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WO2017193141A1 - Biomarqueurs de pronostic et compositions antitumorales de traitements thérapeutiques ciblés pour un cancer du sein triple négatif - Google Patents

Biomarqueurs de pronostic et compositions antitumorales de traitements thérapeutiques ciblés pour un cancer du sein triple négatif Download PDF

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WO2017193141A1
WO2017193141A1 PCT/US2017/031624 US2017031624W WO2017193141A1 WO 2017193141 A1 WO2017193141 A1 WO 2017193141A1 US 2017031624 W US2017031624 W US 2017031624W WO 2017193141 A1 WO2017193141 A1 WO 2017193141A1
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dedd
tnbc
inhibitor
expression
protein
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Siyuan Zhang
Yingjia NI
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates generally to the field of ER-negative breast cancers, and methods for treating a subject identified ER-negative breast cancer and TNBC.
  • the invention also relates to the field of genetic markers, as a DEDD biomarker for use in the selection of appropriate treatment modalities for an ER- negative patient and/or TNBC patient, as well as for use in the selection/screening of therapeutic treatments for these diseases, are provided.
  • TNBC Triple negative breast cancer
  • ER estrogen receptor
  • PR progesterone hormone receptor
  • HER2 human epidermal growth factor receptor 2
  • TNBC Because of absence of both hormones receptors (ER " , PR) and lack of HER2 (HER), women with TNBC do not benefit from endocrine therapies or HER2 inhibitors such as trastuzumab.
  • Current treatment strategy for TNBC patients is mainly chemotherapy with standard cytotoxic agents, although they have worse outcome after chemotherapy compared to patients with breast cancers of other subgroups. Due to its heterogeneous molecular profiling, there is no well-established etiology or target therapy for TNBC.
  • the epidermal growth factor receptor is a key member of the ErbB family of receptors, a subfamily of four closely related receptor tyrosine kinases (RTK). Activation of EGFR stimulates its downstream signaling like MAPK, Akt and JNK pathways, leading to DNA synthesis and cell proliferation.
  • EGFR amplification or hyper-activity has been linked to the growth of a number of human epithelial malignancies, including head and neck squamous- cell carcinoma (FfNSCC), non-small-cell lung carcinoma (NSCLC), metastatic colorectal cancer (CRC), TNBC, pancreatic cancer and brain cancer.
  • FfNSCC head and neck squamous- cell carcinoma
  • NSCLC non-small-cell lung carcinoma
  • CRC metastatic colorectal cancer
  • TNBC pancreatic cancer and brain cancer.
  • Anti-EGFR treatment has been reported to confer clinical benefit with improved symptoms in patients with NSCLC, metastatic CRC and HNSCC.
  • TNBC patients frequently having
  • TNBC has the worse prognosis and tends to relapse early and aggressively compared with other subtypes of breast cancer 2 . Because of the absence of hormone receptors for ER and PR, and the absence of HER 2 , women with TNBC do not benefit from endocrine therapies or HER2 inhibitors such as trastuzumab 3 .
  • Current treatment strategy for TNBC patients is mainly chemotherapy with standard cytotoxic agents. These patients have a worse outcome after chemotherapy compared to patients with breast cancers of other subgroups 4,5 . However, due to its heterogeneous molecular profiling, there is no well-established etiology or target therapy for TNBC.
  • EGFR epidermal growth factor receptor
  • RTK receptor tyrosine kinases
  • Activation of EGFR stimulates its downstream signaling like MAPK, Akt and JNK pathways, leading to DNA synthesis and cell proliferation.
  • EGFR amplification or hyper-activity has been linked to the growth of a number of human epithelial malignancies, including head and neck squamous-cell carcinoma (HNSCC), non-small-cell lung carcinoma (NSCLC), metastatic colorectal cancer (CRC), TNBC, pancreatic cancer and brain cancer 6 .
  • HNSCC head and neck squamous-cell carcinoma
  • NSCLC non-small-cell lung carcinoma
  • CRC metastatic colorectal cancer
  • TNBC pancreatic cancer and brain cancer 6 .
  • Anti-EGFR treatment has been reported to confer some clinical benefit in patients with NSCLC, metastatic CRC and HNSCC, particularly in previously treated cases 7 .
  • DEDD Death domain containing DNA binding protein
  • DEDD has not been specifically implicated in the prognosis or pathology of the progression of cancer generally, or breast cancer in particular.
  • DEDD is recognized to induce autophagy and mild apoptosis through binding to other cell death pathway regulators.
  • DEDD has the potential to act as a tumor suppressor and inhibit tumor growth 14,15 .
  • the Retinoblastoma gene is a tumor suppressor gene that was first identified in a malignant tumor of the retina known as retinoblastoma. Since its encoded protein has been identified as a universal cell cycle regulator with central role in controlling the commitment of a cell to initiate DNA replication and divide which are the critical events triggering tumor outgrowth, studies have focused on the mechanisms of Rb protein on cell cycle regulation 18 .
  • Rb protein is responsible for a major Gl checkpoint (restriction point) blocking S-phrase entry and cell growth, promoting terminal differentiation by inducing both cell cycle exit and tissue- specific gene expression 19 .
  • Rb protein is hypophosphorylated in resting GO cells which allows Rb protein bind to E2F transcription factors.
  • Rb-E2F interaction blocks the transcriptional activity of E2F factors, and in some cases, it converts E2F to transcriptional repressors.
  • Rb protein is increasingly phosphorylated by cyclin/cdk complexes during the cell cycle progression through Gl and is maintained in a hyperphosphorylated state until late mitosis 19"21 .
  • the gradually increased phosphorylation of Rb protein makes it unable to bind to E2F transcription factors, which allows E2F be activated and cell cycle progresses.
  • Post-translational modifications play a critical role in modulating the activities of tumor suppressors.
  • a variety of amplified E3 ubiquitin ligases in cancers have been reported down- regulating tumor suppressors such as p53 or PTEN through ubiquitination- dependent degradation mechanisms 28"31 .
  • Rb protein is the first reported human tumor suppressor and its functions on cell cycle regulation and proliferation have been well established, the post-translational regulation of Rb protein has yet to be well addressed 19 ' 20 . It has been reported that viral protein have the capability to interact with and degrade Rb protein 32"34 .
  • Despite frequent genetic deletions of Rb protein expression in human tumors only 15% of breast cancer patients show deletions or downregulation of Rb gene expression 16 ' 17 .
  • the present invention provides a prognostic clinical tool that employs the level of DEDD gene expression (overexpression) in a subject identified to have an ER " breast cancer, such as a triple negative breast cancer (TNBC), to identify and select a subject having a higher probability of demonstrating reduced tumor growth or inhibition of cancer progression in response to a defined therapy.
  • the therapy may comprise an EGFR inhibitor (anti-EGFR agent), a CDK-4/6 inhibitor (anti-CDK-4/6 agent), or a combination of both of these, in a pharmaceutically acceptable carrier. The combination is shown to have synergistic effects for inhibiting tumor growth.
  • a method of using the clinical tool comprises selecting a treatment for an ER " breast cancer subject, such as a TNBC subject, having an elevated DEDD expression level, the treatment comprising a combinatorial treatment of an EGFR inhibitor (anti-EGFR agent) and a CDK-4/6 inhibitor (anti-CDK-4/6 agent).
  • a treatment for an ER " breast cancer subject such as a TNBC subject, having an elevated DEDD expression level
  • the treatment comprising a combinatorial treatment of an EGFR inhibitor (anti-EGFR agent) and a CDK-4/6 inhibitor (anti-CDK-4/6 agent).
  • the invention provides a method for screening agents for treating a TNBC patient using a TNBC cell that has been resensitized to EGFR.
  • the resensitized TNBC cell line is a prepared as a knockdown of a DEDD gene in a TNBC cell by a lentivirus transfection of anti-DEDD shRNAs, providing transfected TNBC cells that are sensitive to anti-EGFR therapy.
  • the TNBC cells may comprise a TNBC cell line, such as a TNBC cell line HCC1806, HCC38 and MDA-MB-468.
  • a biomarker for breast cancer provides for the identification of a patient and/or subject having a high (at least 50%, or 60% or 70% or greater) probability of positively responding to a combinatorial therapy of an EGFR inhibitor and a CDK-4/6 inhibitor.
  • the biomarker will identify a TNBC patient or subject, or other ER " patient and/or subject, having a high (at least 50%, or 60% or 70% or greater) probability of positively responding to a combinatorial therapy of an EGFR inhibitor and a CDK-4/6 inhibitor, in a pharmaceutically acceptable carrier.
  • the breast cancer biomarker is an ER ⁇ breast cancer biomarker.
  • the breast cancer biomarker is a TNBC biomarker.
  • the breast cancer biomarker is a DEDD biomarker.
  • the breast cancer biomarker is further described as comprising an elevated expression level of a Death domain containing DNA binding protein (DEDD) that is at least two (2)- fold greater than the DEDD expression level of a reference triple negative breast cancer (TNBC) population.
  • DEDD Death domain containing DNA binding protein
  • the breast cancer biomarker may be described as an elevated expression level of a DEDD gene that is at least two (2) standard deviations (SD) away from (greater) than a mean DEDD expression level of a reference TNBC population.
  • the enhanced DEDD expression level may be an mRNA level.
  • the enhanced DEDD expression level may be a protein level. Comparative values may be calculated based on the relative DEDD "z" value of the TNBC population, as described herein.
  • a TNBC biomarker standard may be provided according to the present invention on a solid substrate, together with a control marker, as part of a kit for commercial and/or clinical use.
  • An instructional insert may also be included.
  • kits for assessing DEDD overexpression is also provided.
  • the kit may be described as a DEDD gene breast disease detection kit comprising a standard, the standard curve to be provided from a volume of a set of Death domain containing DNA binding domain (DEDD) reagents provided with the kit, a volume of a positive control reagent and a volume of a negative control reagent, a volume of an extraction buffer; and an instructional insert.
  • the set of DEDD standard reagents are a set of RNA cell line lysates, having a concentration of about 1 ⁇ g/ ⁇ l RNA, and the extraction buffer is an RNA buffer.
  • the kit may also include qRT-PCR DEDD primers, control primers and an enzyme master mix.
  • the set of cell line lysates are made up of a lysate of each of the cell lines MDA-MB-231, BT20, HCC1806, MDA-MB-468, MBA-MB-436, and HCC38.
  • the DEDD gene breast disease detection kit is a DEDD protein measurement kit, comprising a set of protein standards to make up a standard curve, a positive control reagent and a negative control reagent.
