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WO2007044571A2 - Procedes permettant d'identifier des proteines essentielles a la proliferation des cellules humaines, et agents therapeutiques diriges contre ces proteines - Google Patents

Procedes permettant d'identifier des proteines essentielles a la proliferation des cellules humaines, et agents therapeutiques diriges contre ces proteines Download PDF

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WO2007044571A2
WO2007044571A2 PCT/US2006/039235 US2006039235W WO2007044571A2 WO 2007044571 A2 WO2007044571 A2 WO 2007044571A2 US 2006039235 W US2006039235 W US 2006039235W WO 2007044571 A2 WO2007044571 A2 WO 2007044571A2
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cont
cells
kinases
kinase
cell
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WO2007044571A3 (fr
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Dorre Grueneberg
Ed Harlow
Joan Davies
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Harvard University
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    • 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
    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • This invention relates to the fields of kinases, cancer, and pharmaceutical compounds.
  • Tissue culture cells are superb systems for many biochemical and cell biological studies, such as the study of signal transduction pathways or the analysis of protein translocation. They have also been excellent sources from which purified systems for the in vitro study of many cell processes have been developed. However, mammalian tissue culture systems have not generally been useful for complex genetic studies.
  • RNA interference RNA interference
  • proteome Ziauddin and Sabitini, 2001; Chanda et al. 2003; Matsuda et al., 2003; Berns et al., 2004; Kittler et al. 2004; Paddison et al., 2004; Zheng et al., 2004; MacKeigan et al., 2005; Pelkmans et al., 2005).
  • siRNA-based screens have looked at cell survival (Kittler et al., 2004), induction of apoptosis (MacKeigan et al, 2005), or changes in endocytosis (Pelkmans et al., 2005). All of these siRNA screens have been done using parallel transfection of oligonucleotide siRNAs into appropriate cells.
  • the siRNAs were generated by RNAseIII cleavage of double strand RNA precursors made by transcription of cDNAs for the target genes.
  • the siRNAs were prepared synthetically and purchased from a commercial supplier.
  • the invention features methods for identifying and utilizing kinases, and other proteins, whose down-regulation induces a desirable outcome.
  • the methods provided may be employed to identify kinases that are essential to proliferation in cancer cells.
  • the methods for identifying kinases employ contacting cells with one or more shRNAs that target a particular protein and determining the effect of down-regulation.
  • the invention also features a method of identifying a therapeutic compound for treating cancer by assaying a candidate therapeutic compound for inhibition of the activity of a kinase selected from the group consisting of ANPB, RSK2, PAK6, NLK, CONT PACGFP, ULK4, DDR2, PDGFRB, TRRAP, EPHBl, TSSK2, CAMLCK, MST2, CONT TIE, CK2A2, TAKl, CONT KIT, JNK3-1937, CDKI l, ALK, NEK7, STK33, FYN, PAK3, PAK3, CONT ERBB2, HER3, PKD2, CDKlO 5 MAP3K8, PKNl, MYO3B, EPHB4, BRD2, CDK3, JNK2, HER3, PKD3, PITSLRE, PEK, CDK7, JNK3-, SLK, SLK, CONT BCR, PLKl, MELK, PCTAIRE, CKlE, CONT MTOR 5 CDK9, PITS
  • the assaying for example can include contacting the candidate therapeutic compound with the kinase protein in vitro or in vivo and measuring the activity of the kinase.
  • the assaying includes contacting the therapeutic compound with a cell capable of transcribing the mRNA encoding the kinase and measuring the level of expression of the mRNA or other characteristics associated with a change in the level of biological activity of the encoded kinase or the level of the kinase so encoded.
  • Assays may also be performed in animal models. Assays known in the art for monitoring kinase activity, transcription, translation, protein stability, and availability may all be used in the methods of the invention.
  • the invention further features a method of generating a database using data obtained from methods that include contacting cells expressing a class of mRNA, each member having known sequence, with one or more shRNAs specific to each sequence of the class; measuring the ability to proliferate of the cells, wherein a reduction in the ability identifies an mRNA as essential to proliferation; and creating a record in the database, wherein the record includes the identity of an mRNA of the class that is essential.
  • a record may also include other information on the mRNA (and the protein it encodes), such as the phenotypic effect of reducing the activity of the protein.
  • a class of mRNA is meant a class so grouped by the user, typically based on the function of the encoded proteins. Exemplary classes include kinases, receptors, phosphatases, and transcription factors.
  • the invention also features databases containing information on proteins, such as kinases, obtained as described herein.
  • Exemplary Genebank accession numbers for the human form of these genes are also set forth in Table 2.
  • genes listed above is also meant a nucleic acid with at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% percent sequence identity to any of the genes listed above as determined by the NCBI BLAST program.
  • a gene listed above is defined as a nucleic acid that hybridizes under high stringency conditions to a nucleic acid of a gene listed above.
  • the "percent sequence identity" of two nucleic acid or polypeptide sequences can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, Academic Press, 1987', and Sequence Analysis Primer, Gribskov, and Devereux, eds., M. Stockton Press, New York, 1991; and Carillo and Lipman, SIAM J. Applied Math. 48:1073, 1988.
