an interaction results in an acti vity (which produces a biological effect) of one or both of said molecules.
The "iron-sulfur cluster biogenesis pathway" refers to the full set, or relevant subsets thereof, of proteins required for generating iron-sulfur (Fe-S) clusters composed of iron and inorganic sulfur for itse as cofactors in generating Fe-S proteins (see, for example, Lill et al'. (2012) Biochmt Biophys. Acta 1823: 1491 - 1508; Lill and MuSenhoff (2005) Trends Biochem. Set 30: 133- 141 ; Renault (2012) Dm. Mode! Meek 5: 155-164, Ye and Touault (2010) Biochem. 49:4945-4956, and Rouauh and Tong (2005) Nat. Rev. Mot. Cel! Biol. 6:345-351 , Iroii-sulfur clusters are critical for the production of a subset of enzymes invol ved in critical cellular processes, such as oxidative phosphorylation, the citric acid cycle, heme biosynthesis, iron homeostasis, and DNA repair. Figure 1 shows an exemplary schematic diagram of the pathway. Members of the pathway, including terminology, sequences, and function, are well known in the art. For example, "NFSl" refers to the nitrogen fixation i homolog cysteine desulfitrase member of the class- V family of yridoxal phosphate-dependent aminotransferase family and is alternatively known as "iscS," " PS, and "HUSSY-OB.'" NFSl , whose structure- function relationship is known, supplies inorganic sulfur to iron-sulfur clusters by removing the sulfur from cysteine thereby creating alanine in the process ( Fartian et ai (2014) Mol. Genet. G no . Med. 2:73-80; urihara el ai. (2003) Biochim. Biophys. Acta 1647:303-309; Cupp- Viekery et al. (20 3) J. Mol. Biol. 330:1049-1059). The NFS1 gene uses alternate in- frame translation initiation sites to generate mitochondrial forms and eytopiasmie nuelear forms. Selection of the alternative initiation sites is determined by the cytosolic pH. In one embodiment, mitochondrial forms are itsed according to the present invention, in another embodiment, cytoplasmic/nuciear forms are used according to the present invention. At least two splice variants encoding two distinct hitman mitochondrial SFI isoforms exist and sequences are publicly available on the GcnBank database maintained by the U.S. National Center for Biotechnology Information. For example, human NSF I transcript variant I (NMJ)21 .100.4) encodes the long human NSF I isoform .1 (NPJ)66923.3).
Human NSF I transcript variant 2 (NM 001 1 8989.1) lacks an in -frame exon in the 5' coding region compared to variant 1 , resulting in an isoform (NP 001 185 18.1 ) that is shorter compared to isoform 1. Nucleic acid and polypeptide sequences of N FS orfhologs in species other than humans are also well known and include, for example, monkey NFS 1 (XMJ)0! 0976989.2, XPJ10.1097699.1, XMJKH 097983.2, and .XPJMM097983. l X dog NFS! (XM 534405.4, XP 534405.2, XMJKB433251.2, and XPJJ0343329 .5 ), cow NFS1 (NMJ)01099001 .1 and NPJMJ 1.092471.1 ), mouse NFS! (NM JH 1 1 .2 and NPJB504L2), and rat NFSl (NMJ)53462,2 and NP 45 14.2). Representative sequences of NFSl orfhologs are presented below in Table 1 , Anti-NFS 1 agents, including antibodies, nucleic acids, and the like are well-known in the art and include, for example, iron, L-alanine, L-cysteine, pyridoxal 5 '-phosphate and derivatives. It is to be noted that the term can further be used to refer to arty combination of features described herein regarding NFS I molecules. For example, an combination of sequence composition, percentage identify, sequence length, domain structure, functional acti vity, etc. can be used to describe an NFS 1 molecule of the present invention.
As used herein, "LYR 4" refers to the LYR motif containing 4 and is alternatively known as "homolog of yeast Isdl 1 " and "mitochondrial matrix Nfsl interacting protein," The LYRM4 gene encodes the ISDl I protein that forms a stable complex in vim with the human cysteine desulfitrase !SCS to generate the inorganic sulfur needed for iron- sulfur protein biogenesis (Shi et l. (2009) Hum. Mai, Genet .1 :3014-3025). At least three splice variants encoding three distinct human LYRM4 isoforms exist and sequences are publicly available on the GenBank database maintained by the U.S. National Center for
Biotechnology Information. For example, human LYRM4 transcript variant i
(NM 020408.5) encodes the short human LYRM'4 isoform 1 (NP 065141 .3), Human LYR 4 transcript variant 2 (NMJ)Ol 164840.2) contains an alternate 3' terminal exon to create a different 35 coding region and 3' UTR compared to variant 1 and to thereby encode an isoform (NP 001 1583.12.1 ) having a distinct C-terminiis and a longer sequence than that of isoform 1 . Human LYRM4 transcript variant 3 (NM 001 164841.2) includes an additional exon that results in an altemate 3' coding region and ' UTR compared to variant 1 to thereby encoded an isoform f NPJK) 1 158313.1) having a distinct C-terminus and a longer sequence than that of isoform 1 . Each of the isoforms is functional. Nucleic acid and polypeptide sequences of LYRM4 orthologs in species other than humans are also well known and include, for example, monkey LYRM4 (XM_00.1095995.2 and
XP .00iO95995.2X dog LYRM4 (XM .005640157.1 and XP..005640214.1 ), cow LYRM4 (NM..001076306.1 and NP .00! 069774.1), mouse LYR 4 (NM...201358.2 and
NP_958746.l), and chicken LYRM4 (NM„001 198888.1 and NPJK) 11 85 17. J),
Representative sequences of LYRM4 orthologs are presented below in Table 1. Anti- LYRM4 agents, including antibodies, nucleic acids, and the like are well-known in the art and include, for example, dominant-negative binding proteins such as versions of NFS 1 without a catalytic domain, it is to be noted that the term can further be used to refer to any combination of features described herein regarding LYRM4 molecules. For example, any combination of sequence composition, percentage identify, sequence length, domain structure, functional activity, etc. can be used to describe an LYRM4 molecule of the present invention.
As used herein, "ISCU" refers to the iron-sulfur cluster assembly enzyme and is alternatively known as "iSU2/; "NIFU," and "NIFUN." The ISCU gene encodes two isomeric forms, ISCU I and ISCU2, of the iron-sulfur cluster scaffold protein and the structures of the proteins in complex with other iron-sulfur cluster assembly proteins is biown (see, for example, ajewska er a!, (2013) J, Biol Chem. 288:29134-29142). In one embodiment, ISCU l is used according to the present invention. In another embodiment, ISCU2 is used according to the present invention. In still another embodiment, both ISCU l and ISCU2 are used n combination according to the present invention. At least two splice variants encoding the two distinct human ISO.) isofomis exist and sequences are publicly available on the GenBank database maintained by the U.S. National Center for
Biotechnology Information. For example, human ISCU transcript variant 1
(NMJ)14301.3) contains an alternate segment in the 5' coding region and uses a
downstream start codon compared to variant 2 such that the ISCU 1 isoform (MP 0551 1 .1 ) has a shorter and distinct N-terrainus compared to the 1SCU2 isoform. The ISCU i isoform is found, io the eytosol and nucleus. Human ISCU transcript variant 2 (NM_213595.2) encodes the longer ISCU2 isoform (NP 998760.1 ), which is found in mitochondria.
Nucleic acid and polypeptide sequences of NFS orthologs is species other than humans are also well known and. include, for example, monkey ISCU (NM 001261474.1 and
NPJK>1248403.1), cow ISCU (NM 001.075683.2 and NPJX) 1069151.1 ), mouse ISCU (NM 025526.4 and NP 079802, 1), rat ISCU (MM..001 105936.1 and NPJX) 1099406, 1), and chicken ISCU (XJVi J303642 ! 82.2 and XPJXB642230.2), Representative sequences of ISCU orthoiogs are presented below in Table 1. Anti-ISCU agents, including antibodies, nucleic acids, and the like are well-know in the art and include, for example, iro and derivati ves thereof. It is to be noted that the term can further be used to refer to any combination of features described herein, regarding ISCU molecules. For example, any combination of sequence composition, percentage identify, sequence length, domain structure, functional activity, etc. can be used to describe an ISCU molecule of the present invention.
As used herein, "FXN" refers to frataxin and is alternatively known as "CyaY" and "FARR." The FXN gene encodes the mitochondrial frataxin protein that functions in regulating mitochondrial iron transport and respiration (Stemmler ei al. (2010) J. Biol. Chem. 285: 26737-26743; Gentry ei al. (20.13) Biochem. 52:6085-6096; Abruzzo m al (2013) BioMed R . Intl. 20 3, article ID 276808; and Pastore and Puccio (2013} J.
Neurochem. 126:43-52). At least three splice variants encoding three distinct human FXN isofomis exist and sequences are publicly available on the GenBank database maintained by the U.S. National Center for Biotechnology Information. For example, human FXN transcript variant I (NMJiOO.144.4) encodes the long human FXN isoform I
(NP 00 135.2). The mature peptide is represented by residues 56- 10 and the proprotein is represented by residues 42-210, Human FXN transcript variant 2 (NM. 181425.2) uses an alternate splice site in the 3' coding region compared to variant 1 resulting in a frameshift and encodes isoform 2 (NPJ$52090.l) that is shorter and has a distinct C-terminus compared to that of isoform .1. The mature peptide is represented by residues 56-1 6 and the proprotcirt is represented by residues 42-1 6. Human FXN transcript variant 3
(NM_00.1 161706.1) uses an alternate ex on in the 3' coding region compared to variant 1 that results irt a .frame-shift and encodes isoform 3 (NP_001 155178,1) that is shorter and has a distinct C-tertninus compared to that of isoform 1. The mature peptide is represented by residues 56-171 and the proprotetn is represented by residues 42-171, Each of the isoforms is functional. Nucleic acid and polypeptide sequences of FXN orthologs in species other than humans are also well known and include, for example, chimpanzee FXN
(XM 001 137864.2 and XP .001 137864.2), monkey FX (NM 001260741.1 and
NP 001247670.1), dog FXN (NM 001 109958.1 and NP 001 103428.1), cow FXN
(NMJX) J 080727.1 and NP 0010741 6.1), mouse FXN (NM 008044.2 and P 03207Q.1 ), rat FXN (MM 001 1 1 52.1 and NP 0 1 178881.1), and chicken FXN (XM .424827.4 and XP 424827.3). Representative sequences of FXN orthologs are presented below in Table 1 . Anti-FXN agents, including antibodies, nucleic acids, and the like are well-known irt the art and include, for example, iron and heme, it is to be noted that the term can further be used to refer to any combination of features described herein -regarding FXN molecules. For example, any combination of sequence composition, percentage identify, sequence length, domain structure, functional activity, etc. can be used to describe an FXN molecule of the present invention.
Other members of the iron-sulfur cluster bfosynthetic pathway are well-known. For example, NFXJ l encodes a protein that is localized to mitochondria and plays a critical role in iron-sulfur cluster biogenesis (Li et ah (2013) Biachem, 52:4904-4913). The encoded protein assembles and transfers 4Fe-4S clusters to target apoproteins including succinate dehydrogenase and lipoic acid synthase. Nucleic acid and polypeptide sequences of FU 1 orthologs in species including humans are also well known and include, for example, human NFUl (NMJM 5700.3, NPJ>56S! 5.2f NMJM002755.2, NPJX)l002755.i (mature peptide represented by residues 10-254), NMJ.IOl 002756.2, and NP .001.002756, 1 , all of which isoforms are fotiettonal.i, cow NFUl (NM. 001 46566.2 and P1 001040031 .1), mouse NFU 1 (NMJ)01170591 J , NP_001 1.64062.1 , NM_020045.3, and NP_064429.2), rat NFU l (NM. 001 106606.2 and NP 001100076,2), and chicken NFUl (NM 001006305.2 and NP . OOl 006305.2 ). GL.RX5 encodes a mitochondrial protein, whose crystal structure- function relationship is known, that i involved in the biogenesis of iron-sulfur clusters and is required for normal homeostasis (Ye ei al. (2010) J, Clin. Invest 120; 1749- 17 1 and Johansson et al. (201 1) Biochem J. 433:303-31 1 ). Nucleic acid and polypeptide sequences of GLRX5 orthologs in species including humans are also well known and include, for example, human GLRX5 (NM 016417.2 and NP. 057501.2 (mature peptide represented by residues 32- 157)}, chimpanzee GLRX5 (ΧΜ 0Θ 1 154482.1 and XP OOl ! 54482.1 ), monkey GLRX5 (N JX)1265635.2 and NP_001252564.1 ), cow GLRXS (NMJ)Ol 100303.1 and NP..001093773J ), mouse GLRX5 <NMJ)28419.2 and NP 082695.1 ), rat GLRX5
(NM. 001 108722.1 and NP 001 102192.1 ), and chicken GLR 5 (NM. 001008472.1 and NPJ)01008472.1).
BOLA3 encodes a protein, for which the structure-functi n relationship is known, that plays an essential, role in the production of iron-sulfur (Fc-S) clusters for the normal maturation of itpoate-containing 2-oxoacid dehydrogenases, and for the assembly of the mitochondrial respirators' chain complexes (Cameron et al (201 1 ) Am. J. Hum. Genet. 89:486-495; Zhou et al. (2008) Afo/. Ceil. Biochem. 317:61-68; and asai et al (2004) Protein Set 13:545-548), Two alternatively spliced transcript variants encoding different isoforms with distinct subcellular localization are known. Isoform 1 (NM_212552.2 and NP_9977.I 7.2) are mitochondrial, whereas isoform 2 (NM_0010 5505. 1 and
NP_00i030582, l) are cytoplasmic. Nucleic acid and polypeptide sequences of BOLA3 orthologs in species other than humans are also well known and include, for example, chimpanzee BOLA3 (XM J)01 153666.2, XP_00? 153666.1, XM_515554.2, and
XPJ 15554.1), monkey BOLA3 (NMJM) 126565 L. I and NP .001252580.1 ), cow BOLA3 (NM 0 1035452.2 and NP 001030529, 1 ), mouse BOLA3 (NM. J 75277.4 and
NPJ780486.5 ), and rat BOLA3 ( MjOOl 106601.1 and NP_ 00i 100071.5 ).
HSCB, also known as the HscB iron-sulfur cluster co-ehaperone homolog, encodes a protein, for which the structure-function relationship is known, that is an integral component of the human iron-sulfur cluster biosynthetic machinery (Uhrigshardt et al. (2010) Hum. Mol Genet. 19:3816-3834 and Bitto et al (2008) ,/. Biol. Chem. 283:30184- 301.92). Nucleic acid and polypeptide sequences of HSCB orthologs in species including humans are also well known and include, for example, human HSCB (NM 172002.3 and NP..741 9 .3 (mature peptide represented b residues 30-235)), chimpanzee HSCB
(XM„515052.3, XP_ 515052.2, XMJX6953858.1, an XPj>03953907J ). monkey HSCB (NMJIOl 194228.1 and NPJXH 181157.1 ), dog HSCB (XM.J34725.4 and XPJ34725.2), cow HSCB (NMjOOl 102340.1 and NP^OO 109581.0.1), mouse HSCB (KM J 53571.2 and P .705799,2), rat HSCB (NM 001 108340 J and P .001 101810. 1), and chicken BSCB (XM_003 42207.2 artel XPJ)03642255J).
HSPA9, also known as mortalm, encodes a member of the heat shock protein 70 gene family. The encoded protein, for which the structure-function relationship is known, is primarily locaiized to the mitochondria but is also found in the endoplasmic reticulum, plasma membrane and cytoplasmic vesicles (Lao el al (2010) Protein. Expr, Purif. 72:75- B 1 and Craig and Marszalek (2002) Cell Mol Life Scl 59; 1 58-1 65) . Nucleic acid and polypeptide sequences of HSPA9 orthologs in species including humans are also we!i known and include, for example, human HSPA9 (NM 004134.6 and MP 004125.3 (mature peptide represented by residues 47-679)), chimpanzee HSPA9 (XMJXM 171426.3 and XPJ)01171426.2), dog HSPA9 (XM 531923.4 and XPJ31923.2), cow HSPA9
(NM 00103452 ,2 and N JIOI 029696.1 ), mouse HSPA9 (NMJ)i048i.2 and
NPJB4 11 .2), rat HSPA9 (NM 001 1006 .2 and N J)01 94.128.2), and chicken HSPA9 (NM 00100 1 7.1 and NP .001 06147.1 }.
ISCA 1 , also known as iron-sulfur cluster assembly 1 , encodes a mitochondrial protein involved in the biogenesis and assembly of iron-sulfur clusters, which play a role in electron-transfer reactions. The encoded protein, for which the structure-func tion relationship is known, is primarily localized to the mitochondria but is also found in the endoplasmic reticulum, plasma membrane and cytoplasmic vesicles (Cozar-Casteliano el al. (2004) Biochim. Biop ys. Acta. 1700: 179-188; Lu et l. (2010) Blochem. J. 428: 1 25- 131; and Song ei al. (2009) J. Biol. Chem. 284:35297-35307). Nucleic acid and polypeptide sequences of ISCA 1 orthologs in species including humans are also well known and include, for example, human ISCA 1 (NM_030940.3 and N1 12202.2 (mature peptide represented by residues 13-129», chimpanzee ISC 1 (NM_O01242612.1 and NP„001229541.1), dog ISCA1 (XMJS44342.3 and XT 49435.2), cow ISCA1
(NMJM034470.2 and NP J)() 1029642.1), mouse 1SCA1 (NM..026921.4 and
NPJ)8 i 197.1), rat !SCAi {NM J 81626.3 and NP .853657.1), and chicken ISCAi (NMJ)01271936.1 and NPJXH 258865.1 ).
ISCA2, also known as iron-sulfur cluster assembly 2, encodes an A-type iron-sulfur cluster mitochondrial protein in volved in the maturation of mitochondrial iron-sul for proteins (Sheftel el al (2012) Mol. Biol Cell. 23: 1 157-1166 and Hendrickson ei al (2010) PLoS 5:el2862). Two alternatively spliced human transcript variants encoding different isoforms are known, Isoform .1 ( M_ 1 4279.3 and NPJ? .1 255.2 (mature peptide represented by residues 9-154)) represents the longer isoform and isoform 2
(NMJ)Ol 272007. i and NPjOOl 258936 J (mature peptide represented by residues 9-60)) is encoded by a nucleic acid that lacks an alternate coding exon compared to transcript variant. 1 resulting in a frameshift and a. shorter isoform having a distinct C-termtnus relative to isoform 1. Each isoform is functional. Nucleic acid and polypeptide sequences of ISCA2 orthologs in species other than humans are also well known and include, for example, chimpanzee ISCA2 (XM..001. 143075.3 and XP...001 143075.2), monkey ISCA2
(NM ...00126 Ϊ 007.1 and NP 0012547936. Ϊ ), dog 1SCA2 (XM...547905.4 and
XPJ47 05.3), cow JSCA2 (NM 001038683.2 and NP 0 1033772.1 ), mouse ISCA2 (NMJ>28863.1 and NPJ183139.1), and rat ISCA2 (NMjK)l 109278.2 and
ΝΡ_0Ο1 102748.1).
IBA57, also known as MMDS 3, encodes a protein involved in iron-stiifur protein biosynthesis and normal heme biosynthesis (BoJar t al. (2013) Hum. MoL Genet. 22:2590- 2602; Sheftel etai (2012) MoL Bioi. Ceil, 23: 1157-1 166; and NHsson et ai. (2009) Ceil Mei boi 10: 1 19-130). Nucleic acid and polypeptide sequences of ISCA1 ortlioiogs m species including humans are also well known and include, for example, human iSCA 1 (NM_00.1010867.2 and NP_0 1.010867.1 ; the mature peptide is represented by residues 40- 356 since residues 1-39 represent a transit, peptide), chimpanzee ΪΒΑ57 (XMJ514253.3 and X"P_514253.2), monkey 1BA57 (XM 001083460.2 and XP . 01083460.1), co 1BA57 (NMJ)0120S580J and NP 001 192509.1 ), mouse 1BA57 (NM J 73785.6, NP_77614f>.1 , NM 0012707 1.1 , arid NP 001257720.1), rat 1BA57 (KM JXU 1.08827..1 and
NP 001 102297.1 ), and chicken 1BA57 (NM. 001030958.2 and NP 001026129.2).
NUBPL, also known as IND l and HU'LND l , encodes a member of the Mrp NBP35
ATP-binding proteins family. The encoded protein is required for the assembly of the respirators' chain NA.DH dehydrogenase (complex I)„ an oligomeric enzymatic complex located in the inner mitochondrial membrane. The respiratory complex 1 consists of 45 subunits and 8 iron-stilfur (Fe/S) clusters. This protein is an Fe/S protein that plays a critical role in the assembly of .respiratory comple i, likely by transferring Fe/S into the Fe/S-contairang complex 1 subuniis (Sheftel et ai (2009) MoL Bioi Cell. 29:6059-6073; Calvo et ai. (2010) Nat. Genet. 42:851-858; and Kcveiam et i (2013) Neurol 80: 1577- 1583). Three alternatively spliced human transcript variants encoding different isoforms are known. Isoform i (NMJI25152.2 and N.PJ)79428.2 (mature peptide represented by residues 39-319)) represents the longest isoform, whereas isoform 2 (NMJK) 120.1573.1 and NP ...001 188502 J ) is encoded by a -nucleic acid that lacks two exons from the 5' end and has an alternate 5' exon resulting in an isoform having a shorter N-temumis as compared to isoform 1 and isoform: 3 (NMJIO 1201574.1 and. NPJiO.l 188503.1) is encoded b a nucleic acid that lacks several exoos from the 5' end and has an alternate 5" exon resulting in an isoform having a much shorter N-terminus as compared to isoform t . Nucleic acid and polypeptide sequences of NUB PL orthologs in species other than humans are also well known and include, for example, monkey NUBPL (XM 001 108145,2 and
XP .001 108145.2), cow NUBPL (NM .0 1 1 3042. i and NP 001 1 9971 .1 ), mouse NUBPL (NM. 029760.2 and NP 084036.2). and rat NUBPL (NM 001 185025.1 and NP_ 00U 71954.1).
SLC2SA28, also known as solute carrier family 25 (mitochondrial iron transporter) member 28 and mitofe.rr.m2 and MRS3/4, encodes a mitochondrial iron transporter that mediates iron uptake and is required for heme synthesis of hemoproteins and Fe-S cluster assembl in non-etytliroid ceils. The iron delivered into the mitochondria, presumably as Fe(2+), is then delivered to ferroeheiatase to catalyze Fe(2+) incorporation into
protoprophyrin IX to make heme (Li et al. (2001 ) FEES Lett. 49 : 79-84; Pal mie i (2013) Mol Aspects Med 34:465-484; and Hung et al (2013) J. Biol. Chem. 288:677-686).
Nucleic acid and polypeptide sequences of SLC25A28 orthologs in species including humans arc also well known and include, for example, human SLC25A2S (NM 031212,3 and NPJ 12489.3), monkey SLC25A28 ( M 001265757. 1 and NP_00! 252686.1), dog SLC25A28 (XM 846248.3 and XP..851341.2), cow SLC25A28 (NM.001205552.1 and NP .001192481.1 mouse SLC25A28 (NM .1451 6.1 and NP .6601 8.1 ), rat SLC25A28 (NMJKi l 109 15.1 and NPJIOI 102985.1), and chicken SLC25A28 (XM_421702.3 and XM_421702.3).
FD.X L, also known as ferrodoxin reductase, encodes a mitochondrial flavoprotein that initiates electron transport for cytochromes P450 receiving electrons froni ADPR (Slit et al (2012) Biochim. Biophys. Acta 1 823:484-492; Lin et al ( 1990) Proa Natl. Acad. Set. U.S.A. 87:8516-8520; and Liu and Chen (2002) Oncogene 21:7195-7204)... Seven alternatively spliced human transcript variants encoding seven different isoforms are known (NM .024417,3, NP 077728.2 (mature peptide represented by residues 33-4 1),
NMJ0041 10.4, NPJ'M IO I .2 (mature peptide represented, by residues 30-493), NMJK) 1258012.2, NPJMM244941.1 (mature peptide represented by residues 33-534), NMJXU258013.2, NPJX) 1244942.1 , NMJK) 1258014.2, NPJX) 1244943.1 (mature peptide represented, by residues 33-483), M 00125801 .2, NP 001244944. i (mature peptide represented by residues 33-4 1), NMJK) 1258 16.2, and NPJX) 1244945.1 ), each of which is functional. Nucleic acid and polypeptide sequences of FDXR orthologs in species other than humans are also well known and include, for example, chimpanzee FDXR
£XM_511666.4 arid XPJ 1 1666,3), monkey FDXR (XMJX)1091261.2 arid
XP 001091261 .2), cow FDXR (NMJ 74691.1 and NPJ777 i 16.1), mouse FDXR
(NMJXJ7997.1 and NP 032023.1 >, and rat FDXR (N J)24J 53J and NPJ)77067. i).
FDX2, also known as ferrodoxin 1 like, encodes a mitochondrial ferrodoxin required for iron-sulfur protein biogenesis and cellular iron homeostasis as F.D.X2 deficienc leads to increased cellular iron uptake, iron accumulation in mitochondria, and impaired Fe/S protein biogenesis (Sheftel et oi (2010) Prae. Nail. Acad Set. U.S.A.
107:1 1775- 1 1780 and Qi ef i. (2013) Dahon I'mm. 42:3088-3091 ). Nucleic acid and polypeptide sequences of FDX2 orthologs in species including humans are well known and include, for example, human FDX2 (NM 001031734.2 and NP 001026904.1 (mature peptide represented by residues 53-183)), chimpanzee FDX2 (XM. 512366.4 and
XP_512366.3), monkey FDX2 (XMJ)O i 105309.2 and XPJX)l 105309.2), dog FDX2 (XMJ42073.4 and XPJ42073.1), cow FDX2 (NMJK) 1080226.2 and NPJ)0i 073695.1 ), mouse FDX2 (NMJK) 1 39824.2 and NPJK) 1034 13.1 ), and rat FDX2 (NM J)0.1 1 8002.1 and NP 001 101472.1 ),
An "'isolated protein" refers to a protein that is substantially free of other proteins, cellular material, separation medium, and culture medium: when isolated from cell or produced by recombinant DNA techniques, or chemical precitrsors or other chemicals when chemically synthesized. An "isolated" or "purified" protein or biologically active portion thereof is substantiall free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody, polypeptide, peptide or fusion protein i deri ved, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of a biomarker polypeptide or fragment thereof in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of a biomarker protein or fragment thereof, having less than about 30% (by dry weight) of non-biomarker protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-biomarker protein, stil! more preferably less than abo ut 10% of non-biomarker protein,, and most preferably less than about 5% non- biomarker protein. When antibody, polypeptide, peptide or fusion protein or fragment thereof, e.g. , a biologically active fragment thereof, is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
A "kit" is any manufacture (e.g. a package or container) comprising at least one reagent, e.g. a probe or small molecule, for specifically detec ting and/or affecting the expression of a marker of the invention. The kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention. The kit may comprise one or more reagents necessary to express a composition useful in the methods of the present invention, in certain embodiments, the kit may further comprise a reference standard, e.g., a nucleic acid encoding a protein that does not affect or regulate signaling pathways controlling cell growth, division, migration, survival or apoptosis. One skilled in the art can envision many such control proteins, including, but not limited to, common molecular tags {e.g., green fluorescent protein and heta-gaiaetosidase), proteins not classified in any of pathway encompassing cell growth, division, migration, survival or apoptosi by
GeneOntology reference, or ubiquitous housekeeping proteins. Reagents in the kit may be provided in individual containers or as mixtures of two or more reagents in a single container. In addition, instructional materials which describe the use of the compositions within the kit can be included.
The term "neoadjuvant therapy" refers to a treatment given before the primary treatment. Examples of neoadjuvant fherapy can include chemotherapy, radiation therapy, and hormone therapy. For example, in treating breast cancer, neoadjuvant therapy can allows patients with large breast cancer to undergo breast-conserving surgery.
The term "NFS! pharmacodynamic biomarkers" refers to biomarkers and related assays whose modulation is correlated with that of NFS 1 such that they can be used as surrogates, combinations, or other readouts associated with NFS] modulation.
Representative examples include, without limitation 1) decreased conversion of cysteine to alanine or methylene blue; 2) induction and/or promotion of mitochondrial dysfunction, such∑ts a) a decrease in aconitase copy number, amount, and/or activity and/or b) a decrease in succinate dehydrogenase copy number, amount-, and/or activity; 3} induction and/or promotion of iron regulatory protein dysfunction, such as a) a decrease in ferritin copy number, amount, and/or activity and/or b) an increase in transfcrria-rccep or copy number, amount, and/or activity and/or c) a decrease in Hif2alpha copy number, amount, and/or activity; and 4) induction and/or promotion of ferroptosis, such as a) an increase and/or accumulation of lipid reactive oxygen species (ROS) and/or b) art increase in PTGS2 (COX2) copy number, amount, and/or activity.
Molecules and reagents useful as NFS !, pharmacodynamic biomarkers are well known in the art.
For e-xainpie, "aeonitases"" are iron-sulfur proteins that function to catalyze the conversion of citrate to isoeitrate. When cellular iron levels are low, the protein binds to iron -responsive elements (IREs), which are stem-loop structures found in the 5' UTR. of ferritin mRNA, and in the 3' UTR of transferrin receptor mRNA. When the protein binds to IRE, it resul ts in repression of translation of ferritin mRNA, and inhibition of degradation of the otherwise rapidly degraded transferrin receptor mRNA. There are two forms of aconitascs in mammalian cells, including a cytoplasmic aconitase encoded by Acol and a mitochondrial aconitase encoded by Aco2. In certain embodiments, the term "aconiiase" encompasses the combination of nucleic acids and/or proteins of Acol and Aco2. in other embodiments, the term ""aconitase" encompasses the nucleic acids and/or proteins of Acol alone or of Aco2 alone.