  • Each protein standard comprising at least 100 ⁇ of DEDD protein.
  • Each protein standard may be prepared from a cell line, such as from each of the cell lines described above. Other cell lines apart from those identified here may also be used to create the lysates for use as part of a standard curve, for assessment of DEDD gene expression and/or protein concentration, according to those techniques known to those of skill ion the art without an undue amount of experimentation and error, given the information provided in the present teachings.
  • Combination Therapies/TNBC Patients, ER " Elevated DEDD Expression Levels Novel combinatorial therapy preparations of anti-EGFR and anti-CDK4/6 inhibitors in a pharmaceutically acceptable carrier are provided.
  • the combinatorial therapy preparations dramatically suppress cancer and/or tumor cell proliferation, presenting a reduction in tumor size in TNBC tumor tissue.
  • a pharmaceutical preparation comprising a combination of a CDK-4/6 inhibitor and an EGFR-inhibitor in a pharmaceutically acceptable carrier for use in a TNBC subject is provided.
  • the CDK-4/6 inhibitor is abemaciclib, Palbociclib, Ribociclib, Abemacicilib, Trilaciclib, or a combination thereof.
  • the EGFR inhibitor is Lapatinib, Erlotinib, Gefitinib, Osimertinib,
  • Vandetanib Afatinib, Neratinib, Olmutinib, Canertinib, Dacomitinib, Sapitinib, Pelitinib, - Icotinib, or a combination thereof.
  • the CDK-4/6 inhibitor is palbociclib and the EGFR inhibitor is lapatinib.
  • the CDK-4 inhibitor is ribociclib and the EGFR inhibitor is lapatinib.
  • the CDK-4/6 inhibitor is abemaciclib and the EGFR inhibitor is lapatinib.
  • the preparations may further include other chemotherapeutic agents, sterilants, salts, stabilizers, and the like, common to the preparation of pharmaceutical preparations, and/or for the use in the treatment of a cancer patient.
  • the present invention also provides a method for selecting a therapy for a breast cancer subject and/or patient, particularly an ER " , PR " and HER " subject, such as a TNBC subject/patient
  • the method comprises comparing a level of DEDD expression in a specimen from the breast cancer subject to a TNBC reference level of DEDD expression, said TNBC reference level derived from a TNBC population of DEDD measurements, and then selecting a breast cancer subject having a DEDD expression level that is higher than the reference TNBC DEDD expression level, for treatment.
  • the treatment to be provided to the selected breast cancer subject may comprise a pharmaceutical preparation of an EGFR inhibitor or a combination of a CDK-4/6 inhibitor and an EGFR inhibitor, so as to provide a treated breast cancer subject.
  • tumor size by volume in the treated breast cancer subject may be reduced at least about 25%, or 30% or up to about 300%, compared to tumor size in a breast cancer subject having a similarly high DEDD expression level not provided the treatment.
  • a higher level of DEDD gene expression in a selected breast cancer patient such as a selected TNBC patient, can be identified by reference to the patient /subject "z" score for DEDD expression, where the patient "z" score is two (2) standard deviations (SD) or greater than (away from) (even as much as 4 SD away from) a reference TNBC population z score.
  • SD standard deviation
  • a standard deviation (SD) is 0.54 as calculated for the MDA-M-231 DEDD z-score.
  • the pharmaceutical preparation of the treatment comprises a combination of the CDK-4/6 inhibitor ribociclib and the EGFR inhibitor lapatinib, and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable preparation is a combination of the CDK-4/6 inhibitor palbociclib and the EGFR inhibitor lapatinib, and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable preparation may comprise a CDK-4/6 inhibitor abemaciclib, and the EGFR inhibitor Lapatinib.
  • the pharmaceutically acceptable preparation may comprise an EGFR inhibitor such as Lapatinib, Erlotinib, Gefitinib, Osimertinib, Vandetanib, Afatinib, Neratinib, Olmutinib, Canertinib, Dacomitinib, Sapitinib, Pelitinib, Icotinib, or a combination thereof.
  • an EGFR inhibitor such as Lapatinib, Erlotinib, Gefitinib, Osimertinib, Vandetanib, Afatinib, Neratinib, Olmutinib, Canertinib, Dacomitinib, Sapitinib, Pelitinib, Icotinib, or a combination thereof.
  • the CDK-4 inhibitor may comprise abemaciclib, Palbociclib,
  • tumor growth in the treated TNBC subject is reduced and/or inhibited 4-fold to 20-fold compared to tumor growth in a TNBC subject having a similarly high DEDD expression level not provided the treatment.
  • a method of treating a TNBC subject comprises assaying a breast cancer sample derived from a triple negative breast cancer subject to determine the level of expression in said sample of a DEDD biomarker, detecting a high expression level of said biomarker in said sample relative to a TNBC reference DEDD expression level; and administering a therapeutically effective amount of a CDK-4 inhibitor, an EGFR-inhibitor, or a combination of a CDK-4 inhibitor and an EGFR inhibitor, to said subject.
  • a TNBC subject is defined as a subject/patient that is Estrogen Receptor (ER) negative, Progesterone Receptor (PR) negative, and HER-2 negative.
  • the level of expression of the DEDD biomarker is determined at the nucleic acid level.
  • the level of expression of the DEDD biomarker is determined by detecting cDNA, mR A, miRNA or DNA.
  • the level of expression of the biomarker may be determined by using a technique selected from the group consisting of polymerase chain reaction (PCR) amplification reaction, reverse-transcriptase PCR analysis, quantitative reverse-transcriptase PCR analysis, Northern blot analysis, RNAase protection assay, digital RNA detection/quantitation, and combinations or sub-combinations thereof.
  • PCR polymerase chain reaction
  • the level of expression of the DEDD biomarker may be determined at the protein level
  • the presence of the protein may be detected using an antibody or antigen binding fragment thereof, which specifically binds to the protein.
  • the antibody or antigen binding fragment thereof may be selected from the group consisting of a murine antibody, a human antibody, a humanized antibody, a bispecific antibody, a chimeric antibody, a Fab, Fab', F(ab').sub.2, ScFv, SMIP, affibody, avimer, versabody, nanobody, a domain antibody, and an antigen binding fragment of any of the foregoing.
  • the antibody or antigen binding fragment thereof is labeled.
  • the label may be any number of labels known to those of skill in the art, such as a radio-label, a biotin-label, a chromophore-label, a fluorophore-label, and an enzyme-label.
  • inventions of the method may provide for the measurement of the level of expression of the DEDD biomarker by using a technique such as an immunoassay, a western blot analysis, a radioimmunoassay, immunofluorimetry, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence immunoassay (ECLIA), ELISA assay, immunopolymerase chain reaction and combinations or sub-combinations thereof.
  • a technique such as an immunoassay, a western blot analysis, a radioimmunoassay, immunofluorimetry, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence immunoassay (ECLIA), ELISA assay, immunopolymerase chain reaction and combinations or sub-combinations thereof.
  • the immunoassay may be described as (a) a solution-based immunoassay selected from the group consisting of electrochemiluminescence, chemiluminescence, fluorogenic chemiluminescence, fluorescence polarization, and time-resolved fluorescence; or (b) a sandwich immunoassay selected from the group consisting of electrochemiluminescence, chemiluminescence, and fluorogenic chemiluminescence.
  • the invention provides pharmaceutically acceptable combinatorial preparations of a CDK-4/6 inhibitor, an EGFR-inhibitor and a pharmaceutically acceptable carrier.
  • the pharmaceutical preparation comprises a combination of a CDK-4/6 inhibitor and an EGFR-inhibitor in a pharmaceutically acceptable carrier for use in a selected ER " subject, such as a TNBC subject.
  • the CDK-4/6 inhibitor comprises abemaciclib, Palbociclib, Ribociclib, Abemacicilib, Trilaciclib, or a combination thereof.
  • the EGFR inhibitor comprises Lapatinib, Erlotinib, Gefitinib, Osimertinib, Vandetanib, Afatinib, Neratinib, Olmutinib, Canertinib, Dacomitinib, Sapitinib, Pelitinib, Icotinib, or a combination thereof.
  • the pharmaceutical preparation comprises the CDK-4/6 inhibitor palbociclib and the EGFR inhibitor lapatinib. In yet another embodiment, the pharmaceutical preparation comprises the CDK-4/6 inhibitor ribociclib and the EGFR inhibitor lapatinib. In another embodiment, the pharmaceutical preparation comprises the CDK-4/6 inhibitor abemaciclib and the EGFR inhibitor lapatinib.
  • IB - Western blots show EGFR as well as Akt and Erk (two primary downstream signaling of EGFR) expression in TNBC cell lines 2 hours post-treatment of Lapatinib.
  • 1C Flow scheme of a genome-wide barcode shRNA screening procedure.
  • IE - qRT-PCR assay shows DEDD mRNA expression levels 2 weeks after Lentiviral infection of different anti-DEDD shRNAs separately in TNBC cell lines.
  • IF - Cell counting assay shows cell viability of DEDD knockdown TNBC cell lines under 6 days lapatinib (2 ⁇ ) treatment.
  • Figure 3 A - 3B; 3 A - Western blots show retinoblastoma protein expression 6 hours after FBS stimulation in DEDD knockdown HCC1806 cells.
  • 3B - Western blots show Rb protein expression on Ohr, 4hr, 7hr and lOhr under MG-132 treatment in DEDD knockdown HCC1806 cells.
  • EGFR/HER2 targeted therapy 4A - Schematic representation of the synthetic lethal dropout screen using barcoded shRNA under EGFR inhibitor Lapatinib treatment (LAP). Triple negative breast cancer (TNBC) cell line HCC1806 with EGFR overexpression was used for the shRNA drop-out screen. Decipher Lentiviral shRNA Library Screening. 4B - Barcoded shRNAs differentially expressed between DMSO and Lapatinib treated HCC1806 cells. Individual bars represent the difference in expression in DMSO versus Lapatinib treated HCC1806 cells for a barcoded shRNA.
  • LAP Lapatinib treatment
  • TNBC Triple negative breast cancer
  • Barcoded shRNAs that were depleted in Lapatinib treated HCC1806 cells compared to DMSO treated HCC1806 cells were identified as synthetic lethal (bottom side of y axis, blue bars). Barcoded shRNAs whose expression decreased by 2-fold or greater upon treatment with Lapatinib were considered as drop-out hits (colored yellow). SI. Log2 fold change between barcoded shRNAs expression of DMSO and Lapatinib groups is shown along the x axis). 4C - Expression of Top 10 genes among 95 drop-out hits that overexpressed in the Cancer Genome Atlas (TCGA) clinical basal-like breast carcinomas tissues. Clinically, the basal-like breast cancer are mostly TNBC.