  • Computer program methods to determine identity are available in publicly available computer programs.
  • Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux et al., Nucleic Acids Research 12:387, 1984), BLASTP, BLASTN, and FASTA (Altschul et al., J. MoI. Biol. 215:403, 1990).
  • the well known Smith Waterman algorithm may also be used to determine identity.
  • the BLAST program is publicly available from NCBI and other sources (BLAST Manual, Altschul, et al., NCBI NLM NIH Bethesda, Md. 20894).
  • Searches can be performed in URLs such as the following: http ://www.ncbi.nlm.nih.gov/BLAST/unfmishedgenome.html; or http ⁇ /www.tigr.org/cgi-bin/BlastSearch/blast.cgi.
  • Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • hybridize pair to form a double-stranded complex containing complementary paired nucleobase sequences, or portions thereof, under various conditions of stringency.
  • stringency See, e.g., Wahl. and Berger, Methods Enzymol 152:399 (1987); Kimmel, Methods Enzymol 152:507 (1987)
  • hybridizes under high stringency conditions is meant under conditions of stringent salt concentration, stringent temperature, or in the presence of formamide.
  • stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.
  • SDS sodium dodecyl sulfate
  • hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 ⁇ g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 ⁇ g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
  • wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
  • stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and most preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C.
  • wash steps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a most preferred embodiment, wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art.
  • Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis ⁇ Science 196:180 (1977)); Grunstein and Hogness ⁇ Proc Natl Acad Sci USA 72:3961 (1975)); Ausubel et al. ⁇ Current Protocols in Molecular Biology, Wiley Interscience, New York (2001)); Berger and Kimmel ⁇ Guide to Molecular Cloning Techniques, Academic Press, New York, (1987)); and Sambrook et al. ⁇ Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York).
  • hybridization occurs under physiological conditions.
  • complementary nucleobases hybridize via hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.
  • hydrogen bonding may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.
  • adenine arid thymine are complementary nucleobases that pair through the formation of hydrogen bonds.
  • inhibition of kinase activity is meant any reduction of the biological activity of the kinase attributable to any mechanism, including reduction in transcription, translation, or stability of the mRNA encoding the kinase or by binding, degrading, or otherwise inhibiting the enzymatic activity of the kinase or its substrate.
  • kinase activity is meant the activity whereby an enzyme phosphorylates an amino-acid residue on a substrate (e.g., a protein, lipid, or carbohydrate substrate).
  • a substrate e.g., a protein, lipid, or carbohydrate substrate.
  • a “compound,” “candidate compound,” or “factor” is meant a chemical, be it naturally occurring or artificially derived.
  • Compounds may include, for example, peptides, polypeptides, synthetic organic molecules, naturally occurring organic molecules, nucleic acid molecules, and components or combinations thereof.
  • Figure 1 is a graph showing the correlation between the relative growth of cells in two screening assays.
  • Figure 2 is a graph showing the correlation between the relative growth of cells in two screening assays.
  • Figure 3 is a diagram showing the overlap of shRNA hits in two different cell lines.
  • Figure 4 is a graph showing growth of cells transfected with virus containing shRNA constructs in the presence and absence of puromycin.
  • Figure 5 is a histogram showing the titer of viruses produced in uninfected and infected cells.
  • Figure 6 is a set of two histograms showing the number of cells containing the indicated relative DNA content.
  • the left panel shows the DNA content of cells transfected with a luciferase construct and the right panel shows the DNA content of cells transfected with a shRNA construct.
  • Figure 7 is a set of two histograms showing the growth rate of cells expressing both an shRNA construct against ErbB3 and a cDNA.
  • Figure 7 is a set of photomicrographs of cells expressing both shRNA against ErbB3 and a cDNA encoding MAP3K14 (bottom left and bottom center panels), or control cells (bottom right panel).
  • Figure 8 is a diagram showing an experimental plan for identifying and characterizing essential kinases.
  • Figure 9 is a diagram showing follow up experiments for proteins identified in the screens of the invention.
  • Figure 10 is a photomicrograph showing MAP LC3 staining of FIELA cells.
  • Figure 11 is a photomicrograph showing MAP LC3 staining of HDELA cells.
  • Figure 12 is a photomicrograph showing SA-beta Gal staining of HELA cells.
  • Figure 13 (left panel) is a photomicrograph MAP LC3 staining of HELA cells;
  • Figure 13 (right panel) is a photomicrograph of Activated Caspase-3 staining of HELA cells.
  • Figure 14 is a set of four histograms showing the number of cells containing the indicated relative DNA content in cells transfected with the indicated shRNA.
  • RNA interference RNA interference-based approach, in which small hairpin RNAs (shRNAs) directed against specific gene targets and introduced into cells, e.g., via a lentiviral vector, cause gene- specific knock-down of mRNA and protein levels.