Acol encodes a bifonctionai, cytosolic protein that functions as an essential enzyme in the TCA cycle and interacts with mRNA to control the levels of iron inside cells. When cellular iron levels are high, this protein binds to a 4Fe-4S cluster and functions as an acom'fcse (Philpott et al. (1 94) Free. Nail Acad. Set. U.S.A. 91:7321-7325; Bra-ssolotto et al (1999) J. Biol. Chem. 274:21625-21630; aptain et al. (1991) Proc. Natl Acad. Set U.S.A. 88: 10109-101 13; and Li el al. (2006) J. Biol Chem. 281 : 1 2344-12351 ). Two alternatively spliced human transcript variants encoding the same isoforras are known. Transcript variant 1 (NM 001278352. J ) represents the longer transcript and transcript variant 2 (NM 0021 7,2) differs from transcript variant 1 b having a different 5' UTR despite the fact that both transcript variants encode the same protein (NP_002'l88.'l and NP 00 i 265281.1 ). Nucleic acid and polypeptide se uences of Aco 1 orthologs in species other than humans are also well known and include, for example, chimpanzee Acol (XM^OOH 56102.3 and XPJ)0 I I 56102.1), monkey Acol (NMJXH25786S.J and NPJM) 1244794.1 ), dog Acol (XM„538698.4 and XP 38698.2), cow Acol
(NMJM!1075591 J and NPJXH 069059.1 ), mouse Acol ( MJ107386.2 and
NP 031412.2), rat Acol (NM.. 017321.1 and NP 059017.1), and chicken Acol
( MJK) 5030536. i and MP JX> J 025707.1).
Aco2 encodes a Afunctional, mitochondrial protein that catalyzes the
intereonversion of citrate to isocttrate via cis-aconitate in the second step of the TCA cycle. This protein is encoded in the nucleus and functions in the mitochondrion (Mire! et al (1998) Gene 213:205-218; Kkusner and Rouault ( 1.993) Mol. Biol. ¾// 4: 5 -5: and Gmer et tl ( 5 97) Trends Biochem. Sci. 22:3-6). Nucleic acid and polypeptide sequences of Aco2 ortho!ogs in species including humans are well known and include, for example, human Aco2 (NM 001098.2 and NP 001089.1 (mature peptide represented by residues 28-780)), monkey Aco2 (NMJ1 1261 164.2 and NP^OO 1248093.1), dog Aco2 (XMJJ44073.3 and XP 84 166.1), cow Aco2 (NM 173977.3 and NP 776402.1), mouse Aco2 (NMJ>80633.2 and NP J42364.1 ), rat Aeo2 (NM 024398.2 and NP 077374.2), chicken Aco2
(NM _204188.2 and NP_989559.1 ), and zebrafish Aco2 (N _ 198908.1 and NP_9445 0.1 ).
"Succinate dehydrogenase," also known as SDH, succinate-coenzyme Q
reductase (SQ ), and respiratory Complex 11, is a well-known enzyme complex that exists in. bound form on the inner mitochondrial membrane of mammalian mitochondria
(Yankovskaya ef L (2003) Science 299:700-704; Cheng et ai (2008) Biochem is iiy 47: 6107). In the citric acid cycle, SDH catalyzes the oxidation of succinate to marate with the reduction of ubiquinone to ubiquinol. Mammalian and mitochondrial SDH are composed of four subunits: two hydrophilic and two hydrophobic. The first two subtmits. a t!avoprotein (SdhA) and an iron-sulfur protein ( SdhB), are hydrophilic. SdhA contains a covalentiy attached flavin adenine dinucleotide (FAD) cofaeior and the succinate binding site and SdhB contains three iron-sulfur clusters: [2Fe-2S], |4Fe«4S], and j.3Fe-4S'j. The second two subunits are hydrophobic membrane anchor subunits, SdhC and SdhD. Human mitochondria contain two distinct isoform of SdhA (Fp subunits type I and type II). The subunits form a membrane-bound cytochrome b complex with six transmembrane helices containing one heme b group and a ubiquinone-btnding site. Two phospholipid molecules, one cardiolipin and one phosphatidylethanolamine, are also found in the SdhC and SdhD subunits and serve to occupy the hydrophobic space below the heme. There are two distinct classes of inhibitors of complex H: those that bind in the succinate pocket and those that bind in the ubiquinone pocket. UBQ inhibitors include carboxin and the noytaifiuoroacetone. Succinate-analogue inhibitors include the synthetic
compound malonate as well as the TCA cycle intermediates, malate and oxaloaeetate. indeed, oxaloacetate is one of the most potent inhibitors of Complex 11. in addition, assays for analyzing SDH activity are well known in the art and include, for example,
specirophotometric analysis of enzyme reactions, analysis of reduction of artificial electron acceptors such as 2,6 dichlorophenolindophenoi (DCJP) (see, for example, Jones t al. (2013) Anal Biochem. 442: 19-23).
"Ferritin" refers to a well-known intracellular protein that stores and releases iron and exists as a globular protein complex consisting of 12 or 24 protein subunits wherein the submits associate to form a spherical nanocage. Ferritin that is not combined with iron is referred to "apoferritin." A "ferritin protein subunif is defined as one of the 12 or 24 polypeptide subunits that make up a ferritin protein. The numbering system used herein for the identification of amino acids within, ferritin sobitriiis is based on the original sequence of horse spleen L ferritin (Swiss Protein Database Accession Number P027 ), The horse spleen numbering system can be easily converted to a numbering system based on the human H sequence (Swiss Protein Database accession number P02794; the human L sequence accession number is P02792), which has four additional amino acids at the N- terminus. The human H sequence numbering therefore adds 4 to the corresponding amino acid number in horse spleen ferritin. For example, LI 34 by horse spleen numbering corresponds to Li 38 by human li sequence numbering. Alignments of ferritin suburtit sequences can be found, e.g., in Theil, E. , in Handbook of Metalloproieins,
( esserschmidt, A. et al, eds.), John Wiley & Sons. Chichester, UK, pp. 771 -81. 2001 ; Waldo, G. S. and Theil, E.€., in Comprehensive Supraraoleeular Chemistry, Vol. 5, (K. S. Suslick, ed,), Pergamon Press, Oxford, UK, pp. 65-89, 1 96; Orino Koichi et al, Veterinary Biochem. 42:7-1 i (2005); Accession number: 06A.006486; and U.S. Paf. Pubis. 2013- 026704! and 201 .1 -0287033. In vertebrates, the subunits are both the fight (L) and the heavy (H) type with an apparent molecular weight of 19 kDa or 21 kDa respectively. Some ferritin complexes in vertebrates are hete.tO-oitgom.ers of two highly related gene products with slightly different physiological properties. The ratio of the two homologous proteins in the comple depends on the relative expression levels of the two genes. Assays for analyzing ferritin present, amount, and acti vity are well known in the art as described above. 'Transferrin receptors" are carrier protein for transferrin. When cellular iron levels are lo , ceils increase the level of transferrin receptor produced in order to increase iron intake and this process is regulated by iron via iron response/regulatory element binding protein (IRE-BP or IRP) that binds to the hairpin structure of the iron response element loc ated in the 3' UTR of the transferrin receptor-encoding gene. When the protein binds to IRE, it results in repression of translation of ferritin m'RNA, and inhibition of degradation of the otherwise rapidly degraded transferrin receptor mRNA. There are two forms of transferrin receptor in mammalian cells, TFR i and TPR2. i certain embodiments, the term "transferrin receptor" encompasses the combination of nucleic acids and/or proteins of TFR1 and TFR2. In other embodiments, the term "transferrin receptor" encompasses the nucleic acids and/or proteins ofTFR! alone or of TFR2 alone.
"TFRL" also known as CD? I , encodes a type-II receptor that resides on the outer cell membrane and cycles into acidic autosomes into the cell in a. cl ihrin/dynamin- dependent manner (Worthen and Eons (2014) Front Pharmacol. 5:34; Frazer and
Anderson (2014) Biofaclon 40:206-214; Darnels el al (201 2) Biochim. Biophys, Ada 1820:2 1-317). Two alternatively spliced human transcript variants encoding the same isoforms are known. Transcript: variant .1 (NM 003234.2) represents the longer transcript and transcript variant 2 ( M_001 12 148.1) differs from transcript variant I by having a different 5" UTR despite the fact that both transcript variants encode the same protein ( PJ)03225.2 and NPJXM 121620.1 ; mature peptide represented by residues 101-760)). Nucleic acid and polypeptide sequences of TFR 3 orthologs in species other than humans are also well known and include, for example, monkey TFR 1 ( M_001257303.1 and
NPJM) 1244232. i k dog TFR 1 (NM. 0010031 11.1 and NPJM) 10031 1 1.1 ), cow TFR 1 (NM 001206577.1 and NP ..001 193506.1 ), mouse TFR1 (NM_01 1638.4 and
NPJB5768.1 ), and chicken TFR 1 (NM_20525 J and P_990S87.1).
"TFR2" encodes a transferrin receptor thai is highly homologous to TFR! that mediate cellular uptake of tiansfemn-boitnd iron but whose expression is largely restricted to hepatocytes (Daniels ei al. (2006) Gin. Immunol. 121 : 144- 158 and Zhao ei al. (2013) Biochem. 52:3310-3319). Two alternatively spliced human transcript variants encoding different isoforms are known. isoform 1 (NM_003227,3 and Νί )03218.2) represents the longer isofomi and isofbrm 2 (NM 001206855.1 and NP..001 193784, 1) i encoded by a nucleic acid that differs at the 5' end compared to variant 1 and initiates translation from, an in-frame downstream AUG resulting in an isoform with a shorter N- terminus and lacking the transmembrane domain relati ve to isoform 1 . Each isoform is functional. Nucleic acid and polypeptide sequences of TFR2 orthoiogs in species other than humans are also well known and include, for example, chimpanzee TFR2 (X 003318650.1 and
XPJ)033.18698.1), monkey T.FR2 (XM J)0 I I I 3.151.2 and PjOOl 1 13151 . ! }, cow TF 2 ( MJXM 177741.1 and NPjKH 171212.1), mouse TFR2 iNM._ 001289507.1,
NP 001276436.1 , NMJX) 1289509, 1 , NM 00128951 1 ,1 , NM 01.5799.4, and
NPJ}56614.3), rat TFR2 (NM jO t 105916.1 and NP OO10993S6.1 ), and zebrafish TFR2 (NMJM00 916.1 and PjOOl 09916.1 }.
i4Hif2a," also known as endothelial PAS domain protein 1 , encodes a transcription factor invoi ved in the induction of genes regulated by oxygen, which is induced as oxygen levels fall (Mastrogiannaki et at (2013} Blood 122:885-892 and Haase (2010) Am. J.
Physiol Renal. Physiol. 299;Fi -P.13). The encoded protein contains a basic-helix-loop- helix domain protein dimerizafioti domain as well as a domain found in proteins in signal transduction pathways which respond to oxygen levels. Nucleic acid and polypeptide sequences of.H.iSa orthoiogs in species including humans are well known and include, for example, human Hi£2a (NM. 001430.4 and NP .001421.2), chimpanzee Flifia
(XM...001 1.47219.3 and XP . Q01 147219.1), monkey Hif2a (XM..001 1 12947.2 and
XP_00I U 2947.2), dog Mif2a (XM_005626080.1 and XP_ 005626 i 37. i), cow Hif2a (NM_1.74725.2 and NPJ777150.1), mouse Hif2a (NMJ)i0137.3 and NP_ 034267.3), rat Hif2a (NMJ!23090.1 and NP J>75578.1 )f and chicken Hif2a (NM„204807.1 and
NP_990138.1).
"PTGS2" refers to a specific isozyme of the prostaglandin-endoperoxicle synthase (PTGS), also known as cyciooxyge.nase-2, which is the key enzyme in prostaglandin biosynthesis and acts as both a dioxygenase and as a peroxidase. There are two isozymes of PTGS: a constituti e PTGS 1 and an inducible PTGS2, which differ in their regulation of expression and tissue distribution. PTGS2 encodes the inducible isozymes and is regulated by specific stimulatory events, indicating that it is responsible for the prostanoid biosynthesis .tnvoived in inflammation, and mitogencsis (Percy et l. ( 1998) Analyst 123:41- 50), Nucleic acid and polypeptide sequences of PTGS2 orthoiogs in species including humans are well known and include, for example, human PTGS2 (NM_J)00963.3 and NP 000954.1 (signal peptide sequence represents residues 1-23), chimpanzee PTGS2 (XM .524999.4 and XP 524999.3), monke PTGS2 (XM JIOI 107538.2 and
XPJM) J 107538.2), dog TGS2 (NMJX>1003354.1 and PJX)1003354..l), cow PTGS2 (NMJ 74445.2 and NPJ77687G. I ), mouse PTGS2 (NMJ>1.1 198.3 and NPJ)35328.2), and rat PTGS2 (NM J) 1.7232.3 and NP_ 058928.3).
"Lipid reactive oxygen species (ROS)'' refer to lipids that can participate in reactions that give rise to free radicals to thereby cause oxidative damage. Lipids are prone 5 to oxidative damage since ROS species can act on unsaturated lipids to yield reactive
unsaturated aldehydes. These unsaturated aldehydes can react with other cellular components, such as membrane-bound or associated proteins and nucleic acids, thereby erossiinkmg them to the lipid. Oxidized lipids may be identified by presence of lipid peroxides. Exemplary ROS include hydroxy! radicals (OH,), superoxide radical (02.-), it) nitric oxide (NO.), thyi (RS,), peroxyi (R02.), and lipid peroxyi (LOO,). Lipids can form lipid ROS when present in conditions of oxidative challenge or stress, wherein lipids are vulnerable to oxidative damage. An oxidative challenge can involve the introduction of free radicals, ROS, or reacti ve nitrogen species, such as to RBC or lysed RBC, for example in an assay of antioxidant activity. The oxidative challenge may be created by adding a free
1.5 radical generator, such as hydrogen peroxide or AAPH. Assays for detection of lipid ROS are well known in the art (see, for example, U.S. Pat. Pubis. 2014-0017341 and 2013- 0260418 and Dixo et al (2012) CHI 149: 1060- 1072).
The "norma!" level of expression, of a biomarker is the level of expression of the biomarker in cells of a subject, e.g., a human patient, not afflicted with a cancer. An "over-
20 expression" or "significantly higher le vel of expression" of a biomarker refers to an
expression level in a test sample that is greater than the standard error of the assay employed to assess expression, and is preferably at least twice, and more preferably 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, UK 10,5, 1 1, 12, 13, 14, 1 , 16, 17, 18, 19, 20 times or more higher than the expression activity or
25 level of the biomarker in a control sample (e.g. , sample from a healthy subject not having the biomarker associated disease) and preferably, the average expression level of the biomarker in several control samples. A "significantly lower level of expression" of a biomarker refers to an expression level, in a test sample that is at least twice, and more preferably 2.1 , 2,2, 2,3, 2,4, 2,5, 2,6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6,5, 7, 7.5, 8, 8.5, 0 9, 9.5, 10, 10.5, .1 1 , 12, 13, 14, 15, 16, 1.7, 18, 19, 20 times or more lower than the
expression level of the biomarker in a control sample (e.g. , sample from a healthy subject not having the biomarker associated disease) and preferably, the average expression level, of the biomarker in several control samples. Such "significance" levels can also be applied to an other measured parameter described herein, such as for expression, inhibition,
cytotoxicity, ceil growth, and the like.
The term "predictive"' incl udes the use of a biomarker nucleic acid and/ or protein status, e.g., over- or under- activity, emergence, expression, growth, remission, recurrence or resistance of tumors before, during or after therapy, for determining the likelihood of response of a cancer to anti-cancer therapy, such as iron-sulfur cl uster biosynthesis pathway inhibitor treatment (e.g., NFS ! inhibitors). Such predictive use of the biomarker may be confirmed by, e.g., (1 ) increased or decreased copy number {e.g., by FISH, FISH plus SKY, single-molecule sequencing, e.g.. as described in the ar at least at J. BiotechnoL, 86:289- 30 i , or qPCR), overexpression or underexpression of a biomarker nucleic acid (e.g., by ISH, Northern Blot, or qPCR), increased or decreased biomarker protein (e.g., by !HC) and/or biomarker target, or increased or decreased activity, e.g., in more than about 5%, 6%, 7%, 8%, 9%, 10%, I i%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more of assayed human cancers types or cancer samples; (2) its absolute or relatively modulated presence or absence in a biological sample, e.g., a sample containing tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool or bone marrow, .f om a subject, e.g. a human, afflicted with cancer; (3) its absolute or relatively modulated presence or absence in clinical subset of patients with cancer (e.g., those responding to a particular anti-cancer therapy (e.g. , iron-sulfur cluster biosynthesis pathway inhibitory therapy) or those developing resistance thereto).
The terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the probability of developing a disease, disorder, or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder, or condition.
The term "probe" refers to any molecule which is capable of selectively binding to a specifically intended target molecule, for example, a nucleotide transcript or protein encoded by or corresponding to a biomarker nucleic acid. Probes can be either synthesized by one skilled in the art, or derived from appropriate biological preparations. For purposes of detection of the target molecule, probes may be specifically designed to be labeled, as described herein. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.
The term "prognosis" includes a prediction of the probable course and outcome of cancer or the likelihood of recovery from the disease, in some embodiments, the use of statistical algorithms provides a prognosis of cancer in an individual. For example, the prognosis can be surgery, development of a clinical subtype of cancer (e.g., solid tumors, such as lung cancer, melanoma, and renal cell carcinoma), development of one or more clinical factors, development of intestinal cancer, or recovery from the disease.
The term: "response to anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy)" relates to any response of the hyperproltferattve disorder (e.g., cancer) to an anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy), such as anti-NFS 1 inhibitor therapy), preferably to a change in tumor mass and/or volume after initiation of neoadjuvant or adjuvant chemotherapy. Hyperproliferative disorder response may he assessed , for example for efficacy or in a neoadju vant or adjuvant situation, where the size of a tumor after systemic intervention can be compared to the initial size and dimensions a measured by CT, PET, mammogram:, ultrasound or palpation. Responses may also be assessed by caliper measurement or pathological e amination of the tumor after biopsy or surgical resection. Response may be recorded in a quantitative fashion like percentage change in tumor volume or in a qualitative fashion like "pathological complete response" ipCRk "clinical complete remission'' (eCR), "clinical partial remission' (cPR), "clinical stable disease" (cSD), "clinical progressive disease" (cPD) or other qualitative criteria. Assessment of hyperproHferative disorder response may be done early after the onset of neoadjuvant or adjuvant therapy, e.g., after a few hours, days, weeks or preferabl after a few months. A typical endpoint for response assessment is upon termination of neoadjuvant chemotherapy or upon surgical removal of residual tumor cells and/or the tumor bed. This is typically three months after initiation of
neoadjuvant therapy. In some embodiments, clinical efficacy of the therapeutic treatments described herein may be determined by measuring the clinical benefit rate (CBR). The clinical benefit rate is measured by determining the sum of the percentage of patients who are in complete remission (CR), the number of patients who are in partial remission (PR) and the number of patients having stable disease (SD) at a time point at least 6 months out from the end of therapy. The shorthand for this formula is CBRs R+PR+SD over 6 months. In some embodiments, the CBR for a particular cancer therapeutic regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more. Additional criteria for evaluating the response to cancer therapies are related to "survival," which includes all of the following: sitrvi va! until mortality, also known as overall survival (wherein said mortality may be either irrespective of cause or tumor related); "recurrence- free survival" (wherein the terra recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the terra disease shall include cancer and diseases associated therewith). The length of said survival may be calculated by reference to a defined start point (e.g., time of diagnosis or start of treatment) and end point (e.g., death, recurrence or metastasis). In addition, criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence, for example, in order to determine appropriate threshold values, a particular cancer therapeutic regimen can be administered to a population of subjects and the outcome can be correlated to biomarker measurements that were determined prior to administration of any cancer therapy. The outcome measurement may be pathologic response to therapy gi en in the neoadjuvant setting. Alternatively, outcome measures, such as overall survival and disease- free survival can be monitored over a period of time for subjects following cancer therapy for whom biomarker measurement values are known. In certain embodiments, the doses administered are standard doses known in the art for cancer therapeutic agents. The period of time for which subjects are monitored can vary. For example, subjects may be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 51 or 60 months. Biomarker measurement threshold values that con-elate to outcome of a cancer therapy can be determined using well-known methods in the art, such as those described in the Examples section.
The term "resistance" refers to an acquired or natural resistance of a cancer sample or mammal to a cancer therapy ( i.e., being nonresponsive to or having reduced or limited response to the therapeutic treatment), such as having a reduced response to a therapeutic treatment by 25% or more, for example, 30%, 40%, 50%, 60%, 70%, 80%, or more, to 2- foid, 3-fold, 4-foSd, 5-fold, 10-fold, 15-fold, 20-fold or more. The reduction in response can he measured by comparing with the same cancer sample or mammal before the resistance is acquired, or by comparing with a different cancer sample or a mammal who is known to have no resistance to the therapeutic treatment. A typical acquired resistance to chemotherapy is called "multidrug resistance." The multidrug resistance can be mediated by P-glycoprotein or can be mediated by other mechanisms, or it can occur when a .mammal is infected with a mute -drug-resistant microorganism or a combination of microorganisms. The determination of resistance to a therapeutic treatment is routine in the art and within the skill of an ordinarily skilled clinician, for example, can be measured by cell proliferative assays and ceil death assays as described herein as "sensitizing." In some embodiments,, the term "reverses resistance'" means that the use of a second agent in combination with a primary cancer therapy ( g., chemotherapeutic or radiation therapy) is able to produce a significant decrease in tumor volume at a level of statistical significance (e.g., p<0.05) when compared to tumor volume of untreated tumor In the circumstance where the primary cancer therapy (e.g. , chemotherapeutic or radiation therapy) alone is unable to produce a statistically significant decrease in tumor volume compared to tumor volume of untreated tumor. This generally applies to tumor volume measurements made at a time when the untreated tumor is growing log rhythmically.
The terms "response" or "responsiveness" refers to an anti-cancer response, e.g. in the sense of reduction of tumor size or inhibiting tumor growth. The terms can also refer to an improved prognosis, for example, as reflected by art increased time to recurrence, which is the period to first recurrence censoring for second primary cancer as a first event or death, without evidence of rec urrence, or an increased overall survival, which is the period from treatment to death from any cause. To respond or to have a response means there is a. beneficial endpoint attained when exposed to a stimulus. Alternatively, a negati ve or detrimental symptom is minimized, mitigated or attenuated on exposure to a stimulus. It will be appreciated that evaluating the likelihood that a tumor or subject will exhibit a favorable response is equivalent to evaluating the likelihood that the tumor or subject will not exhibit favorable response {i.e. , will exhibit a lack of response or be iion-responsi ve).
An "R A interfering agent" as used herein, is defined as any agent which interferes with or inhibits expression of a target biomarker gene by RNA interference (RNAi). Such RNA interfering agents include, but are not limited to, nucleic acid molecules including RNA molecules which are homologous to the target biomarker gene of the invention, or a fragment thereof, short interfering R (siR A), and small molecules which interfere with or inhibit expression of a target biomarker nucleic acid by RNA interference (RNAi).
"RNA interference (RNAi)" is an evolutionally conserved process whereby the expression or introduction of RNA of a sequence that is identical or highly similar to a target biomarker nucleic acid results in the sequence specific degradation or specific post- transcriptiona! gene silencing (PIGS) of messenger RN (mRNA) transcribed from that targeted gene (see Cobnrn, G. and Cullen, B. (2002) J, ofVimlogy 76(18):9225), thereby inhibiting expression of the target biomarker nucleic acid, in one embodiment, the RNA is double stranded RNA fdsRNA). This process has been described in plants, invertebrates, and mammalian cells. In nature, RNAi is initiated by the dsRNA-specifie endonac!ease Dicer, which promotes processive cleavage of long dsRNA into double-stranded fragments termed siRNAs. siRNAs are incorporated into a protein complex that recognizes and cleaves target mRNAs. RNAi can also be initiated by introducing nucleic acid molecules, e.g., synthetic siRNAs, shR As, or other RNA interfering agents, to inhibit or silence the expression of target faiomarker nucleic acids. As used herein, "inhibition of target biomarker nucleic acid expression" or "inhibition of marker gene expression" includes any decrease in expression or protein activity or level of the target biomarker nucleic acid or protein encoded by the target biomarker nucleic acid. The decrease may be of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as compared to the expression of a target biomarker nucleic acid or the activity or level of the protein encoded by a target biomarker nucleic acid which has not been targeted b an RNA interfering agent.
The term "sample" used for detecting or determining the presence or le vel of at least one biomarker is typically whole blood, plasma, scrum, saliva, urine, stool (e.g., feces), tears, and any other bodily fluid (e.g., as described above under the definition of "body fluids"), or a tissue sample (e.g., biopsy) such as a small intestine, colon sample, or surgical resection tissue, in certain instances, the method of the present in vention further comprises obtaining the sample from the individual prior to detecting or determining the presence or level of at least one marker in the sample.
The term "sensitize'" means to alter cancer cells or tumor cells in a way that allows for more effecti ve treatment of the associated cancer with a. cancer therapy (e.g., iron-sulfur cluste biosynthesis pathway inhibitor, chemotherapeutic, and/or radiation therapy). In some embodiments, normal cells are not affected to an extent that causes the normal cells to be unduly injured by the anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy). An increased sensitivity or a reduced sensitivity to a therapeutic treatment is measured according to a known method in the art for the particular treatment and methods described herein below, including, but not limited to, cell proliferative assays (Tanigawa N, Kern D H, Kikasa Y, Morton D L, Cancer Res 1 82; 42: 2159-2164), ceil death assays (Weisenthal L M, Shoemaker R H, Marsden J A, Dill P L, Baker j A, Moran E M, Cancer Res 1984; 94: 161 - 173; Weisenthal L M, Lippman M E, Cancer Treat Rep 1985; 69: 615-632; Weisenthal L , in: Kaspers G J L, Pieters R, Twenfy an P R, Weisenthal L M, Veerman A J P, eds. Drug Resistance in Leukemia and Lymphoma, Langhorne, P A: Har ood Academic Publishers, 1993: 415-432; Weisenthal L M, Contrib Gynecol Obstet 1 94: 19: 82-90), The sensitivity or resistance may also be measured in animal by
measuring the tumor size reduction over a period of time,, for example, 6 month for human and 4-6 weeks for mouse. A composition or a method sensitizes response to a therapeutic treatment if the increase in treatment sensitivity or the reduction in resistance is 25% or more, for example, 30%, 40%, 50%, 60%, 70%, 80%, or more, to 2 -fold, 3-fold, 4-fold, 5- fcld, 10-fold, 15-fold, 20-fold or more, compared to treatment sensitivity or resistance n the absence of such composition or method. The determination of sensitivity or resistance to a therapeutic treatment is routine in the art and within the skill of n ordinarily skilled clinician, ft is to be understood that any method described herein for enhancing the efficacy of a cancer therapy can be equally applied to methods for sensitizing hyperproh erative or otherwise cancerous cells (e.g., resistant cells) to the cancer therapy.
The term: "synergistic effect" refers to the combined effect- of two or more iron- sulfur cluster biosynthesis pathway inhibitor agents can be greater than the sum of the separate effects of the anticancer agents alone,
"Short interfering RNA" (siRNA), also referred to herein as "small interfering
RNA" is defined as an agent: which functions to inhibit expression of a target bioroarker nucleic acid, e.g. , by RNAi. An siRNA may be chemically synthesized, ma be produced by in vitro transcription, or may be produced within a host cell. In one embodiment, siR A is a double stranded RNA (dsRNA) molecule of about 15 to about 40 nucleotides in length, preferably about 15 to about 28 nucleotides, more preferably about 1 to about 25
nucleotides in length, and more preferably about 1 , 20, 21 , or 22 nucleotides in length, and may contain a 3' and/or 5' overhang on each strand having a length of about 0, 1 , 2, 3, 4, or 5 nucleotides. The length of the overhang is independent between the two strands, i.e., the length of the overhang on one strand is not dependent on the length of the o verhang on the second strand. Preferably the siRN A is capable of promoti ng RN A interfere nce through degradation or specific post-transcriptional gene silencing (PTGS) of the target messenger RNA (raRNA).
In another embodiment, an siRNA is a small hairpin (also called stem loop) RNA (shRNA). In one embodiment, these shRNAs are composed of a short (e.g., 19-25
nucleotide) antisense strand, followed by a 5-9 nucleotide loop, and the analogous sense strand. Alternatively, the sense strand may precede the nucleotide loop structure and the antisense strand may follow. These shRNAs may be contained in plasmtds, retroviruses, and lentiviruses and expressed from, for example, the pel III U6 promoter, or another promoter (see, e.g., Stewart, ei ai. (2003) RNA Apr;9(4):493-501 incorporated fay reference herein).
RNA interfering agents, e.g.. siRNA molecules, may be administered to a patient having or at risk for having cancer, to inhibit expression of a biomarker gene which is overexpressed in cancer and thereby treat, prevent, or inhibit cancer in the subject.
The term "subject" refers to any healthy animal, mammal or human, or any animal, mammal or human afflicted with a cancer, e.g., lung, ovarian, pancreatic, liver, breast, prostate, and colon carcinomas, as well as melanoma and multiple myeloma. The term "subject" is interchangeable with "patient."
The ton "survival" includes all of the following: survi val until mortality, also known as overall survival (wherein said mortality may be either irrespective of cause or tumor related); "recurrence-free survival" (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith). The length of said survival may be calculated by reference to a defined start point (e.g. time of diagnosis or start of treatment) and end point (e.g. death, recurrence or metastasis), in addition, criteria for efficacy of treatment ca t be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.
The term: "therapeutic effect" refers to a local or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance. The term thus means any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental development and conditions in an animal or human. The phrase "therapeutical iy- effeefive amount" means that amount of such a substance that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. In certain embodiments, a therapeutically effective amount of a compound will depend on its therapeutic index, solubility, and the like. For example, certain compounds discovered by the methods of the present invention may be administered in a sufficient amount to proditce a reasonable benefit/risk ratio applicable to such treatment.
The tons "therapeutically-efJfective amount" and "effective amount" as used herein means that amount of a compound, material, or composition comprising a compound of the present invention which is effective for producing some desired therapeutic effect in at ieast a sub-population of ceils in an animal at a reasonable benefit/risk ratio applicable to any medical treatment. Toxicity and therapeutic efficacy of subject compounds may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, £?.g. , for determining the L so and the EDsu. Compositions that exhibit large therapeutic indices are preferred. In some embodiments, the L¾> (lethal dosage) can be measured and can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more reduced for the agent relative to no administration of the agent. Similarly, the ED¾> (i.e. , the concentration which achieves a half-maximal inhibition of symptoms) can be measured and can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increased for the agent relative to no administration of the agent. Also, Similarly, the IC>« (i.e., the concentration which achieves haif-maximaJ cytotoxic or cytostatic effect on cancer cells) can be measured and can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increased for the agent relative to no administration of the agent, in some embodiments, cancer ceil growth in an assay can he inhibited by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,, 65%, 70%, 75%,, 80%, 85%, 90%,, 95%, or even 100%. to another embodiment, at least about a 1.0% , 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 1.00% decrease in a solid malignancy can. be achieved.