  • TCGA Cancer Genome Atlas
  • DEDD is overexpressed in 78% of 107 PAM50 basal-like breast cancer patients.
  • PAM50 basal type breast invasive carcinoma TCGA, Cell 2015, n-107). 4D - DEDD genomic amplification at chromosome lq23.3 in basal-lie (TNBC) breast cancer patient tissue samples.
  • TCGA breast invasive carcinoma segmented copy number (delete germline CNV). 4E - Cell counting assay validating knockdown DEDD sensitizes TNBC cell line to Lapatinib (LAP) treatment. TNBC cell lines treated with Lapatinib (2 ⁇ ) for 6 days. Error bars represent means ⁇ s.e.m. All quantitative data were generated from a minimum of three replicates.
  • LAP Lapatinib
  • FIG. 5A-5G Cytosolic DEDD drives a non-oncogene addiction to Gl/S cell cycle transition in TNBC cells: 5A - Relative mRNA expression level of TNBC lines. mRNA expression levels were extracted from Cancer Cell Line Encyclopedia (Novartis/Broad, Nature 2012, cBioportal). ESRl: estrogen receptor; PGR: progesterone receptor. 5B - Cell proliferation assay examining TNBC cell viability after DEDD knockdown. TNBC cells with overexpressed DEDD (HCC38 and MDA-MB-468) are more sensitive to DEDD depletion by shRNA DEDD (shDEDD-1, shDEDD-2).
  • 5C EdU-incorporation flow cytometry (FACS) assay examining the Gl to S transition after DEDD knockdown. FACS showing a decrease of S-phase (EdU+) cell after shDEDD. 5D - Representative image showing decreases of EdU incorporation after shDEDD in HCC38 and MDA-MB-468 cells. 5E - Quantification of EdU incorporation assay. 5F: Schematic for mutation of three nuclear localization signal (NLS) on DEDD gene. 5G - EdU-incorporation flow cytometry (FACS) assay showing the cell cycle changes after overexpression of DEDD with dominate-negative DEDD.NLS (ANLS). Overexpression of ANLS promotes Gl-S transition. This increased Gl-S transition can be reserved by shDEDD.
  • NLS nuclear localization signal
  • FIG. 6A - 6F DEDD mediated Rb degradation and HSC70 recruitment facilities Gl/S transition: 6A - Western blot examining the effect of knocking DEDD in TNBC cells on Rb protein level.
  • shDEDD leads to accumulation of the protein levels of phosphorylated-Rb and total Rb.
  • 6B Time-course western blot assay examining the Rb protein level changes after shDEDD under treatment of proteasome inhibitor MG-132.
  • shDEDD abolishes the Rb accumulation.
  • 6C DEDD dose-dependently induces Rb proteasome-dependent degradation.
  • FALG-DEDD or FALG-ANLS-DEDD was expressed in 293FT cells at different doses.
  • 6D Co-immunoprecipitation assay showing the protein-protein interaction between ANLS-DEDD and Rb protein.
  • 6E Overexpression of FALG-ANLS-DEDD leads to an increased ubiquitination of Rb. Total Rb protein was immunoprecipitated and ubiquitination of Rb protein was examined by anti-ubiquitin (Ubi) western blot.
  • 6F Co-immunoprecipitation assay showing the protein- protein interaction between ANLS-DEDD and HSC70 protein in Rb-deficient TNBC cells.
  • FIG. 7A - 7G Combination of Lapatinib and CDK4/6 inhibitor suppresses TNBC tumor progression.
  • 7A Cell proliferation assay examining the cell viability changes after EGFR inhibitor lapatinib (LAP), CDK4/6 inhibitor Abemaciclib (ABE) or combination treatment (Combo).
  • 7B Heatmap showing the DEDD and EGFR gene expressions in TNBC cell lines corresponding to their response to Combo treatment.
  • 7D Western blot showing the time-dependent impact of the drug treatments on the major cell signaling pathways.
  • Combo treatment synergistically inhibits phosphorylation of Akt.
  • 7E Mammary fad pad xenograft in vivo tumorigenic assay showing tumor growth over 15 days of drug treatments. Top: representative photos showing the tumor size at day 15; bottom: tumor growth curve demonstrates Combo treatment significantly suppresses TNBC HCC-1806 tumor growth (p ⁇ 0.01).
  • 7F Immunohistochemistry (IHC) analysis of tumor cell proliferation (Ki67) and tumor cell apoptosis (cleaved caspase-3).
  • TNBC PDX HCI-001 tumors were transplanted to NOD.SCID mammary fat pad. Once tumor established, the xenograft tumor received daily single treatment or combination treatment for 10 days. LAB: 2mg/day; ABE: 0.5mg/day. Left: IHC analysis of DEDD expression in PDX tumors. Right: Tumor volume changes after 10 days of treatment.
  • FIG. 8 Enhanced therapeutic efficacy by combination treatment of Lapatinib and LEE011 (Ribociclib).
  • TNBC cell lines HCC-38 and MDA-MB-468 were treated with Lapatinib (5 DM), LEE011 (20 DM), or combination (combo) treatment for 48 hours. Cell viability were quantified by MTT assay.
  • FIG. 9 Enhanced therapeutic efficacy by combination treatment of Lapatinib and Palbociclib.
  • TNBC cell lines HCC1806 was treated with Lapatinib (1 DM), Palbociclib (1 DM), or combination (combo) treatment for 4 days.
  • TNBC cell lines MDA-MB-468 was treated with Lapatinib (4 DM), Palbociclib (4 DM), or combination (combo) treatment for 4 days. Cell viability were quantified by MTT assay.
  • patient or “subject” means an individual having symptoms of, or at risk for, cancer or other malignancy.
  • a patient may be human or non-human and may include, for example, animal strains or species used as "model systems" for research purposes, such a mouse model.
  • a patient or subject may include either adults or juveniles (e.g., children).
  • a patient or subject may mean any living organism, preferably a mammal (e.g., human or non-human) that may benefit from the administration of compositions contemplated herein.
  • prognose means predictions about or predicting a likely course or outcome of a disease or disease progression, particularly with respect to a likelihood of, for example, disease remission, disease relapse, disease progression including tumor recurrence, tumor reduction, tumor metastasis and cancer- attributable death (i.e., the outlook for chances of survival), as well as drug resistance of a neoplastic disease.
  • good prognosis or “favorable prognosis,” or like terms, means a likelihood that a patient or subject having a particular cancer, particularly breast cancer, will demonstrate a reduction in tumor size, tumor occurrence, and/or will be disease-free (i.e., cancer-free).
  • poor prognosis or “bad prognosis,” or like terms, means a higher probability or likelihood, or risk, that a patient or subject will experience a cancer relapse or recurrence of an underlying cancer or tumor, increased growth and/or volume in size of an existing or new tumor, a cancer/tumor metastasis or death.
  • a patient or subject classified as having a good prognosis tends to experience a reduction in existing tumor size and/or become free of any existing tumors, or to even become free of the underlying cancer.
  • patients classified as having a bad prognosis tend to experience disease relapse, tumor recurrence and/or tumor growth, metastasis and/or death.
  • prediction means the likelihood that a patient may respond favorably or unfavorably to a therapeutic or therapeutic combination, and also the extent of those responses, or that a patient will survive, following surgical removal of a primary tumor and/or chemotherapy for a certain period of time, without a significant risk of cancer recurrence.
  • the predictive methods described herein can be used clinically to make treatment decisions by facilitating the most appropriate treatment modalities for an individual patient based on molecular genetic factors, such as, for example, expression levels of DEDD.
  • ⁇ means within a statistically meaningful range of a value or values such as a stated concentration, length, molecular weight, pH, sequence identity, time frame, temperature or volume. Such a value or range can be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by “about” will depend upon the particular system under study, and can be readily appreciated by one of skill in the art.
  • tumor means neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and “cancerous” mean a physiological condition in mammals that typically is characterized by unregulated cell growth. Of particular interest is breast cancer.
  • biomarker refers generally to a molecule or substance that is an indicator of a biologic state.
  • the biomarker can be a gene or gene product (protein) that serves as a predictive marker for patient response or resistance to a drug. More specifically, the biomarker is a patient sample expression level of a gene, the DEDD gene, that serves as a predictive marker for a response or resistance to a drug or combination of drugs, in a triple negative breast cancer (TNBC) patient.
  • TNBC triple negative breast cancer
  • the DEDD biomarkers can include polynucleotides comprising the entire or partial sequence of the nucleotide sequence encoding the biomarkers, or the complement of such sequences.
  • polynucleotide means a polymer of nucleic acids or nucleotides that, unless otherwise limited, encompasses naturally occurring bases (i.e., adenine, guanine, cytosine, thymine and uracil) or known base analogues having the essential nature of naturally occurring nucleotides in that they hybridize to single- stranded nucleic acid molecules in a manner similar to naturally occurring nucleotides.
  • nucleic acid polymers of ribonucleotides R A
  • DNA deoxyribonucleotides
  • R A ribonucleotides
  • DNA deoxyribonucleotides
  • the term includes single-stranded nucleic acid polymers, double-stranded nucleic acid polymers, and RNA and DNA made from nucleotide or nucleoside analogues that can be identified by their nucleic acid sequences, which are generally presented in the 5' to 3' direction (as the coding strand), where the 5' and 3' indicate the linkages formed between the 5' hydroxyl group of one nucleotide and the 3 '-hydroxyl group of the next nucleotide.
  • a coding strand presented in the 5 '-3' direction its complement (or non-coding strand) is the strand that hybridizes to that sequence according to Watson-Crick base pairing.
  • the complement of a nucleic acid is the same as the "reverse complement” and describes the nucleic acid that in its natural form, would be based paired with the nucleic acid in question.
  • the gene sequence for the DEDD gene is available in GenBank, and is specifically incorporated herein by reference.
  • nucleic acid As used herein, a "nucleic acid,” “nucleotide” or “nucleic acid residue” are used interchangeably to mean a nucleic acid that is incorporated into a molecule such as a gene or other polynucleotide.
  • the nucleic acid may be a naturally occurring nucleic acid and, unless otherwise limited, may encompass known analogues of natural nucleic acids that can function in a similar manner as naturally occurring nucleic acids.