  • shRNAs small hairpin RNAs
  • genes identified in (1) further by grouping the genes into broad categories that describe how disruption of the genes affects viability and/or proliferation. These modes of action include induction of cell death (apoptosis), induction of autophagy, and/or induction of cellular senescence in the presence of a shRNA that disrupts the gene target. 3. Identifying and developing drugs that disrupt the function of those target proteins identified in the screen as good candidates, e.g., based on the fact that they are essential in cancerous cells but not in corresponding normal cells.
  • any class of genes e.g., those encoding proteins from given receptor class, phosphatases, and transcription factor families, could be tested and grouped in a similar manner using these methods
  • a set of genes essential for viability and/or proliferation of two different cell lines were identified using a collection of more than 2,000 shRNAs directed against a set of 416 kinase-encoding genes (the shRNAs were developed by The RNAi Consortium).
  • the set of genes identified as essential in either cell line were used to screen normal (i.e., non-transformed) and cancerous (i.e., transformed) cell lines believed to be relevant to specific cancer types (for example, normal MCFlOA and tumorogenic MCF 7 cells originating from breast tissue).
  • the screen may employ transfection of the cell lines and an alamar blue readout of proliferation/viability.
  • screens may employ lentiviral transduction and alamar blue readout, followed by secondary assays, e.g., for apoptosis, autophagy, and senescence (anti-activated-Caspase-3 immunofluorescent detection, anti-LC3 immunofluorescent detection, and SA- beta-Gal assays, respectively).
  • secondary assays e.g., for apoptosis, autophagy, and senescence (anti-activated-Caspase-3 immunofluorescent detection, anti-LC3 immunofluorescent detection, and SA- beta-Gal assays, respectively).
  • the readout may be generated using any number of systems known to those skilled in the art.
  • kinases shown to be essential in cancerous but not noncancerous cells from the same tissue type are considered better targets than kinases essential for viability in both cell types since the ideal therapeutic approach is to kill cancerous cells without affecting normal cells.
  • the accumulation of data on many different cell types should lead to identification of a sub-set of kinases essential in most, or many, cell types and, conversely, a sub-set of kinases that are essential only in a small sub-set of cells.
  • the accumulation of data on many different cell types allows one to group the action of kinases as generally acting through a particular mode or modes of action, or as acting in different modes in different cell types.
  • genes that act via one mode or modes have better potential for cancer therapies than genes that act in a different mode or modes (for example, a reasonable a priori guess is that proteins whose knock-down via RNAi leads to apoptosis may be better candidates for the development of targeted cancer therapies than those that work via senescence).
  • RNA interference RNA interference
  • Many eukaryotic cells have enzymatic machinery that recognizes double strand RNA sequences, processes them to active short duplex sequences, and then uses them to modify gene expression.
  • RNAi has been reviewed extensively (for example see Huppi et al., 2005, Tomari and Zamore, 2005).
  • siRNA are short RNA duplexes typically between 19 and 27 nucleotides in length that are recognized by a multimeric protein complex known as the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • the RISC complex unwinds the duplex, uses one strand of the duplex for recognition of homologous sequences in mRNA, and then cleaves the niRNA. The outcome is the lowering of specific mRNA levels and the subsequent reduction of specific protein levels.
  • Two methods are available to generate siRNAs. The more straightforward but expensive approach is to synthesize both strands of the siRNA, hybridize the two strands to form a duplex, and transfect the oligonucleotides into cells. Oligonucleotides transfect more readily than longer double strand sequences making this an efficient process in many cells.
  • the other popular method is introducing an expression construct into cells that transcribes a short hairpin RNA (shRNA) where the duplexed sense and antisense RNA sequences are connected by a small loop.
  • shRNA short hairpin RNA
  • Dicer a cellular enzyme that removes the hairpin and releases the siRNA, which in turn is processed by RISC.
  • RNAi is amenable to high throughput approaches and can be used to test large collections of siRNAs or shRNAs for cellular changes (Berns et al., 2004; Paddison et al., 2004; Zheng et al., 2004; Kittler et al., 2005; Mackeigan et al., 2005; Pelkmans et al., 2005).
  • siRNAs are transfected into cells or shRNA-expressing vectors are transfected or transduced into cells, and phenotypic changes are measured. Because it may not be possible to predict which siRNA/shRNA sequences will most efficiently lead to mRNA degradation, multiple siRNA/shRNA for each gene are often used in these screens.
  • RNAi' s can be added in a pool, and the RNA that is driving the change identified in subsequent deconvolution experiments, or each RNAi can be introduced separately in parallel, and tested individually.
  • the types of cell- based assays that can be utilized are considerably more informative when using parallel strategies. Pooling selects for stronger phenotypes given that background RNAi' s can dilute out weaker phenotypes. With pooling, the assay is frequently limited to positive selections, and therefore deleterious outcomes cannot easily be scored. With parallel screening, both strong and weak phenotypes can be scored. Finally, when using parallel screening, no deconvolution steps are required, and the results of each RNAi can be recorded for comparison.
  • shRNA-expression vectors are prepared from a plasmid resource and therefore are in an inexhaustible supply.
  • the plasmids are prepared using standard lab methods, making them less expensive to use than commercial siRNAs. For high throughput analysis, this is an important parameter when considering the supply costs encountered with a range of screens and the needed repetitions to establish statistical significance.