A "transcribed polynucleotide" or "nucleotide transcript" is a polynucleotide (e.g. an raRNA, hnRNA, a cDNA, or an analog of such RNA or cDNA) which is complementary to or homologous with all or a portion of a mature mRNA made by transcription of a bio-marker nucleic acid and normal post-rranscriprional processing (e.g. splicing), if any, of the RNA transcript, and reverse transcription of the RNA transcript.
There is a known and definite correspondence between the amino acid sequence of a particular protein and the nucleotide sequences that can code for the protein, as defined by the genetic code (shown below). Likewise, there is a known and definite correspondence between the nucleotide sequence of a particular nucleic acid and the amino acid sequenee encoded by that nucleic acid, as defined by the genetic code.
Figure imgf000047_0001
An important and well known feature of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one coding nucleotide triplet may be employed (illustrated above). Therefore, a number of different nucleotide sequences may code tor a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrmridine may be found in a given nucleotide sequence. Such methylations do not affect the coding relationship between the trinucleotide codon and the corresponding amino acid. iii view of the foregoing, the nucleotide sequence of a D A or R A encoding a biomarker nucleic acid (or any portion thereof) can be used to derive the polypeptide amino acid sequence, using the genetic code to translate the DNA or RNA into an amino acid sequence. Likewise, for polypeptide amino acid sequence, corresponding nucleotide sequences that can encode the polypeptide can be deduced from the genetic code (which, because of its redundancy, will produce multiple nucleic acid sequences for any given amino acid sequence). Thus, description and/or disclosure herein of a nucleotide sequence which encodes a polypeptide should be considered to also include description ancl or disclosure of the amino acid sequence encoded by the nucleotide sequence. Similarly, description and/or disclosure of a poly peptide amino acid sequence herein should be considered to also include description and/or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.
Finally, nucleic acid and amino acid sequence information for the loci and biomarkers of the present invention and related biomarkcrs (e.g. biomarkcrs listed in Table 1 ) are well known in the art and readily available on publicly available databases, such as the National Center for Biotechnology information (NCBl). For example, exemplary nucleic acid and amino acid sequences derived from publicly available sequence databases are provided below.
Representative sequences of the biomarkers described above are presented below in Table 1. It is to be noted that the terms described above can further be used to refer to any combination of features described herein regarding the biomarkers. For example, any combination of sequence composition, percentage identify, sequence length, domain structure, functional activity, etc. can be used to describe a biomarker of the present invention.
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
5
W
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20
25
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35
40
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50
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
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Human succinate dehydrogenase
Human ferritin
Lipid reactive oxygen species
1) Decreased conversion, of cysteine to alanine or methylene blue
2) induction and/or promotion of mitochondrial dysfunction:
a.) Decrease in aconitase copy number, amount, and/or acti vity
b) Decrease in succinate dehy drogenase copy number, amount, and/or activity
3) Induction and/or promotion of iron regulatory protein dysfunction:
a) Decrease in ferritin copy number, amount, and/or activity
b) Increase in transfcrrin-receptor copy number, amount, and/or activity
c) Decrease in Hifiaipha copy number, amount, and/or activity
4) Induction and/or promotion of ferroptosis
a) increase and/or accumulation of lipid reactive oxygen species (ROS)
b) Increase in PTGS2 (COX2) copy number, amount, and/or activity * included in Table 1 are RNA nucleic acid molecules (e.g., thy mines replaced with uredines), nucleic acid moiecuies encoding orthologs of the encoded proteins, as well as DNA or RNA nucleic acid sequences comprising a nucleic acid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with the nucleic acid sequence of any SEQ ID NO listed in Table I , or a portion thereof. Such nucleic ac id molecules can have a function of the full-length nucleic acid as described further herein.
* Included in Table 1 are orthologs of the proteins, as well as polypeptide moiecuies comprising an amino acid sequence having at least %, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with an amino acid sequence of any SEQ ID NO listed in Table 1 , or a portion thereof. Such polypeptides can have a function of the full-length polypeptide as described further herein.
II, Subjects
la one embodiment, the subject for whom cancer treatment is administered or who is predicted likelihood of efficacy of an anti-cancer therapy (e.g. , iron-sulfur cluster biosynthesis pathway inhibitory therapy) is determined, is a mammal (e.g., mouse, rat, primate, non-human mammal, domestic animal such as dog, cat, cow, horse), and is preferably a human.
In another embodiment of the methods of the invention, the sub ject has not undergone treatment, such as chemotherapy, radiation therapy, targeted therapy, and/or anti-cancer therapy (e.g., iron-stilmr cluster biosynthesis pathway inhibitory therapy), in still another embodiment, the subject has undergone treatment, such as chemotherapy, radiation therapy, targeted therapy, and/or anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy).
in certain embodiments, the subject has had surgery to remove cancerous or precancerous tissue. In other embodiments, the cancerous tissue has not been removed, e.g. , the cancerou tissue may be located in an inoperable region of the body, such as in a tissue that is essential for life, or in a region where a surgical procedure would cause considerable risk of harm to the patient.
The methods of the invention can be used to determine the responsiveness to anticancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy) of many different cancers in subjects such as those described above, in one embodiment, the cancers are hematologic cancers, such as leukemia. In another embodiment, the cancers are solid tumors, such as lung cancer, melanoma, and/or renal ceil carcinoma. In another embodiment, the cancer is an epithelial cancer such as, but not limited to, brain cancer (e.g., glioblastomas) bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer.
III. Sample Collection. Preparation and Separation
In some embodiments, biomarker presence, absence, amount, and/or activity measurements) in a sample from a subject is compared to a predetermined control (standard) sample. The sample from the subject is typically from a diseased tissue, such as cancer ceils or tissues. The coturoi sample can be from the same subject or from a different subject. The control sample is typicall a normal non-diseased sample. However, in some embodiments, such as for staging of disease or for evaluating the efficacy of treatment, the control sample can be from a diseased tissue. The control sample can be a combination of sample from several different subjects. n some embodiments, the biomarker amount and/or activity measurements) from a subject is compared to a pre-determined level. This predetermined level is typically obtained from normal samples, such as the normal copy number, amount, or activity of a biomarker in the cell or tissue type of a member of the same species as from which the test sample was obtained or a non-diseased cell or tissue from the subject from which the test samples was obtained. As described herein, a "predetermined" biomarker amount and/or activity measurements) may be a bioniarker amount and/or activity measurements) used to, by way of example only, evaluate a sub jec t that may be selected for treatment, evaluate a response to an anti-cancer therapy (e.g., iron- sulfur cluster biosynthesis pathway inhibitory therapy), and/or evaluate a response to a combination anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory- therapy plus immunoinhibitory inhibitor therapy). A pre-determined biomarker amount and/or activit measurement8) ma be determined in populations of patients with or without cancer. The pre-determined biomarker amount and/or activity measurements) can be a single number, equally applicable to every patient, or the pre-determined biomarker amount and/or activit measurements) can vary according to specific subpopitlations of patients. Age, weight, height, and other factors of a subject may affect the pre-determined biomarker amount and/or activity measurements) of the individual. Furthermore, the predetermined biomarker amount and/or activity can be determined for each subject individually, in one embodiment, the amounts determined and/or compared in a method described herein are based on absolute measurements. In another embodiment, the amounts determined and/or compared in a method described herein are based on relative measurements, such as ratio (e.g., biomarker expression normalized to the expression of a housekeeping gene, or gene expression at various time points).
The pre-determined biomarker amount and/or activity measurement's) can be any suitable standard. For example, the pre-determined biomarker amount and/or activity measurements) can be obtained from the same or a different human for whom a patient selection is being assessed, in one embodiment, the pre-determined biomarker amount and/or activity measurements) can 'be obtained from a previous assessment of the same patient In such a maimer, the progress of the selection of the patient can be monitored over time. In addition, the control can be obtained from an assessment of another human or multiple humans, e.g. , selected groups of humans, if the subject is a human, in such a manner, the extent of the selection of the human for whom selection is being assessed can be compared to suitable other humans, .g., other humans who are in a similar situation to the human of interest, such as those suffering from similar or the same conditions) and/or of the same ethnic group.
In some embodiments of the present invention the change of biomarker amount and/or activity measurements.) from the predetermined level is about 0.5 fold, about 1 ,0 fold, about 1 .5 fold, about 2.0 fold, about 2.5 fold, about 3.0 fold, about 3,5 fold, about 4.0 fold, about 4.5 fold, or about 5.0 fold or greater. In some embodiments, the fold change is less than about 1 , less than about 5, less than about 10, less than about 20, less than about 30, less than about 40, or less than about 50. io. oilier embodiments, the fold change in biomarker amount and/or activity' measurements) compared to a predetermined level is more than about 1 , more than about 5, more than about 10, more than about 20, snore than about 30, more than about 40, or more than about 50,
Biological samples can be collected from a variety of sources from a patient including a body fluid sample, cell sample, or a tissue sample comprising nucleic acids and/or proteins. "Body fluids" refer to fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g. , amniotic fluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, co per's fluid or pre-ejaculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomit). In a preferred embodiment, the subject and/or control sample is selected from the group consisting of cells, cell lines, histological slides, paraffin embedded tissues, biopsies, whole blood, nipple aspirate, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bone marrow. In one embodiment, the sample is serum, plasma, or urine, in another embodiment, the sample is scrum,
The samples can be collected from individuals repeatedly over a longitudinal period of time (e.g., once or more on the order of days, weeks, months, annually, biannually, etc.). Obtaining numerous samples from an indi vidual over a period of time can be used to verify results from earlier detections and/or to identify an alieration in biological pattern as a result of, for example,, disease progression, drug treatment etc. For example, subject samples can be taken and monitored every month, every two mouths, or combinations of one, two, or three month intervals according to the invention, in addition, the biomarker amount and/or activity measurements of the subject obtained over time can be conveniently compared with each other, as well as with those of normal control during the monitoring period, thereby providing the subject's own values, as an internal, or personal, control for long-term monitoring.
Sample preparation and separation can involve any of the procedures, depending on the type of sample collected and/or analysis of biomarker measurements). Such procedures include, by way of example only, concentration, dilution, adjustment of pH, removal of high abundance polypeptides {e.g., albumin, gamma globulin, and transferrin, etc.), addition of preservatives and calibrants, addition of protease inhibitors, addition of denaturants, desalting of samples, concentration of sample proteins, extraction and purification of lipids.
The sample preparation can also isolate molecules that are bound in non-eovalent complexes to oilier protein (e.g., carrier proteins). This process may isolate those molecules bound to a specific carrier protein (e.g. , albumin), or use a more general process, such as the release of hound molecules from. all. carrier proteins via. protein denaturation, for example using an acid, followed by removal of the carrier proteins.
Removal of undesired proteins (e.g., high abundance, uninforrnative, or
undetectable proteins) from a sample can be achieved using high affinity reagents, high, molecular weight filters, uStraeentrifugatton and/or e!eeirodia!ysis. High affinity reagents include antibodies or other reagents (e.g., aptamers) that selectively bind to high abundance proteins. Sample preparation could also include ion exchange chromatography, metal ton affinity chromatography, gei filtration, hydrophobic chromatography, chromatofocusing, adsorption chromatography, isoelectric focusing and related techniques. Molecular weight filters include membranes that separate molecules on the basis of size and molecular weight. Such filters may further employ re verse osmosis, nanofiltration, ultrafiltration and microfiltratton.
Ultracenrrifugation is a method for removing undesired polypeptides from a sample,
Uliraeentrifugation is the eentrifisgation of a sample at about 15,000-60,000 rpm while monitoring with an optical system the sedimentation (or lack thereof) of particles.
Electrodialysis is a procedure which uses an eiectromembnme or semipermable membrane in a process in which ions are transported through semi-permeable membranes from one solution to another under the influence of a potential gradient. Since the membranes used in electrodialysis may have the ability to selectively transport ions having positive or negative charge, reject ions of the opposite charge, or to allow species to migrate through a semipermabie membrane based on size and charge, it renders electrodialysis useful for concentration, removal, or separation of electrolytes.
Separation and purification in the present invention may include any procedure known in the art, such as capillary electrophoresis (e.g., in capillary or on-chip) or
chromatography (e.g., in capillary, column or on a chip). Electrophoresis is a method which can be used to separate ionic molecules under the influence of an electric field. Electrophoresis can be conducted in a gel, capillars', or in a mierocharmel on a chip.
Examples of gels used for electrophoresi include starch, aerylamide, polyethylene oxides, agarose, or combinations thereof. A gel can be modified fay its cross-linking, addition of detergents, or denatnrants, immobilization of enzymes or antibodies (affinity
electrophoresis) or substrates (kymography) and incorporation of a pH gradient. Examples of capillaries used for electrophoresis include capillaries that interface with an electrospray.
Capillary electrophoresis (CE) is preferred for separating complex hydrop Hc molecules and highly charged solutes. CE technology can also be implemented on.
microfluidic chips. Depending on the types of capillary and buffers used. CE can be further segmented into separation techniques such as capillary zone electrophoresi (CZE), capillary isoelectric focusing (CIEF), capillary isotachophoresis (clTP) and capillary eleetroeliromatography (CEC). An embodiment to couple CE techniques to electrospray ionization involves the use of volatile solutions, for example, aqueous mixtures containing a volatile acid and/or base and an organic such as an alcohol or aceiomtrile,
Capillary tsotachophoresis (eiT.P) is a technique in which the analytes move through the capillary at a constant speed but are nevertheless separated by their respective mobilities. Capillary zone electrophoresis (CZE), also known as free-solution CE (PSCE), is based on differences in the eleetrophoretie mobility of the species, determined by the charge on the molecule, and the frictional resistance the molecule encounters during migration which is often directly proportional to the size of the molecule. Capillary isoelectric focusing (CIEF) allows weakly-iouizabSe amphoteric molecules, to be separated by electrophoresis in a pH" gradient, CEC is a hybrid technique between traditional high, performance liquid chromatography (HPLC) and CE. Separation and purification techniques used in die present- invention include any chromatography procedures known in the art. Chromatography can be based on the differential adsorption and elution of certain anaiytes or partitioning of analy es between mobile and stationary phases. Different examples of chromatography include, but not limited to, liquid chromatography (LC), gas chromatography (GC), high performance liquid chromatography (H.PLC), etc,
IV. Biomarkcr Nucleic Acids and Polypeptides
One aspect: of the presen t invention pertains to the use of isolated nucleic acid moiecuies that correspond to biomarker nucleic acids that encode a biomarkcr polypeptide or a portion of such a polypeptide. As used herein, the term "nucleic acid molecule" is intended to include DNA moiecuies (e.g., cDNA or genomic DNA) and RNA molecules (eg., tn'RNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is doubie- stranded DN A.
An "isolated" nucleic acid molecule is one which is separated from other nucleic acid moiecuies which are present in the natural source of the nucleic acid molecule.
Preferably, an "isolated" nucleic acid molecule is free of sequences (preferably protein- encoding sequences) which naturally flank the nucleic acid {i.e. , sequences located at the 5* and 3' ends of the nucleic acid) in the genomic DNA of the organism from: which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kB, 4 kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the eel! from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized,
A biomarker nucleic acid molecule of the present invention can be isolated using standard molecular biolog techniques and the sequence information in the database records described herein. Using all or a portion of such nucleic acid sequences, nucleic acid molecules of the invention can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al. y ed„ Molecular Cloning: A Laboratory Manual, 2nd d., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). A nucleic acid molecule of the invention can be amplified using cDNA, mR A, or genomic DNA as a template and appropriate oligoiiueieotide primers according to standard PGR amplification techniques. The nucleic acid molecules so amplified cao he cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotide* corresponding to ail or a portion of a nucleic acid molecule of the invention can be prepared by standard synthetic techniques, e.g., using an automated DN A synthesizer.
Moreover, a nucleic acid molecule of the in vention can comprise only a portion of a nucleic acid sequence, wherein die fail length nucleic acid sequence comprises a marker of the invention or which encodes a polypeptide corresponding to a marker of the invention. Such nucleic acid molecules can be used, for example, as a probe or primer. The probe/primer typically is used as one or more substantially purified oligonucleotides. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 7, preferably about 15, more preferably about 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or 400 or more consecutive nucleotides of a biomarker nucleic acid sequence. Probes based on the sequence of a biomarker nucleic acid molecule can be used to detect transcripts or genomic sequences corresponding to one or more markers of the invention.. The probe comprises a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
A biomarker nucleic acid molecules that differ, due to degeneracy of the genetic code, from the nucleotide sequence of nucleic acid molecules encoding a protein which corresponds to the biomarker, and thus encode the same protein, are also contemplated.
in addition, it will be appreciated by those skilled in the art thai DNA sequence polymorphisms that lead to changes in the amino acid sequence can exist within a population (e.g., the human population). Such genetic polymorphisms can exist among individuals within a population due to natural allelic variation. An allele is one of a group of genes which occur alternatively at a given genetic locus, in addition, it will be appreciated that D A polymorphisms that affect R A expression levels can also exist that may affect the overall expression level of that gene (e.g., by affecting regulation or degradation).
The term "allele," which is used interchangeably herein with "allelic variant" refers to alternative forms of a gene or portions thereof Alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be homozygous for the gene or aiiele. When a subject has two different alleles of a gene, the subject- is said to be heterozygous for the gene or aiiele. For example, biomarker alleles can differ from each other in a single nucleotide, or several nucleotides, and can include substitutions, deletions, and insertions of nucleotides. An allele of a gene can also be a fbrin of a gene containing one or more mutations.
The term "allelic variant of a polymorphic region of gene" or "allelic variant", used interchangeably herein, refers to an alternative form of a gene having one of several possible nucleotide sequences found in that region of the gene in the population. As used herein, allelic variant: is meant to encompass functional allelic variants, non-functional allelic variants, SNPs, mutations and polymorphisms.
The term "single nucleotide polymorphism" (SNP) refers to a polymorphic site occupied b a single nucleotide, which is the site of variation between allelic sequences. The site is usually preceded by and followed by highly conserved sequences of the allele (e.g., sequences that vary in less than 1/100 or 1/1000 members of a population). A S P usually arises due to substitution of one nucleotide for another at the polymorphic site. SNPs can also arise from a deletion of a nucleotide or an insertion of a nucleotide relative to a reference allele. Typically the polymorphic si te is occupied by a base other than the reference base. For example, where the reference allele contains the base "T" (thymidine) at the polymorphic site, the altered aiiele can contain a "C" (cytidhie), "G" (guanine), or "A" (adenine) at the polymorphic site. SNP's may occur in protein-coding nucleic acid sequences, in which, ease they may give rise to a defective or otherwise variant protein, or genetic disease. Such a SNP may alter the coding sequence of the gene and therefore specify another amino acid (a "missense" SNP) or a SNP may introduce a stop codon (a "nonsense" SNP). When a SNP does not alter the amino acid sequence of a protein, the SNP is called "silent." SNP's may also occur in noncoding regions of the nucleotide sequence. This may result in defective protein expression, e.g., as a result of alternative spicing, or it may have no effect on the function of the protein.
As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide corresponding to a marker of the invention. Such natural allelic variations can typically result in 1 -5% variance in the nucleotide sequence of a gi ven gene. Alternati ve alleles can be identi fied by sequencing the gene of interest in a number of different individuals. This can be readily carried out by using hybridization probes to identify the same genetic locus in a variety of individuals. Any and all such nucleotide variations and resulting amino acid
polymorphisms or variations that are die resu lt of natural allelic variation and that do not alter the functional activity are intended to be within the scope of the in vention,
in another embodiment, a biomarker nucleic acid molecule is at least 7, 15, 20, 25, 30, 40, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 550, 650, 700, 800, 900, 1.000, i 100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1 00, 2000, 2200, 2400, 2600, 2S00, 3000, 3500, 4000, 4500, or more nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule corresponding to a. marker of the invention or to a nucleic acid molecule encoding a protein corresponding to a marker of the invention. As used herein, the ton "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% (65%, 70%, 75% 80%, preferably 85%) identical to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in sections 6,3.1 -6.3,6 of Current Protocols in Molecular Biology, John Wiley & Sons, N,Y, (1989). A preferred, non-limiting example of stringent hybridization conditions are hybridization i 6X sodium chloride/sodium citrate (SSC) at about 45:'C, followed by one or more washes in 0.2X SSC, 0.1 % SDS at 50-65°C.
in addition to naturally-occurring allelic variants of a nucleic acid molecule of the invention that can exist in the population, the skilled artisan will further appreciate that sequence changes can be introduced by mutation thereby leading to changes in the amino acid sequence of die encoded protein, without altering the biological activity of die protein encoded thereby. For example, one can make nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues, A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. For example, amino acid residues that are not conserved, or only semi -conserved among homologs of various species may be non-essential for activity and thus would be likely targets for alteration. Alternatively, amino acid residues that are conserved among the homologs of various species (e.g., murine and human) may be essentia! for activity and thus would not be likely targets for alteration.
Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding a polypeptide of the invention that contain changes in amino acid residues that are not essential for activity. Such polypeptides differ in amino acid sequence from the natunuiy-oceitrriog proteins which correspond to the markers of the invention,, yet retain biological activity- In one embodiment, a biomarker protein has an amino acid sequence that is at least about 40% identical, 50%, 60%, 70%, 75%, 80%, 83%, 85%, 87,5%, 90%, 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or identical to the amino acid sequence of a biomarker protein described herein.
An isolated nucleic acid molecule encoding a variant protein can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of nucleic acids of the invention, such that one or more amino acid residue substitutions, additions, or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis- Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidrae), acidic side chains (e.g. , aspartic acid, glutamic acid), uncharged polar side chains (e.g. , glycine, asparagine, giuta.oii.oe, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidirte). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly and the activity of the protein can be determined,
in some embodiments, die present invention further contemplates the use of anii- biomarker antisense nucleic acid molecules, i.e., molecules which are complementary to a sense nucleic acid of the invention, e.g., complementary to the coding strand of a double- stranded cDNA molecule corresponding to a marker of the invention or complementar to an ra' A sequence corresponding to a marker of the invention. Accordingly, an antisense nucleic acid molecule of the invention can hydrogen bond to (i.e. anneal with) a sense nucleic acid of the invention. The antisense nucleic acid can be complementary to an entire coding strand, or to only a portion thereof, e.g., ail or part of the protein coding region (or open reading frame). An antisense nucleic acid molecule can also be antisense to ail or part of a non-coding region of the coding strand of a nucleotide sequence encoding a polypeptide of the invention. The non-coding regions ("5' and 3' untranslated regions") are the 5' and 3' sequences which flank" the coding region and are not translated into amino acids.
An antisense oligonucleotide can be, for example, about 5, 10„ 15, 20, 25, 30, 35,
40, 45, or 50 or more nucleotides in length. An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5- fkiorouracii, S-broroouraci!, 5-ehlorouraeil, 5-iodouracil, hypoxanthine, xanthine, 4- aeety!eytosine, 5-{carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, S-earboxyrnetlwlaminoniethxdwacil, dihydrouracii, beta-D- alactosyk ucosinc, inosine, 6-isopentenyladenme, 1 -niethyiguaiiirie, l-meth Sinosirie, 2,2-diniethylgnanine,
2- methyladentne, 2-mediylguanine, 3-methylcytosine, 5-methylcytosine, 6-adenme, 7- meth lguanme, 5-methyiaminomethylnraeil, 5-niethoxyaminomethyi-2-thiouracii, beta-D- mannosylqueosine, S'-methoxycarboxymethyiuracii, S-methoxyuracil, 2«methylthio-N6- isopcntcnyladenine, uractl-5-oxyace ic acid (v), wybutoxosine, pseudouraciJ, queosine, 2- thiocytosine, 5-methyl-2-thiouracil5 2-ihiouraeil, 4-thiouracil, 5-meihyluracil, uracil-S- oxyacetic acid methylester, uracil-5~oxyaeetic acid (v), 5-methyi-2-thiouracil, 3-(3-amino-
3- N-2-earboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been sub-cloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orien tation to a target nucleic acid of interes t, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic D A encoding a polypeptide corresponding to a selected marker of the invention to thereb inhibit expression of the marker, e.g. , by inhibiting transcription and/or
translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. Examples of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site or infusion of the antisense nucleic acid into a blood- or bone marrow-associated body fluid. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by
Unking the antisense nucleic acid molecules to peptides or antibodies which bind to eel! suriace receptors or antigens. The antisense nucleic acid molecules can a! so be deli vered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under die control of a strong pol 11 or poi ill promoter are preferred.
An antisense nucleic acid molecule of the invention can be an a-anomcric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ot-unsts, the strands run parallel to each other (Gaultier el at, 1987, Nucleic Ackis Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2†-c~methylribonucleotide (inoue et al, 1987, Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (inoue et L imi. FEBSLeit. 215:327-330).
The present invention also encompasses ribozymes. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes as described in Haselhoff and Gerlach, 1 88, Nature 334:585-59 i ) can be used to catalytically cleave mRNA transcripts to thereby inhibit translation of the protein encoded, by the mRNA. A ribozyme having specificity for a nucleic acid molecule encoding a polypeptide corresponding to a marker of the invention can be designed based upon the nucleotide sequence of a cDN A corresponding to the marker. For example, a deri vative of a Tetr hymen L-1 1'VS RN A can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved (see Cecil el el U ,S. Patent No. 4,987,0? I ; and Cech el o!. U.S. Patent No. 5, 116,742), Alternatively, an mRNA encoding a polypeptide of the invention can be used to select a catalytic R A having a specific ribonuclease acti vity from a pool of RNA molecules (see, e.g., Bartel and Szostak, 1993, Science 261 :141 1-1418).
The present invention also encompasses nucleic acid molecules which form triple helical structures. For example, expression of a biomarker protein can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the gene encoding the polypeptide (e.g. , m promoter and/or enhancer) to form triple helical structures thai prevent transcription of the gene in target cells. See generally Helene (1 91) Anticancer Drug m. 6(6):56 -84; Helene (1 92) A n. N. Y. Acad. Sci. 660:27-36; and Maher (1992) Biamscm 1 (12): 807- 1.5.
in various embodiments, the nuc eic acid molecules of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acid molecules (see Hyrup et al, 1996, Bioorganic & Medicinal Chemistry 4(1 ): 5- 23). As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid, mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DN A and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard, solid phase peptide synthesis protocols as described in Hyrup et al. (1996),, supra; Perry-O' eefe et al. (1996) Proc. Na . Acad. Sci. USA 93: 14670-675.
PNAs can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs can also be used, e.g. , m the analysis of single base pair mutations in a gene by, e.g. , PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., SI nucleases (Hyrup (1 96), supra; or as probes or primers for DMA sequence and .hybridization (Hyrup, 1 96, supra; Perry-O'Keefe et al., 1996, Proc. Natl. Acad. Sci. USA 93: 14670-675).
in another embodiment, PNAs can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-D A chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras can be generated which can combine the advantageous properties of P A ami DNA, Such chimeras allow DNA recognition enzymes,, e.g., RNASE H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras cart be linked using linkers of appropriate lengths selected in terms of base stacki ng, number of bonds between the nucleobases, and orientation (Hyrup, 1 96, supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996), supra, and Finn et al { 1 96) Nucleic Acids Res. 24( 17):3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosp oramulitc coupling chemistry and modified nucleoside analogs. Compounds such as 5'-(4-methoxytrityi)an5iuo-5'-deoxy- thymidine phosphoramidite can be used as a link between the PNA and the 5' end of DNA (Mag et al, 1989, Nucleic Acids lies. 17:5973-88). PN A monomers are then coupled in a step-wise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn el at, \ 996, Nucleic Acids Res. 24( 57): 3357-63), Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterscr et al. , 1 75 , Bioarganic Med. Chem. Lett. 5: 1 1 19-1 1124).
in other embodiments, the oligonucleotide can include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. S t. USA 86:6553-6556; Lemaitre et al, 1987, Proc. Natl Acad. Set. USA 84:648-652; PCT
Publication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89. 10134). in addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., rol et at, 1988, Bio/Techniques 6:958-976) or intercalating agents (see, e,g„ Zon, 1988, Pharm. Res. 5:539-549). To this end, the oligonucleotide can be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
Another aspect of the present invention pertains to the use of biomarker proteins and biologically active portions thereof. n one embodiment, the native polypeptide corresponding to a marker can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques, in another embodiment, polypeptides corresponding to a marker of the invention are produced by recombinant DNA techniques. Alternative to recombinant expression, a polypeptide corresponding to a marker of the invention can be synthesized chemically using standard peptide synthesis techniques. An "isolated" or '"purified" protein or biologically acti ve portion thereof is
substantially free of ceiluiar material or other contaminating proteins from the cell or tissue source from which the protein is deri ved, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparation of protein in which the protein is separated from ceiluiar components of the ceils from which it is isolated or recombmantly produced. Thus, protein that is substantially free of cellular materia! includes preparations of protein having less than about 30%, 20%, .1 %, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein"). When die protein or biologically acti e portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, Le., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation. When the protein is produced by chemical synthesis,, i is preferably substantially free of chemical precursors or other chemicals, i.e. , it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly such preparations of the protein ha ve less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide of interest.
Biologically active portions of a biomarker polypeptide include polypeptides comprising amino acid sequences sufficiently identical to or deri ved from a biomarker protein amino acid sequence described herein, but which include fewer amino acids than the full length protein, and exhibit at least one activity of the corresponding full-length protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the corresponding protein. A biologically active portion of a protein of the invention can be a polypeptide which is, for example, 1 , 25, 50, 100 or more amino acids in length . Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared b recombinant techniques and evaluated for one or more of the functional activities of the native form of a polypeptide of the invention.
Preferred polypeptides have an amino acid sequence of a biomarker protein encoded by a nucleic acid molecule described herein. Other useful proteins are substantially identical (e.g., at least about 40%, preferably 50%, 60%, 70%, 75%, 80%, 83%, 85%, 88%, 90%, 1 , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) to one of these sequences and retain the functional activity of the protein of the corresponding naturally-occurring protein yet differ in amino acid sequence due to natural allelic variation or mutagenesis. To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity ::: # of identical positions/total # of positions (e.g., overlapping positions) xi 00). In one embodiment the two sequences are the same length.
The determination of percent identity between two sequences can he accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of arlin and Altsehul ( 1.990) Pmc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altsehul (1.993) Pmc. Nad. Acad Sci. USA 90:5873-5877. Such an algorithm is
incorporated into the NBLAST and XBLAST programs of Altsehul, ei o!. ( 1 90) J. Mol. Biol 215:403-410. BLAST nucleotide searches can. be performed with the NBLAST program, score - 100, wordlength - 12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the inven tion. BLAST protein searches can be performed with the XBLAST program, score ~-: 50, wordlength ~- 3 to obtain amino acid sequences homologous to a protei molecules of the invention. To obtain gapped alignments for comparison purposes. Gapped BLAST can be utilized as described in Altsehul ei ai. ( 1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-BIast can be used to perform an iterated search which defects distant relationships between molecules. When utilizing
BLAST, Gapped BLAST, and PSI -Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See the National Center for Biotechnology Information (NCBI) website at ncbi.nlm.nih.gov. Another preferred, non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, (1988) C mpui Appf Biosci, 4:1 1-7. Such an algorithm is incorporated into the ALIGN program, (version 2.0) which is part of the GCO sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a P AMI 20 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm as described in Pearson and Lipraan (1 88) Proa Natl Acad Sei. USA 85:2444-2448, When using the FASTA algorithm for comparing nucleotide or amino acid sequences, a PA 120 weight residue table can, for example, be used with a A-tuple value of 2.