  • nucleic acids include any of the known base analogues of DNA and RNA such as, but not limited to, 4 acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5 (carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5 carboxymethylaminomethyl-2- thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1 methylinosine, 2,2 dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6- methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5 methoxyaminomethyl-2- thiouracil, beta-D-
  • the biomarkers can include DNA, RNA, cDNA, cRNA, or iR A comprising an entire or partial nucleotide sequence suitable for use as an indicator molecule as provided herein. It is contemplated that in some instances, a native or non-native (modified) amino acid sequences of the biomarker as provided herein may be used.
  • the biomarkers can include not only the entire biomarker sequence but also fragments and/or variants thereof.
  • fragment or “fragments” means a portion of the nucleic or amino acid sequence of the biomarker.
  • Polynucleotides that are fragments of a biomarker nucleic acid sequence generally comprise at least about 10, 15, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,200 or 1,500 contiguous nucleotides, or up to the number of nucleotides present in a full-length biomarker polynucleotide disclosed herein.
  • a fragment of a biomarker polypeptide comprises at least about 15, 25, 30, 50, 100, 150, 200 or 250 contiguous amino acids, or up to the total number of amino acids present in a full-length biomarker protein.
  • variants or “variants” means substantially similar sequences. Generally, variants of a particular biomarker have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity (preferably over the full length) to a biomarker as determined by sequence alignment programs.
  • variants can be constructed via modifications to either the polynucleotide or polypeptide sequence of the biomarker and can include substitutions, insertions (e.g., adding no more than ten nucleotides or amino acid) and deletions (e.g., deleting no more than ten nucleotides or amino acids).
  • substitutions e.g., adding no more than ten nucleotides or amino acid
  • deletions e.g., deleting no more than ten nucleotides or amino acids.
  • the biomarkers of the kits and methods provide for the measure of DEDD gene expression levels in a subject patient test specimen, and provides a specific indication that the particular TNBC subject and/or patient likely or not likely benefit from an anti-CDK-4/6, anti- EGFR, or combined anti-CDK-4/6 and anti-EGFR regimen.
  • a TNBC subject and/or patient will be considered to have a high probability of demonstrating a positive response (such as a reduction in tumor growth or remission of the cancer) from a regimen as described herein where a DEDD expression score from the patient/subject specimen (the test specimen) is high (TNBC sample demonstrates a high, several fold higher, expression level of DEDD gene), compared to a control and/or reference DEDD specimen expression level, using the present kits, methods and compositions of the present technology.
  • a TNBC patient will not be considered to benefit from the regimen where the expression level of the DEDD gene is relatively the same or corresponds to a z score that is not more than about 1 or 2 standard deviations (SD) away from a z score of a reference TNBC population for DEDD gene expression.
  • SD standard deviations
  • kit or “kits” means any manufacture (e.g., a package or a container) including at least one reagent, such as a nucleic acid probe or the like, for specifically detecting the expression of the biomarkers described herein.
  • probe means any molecule that is capable of selectively binding to a specifically intended target biomolecule, for example, a nucleotide transcript or a protein encoded by or corresponding to a biomarker, such as a biomarker for the DEDD gene. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies and organic molecules.
  • the kit in some embodiments includes an instructional insert or instructions for use on a label or other surface available for print on the product.
  • Methods of synthesizing polynucleotides are well known in the art, such as cloning and digestion of the appropriate sequences, as well as direct chemical synthesis (e.g., ink-jet deposition and electrochemical synthesis). Methods of cloning polynucleotides are described, for example, in Copeland et al. (2001) Nat. Rev. Genet. 2:769-779; Current Protocols in Molecular Biology (Ausubel et al. eds., John Wiley & Sons 1995); Molecular Cloning: A Laboratory Manual, 3rd ed. (Sambrook & Russell eds., Cold Spring Harbor Press 2001); and PCR Cloning Protocols, 2nd ed.
  • Methods of direct chemical synthesis of polynucleotides include, but are not limited to, the phosphotriester methods of Reese (1978) Tetrahedron 34:3143-3179 and Narang et al. (1979) Methods Enzymol. 68:90-98; the phosphodiester method of Brown et al. (1979) Methods Enzymol. 68:109-151; the diethylphosphoramidate method of Beaucage et al. (1981) Tetrahedron Lett. 22:1859-1862; and the solid support methods of Fodor et al. (1991) Science 251:767-773; Pease et al. (1994) Proc.
  • Kits can be promoted, distributed or sold as units for performing the methods described below. Additionally, the kits can contain a package insert describing the kit and methods for its use.
  • the insert can include instructions for correlating the level of biomarker expression measured with a patient's likelihood of cancer recurrence, long-term survival, and the like, and select the most appropriate treatment option accordingly.
  • kits therefore can be used for identifying a TNBC patient with biomarkers at the nucleic acid level.
  • kits are compatible with both manual and automated nucleic acid detection techniques (e.g., gene arrays, Northern blotting or Southern blotting).
  • These kits can include a plurality of probes, for example, from 5 to 100 nucleic acid probes that specifically bind to distinct biomarkers, fragments or variants thereof.
  • kits can contain at least 5 probes, at least 10 probes, at least 15 probes, at least 20 probes, at least 30 probes, at least 40 probes, at least 50 probes, at least 80 probes, at least 90 probes, at least 100 probes, at least 110 probes, at least 120 probes, at least 150 probes, or at least 200 probes.
  • kit reagents can be provided within containers that protect them from the external environment, such as in sealed containers.
  • Positive and/or negative controls can be included in the kits to validate the activity and correct usage of reagents employed in accordance with the technology.
  • Controls can include samples, such as tissue sections, cells fixed on glass slides, RNA preparations from tissues or cell lines, and the like, known to be either positive or negative for the presence of at least five different biomarkers. The design and use of controls is standard and well within the routine capabilities of one of skill in the art.
  • sample means any collection of cells, tissues, organs or bodily fluids in which expression of a biomarker can be detected.
  • samples include, but are not limited to, biopsy specimens of cells, tissues or organs, bodily fluids and smears.
  • the sample can include, but is not limited to, breast cells, particularly breast tissue from a biopsy, such as a breast tumor tissue sample.
  • Biopsy specimens can be obtained by a variety of techniques including, but not limited to, scraping or swabbing an area, using a needle to aspirate cells or bodily fluids, or removing a tissue sample. Methods for collecting various samples/biopsy specimens are well known in the art.
  • a breast tissue sample is obtained by, for example, fine needle aspiration biopsy, core needle biopsy, or excisional biopsy. Fixative and staining solutions can be applied to, for example, cells or tissues for preserving them and for facilitating examination.
  • Samples can be transferred to a glass slide for viewing under magnification.
  • the sample is a breast tumor tissue sample, and can be a FFPE breast tumor tissue sample, a fresh breast tumor tissue sample or a fresh frozen breast tissue sample.
  • the breast tissue sample is in some embodiments particularly a primary breast tumor tissue cancer sample.
  • the methods After collecting and preparing the specimen from the patient, the methods then include detecting expression of the biomarkers.
  • detecting expression means determining the quantity or presence of a biomarker polynucleotide or its expression product. As such, detecting expression encompasses instances where a biomarker is determined not to be expressed, not to be detectably expressed, expressed at a low level, expressed at a normal level, or overexpressed.
  • Expression of a biomarker can be determined by normalizing the level of a reference marker/control, which can be all measured transcripts (or their products) in the sample or a particular reference set of RNA transcripts (or their products). Normalization can be performed to correct for or normalize away both differences in the amount of biomarker assayed and variability in the quality of the biomarker type used. Therefore, an assay typically measures and incorporates the expression of certain normalizing polynucleotides or polypeptides, including well known housekeeping genes, such as, for example, GAPDH and/or actin. Alternatively, normalization can be based on the mean or median signal of all of the assayed biomarkers or a large subset thereof (global normalization approach).
  • a sample from the subject of interest were analyzed to determine the level of DEDD expression present. This value obtained with the sample may then be compared against a reference value for DEDD expression calculated from a population of TNBC patients.
  • determining biomarker overexpression requires no comparison between the sample and a corresponding sample that originated from a population of TNBC patients. For example, detecting overexpression of a DEDD biomarker indicative of a poor prognosis as determined using a calculated mean value for DEDD obtained for this population of TNBC subjects may preclude the need for a separate numerical comparison step.
  • Methods of detecting and quantifying polynucleotide biomarkers in a sample are well known in the art. Such methods include, but are not limited to gene expression profiling, which are based on hybridization analysis of polynucleotides, and sequencing of polynucleotides.
  • gene expression profiling which are based on hybridization analysis of polynucleotides, and sequencing of polynucleotides.
  • the most commonly used methods art for detecting and quantifying polynucleotide expression in include northern blotting and in situ hybridization (Parker & Barnes (1999) Methods Mol. Biol. 106:247-283), RNAse protection assays (Hod (1992) Biotechniques 13:852-854), PCR-based methods, such as RT-PCR (Weis et al.
  • OLISA oligonucleotide-linked immunosorbent assay
  • RNA extraction from paraffin-embedded tissues also are well known in the art. See, e.g., Rupp & Locker (1987) Lab Invest. 56:A67; and De Andres et al. (1995) Biotechniques 18:42-44.
  • isolation/purification kits are commercially available for isolating polynucleotides such as RNA (Qiagen; Valencia, CA). For example, total RNA from cells in culture can be isolated using Qiagen RNeasy® Mini-Columns. Other commercially available RNA isolation/purification kits include MasterPureTM Complete DNA and RNA Purification Kit (Epicentre; Madison, WI.) and Paraffin Block RNA Isolation Kit (Ambion; Austin, TX). Total RNA from tissue samples can be isolated, for example, using RNA Stat-60 (Tel-Test; Friendswood, TX). RNA prepared from a tumor can be isolated, for example, by cesium chloride density gradient centrifugation. Additionally, large numbers of tissue samples readily can be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski (US Patent No. 4,843, 155).
  • the polynucleotide such as mRNA
  • hybridization or amplification assays including, but not limited to, Southern or Northern blotting, PCR and probe arrays.
  • One method of detecting polynucleotide levels involves contacting the isolated polynucleotides with a nucleic acid molecule (probe) that can hybridize to the desired polynucleotide target.
  • probe nucleic acid molecule
  • the nucleic acid probe can be, for example, a full-length DNA, or a portion thereof, such as an oligonucleotide of at least about 10, 15, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400 or 500 nucleotides or more in length and sufficient to specifically hybridize under stringent conditions to a polynucleotide such as an mRNA or genomic DNA encoding a biomarker of interest. Hybridization of a polynucleotide encoding the biomarker of interest with the probe indicates that the biomarker in question is being expressed.