  • the shRNAs that may be used in these studies include those in a lentivirus backbone which can thus be used to transduce almost all cell types, whether growing or arrested. This eliminates the concerns which accompany working with cells or under conditions where transfection of oligonucleotides may fail.
  • stable viral integration of the shRNA enables protein down-regulation over the life span of the cell. This is especially useful when protein turnover is slow.
  • TRC shRNA vectors are based on a lentiviral backbone, allowing infection of essentially all human cells. Although this vector is easily used as a lentivirus, it also works quite well as a plasmid when transfected into cells, albeit with the limitations of transfection.
  • RNAi has technical limitations of its own. It is now well documented that siRNAs and shRNAs often induce off-target effects (Jackson et al., 2003; Jackson and Linsley, 2004). These off-target effects are thought to come about by the unexpected recognition of mRNAs by RISC loaded with the siRNA. No clear rules exist on how to predict these off- target effects, and this emphasizes the value of validation and follow-up experimentation in assigning a functional role to a protein based on a siRNA/shRNA hit.
  • Kinases are important for reasons including: (1) their established roles in many important cell pathways, (2) the extensive knowledge of their structure and function, and (3) the ability to find and design small molecular inhibitors that specifically block kinase activity. In addition, although there has been extensive work in the study of kinases, there remains a great deal which is unknown or poorly understood. One hundred one kinases in our current target list have never been mentioned in a refereed publication, and 260 are discussed in 5 or fewer publications.
  • Non-protein kinases like the protein kinases, also help control cell metabolism. Any splice isoform of a kinase may be assayed.
  • shRNAs to 434 kinases were individually transfected into both 293T and HeLa cells.
  • the DNA was introduced using a standard lipid carrier (Fugene), and the cells transferred to drug selection at 24 hrs. At 4 or 5 days, the number of viable cells was measured by Alamar blue assay.
  • the Alamar blue method was chosen after comparison with tetrazolium salt assays, such as MTT or MTS, or other more specific tests for individual phenotypes, e.g. apoptosis measured by DNA fragmentation, because of its robust nature, sensitivity, lack of toxicity, ease of use, low cost, and ability to identify a number of interesting classes of response.
  • HeLa and 203T lines were chosen for two reasons. First, they are among the most prevalent cell lines used for mammalian cell experimentation. Second, both of these cell lines are known to be easily transfectable. In the development of these assays HeLa transfections have now been done three times and the 293 T transfections have been done four times to establish methods for analysis and to test for reproducibility. Within one experiment, each transfection is done in triplicate, and the results are averaged. Figure 1 compares the values recorded for the second and third trials in HeLa cells. The reproducibility of this assay is high, as demonstrated by a correlation coefficient of 0.897, between these two data sets.
  • the shRNA vector backbone was designed by the TRC to allow lentivirus production. This is a useful viral vector system that allows infection of almost every cell line, whether proliferating or post-mitotic.
  • One of the underlying reasons we began screening essential kinases by transfection was the concern that growth-inhibiting hairpins, targeting essential kinases, might kill the 293 T viral packaging cells before adequate virus was harvested. This concern proved valid.
  • Some of the strongest hits from the 293T set were used to test lentiviral production in 293 T cells, and in a fraction of the cases the resulting supernatants did not contain infectious virus particles.
  • the "no viral" phenotype was determined by the cells ability to grow in the absence but not the presence of puromycin ( Figure 4).
  • Standard methodologies for titering virus are laborious and slow.
  • the number of infectious particles per ml can be determined by performing serial dilutions of the viral stock followed by infection of NIH3T3 cells. Twenty-four hours after infection, cells are incubated with puromycin and over time singly dispersed cells grow into resistant colonies that can be visualized with crystal violet staining and counted.
  • GFP-expressing viruses can be used, and the ratio of GFP-positive and GFP-negative infected cells as determined by flow cytometry used to calculate titer.
  • the next step for analyzing the shRNA hits is to determine the aspect of cellular physiology that is affected.
  • Our strategy is to use flow cytometry analysis to first identify which shRNAs can cause cell cycle blocks.
  • Figure 6 shows the results of one round of flow cytometry analysis with a test set of 30 shRNAs identified in the primary screens ( Figure 6). We find that greater than 25% of the kinase shRNA hits when transfected into cells give a specific cell cycle block, and about 10% lead to increased apoptosis.
  • the G2 cell cycle block shown in Figure 5 is caused by an shRNA to PDGFRb, a kinase not previously known to act in G2.
  • cDNA/shRNA screens can be used to find downstream events that rescue blocks
  • the methods of the invention may be used to identify the cellular context in which newly identified kinases function by establishing and ordering them in signal transduction pathways.
  • One method to determine these types of relationships is to look for genetic interactions. This may be done by employing the equivalent of genetic modifier screens using the shRNA-induced phenotype as an initial alteration.
  • a cDNA expression construct or a second shRNA is then introduced to permit a search for genes that suppress or enhance the initial shRNA-dependent block.