The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.
The invention also provides chimeric or fusion proteins corresponding to a
biomarker protein. As itsed herein, a "chimeric protein'" or "fusion protein" comprises all or part (preferably biologically active part) of a polypeptide corresponding to a marker of the invention operably linked to a heterologous polypeptide (i.e., a polypeptide other than the polypeptide corresponding to the marker). Within the fusion protein, the term
"operably linked" is intended to indicate that the polypeptide of the invention and the heterologous polypeptide are fused in-frame to each other. The heterologous polypeptide can be fused to the araino-terarinus or the carboxyi-teoiiinus of the -polypeptide of the invention.
One useful fusion protein, is a GST fusion protein in which a polypeptide corresponding to a marker of the invention is f sed to the carboxyl terminus of GST sequences. Such fusion proteins can facilitate the purification of a recombinant polypeptide of the invention.
in another embodiment, the fusion protein contains a heterologous signal sequence, immunoglobulin fusion protein, toxin, or other useful protein sequence. Chimeric and fusion proteins of the invention can be produced by standard recombinant DNA techniques, in another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PGR amplification of gene
fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see, e.g., Ausubel et ai, supra). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A nucle ic acid encoding a polypeptide of the invention can be cloned into such an expression vector such ihat the fusion moiety is linked in-frame to the polypeptide of the invention. A signal sequence can be used to facilitate secretion and isolation of the secreted protein or other proteins of interest. Signal sequences are typicall characterized b a core of hydrophobic amino acids which arc generally cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides contain processing sites that 5 allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway . Thus, the invention pertains to the described polypeptides having a signal sequence, as well as to polypeptides from which the signal sequence has been proteoiytieaiSy cleaved ( .£?., the cleavage products)- hi one embodiment, a nucleic acid sequence encodin a signal sequence can be operably linked in an expression vector to a it) protein of interest, such as a protein which is ordinarily not secreted or is otherwise difficult to isolate. The signal sequence directs secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed,, and the signal sequence is subsequently or concurrently cleaved. The protein can then be readily purified from the extracellular medium by art recognized methods. Alternatively, the signal sequence can be
15 linked to ihe protein of interest using a sequence which facilitates purification, such as with a GST domain.
The presen t invention also pertains to variants of the biornarker polypeptides described herein. Such variants have an altered amino acid sequence which can function as either agonists (mimetics) or as antagonists. Variants can he generated bv mutagenesis.
20 e.g. , discrete point mutation or truncation. An agonist can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of the protein. An antagonist of a protein can inhibit one or more of the activities of the naturally occurring form of the protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the protein of interest. Thus,
25 specific biological effects can be elicited by treatment with a variant of limited function.
Treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein can have fewer side effects in a subject relative to treatment with the naturally occurring form of the protein.
Variants of a biornarker protein which function as ei ther agonists (mimetics) or as 0 antagonists can be identified by screening combinatorial libraries of mutants, e.g. ,
truncation mutants, of the protein of the invention for agonist or antagonist activity. In one embodiment, a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of variants can be produced by, for example, enzymatically iigatimg a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display). There are a variety of methods which can be used to produce libraries of potential variants of the polypeptides of the invention from a
degenerate oligonucleotide sequence. Methods for synthesizing degenerate
oligonucleotides are known in the art (see, e.g., Narang, 1983, Tetrahedron 39:3; Itakura ttf a!. , 19 , Af>n . Rev. Biochem. 53:323; liakura et oL, 1 84, Science 198: 1056; ike et ttl, 1983 Nucleic Acki Res. 1 1 :4?7).
in addition, libraries of f agments of the coding sequence of a polypeptide corresponding to a marker of the invention can be used to generate a variegated population of polypeptides for screening and subsequent selection of variants. For example, a library ofcodiiig sequence fragments can be generated by treating a double stranded PGR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/an tiseose pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligatmg the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes amino terminal and Internal f agments of various sizes of the protein of interest.
Several techniques are known in the art for screening gene products of
combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into repiieabie expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions In which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify variants of a protein of the invention. (Arkin and Yourvan, 1 92, Pr e. Nail Acad Set. USA §9:781 1-7815; Dcigrave et ai, 1 93, Protein Engineering 6{3};327- 331). The production and use of biomarker nucleic acid and/or biomarker polypeptide molecules described herein can be facilitated by using standard recombinant techniques, in some embodiments, such techniques use vectors, preferably expression vectors, containing a nucleic acid encoding a biomarker polypeptide or a portion of such a polypeptide. As 5 used herein, die term: "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "p!asmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be !igated Another type of vector is a viral vector, wherein additional DN A segments can be !igated into the viral genome. Certain vectors are capable of autonomous replication in a it) host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g. , rton-episoroal
mammalian vectors) are integrated into the genome of a host ceil upon introduction into the host cell, and thereby arc replicated along with the host genome. Moreover, certain vectors, namely expression vectors, are capable of directing the expression of genes to which they
15 are operabiy linked. In general, expression vectors of utility in recombinant DNA
techniques are often in the form of plasroids (vectors). However, the present invention is intended to include such other forms of expression vectors, such as viral vectors (e.g. , replication, defective retroviruses, adenoviruses and adeno-associated viruses), which serve eq uival e nt functions .
20 The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell. This means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operabiy linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operabiy
25 linked" is intended to mean that the nucleotide sequence of interest is linked to the
regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when tie vector is introduced into the host cell). The term, "regulatory sequence" is intended to include promoters, enhancer's and other expression control elements (e.g., polyadenylatton signals). 0 Such .regulatory sequences are described, for example, in Goeddei, Me (hods in Enzym log :
Gene Expression Teehnoi'ogy vol.185, Academic Press, San Diego, CA (1 1). Regulatory seq uences include those which, direct constitutive expression of a nucleotide sequence in many types of host ceil and those which direct expression of the nucleotide sequence only in certain host ceils (e.g. , tissue-specific regulatory sequences), it will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as die choice of die host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.
The recombinant expression vectors for use in the invention can be designed tor expression of a polypeptide corresponding to a marker of the invention in prokaryotie (e.g., E. coli) or eukaryotic cells (e.g., insect cells (using baculovirus expression vectors}, yeast cells or mammalian ceils). Suitable host ceils are discussed further in Goeddel, supra.
Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in E. coii with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins, fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein . Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the sorubiUiy of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a iigand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1 88, Gene 67:3.1 -40), pMAL (New England Biolabs, Beverly, MA) and pRlT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
Examples of suitable inducible non-fusion E, coii expression vectors include p'frc (Amann et al, 1 88, Gene 69:301 -3 i 5) and pET i Id ( Srudier et al , p. 60-89, In Gene Expression Technology: Methods in Emymo gy vol.185, Academic Press, San Diego, CA, 1 91). Target biomarker nucleic acid expression from the pTrc vector relies on host A polymerase transcription from a hybrid trp-lac fusion promoter. Target btoniarker nucleic- acid expression from the pET 11 d vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a co-expressed viral R. A polymerase (T7 gni). This viral polymerase is supplied by host strains BL21 (DE3) or HMS.174{DB3) from: a resident prophage harboring a T7 gnl gene under the transcriptional control of Ac lacliV 5
promoter.
5 One strategy to maximize recombinant protein expression in E. coli is to expres the protein in a host bacterium with, an impaired capacity to proteoiytieaily cleave the
recombinant protein (Gottesnian, p. i 19-128, in Gene Expression Technology: Methods in Enzymo gy vol. 185, Academic Press, San Diego, CA, 1990. Another strategy is to alter the nucleic acid sequence of the nucieic acid to be inserted into an expression vector so that it) the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al, 1992, Nucleic Acids Res. 20:21 1 .1 -2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
io. another embodiment, the expression vector is a yeast expression vector.
Examples of vectors for expression in yeast S. cerevtsiae include p YepSec l (Batdari et ol,
1.5 1987, BMBOJ. 6:229-234), pMFa (Kurjan and Hersko itz. 1 82, Cell 30:933-943),
pJRY88 (Schultz et a!., 1987, Gem 54: 1 13-1 3), pYES2 (Invitrogen Corporation, San Diego, CA), and pPieZ (Invitrogen Corp, San Diego, CA).
Alternatively, the expression vector is a baculovirus expression vector. Baculovirus vectors available for expression of proteins in cultured insect ceils (e.g.} Sf 9 cells) include
20 the pAc series (Smi th et αί. , 1.983, MoL Cell Biol. 3:2156-2165) and the pVL series
(Lucklow and Summers, 1989, Virology 170:31 -39).
in yet another embodiment., a nucleic acid of the present invention is expressed in mammalian cell using a mammalian expression vec tor. Examples of mammalian expression vectors include pCDM8 (Seed, 1987, Nature 329:840) and pMT2PC (Kaufman
25 et ai, 1987, EMBO J. 6: 187-195). When used in -mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly- used promoters are derived from polyoma. Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic ceils see chapters 16 and 17 of Sambrook et a' supra,
0 in another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g. , tissue- specific regulatory elements are used to express the nucleic acid). Tissue-specific
regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pi«kert <?/' al., 1987, Genes Dev. 1 :268-277), iymphoid-speeifk promoters (Calame and Eaton, 1.988,, Adv. Immunol, 43:235- 275), in particular promoters of T cell receptors (Winoto and Baltimore, 1 89, EMBOJ. 8:729-733) an immunoglobulins (Banerji i al.., .1 83, Cell 33:729-740; Queen and Baltimore, .1 83, Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1 89, Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas- specific promoters (Edlimd el al, 1985, Science 230:912-916), and mammary gland- specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264, 166). DevelopmentaUy-regulated promoters are also encompassed, for example the murine hox promoters ( essel and Gruss, 1 90, Science 249:374-379} and the a-£etoprote½ promoter (Camper and Tilghman, 1989, Genes Dev. 3:537-546).
The present invention further provides a recombinant expression vector comprising a DNA molecule cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operabiy linked to a regulator}'' sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to the raRNA encoding a polypeptide of the invention. Regulatory sequences operabiy linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of eel! types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue-specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant piasmid, phagemid, or attenuated virus in which antisense nucleic acids are produced, under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which, the vector is introduced. For a discussion of the regulation of gene expression using antisense genes (see Weiutraub ei al., 1986, Trends in Genetics, Vol. 1( 1)).
Another aspect of the present invention pertains to host cells into which a
recombinant expression vector of the invention lias been introduced. The terms "host cei and "recombinant host celF are used interchangeably herein. It i understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environ mental influences, such progen may not, in fact, be identical to the parent cel l, but are still included within the scope of the term as used herein. A host cell can be any prokaryotic {e.g., E. call) or eukaryotic cell (e.g. , insect cells, yeast or mammalian ceils).
Vector D A can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techttiques. As used herein, the terms "transformation" and 'transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid into a. host cell, including calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, !ipofeetiou, or eiectroporation.
Suitable methods for transforming or transfecting host cells can be found in Sambrook, et l {supra), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome, in order to identify and select these
integrants, a gene that encodes a selectable marker (e.g. , for resistance to antibiotics) is generally introduced into the host ceils along with the gene of interest Preferred selectable markers include those which confer resistance to drugs, such as G 1 8, hygromycin and methotrexate. Ceils stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
V. Anal zing B iomarker Nuc leic Acids and Poiypepti des
Biomarker nucleic acids and/or biomarker polypeptides can be analyzed according to the methods described herein and techniques known to the skilled artisan to identify such genetic or expression alterations useful for the present invention including, but not limited to, 1 ) an alteration in the level of a biomarker transcript or polypeptide, 2) a deletion or addition of one or more nucleotides from a biomarker gene, 4} a substitution of one or more n ucleotides of a biomarker gene , 5) aberrant modification of a biomarker gene, such as an expression regulatory region, and the like,
a. Methods for Detection of Copy Number and/or Genomic Nucleic Acid Mutations Methods of evalua ting the copy number and/or genomic nucleic acid status (e.g., mutations) of a biomarker nucleic acid are well known to those of skill in the art. The presence or absence of chromosomal gain or loss can be e val uated simply by a
determination of copy number of the regions or markers identified herein. in one embodiment, a biological samp!e is tested for die presence of copy number changes in genomic loci containing die genomic marker. In some embodiments, the increased copy number of at least one biomarker listed in Tabic i is predictive of better outcome of iron-sulfur cluster biosynthesis pathway inhibitory therapy. A copy number of at least 3, 4, 5, 6„ 7, 8, , or .10 of at least one biomarker listed in Table 1 is predictive of likely responsive to iron-sulfur cluster biosynthesis pathway inhibitory therapy.
Methods of evaluating the copy number of a. biomarker locus include, but are not limited to, hybridization-based assays. Hybridization-based assays include, but are not limited to, traditional "direct: probe" methods, such as Southern blots, in situ hybridization (e.g., FISH and FISH plus SKY) methods, and "comparative probe" methods, such as comparative genomic hybridization (CGH), e.g., cDNA-based or oligonueleotide-based CGH. The methods can be used in a wide variety of formats including, but not limited to, substrate (e.g. membrane or glass) bound methods or array-based approaches.
In one embodiment, evaluating the biomarker gene copy number in a sample involves a Southern Blot. In a. Southern Blot, the genomic DNA (typically fragmented, and separated on an deeirophoreiie gel) is hybridized to a probe specific for the target region. Comparison of the intensity of the hybridization signal from the probe for the target region with control probe signal from analysis of normal genomic DN A (e.g., a non-amplified portion of the same or related cell, tissue, organ, etc.) provides an estimate of the relative copy number of the target nucleic acid. Alternatively, a Northern blot may be utilized for evaluating the copy number of encoding nucleic acid in a sample. In a. Northern blot, mRNA is hybridized to a probe specific for the target region. Comparison of the intensity of the hybridization signal from the probe for the target region with control probe signai from analysis of normal R A (e.g., a non-amplified portion of the same or related cell, tissue, organ, etc.') provides an estimate of the relative copy number of the target nucleic acid. Alternati ely, other methods well known in the art to detect RNA can be used, such that higher or lower expression relative to an appropri te control {e.g., a non-amplified portion of the same or related cell tissue, organ, etc..) provides an estimate of the relative copy number of the target nucleic acid.
An alternative means for determining genomic copy number is in situ hybridization
(e.g., Angercr (1 87) M th. Ensymol 152: 649). Generally, in site hybridization comprises the following steps: (I) fixation of tissue or biological structure to be analyzed; (2) prehybridization treatment of the biological structure to increase accessibility of target DNA, and to reduce nonspecific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological structure or tissue: (4) post-hybridization washes to remove nucleic acid fragments not bound in the hy bridization and (5) detection of the hybridized nucleic acid fragments. The reagent used in each of these steps and the
conditions for use vary depending on the particular application, in a typical In situ hybridization assay, ceils are fixed to a solid support, typically a glass slide, if a nucleic acid is to be probed, the cells are typically denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of labeled probes specific to the nucleic acid sequence encoding the protein. The targets (e.g., cells) are then typically washed at a predetermined stringency or at an increasing stringency until an appropriate signal to noise ratio is obtained. The probes are typically labeled, e.g., with radioisotopes or fluorescent reporters, in one embodiment, probes are sufficiently long so as to specifically hybridize with the target nucleic acid(s) under stringent conditions. Probes generally range tn length from about 200 bases to about 1 00 bases, in some applications it is necessary to block the hybridization capacity of repetitive sequences. Thus, in some embodiments, tRNA, huma genomic DNA, or Cot-I DNA is used to block non-specific hybridization.
An alternative means for determining genomic copy number is comparative genomic hybridization. In general, genomic DNA is isolated from normal reference cells, as well as from test cells (e.g., tumor cells) and amplified, if necessary. The two nucleic acids arc differentially labeled and then hybridized in situ to metaphasc chromosomes of a reference ceil. The repetitive sequences in both the reference and test DNAs are either removed or their hybridization capacity is reduced by some means, for example by prehybridization with appropriate blocking nucleic acids and/or including such blocking nucleic acid sequences for said repetitive sequences during said hybridization. The bound, labeled DNA sequences are then rendered in a visual izable form, if necessary.
Chromosomal regions in the test cells which are at increased or decreased copy number can be identified by detecting regions where the ratio of signal from the two DNAs is altered. For example, those regions that have decreased in copy number in the test cells will show relatively lower signal from the test DNA than the reference compared to other regions of the genome. Regions that have been increased fa copy number in the test cells will show relatively higher signal from, the test DN A. Where there arc chromosomal deletions or multiplications, differences in the ratio of the signals from the two labels will be detected and the ratio will provide a measure of the copy number. In another embodiment of CGH, array CGH (aCGH), the immobilized chromosome element is repiaced with a collection of solid support bound target nucleic acids on an array, allowing for a large or complete percentage of the genome to be represented in the collection of solid support bound targets. Target nucleic acids may comprise c-DNAs, genomic DNAs, oligonucleotides (e.g., to detect single nucleotide polymorphisms) and the like. Array-based CGH may also be performed with single-color labeling (as opposed to labeling the control and the possible tumor sample with two different dyes and mixing them prior to hybridization, which will yield a ratio due to competitive hybridization of probes on the arrays). In single color CGH, the control is labeled and hybridized to one array and absolute signals arc read, and the possible tumor sample is labeled and hybridized to a second array (with identical content) and absolute signals are read. Copy number difference is calculated based on absolute signals from the two arrays. Methods of preparing immobilized chromosomes or arrays and performing comparative genomic hybridization are well known in the art (see, e.g., U.S. Pat. Nos: 6,335,167; 6,197,501 ; 5,830,645; and 5,665,549 and Albertson { 1 84) EMBOJ. 3: 1227-1234; Pinkei ( 1988) Pwc. NatL Acad Set. USA 85: 9138-9142; EPO Pub. No, 430,402; Methods in Molecular Biology, Vol. 33: In situ Hybridization Protocols, Choo, ed., Humana Press, Totowa, N.i. (1994 , etc.) in another embodiment, the
hybridization protocol of Pinkef ei l. ( 1 98) Nature Genetics 20: 207-21 i, or of
Kaliioniemi (1992) /Voe. Natl Acad Set USA 89:5321.-5325 (1992) is used.
in still another embodiment, amplification-based assays can be used to measure copy number, in such amplification-based assays, the nucleic acid sequences act as a template in an amplification reaction (e.g.. Polymerase Chain Reaction (PCR)). In a quantitative amplification, the amount of amplification product will be proportional to the amount of template in the original sample. Comparison to appropriate controls, e.g. healthy tissue, provides a measure of the copy number.
Methods of^quanfitat ve" amplification are well known to those of skill in the art. For example, quantitative PCR involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction. Detailed protocols for quantitative PCR are provided in Innis, et l. (1990) PGR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.). Measurement of DNA copy number at mtcrosatelltte loci using quantitative PCR analysis is described in Gmzonger, ei al. (2000) Cancer Research 60:5405-5409. The known nucleic acid sequence for the genes is sufficient to enable one of sk il io the art to routinely select primers to amplify any portion of the gene. Fluorogenic quantitative PCR may also be used in the methods of the invention, in fluorogenic quantitative PCR, quantitation is based on amount of fluorescence signals, e.g., Taq an and SY.8R green.
Other suitable amplification methods include, but are not limited to, ligase chain reaction (LCR.) (see Wu and Wallace (1989) Genomics 4: 560, Landegrea, et al. (3988) Science 241 : 1077, and Barringer et al. ( 1.990) Gene 89: 1.17), transcription amplification (Kwoh, et al. (1 89) Proc. Mat!. Acad. Set USA 86: 1 173), self-sustained sequence replication (Guateili, ef ai. (1990) Proc. Na Aca Sci. USA 87: 1874), dot PCR, and linker adapter PCR, etc.
Loss of heterozygosity (LOH) and major copy proportion (MCP) mapping ( Wang, Z.C., et al. (2004) Cancer Res 64(1);64-71 ; Seymour, A. B., ei ei (1994) Cancer Res 54, 2763-4; Hahn, S. A,, et at. (3995) Cancer Res 55, 4670-5; Kimuta, M., et al. (3996) Gems Chromosomes Cancer 17, 88-93; Li et al, (2008) MBC Bkmfbrm. 9, 204-219) may also be used to identify regions of amplification or deletion.
b. Methods for Detection of Biomarker Nucleic Acid Expression
Biomarker expression may be assessed by an of a wide variety of well-known methods for detecting expression of a transcribed molecule or protein. Non-limiting examples of such methods include immunological methods for detection of secreted, celi- surface, cytoplasmic, or nuciear proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods.
in preferred embodiments, activity of a particular gene is characterized by a measure of gene transcript (e.g. mRNA), by a measure of the quantity of translated protein, or by a measure of gene product activity. Biomarker expression can be monitored in a variety of ways, including by detecting mRNA levels, protein levels, or protein activity, any of which can be measured using standard, techniques. Detection can involve quantification of the level of gene expression (e.g., genomic DNA, cDNA, mRNA, protein, or enzyme activity), or, alternatively, cati. be a qualitative assessment of the level of gene expression, in particular in comparison with a control level. The type of level being detected will be clear from the context.
In another embodiment, detecting or determining expression, levels of a biomarker and. functionally similar homoiogs thereof, including a fragment or genetic alteration
- 3.00 - thereof < 'e.g. , in regulatory or promoter regions thereof) comprises detecting or determining RNA levels for the marker of interest In one embodiment, one or more cells from the subject to be tested are obtained and RNA is isolated from the cells. In a preferred embodiment, a sample of breast tissue cells is obtained from the subject.
in one embodiment, RNA is obtained from a single cell. For example, a cell can be isolated from a tissue sample by laser capture microdissection (LCM). Using this technique, a cell can be isolated from a tissue section, including stained tissue section, thereby assuring that the desired cell is isolated (see, e.g. , Bonner et al. (1 97) Science 278: 1481; Emmert-Buck et al ( 1996) Science 274:998; Fend et al. (1999) Am. J. Path. 154: 61. and Murakami et al. (2000) Kidney int. 58; 1346), For example, Murakami et al,, supra, describe isolation of a cell from a previously imimmostained tissue section.
it is also be possible to obtain cells from a subject and culture the cells in vitro, such as to obtain a larger population of cells from which RNA can. be extracted. Methods for establishing cultures of non-transformed cells, i.e., primary cell cultures, are k own in the art.
When isolating RNA from tissue samples or cells from individuals, it may be important to prevent any further changes in gene expression after the tissue or cells has been removed from the subject. Changes in expression levels are known to change rapidly following perturbations, .g., heat shock or activation with lipopoiysaecharide (LPS) or other reagents, in addition, the RNA in the tissue and cells may quickly become degraded. Accordingly, in a preferred embodiment, the tissue or cells obtained from a subject is snap frozen as soon as possible.
RNA can be extracted from: the tissue sample by a variety of methods, e.g., the guanidium thiocyanate lysis followed by CsCl centrifugation (Chirgwin et al., 1979, Biochemistry i 8:5294-5299). R from single cells can be obtained as described in methods for preparing cD A libraries from single cells, such as those described in Dulac, C. (.1998) Curr. Top. Dev. Biol. 36 245 and Jena et al. (1996) J. Immunol. Methods 1 0:1 9, Care to avoid RNA degradation must be taken, e.g. , b inclusion of RNA sin.
The RNA sample can then be enriched in particular species. In one embodiment, poly(A)-*- RNA is isolated from the RNA sample, in general, such purification takes ad vantage of the poiy-A. tails on mR A. In particular and as noted above, poly-T oligonucleotides may be immobilized within on a sol id support to serve as affinity ligands
- 1.01 - for mRNA. Kits for this purpose are commercially available,, e.g., the MessageMaker kit (Life Technologies, Grand Island, NY).
in a preferred embodiment, the RNA population is enriched in marker sequences. Enrichment can be undertaken, e.g., by primer-specific c'DNA synthesis, or multiple rounds 5 of linear amplification based on cDNA synthesis and template-directed in vitro
transcription (see, e.g.., Wang et al. (1989) PNAS 86, 9717; Dutac et a!., supra, and Jena et al, supra).
The population of RNA, enriched or not in particular species or sequences, ca farther be amplified. As defined herein, an "amplification process" is designed to it) strengthen, increase, or augment a molecule within the RNA. For example, where RNA is mRNA, art amplification process suc as RT-PCR can be utilized to amplify the mRNA, such that a signal is detectable or detection is enhanced. Such art amplification process is beneficial particularly when the biological, tissue, or tumor sample is of a small size or volume.
15 Various amplification and detection methods can be used. For example, it is within the scope of the present invention to reverse transcribe mRNA into cDNA followed by polymerase chain reaction (RT-PCR); or, to use a single enzyme for both steps as described in. U.S. Pat. No. 5,322,770, or reverse transcribe mR A into cDNA followed by symmetric gap ligase chain reaction (RT-AGLCR) as described by R. L. Marshall; e al, PCR
20 Methods and Applications 4: 80-84 (1994). Real time PCR may also be used.
Other known amplification methods which can be utilized 'herein include but are not limited to the so-called "NASBA" or "3SR" technique described in PNAS USA 87: 1874- 1878 (1990) and also described in Nature 350 (No. 6313): 91 -92 (1991 ); Q-beia
amplification as described in published European Patent Application (EPA) No. 4544610;
25 strand displacement amplification (as described in G. T. Walker et al., Clin. Chem. 42: 9-13 (1996) and European Patent Application No. 684315; target mediated amplification, as described by PCT Publication W0932246! ; PCR; ligase chain reaction (LCR) (see. e.g. , Wit and Wallace, Genomics 4, 560 (1989), Landegren et al.s Science 241 , 1077 ( 1988)); self-sustained sequence replication (SSR) (see, e.g., Guatelti et al., Proc. Nat. Acad. Sci. 0 USA, 87, 1874 ( 1.990)); and transcription amplification (see, e.g. , Kwoh et al., Proc. Natl Acad. Sci. USA 86, 1173 ( 1 89)).
Many techniques are kno wn in the state of the art for determining absolute and relative levels of gene expression, commonly used techniques suitable for use in the present invention include Northern analysis, RNase protection assays (RPA), microarravs and PCR- based techniques, such as quantitative PCR and differential di play PCR. For example. Northern blotting 'involves running a preparation of RNA on a denaturing agarose gel, and transferring it to a suitable support, such as activated cellulose, nitrocellulose or glass or nylon membranes. Radiolabeled cD A or RNA is then hybridized to the preparation,, washed and analyzed by autoradiography.
In situ hybridization visualization may also be employed, wherein a radioaetively labeled ami sense RNA probe is hybridized with a thin section of a biopsy sample, washed, cleaved with RNase and exposed to a sensitive emulsion for autoradiography. The samples may be stained with hematoxylin to demonstrate the histological composition of the sample, and dark field imaging with a suitable light filter shows the developed emulsion. Non-radioactive labels such as digoxigenin may also be used.
Alternatively, mRNA expression can be detected on a DNA array, chip or a microarray. Labeled nucleic acids of a test sample obtained from subject may be hybridized to a solid surface comprising biomarker DNA. Positive hybridization signal is obtained with the sample containing biomarker transcripts. Methods of preparing DNA arrays and their use are well known in the art (see, e.g., U.S. Pat. Nos: 6, 18, 796;
6,379,897; 6,664,377; 6,451 ,536; 548,257; U.S. 20030157485 and Schena et at (1995) Science 20, 467-470; Gerhold et al. (1 99) J'rends In Biochem. Set 24, .168-173; and Lennon et al. (2000) Drug Discovery Today 5, 59-65, which are herein incorporated by reference in their entirety). Serial Analysis of Gene Expression (SAGE) can also be performed (See for example U.S. Patent Application 20030215858).
To monitor mRNA levels, for example, raRNA is extracted from the biological sample to be tested, reverse transcribed, and fiuorescentiy-labeled cDNA probes are generated. The microarravs capable of hybridizing to marker cDNA are then probed with the labeled cDNA probes, the slides scanned and fluorescence intensity measured. This intensity correlates with the hybridization intensity and expression levels.
Types of probes that can be used in the methods described herein include cDNA, riboprobes, synthetic oligonucleotides and genomic probes. The type of probe used will generally be dictated by the particular situation, such as riboprobes for in siiu hybridization, and cDNA for Northern blotting, for example. I one embodiment, the probe is directed to nucleotide regions unique to the RN A. The probes ma be as short as is required to differentially recognize marker mRNA transcripts, and may be as short as, for example, 15 bases: however, probes of at least ! 7„ 18, 19 or 20 or more bases can be used. In one embodiment, the primers and probes hybridize specifically under stringent conditions to a DNA fragment having the nucleotide sequence corresponding to the marker. As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% identify in nucleotide sequences, in another embodiment, hybridization under
"stringent conditions" occurs when there is at least 97% identity between the sequences.
The form of labeling of the probes may be any that is appropriate, such as the use of radioisotopes, for example, J~P and *,5S. Labeling with radioisotopes may be achieved, whether the probe is synthesized chemically or biologically, by the use of sui tably labeled bases.
In one embodiment, the biological sample contains polypeptide molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.
In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting marker polypeptide, mRNA, genomic DNA, or fragments thereof, such that the presence of the marker polypeptide, mRNA, genomic DNA, or fragments thereof, is detected in the biological sample, and comparing the presence of the marker polypeptide, mRNA, genomic DNA, or fragments thereof, in the control sample with the presence of the marker polypeptide, mRNA, genomic DNA, or fragments thereof in the test sample,
c. Methods for Detection of Biomarker Protein Expression
The activity or level of a biomarker protein can be detected and/or quantified by detecting or quantifying the expressed polypeptide. The polypeptide can be detected and quantified by any of a number of means well known to those of skill m the art Aberrant levels of polypeptide expression of the polypeptides encoded by a biomarker nucleic acid and functionally similar homologs thereof, including a fragment or genetic alteration thereof (e.g., in regulatory or promoter regions thereof) are associated with the likelihood of response of a cancer to an anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy). Any method known in the art for detecting polypeptides can be used, Such methods include, but are not limited to, immunodiffusion, immunoeiectrophoresis, radioimmunoassay ( IA), enzyme-linked immunosorbent assays (ELlSAs),
immunoflttorescent assays, Western blotting, binder-ltgand assays, immuaohistochcraical techniques, agglutination, complement assays, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography; and the like (e.g., Basic and Clinical Immunology, Sites and Terr, eds., Appleton and Lange, Norwaik, Conn, pp 217-262, 1 1 which is incorporated by reference). Preferred are binder-iigand immunoassay methods including reacting antibodies with an epitope or epitopes and competitively displacing a labeled polypeptide or derivative thereof.