  • Stringent hybridization conditions typically include low ionic strength and high temperature for washing and can be defined as hybridizing at 68°C in 5x SSC/5x Denhardt's solution/1.0% SOS, and washing in 0.2x SSC/0.1 % SOS +/- 100 ⁇ / ⁇ _1 denatured salmon sperm DNA at room temperature (RT).
  • Moderately stringent hybridization conditions include conditions less stringent than those described above (e.g., temperature, ionic strength and % SOS) and can be defined as washing in the same buffer at 42°C.
  • Another method of detecting polynucleotide expression levels involves immobilized polynucleotides on a solid surface such as a biochip or a microarray and contacting the immobilized polynucleotides with a probe, for example by running isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose.
  • the probes can be immobilized on a solid surface and isolated mRNA is contacted with the probes, for example, in an Agilent GeneChip Array or Affymetrix GeneChip.
  • biochip or “microarray” can be used interchangeably to mean a solid substrate comprising an attached probe or plurality of probes as described herein, wherein the probe(s) comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200 or more probes.
  • the probes may be capable of hybridizing to a target sequence under stringent hybridization conditions.
  • the probes may be attached at spatially defined address on the substrate. More than one probe per target sequence may be used, with either overlapping probes or probes to different sections of a particular target sequence.
  • the probes may be capable of hybridizing to target sequences associated with a single disorder.
  • the probes may be attached to the biochip/microarray in a wide variety of ways, as were appreciated by one of skill in the art.
  • the probes may either be synthesized first, with subsequent attachment to the biochip, or may be directly synthesized on the biochip/microarray.
  • the solid substrate may be a material that may be modified to contain discrete individual sites appropriate for the attachment or association of the probes and is amenable to at least one detection method.
  • substrates include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon®, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses and plastics.
  • the substrates may allow optical detection without appreciably fluorescing.
  • the substrate may be planar, although other configurations of substrates may be used as well. For example, probes may be placed on the inside surface of a tube, for flow-through sample analysis to minimize sample volume.
  • the substrate may be flexible, such as a flexible foam, including closed cell foams made of particular plastics.
  • the biochip/microarray and the probe can be derivatized with chemical functional groups for subsequent attachment of the two.
  • the biochip/microarray may be derivatized with a chemical functional group including, but not limited to, amino groups, carboxyl groups, oxo groups or thiol groups.
  • the probes can be attached using functional groups on the probes either directly or indirectly using a linker.
  • the probes may be attached to the solid support by either the 5' terminus, 3' terminus, or via an internal nucleotide.
  • the probe may also be attached to the solid support noncovalently.
  • biotinylated oligonucleotides can be made, which may bind to surfaces covalently coated with streptavidin, resulting in attachment.
  • probes can be synthesized on the surface using techniques such as photopolymerization and photolithography.
  • microarrays can be used to detect polynucleotide expression. Microarrays are particularly well suited because of the reproducibility between different experiments. DNA microarrays provide one method for the simultaneous measurement of the expression levels of large numbers of polynucleotides. Each array consists of a reproducible pattern of capture probes attached to a solid support. Labeled R A or DNA is hybridized to complementary probes on the array and then detected by laser scanning. Hybridization intensities for each probe on the array are determined and converted to a quantitative value representing relative gene expression levels. See, e.g., US Patent Nos. 6,040, 138; 5,800,992; 6,020, 135; 6,033,860 and 6,344,316.
  • High-density oligonucleotide arrays are particularly useful for determining expression profiles for a large number of polynucleotides in a sample.
  • the methods described herein used a microarray and 4 or 5 probes including 212022_s_at (MKI67), 203145_at (SPAG5), 204817_at (ESPL1), 202240_at (PLK1).
  • arrays can be nucleic acids (or peptides) on beads, gels, polymeric surfaces, fibers (such as fiber optics), glass or any other appropriate substrate. See, e.g., US Patent Nos. 5,770,358; 5,789,162; 5,708,153; 6,040,193 and 5,800,992.
  • PCR-amplified inserts of cDNA clones can be applied to a substrate in a dense array.
  • a substrate for example, at least about 10,000 nucleotide sequences can be applied to the substrate.
  • the microarrayed genes, immobilized on the microchip at 10,000 elements each, are suitable for hybridization under stringent conditions.
  • Fluorescently labeled cDNA probes can be generated through incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from tissues of interest. Labeled cDNA probes applied to the chip hybridize with specificity to each spot of DNA on the array. After stringent washing to remove non-specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera. Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance.
  • microarray analysis can be performed by commercially available equipment, following manufacturer's protocols, such as by using the Affymetrix® GeneChip Technology, or Agilent® Ink-Jet Microarray Technology.
  • Affymetrix® GeneChip Technology or Agilent® Ink-Jet Microarray Technology.
  • Agilent® Ink-Jet Microarray Technology The development of microarray methods for large-scale analysis of gene expression makes it possible to search systematically for molecular markers of cancer classification and outcome prediction in a variety of tumor types.
  • Another method of detecting polynucleotide expression levels involves a digital technology developed by NanoString® Technologies (Seattle, WA) and based on direct multiplexed measurement of gene expression, which offers high levels of precision and sensitivity ( ⁇ 1 copy per cell).
  • the method uses molecular "barcodes" and single molecule imaging to detect and count hundreds of unique transcripts in a single reaction. Each color- coded barcode is attached to a single target-specific probe corresponding to a gene of interest. Mixed together with controls, they form a multiplexed CodeSet. Two ⁇ 50 base probes per mRNA can be included for hybridization.
  • the reporter probe carries the signal, and the capture probe allows the complex to be immobilized for data collection.
  • nCounter® Cartridge After hybridization, the excess probes are removed and the probe/target complexes aligned and immobilized in an nCounter® Cartridge. Sample cartridges are placed in a digital analyzer for data collection. Color codes on the surface of the cartridge are counted and tabulated for each target molecule.
  • nucleic acid amplification for example, by RT-PCR (US Patent No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci.
  • biomarker expression can be assessed by quantitative fluorogenic RT-PCR (i.e., the TaqMan® System).
  • PCR analysis methods and software are available to determine primer sequences for use in the analysis. These methods are particularly useful for detecting polynucleotides present in very low numbers.
  • RNA blot such as used in hybridization analysis such as Northern or Southern blotting, dot, and the like
  • microwells sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids).
  • Polynucleotide biomarker expression also can include using nucleic acid probes in solution.
  • SAGE Another method of detecting polynucleotide expression levels involves SAGE, which is a method that allows the simultaneous and quantitative analysis of a large number of polynucleotides without the need of providing an individual hybridization probe for each transcript.
  • a short sequence tag (about 10-14 bp) is generated that contains sufficient information to uniquely identify a transcript, provided that the tag is obtained from a unique position within each transcript.
  • many transcripts are linked together to form long serial molecules that can be sequenced, revealing the identity of the multiple tags simultaneously.
  • the expression pattern of any population of transcripts can be quantitatively evaluated by determining the abundance of individual tags and identifying the gene corresponding to each tag. See, Velculescu et al. (1995), supra.
  • microbead library of DNA templates can be constructed by in vitro cloning. This is followed by assembling a planar array of the template- containing microbeads in a flow cell at a high density (typically greater than 3.0 x 106 microbeads/cm2).
  • the free ends of the cloned templates on each microbead are analyzed simultaneously, using a fluorescence-based signature sequencing method that does not require DNA fragment separation. This method has been shown to simultaneously and accurately provide, in a single operation, hundreds of thousands of gene signature sequences from a yeast DNA library.
  • the method described herein After measuring expression levels of the biomarkers, the method described herein then includes correlating the expression levels of the biomarkers in the patient sample to a reference/control set to determine the prognosis of the patient.
  • present method may also be implemented through the use of a computer.
  • present method may employ a computer running a software program that can analyze DEDD biomarker expression level data from a TNBC patient, compare that data to a reference or control DEDD expression level calculated and/or derived from a population of TNBC patients, and determine whether the TNBC patient's expression levels were sufficiently above the reference DEDD expression level.
  • a computer can be used to generate a report summarizing the patient's biomarker expression levels and/or the patient's suitability for subsequent combinatorial treatment (e.g., anti-CDK-4/6 and anti-EGFR).
  • the computer can perform any statistical analysis of the patient's data or a population of patient's data as described herein in order to generate the status of the patient.
  • the computer program also can normalize the patient's biomarker expression levels in view of a standard or control prior to comparison of the patient's biomarker expression levels to those of the patient population.
  • the computer also can ascertain raw data of a patient's expression values from, for example, a microarray, or the raw data can be input into the computer.
  • Methods for assessing statistical significance include, for example, using a log-rank test, Cox analysis and Kaplan-Meier curves. A p-value of less than 0.05 can be used to establish statistical significance.
  • TNBC biomarker or combination of TNBC biomarkers can be indicative of a poor prognosis for the combinatorial treatment described herein as a viable promising option.
  • indicator of a poor prognosis is intended to mean that altered expression of particular biomarkers or combination of biomarkers is associated with an increased likelihood that be relatively ineffective, and suggest alternative therapeutic regimens be selected.
  • indicator of a good prognosis for the combinatorial therapy treatment refers to an increased likelihood that the TNBC patient will benefit from the combinatorial therapy treatment.
  • “indicative of a good prognosis” may refer to an increased likelihood that the TNBC patient will improve upon combinatorial treatment.
  • polypeptide biomarkers as methods of detecting and quantifying polypeptides in a sample are well known in the art and include, but are not limited to, immunohistochemistry and proteomics- based methods.
  • DEDD is shown here to have a pro- tumorigenic function which can support cancer cells survival and proliferate under the stress of the proliferating signaling being repressed.
  • Therapies for TNBC patients comprising a combination of anti-proliferating therapy(s) that are based on these observations are presented.
  • the DEDD amino acid protein sequence is known, and is published in the available literature and at GenBank and other sources (See UnitProtKB 075618 (DEDD_ HUMAN)).
  • the DEDD protein in human exists in 2 isoforms, a 378 aa isoform and a 348 aa isoform.
  • mRNA sequence information for human DEDD is also publically available, for example, see AF083236 mRNA Translation: AAC33105.1;AF 100341 mRNA Translation AAD16414.1; AF043733 mRNA. Translation AAC80280.1; AJ01973 mRNA. Translation CAA09445.1; AF064605 mRNA Translation AAC17110.3.).
  • the sequence information for murine forms of the DEDD amino acid sequence is also known, and is similarly published in GenBank and other sources. All of these teachings are specifically incorporated herein by reference.
  • the amino acid sequence for the DEDD gene is also provided at the NCBI data base at: https://www.ncbi.nlm.nih.gov/gene/9191.