  • the Moloney virus carries the blasticidin resistance gene and 24 hrs after infection, blasticidin was added. Forty-eight hours post Moloney virus infection, cells were infected with the lentivirus carrying an ErbB3 shRNA and containing a different selectable marker, a puromycin resistance gene.
  • a second type of genetic interaction screen was performed to examine whether similar epistasis studies can be done by transfection.
  • the cell proliferation block imposed by transfection of CDKlO kinase was examined.
  • An shRNA for CDKlO that gives a strong block was co-transfected individually with kinase cDNA expression vectors.
  • the same backbone vector was used for the Moloney virus production discussed in the paragraph above, but used here as simple transfection vectors. Twenty- four hours post- transfection, cells were selected with puromycin to ensure that the shRNA causing growth inhibition was present, and cell number and viability was scored 4 days later.
  • FIGS 8 and 9 outline the process for identifying kinase targets.
  • shRNA expression constructs are used to determine which kinases are essential for survival and proliferation in a small set of commonly used tissue culture cell lines representing both highly transformed and more normal cell lines.
  • the mechanism of action of each of these essential kinases is classified, and those kinases showing a stage-specific cell cycle block when down- regulated are selected for more study.
  • the proteins that have roles in cell death, in regulating the rate of cell proliferation, or in processes that lead to arrests, irrespective of cell cycle position, are also be identified.
  • the context of action of the cell cycle-specific kinases is examined by determining the genetic interactions of the selected kinase with other kinases and by biochemical studies.
  • Genes can be divided into two functional classes — those genes that are essential for proliferation and survival and those genes that are not. Genes can be assigned to either group following assays for cell viability after shRNA expression (Figure 9). Genes whose functions are essential for proliferation or survival can be tentatively identified when shRNA expression leads to inhibition of cell proliferation or loss of viable cells. Cell viability and number can be measured quantitatively in high throughput with agents such as Alamar Blue or tetrazolium salts, both of which measure cellular oxidative metabolism. As with all screens of this type, classification is only tentative and will need to be confirmed by validation studies described below. At this stage a protein is tentatively classified as essential if any one of the five shRNAs for each protein leads to decreased proliferation or survival. False positives will be removed at later stages.
  • HeLa and 293 T cells were transfected with shRNAs targeting 434 human protein kinases. There were approximately 4.5 shRNA constructs per kinase, and each hairpin was assayed independently. Transfected cells were selected with puromycin, allowed to grow for several days, and cultures scored for viability by an Alamar blue assay. Varying the experimental parameters showed that assays carried out 5 days after transfection gave the best balance between maximizing the effects of introduced shRNAs and sensitivity of the Alamar Blue assay. For each experiment, the transfections were done in triplicate, and the entire experiment repeated either three times for HeLa or four times for 293 T.
  • shRNAs may be introduced through infection, e.g., via lentivrus.
  • the group of kinases that are needed for lentivirus production might be of interest in their own right. Some may generate a block that is important for cell survival, but others might be specific for steps in the virus life cycle.
  • a small set of reference cell lines is used to generate a list of essential kinases.
  • this set may include a pair of commonly used cells, e.g., WB 8 and MCFlOA, representing a nonpathological phenotype.
  • WB 8 cells are normal human diploid lung fibroblast cells that undergo senescence through passage in culture.
  • MCFlOA cells are a breast epithelial line commonly used for many comparative experiments with more highly transformed breast cancer cell lines.
  • a pair of cell lines, A549 and MCF7 are used for screening to represent more typical tumor lines.
  • A549 and MCF7 cells were chosen because they are good representative carcinoma and adenocarcinoma cell lines, respectively, that have been analyzed for mRNA profiling and sequencing of protein tyrosine kinase mutations.
  • A549 are from a lung tumor and MCF7 from a breast tumor.
  • HeLa and 293T cells are included to compare the findings from transfections and infections, and they will provide a direct comparison with the shRNA lentivirus results in the other four lines. It will be appreciated that additional cell lines may be employed.
  • the search for essential kinases may also be done lines in a fashion analogous to transfection, except that shRNAs will be introduced by viral transduction.
  • the six cell lines described, or others, may be utilized. Cells are infected independently with each shRNA-expressing lentivirus, and then cultured for 5 days when differences in cell viability and cell number are determined by Alamar blue assays. Each infection will be done in triplicate, and mean values and standard deviations will be used to record the response to each hairpin.
  • shRNAs that lead to decreases in cell viability or proliferation are classified further. There are at least three categories under which these hits are classified— arrest in a specific stage of the cell cycle, increases in cell death, or a general arrest irrespective of cell cycle stage. These three categories can be initially distinguished by, for example, flow cytometry, as described below. Other classes and phenotypes may appear and are readily used in the classified schemes
  • Some of the shRNA-induced cell proliferation and viability blocks will result in cells arrested in specific stages of the cell cycle. These correspond to many cell cycle regulating proteins whose role is required once and only once during the cell cycle. Such experiments have helped establish the technical approaches required here. Treated cells are typically assayed for changes in their cell cycle profiles by flow cytometry after fixation and propidium iodide staining. Requirement for progression through a particular stage is detected by an increase in percentage of cells in one stage compared to controls. Although flow cytometric analysis is not generally considered a high throughput method, it can be done with reasonable efficiency and is considered a gold standard of cell cycle analysis. This approach is preferred, as the assay is robust and the results are readily characterized.
