WO2008085898A2 - Methods for identifying essential proteins and therapeutic agents - Google Patents

Abstract

The invention features methods of identifying compounds that selectively interfere with anchorage independent cell survival and methods of diagnosing a subject as having, or having the propensity to develop, a proliferative disorder (e.g., cancer).

Description

METHODS FOR IDENTIFYING ESSENTIAL PROTEINS AND THERAPEUTIC AGENTS

BACKGROUND OF THE INVENTION

This invention relates to the fields of kinases, cancer, and pharmaceutical compounds.

Mammalian cells in culture have provided a powerful and immensely useful system to study many aspects of eukaryotic cell physiology. 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. Genetically tractable organisms have provided powerful models for the study of many aspects of biology in an unbiased manner. When genetic screens are done to saturation in these systems, they allow comprehensive identification of the genetic components which play a role in the biological event under study. Mammalian tissue culture cells have historically not been tremendously useful for such genetic screening studies for several reasons. It is difficult to construct and test homozygous null genetic backgrounds in mammalian cells because they are diploid and there is no ready system for mating or direct exchange of genetic information between cells. Many of the cells that grow well in culture are genetically unstable, making any long- term selection or assessment more difficult. Recent advances in methods for manipulation of cDNA clones and the development of RNA interference (RNAi) made cells in culture more amenable to genetic manipulation. Using RNAi it is possible to alter the levels of a given protein and measure the resulting phenotype. Protein levels can be raised by synthesis from cDNA expression vectors or reduced by introducing an inhibitory RNA. These types of changes can be done in high throughput to screen large numbers of proteins, with the ultimate goal of screening the entire coding potential of the genome, termed the proteome. Transformed cells and cancer cell lines often exhibit enhanced anchorage- independent survival when compared to normal epithelial cells. Resistance to anoikis, or apoptosis induced by matrix detachment, may critically contribute to processes such as invasion and metastasis of tumor cells. The pathways and signaling molecules involved in anoikis-resistance conferred by specific oncogenes have yet to be comprehensively elucidated.

SUMMARY OF THE INVENTION

The invention features methods for identifying and utilizing kinases, and other proteins, whose down-regulation induces a desirable outcome. For example, the methods provided may be employed to identify kinases that are essential to the anchorage-independent survival of cells associated with cancer metastasis. The methods for identifying kinases involve utilizing one or more siRNAs to target a particular protein and determining the effect of down-regulation on anchorage- independent survival. The invention also features methods of identifying compounds useful for treating proliferative disorders and methods of diagnosing a subject as having, or having a propensity to develop, a proliferative disorder (e.g., cancer).

In one aspect, the invention features a method of identifying a therapeutic compound (e.g., a siRNA or antibody) for treating a proliferative disease (e.g., cancer). This method includes the steps of assaying a candidate therapeutic compound for inhibition of the kinase activity of at least one (e.g., at least 2, 3, 4, 5, 6, 7, or 8) kinase selected from: ACVR2B, ADCK2, ADRBKl, AKl, AK5, AKAP12, AKAP3, AKAP9, AKTl, AKT2, ALS2CR2, ALS2CR7, BLK, BMPRlA, BMX, CAMK2B, CAMKIINalpha, CARKL, CDC42BPB, CDK3, CDK5R2, CDKL3, CHK, CIB3, CLK3, CSNKlAl , CSNKlD, DAPK3, DGKE, DOKl, DYRKlB, DYRK4, EGFR, EPHB6, ERK8, FLTl, HIPKl, HKl, HRI, IKBKE, ILK, ITK, ITPKA, KIP2, LAK, MAK, MAP2K1IP1, MAP3K3, MAP3K7IP1, MAP4K2, MAP4K4, MAPK8IP3, MAPKAPl, MAPKAPK2, MAPKAPK3, MARKl, MARK3, MAST2, MK-STYX, NAGK, NEK8, NEK9, NIPA, NME3, NTRKl, NTRK3, NUCKS, PAK4, PFKFBl, PFKFB4, PFKM, PHKGl, PIK3CD, PIP5K1C, PIP5K2A,