  • a fusion protein was created by adding a 3X-FLAG tag to the N- terminal of the DEDD sequence, using conventional molecular biology techniques known to those of ordinary skill in the art. This fusion protein was created because there is no available antibody that was suitable for reliably detecting wild type DEDD.
  • DEDD Death Effector Domain Containing
  • TNBC cell lines which have different expression levels of DEDD, including HCC1806, HCC38, MDA-MB-231 and MDA-MB-468.
  • HCC1806 has average DEDD expression
  • MDA-MB-231 have relatively lower DEDD expression
  • HCC38 and MDA-MB-468 have relatively high DEDD expression 16,17 .
  • HER2 positive cell line BT-474 as a control cell line to confirm the inhibition efficiency of an anti- proliferating drug Lapatinib (HER2 and EGFR dual inhibitor).
  • 293FT cells were used as transient vector expression cell line and virus packaging cell line.
  • the 293FT cell line was a particularly suitable host for generating lentiviral constructs using the lentiviral expression system.
  • the HCC1806, HCC38, MDA-MB-231, MDA- MB-468, BT-474 and 293FT cell lines were from ATCC.
  • 293FT cell line was cultured in DMEM high glucose medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin streptomycin (Pen-Strep).
  • FBS fetal bovine serum
  • Pen-Strep penicillin streptomycin
  • BT-474, MDA-MB-231 and MDA-MB-468 cell lines were cultured in DMEM/F-12 medium supplemented with 10% FBS and 1% Pen-Strep.
  • HCC1806 and HCC38 cell lines were cultured in RPMI1640 medium supplemented with 10% FBS and 1% Pen-Strep. All cell lines were maintained at 37°C in a 5% C0 2 humidified environment and sub- cultured upon reaching approximately 90% confluence. 24 hours prior to experiments, cells were re-plated into new petri dishes with 10% FBS medium at about 50%» confluency.
  • mice For an in vivo model system, Rag- 1 -deficient mice were used because these mice have small lymphoid organs that do not contain mature B and T lymphocytes 37 . This allowed human cancer xenografts to grow and form visible solid tumors on Rag- 1 -deficient mice.
  • the HCC- 1806 and MDA-MB-468 cell line was used to form human xenograft on mice because they stably formed tumors and metastases in immune deficient mice 38 . All animal experiments are conducted in accordance with the federal and local institutional rules for animal experimentation.
  • Rag-1- deficient mice (8-10 weeks old) were randomly divided into indicated experimental groups with each group containing 10 mice.
  • mice were subcutaneously injected with the indicated cells in mixed injection medium (1:1 mixture ratio of Matrigel and DMEM/F-12 medium without FBS) and implanted s.c. on one side of the abdominal region of a female mouse.
  • mixed injection medium (1:1 mixture ratio of Matrigel and DMEM/F-12 medium without FBS
  • cells were inoculated on both left and right side.
  • Tumors were allowed to grow to 5 to 10 mm in diameter (4 weeks) before the mice (30 in total) before sacrificed and subjected to tumor analysis.
  • Tumor sizes were measured by caliper measurements of the largest longitudinal (length) and transverse (width) diameter.
  • PFA paraformaldehyde
  • EXAMPLE 2 Amplification or Up-regulation of Death Effector Domain-Containing Protein confers vulnerability to anti-EGFR/HER2 targeted therapy
  • TNBC cell lines including HCC-1806, HCC-38 and MDA-MB-468 cell lines were tested with increasing concentrations of an EGFR tyrosine kinase inhibitor, Lapatinib (LAP).
  • LAP Lapatinib
  • a Her2 positive cell line BT474 was used as a control cell line ( Figure 1A). While BT474 cells were highly sensitive to LAP treatment, all of three TNBC cell lines showed consistent resistance to LAP.
  • EGFR mutations have been identified in several other cancers 1 ⁇ 3 . Therefore, the no-responding phenotype of TNBCs was examined to determine if it was due to an EGFR mutation(s).
  • TNBC cell lines were treated with increasing dosages of EGFR inhibitor from 0.25 ⁇ to 4 ⁇ 2 hours prior to harvest the cells. Then western blots (WB) were performed to check EGFR as well as two downstream signaling Akt and Erk.
  • the WB results showed decreased expression of phosphorylated EGFR (p-EGFR), phosphorylated Akt (p-Akt) and phosphorylated Erk (p-Erk) at all dosages used in all TNBC cell lines under 2 hours treatment (Figure IB), indicating LAP can effectively block EGFR signaling in TNBC cell lines.
  • a redundant pathway(s) was opined to exist in TNBCs, contributing to the resistance to anti-EGFR therapies.
  • DEDD a Death Effector Domain Containing protein
  • a previously known scaffold protein which interacts with caspase family members and regulates cellular death pathways decreased the cell numbers of HCC1806 in LAP treatment group significantly (4 of 5 shRNAs depleted in Lap group).
  • DEDD has been shown amplified expression over 70% of all basal-like invasive breast cancers (78% in basal vs 44% in all breast tumors).
  • three top hits, DEDD, ABCB10 and UAPl identified from the functional synthetic screen are located at chromosome lq, which amplified in breast cancer patients (Figure 4D).
  • the large initial pool of potential hits may include false positive candidates which do not contribute to the phenotype of resistance to anti-EGFR therapies in TNBCs. Therefore, to validate the screening result, a lentivirus shRNA transfection assay was performed to knock down DEDD in TNBC cell lines, followed by mRNA expression validation using quantitative RT-PCR assay. To complement the pharmacological studies, which have the potential for unanticipated "off-target” effects, multiple different short hairpin RNA (shRNA) were used to target DEDD in TNBC cell lines. This reduced the abundance of DEDD mRNA by -60-90% ( Figure IE). A cell counting assay was then performed to check cell viability. Knockdown DEDD reliably re-sensitized TNBC cell lines to the LAP treatment ( Figure IF; Figure 4E).
  • DEDD cell cycle regulators dominate the interaction network, and further biological process enrichment analysis showed cell cycle regulation pathways were significantly enriched.
  • DEDD was shown to have the ability to directly bind to cyclin Bl and to reduce cyclin Bl/Cdkl complex activity in 293FT cells. 6
  • EdU 5-ethynyl-2' -deoxyuridine
  • DEDD mainly expresses in the nucleus, and this nuclear localization may lead to caspase activation and the inducement of apoptosis. Abolishing DEDD's nuclear translocation capability may decreases the apoptosis potential of DEDD 7-9 .
  • DEDD has been reported to physically interact with a transcription factor Smad3 and prevent Smad3 phosphorylation, leading to the reduced expression of Smsd3-targeted gene transcription 10 . It has been suggested that cytoplasmic DEDD may have different function(s) besides regulation of cell death pathways 11 .
  • DEDD Stable TNBC cell lines with genetic loss or gain of DEDD expression.
  • DEDD demonstrates amplified expression in about 78% of TNBC patients, although TNBC cell lines have different DEDD expression status 16 ' 17 .
  • two relatively moderate DEDD expression TNBC cell lines were examined in the present studies, HCC1806 and MDA-MB-468. This permitted the examination of the function(s) of wild type DEDD in these TNBC cell lines as well as provided a higher expression window to express exogenous DEDD.
  • HCC1806 and MDA-MB-468 two relatively moderate DEDD expression TNBC cell lines were examined in the present studies, HCC1806 and MDA-MB-468. This permitted the examination of the function(s) of wild type DEDD in these TNBC cell lines as well as provided a higher expression window to express exogenous DEDD.
  • Through loss of DEDD function experiments it was established that DEDD is necessary for the cell cycle regulation phenotype. By gain of DEDD function experiments, it was established that DEDD is sufficient to alter cell cycle profiles in TNBC
  • Stable DEDD knockdown TNBC cell lines were prepared through Lentiviral transfection of plasmids reliably expressing anti-DEDD shR As.
  • a lentivirus shR A transfection assay was performed to knock down DEDD in TNBC cell lines (HCC1806 and MDA-MB-468, available in GenBank).
  • the PLKO.l empty plasmid was used as a control shRNA target plasmid, PLK0.1-puro-shRNA-DEDD493 and PLKO.l-puro-shRNA- DEDD1056 as two DEDD knockdown plasmids (glycerol stocks from Sigma).
  • the TNBC cell lines were cultured in 10% FBS medium containing ⁇ g/ml Puromycin for 4 days (change medium every 48 hours) to enrich the infected cells. After that, the cells were allowed to passage 1 generation before further study. To validate the knockdown efficiency, a RT-PCR assay was performed to detect DEDD mRNA levels in TNBC cell lines (no reliable antibodies for detecting endogenous DEDD protein).
  • Stable DEDD overexpressing TNBC cell lines through Lentiviral transfection of plasmids reliably expressing 3X-FLAG-DEDD.
  • a fusion protein was created by adding a 3X-FLAG tag on to the N-terminal of DEDD, due to the fact that there is no available antibody that can reliably detect wild type DEDD.
  • the infected TNBC cell lines were cultured and treated the same as previously described herein.
  • TNBC cell lines established here were used in the present studies.
  • the medium of the cells was replaced with medium without FBS. This will allow the cells to synchronize their cell cycle majorly in GO.
  • the cells were treated with lng/ml epidermal growth factors (EGF) to induce cell cycle transition.
  • EGF epidermal growth factors
  • Cyclin-dependent kinases are serine/threonine kinases and their catalytic activities are modulated by interactions with cyclins and CDK inhibitors (CKIs) 43 .
  • Cyclins belong to a remarkably diverse group of proteins classified solely on the existence of a cyclin box that mediates binding to CDK 44 .
  • Cyclin A controls S phase of cell cycle in complex with CDK2 or CDK1.
  • Cyclin B controls M phase of cell cycle in complex with CDK1.
  • Cyclin D controls Gl phase of cell cycle in complex with CDK4 or CDK6.
  • Cyclin E controls Gl-S phase of cell cycle in complex with CDK2 45 .
  • TNBC cell lines established as described herein were used to investigate the dynamic cyclins expression profile alteration by Western Blotting in DEDD knockdown TNBC cell lines.
  • the culture medium of the cells was replaced with medium without FBS. This will allow the cells to synchronize their cell cycle in GO.
  • the cells were treated with lng/ml epidermal growth factor (EGF) to induce cell cycle transition.
  • EGF epidermal growth factor
  • the cells were harvest and subjected to Western Blot analysis.
  • Western blot analyses was performed with precast gradient gels (4-20%)(Bio-Rad) using standard methods.