  • the second class of essential kinases includes those that stop or greatly slow cell division, but do so without specificity for cell cycle position. These include kinases involved in energy regulation, general transcription, or other events that are important throughout a division cycle. Such kinases are identified by virture of flow cytometry profiles that resemble the profiles untreated cell cultures.
  • the final class of essential kinases identified include those that induce cell death. Most types of cell death including all types of apoptosis can be initially identified by flow cytometry profiles as cell bodies with chromosome numbers below the Gl population of the untreated cultures. All of the hits from the essential kinase screens are tested for cell death by histone H3 staining for chromosome condensation that accompanies cell death. Further apoptotic assays which may be used are known in the art and include assays for caspase activation and DNA breaks.
  • shRNA hits may also be validated to determine whether the observed phenotype is the result of the kinase's action on its intended gene target or an off-target effect.
  • a traditional approach for validation is the measurement of changes in mRNA and protein levels using quantitative RT-PCR (for example, TaqMan) and western blotting assays, respectively.
  • validation of these hits may be done by establishing similar phenotypes with a second independent shRNA (or siRNA). Having two distinct shRNAs that target the same transcript generating the same phenotype is a clear indicator that the loss the targeted mRNA is responsible for the phenotype. Whenever possible a second shRNA is targeted to the non-coding regions of the mRNA to enable rescue experiments described below.
  • RNAi to the non- coding regions of the protein
  • rescue the phenotype by expressing the protein from an available cDNA expression libraries. This can be done on a large number of shRNA hits in a high throughput manner.
  • a second manner of rescue is to make a mutant kinase cDNA where the mutation destroys or reduces the recognition by the shRNA. Either approach is sufficient to establish validation — two shRNAs to different regions of the mRNA both independently inhibit growth, and this proliferation defect can be overcome by over-expression of the targeted protein.
  • the second shRNA is not in the untranslated region or is not readily available, direct measurements in mRNA levels will be performed on the single hairpin hit using TaqMan analysis. Where available, antibodies to the targeted protein are also used to determine changes in protein levels by western blotting. The combination of these approaches provides multiple levels of validation without undue efforts.
  • Stage-specific blocks are characterized in more detail, in particular to determine as accurately as possible the precise point at which the block has occurred and find any unusual features of the block. This is done both biochemically and immunochemically by examining other cell cycle events associated with the arrest phenotype.
  • the state of chromosomal DNA in the blocked cells is further characterized.
  • cells are, for example, labeled with 5-bromo-2'-deoxyuridine (BrdU), which is incorporated into DNA and identifies cells replicating their DNA in S phase.
  • PrdU 5-bromo-2'-deoxyuridine
  • G2 blocks with mitotic blocks As for G2/M, cell cycle analysis using flow cytometry and PI staining clusters G2 blocks with mitotic blocks. To distinguish these different blocks and fully classify the mitotic arrest into prophase, metaphase, anaphase, or telophase, fluorescence microscopy are performed using the nuclear stain Hoechst in combination with a phosphorylation-specific antibody against histone H3 conjugated to fluorescein. Phosphorylation-specific antibodies to histone H3 detect cells in late G2 (speckled pattern) and mitosis, and is a useful marker for mitosis and chromosome condensation.
  • G2 and mitotic arrests can be characterized by an overall increase in mitotic index (% cells in mitosis) coincident with a homogenous nuclear morphology (all speckled G2, all in prophase, all in metaphase, etc.) showing mitosis stage specific blocks. Blocks in telophase and cytokinesis defects are monitored by including standard counterstains for microtubules or actin.
  • cdk2 is a late Gl event
  • cdk4 indicates a mid-to-late Gl phase has been passed
  • cdc2 activation is a late G2 to M event.
  • the activity of other kinases such as the PLK and aurora kinases can also help characterize G2 or M phase blocks.
  • These kinase activity assays are conveniently done by immunoprecipitation with specific antibodies from shRNA treated cells followed by an in vitro kinase assay with the addition of appropriate kinase substrate.
  • the kinase activity can be monitored by microscopy and immunofluorescence with a phosphospecific antibody that is conjugated to a fluorochrome.
  • G2 there are important phosphatases whose activity is carefully controlled by cell cycle position. The activities of G2-specific phosphatases can be monitored in extracts from shRNA-treated cells with antibodies specific to the appropriate phosphorylated substrates.
  • stage-specific transcription events can help characterize the specific cell cycle blocks.
  • the expression of several cyclins has been well characterized and can provide useful cell cycle landmarks.
  • other cell cycle specific transcripts have been found in recent years by the use of expression profiling; the exact candidates that are employed will depend upon the stages of cell cycle blocks that are obtained. This can be measured by synchronizing cells and testing the timing of gene activation by standard quantitative measure of RNA levels. Synchronizing cells can be done by a number of methods depending on cell type.