PIP5K2B, PKNbeta, PLKl, PRKABl, PRKACA, PRKACG, PRKAG3, PRKAR2A, PRKCABP, PRKCE, PRKCQ, PRKX, PTK6, PXK, RIOK3, RIPKl, ROCK2, ROR2, SMGl, STK16, STK22C, STK22D, STK25, STK29, STK35, STK38L, TKl, TK2, and TNNI3K. In preferred methods, the at least one kinase is selected from ACVR2B, CAMK2B, CHK, CSNKlAl, DGKE, IKBKE, MAP4K4, PIP5K2A, PRKACG, PRKCQ, PTK6, and ROR2. In this method the level of inhibition is indicative of the therapeutic efficacy for treating the proliferative disease with the candidate compound. In a desirable embodiment of the method, the candidate compound mediates a reduction (e.g., at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%) in the kinase activity of at least one (e.g., at least 2, 3, 4, 5, 6, or 7) of the above listed kinases compared to kinase or sample untreated with the candidate compound. In the above embodiment, the assay may include contacting the candidate therapeutic compound with the kinase protein in vitro and measuring the kinase activity of the kinase protein (e.g., in vitro kinase assay). In any of the forgoing embodiments the assay may also include contacting the therapeutic compound with a cell capable of transcribing the mRNA encoding the kinase and measuring the level of expression of the mRNA (e.g., via Northern blot or reverse transcription polymerase chain reaction) or the kinase encoded by the mRNA (e.g., via Western blot). Any of the above described methods may also include the steps of contacting cells (e.g., cells expressing one or more of the genes selected from: IGF-IR, ErbB2, and phosphatidylinositol 3 '-kinase alpha) with the candidate therapeutic compound followed by measuring the ability to survive under anchorage- independent conditions of the cells after the contacting. In this embodiment, the level of reduction in the ability to survive under anchorage-independent conditions is indicative of the therapeutic efficacy for treating the proliferative disease with the candidate compound. In another aspect the invention features a method of generating a database.

This method includes contacting cells expressing a class of mRNA, each member having known sequence, with one or more siRNAs specific to each sequence of the class. This step is followed by measuring the ability of the cells to survive under anchorage-independent conditions. In this method, a reduction in the ability to survive under anchorage-independent conditions identifies an mRNA as essential to anchorage-independent survival. This method also includes creating a record in the database, wherein the record includes the identity of an mRNA of the class that is essential. In this aspect, the record can further include the identity of the phenotypic effect of reducing the activity of the protein encoded by the mRNA.

In another aspect, the invention features a database including data relating to biological activity of at least two (or, for example, at least 3, 4, 5, 6, 7, 8, 10, or 80) of the kinases selected from the group of ACVR2B, ADCK2, ADRBKl , AKl , AK5, AKAP12, AKAP3, AKAP9, AKTl, AKT2, ALS2CR2, ALS2CR7, BLK, BMPRlA, BMX, CAMK2B, CAMKIINalpha, CARKL, CDC42BPB, CDK3, CDK5R2, CDKL3, CHK, CIB3, CLK3, CSNKlAl, CSNKlD, DAPK3, DGKE, DOKl, DYRKlB, DYRK4, EGFR, EPHB6, ERK8, FLTl , HIPKl, HKl, HRI, IKBKE, ILK, ITK, ITPKA, KIP2, LAK, MAK, MAP2K1IP1, MAP3K3, MAP3K7IP1, MAP4K2, MAP4K4, MAPK8IP3, MAPKAPl, MAPKAPK2, MAPKAPK3, MARKl, MARK3, MAST2, MK-STYX, NAGK, NEK8, NEK9, NIPA, NME3, NTRKl, NTRK3, NUCKS, PAK4, PFKFBl, PFKFB4, PFKM, PHKGl, PIK3CD, PIP5K1C, PIP5K2A, PIP5K2B, PKNbeta, PLKl, PRKABl, PRKACA, PRKACG, PRKAG3, PRKAR2A, PRKCABP, PRKCE, PRKCQ, PRKX, PTK6, PXK, RI0K3, RIPKl, ROCK2, ROR2, SMGl, STKl 6, STK22C, STK22D, STK25, STK29, STK35, STK38L, TKl, TK2, and TNNI3K. In a preferred embodiment, the database includes data relating to the^' biological activity of at least two of the kinases selected from ACVR2B, CAMK2B, CHK, CSNKlAl, DGKE, IKBKE, MAP4K4, PIP5K2A, PRKACG, PRKCQ, PTK6, and ROR2.