  • cells were lysed in M2 complete lysis buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris-Cl (pH 8.0) and 1% Triton-X) containing protease/phosphatase inhibitor cocktail (Cell Signaling). Proteins were separated by SDS-PAGE and blotted onto a 0.45 ⁇ nitrocellulose membrane (VWR). Membranes were probed with the specific primary antibodies and then with peroxidase conjugated secondary antibodies. The bands were visualized by SuperSignal west pico chemiluminescence substrate (ThermoFischer Scientific).
  • the following antibodies were used: antibodies against Beta-actin (1:2000, Cell Signaling), Cyclin A2 (1:1,000, Cell Signaling, #4656, clone BF683), Cyclin Bl (1:1000, Cell Signaling #4138), Cyclin Dl (1:1000, Cell Signaling #2978, clone 92G2), Cyclin El (1:1000, Cell Signaling #4129, clone HE12).
  • the ImageJ program http://rsbweb.nih.gov/ij/download.html was used for densitometric analyses of western blots, and the quantification results was normalized to the loading control.
  • the peak of each cyclin expression is expected to be delayed in DEDD knockdown TNBC cell lines, which associated with the phenotype that knockdown of DEDD impairs cell cycle transition.
  • the peak of each cyclin expression is expected to be shown at an earlier time point in DEDD overexpressed TNBC cell lines, which is consistent with the phenotype that DEDD promotes cell cycle transition.
  • RT-PCR assay to test Rb gene messenger R A level to test whether DEDD reducing Rb protein expression is through decreasing its mRNA expression. CalFectin transfection was performed when 293FT cells in 10 cm plates reached about 50% confluence. 2 ⁇ g of plasmids encoding 3X-FLAG-DEDD was used in transfection. The control group of 293FT cells was transfected with empty expression vectors (pLove empty). 48 hours later, the cells were treated with lng/ml of EGF. 6 hours after EGF inducement, cell lysates were subjected to western blotting.
  • Total cell RNA was prepared using TriZol reagent (In itrogen) following the manufacturer's instructions, lmg of total RNA was subjected to reverse transcription to synthesize cDNA using the Verso cDNA kit (ThermoFischerScientific). A 20 ⁇ volume reaction consisted of 2 ⁇ reverse transcription product and 20 nM of each primer.
  • the cells were treated with the protein synthesis inhibitor cycloheximide (CHX) (Sigma, 10 ⁇ g/ml) for the indicated durations (0, 3, 6, 9, 12, and 15 hours) before collection.
  • CHX protein synthesis inhibitor cycloheximide
  • DEDD Knockdown of DEDD causes Rb protein expression in the HCC1806 cell line ( Figure 2A), which suggests an inverse expression correlation between DEDD and Rb protein.
  • DEDD has a death-effector domain and a DNA binding domain which can directly interact with other nuclear proteins and activate their functions such as caspase-6 and Smad3 46 ' 47 .
  • Rb protein regulates different pathways through the stimulation or inhibition of the activity of interacting proteins. These suggest that both DEDD and Rb protein can serve as a scaffold proteins which has protein binding capability to interact with each other.
  • Human Rb protein contains 928 amino acids and is commonly described as having three domains.
  • the central domain was identified as the minimal region necessary to bind viral oncoproteins, such as adenovirus E1A, SV40 Tag and human papilloma virus E7, and it was named the 'pocket' 48 .
  • the pocket comprises two subdomains, A and B, each resembling a cyclin fold MB CDK4 with three additional helices.
  • Approximately the last 150 residues of Rb protein form the carboxy-terminal domain (RBC), which is intrinsically disordered.
  • RBC carboxy-terminal domain
  • the linker sequences between the pocket subdomain B and RBC are notable because they contain CDK dependent phosphorylation sites that have a critical role for the inactivation and degradation of Rb protein 18 .
  • Rb protein can be degraded through proteasome- ubiquitin pathway by NRBE349.
  • Rb protein needs to be hypophosphorylated to become activated which allows Rb protein bind to E2F transcription factors during GO phrase and increasing phosphorylation of hypophosphorylated Rb protein by cyclin/cdk complexes to become hyperphosphorylated status is necessary for Rb protein inactivation during Gl-S transition 19-21 .
  • Rb protein may also need to be phosphorylated before being recognized by DEDD. Taken all of these together, DEDD and Rb protein can physically interact with each other. In addition, this DEDD-Rb protein interaction may be critical for Rb protein destabilization.
  • cell lysates were incubated with ⁇ g of Rb (4H1) antibody overnight (18 hours). 1% of cell lysates was saved as the input control. After that, the samples were incubated with 50 ⁇ of protein A/G agarose beads (Santa Cruz) at 4 °C for 2 hours. The immunocomplexes was then washed with M2 lysis buffer three times. The proteins bound to beads was released by boiling in 30 ⁇ of 5X SDS-PAGE sample buffer at 95 °C for lO min. The samples were resolved by 4-20% gradient SDS-PAGE gels followed by immunoblot analysis using a monoclonal anti- FLAG antibody.
  • Mouse IgG pull down was used as a non-specific pull down control. Both 1% input of lysates and immunoprecipitates was examined using the indicated primary antibodies followed by detection with the related secondary antibody and the SuperSignal west pico chemiluminescence substrate (ThermoFischerScientific). Co-expression of HA-Ubiquitin and 3X-FLAG-DEDD followed with Rb protein pull down assay to test whether DEDD promotes Rb protein ubiquitination. 293FT cells were co- transfected with HA-Ub and 3X-FLAG-DEDD plasmids. Immunoprecipitation was performed with Rb (4H1) antibody or mouse IgG, and the ubiquitination of Rb protein was evaluated by Western blotting with anti-HA antibody. Cells were collected and processed as previously described herein.
  • DEDD mainly expresses in nuclearly, and that this nuclear localization leads to caspase activation and induction of apoptosis. Abolishing DEDD's nuclear translocation capability decreases its apoptosis potential. 47, 51 ' 52 Previous researches showed DEDD can physically interact with a transcription factor Smad3 and prevent Smad3 phosphorylation which leads to the reduced expression of Smsd3-targeted genes transcription, addressing the importance of the nuclear function of DEDD. However, it has been suggested that cytoplasmic DEDD may have different function(s) besides regulation of cell death pathways 52 . Besides, the proteasome functions primarily in cytoplasm, which suggests that DEDD potentiates Rb protein degradation through its cytoplasmic function.
  • cells were incubated with the indicated anti-FLAG and Rb antibodies (dilution 1:50) for 18 h at 4 °C, followed by incubation with TRITC-conjugated or FITC-conjugated secondary antibody (dilution 1: 100) for 1 h at 25 °C.
  • the nuclei were stained with DAPI (Sigma), and images were visualized with an Olympus FV1000 2-Photon Confocal Microscope (IUSM-SB).
  • a fusion protein was created by adding a 3X-FLAG tag on to the N- terminal of mutated DEDD using the pENTR-TOPO/SD-MUT-DEDD as the template. After the correct right clone of the mutated DEDD was obtained, the new construct was expressed in 293FT cells.
  • Lapatinib single drug treatment has not been shown to provide any significant clinical efficacy in triple negative breast cancer patients 54 ' 55 .
  • Knockdown of DEDD is shown in the present invention to dramatically enhance the sensitivity of Lapatinib-resistant TNBC cell lines in vitro.
  • the combination of a DEDD signaling inhibitor with a CDK-4/6 inhibitor, such as lapatinib provides an effective treatment to inhibit tumor growth in TNBC patients.
  • the genome-wide functional screening and the following validation results showed DEDD potentiates drug resistance to anti-EGFR therapy in TNBC cell lines ( Figure IF).
  • targeting DEDD signaling activity is expected to enhance the therapeutic efficacy of anti-EGFR treatments of TNBCs, such as lapatinib.
  • DEDD has been reported to be a scaffold protein 13,51 . This information brings about the challenge that DEDD signaling may not be suitable for targeted therapy development since most artificially synthetic drugs in cancer treatment are small molecules targeting enzymatic function of kinases 56 . Thus, the downstream signaling (s) of DEDD may be examined for drug discovery.
  • One of the first steps in inactivation of Rb protein appears to be phosphorylation of the C-terminus by cyclin D/cdk4 39 . This inactivation causes Rb protein degradation and turnover. In malignant cells, genetic changes can result in loss of controlled cell cycle progression by impairing Rb protein function 57 ' 58 .
  • mice were treated in the 4 treatment groups (10 mice per group) by oral gavage. The mice were treated as follows: either vehicle, CDK 4/6 inhibitor abemaciclib only, lapatinib only, or abemaciclib plus lapatinib combination).
  • the present therapeutic methods may be provided to a TNBC patient, or other non-ER+ - patient, identified to be in need thereof, using virtually any agent identified to have an anti- EFGR activity (e.g., EGFR Inhibitors) or an anti-CDK4 activity (e.g., CDK-4 Inhibitors) described herein.
  • an anti- EFGR activity e.g., EGFR Inhibitors
  • an anti-CDK4 activity e.g., CDK-4 Inhibitors
  • antibody-based drugs may also be administered as a component of the present invention. These antibody-based drugs may be used to target EGFR as a component of the present inventive combinatorial regiments and methods. A combination of an antibody based drug to target EGF with a CDK4/6 inhibitor may also be used.
  • the present example demonstrates the utility to the present combinatorial treatment methods of a CDK-4/6 inhibitor, Palbociclib, with the EGFR inhibitor, Lapatinib.
  • the TNBC cells HCC1806 and MDA-MB-468 were plated in 96- well plates 24 hours prior to the treatment. Then the cells were treated with DMSO, Lapatinib, Palbociclib or Lapatinib+Palbociclib for 4 days (replace medium containing indicated concentration of drugs every 48 hours). The cell viability was tested by MTT assay. The result shows the combination treatment significantly increased inhibition of cell growth in HCC1806 and MDA-MB-468 cell lines compared to single drug treatments (p ⁇ 0.05) ( Figure 9).
  • a proliferation assay was performed to test the efficacy of a combination treatment of a CDK4/6 inhibitor Abemaciclib (ABE) and an EGFR inhibitor, lapatinib (LAP), in seven different TNBC cell lines (Fig 7A). Except for the MDA-MB-231 cell line, six TNBC cell lines showed a synergistic effect on inhibition of cell proliferation with treated with combination treatment (Combo vs single treatment). In addition, the therapeutic response to the combination treatment was closely associated with DEDD expression ( Figure 7B).
  • the in vivo therapeutic efficacy of the combination treatment of an EGFR inhibitor and a CDK-4/6 inhibitor was also examined.