For example, ELISA and A procedures may be conducted such that a desired biomarker protein standard is labeled (with a radioisotope such as ' or " ~S, or art assayable enzyme, such as horseradish peroxidase or alkaline phosphatase), and, together with the unlabelled sample, brought into contact with the corresponding antibody, whereon a second antibody is used to bind the first, and radioactivity or the immobilized enzyme assayed (competitive assay). Alternatively, the biomarker protein in the sample is allowed to react with the corresponding immobilized antibody, radioisotope- or enzyme-labeled anti-biomarkcr protcinantibody is allowed to react with die system, and radioactivity or the enzyme assayed (ELiSA-sandwieh assay). Other conventional methods may also be employed as suitable .
The above techniques may be conducted essentially as a "one-step" or "two-step" assay. A "one-step'' assay involves contacting antigen with immobilized antibody and, without washing, contacting the mixture with labeled antibody. .4 "two-step" assay involve washing before contacting, the mixture with labeled antibody. Other conventional method may also be employed as suitable.
in one embodiment, a method for measuring biomarker protein levels comprises the steps of: contacting a biological specimen with an antibody or variant (e.g., fragme t) thereof which selectively binds the biomarker protein, and detecting whether said antibody or variant thereof is bound to said sample and thereby measuring the levels of the biomarker protein.
Enzymatic and radiolabeling of biomarker protein and/or the antibodies may be effected by conventional means. Such means will generally include covaiest linking of the enzyme to the antigen or the antibody in question, such as by g!utara!dehyde, specifically so as not to adversely affect the activity of the enzyme, by which, is meant that the enzyme must still be capable of interacting with its substrate, although it is not necessary for all of the enzyme to be active, provided that enough remains active to permit the assay to be effected. Indeed, some techniques for binding enzyme are non-specific (such as using formaldehyde), and will only y ield a proportion of active enzyme. it is usually desirable to immobilize one component of the assay system: on a support, thereby allowing other components of the system to be brought into contact with the component and readily removed without laborious and time-consuxnvng labor. It is possible for a second phase to be immobilized away from the first, but one phase is usually sufficient,
it is possible to immobilize the enzyme itself on a support, but if sol id-phase enzyme is required, then this is generally best achieved by binding to antibody and affixing the antibody to a support, models and systems for which are well-known in the art. Simple polyethylene may provide a suitable support.
Enzymes employable for labeling are not particularly limited, but may be selected from the members of the oxidase group, for example. These catalyze production of hydrogen peroxide by reaction with their substrates, and glucose oxidase is often used for its good stability, ease of availabiiitv and cheapness, as well as the ready availabiiitv of its substrate (glucose). Activity of the oxidase may be assayed by measuring the concentration of hydrogen peroxide formed after reaction of the enzyme-labeled antibody with the substrate under controlled conditions well-known in the art
Other techniques ma be used to detect bioraarker protein according to a practitioner's preference based upon the present disclosure. One such technique is Western blotting (Towbin et at., Proe. Nat, Acad. Sci, 76:4350 (1979)), wherein a suitably treated sample is run on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose filter, Aoti-biomarket protein antibodies (unlabeled) are then brought into contact with the support and assayed by a secondary immunological reagent, such as labeled protein A or anti-iniraitnoglobulin (suitable labels including i2' l. horseradish peroxidase and alkaline phosphatase). Chromatographic detection may also be used.
immunohistochemistry may be used to detect expression of btomarker protein, e.g., in a biopsy sample. A suitable antibody is brought into contact with, for example, a thin layer of cells, washed, and then contacted with a second, labeled antibody. Labeling ma be by fluorescent markers, enzymes, such as peroxidase, avidin, or radioiabeliing. The assay is scored visually, using microscopy.
Anti-biomarker protein antibodies, such as intrabodies, may also be used for imaging purposes, for example, to detect: the presence of btomarker protein in cells and tissues of a subject. Suitable labels include radioisotopes, iodine ( 5I, !J !i), carbon (i C.I, sulphur ( > }. tritium ( *H), indium (u¾n), and technetium ( 'vm7c), fluorescent labels, such as fluorescein and rhodamine, and biotin.
For in viva imaging purposes, antibodies are not detectable, as such, from outside the body, and so must be labeled. Of otherwise modified, to permit detection. Markers for this purpose may be any that do not substantially interfere with the antibody binding, but which allow externa! detection. Suitable markers may include those that may be detected by X-radiography, NMR or MR!. For X-radiographie techniques, suitable markers include any radioisotope that emits detectable radiation but that is not overtly harmful to the subject, such as barium or cesium, for example. Suitable markers for NMR and MR! generally include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by suitable labeling of nutrients for the relevant hybridoma, for example.
The size of the subject, and the imaging system used, will determine the quantity of imaging moiety needed to produce diagnostic images, in the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of technetium- 9. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain biomarker protein. The labeled antibody or antibody fragment can then be detected using known techniques.
Antibodies that may be used to detect biomarker protein include any antibody, whether natural or synthetic, full length or a fragment thereof, monoclonal or polyclonal, that bind sufficiently strongly and specifically to the biomarker protein to be detected. An antibody may have a ¾ of at most about 10* M. ΚΓ M, 10'8 M, ]( u, H)"!0 M, ΗΤ Μ, or JO'^M. The phrase "specifically binds" refers to binding of, for example, an antibody to an epitope or antigen or antigenic determinant in such a manner that binding can be clispiaeed or competed with a second preparation of identical or similar epitope, antigen or antigenic determinant. An antibody may bind preferentially to the biomarker protein relative to other proteins, such as related proteins.
Antibodies are commercially available or may be prepared according to methods known in the art.
Antibodies and derivatives thereof that may be used encompass polyclonal or monoclonal antibodies, chimeric, human, humanized, primatized (CDR -grafted), veneered or single-chain antibodies as well as functional fragments, i.e., biomarker protein binding fragments, of antibodies. For example, antibody fragments capable of binding to a biomarker protein or portions thereof, including, but not limited to, Fv, Fab, Fab' and F(ab') 2 fragments can be used. Such fragments can be produced b enzymatic cleavage or by recombinant techniques. For example, papain or pepsin cleavage can generate Fab or F(ah') 2 fragments, respectively. Other proteases with the requisite substrate specificity can also be used to generate Fab or F(ab') 2 fragments. Antibodies can also bo produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab') 2 heavy chain portion can be designed to include DNA sequences encoding the€H, domain and hinge region of the heavy chain.
Synthetic and engineered antibodies are described in, e.g., Cabilly et a U.S. Pat. No. 4,816,56? Cabiiiy et aL European Patent No, 0,125,023 Bl ; Boss et aL US, Pat. No. 4,816,397; Boss et a!., European Patent No. 0,120,694 81 ; Neuberger, M. S. et aL WO 86/01533; Neuberger, M. S. et al., European Patent No. 0, 1 4,276 Bl ; Winter, U.S. Pat. No. 5,2.25,539; Winter, European Patent No. 0,239,400 Bl ; Queen ct al, European Patent No. 0451216 Bl ; and Pad!an, E. A. et al, EP 0519596 Al . See also, Newman, , et al, Biotechnology, 10: 1455-1460 { 1 92), regarding primatized antibody, and Ladner et aL U.S. Pat. No. 4,946,778 and Bird, . E. et a!., Science, 242: 423-426 ( 1 88)) regarding single-chain antibodies. Antibodies produced from a library, e.g., phage display library, may also be used.
in some embodiments, agents that specifically bind to a biomarker protein other than antibodies are used, such as peptides. Peptides that specifically bind to a biomarker protein can be identified by any means known in the ait. For example, specific peptide binders of a biomarker protein can be screened for using peptide phage display libraries. d. Methods for Detection of Biomarker Structural Alteration
The following illustrative methods can be used to identify the presence of a structural alteration in a biomarker nucleic acid and/or biomarker polypeptide molecule in order to, for example, identify sequences or agents that affect translation of iron-sulfur cluster biosynthesis-related genes.
in certain embodiments, detection of the alteration involves the use of a
probe/primer in a polymerase chain reaction (PCR) (see, e.g. , U.S. Pat, os, 4,683, 1 5 and 4,683,202), such as anchor PCR or RACE. PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et L (1988) Science 241 : 1077-1080; and akazawa et al. (1994) Proc. Natl. Acad. Set. USA 1 :360-364.), the latter of which can be particularly useful for detecting point mutations in a biomarker nucleic acid such as a biomarker gene (see Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). 'This method can include the steps of collecting a sample of ceils from a subject, isolating nucleic acid (#.g.5 genomic, mR A or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a biomarker gene under conditions such that hybridization and amplification of the biomarker gene (if present) occ urs, and detecting the presence or absence of an amplification product, or defecting the size of the amplification product and comparing the length to a control sample, it is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
Alternative amplification methods include: self sustained sequence replication
(Guatelli, J. C. et al. (.1990) Proc. Natl. Acad. Sci. USA 87: 1 874- 1.878), transcriptional amplification system (Kwoh, D. Y. et al. (.1 89) Proc. Natl. Acad. Sci. USA 86: 1 .1 3-1177)„ Q-Beta Rcplicasc (Lizardi, P. M. et al. (1 88) Bio-Technology 6: 3 1 7), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers,
in an alternative embodiment, mutations in a biomarker nucleic acid from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonuc!eases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Pat, No, 5,498,531 ) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
In other embodiments, genetic mutations in biomarker nucleic acid can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA„ to high density arrays containing hundreds or thousands of oligonucleotide probes (Crontn, M. T, et al. ( 1996) Hum. Mutat 7:244-255; Kozal M. J, et al. ( 1996) Nat. Med,. 2:753-759). For example, biomarker genetic mutations can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin et al (1 96) supra. Briefly, a first hybridization array of probes can be used, to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected . Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene. Such biomarker genetic mutations can be identified in a. variety of contex ts, including, for example, germline and somatic mutations.
In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence biomarker gene and detect mutations by comparing the sequence of the sample biomarker with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxam and Gilbert ( 1977) Pro . Nad Acad. ScL USA 74:560 or Sanger (.1977) . Proc. Natl. Acad Set USA. 74:5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve (1995)
Biotech liiq es 19:448-53), including se uencing by mass spectrometry (see, e.g. , PCX
International Publication No. WO 94/1 101; Cohen el al. (1996) Adv. Chromatogr. 36:127- 1 2; and Griffin et al ( 1 93) Appl. Bi c m. Biot cfmoi 38: 147-159).
Other methods for detecting mutations in a biomarker gene include methods in. which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heterodupiex.es (Myers et al ( 1985) Science 230: 1242). In general, the art technique of "mismatch cleavage" starts by providing heteroduplexes formed by hybridizing (labeled) R A or D A containing the wild-type biomarker sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to base pair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA DNA hybrids treated with SI nuclease to enzymatically digest the mismatched regions. In other
embodiments, either DMA/DMA or R A/D A ditpiexes can be treated with hydroxyiaraine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl. Acad. Set. "USA 85:4397 and Saleeba et al ( 1992) Methods
- no - Enzymoi. 217:286-295. In a preferred embodiment, the control DMA or KNA can be labeled for defection.
in still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DMA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in biomarker cD As obtained from samples of cells. For example, the mutY enzyme of colt cleaves A at G/A mismatches and the thymidine DNA glyeosylase from HeLa ceils cleaves T at G/T mismatches (Hsu et al. (1 94) Carcinogenesis 15: 657-1662). According to an exemplary embodiment, a probe based on a biomarker sequence, e.g. , a wild-type biomarker treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like (e.g., U.S. Pat. No. 5.459.039.) in other embodiments, alterations in eiectrophoretic mobility can be used to identify mutations in biomarker genes. For example, single strand conformation polymorphism iSSCP) may be used to detect differences in eiectrophoretic mobility between mutant and wild type nucleic acids (Orita ei al. (1989) Proe Natl. Acad. Set USA 86:2766; see also
Cotton (i ) MiU t Res. 285: 125-144 and Hayashi (1992) Genet Anal Tech. Appl 9:73- 79), Single-stranded DNA fragments of sample and control biomarker nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in eiectrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather tha DNA), in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject- method 'utilizes heteroduplcx analysis to separate doable stranded heteroduplcx molecules on the basis of changes in eiectrophoretic mobility (Keen et al. (19 1 ) Trends Genet. 7:5).
In yet another embodiment the movement of mutant or wild-type fragments i polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DOGE) (Myers et i. (1 85) Nature 313:495). When DOGE is used as the method of analysis, DNA will be modified to ensure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp o higb- melting GC-rich DNA by PGR, In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobil ity of control and sample DNA (Rosenbaum and Reissner (1987) Bi phys. Chem. 265:12753), Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared, in which the known mutation is placed centrally and then hybridized to target DNA. under conditions which permit hybridization onl if a perfect match is found (Saiki et al. (1 86) Nature 324:163; Saiki et ah ( 1989) Proc Natl. Acad. Set USA 86:6230). Such allele specific oligonucleotides arc hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hy bridizing membrane and hybridized with labeled target DNA.
Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differentia! hybridization) (Gibbs el al. ( 1989) Nucleic Ackk Res'. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibiech 1 1 :238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6: 1 j. it is anticipated that in certain embodiments amplification ma also be performed using Taq ligase for amplification (Barany (1991) Proc, Natl. Acad. Sci USA 88:189). In such cases,, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
3. Anti-Cancer Therapies
The efficacy of anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy) is predicted according to biomarker presence, absence, amount and/or activity associated with a cancer (e.g., cancer) in a subject according to the methods described herein. In one embodiment, such anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy) or combinations of therapies (e.g. , anti-PD- 1 and anti-immunoiub.ibitory- therapies) can be administered to a desired subject or once a subject is indicated as being a likely responder to anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy), in another embodiment, such anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy) can be avoided once a subject is indicated as not being a likely responder to the anti-cancer therapy (e.g., iron- sulfur cluster biosynthesis pathway inhibitory therapy) and an alternative treatment regimen, such as targeted and/or untaxgeted anti-cancer therapies can be administered. Combination therapies are also contemplated and can comprise, for example, one or more chemotherapeutie agents and radiation, one or more chemotherapeutie agents and
immunotherapy, or one or more chemotherapeutie agents, radiation and chemotherapy, each combination of which can be with or without anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy).
The iron-sulfur cluster biosynthesis pathway and exemplary agents useful for inhibiting the iron-sulfur cluster biosynthesis pathway, or other hiomarkers described herein, have been described above.
The term: "targeted therapy" refers to admini tration of agents that selectively interact with a. chose biomolccule to thereby treat cancer. For example, targeted thcrepy regarding the inhibition of immune checkpoint inhibitor is useful in combination with the methods of the present invention. The term "immune checkpoint inhibitor" means a group of molecules on the cell surface of CD * and/or CD8-¾- T ceils that fine-tune immune responses by down-modulating or inhibiting an anti-tumor immune response. Immune checkpoint proteins are well known in the art and include, without limitation, CTLA-4, Pi 1, VISTA, 87-112, B7-H3, PD-Ll , Β7-Ϊ Ι4, B7-H6, 2B ICOS, HVEM, PD-L2. CD 160. gp49B, PIR-B, K.1 L famil receptors, TIM- 1 , ΤΪΜ-3, ΤΪΜ-4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7 B7.2, iLT-2, iLT-4, TiGIT, and A2aR (see, for example, WO 2012/177624). Inhibition of one or more immune checkpoint inhibitors can block or otherwise neutralize inhibitory signaling to thereb upregulate an immune response in order to more efficaciously treat cancer.
immunotherapy is one form of targeted therapy that may comprise, for example, the use of cancer vaccines and/or sensitized antigen presenting cells. For example, an oncolytic virus is a virus that is able to infect and lyse cancer cells, while leaving normal cells unharmed, making them potentially useful in cancer therapy. Replication of oncolytic viruses both facilitates tumor cell destruction and also produces dose amplification at the tumor site. They may also act as vectors for anticancer genes, allowing them to be
specifically deli vered to the tumor site. The mununotherapy can involve passi ve immunity for short-term protection of a host, achieved by the administration of pre-formed antibody directed against a cancer antigen or disease antigen {<?.#., administration of a monoclonal antibody, optionally linked to a chemotherapeutie agent or toxin, to a tumor antigen). For example, anti-VEGF and raTOR inhibitors are .known to be effective in treating renal cell carcinoma. Immunotherapy can also focus on using the cytotoxic lymphocyte-recognized epitopes of cancer ceil lines. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides and the like, can be used to selectively modulate biomolecules that are linked to the initiation, progression, and/or pathology of a rumor or cancer.
The term "nn targeted therapy" referes to admi nistration of agents that do not selectively interact with a chosen biomolccule yet treat cancer. Representative examples of untargeted therapies include, without limitation, chemotherapy, gene therapy, and radiation therapy.
in one embodiment, mitochondrial coiactor therapy is useful For example, vitamin E is known to block cell death via ferroptosis such that mitochondrial coiactor therapy can alleviate or improve any toxicity associated with ISC biosynthesis pathway inhibition. Mitochondrial cofaetor therapies are well known in the art and include, for example, coenzyme Q10 (ubiquinone), riboflavin, thiamin, niacin, vitamin K (phylloqulnone and menadione), creatine, carnitine, and other antioxidants such as ascorbic acid and lipoic acid (see, for example, M.an-iage el. al (2003) ,/ Am. Diet. Assoc. 103:1029-1038 and Parik el til. (2009) Curr. Trea Options Neurol. 1 :4.14-430).
in one embodiment, chemotherapy is used. Chemotherapy includes the
administration of a chemotherapeutie agent. Such a chemotherapeutie agent may be, but is not limited to, those selected from among the following groups of compounds: platinum compounds, cytotoxic antibiotics, antimetabolities, an ti -mitotic agents, alkylating agents, arsenic compounds, D A topoisomerase inhibitors, taxanes, nucleoside analogues, plant alkaloids, and toxins; and synthetic derivatives thereof. Exemplary compounds include, but are not limited to, alkylating agents: cispfatin, treosulfan, and trofosfamide; plant, alkaloids: vinblastine, paclitaxel, docetaxol; DNA topoisomerase inhibitors: teniposide, crisnatol, and mitomycin; anti-folates: methotrexate, mycophetiolic acid, and hydroxyurea; pyrimidine analogs: 5-fluorouracil, doxiiliiridine, and cytosine atabinoside; purine analogs:
mercaptopurine and thioguanine; DNA antimetabolites: 2'-deoxy-5-fl uorouridine,
aphidicolin glycinate, and pyrazoioiniidazole; and antimitotic agents: halichondrim colchicine, and rhi oxin. Compositions comprising one or more chemotherapeutie agents (e.g., FLAG, CHOP) may also be used. FLAG comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF, CHOP comprises cyclophosphamide, vincristine, doxorubicin, and prednisone. In another embodiments, PARP (t\g., PARP-l and/or PARP-2) inhibitors are used and such inhibitors arc well known in the art (e.g., Olaparib, ABT-888, BS!-20! , BGP-15 (N-Gene Research Laboratories, inc.); lNO-1 0'i (Inoiek Pharmaceuticals Inc.); PJ34 (Soriano et al., 2001 ; Paeher et ai, 2002b): 3-aminoberizamide (Trevigen); 4-amirto- l ,8-o.aphthalimide; (Trevigen); 6(5H)-phetianthridinone (Trevigen); benzamide (U.S. Pat, Re. 36,397); and Nil 1025 (Bowman et al). The mechanism of action is generally related to the ability of PARP inhibitors to bind PARP and decrease its activity. PARP catalyzes the conversion of .beta.-uicotiuamide adenine diuuc!eotide (NAD+) into nicotinamide and poly-ADP-ribose (PAR). Both poly (ADP-ribose) and PARP have been linked to regulation of transcription, cell proliferation, genomic stability, and carcinogenesis (Bouchard V. J. et.al. Experimental Hematology, Volume 31, Number 6, June 2003, pp. 446-454(9); Hereeg 2.; Wang Z.-Q, Mutation Research/Fundamental and Molecular
Mechanisms of Mutagenesis, Volume 477, Number 1, 2 jun, 2001 , pp. 97-1 10(14)).
Poly(ADP-ribose) polymerase i (PARP!) is a key molecule in the repair of DNA single- strand breaks (SSBs.) (de Murcia J. et al. 1997. Proc Nail Acad Sci USA 94:7303-7307; Schreiber V, Danteer F, Ame J C de Murcia G (2006) Nat Rev Mol Ceil Biol 7:517-528; Wang Z Q, et al. ( 1997) Genes Dev 1 1 :2347-2 58 . Knockout of SSB repair by inhibition of PARPl function induces DNA double-strand breaks (DSBs) that can trigger synthetic lethality in cancer cells with defective homology-directed DSB repair (Bryant H E„ et al. (2005) Nature 434:913-917; Fanner H, et al. (2005) Nature 434:917-921). The foregoing examples of ehemotherapeittie agents are illustrative, and are not intended to be limiting.
In another embodiment, radiation therap is used. The radiation used in radiation therapy can be ionizing radiation. Radiation therapy can also be gamma rays. X-rays, or proton beams. .Examples of radiation therapy include, but are not limited to, external-beam radiation therapy, interstitial implantation of radioisotopes (1-125, palladium, iridium), radioisotopes such as strontium-89f thoracic radiation therapy, intraperitoneal P-32 radiatton therapy, and/or total abdominal and pelvic radiation therapy. For a general overview of radiation therapy, see He!imaa, Chapter 1 : Principles of Cancer Management: Radiation Therapy, 6th edition, 2001, DeVita et ai., eds., J. B. Lippencott Company, Philadelphia. The radiation therapy can be administered as external beam radiation or teletherapy wherein the radiation is directed from a remote source. The radiation treatment can aiso be administered as internal therapy or brachytherapy wherein a radioactive source is placed inside the body close to cancer cells or a tumor mass. Also encompassed is the use of photodynamic therapy comprising the administration of photosensitizers, such as hematoporphyrin and its derivatives, Vertoporfrn (BPD- A), phthalocyanine,
photosensitizer Pc4, demethoxy-'hypocrellin A; and 2BA-2-DMHA.
in another embodiment, hormone therapy is used. Hormonal therapeutic treatments can comprise, for example, 'hormonal agonists, ormonal antagonists (e.g- , flutanride, bicaiutamide, tamoxifen, raloxifene, leupro!icle acetate (LUPR.ON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, and steroids (e.g. , dexamethasone, retinoids, deltoids, betamethasone, Cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorttcoids, estrogen, testosterone, progestins), vitamin A derivatives (e.g., all-trans retinoic acid (ATRA)); vitamin 1)3 analogs; antigestagens (e.g., mifepristone, onapristone), or antiandrogens i o.g. t cyproterone acetate).
in another embodiment, hyperthermia, a procedure in which body tissue is exposed to high temperatures (up to i OtV .) is used. Heat may help shrink tumors by damaging cells or depriving them of substances they need to live. Hyperthermia therapy cart be local, regional, arid whole-body hyperthermia, using external and internal heating devices.
Hyperthermia is almost always used with other forms of therapy (e.g., radiation therapy, chemotherapy, and biological therapy) to try to increase their effectiveness. Local hyperthermia refers to heat that is applied to a very small area, such as a tumor. The area ma be heated externally with high-frequency waves aimed at a tumor from a device outside the body. To achieve internal heating, one of several types of sterile probes may be used, including thin, heated wires or hollow tubes filled with warm water; implanted microwave antennae; and radiofrequency electrodes. In regional hyperthermia, an organ or a !imb is heated. Magnets and devices that produce high energy are placed over the region to be heated, in another approach, called perfusion, some of the patient's blood is removed- heated, and then pumped, (perfused) into the region that is to be heated internally. Whole- body heating is used to treat metastatic cancer that has spread throughout the body. It can be accomplished using warm-water blankets, hot wax, inductive coils (like those in electric biatikets), or thermal chambers (similar to large incubators). Hyperthermia does not cause any marked, increase in radiation side effects or complications. Heat applied directly to the skin, however, can cause discomfort or e ven signi ficant local pain in about half the patients treated, it can also cause blisters, which generally heal rapidly . i still another embodiment, photodynamic therapy (also called PDT, photoradiation therapy, phototherapy, or photochemotherapy) i used for the treatment of some types of cancer. It is based on the discovery that certain chemicals known as photosensitizing agents can kill one-celled organisms when the organisms are exposed to a particular type of light. PDT destroys cancer cells through the use of a fixed- frequency laser light in combination with a photosensitizing agent, in PDT, the photosensitizing ag nt is injected into the bloodstream and absorbed by cells all over the body. The agent remains in cancer ceils for a longer time than it does in normal cells. When the treated cancer cells are exposed to laser f ght, the photosensitizin agent absorbs the light and produces an acti e form of oxygen thai destroys the treated cancer ceils. Light exposure must be timed carefully so that it occurs when most of the photosensitizing agent has left healthy cells but is still present in the cancer cells. The laser light used in PDT can be directed through a fiberoptic (a very thin glass strand). The fiber-optic is placed close to the cancer to deliver the proper amount of light The fiber-optic can be directed through a bronchoscope into the lungs for the treatment of lung cancer or through an endoscope into the esophagus for the treatment of esophageal cancer. An advantage of PDT is that it causes minima] damage to healthy tissue. However, because the laser light currently in use cannot pass through more than about 3 centimeters of tissue (a l ittle more than one and an eighth inch), PDT is mainly used to treat tumors on or just under the skin or on the lining of internal organs.
Photodynamic therapy makes the skin and eyes sensitive to light for 6 weeks or more after treatment. Patients are advised to avoid direct sunlight and bright indoor light for at least 6 weeks. If patients must go outdoors, they need to wear protective clothing, including sunglasses. Other temporary side effects of PDT are related to the treatment of specific areas and can include coughing, trouble swallowing, abdominal pain, and painful breathing or shortness of breath. In December 1 95, the U.S. Food and Drug Administration (FD A) approved a photosensitizing agent called porftmer sodium, or PhotofriniK), to relieve symptoms of esophageal cancer that is causing an obstruction and for esophageal cancer thai cannot be satisfactorily treated with lasers alone. In January 1998, the FDA approved porfimer sodium for the treatment of early aonsmall ceil long cancer in patients for whom. the usual treatments for lung cancer are not appropriate. The National Cancer Institute and other institutions are supporting clinical trials (research studies) to evaluate the use of photodynamic therapy for several types of cancer, including cancers of the bladder, brain, larynx, and oral cavity. in yet another embodiment laser therapy is used to harness high-intensity light to destroy cancer cells. This technique is often used to relieve symptoms of cancer such as bleeding or obstruction, cspecialiv when the cancer cannot be cured by other trcatnicnis. It may also be used to treat cancer by shrinking or destroying tumors. The term "laser" stands for light amplification by stimulated emission of radiation. Ordinary light, such as that from a light bulb, has many wavelengths arid spreads in ail directions. Laser light, on the other hand, has a specific wavelength arid is focused in a narrow beam. This type of high- intensity light contains a lot of energy. Lasers are very powerful and may be used to cut through steel or to shape diamonds. Lasers also can be used for very precise surgical work, such as repairing a damaged retina in the eye or cutting through tissu e (in place of a scalpel}. Although there are several different kinds of lasers, only three kinds have gained wide use in medicine: Carbon dioxide (CO;) iaser-This type of laser can remove thin layers from the skin's surface without penetrating me deeper layers. This technique is particularly useful in treating tumors that have not spread deep into the skin and certain precancerous conditions. As an alternative to traditional scalpel surgery, the CO> laser is also able to cut the skin. The laser is used in this way to remove skin cancers.
eodytmum:yttriuin-aluminiu»--garnet (Nd:YAG) laser— Light from this laser can penetrate deeper into tissue than light from the other types of lasers, and it can cause blood to clot quickly. It can be carried through optical fibers to less accessible parts of the body. This type of laser is sometimes used to treat throat cancers. Argon laser- This laser can pass through only superficial layers of tissue and is therefore useful in dermatology and in eye surgery. It also is used with light-sensitive dyes to treat tumors in a procedure known as phoiodynamic therapy (PDT). Lasers have several advantages over standard surgical tools, including: Lasers are more precise than scalpels. Tissue near an incision is protected, since there is little contact with surrounding skin or other tissue. The hear produced by lasers sterilizes the surgery site, thus reducing the risk of infection. Less operating time may be needed because the precision of the laser allows for a smaller incision. Healing time is often shortened; since laser heat seals blood vessels, there is less bleeding, swelling, or scarring. Laser surgery may be less complicated. For example, with fiber optics, laser light can be directed to parts of the body without making a large incision. More procedures may¬ be done on an outpatient basis. Lasers can be used in two ways to teat cancer; by
shrinking or destroying a tumor with heat, or by activating a chemical—known as a photosensitizing agent~~that destroys cancer ceils, in PDT, a photosensitizing agent is retained in cancer cells and can be stimulated by light to cause a reaction that kills cancer cells. CO? and d:YAG lasers are used to shrink or destroy tumors. They may be used with endoscopes, tubes that allow physicians to see into certain areas of the body, such as the bladder. The light from some lasers cm be transmitted through a flexible endoscope fitted with fiber optics. This allows physicians to see and work in parts of the body that could not otherwise be reached except by surgery and therefore allows very precise aiming of the la ser beam. Lasers also may be used with low-power microscopes, giving the doctor a clear view of the site being treated. Used with other instruments, laser systems can produce a c utting area as small a 200 microns in diameter— less than the width of a very fine thread. Lasers are used to treat many types of cancer. Laser surgery is a standard treatment for certain stages of glottis (vocal cord), cervical, skin, lung, vaginal, vulvar, and penile cancers. In addition to its use to destroy the cancer, laser surgery is also used to help relieve symptoms caused by cancer (palliative care). For example, lasers may be used to shrink or destroy a tumor that is blocking a patient's trachea (windpipe), making it easier to breathe. It is also sometimes used for palliation in colorectal and anal cancer. Laser- induced interstitial thermotherapy (LOT) is one of the most recent developments in laser therapy. LOT uses the same idea as a cancer treatment called hyperthermia; that heat may help shrink tumors by damaging cells or depriving them of substances they need to live, in this treatment, lasers are directed to interstitial areas (areas between organs) in the body. The laser light then raises the temperature of the tumor, which damages or destroys cancer cells.
The duration and/or dose of treatment with anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy) may vary according to the particular iron- sulfur cluster biosynthesis pathway inhibitor agent or combination thereof. An appropriate treatment time for particular cancer therapeutic agent will be appreciated by the skilled artisan. The invention contemplates the continued assessment of optimal treatment schedules for each cancer therapeutic agent, where the phenotype of the cancer of the subject as determined b the methods of the invention is a factor in determining optimal treatment doses and schedules.