  • the blocks imposed by these shRNAs are challenged by expression of a protein kinase from the cDNA expression vectors described above.
  • the experimental protocol is to infect cells with the shRNA-expressing lentivirus (marked with the puromycin resistance gene) and a cDNA-expressing Moloney virus (marked with the blasticidin resistance gene) and select with both puromycin and blasticidin.
  • Our results indicate that the best rescues occur when the cDNA-expression is established prior to the infection with the shRNA-expression lentivirus.
  • Cell proliferation is measured by Alamar blue assays. While subsets of the kinase cDNA expression libraries are used (for example, only those that correspond to the shRNA hits), the entire kinase cDNA repository only numbers about 500.
  • shRNA expression is also possible and will identify downstream negative regulators. All of the shRNA constructs have been screened for their ability to block cell cycle but not for their ability to overcome blocks by other shRNAs. Epistasis and biochemical analysis.
  • the other mutant that is planned is changing the conserved aspartic acid, phenylalanine, glycine sequence (DFG) in the active site to asparagine, phenylalanine, glycine (RFG). These mutants may be used to study the action of the chosen pathways.
  • a second rescuing kinase rescues the block without the activation of the cdk nor phosphorylation of P. This suggests that Y acts downstream of X.
  • a second rescuing kinase Z may rescue with phosphorylation of protein P but without cdk activity, suggesting that Z acts downstream of X, and further that the well-known substrate of this cdk may also be the substrate of a second kinase, possibly even kinase Z itself. Any feature of the arrested cell identified in Figure 9 above can be studied in this way.
  • the basal level of phosphorylation of a rescuing kinases is established first.
  • Cells are infected with the kinase as a fusion protein (both amino- and carboxy-terminal fusion proteins with a number of tags are available for optimization) of the kinase dead version of the kinase, and then the tagged protein will be precipitated from lysates of the cells and a phosphopeptide tryptic map of the protein established by mass spectroscopy. Changes in the pattern of phosphorylation are then examined by co-infecting with the cDNAs or shRNAs for the rescue kinases in the set.
  • rescuing kinases were selected by their ability to overcome the initial cell cycle block, the action of these kinases as studied is robust.
  • Rescue kinases either over-expressed by cDNAs or down-regulated by shRNAs, are tentatively classified as working upstream of given kinase if they induce changes in the phosphorylation pattern. Any relationships detected in these experiments are considered putative ordering of the pathway.
  • These types of kinase and downstream substrate tests are then carried out either in the presence or absence of the original blocking shRNA or in stable or transient assays.
  • kinase function using shRNAs provides a starting point to examine their roles in cellular events such as proliferation and survival.
  • the same technical approaches developed here for 650 kinases can be expanded to studies of other protein families and eventually to large proteome- scale studies. While shKNA-induced loss of a protein from cells is a dramatic change, these types of larger-scale screens tentatively classify protein function and serve as useful starting points for more detailed biochemical studies.
  • the methods described herein may also be employed to generate a database containing the identify of proteins, whose down-regulation produces a particular result, either desirable or undesirable.
  • kinases that are essential for tumor cell growth may be examined for possible new drug development targets. Examples of desirable characteristics would be kinases for use as a development target are, for example, that the kinase is required in tumor cells but not in non-pathological cells and that the kinase has a role in cellular proliferation which was not previously appreciated.
  • target genes identified by assaying with shRNA is exploited to identify candidate therapeutic compounds for cancer or other hyperproliferative disorders, e.g., to inhibit tumor growth, to inhibit angiogenesis, to decrease inflammation associated with a lymphoproliferative disorder, to inhibit graft rejection, or neurological damage due to tissue repair.
  • Other hyperproliferative disorders include atherosclerosis, graft coronary vascular disease after transplantation, vein graft stenosis, peri-anastomatic prosthetic graft stenosis, restenosis after angioplasty or stent placement, psoriasis, and endometriosis.
  • Tumors of interest include carcinomas, e.g.
  • Cancers of interest include breast cancers, (e.g., ductal carcinoma in situ, infiltrating (or invasive) ductal carcinoma (IDC), infiltrating (or invasive) lobular carcinoma (ILC)), non-small cell lung carcinoma including epidermoid carcinoma (also called squamous cell carcinoma), adenocarcinoma , large cell carcinoma, carcinoid, cylindroma, mucoepidermoid, malignant mesothelioma, and skin cancer (e.g., melanoma).
  • Methods for identifying therapeutic compounds that inhibit the activity of a kinase typically detect the level of expression of the kinase or its activity. Essentially, any compound that reduces the activity of a kinase that is essential to proliferation in hyperproliferative cells is of interest. Mechanisms for reducing activity include reducing transcription, translation, or post- transcriptional alteration of the kinase, and inhibiting the binding of the kinase substrate, e.g., by competitive inhibition or covalent modification of the kinase. For screening purposes, the chemical identity of the candidate therapeutic compounds is not critical. Compounds may be randomly screened for activity, or rational drug design may be employed based on the nucleic acid sequence encoding the kinase or the structure of the protein.