  • the HCC1806 mammary fat pad tumor formation assay was employed for this purpose.
  • the HCC1806 xenograft tumor progressed aggressively with an ⁇ 8 fold increase of tumor size within 15 days post treatment in vehicle, LAP and ABE treated group.
  • the combination treatment suppressed the tumor proliferation with a 7 fold reduction of tumor size ( Figure 7E).
  • Further immunohistochemistry analysis revealed that the combination treatment abolished active cell proliferation as indicated by Ki-67 staining.
  • TNBC patient derived xenograft (PDX) model data was obtained ( Figure 7G).
  • the PDX tumor sample was found to express high levels of DEDD, as measured by immunohistochemistry ( Figure 7G, left).
  • the EGFR inhibitor eg, ABE
  • the combined treatment with a CDK-4/6 inhibitor demonstrated a -2.8 fold decrease in overall tumor size, compared with the vehicle control group.
  • the present demonstrates the utility of the present combinatorial treatment methods of a CDK-4/6 inhibitor, Ribociclib, with the EGFR inhibitor, Lapatinib.
  • the data obtained employing this combinatorial treatment is provided at Figure 8.
  • TNBC is one of the most aggressive types of breast cancer, with limited targeted therapy options, the present approach presents a more effective treatment avenue for clinical management of the TNBC patient.
  • TNBC Targeted therapy Polyadenosine diphosphate-ribose polymerase (PARP) inhibitor has been clinically tested for TNBC patient.
  • PARP Polyadenosine diphosphate-ribose polymerase
  • TNBC and basal-like cancers are associated with a younger age at presentation, having a mean age of 53 years old, compared to 58 years old for other subgroups 59 ' 60 ' 61 .
  • EGFR therapy approached remained unsuccessful for the entire TNBC population.
  • the present methods provide a technique where a sub- population of TNBC patients may be provided and EGFR-inhibitor therapy successfully, together with a CDK-4/6 inhibitor therapeutic, to treat this invasive, and most times terminal, cancer.
  • a specimen from a patient having been identified as a triple negative breast cancer subject will be obtained. This specimen will be assayed to determine the expression level of the DEDD gene. The expression level of the DEDD gene in the patient's sample will be compared to the DEDD expression level in other TNBC patient samples. These other DEDD expression levels will be considered as follows: When evaluating patient tumor DEDD expression to the Breast Cancer TCGA dataset, TNBC patient tumor DEDD mRNA expression EXP(z-score) > 2 will be considered overexpressed. This group of the patients will most likely respond to the combinational treatment of an EGFR inhibitor and a CDK4/6 inhibitor.
  • DEDD-REF DEDD expression reference scale
  • PDX patient- derived xenograft
  • a clinical breast cancer tumor biopsy will be collected from the TNBC patient, and mRNA expression of DEDD gene in the biopsy will be assessed using qRT-PCR.
  • the z-score of mRNA expression data for each TNBC patient tumor biopsy specimen will be computed using the relative expression of the DEDD gene in the specimen compared to DEDD's expression distribution in all samples in a DEDD-REF. (population of reference TNBC tissue samples).
  • the returned z score value indicates the number of standard deviations (SD) away from the mean of expression in a reference population.
  • SD standard deviations
  • the TNBC patient from whom the specimen was obtained will then be identified as having a higher probability of positively responding to a CDK-4/6 inhibitor, an EGFR inhibitor, or a combination of these as part of a "combination" therapeutically effective pharmaceutical preparation comprising a CDK-4/6 inhibitor and a EGFR inhibitor.
  • a TNBC patient that demonstrates a z score of 1, or of 0, would not be considered to be overexpressing the DEDD gene, and therefore would not be expected to demonstrate a particularly positive response to the combination therapies described herein.
  • a z-score for a test subject/patient sample indicates the number of standard deviations away from the mean of expression in the reference.
  • the formula is:
  • mRNA and microRNA expression data what is typically computed is the relative expression of an individual gene and tumor to the gene's expression distribution in a reference population.
  • the reference population in the present case is all tumors that are diploid for the DEDD gene.
  • CDK-4/6 inhibitors have only been described and developed for ER+ and HER+ breast cancer populations, and have therefore specifically excluded the population of TNBC patients, as well as ER- patient and HER- patient groups.
  • the human equivalent dose of an EGFR inhibitor and a CDK-4/6 inhibitor employing a conventional conversion for dosage form known to those of skill in the dedicinal arts 62 , is calculated to be as follows:
  • the present example presents both a nucleic acid based test kit and a protein based test kit for assessing the presence of DEDD gene and/or DEDD gene product protein in a breast tissue specimen or material derived from a breast tissue specimen.
  • Standardized DEDD biomarker kits are provided to evaluate DEDD expression in a tumor tissue biopsy or material derived from a tumor tissue biopsy.
  • the DEDD Breast Disease Diagnostic Kit is provided in at least two different embodiments. These embodiments include 1) A DEDD-mRNA measurement kit; and 2) A DEDD-protein measurement kit.
  • the DEDD-mRNA kit will comprise:
  • RNA-samples - or number sufficient to provide a standard curve
  • RNA standards will comprise a set of 8 RNA samples (1 ⁇ g/ ⁇ l) extracted from six (6) cell lines and two (2) Control standards, so as to create a standard curve:
  • RNA from each of the above cell lines and for each of the above tumors will extracted according to techniques well known to those of skill in the art (such as described in standard biotechnology assay texts both in print and on line 63 , and will be provided as part of the kit. Instructions will be provided on the construction of the standard curve from high to low DEDD using these RNA sample. The RNA from the specimen will then be extracted, and the amount of DEDD mRNA assessed. This amount will then be compared against the standard curve. Tumor biopsies (Bi ...n ) will be homogenized.
  • RNA The total RNA of each sample will be extracted according to http://cshprotocols.cshlp.Org/content/2010/6/pdb.prot5439.long. Purified RNA will be quantified and diluted into final concentration of 1 ⁇ g/ ⁇ l. Sample Bi ...n and 8 RNA standards will be examined by using DEDD and control primers set by qRT-PCR. Relative abundance and DEDD mRNA will be calculated using AACt methods.
  • RNA sample For the RNA sample, relative expression of DEDD mRNA level will then be plotted against a standard curve generated using RNA standards. The Z-score will be calculated. For the sample, relative expression of DEDD protein will be calculated by densitometry
  • the DEDD-protein kit will comprise a set of eight (8) protein standards that comprise a set of eight (8) protein lysate-standards, protein extraction buffer (50 mM Tris- HC1, pH 8.0. 150 mM NaCl. 1% Nonidet P-40 (NP-40) or 0.1% Triton X-100) and a Pre-plotted antibody array (i.e. nitrocellulose-coated slides) which includes DEDD antibody and control antibody (i.e. actin antibody).
  • the protein lysate standards will comprise a set of 8 protein lysates, each lysate having a concentration of about 1 ⁇ g/ ⁇ l. Each protein lysate will be prepared from a cell line.
  • the cell line from which a cell lysate will be prepared for the kit includes the following cell line:
  • xenograft tumor 2 high DEDD, responder.
  • Tumor biopsies (Bi . n ) will be lysed in protein extraction buffer. Protein concentration of total cell lysate will be quantified by BCA assay and normalized to final concentration of 1 ⁇ g/ ⁇ l. Sample protein lysate were denatured by 1% SDS, serial diluted, an spotted on nitrocellulose-coated slides. Each slide was probed with a validated primary antibody plus a biotinconjugated secondary antibody. The signal obtained was amplified using a DakoCytomation-catalyzed system and visualized by 3,30-diaminobenzidine (DAB) colorimetric reaction.
  • DAB 3,30-diaminobenzidine
  • DEDD expression level determination The slides were analyzed using customized image analysis package. Each dilution curve was fitted with a logistic model to standard curve generated from 8 standards. The DEDD expression level in a given tumor biopsy sample with an unknown DEDD protein level will be determined by z-score of DEDD expression. A Z-score will be calculated to determine the relative abundance of DEDD protein in a given biopsy as compared against a reference samples.

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Abstract

La présente invention concerne des préparations pharmaceutiques comprenant un inhibiteur d'EGFR, ou un inhibiteur d'EGFR et un inhibiteur de CDK-4/6, dans un excipient pharmaceutiquement acceptable, pour le traitement d'un sous-ensemble de patients TNBC. L'invention concerne en outre des procédés de sélection et de traitement d'un patient pour réduire la croissance tumorale au moyen des préparations, sur la base de taux élevés d'expression de DEDD de patient. L'expression élevée de DEDD de patient comprend une valeur z qui s'écarte de 2 écarts types ou plus (4 SD) de (au-dessus de) la valeur z d'une population TNBC. L'invention concerne en outre des biomarqueurs (adaptés pour sélectionner un patient/sujet susceptible de répondre au traitement combiné par un inhibiteur d'EGFR et un inhibiteur de CDK-4/6). L'invention concerne en outre des kits comprenant des réactifs (courbe standard) pour évaluer un échantillon de patient pour un taux élevé de DEDD (protéine et ARNm), ainsi que des substrats solides comprenant les biomarqueurs de DEDD élevé.
PCT/US2017/031624 2016-05-06 2017-05-08 Biomarqueurs de pronostic et compositions antitumorales de traitements thérapeutiques ciblés pour un cancer du sein triple négatif Ceased WO2017193141A1 (fr)

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TWI762784B (zh) * 2018-05-23 2022-05-01 大陸商江蘇恒瑞醫藥股份有限公司 Cdk4/6抑制劑與egfr抑制劑聯合在製備治療腫瘤疾病的藥物中的用途
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11395821B2 (en) 2017-01-30 2022-07-26 G1 Therapeutics, Inc. Treatment of EGFR-driven cancer with fewer side effects
US11357779B2 (en) 2018-01-08 2022-06-14 G1 Therapeutics, Inc. G1T38 superior dosage regimes
US12364697B2 (en) 2018-01-08 2025-07-22 Pharmacosmos Holding A/S G1T38 superior dosage regimes
TWI762784B (zh) * 2018-05-23 2022-05-01 大陸商江蘇恒瑞醫藥股份有限公司 Cdk4/6抑制劑與egfr抑制劑聯合在製備治療腫瘤疾病的藥物中的用途
WO2020239051A1 (fr) * 2019-05-30 2020-12-03 江苏恒瑞医药股份有限公司 Utilisations d'un inhibiteur de cdk4/6 conjointement avec un inhibiteur de vegfr dans la préparation d'un médicament pour le traitement d'une tumeur

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