Any means for the introduction of a polynucleotide into mammals, human or non- human, or cells thereof may be adapted to the practice of this invention for the deli very of the various constructs of the invention into the intended recipient. In one embodiment of the invention, the D A constructs are delivered to ceils by transfection, i.e.f by delivery of '"naked" DNA or in a complex with a colloidal dispersion system. A colloidal system includes macromolecule complexes, nanocapsules, microspheres, beads, and lipkl-based systems including oil-in- water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a lipid-eoniplexed or Uposome-formulated DNA. in the former approach, prior to formulation of DNA, e.g., with lipid, a plasmid containing a transgeoe bearing the desired D A constructs may first be experimentally optimized for expression (e.g.t inclusion of an intron in the 5' untranslated region and elimination of unnecessary sequences (Feigner, et al, Ann NY Acad Sci 126-139, 1995). Formulation of DNA, e.g. with various lipid or liposome materials, may then be effected using known methods and materials and delivered to the recipient mammal. See, e.g., Canonieo et ai. Am J Respir Cell Mo I Biol 10:24-29, 1994; Tsan et al, Am J Physiol 268; Alton et al., Nat Genet. 5: 1.35-1 2, 1.993 and U.S. patent No. 5,679,647 by Carson et al.
The targeting of l iposomes can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ- specific, cell-specific, and organelie-speeifie. Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes die natural tendency of liposomes to distribute io ceils of the reticuloendothelial s stem (RES) in organs, which contain sinusoidal capillaries. Active targeting, on the other hand, involves alteration of the liposome by coupling die liposome to a specific iigand such as a monoclonal antibody, sugar, glycoiipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
The surface of die targeted deli very system may be modified in a variety of ways. In the ease of a liposomal targeted delivery system, lipid groups can be incorporated into the lipid bi ayer of the liposome in order to maintain the targeting Iigand in stable association with the liposomal bilayer. Various linking groups can be used for joining the lipid chains to the targeting iigand. Naked DNA or DNA associated with a delivery vehicle, e.g., liposomes, can be administered to several sites in a subject, (see below).
Nucleic acids can be delivered in an desired vector. These include viral or non- viral vectors, including adenovirus vectors, adeno-associatcd virus vectors, .retrovirus vectors, lenti virus vectors, and plasmid vectors. Exemplary types of viruses include HSV (herpes simplex, virus), AAV (adeno associated virus), HIV (human immunodeficiency virus), BIV (bovine immunodeficiency virus), and MLV (murine leukemia virus), Nucieic
- 1.20 - acids can be administered in any desired format that provides sufficiently efficietu delivers' levels, including in virus particles, in liposomes, in nanoparticles, and comp!exed to polymers.
The nucleic acids encoding a protein or nucleic acid of interest may be in a piasmi or viral vector, or other vector as is known in the art. Such vectors are well known and an can be selected for a pariiculat" application. In one embodiment of the invention, the gene delivery vehicle comprises a promoter and a deinethylase coding sequence. Preferred promoters are tissue-specific promoters and promoters which are activated by cellular proliferation, such as the thymidine kinase and thymidilate synthase promoters. Other preferred promoters include promoters which are activatable by infection with a virus, such as the a- and β-iuterferon promoters, and promoters which are activatable by a hormone, such as estrogen. Other promoter which can be used include the Moloney virus LTR, the CMV promoter, and the mouse albumin promoter, A promoter may be constitutive or inducible.
in another embodiment, naked polynucleotide molecules are used as gene delivery vehicles, as described in WO 90/1 1092 and U.S. Patent 5,580,859. Such gene delivery vehicles can be either growth factor DNA or RNA and, in certain embodiments, are linked to killed adenovirus. Curie! et al. Hum. Gene. Ther. 3: 147- 154, 1992. Other vehicles which can optionally be used include D A-hgand (Wit et al., J. Biol Chem.
264:16985- 16987, 1989), hpid-DNA combinations (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413 7417, 1 89), liposomes (Wang et ai„ Proc, Natl. Acad. Sci. 84:7851-7855, 1987) and mieroprojeetiles (Williams et al., Proc. Natl. Acad. Sci. 88:2726-2730, 1991).
A gene delivery vehicle can optionally comprise viral sequences such as a viral ori gin of replication or packaging signal These viral sequences can be selected from viruses such as asirovirus, eoronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornaviats, poxvirus, retrovirus, togavirus or adenovirus. In a preferred embodiment the growth factor gene delivery vehicle is a recombinant retroviral vector. Recombinant retroviruses and various uses thereof have been described in numerous references including, for example, Mann et al., Ceil 33: 153, 1983, Cane and Mulligan, Proc. Natl Acad. Sci. USA 81 :6349, 1.984, Miller et a!., Human Gene Therapy 1 :5-14, 1990, U.S. Patent Nos. 4,405,712, 4,861,719, and 4,980,289, and PCT Application os. WO 89/02,468, WO 89/05,349, and WO 90/02,806. Numerous retroviral gene delivery vehicles can be utilized in the present invention, including for example those described in EP 0,415,731 ; WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Patent No. 5,219,740; WO 931 1 230; WO 9310218; Vile and Hart, Cancer Res. 53:3860-3864. 1 93; Vile and Hart, Cancer Res. 53:962-967, 1 93; Ram et al. Cancer Res. 53:83-88, 1 93; Takamiya et al., J. Neurosci. Res. 33:493-503, 1992; Baba et al., J. Neurosurg.
79:729-735. 1993 (U.S. Patent No. 4,777,127, OB 2,200,651, EP 0,345,242 and
W0 1/02805).
Other viral vector systems that can be used to deliver a polynucleotide of the invention have been derived from herpes vims, e,g., Herpes Simplex Vims (U.S. Patent No. 5,631 ,236 by Woo et al, issued May 20, 1 97 and WO 00/08191 by Neurovex), vaccinia virus (Rklgcway (i 988) Ridge ay, "Mammalian expression vectors," In; Rodriguez R L, Denhardt I) T, ed. Vectors: A survey of molecular cloning vectors and their uses.
Stonehara; Butterworih,; Baichwal and Sugden (1986) "Vectors for gene transfer derived from animal DNA viruses: Transient and stable expression of transferred genes," in:
Kuchcrfapati R, ed. Gene transfer. New York; Plenum Press; Coupar et al. (1988) Gene, 68: 1 -10), and several RNA viruses. Preferred viruses include an aiphavirus, a poxivirus, n arena virus, a vaccinia virus, a polio virus, and the like. They offer several attractive features for various mammalian cells (Friedmann (1989) Science, 244: 1275- 1281;
Ridgeway, 1 88, supra; Baichwal and Sugden, 1986, supra; Coupar et at, 1988; Horwich et. al.(1990) J.Virol., 64:642-650).
in other embodiments, target DNA in the genome can be manipulated using well- known methods in the art. For example, the target DNA in the genome can be manipulated by deletion, insertion, and/or mutation are retroviral insertion, artificial chromosome techniques, gene insertion, random insertion with tissue specific promoters, gene targeting, transposabie elements and/or any other method for introducing foreign DNA or producing modified DNA/modified nuclear D A. Other modification techniques include deleting DNA sequences from a genome and/or altering nuclear DNA sequences. Nuclear DNA sequences, for example, may be altered by site-directed mutagenesis.
In other embodiments, recombinant biomarkcr polypeptides, and fragments thereof can be administered to subjects, in sotne embodiments, fusion proteins can be constructed and administered which have enhanced biological properties. In addition, the biomarker polypeptides, and fragment thereof, can be modified according to well-known
pharmacological methods in the art (e.g., pegylation, glycosylation, oiigotnertzation, etc.) in order to further enhance desirable biological activities, such as increased bioavaiiabiiity aad decreased proteolytic degradation.
4. (.' lineal Efficacy
Clinical efficacy can be measured b any method .known in the art. For example, the response to an aoti-eaoeer therapy (e.g. , iron-sulfur cluster biosynthesis pathway inhibitory therapy), relates to any response of the cancer, e.g., a tumor, to the therapy, preferably to a change in tumor mass and/or volume after initiation of neoadjuvant or adjuvant chemotherapy. Tumor response may be assessed in a neoadjuvant or adj van situation where the size of a tumor after systemic intervention can be compared to the initial size and dimensions as measured by CT, PET, mammogram, ultrasound or palpation and the ceilitlarity of a tumor can be estimated histologically and compared to the cellularuy of a tumor biopsy taken before initiation of treatment. Response may also be assessed by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. Response may be recorded in a quantitative fashion like percentage change in tumor volume or cellularity or using a semi-quantitative scoring system such as residual cancer burden (Syraroans ei i, J. Clin. Oncol. (2007) 25:4414-4422) or Miller-Payne score (Ogston et al, (2003) Breast (Edinburgh, Scotland) 12:320-32?) in a qualitative fashion, like ""pathological complete response" (pCR), "clinical complete remission" (cCR).
""clinical partial remission" (cPR), "clinical stable disease" (eSD), "clinical progressive disease" (cP'D) or other qual itative criteria. Assessment of tumor response may be
performed early after the onset of neoadjuvant or adjuvant therapy, e.g., after a few hours, days, weeks or preferabiv after a few months. A typical endpoint for response assessment is upon termination of neoadjuvant chemotherapy or upon surgical removal of residual tumor cells and/or the rumor bed.
In some embodiments, clinical efficacy of the therapeutic treatments described herein may be determined by measuring the clinical benefit rate (CBR). The clinical benefit rate is measured by determining the sum of the percentage of patients who are m complete remission (CR), the number of patients who are in partial remission (PR) and the number of patients having stable disease (SO) at a time point at least 6 months out from the cod of therapy. The shorthand .for this formula is CBR-CR-i-PR- SD ov er 6 months. In some embodiments, the CBR for a particular tron-sulfur cluster biosynthesis pathway inhibitor therapeutic regimen is at least 25%, 30%, 35%„ 40%, 45%, 50%„ 55%, 60%, 65%„ 70%, 75%. 80%, 85%, or more.
Additional criteria for evaluating the response to anti-cancer therapy (e.g., iron- sulfur cluster biosynthesis pathway inhibitory therapy) are related to "survival;' which includes all of the following: sitrvivai until mortality, also known as overall survival
(wherein said mortality may be either irrespective of cause or rumor related); "recurrence- free survival" (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith). The length of said survival may be calculated by reference to a defined start point (e.g., time of diagnosis or start of treatment) and end point (e.g., death, recurrence or metastasis). In addition, criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.
For example, in order to determine appropriate threshold values, a particular iron- sulfur cluster biosynthesis pathway inhibitor therapeutic regimen can be administered to a populatio of subjects and the outcome can be correlated to biomarker measurements that were determined prior to administration of any anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy). The outcome measurement may be pathologic response to therapy given in the neoadjuvant setting. Alternatively, outcome measures, such as overall sitrvivai and disease-free survival can be monitored over a period of time for subjects following anti-cancer therapy (e.g.. iron-sulfur cluster biosynthesis pathway inhibitors' therapy) for whom biomarker measurement values are known. In certain embodiments, the same doses of iron-su!fur cluster biosynthesis pathwa inhibitor agents are administered to each subject, in related embodiments, the doses administered are standard doses known in the ait for iron-sulfur cluster biosynthesis pathway inhibitor agents. The period of time for which subjects are monitored can vary. For example, subjects ma be monitored for at least 2, 4, 6, 8, 10, 12f 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months. Biomarker measurement threshold values that correlate to outcome of an anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy) can be determined using methods such as those described in the Examples section. 5- 1 rthcr Uses and Methods of the Present Invention
The compositions described herein can be used in a variety of diagnostic, prognostic, and therapeutic applications regarding hiomarkers described herein, such as those listed in 1 able .5 .
a. Screening Methods
One aspect of the present invention relates to screening assays, including non-cell based assays. In one embodiment, the assays provide a method for identifying whether a cancer is likely to respond to anti-cancer therapy (e.g. , iron-sulfur cluster biosynthesis pathway inhibitory therapy) and/or whether an agent cars inhibit the growth of or kill a cancer cell that is unlikely to respond to anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitor}- therapy).
in one embodiment, the invention relates to assays for screening test agents which bind to, or modulate the biological activity of, at least one biomarker listed in Table 1. m one embodiment, a method for identifying such an agent entails determining the ability of the agent to modulate, e.g. inhibit, the at least one biomarker fisted in Table 1.
In one embodiment, an assay is a ceil-free or cell-based assay, comprising
contacting at ieast one biomarker listed in Table 1 , with a test agent, and determining the ability of the test agent to modulate (e.g. inhibit) the enzymatic activity of the biomarker, such as by measuring direct binding of substrates or by measuring indirect -parameters as described below.
In another embodiment, an assay is a cell-free or cell-based assay, comprising contacting at least one biomarker listed in Table 1 , with a test agent, and determining the ability of the test agent to modulate the ability of the biomarker to regulate NFS I or other iron-sulfur cluster biosynthesis pathway member, such as by measuring direct binding of substrates or by measuring indirect parameters as described below.
For example, in a direct binding assay, biomarker protein for their respective target polypeptides or molecules) can be coupled with a radioisotope or enzymatic label such that binding can be determined by detecting the labeled protein or molecule in a complex. For example, the targets can be labeled with % ^S, C, or JH, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, the targets can be enzymatieally labeled with, for example, horseradish peroxidase, alkaline phosphatase, or hiciferase, and the enzymatic label detected by determination of con version of an appropriate substrate to product. Determining the interaction between biomarker and substrate can also be accomplished using standard binding or enzymatic analysis assays. In one or more embodiments of the abo ve described assay methods, it may be desirable to immobilize polypeptides or molecules to facilitate separation of eompiexed from uneomplexed forms of one or both of die proteins or molecules, as well as to accommodate automation of the assay.
Binding of a test agent to a target can be accompli shed in any vessel suitable for containing the reaetants. Non-limiting examples of such vessels include microliter plates, test tubes, and micro-centrifuge tubes, immobilized forms of the antibodies of the present invention can also include antibodies bound to a solid phase like a porous, microporous (with an average pore diameter less than about one micron) or macroporous (with an average pore diameter of snore than about 10 microns) material, such as a membrane, cellulose, nitrocellulose, or glass fibers; a bead, such as that made of agarose or
polyacrylamide or latex; or a surface of a dish, plate, or well, such as one made of polystyrene,
in an alternative embodiment, determining the ability of the agent to modulate the interactio between the biomarker and its natural binding partner can be accomplished by determining the ability of the test agen t to modulate the activity of a polypeptide or other product that functions downstream or upstream of its position within die tron-snlfur chtster biosynthesis pathway.
The present invention further pertains to novel agents identified by the above- described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an antibody identified as described herein can be used in an animal model to determine the mechanism of ac tion of such an agent,
b. Predictive Medicine
The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylaetieally. Accordingly, one aspect of the present invention relates to diagnostic assays for determining the presence, absence, amount, and/or activity level of a biomarker described herein, such as those listed in Table 1, in the context of a biological sample (e.g., blood, serum,, cells, or ti sue) to thereby determine whether an individual afflicted with a cancer is likely to respond to anticancer therapy (eg., iron-sulfur cluster biosynthesis pathway inhibitory therapy }, whether in an original or recurrent cancer. Such assays can be used for prognostic or predictive purpose to thereby prophylactieaily treat an individual prior to the onset or after recurrence of a disorder characterized by or associated with bioraarker polypeptide, nucleic acid expression or activity. The skilled artisan will appreciate that any method can use one or more (e.g., combinations) of biomarkers described herein, such as those listed in Table 1, Another aspect of the present invention pertains to monitoring the influence of agents (e.g., drugs, compounds, and small nucleic acid-based molecules) on the expression or acti vity of a bioraarker listed in Table 1. These and other agents are described in further detail in the following sections.
The skilled artisan will also appreciate that, in certain embodiments, the methods of the present invention implement a computer program and computer system. For example, a computer program can be used to perform the algorithms described herein, A computer system can also store and manipulate data generated by the methods of the present invention which comprises a pluralit of bioraarker signal changes/profiles which can be used by a computer sy stem in Implementing the methods of this invention. In certain embodiments, a computer system receives biomarker expression data; (ii) stores the data; and (iii) compares the data in any number of ways described herein (e.g., analysis relative to appropriate controls) to determine the state of informative biomarkers from cancerous or pre-eaneerous tissue. In other embodiments, a computer system (i) compares the determined expression biomarker level to a threshold value; and (ii) outputs an indication of whether said biomarker level is significantly modulated (e.g., above or below) the threshold value, or a phenotype based on said indication.
i n certain embodiments, such computer systems are also considered part of the present invention. Numerous types of computer systems can be used to implement the analytic methods of this invention according to knowledge possessed by a skilled artisan in the bioinformatics and/or computer arts. Several software components can be loaded into memory during operation of such a computer system.. The software components can comprise both software components that are standard in the art and components that are special to the present invention (e.g., dCHIP software described in Lin el « . (2004) ioinform tics 20, Ϊ 233- 1240; radial basis machine learning algorithms (RBM) known in the art).
- J 77 - The methods of the invention can also be programmed or modeled in
mathematical software packages that allow symbolic entry of equations and high-level Specification of processing, including specific algorithms to be used, thereby freeing a user of the need to procedurally program individual equations and algorithms. Such packages include, e.g., Matlab from Mathworks ( atiek, Mass.), Mathematics from Wolfram Research (Champaign, ill,) or S-Plus from MathSoft (Seattle, Wash,).
in certain embodiments, the eotnpister comprises a database for storage of biomarker data. Such stored profiles can be accessed and used to perform comparisons of interest at a later point in time. For example, biomarker expression profiles of a sample derived from the non-cancerous tissue of a subject and/or profiles generated from population-based distributions of informative loci of interest in relevant populations of the same species can be stored and later compared to that of a sample derived from the cancerous tissue of the subject or tissue suspected effacing cancerous of the subject,
in addition to the exemplary program structures and computer systems described herein, other, alternative program structures and computer systems will be readily apparent to the skilled artisan. Such alternative systems, which do not depart from the above described computer system and programs structures either in spirit or in scope, are therefore intended to be comprehended within the accompanying claims,
c. Diagnostic Assays
The present invention provides, in part, methods, systems, and code for accurately classifying whether a biological sample is associated with a cancer that is likely to respond to anti-cancer therapy (e,g., iron-sulfur cluster biosynthesis pathway inhibitory therapy). In some embodiments, tie present invention is useful for classifying a sample (e.g., from a subject) as associated with or at risk for responding to or not responding to anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy) using a statistical algorithm and/or empirical data (e.g., the amount or activity of a biomarker listed in Table I ).
An exemplary method for detecting the amount or activity of a biomarker listed in Table 1 , and thus useful for classifying whether a sample is likely or unlikely to respond to anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy) involves obtaining a biological sample from a test subject and contacting the biological sample with an agent, such as a protein-binding agent like an antibod or antigen-binding fragment thereof, or a nucleic acid-binding agent like an oligonucleotide, capable of defecting the amount or activity of the biornarker in the biological sample. In some embodiments, at least one antibody or antigen-binding fragment thereof is used, wherein two, three, four, five, six, seven, eight, nine, ten, or more such antibodies or antibody fragments can be used in combination (e.g., in sandwich BLlSAs) or in serial In certain instances, the statistical algorithm is a single learning statistical classifier system. For example, a single learning statistical classifier system can be used to classify a sample as a based upon a prediction or probability value and the presence or level of the biomarker. The use of a single learning statistical classifier system typically classifies the sample as, for example, a likely anti-cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy) responder or progressor sample with a sensitivity, specificity, positive predictive value, negative predictive value, and/or overall accuracy of at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
Other suitable statistical algorithms are well known to those of skill in the art. For example, learning statistical classifier systems include a machine learning algorithmic technique capable of adapting to complex data sets (e.g., parte! of markers of interest) and making decisions based upon such data sets. Irt some embodiments, a single learning statistical classifier system such as a classification tree (e.g., random forest) is used. In other embodiments, a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more learning statistical classifier systems are used, preferably in tandem. Examples of learning statistical classifier systems include, but are not limited to, those using inductive learning (e.g. ,
decision/classification trees such as random forests, classification and regression trees (C& T), boosted trees, etc.), Probably Approximately Correct (PAC) learning, conneetioi!tist learning (e.g., neural networks (NN), artificial neural networks (ANN), neuro fuzzy networks (NFN), network structures, pereeptrons such as multi-layer pereeptrons, multi-layer feed-forward networks, applications of neural networks, Bayesian learning in belief networks, etc,), reinforcement learning (e.g.,, passive learning in a known environment such, as naive learning, adaptive dynamic learning, and temporal difference learning, passive learning in an unknown environment, active learning in an unknown environment, learning action-value functions, applications of reinforcement learning, etc), and genetic algorithms and evolutionary programming. Other learning statistical classifier systems include support vector machines (e.g. , Kernel methods), multivariate adaptive regression splines (MARS), Levenberg-Marquardt algorithms,, Gauss-Newton algorithms. mixtures of Gaussians, gradient- descent algorithms, and learning vector quantization
(LVQ). In certain embodiments, the method of the present invention further comprises sending the sample classification results to a clinician, e.g., an oncologist
In another embodiment, the diagnosis of a subject is followed by administering to the individual a therapeutically effective amount of a defined treatment based upon the diagnosis.
In one embodiment, the methods further involve obtaining a control biological sample (e.g. , biological sample from a subject who does not have a cancer or whose cancer is susceptible to anti-cancer therapy (eg., iron-sulfur cluster biosynthesis path way inhibitory therapy), a biological sample from the subject during remission, or a biological sample from the subject during treatment for developing a cancer progressing despite anticancer therapy (eg., iron-sulfur cluster biosynthesis pathway inhibitory therapy).
d. Prognostic Assays
The diagnostic methods described herein can ftjrthermore be utilized to identify subjects having or at risk of developing a cancer that is likely or unlikely to be responsive to am -cancer therapy (e.g., iron-sulfur cluster biosynthesis pathway inhibitory therapy). The assays described herein, such as the preceding diagnostic assays or the following assays, can. be utilized to identify1 a subject having or at risk of developing a disorder associated with a misreguiatiion of the amount or acti vity of at least one biomarker described in Table .1„ such as in cancer. Alternatively, the prognostic assays can be utilized to identify a. subject having or at risk for developing a disorder associated with a misregulatiori of the at least one biomarker described in Table 1 , such as in cancer.
Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (eg., an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, or other drag candidate) to treat a disease or disorder associated with the aberrant biomarker expression or activity,
e. Treatment Methods
Another aspect of the invention pertains to methods of modulating the expression or activity of one or more biomarkers described herein {e.g., those listed in Table I and the Examples or fragments thereof,) for therapeutic purposes. The biomarkers of the present invention have been demonstrated to correlate with cancers. Accordingly, the activity and/or expression of the biomarker, as well as the interaction between one or more biomarkers or a fragment thereof and its naiurai binding partners) or a fragments) thereof, can be modulated in order to treat cancers.
Modulatory methods of the invention involve contacting a cell with one or more biomarkers of the invention, including one or more biomarkers of the invention, including one or more biomarkers listed in Tabic i and the Examples or a fragment thereof or agent that modulates one or more of the acti vities of biomarker activity associated with the ceil. An agent that modulates biomarker activity can be an agent as described herein, such as a nucleic acid or a polypeptide, a naturally-occurring binding partner of the biomarker, an antibody against the biomarker, a combination of antibodies against the biomarker and antibodies against other immune related targets, one or more biomarkers agonist or antagonist, a peptidomimetie of one or more biomarkers agonist or antagonist, one or snore biomarkers peptide mimetic, other small molecule, or small RNA directed against or a tnitnic of one or more biomarkers nucleic acid gene expression product.
An agent that modulates the expression of one or more biomarkers of the present invention, including one or more biomarkers of the invention, including one or more biomarkers listed in Table 1 and the Examples or a fragment thereof is, e.g., an antisense nucleic acid molecule, Ai molecule, shRNA, mature raiRNA, pre-iniRNA, pri-roiRNA, miRNA*, anri-miRNA, or a miRNA binding site, or a variant thereof or other small RNA molecule, triplex oligonucleotide, ribozyme, or recombinant vector for expression of one or more biomarkers polypeptide. For example, an oligonucleotide complementary to the area around one or more biomarkers polypeptide translation initiation site can be synthesized. One or more antisense oligonucleotides can be added to cell media, typically at 200 ,ug mi, or administered to a pa tient to prevent the synthesis of one or more biomarkers polypeptide. The antisense oligonucleotide is taken up by cells and hybridizes to one or more biomarkers niRNA to prevent translation. Alternatively, an oligonucleotide which binds double- stranded DNA to form a triplex construct to preve t DNA unwinding and transcription can be used. As a result of either, synthesis of biomarker polypeptide is blocked. When biomarker expression is modulated, preferably, such modulation occurs by a means other than by knocking out the biomarker gene.
Agents which modulate expression, by virtue of the fact that they control the amount of biomarker in a cell, also modulate the total amount of biomarker activity in a eel! ,
- 1.31 - hi one embodiment, the agent stimulates one or more activities of one or more biomarkers of the invention, including one or more biomarkers listed in Table I and the Examples or a fragment thereof. Examples of such stimulatory agents include active biomarker polypeptide or a fragment thereof and a nucleic acid molecule encoding the biomarker or a fragment thereof that has been introduced into the cell (e.g., cDNA, mRNA, shR As, siR As, small NAs, mature mi'RNA, pre-tmRNA, pri-miRNA, rm'R'NA*, anti- miRNA, or a miRNA binding site, or a variant thereof, or other functionally equivalent molecule known to a skilled artisan), in another embodiment, the agent inhibits one or more biomarker activities. In one embodiment, the agent inhibits or enhances the
interaction of the biomarker with its natural binding partner{s). Examples of such
inhibitory agents include antisense nucleic acid molecules, anti-bionwker antibodies, biomarker inhibitors, and compounds identified in the screening assays described herein.
These modulatory methods can be performed m vitro (e.g., by contacting the cell with the agent) or, alternatively, by contacting an agent with cells in vivo (e.g., by
administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a condition or disorder that would benefit from tip- or down-modulation of one or more biomarkers of the present invention listed in Table 1 or 2 and the Examples or a fragment thereof, e.g., a disorder characterized by unwanted, insufficient, or aberrant expression or activity of the biomarker or fragments thereof. In one embodiment;, the method involves administering an agent (e.g.. an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) biomarker expression or activity. In another embodiment, the method involves administering one or more biomarkers polypeptide or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted biomarker expression or activity.
Stimulation of biomarker activity is desirable in situations in which the biomarker is abnormally dow.nregulated and/or in which increased biomarker activity is likely to have a beneficial effect. Likewise, inhibition of biomarker activity is desirable in .vi'Zwations in which, biomarker is abnormally upregulated and/or in which decreased biomarker activity is likely to have a beneficial effect.
in addition, these modulatory agents can also be administered in combination therapy with, e.g., chemotherapeutic agents, hormones, antiangiogens, radioiabe!led, compounds, or with surgery, cryotherapy, and/or radiotherapy. The preceding treatment methods can be administered in conjunction with other forms of conventional therapy { .g., standard-of-care treatments for cancer well known to the skilled artisan), either consecutively with, pre- or post-conventional therapy. For example, these modulatory agents can bo administered with a therapeutically effective dose of ehemotherapeutie agent. In another embodiment these modulatory agents are administered in conjunction with chemotherapy to enhance the activity and efficacy of the chemotherapeutie agent. The Physicians' Desk Reference (PD ) discloses dosages of chemotherapeutie agents that have bee used in the treatment of various cancers. The dosing regimen and dosages of these aforementioned chemotherapeutie drugs that are therapeutically effective wii! depend on the particular melanoma, being treated, the extent of the disease and other factors familiar to the physician of skill in the art and can be determined, by the physician.
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In another aspect, the present invention provides pharmaceutically acceptable compositions which comprise a merapeutica.lly~effect.ive amount of an agent that modulates (e.g. , decreases) biomarker expression and/or activity, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) imravagtnally or intrarectaSiy, for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound.
The phrase "iiierapeutically-effeetive amount'' as used herein means that amount of an agent that modulates (e.g., inhibits) biomarker expression and/or activity which is effective for producing some desired therapeutic effect, e.g., cancer treatment, at a reasonable benefit/risk ratio.
The phrase "pharmaceutically acceptable''" is employed herein to refer to those agents, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The phrase "phanraceuticaliy-acceplabie carrier" as used herein means a
pharoiaceuticaliy-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, exeipient, solvent or encapsulating material, involved in carrying or
transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as phan¾aceuticaily-accepiabie carriers include: (1 ) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium earboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) exctpients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil saftlower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11 } poiyols, such as glycerin, sorbitol, mannitol and
polyethylene glycol; (12) esters, such as ethyl oSeaic and ethyl laurate; ( 13) agar; (14) buttering agents, such as magnesium hydroxide arid aluminum hydroxide; (15) alginie acid; (16) pyrogen-fVee water; (17) isotonic saline; ( 18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21 ) other non-toxic compatible substances employed in pharmaceutical formulations.
The term "pharmaceuticaUy-acceptable salts" refers to the relatively non-toxic, inorganic and organic acid addition salts of the agents that modulates (e.g., inhibits) biomarker expression and/or activity. These salts can be prepared in situ during the final isolation and purification of the respiration uncoupling agents, or by separately reacting a purified respiration uncoupling agent in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrohromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oieate, pahnitate, stearate, laurate, bcnzoaie, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, ghieohepfonate, tactobionate, and laurylsulphonate salts and the like (See, for example, Berge et ah (1977) "Pharmaceutical Salts", . Ph rm. SC 66: 1 -19).
In other cases, the agents useful in the methods of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically- acceptable salts with pharraaceutically-acceptable bases. The term "pharmaceutically- acceptable salts" in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of agents that modulates (e.g., inhibits) biomarker expression. These salts can likewise be prepared in situ during the final isolation and purification of the respiration uncoupling agents, or by separately reacting the purified respiration uncoupling agent in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a phamiaceutieaily-aeeeptable metal cation, with ammonia, or with a pharaiaceuticaliy-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include et'hylamine, iethylamine, ethylene-diamine, ethanolaniine, diethanolamine, piperazine and the like (see, for example, Berge et tl., supra).
Wetting agents, emulsifiers and lubricants, such as sodium lauryi sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
Examples of pharmaceuticaily-aceeptable antioxidants include: ( I ) water soluble antioxidants, such as ascorbic acid, cysteine 'hydrochloride, sodium bisu!fate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gaiiate, alp'ha-tocop'herol, and die like: and (3) metal chelating agents, such as citric acid, ethylenediamine tetraaeetie acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
Formulations useful in the methods of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient, which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.