  • kinase activities are known in the art, e.g., Davies et al. Biochem. J. 2000, 351 :95, Bain et al. Biochem. J. 2003, 371 :199, and Copeland Anal. Biochem. 2003, 320:1.
  • therapeutic compounds and kinases may be labeled with FRET moieties to indicate binding, or the rate or amount of phosphorylation of a substrate for the kinase may be monitored.
  • Substrates for a kinase, if unknown, may be identified by methods known in the art, e.g., highthroughput screening of random peptide sequences. Example 1.
  • the “Gene” column lists the gene name. In parentheses is the number of “mode of action” assays in which inhibition of the expression of the indicated gene scored positive. “Mode of action” assays include, for example, autophagy assays (e.g., Figures 10 and 11) SA-beta Gal assays (e.g., Figure 12) and apoptotic assays (e.g., Figures 13 and 14).
  • Shading within a "PUBS" column indicates the number of peer-reviewed journals associated with the indicated gene (i.e., greater than 300, fewer than 10, or fewer than 100).
  • the "normal/tumor” column indicates the effect of inhibition of expression of the indicated gene on the survival of cancer cells relative to the survival of normal cells under the same conditions (i.e., the ratio of survival of the MCFlOA (normal) and MCF7 (cancerous) cell lines.) A value of greater than one indicates that inhibition of the expression of the indicated gene selectively decreases the survival of cancer cells. Shaded entries indicate the greatest selective effect.
  • the genes in Table 1 can be divided into three groups, indicated in Table 2.
  • RNAi screen in human cells identifies new components of the p53 pathway. Nature 428, 431-437.
  • FLEXGene repository from sequenced genomes to gene repositories for high-throughput functional biology and proteomics. MoI Biochem Parasitol 118, 155-165.
  • Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev 15, 2654-2659.
  • An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division. Nature 432, 1036-1040.
  • RNAi screen of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance. Nat Cell Biol. Marsischky, G., and LaBaer, J. (2004). Many paths to many clones: a comparative look at high-throughput cloning methods. Genome Res 14, 2020- 2028.
  • Rasl and a putative guanine nucleotide exchange factor perform crucial steps in signaling by the sevenless protein tyrosine kinase. Cell 67, 701- 716.
  • T24 bladder carcinoma transforming gene is linked to a single amino acid change. Nature 300, 762-765.
  • Radl ⁇ is required for DNA repair and checkpoint responses in fission yeast. MoI Biol Cell 10, 2905-2918.
  • GATEWAY recombinational cloning application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol 328, 575-592.

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Abstract

L'Invention concerne un procédé d'identification des kinases nécessaires à la croissance des cellules cancéreuses. Les kinases identifiées au moyen de ce procédé peuvent être utilisées pour identifier des composés présentant des propriétés anticancéreuses.
PCT/US2006/039235 2005-10-06 2006-10-06 Procedes permettant d'identifier des proteines essentielles a la proliferation des cellules humaines, et agents therapeutiques diriges contre ces proteines Ceased WO2007044571A2 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008007072A3 (fr) * 2006-07-10 2008-05-08 Astrazeneca Ab Procédé d'inhibition cellulaire
WO2010141738A2 (fr) 2009-06-03 2010-12-09 President And Fellows Of Harvard College Compositions et procédé pour inhiber la croissance d'une tumeur
WO2014013231A1 (fr) * 2012-07-16 2014-01-23 The Institute Of Cancer Research: Royal Cancer Hospital Matériaux et procédés pour le traitement d'un cancer à mutation ou déficience en pten
US8652472B2 (en) 2006-12-05 2014-02-18 Oncomed Pharmaceuticals, Inc. Compositions and methods for diagnosing and treating cancer
WO2019033041A1 (fr) * 2017-08-11 2019-02-14 Board Of Regents, The University Of Texas System Ciblage de kinases pour le traitement de métastases cancéreuses

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004082458A2 (fr) * 2003-02-21 2004-09-30 The Johns Hopkins University Tyrosine kinome

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008007072A3 (fr) * 2006-07-10 2008-05-08 Astrazeneca Ab Procédé d'inhibition cellulaire
US8652472B2 (en) 2006-12-05 2014-02-18 Oncomed Pharmaceuticals, Inc. Compositions and methods for diagnosing and treating cancer
WO2010141738A2 (fr) 2009-06-03 2010-12-09 President And Fellows Of Harvard College Compositions et procédé pour inhiber la croissance d'une tumeur
WO2014013231A1 (fr) * 2012-07-16 2014-01-23 The Institute Of Cancer Research: Royal Cancer Hospital Matériaux et procédés pour le traitement d'un cancer à mutation ou déficience en pten
WO2019033041A1 (fr) * 2017-08-11 2019-02-14 Board Of Regents, The University Of Texas System Ciblage de kinases pour le traitement de métastases cancéreuses
EP4036583A1 (fr) * 2017-08-11 2022-08-03 Board of Regents, The University of Texas System Ciblage de kinases pour le traitement de métastases cancéreuses
US11406643B2 (en) 2017-08-11 2022-08-09 Board Of Regents, The University Of Texas System Targeting kinases for the treatment of cancer metastasis

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