Methods of preparing these formulations or compositions include the step of bringing into association an agent that modulates (e.g., inhibits) biomarker expression and/or activity, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a respiration uncoupling agent with liquid earners, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragaeanth), powders, granules, or as solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a
respiration uncoupling agent as an active ingredient. A compound may also be administered as a bolus, electuary or paste.
in solid dosage forms for oral administration (capsules, tablets, pills, dragecs, powders, granules and the like), the active ingredient is mixed with one or more pharmaeeutically-acceptabie carriers, such as sodium citrate or dicalciura phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymeihylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginie acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin : (6) absorption accelerators, such as quaternary ammonium compounds; (?) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium, iauryl sulfate, and mixtures thereof; and ( 10) coloring agents, in the case of capsules, tablets and pills, the
pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high, molecular weight polyethylene glycols and the like. A tablet- may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative,
disintegrant (for example, sodium starch glycol ate or cross-linked sodium earboxyrnethyi cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered peptide or peptido imetie moistened w th an inert liquid diluent.
Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well 'known in the p annaceutieal-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and-'or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient's) only, or preferentially, in a certain portion of the gastrointestinal trac t, optionally, in a delayed manner. Examples of embedding
compositions, which can be used include polymeric substances and waxes. The acti ve ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described exc ipients .
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyi alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions, n addition to the active agent ma contain suspending agents as, for example, ethoxyiated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, raicfocrystalline ccliiilose, aluminum metabydroxidc, bentonite, agar-agar and iragaeanth, and mixtures thereof.
Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more respiration uncoupling agents with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, suppository wa or a salicylate, and which is solid at room
temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal ca vity and release the acti ve agent.
Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such earners as are known in the art to be appropriate.
Dosage forms for the topical or transdermal administration of an agent that modulates (e.g., inhibits) biomarker expression and/or activity include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active component may be mixed under sterile conditions with a plmrmaceuticall -acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
The ointments, pastes, creams and gels may contain, in addition to a respiration uncoupling agent, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacaoth, cellulose deri vatives, polyethylene glycols, silicones, bentomtcs, silicic acid, talc and zinc oxide, or mixtures thereof.
Powders and sprays can contain, in addition to an agent that modulates (e.g., inhibits) biomarker expression and/or activity, excipients sitch as lactose, tale, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as
cliioroiluorohydt'ocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
The agent that modulates (tig., inhibits.) biomarker expression and/or activity, can be alternatively administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A nona ueous (e.g. , fluorocarbon propellant) suspension could be used. Sonic nebulizers are preferred because they minimize exposing the agent to shear, which cart result: in degradation of the compound.
Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically incl ude nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions.
Transdermal patches have the added advantage of providing controlled delivery of a respiration uncoupling agent to the body. Such dosage forms can be made by dissolving or dispersing the agent in the proper medium. Absorption enhancers can also be used to increase the flux of the pcptidomimetic across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the
pcptidomimetic in a polymer matrix or gel.
Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.
Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more respiration uncoupling agents in combination with one or more pharfflaeeuticaily-aceeptable sterile isotonic aqueous or nonaqueou solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents,
Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, etltanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof vegetable oils, such as oiive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as preservati ves, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured b the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like, it may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions, in addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum fnonostearifte and gelatin.
In some cases, in order to prolong the effec of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form.
Alternatively, delayed absorption of a parenteraily-administered drug form is accomplished by dissolving or suspending the drug in an oil -vehicle.
injectable depot forms are made by forming mieroeneapsu!e matrices of an agent that modulates (e.g., inhibits) biomarker expression and/or activity, in biodegradable polymers such as polylaetide-polyglyeolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and polyi anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions, which are compatible with bod tissue.
When the respiration uncoupling agents of the present invention are administered as pharmaceuticals, to humans and animals, the can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier,
Actual dosage levels of the active ingredients in the pharmaceutical compositions of this in ention may be determined by the methods of the present invention so as to obtain an amount of the active ingredient, which i effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.
The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No, 5,328,470) or by stereotactic injection (see e.g. , Chen et aL (1994) Proc. Natl. Acad. ScL USA 91 :3054 3057). Tire pharmaceutical preparation of the gene therapy vector can include the gene therap vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector cat} be produced intact from recombinant ceils, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery s stem:.
The present invention also encompasses kits for detecting and or modulating biomarkers described herein. A kit of the present invention may also include instructional materials disclosing or describing the use of the kit or an antibody of the disclosed invention in a method of the disclosed invention as provided herein. A kit may also include additional components to facilitate the particular application for which the kit is designed. For example, a kit may additionally contain means of detecting the label (e.g., enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a sheep anti-mousc-H P, etc.) and reagents necessary for controls (e.g. , control biological samples or standards), A. kit may additionally include buffers and other reagents recognized for use in a method of the disclosed invention. Non-limiting examples include agents to reduce non-specific binding, such as a carrier protein or a detergent.
Other embodiments of the present invention are described in the following
Examples. The present invention is further illustrated by the following examples which should not be construed as further limiting.
EXAMPLES
Example 1: Materials and Methods for Examples 2-5
a. Cell lines and culture conditions
YD38 was obtained from the Korean Ceil Line Bank; ΪΜ95 was from the Japanese Collection of Research Bioresources Cell Bank; MK 1 and KE97 were from the Riken Cell Bank; SNIJ-L W138 and MDA-M468 were from the American Type Culture Center; MK 74 and YCC1 were provided by the P. Jaime Lab; and NHBE were purchased from Lonza (CC-2540). Cells were cultured in DMEM (YCC I), DMEM plus 10 mg/L insulin (ΪΜ95), RFM1 (MDA-MB469, MK ls MKN74, SNU! , KE97, and. YD38), or EMEM (WB8), All media was supplemented with 10% FBS. NHBE were cultured in BEGM growth medium bullet kit (Lonza CC-3170). b. RNAi and cDNA rescue constructs
NFS1 shRNAs target sequences and corresponding Broad TRC (The RNAi
Consortium) -numbers and match region arc listed in Tabic 2. Negative controls included a non-targeting shRNA (N I), and two separate sbRNAs specific for luciferase (Luc2 and Luc3). sb IFl l, which disrupts mitotic spindie dynamics, served as a positive control for cell death. Pairs of oHgos were synthesized (Eurofitis MWG Operons) for each, target sequence, following the genera! structure of: ACCG-Oligo A and COAA-O!igo B, to introduce sticky Bbsl ends, and where OUgo A is: ACCGG- sense target sequence- GTTAATATTCATAGC(ioop)-antisense seqiiersce-TTTT, and Oiigo B is; the reverse complement of Oligo A . A!i shRNAs were cloned into the Cellecta vectors pRSI6-U6-(sh)- UbiC-TagGFP-2A-Puro (for constitutive shRNA expression) and RSlTi2-U6/TO-(sh}- CMV-TetR-TagRFP-Puro (for Dox-indncihle shRNA expression) at the Bbsl sites.
Insertion of shRNAs was confirmed by sequencing and by Ss l restriction digests.
Overlap PGR was used to generate NFSI-shRNA-resistant cDNA constructs (see Table 4). Wild-type NFS I cDNA. was mutagenized as listed in Table 3 using the primer pairs listed in Table 4, generating silent mutations in NFS I that cause resistance to knockdown by NFS l-shL sh4, or sh6. The sequence-confirmed NFS l-shRNA-resisian: mutant PG products were introduced into the pDONR223 Gateway donor vector, then transferred into a Gateway-adapted pLVX-neo entry vector (by Gateway Cloning, Life Technologies). NFS! -sh5 i an UTR -targeted shRNA and was rescued using wild-type
NFS I cDNA,
Table 2, NFSl and control shRNAs
shRNA Broad TRC Match
shRNA Target Sequence (Sense)
Name Library # Region
NFSi-i GCTACTGAATCCAACAACATA TRCN000014803. CDS
NFS 1-2 GCGCACTCTTCTATCAGGTTT TRCNO CM) 180881 CDS
NFSl-3 CCACAAGCGAATCTCAAAGTT TRCN 000179073 CDS
NFS1-4 CAGTTCCAGAAAGGTATATTT TR.CN0000229753 CDS
NFS 1-5 CI GTGAC TCCACC AGTTATTC TRCN0 00229756 'UTR
NFS 1-6 AGCGGCTGATACA.GAATA.TAA TR.CNG000218827 CDS
NFS 1 -7 GGGACCCTAAGCACCATTATC TRCN0000229754 CDS
NFS 1 -8 TGTGAACiCGTCTTCGAGAAAT TRC 000022975S CDS non- Ti CAACAAGATGAAGAGCACCAA NA targeting shUJ2 CTTCGAAATGTCCGTTCGGTT TR€N0000072:243 CDS sbLUC3 CAAATCACAGAATCGTCGTAT TRCN0000072246 CDS s'hKIFl ί GCGTACAAGAACATCTATAAT TRCNOOOOi 16500 CDS
Table 3. Silent mutations introduced into NFSI coding sequence
Figure imgf000144_0001
Underlined nucleotides are the mutated nucleotides Table 4, Primers used for generating shRNA-resistartt NFSI cDNAs
Figure imgf000144_0002
Bold nucleotides are tlie silent mutations introduced in the shRNA-resistant CDS Italicized nucleotides are Gateway adapter sequences Scheme for overlap PCR method used to generate shNF S i -resistant cDNA clones
Figure imgf000145_0001
293T cells were transfected using Lipofeciamine (Life Technologies) and packaging plas ids carrying VSV'g and delta 8.2, along with shRNA or cDNA expression vector to generate lentiviral particles. Viral supematants were collected and cleared by
eentri&gatioii. Supematants containing cDNA virus were concentrated using Lenti-X Concentrator (Clontech), titered by infection in limiting dilution, and used at 8 to 10 uL per 500,000 cells to achieve an MO! of approximately 0.5. infections using shRNA lertttvirases were carried out using 40 or 50 uL of viral supernatant (using pRS16 or pRSlT12 constructs, respectively) per 100,000 cells, corresponding to an approximate MOi of 3. in generating stable inducible shRNA lines, ceils were selected using puromyc in ( 1 -3 ug ml, depending on cell line) for 5-7 days.
Figure imgf000145_0002
Clonogenic assays were performed for longer-term assessment of proliferative phenotypes. Three days following doxycycline (dox)-inducdon (of stable, inducible shRNA lines using 0.5 ug/mL dox) or infection (for constitutive shRNA expression), cells were trypsinized, counted (Countess Automated Cell Counter, Life Technologies), and re- plated n duplicate or triplicaie in 6-weii dishes at 750 to 3000 cells per well depending on cell line. Cionogenic growth was monitored by R.FP (pRSiT12 vector) or GFP (pRSI6 vector) expression at 1 week intervals using a laser scanning cytometer to measure colony area and numbers of colonies per well (IsoCyte, imageXpress Ve os). Two to three weeks after plating ceils, cells were stained using crystal violet as an additional visualization of colony formation. For shorter-term proliferative assays, cells were also plated onto 96-weil plates using the Ceil Titer-Glo Luminescent Cell Viability Assay (Promega G757J } to measure cell viability on the day of plating and 6 days post-plating.
Aconitase activity was assessed three to eleven days post-infection or dox induction in one million ceils using the Aconitase Activity Coiorimetric Assay Kit (Btovision K716- 100). Succinate dehydrogenase activity was assessed using the Succinate Dehydrogenase Activity Coiorimetric Assa Kit (Biovisio.n K66 -100). e. Da aj Uysis
TCOA (The Cancer Genome Atlas) data were accessed and analyzed via the eBioPoitai for Cancer Genomics (through the Computational Biology Center at Memorial Sloan-Kettering Cancer Center (available on the World Wide Web at ebioportal.org/public- portal/). Growth rate normalization was performed when comparing proliferative
phenotypes of NFS I shRNAs and controls across multiple cell lines. Cell Titer-Glo (CTG) measurements were background-corrected, and the specific growth rate, mu, was calculated as (ln(ave TGday6 eC G,i,y(()) tinie. The data were plotted as mu/rra , where mi is the average mu of control shRN -treated wells, f. R analysis
RNA was purified using the RNeasy Mint Kit (Qiageu), and cD A synthesized using the Superscript® VILO Kit (Life Technologies). Quantitative RT-PCR was carried out using Taqman® Assays (Life Technologies, Hs00738907_mi for NFSi,
HsOOl 53 1.33jni for PTGS2, Hs00824723_m.l for Ubc, a housekeeping gene control) on a ViiA 7 Real Time PCR System (Life Technologies). g. Protein analysis
Cells were lysed in P-40 lysis buffer (Boston BioProducts) supplemented with Ix Halt 'Protease/ Phosphatase inhibitor Cocktail (Pierce Biotechnology, hie), .1 raM
Dithiottireitol, and 1 raM Phenylraethanesulfonyltluoride. Ly sates were cleared by centrifugation and quantified using a BCA Protein Assay Kit (Pierce Biotechnology, Inc.). Lysates were resolved by SDS-PAGE (using Mini-Protean TGX gradient gels, Bio-Rad Laboratories), transferred to nitrocellulose (using the iBIot Gel Transfer Device and Dry Blotting System, Life Technologies) and blocked in 5% nonfat milk (Bbtting-Gra.de Blocker, Bio-Rad). Antibodies for S ! (mouse monoclonal antibody B-7, Santa Cruz Biotechnology, inc.), FTHl (rabbit monoclonal antibody D1D4, Ceil. Signaling
Technology), and GAP'DH (rabbit monoclonal antibody 14CI0, Cell Signaling
Technology) were diluted 1 : 1000 in 5% milk in TBS/Tween-20 (Boston BioProducts). Peroxidase-conjugatcd secondary antibodies (Donkey anti-rabbit !gG or Donkey anti- mouse IgG, Jackson ImmundResearch Laboratories, inc.) were used at I :5000 in 5% blocking solution, and protein bands were detected using Pico SnperSignal West Pico Chemiluminescent substrate (Thermo Scientific, Pierce Biotechnology, Inc.).
Example 2: NFS! and other members of the iron-sulfur cluster biosynthesis pathway are biomarkers of cancer and targets for inhibiting cancer
NFS 1 is a pyrio¾xaI-5 '-phosphate-dependent cysteine desuifurase that removes inorganic sulfur from cysteine, creating alanine as a byproduct. It is primarily localized to mitochondria and is critical for iron-sulfur cluster biosynthesis and thiomodit!cation of transfer RNAs (tRNAs). The accessory protein iSDl i promotes efficient interaction between NFS ! and it substrate cysteine. NFS! exists as a heterodimer with ISDl .1 and binds to ISCU and FX during iron-sulfur cluster biogenesis, FXN (Frataxin) is an iron- binding protein thought to provide iron for Iron-Sulfur Cluster (ISC) formation. FXN also interacts with NFS i , and may facilitate cysteine binding to NFS 1 by exposing its substrate binding sites (Pandey et al (2013) J. Biol. Che , 288(52», ISCU serves as a scaffold on which ISCs assemb le. A numbe r of proteins (HSCB, RSPA9, G RPEL 1/2, GLRX5, BOLA3, ISCA.l/2, 1.BA57, UBPL) act as ehaperones to transfer ISCs from ISCU to apoproteins requiring ISCs for activity, which include component of the citric acid cycle and oxidative phosphorylation, amongst others. FU 1 may serve as another scaffolding protein for ISC formation (Li et a I. (2013) Biochem. 52). A schematic diagram further illustrating the iron-sulfur cluster biogenesis pathway is shown in figure 1.
NFS 1 is frequently amplified in "various human cancers. For example. Figure 2 shows the results of NFS I amplification assessed across available TCGA (The Cancer Genome Atlas) datasets using the cBioPortal for Cancer Genomics (available on the World Wide Web at cbioportal .org/publk-portal - Colorectal md cervical cancers exhibit the highest levels of NFS 1 amplification. Notably, 15% colorectal cancers of a total of 212 cases showed NFS 1 amplification. Approximately 3% stomach cancers of a total of 219 cases also showed NFSl amplification. Approximately 2-4% of tested breast invasive carcinoma, cervical squamous cancer, lung adenocarcinoma, ovarian cystadenorna, prostate carcinoma, sarcoma, and melanoma cases showed NFS l amplification. NFS! amplification within a span of 1 1 genes was also present in recurrent focal amplification in all lung cancers.
in addition to NFSl amplification, additional iron-sulfur cluster biogenesis pathway members are amplified in tumors. Figure 3 shows the results of collective alterations of NFS L LY M4/1SD1 1, ISCU, and FXN evaluated across available TCGA datasets using the cBioPortal for Cancer Genomics (available on the World Wide Web at
cbioportal.org/pitblic-poiiai/). These biomarkers were amplified in many different cancers and an even larger percentage of tumors have overexpression of NFS! and overexpression of other iron-sulfur cluster biogenesis pathway members despite the fact thai they lack amplifications. Colorectal cancer, ovarian cancer, and sarcomas exhibit the highest levels of iro -sulfur cluster biogenesis pathway dysregnlation.
Similarly, Figure 4 shows the results of mutation analyses of solute carrier family 25 mitochondrial iron transporter, member 28 (SLC25A28), a mediator of iron uptake, assessed across TCGA samples using datasets from the cBioPortal for Cancer Genomics (available on the World Wide Web at cbiopoitai.org/pubiic-portal/) and the Memorial Sloan-Kettering Cancer Center (MS CC). AH markers shown represent missense
mutations, except for the seventh marker from the left located at the -terrainus of the Mito carr domain, which represents a frameshift deletion. SLC25A28 Rl I 2C/M is a recurring mutation in colorectal and gastric cancers. S.LC25A28 gatn-of-function mutations are believed to be particularly dependent on NFS 1.
E xample 3 : Knockdown of NFSl inhibits clonogenic cell growth
Knockdown of NFS! inhibits clonogenic cell growth. For example, inducible knockdown of NFS 1 using sliRNAs causes potent growth inhibition in cells of the gastric cancer cell Sine, K /4. NFS l is amplified in the MKN /4 ceil line. Other ceil lines that are dependent on NFSl overexpression are well known in the art and include, for example, the AGS and KE39 ceil lines. Such ceil lines, including MKN74, can grow as xenografts in SG mice. MK.N74 stable cell lines carrying dox-inducible shRNAs for Kifl 1 (positive kill control), luciferase (Luc2 or Luc3: negative controls), or NFS 1 shRNAs (NFS1-L -4, - 5, -6) were induced to express shRNAs by addition of dox (0,5 lig/mL) io the culture media, or left un-induced (no dox). Three days post-dox-ind crion, cells were trypsinized.
counted, and plated at low density (750 cells per well of a 6- well dish) for assaying clonogenic growth. Additional cells were also collected and assessed by q-RT-PCR for NFS'! knockdown, Clonogeme growth (relative colony area shown in Figure 5, as well as the number of objects) was monitored by red fluorescent protein (RFP) expression over two weeks scanning the plates on a laser scanning cytometer (I soCyte, ImageXpress Velos), The average coiony area at days 7 or 14 post-plating from 2 replicate wells was normalized to the average of the negative controls (Figure 5). Clonogenic growth was also visualized by crystal violet (CV) staining at day 14 (Figure 5; bottom panel).
In order to verify the specificity of the growth inhibition effects to FS1
knockdown. cDNA rescue experiments were conducted to confirm on-target activity of NFS1 shRNAs. Stable NFSI-shRNA ( FSl -sh5) or shRNA. control lines were infected with lentivirus expressing wild-type NFS 1 cDNA, and compared to uninfected
counterparts. Cells were cultured in the presence or absence of 0,5 g/rnL dox for 3 days, then replated to assay for clonogenic growth. NFS 1 knockdown was confirmed by q-RT- PCR, and colony growth was also monitored on a laser scanning cytometer. Ceils were fixed and stained with crystal violet at day 14 post-plating. Stable MKN74-shNFSl cells induced with dox were significantly impaired in eolouy formation (see, for example, the left boxed images of Figure 6) relative to the negative controls, while the addition of NFS! cDNA restores colony formation (see, for example, the right boxed images of Figure 6). Similar results were obtained from other rescue experiments performed with additional NFS! shRNAs (shl , sh.4, sh6) with corresponding NFS! cD A constructs resistant to knockdown by each shRNA (see Example I).
NFS! knockdown was also determined to cause differential effects on cell growth across a panel, of different cell lines. A panel of eel! lines were infected with constitutive NFS1 shRNAs (sh4, sh5, sli6)« negati ve control shRN As (for Luc2, Luc3), and positive kill. control shRNA. (for Kifl 1 ). Three days post- infection, cells were trypsinized, counted, and re-plated in 96-well plates. Cell viability was measured on the day of re-plating and at day 6 post-re-pi ating by Cell-Titer® Glo, and. the data was growth rate normalized (see Example I). Cells collected on the day of re-plating were assessed for NFS! D by cj-RT- PC R and Figure 7 shows that NFSi knockdown yielded a range of NFS 1 -dependency with respect to modulation of cell growth.
Finally, it was determined that shutting off NFSI shRNA function restored clonogenic growth. For example, stable YD38 sh'RNA lines were cultured in the presence or absence of 1 ug/mL Dox for 3 days, then trypsinized and replated to assay for clonogenic growth with 1) continued Dox, 2) continued absence of Dox., or 3) withdrawal of Dox.
Knockdown of NFS! was assessed on the day of replating. At day 1 1 post-re-plating, colonies were visualized and quantified with a laser scanning cy ometer, and colony area was normalized to the average of the negative controls. Figure 8 shows that withdrawal of Dox allowed increased clonogenic growth for sh4, $h5, and sh6. Thus, growth inhibition induced by NFSI knockdown in cell lines like YD38 can be reversed by shutting-off shNFS l expression.
Example 4: Pharmacodynamic markers related to NFSI act as biomarkers for NFSI activity and cell growth modulation and ceil-free and cell-based screening strategies for identifying inhibitors of NFSI and/or other biomarkers listed in Table 1 Previous studies indicate that iron-sulfur dependent mitochondrial enzymes, such as succinate dehydrogenase (SDH) and aconitase (Aco), act as pharmacodynamic biomarkers for NFSI function. For example. Figure 9 shows that SDH and Aco activity are coordinately modulated according to NFSI activity (Majewska et aL (2013) J. Biol. Chem. 288 ;2 134-29142) and Figure 10 further shows thai SDH and Aco activity are significantly decreased following NFSI knockdown.
in addition, it is believed that cell death due to NFSI knockdown is due, at least in part, to dysregulation of free iron levels through a non-apoptotic, iron-dependent form of cell death known as ferroptosis (Figure 1 1 ). in particular, Erastin, 1 S3R-RSL3, & PE induce ferroptosis and PTGS2 (Cox2) is a known biomarker of ferroptosis, as wel l as bio marker of oxidative stress, which is a hallmark of ferroptosis (Figure 12 and Yang et aL (2014.) Cell 156:317-331 ), The implication of modulation of NFS! and free iron levels are also belie ved to involve the change in expression of ferritin and transferrin receptors, whose translation is regulated by iron-response-proteins.
Moreover, HlF2a is modulated via NFS I modulation. HIF2a is an "undruggab!e" transcription factor in oncology and inflammation that is activated by mutations (either directly or indirectly) in a subset of cancers. For example, Thompson el ctl. (2014) Blood 1 :366-376 demonstrate that HlF2a regulates neutrophil longevity and modulates inflammation. Figure 13 shows that iron-regulatory protein 1 (IRP.1 ) inhibits HIF2a translation ant! activity such that ! Pi activators would downregulate the HlF2a pathway. NFS! inhibition leads to depletion of Pe~S clusters. It has been detemiined herein that such depletion leads to acti vation of Iron Response Element Binding Protein (IRE-BP) since modulation of genes regulated by the Iron Response Element, such as the transferrin receptor and ferritin, was observed in the shRNA knockdown lines described. Thus, it is believed that HlF2a is modulated by this same regulator}' mechanism and that NFS I inhibition leads to decreased HfF2a levels. Zi mer er ai (2008) Mol. Cell 32:838-848 demonstrate that compounds that induce IRE-BP activity decrease HIF2a mRNA and protein levels and it is expected that NFS 1 inhibition has the same consequences. HIP 2a gain-of-function mutations have been described in congenital polycythemia and number of cancers including paragangliomas and these mutations are believed to be driver imitations. Thus, NFS ! inhibition not only affects fundamental iron metabolism in tumors, but also directly shuts down signaling from a mutated driver mutation. Mutations in HIF2a or other mutations that activate F1IF levels, such as imitations in succinate dehydrogenase, are thus predictive biomarkers for NFS 1 therapy.
Additionally, HlF2a has been demonstrated to be important for myeloid cell function, in particular neutrophils (Thompson el al. (2014) Blood .123:366-376), and it is expected that NFS! will modulate activity of myeloid cells within the hypoxic tumor niieroeiiviroBirient. Tumor-associated myeloid cells have been demonstrated to inhibit the anti-tumor immune response such that inhibition of NFS 1 is expected to lead to increased apoptosis of tumor associated myeloid cell which can have therapeutic benefit. These expected anti-tumor mechanisms of NFS 1 inhibition (via modulation of RlF2a) could be achieved without chronic long-term NFS ! inhibition.
In order to confirm that such pharmacodynamic markers associated with NFS I can be used as adjuncts or surrogate markers for directly assessing NFS 1 modulation, several experiments were performed. Figure 14 shows the results of M N74 stable ceil lines carrying dox-indueible shRNAs for ift 1 (positive kill control), !ueiferase (Lue2 or Luc3: negative controls), or NFS I. shRNAs (NFS! -4, -5, ~6) that were cultured in the presence or absence of doxycyciine (0.5 ug/mL). Three days posi-dox-induetion, ceils were
trypsimzed, counted, and some cells assessed by Western blot for NFS.! knockdown and ferritin (FTH1 : ferritin heavy chain 1 ) levels with GAPDH served as loading control (left panel). Aeoniiase activity was also assessed at this time point using .1 million ceils per condition (middle panel). The stable lines were repiated at 3 days post-dox, cultured for 3 days further, and assessed, for NFS1 mRNA knockdown (top right panel) or PTGS2 (Cox2) niRNA levels (bottom right panel) by q-RT-PCR Figure .14 shows that NFS ! knockdown results in decreased aconitase levels and ferritin levels. In addition, PTGS2 (COX2) mR A was induced and 1RP1 was activated as a result of NFS 1 inhibition.
Similarly, after 7 days of NFS 1 knockdown, decreases in aconitase activity and ferritin protein levels, along with upregulation of T.F C protein were observed (Figure 15). Specifically, MKN74 cells stably carrying individual inducible NPS1 shRNAs, negative controls (shLuc2 or shLuc3) or positive kill control (s'hKif 1 1 ), were induced with
doxycycline for 7 clays. These cells were then either lysed in RIPA buffer, quantified, and equall loaded on a SDS-PAGE gel tor analysis by Western by staining for TfRC, NFS1 , FTL, or GA'PDH (loading control); or trypsintzed, counted, lysed, and analyzed for aconitase activity. Aconitase activity was normalized to the protein concentration of each lysate. After .1 1 days of NFS 1 knockdown, decreases in aconitase activity were maintained along with decreases in succinate dehydrogenase (SDH) activity as determined by the readout of succinate conversion to iuraarate arid as normalized against protein
concentration of each analyzed lysate. Specifically, stable M .N74 shRN lines were induced to express NFS ! or negati ve control shRNAs by doxycycline treatment for 1 .1 days. These cells were then either lysed in R PA buffer, quantified, and equally loaded on a SDS- PAGE gel for analysis by Western and staining for NFS1 and GA'PDH; or trypsioized, counted, lysed and analyzed for succinate dehydrogenase activity and aconitase activity. SDFl activity correlated with aconitase activity and NFS! knockdown and was consistent in repli cate experiments,
As described above, HTF2a is an "undruggable" transcription factor that is active in a subset of cancers, regulates neutrophil longevity, and modulates inflammation (Thompson et l. (2014) Blood .16:366-376). NFS. I knockdown was determined to be associated with down-regulation of HlF2a protein levels (Figure 16), which also correlated with the down- regulation ofFTHl protein levels, with, the effects on both proteins likely due to the activation of iron-regulatory protein 1 (1R.P1). Specifically, stable shRNA lines of the
NFS 1 -amplified colorectal line C2BBE 1 were established following infection and
puromycin-selection for each respective shRNA constructs (individual inducible NFS 1 shRNAs: slil , sh2, sh4, sh5, sh6; negative (shLuc2 or shLue3); or positive kill control (shKi.fl 1.)). These stable lines were induced with doxyeycline For 2 weeks, and split 3 times during the course of culture. These cells were then lysed in the plate with ice cold RIPA buffer, quantified, and equally loaded on a S'DS-PAGE gel for analysis by Western analyses. Treatment of parental C2.8BEI cells with the hypoxia mimicking agent, cobalt 5 chloride, showed modulation of HIF2alpha level relative to untreated parental C2BBE.1 cells, as expected, and highlighted the correct molecular weight band to monitor for modulation fay Nf Si KD, Western analyses were carried out using antibodies specific for NFS'I , FTH1 , RlF2aipha, GAPDH and vineulin, with the latter two proteins serving as loading controls.
it) Thus, candidate biomarkers for iron-sulfur cluster biosynthesis pathway modulation and iron-dependent cell death (ferroptosis) correlate with NFS 1 inhibition.
Example 5: cD A rescue of NFS1 confirms on -target activity of NFS1 sh NAs
As described above, cDNA rescue experiments are useful- for confirming the
15 specificity of effects observed with modulating the expression of a gene such as NFS1 by shRNAs (Figure 6). Figure 17A confirms thai the KN74 gastric cell line harbors amplifications of NFS I, as detern nedby fluorescent in situ hybridization (FISH) analyses relative to C.EP20, a chromosome 20 centromere marker, and DAP! used to stain genomic DNA. Stable NFS1 shRNA cell lines were allowed to undergo clonogenic growth and were
20 then analyzed thirteen days post-doxycyeline activation of the shRNA expression constructs in addition to cDNA rescue. Figure 17B shows the results of the cells wherein the cells were either rescued with wild-type NFS 1 (WT NFS 1 } or a mutant NFS1 that is eatalyikaUy dead due to a C381 A mutation (NFS lcm ). NFS 1 cmA acts as a dominant negative mutant with growth inhibitory effects comparable to that of NFSl -shS, Specifically, the stable
25 MK. 74 shRN A lines (for the negative control shRNAs NT. I , l. c2, or Luc3, or the NFS 1 shRNA) were each infected with lentivirus (generated using the pLVX-neo vector) at an approximate MOI of 3 to constitutively over-express GFP as a negative control, WT NFS! , orNFSla>Wi\ and selected for one week using neomycin. These selected lines were then treated with do ycycline to induce shRNA expression, and at day 3 post-doxycyeline, these 0 cells were trypsinized and replated to assess their clonogenic growth in duplicate in 6- well plates. Figure Γ7Β shows that impaired clonogenic growth mediated by NFSI-sh.5, a UTR- targeted shRNA: KD » 87% by q PCR, was restored by WT NFS 1 , but not by NFS l slA, The combination of FSl-shS andNFS'lt'">,>lA completely inhibited clonogenic growth. Similarly, Figure 18 shows that cDNA rescue with WT NFS! (in the same clonogenic growth experiment: described in Figure 17} restored the S ! -shS-dependent effect on aconitase activity. Specifically, cell lysates were prepared from 100cm plates of the same sets of infected ceils described in Figure 17, and analyzed for aconitase activity ten days 5 after doxycyciine treatment. Aconitase activity was normalized to protein concentration of each lysate. Similar experiments with NFS !'" "" Α caused insufficient number of cells for analyses since the mutant MP Si strongly inhibited ceil growth.
WT NFS 1 , but not NFSl MA. also rescued the NFS i~sh5~dependent inhibition of FTH 1 protein levels and the up-reguiation of TfRc protein levels (Figure 19), Specifically, it) the same infected ceils described in Figure 1? were used in this experiment. These ceils were cultured in 10 cm plates, induced with doxycyciine for 7 days to induce sh'RNA expression, and lysed in cold RI.PA buffer. 'Equal quantities of protein was loaded onto SDS-PAGE gels, and Western analysis done to assess protein levels of NFS l, FTH1 and TfRC, with vmculiB and GAPDH staining also done as loading controls. Bands on
1.5 Westerns were quantified by densitometry, and graphed relative to loading controls. Again, the NFSl Mt,A effects were similar to those of MFS l-sb,5 on biomarker modulation.
it was further determined that the NFS 1Ϊ > ' catalytic mutant causes a decrease in aconitase activity and ferritin levels comparable to that with FS l-s i or NFS i -sh5 (Figure 20). M 74 cells were infected at an approximate MOI of 3 at time of cell replating into
20 10cm dishes, using lendvirus expressing either constitutive shRNAs (from pRSI6 vector:
NFSl -shl , NFSl -sh.5, or negative control shRNAs, shhie2 or shluc3), or constitutive cDNAs (from pLVX-neo vector: WT NFS I , C381 A catalytic mutant NFS 1 , or GFP (negative control). Selection of infec ted cells was carried out using puromycin (for pRSI6 vectors) or neomycin (for pLVX-neo vectors), beginning 2 days after infection. Seven days
25 post-infection, cells were either lysed for further analysis of aconitase activity, or lysed with cold RIPA buffer, and lysates were then quantified and loaded in equal protein quantities onto an SDS-PAGE gel. Western analysis was carried out to detect NFS! , FTHl, and vtnctiltn (loading control) levels. Aconitase activity was normalized to protein
concentrations.
0
Example 6 Biochemical assays reporting NFS! activity
As described above in the specification, iron-sulfur cluster biosynthesis pathwa members and pharmacodynamic markers related to same can be used in various screening assay to identify modulators (eg., inhibitors) of iron-sulfur cluster biosynthesis pathway members of interest,
in certain embodiments, biochemical screening for evaluating expression arid activity of a biomarker listed in Table I , such as due to application of inhibitors of a biomarker listed in Table I (e.g., NFS I inhibitors), are presented (see, for example, Li et al. (2004) Am. J. Physiol. Cell Physiol. 28?:C1547-C1559; Tsai and Baroadeau (2010)
Biochem. 49:9132-9139; Schmueker et al. (2011 ) PLoS ONE 6:et6199). Protein for use in the assays can be purified from any number of natural or recombinant sources (see, for example, Majewska et I. (2013) J. Biol Chem. 288:29134-29142). For example, . call expressing a bicistronic expression vector encoding both NFS! and ISD11 can be used to readily purify a complex of NFS! and 1SD1 1 by, for example, using a tagged protein (see, for example, Marelja et al. (2008) J. Biol. Chem, 283:25178-25185). In addition, the expression vector or post-translation modifications can be engineered to remove mitochondrial signal peptides and oilier domains that are not associated with protein function. The activity of a biomarker of interest, such as the effect of a test agent or compound on a purified protein, can be analyzed using direct or indirect enzymatic assays. in an indirect method, analysis of an enzymatic reaction product can he analyzed as a surrogate for directly measuring enzyme action. For example, sulfide-based (e.g., methylene blue assays or fluorogenic sulfide probes, such as AzMC to AMC detection) or alanine-hased (e.g., alanine dehydrogenase activity) detection methods can be used to analyze FSl readout (Figure 21). in one embodiment, a reaction mixture containing a buffer, purified NFS 1/ISDl 1 , cysteine, and PLP cofactor can be created in the presence or absence of a test agent or compound and any enzymatic reaction can be stopped with the addition of ,N-dimeihyl-p-phenylenediami«e and FeC¾ in HQ solution such that monitoring of the production of methylene blue at an absorbauce of 670 am will indicate the extent of inhibition. The methylene blue-cysteine desulfurase assay is a standard coloriraetrk assa that is well known in the art (see, for example, Pandey el al (201 1) ./. Biol. Chem. 286:38242-38252). However, the assay has not heretofore been adapted for use in high-throughput format, such as for use in high- capacity plates containing 96 wells. figure 22A provides a representative methylene blue assay suitable for a high-throughput format, This was accomplished by making a reaction buffer having 100 niM Tris, pH 8.0, 200 ffl:M NaCl, 100 uM pyrodoxial phosphate, 100 uM DTT, and 100 μΜ of L-Cysteine. This mixture was then added to wells of a clear 96-weSl plate. After, a range of concentrations of sodium sulfide could be added for use as a standard. To defect the sulfide, 10 μί,- ofN,NHjiraethyl-p-phenylenediarame and 10 μΐ of PeCij were added, and after an incubation time of up to one hour, the absorbance of the well was measured at 670 am using an BnSpire® plate reader. Figure 22B demonstrates a representative sulfide detection range, wherein reactions were run with DMPPDA sulfide probe and a standard curve of sodium sulfide concentration as substrate was used to generate methylene blue. Here, the same protocol that was described for Figure 22A was used. Briefly, wells were set up with the reaction buffer, sodium sulfide was diluted serially to a range of uM and rsM concentrations and added to the wells, then, methylene blue production was detected after dimethyl-p-phenylenediamme and FeC¾ bad been added via a plate reader. The
"blanks" for the experiment were wells that recei ved the same reagents, without any sodium sulfide. T values were calculated for each sulfide concentration as a measurement of how robust the potential assay would be. Sulfide detection and Z* values at 5 uM and above were especially robust.
Since hydrogen sulfide does not remain in solution over time, standard curves can become unreliable such that sulfide production is underestimated and sulfide generated by NFSl /fscS can be lost during the reaction (Figure 23). In order to avoid sulfide loss, other indirect detection assay can be used. In one embodiment, a fiuorogenic probe can be used, such as ?-a2ido-4-methylcoitinarin (AzMC) (Thorson el at. (2013) Angewantite Chemie. Intl. Ed. 52:4641 -4544). AzMC reacts with and detects sulfide as NFS 1 produces it, so sulfide loss is minimized relative to a methylene blue assa in which DMPPDA isn't added until the NFS ! reaction is complete (e.g., for at least 20 minutes). AzMC assays have not heretofore been adapted to measure NFS I acti vity . Figure 24 A provides a representative AzMC assay suitable for a high-throughput format. This was accomplished by making a reaction buffer having 100 mM Tris, pH 8.0, 200 mM NaCL 100 μ pyrodoxial phosphate, 5 mM glutathione, 100 uM of L-cysteine and 0,5 mg/mL ofBSA. This mixture was then added to wells of a. black, clear-bottom 96-well plate. "The AzMC probe was then added to each well to a final concentration of .1 μΜ. Then, a range of Sodium Sulfide
concentration could be added for use as a standard. The reaction was either incubated in the dark at room temperature for at least an hour prior to measuring the fluorescence, or a time course protocol was used via the plate reader to measure the fluorescence over time. Fluorescence was measured at an excitation of 365 nm with an emission of 450 nm. Figure 24B demonstrates a representative sulfide detection range, wherein reactions were run with AzMC sulfide probe and a standard curve of sodium sulfide concentration as subs trate was used to generate die fluorophoref AMC. The same protocol that was described for Figure 24A was used. Briefly, wells were set up with the reaction buffer, sodium sulfide was diluted serially to a range of μΜ and «M concentrations and added to the weils, the Az C probe wa added, the reaction was incubated at room temperature for one hour in the dark, after which, the fluorescence was measured via. a. plate reader. The "blanks" for the experiment, were wells that received the same reagents, without any sodium sulfide. Z' values were calculated for eac sulfide concentration as a measurement of how robust the potential assay would lie. Sulfide detection and Z' values at 500 nM and above were especially robust. Figure 24C demonstrates enzyme kinetics of IscS using the AzMC assay optimized for high-throughput analyses. To study the enzyme kinetics of the IscS protein via the AzMC Assay, the same reaction buffer described for Figure 24A was made, without L-cystcinc. The reaction mixture was thai added to a welis of a black, clear-bottom 96- well plate. Then, the AzMC probe was added, sodium sulfide standards were made as described above, and L-cysteine was added to different wells at different concentrations. The react ion was then carried out at 37eC with different concentrations of L-cysteine and at several time points in order to find the initial velocities for each substrate concentration. The final IscS concentration used was 250 n. . After calculating the initial 'velocities, a Michaelis-Mmten Graph and a Lineweaver-Burk Plot were made, and the Km and VstsiK were subsequently determined.
in another embodiment, alanine assays cati. be used (Colin et ah (2013) J. Amer. Chem. Sac. 1.35:733-740; Tsai and Baroadeau (201.0) Biechem. 49: 132-9139; Anthony et al. (20! I ) P oS ONE 6:e20374). As with the AzMC assay described herein, alanine assays have not heretofore been adapted to measure NFS! activity. Figure 25 A provides a representative alanine assay suitable for a high-throughput format. This was accomplished by making an NFS1 reaction buffer having 100 mM Tris, pH 8.0, 200 mM aCl, 100 μΜ pyrodoxiai phosphate, 100 μ ' TT, 100 μΜ of L-cysteine. "This mix ture was then added to wells of a clear, UV- transparent 96- well plate. If protein was being tested, it would be added at this point and incubated for the appropriate amount of time. For the alanine dehydrogenase reaction, the pH was increased to 10 by adding a reaction mixture of 100 mM sodium carbonate buffer with the same concentrations of NaCl, pyrodoxiai phosphate, and DTT as described. As a co-substrate, 1 mM NAD* was added. A range of L-alanine concentrations were used as standards, and were added to the same reaction mixture described above. Alanine dehydrogenase was added to a final concentration of 0.03 units/raL to each well, and this reaction was run for 30 minutes at room temperature. The alanine dehydrogenase converts alanine and NAD' to NADH, which fluoresced and could be measured at an excitation of 340 am artel an emission of 460 nm. The blank for this reaction would be the reaction mixture without L-alanine and Figure 25B demonstrates a representative NADH detection range, wherein reactions were run with alanine
dehydrogenase (AlaDH) and a standard curve of alanine concentration as substrate was used to generate NADH, which fluoresces at 460 am. The same protocol that was described for Figure 25A was used. Briefly , wells were set up with the NFS I reac tion buffer, L-alanine was diluted serially to a range of u and aM concentrations and added to the wells, the AlaDH reaction buffer and subsequently the AlaDH were added, and the reaction was incubated at room temperature for 30 minutes, after which the fluorescence was measured via a plate reader. The "blanks'" for the experiment were wells that received the same reagents without any L-alanine. Z> values were calculated for each L-alanine concentration as a measurement of how robust the potential assay would be. Alanine detection and Z* values at 500 nM and above were especially robust. Figure 25C demonstrates enzyme kinetics of IscS using the alanine assay optimized for high- throughput analyses. To study the enzyme kinetics of the IscS protein via the alanine assay; the same NFS! reaction buffer described for Figure 25A was made without L-eysteine. The reaction mixture was then added to a series of PC tubes. Then, L-aianine was added to the standards tubes, and L-cysteine was added to different tubes at different concentrations. The IscS protein was added to a final concentration of 250 nM The IscS reaction was then carried out at 37*C with different concentrations of L-eysteine and at different time points, in order to find the initial velocities for each substrate concentration. The reactions at each time point would be halted via heat inactivation at 9S°C, and after ail reactions were finished, the AlaDH reaction was run under the conditions described in Figure 25A. After measuring the fluorescence and calculating the alanine concentration, the initial velocities were found, a Michaeiis-Menten Graph and a Lineweaver-Burk Plot were made and the Km and Vj„;a were subsequently determined.
By contrast, a direct enzymatic assay, such as that using the same enzymatic reaction mixture described above, but actively monitoring the production of alanine via a mass-spectrometer (e.g., such as the Agilent RapklFire# platform), can be used to identif the extent- or inhibition. Test agents or compounds that reduce the production of alanine, for example, can be identified as NFS I inhibitors.
in other embodim nts, celS-based screening methods to identify modulators of a bio marker listed in Table 1 (e.g., Nf S I inhibitors) are presented (see, for example, Li et al. 5 (2004) Am. J. Physiol. Ceil Physiol. 287:C 1547-C.l 559). Cells treated with or without a test agent or compound can be monitored for the. modulation of Fe-S dependent enzymes such as by assaying decreases in aconitase activity or modulation of other activities listed in Table I . Cells treated with or without a test agent or compound can also be monitored for the modulation of iron-responsive reporter genes. For example, antibodies can be used to it) detect levels of endogenous IRE-moduiated proteins to provide readout of iron-sulfur
cluster depletion which will result from NFS 1 inhibition. This assay could be configured as a high content screen (Weerapana et al. {2010) Nature 468:790-795), Alternatively, stable cell lines expressing reporter gene (e.g.. lueiferase, GFP, etc.) with t« NA context that contains iron response elements that will increase mRNA stability and ultimately reporter
15 gene activity ca be used. The use of a destabilized reporter gene would likely increase sensitivity. In one embodiment, the mRNA context of transferrin receptor can be used as model, wherein iron response elements in 3' UT that increase stabilit of mRNA under low Fe-S levels.
in still another embodiment, the fact that IREs in 3' UT stabilize mRNA, while 20 IRE in 5' UT reduces translation, can be exploited by generating at least two reporters, one that will be upregulated and one that will be down regulated upon inhibition, such as NFS ! inhibition (Fe-S cluster depletion), and the ratio of at least two reporters can be monitored. This can be done with either fluorescent proteins or lueiferase proteins or a combination of both. Figure 26 shows art exemplary schematic diagram illustrating
25 possible reporter constructs. For example, the first reporter can have a 5 ' UTR arrangement similar to ferritin mRNA that will have low protein levels upon NFS I inhibition (e.g., 5 "UTR of ferritm-firefJy luficerase-2A-GFP reporter). The second reporter can have a 3' UTR arrangement similar to transferrin receptor mRNA that will have increased protein levels of the report with increases in NFS 1 inhibition (e.g., Renil!a luciferase-2A-RFP
0 reporter-3' UTR of transferrin receptor). Drug selection markers, such as puromycin, can also be used. Appropriate control constructs, such as those illustrated in Figure 26, can also be used. The readout would be the ratio of at least two reporters. For example, NFS ! inhibition will increase the ratio of Renilla-to-firefly lueiferase ratios and or RFP-to-GFP ratios. Similarly, simplified reporters with just FTL.iron response element (IRE)-iuciferase and a corresponding control can be used. The engineered constructs can be expressed via any number of well -known vectors, including viruses, such as lentiviral vectors shown in Figure 25, plasmids, and the like. Such a strategy, in addition to allowing the use of the luciferase ratio to identify NFSl inhibitors, could also be adapted for live ceil imaging using the ratio of, for example, two fluorescent markers.
Example 7: Microbial pathogen growth inhibited using inhibitors of NFS! homologs
Inhibitors of NFS 1 homologs in microbial pathogens are believed to have therapeutic potential as inhibitors of microbial pathogenic growth since the catalytic cysteine residue is always conserved. Such NFS! homologs are essential for growth in a variety of bacterial/fungal species including helicohacier pylori via its NFSl homolog, NifS (Olson ei a!. (2000) Bioc em. 30: 16213-16219; mycobctcierium tuberculosis via its NFSl homolog, IscSMtb (Rybmker et al (2014) Biochem. J. Feb.1 e-pub: and yeast v a its NFSl homolog, fslp (Li el al. (1999) J. Biol. Chem. 274:33025-22034). Human NFS 1 is -8 % similar to the E, coli homolog and the conserved deep substrate pocket and conservation of the catalytic cysteine support draggability of NFS i homologs using agents such as small molecules and eovalent inhibitors. incorporation by Reference
All publications, patents, and patent applications mentioned 'herein are hereby incorporated by reference in their entirety as if each Individual publication, patent o patent application was specifically and indi vidual! indicated to be Incorporated by reference, in case of conflict, the present application, including any definitions herein, will control.
Also incorporated by reference in their entirety are any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) on the World Wtde Web and/or the National Center for Biotechnology information (NCBi) on the World W ide Web.
- i 59 - Equivalents
Those skilled in the art will recognize, or be able to ascertain using no snore than routine experimentation, many cquivaleiits to the specific embodiments of the invention described herein. Such equivaients are intended to be encompassed by the following claims.

Claims

What- is claimed is;
1 . A method of treating a subject afflicted with a cancer comprising sdmmisteriiig to the subject an agent that inhibits the copy number, amount, and/or activity of at least one biomarker listed in Tabic 1 , thereby treating the subject afflicted with the cancer.
2. The method of claim i , wherein the a ent is administered in a pharmaceutically acceptable formulation.
3. The method of claim 1, whereiii the agent directly binds the at least one biomarker listed in Table L
4. The method of claim 1 , wherein the at least one biomarker listed in Table I is toman NFS 1 or an ortholog thereof.
5. The method of claim t , further comprising administering one or more additional anti-cancer agents, optionally comprising mitochondrial eofaetor therapy.
6. A method of inhibiting hyperproliferative growth of a cancer ceil or cells, the method comprising contacting the cancer ceil or cells with an agent that inhibits the copy number, amount, and/or activity of at least one biomarker listed in Table 1 , thereby inhibiting hyperproiiferative growth of the cancer cell or cells.
7. The method of claim 6, wherein the step of contacting occur in vivo, ex vivo, or in vitro.
8. The method of c!aim 6, wherein the agent is administered in a pharmaceutically acceptable formulation.
9. The method of claim 6, wherein the agent directly binds the at least one biomarker listed in Table .1 .
I.0. The method of claim 6, wherein the at least one biomarker listed in Table I is human NFS! or an ortholog thereof,
I I . The method of claim 6, further comprising administering one or more additional anti-cancer agents, optionally comprising mitochondrial eofaetor therapy.
1.2. A method of determining whether a subject afflicted with a cancer or at risk for developing a cancer would benefit from iron-sulfur cluster (ISC) biosynthesis pathway inhibitor therapy, the method comprising:
a) obtaining a biologies! sample from the subject;
b) determining the copy number, amount, and/or activity of at least one biomarker listed in Tabic 1 in a subject sample;
c) deter.oii.uing the copy number, amount, and or activity of the at least one biomarker in a control; and
d) comparing the copy .number, amount, and/or activity of the at least one biomarker detected in steps b) and c);
wherein a significant increase in the copy number, amount and/or activity of the at least one biomarker in the subject sample relative to the control copy number, amount, aad or acti vit of the at least one biomarker indicates that the subject afflicted with the cancer or at risk for developing the cancer would benefit from ISC biosynthesis pathway inhibitor therapy.
13. The method of claim 1 2, further comprising recommending, prescribing, or administering ISC biosynthesis pathwa inhibitor therapy if the cancer is determined to benefit from ISC biosynthesis pathway inhibitor therapy.
14, The method of claim 12, further comprising recommending, prescribing, or administering anti-cancer therapy other than ISC biosynthesis pathway inhibitor therapy if the cancer is determined, to not benefit from ISC biosynthesis pathway inhibitor therapy.
! 5. The method of claim 14, wherein the anti-cancer therapy is selected from the group consisting of targeted therapy, chemotherapy , radiation therapy, aad/or
hormonal therapy.
16. The method of any one of claims 1.2-15, wherein the control sample is determined from a cancerous or non-cancerou sample from either the patient or a member of the same species to which the patient belongs.
17. The method of any one of claims 12-16, wherein the control sample comprises cells.
18. The method of any one of claims 12- i7, further comprising determining responsiveness to ISC biosynthesis pathway inhibitor therapy measured by at least one criteria selected from the group consisting of clinical benefit rate, survival until mortality, pathological complete response, semi-quantitative measures of pathologic response, clinical complete remission, clinical partial remission, clinical stable disease, recurrence-free survival, metastasis free survival, disease free survival, circulating tumor ceil decrease, circulating marker response, and ECIST criteria,
1.9. A. method of assessing the efficacy of an agent for treating a cancer in a subject, comprising:
a) detecting in a first subject sample and maintained in the presence of the agent the copy number, amount or activity of at least one biomarker listed in Table I:
b) detecting the copy number, amount, and/or activity of the at least one biomarker listed in Tabic 1 hi a second subject sample and maintained in the absence of the test compound; and
c) comparing the copy number, amount, and/or activ ity of the at least one biomarker listed i Table 1 from steps a) and b), wherein a significantly increased copy number, amount, and/or activity of the at least one biomarker listed in Table 1 in the first subject, sample relative to the second subject sample, indicates that the agent treats the cancer in the subject.
20. A method of monitoring the progression of a cancer in a sub jec t, comprising;
a) detecting in a subject sample at a first point in time the copy number, amount, and/or activity of at least one biomarker listed in Table 1 ;
b) repeating step a) during at least one subsequent point in time after administration of a therapeutic agent; and
c) comparing the copy number, amount, and/or acti vity detected in steps a) and b), wherein a significantly increased copy number, amount, and/or activity of die at least one biomarker listed in Table I in the first subject sample relative to at least one subsequent subject sample, indicates that the agent treats the cancer in the subject.
2.1. The method of claim 20, wherein between the first point in time and the subsequent point in time, the subject has undergone treatment, completed treatment, and/or is in remission for the cancer.
22. The method of claim 20 or 21 , wherein between the first point in time and the subsequent point in time, the subject has undergone ISC biosynthesis pathway inhibitor therapy.
23. The method of any one of claims 20-22, wherein, the first and/or at least one subsequent sampie is selected from the group consisting of ex vivo and in vivo samples.
24. The method of any one of claims 20-23, wherein the first and/or at least one subsequent sampie is obtained from an animal model of the cancer.
25. The method of any one of claims 20-24, wherein the first and/or at least one subsequent sampie is a portion of a single sample or pooled samples obtained from, the subject.
26. A cell-based method for identifying an agent which inhibits a cancer, the method comprising;
a) contacting a ceil expressing at least one biomarker listed in Table 1 with test, agent; and
b) determining the effect of the test agent on the copy number, .level of expression, or level of activity of the at least one biomarker listed in Table 1 to thereby identify an agent that inhibits the cancer.
27. The method of claim 26, wherein said ceils are isolated from an animal model of a cancer.
28. The method of claim 26 or 27, wherein said cells are from a subject: afflicted with a cancer.
29. The method of any one of claims 26-28, wherein said ceils are unresponsive to ISC biosynthesis pathway inhibitor therapy.
30. The method of any one of claims 26-29, wherein the step of contacting occurs in vivo, ex vivo, or in vitro.
31. The method of any one of claims 26-30, further comprising determining the ability of the test agen t to bind to the at least one biomarker listed in Table i before or after determining the effect of the test agent on the copy number, level of expression, or level of activity of the at least one biomarker listed in Table 1.
32, The method of any one of claims 12- 1, wherein the sample comprises ceils, cell hues, histological slides, paraffin embedded tissue, fresh frozen tissue, fresh tissue, biopsies, blood, plasma, serum., buccal scrape, saliva, cerebrospinal fluid, urine, stool, mucus, or bone marrow, obtained from the sub ject.
33, A ceil-free method for identifying a compound which inhibits a cancer, the method comprising:
a) determining the effect of a test compound on the amount or activity of at least one biomarker listed in Table 1 contacted with a test compound;
b) determining the amount or activity of the at least one biomarker listed in Table 1 maintained in the absence of the test compound; and
c) comparing the amount and/or activity of the at least one biomarker listed in Table 1 from steps a) and b), wherein a significantly increased amount, and/or activity of the at least one biomarker listed in Table 1 in step a) relative to step b), identifies a compound which inhibits the cancer.
34, The method of claim 33, further comprising determining the ability of the test compound to hind to the at least one biomarker listed in Table 1 betore or after determining the effect of the test compound on the amount or activity of the at least one biomarker,
35, The method of claim 33 or 34, wherein steps a) and b) are selected from the group consisting of a methylene blue assay, a 7-azido-4-raethylcourriarin (A C) assay, an alanine assay, and a mass spectrometry assay,
36, The method of claim 35, wherein the methylene blue assay comprises i) reacting the at least one biomarker listed in Tabic 1 in a buffer comprising a) cysteine, b) a pyridoxai. phosphate cofactor, and c) optionally the test compound; it) stopping the reaction by adding , -difflethyl-p-phenylenediamine and iron chloride (FeCB) in hydrogen chloride (HCl) solution, and iii) determining the production of methylene blue via absorbartce of light having a wavelength of 670 nm.
37. The method of claim 35„ wherein the AzMC assay comprises i) reacting the at least one biomarker listed in Table i in a buffer comprising a) cysteine,, b) a pyridoxal phosphate cofactor, c) glutathione as reducing agent, d) bovine serum albumin, e) 7-azido-4- methylcoumarin, and f) optionally, the test compound; and ii) fluoromefrically monitoring the reaction product, 7-amino-4-methyleoumarin.
38. The method of claim 35, wherein the alanine assay comprises i) reacting the at least one biomarker listed i Table 1 in a buffer comprising a) cysteine, b) a pyridoxal ptiosphate cofactor, c) DTT as reducing agent, and d) optionally, the test compound; ii) performing a secondary reaction to measure alanine production in a buffer containing a) NAD
{nicotinamide adenine drnueJeotide) and b) alanine dehydrogenase enzyme; and tii) fluorometrica!ly measuring the reaction product, ADH.
39. The method of claim 35, wherein the mass spectrometry assay comprises i) reacting the at least one biomarker listed in Table .1 in a buffer comprising a) cysteine, b) a pyridoxal phosphate cofactor, and c) optionally the test compound; and ii) determining the production of alanine using mass spectrometry.
40. The method of any one of claims 1.2-39, wherein the copy number is assessed by microarray. quantitative PGR (qPCR). high-throughput sequencing, comparative genomic hybridization (CGH), or fluorescent in situ hybridization (FISH).
41. The method of any one of claims 12-39, wherein the amount of the at least one biomarker is assessed by detecting the presence in the samples of a. polynucleotide molecule encoding the biomarker or a portion of said polynucleotide molecule.
42. The method of claim 41 , wherein the polynucleotide molecule is a mR A, cDNA, or functional variants or fragments thereof
43. The method of claim 41 , wherein the step of detecting further comprises amplifying the polynucleotide molecule,
44. The method of any one of claims 12-43, wherein the amount of the at least one biomarker is assessed by annealing a nucleic acid probe with the sample of the
polynucleotide encoding the one or more biomarkers or a portion of said polynucleotide molecule under stringent 'hybridization conditions.
45. The method of any one of claims 12-39, wherein the amount of the at least one biomarker is assessed b detecting the presence a polypeptide of the at least one biomarker.
46. The method of claim 5, wherein the presence of said polypeptide is detected using a reagent which specifically binds with said polypeptide.
47. The method of claim 46, wherein the reagent is selected from the grou consisting of an antibody, art antibody derivative, and an antibody fragment.
48. The method of any one of claims 12-39, wherein the activit of the at least one biomarker is assessed by determining the magnitude of modulation of at least one NFS ! pharmacodynamic biomarker listed in Table 1,
49. The method of any one of claims 12-39, wherein the activity of the at least one biomarker is assessed by determining the magnitude of modulation of the activity or expression level of at least one downstream target of the at least one biomarker,
50. The method of any one of claims 1 -49, wherein the ISC biosynthesis pathway inhibitor agent or test compound modulates a biomarker selected from the group consisting of human NFS.l , human LYRM4, human ISCU, human FXN, human NFUl, human
GLRXS, human BOLA3, human liSCB, human HSPA9, human 1SCA1 , human ISCA2, human ΪΒΑ57, human NUBPL, human SLC25A28, human FDXR, human FDX2, and ortho!ogs of said hiomarkers thereof.
51. The method of any one of claims 1-50, wherein the ISC biosynthesis pathway inhibitor agen t or test compound is an inhibitor selected from the group consisting of a small molecule, antiseuse nucleic acid, interfering R A, shR A, siR A, aptamer, rtbozyme, dominant-negative protein binding partner, and combinations thereof
52. The method of any one of claims 1-51 , wherein the at least one biomarke is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, or .more hiomarkers.
53. The method of any one of claims 1 -52, wherein the at least one biomarker is selected from the group of ISC biosynthesis pathway hiomarkers listed in Tabic Ϊ .
54. The method of claim 53„ wherein the iSC biosynthesis pathway biomarkers listed in Table 1 are seiected from the group consisting of human NFSi , human LYRM4„ human iSCU, human FXN, human NFti l, human GLRX5, human BOLA3, human HSCB, human HS.P 9, human JSCA1, human ISCA2, human ΪΒΑ57, human NIJBPL, human SLC25A28, human FDXR, human FDX2, and orthoiogs of said biomarkers thereof.
55. The method of any one of claims 1 -52, wherein the at least one biotnarker is selected from the group of NFS 1 pharmacodynamic biomarkers listed in Table I .
56. The method of claim 5.5, wherein the NFS1 nbanmcodynamic biomarkers listed in Table 1 are selected from the group consisting of human aconitase, human succinate dehydrogenase, human ferritin,, human transferrin -receptor, human Hi†2alphaf human PTGS2, and lipid reactive oxygen species (ROS).
57. The method of any one of claims ί -56, wherein the cancer is selected from the group consisting of paragangliomas, colorectal cancer, cervical cancer, lung
adenocarcinoma, ovarian cancer, and myeloid cancer within a hypoxic tumor
microenvironraent.
58. The method of any one of claims I -57, wherein the subject is a mammal
59. The method of claim 58, wherein the mammal is an animal model of cancer.
60. The method of claim 58, whereio the mammal is a human.
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US11060149B2 (en) 2014-06-18 2021-07-13 Clear Gene, Inc. Methods, compositions, and devices for rapid analysis of biological markers
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