This database may also include data relating to the biological effect of a compound on the at least two (e.g., at least 3, 4, 5, 6, or 7) of the kinases listed. For example, the database may include data relating to at least two (e.g., at least 3, 4, 5, or 6) biological activities of the at least two of the kinases listed or data on the biological activity of the at least two kinases in at least two (e.g., at least 3, 4, 5, or 6) types of cells, wherein one type has pathological characteristics and the other does not.

In another aspect, the invention features a method of diagnosing a subject as having, or having a propensity to develop, a proliferative disorder (e.g., cancer). This method includes isolating cells from the subject and measuring the kinase activity of at least one kinase (e.g., at least 2, 3, 4, 5, 6, 7, 10, or 80 kinases) selected from the group of: ACVR2B, ADCK2, ADRBKl, AKl, AK5, AKAP 12, AKAP3, AKAP9, AKTl, AKT2, ALS2CR2, ALS2CR7, BLK, BMPRlA, BMX, CAMK2B, CAMKIINalpha, CARKL, CDC42BPB, CDK3, CDK5R2, CDKL3, CHK, CIB3, CLK3, CSNKlAl, CSNKlD, DAPK3, DGKE, DOKl, DYRKlB, DYRK4, EGFR, EPHB6, ERK8, FLTl, HIPKl, HKl, HRI, IKBKE, ILK, ITK, ITPKA, KIP2, LAK, MAK, MAP2K1IP1, MAP3K3, MAP3K7IP1, MAP4K2, MAP4K4, MAPK8IP3, MAPKAPl , MAPKAPK2, MAPKAPK3, MARKl, MARK3, MAST2, MK-STYX, NAGK, NEK8, NEK9, NIPA, NME3, NTRKl, NTRK3, NUCKS, PAK4, PFKFBl, PFKFB4, PFKM, PHKGl , PIK3CD, PIP5K1C, PIP5K2A, PIP5K2B, PKNbeta, PLKl, PRKABl, PRKACA, PRKACG, PRKAG3, PRKAR2A, PRKCABP, PRKCE, PRKCQ, PRKX, PTK6, PXK, RIOK3, RIPKl, ROCK2, ROR2, SMGl, STKl 6, STK22C, STK22D, STK25, STK29, STK35, STK38L, TKl, TK2, and TNNI3K. In a preferred embodiment of the above method, the least one kinase is selected from the group of ACVR2B, CAMK2B, CHK, CSNKlAl, DGKE, IKBKE, MAP4K4, PIP5K2A, PRKACG, PRKCQ, PTK6, and ROR2. In this method, an increase in the kinase activity of the kinase or kinases, as compared to the level in a reference sample, is a diagnostic indicator of a proliferative disorder or a propensity to develop a proliferative disorder.

In yet another aspect the invention includes a method of diagnosing a subject as having, or having a propensity to develop, a malignant proliferative disorder. This method includes isolating cells from the subject and measuring the kinase activity of at least one kinase (e.g., at least 3, 4, 5, 6, 7, 8, 10, or 80 kinases) selected from the group consisting of: ACVR2B, ADCK2, ADRBKl, AKl, AK5, AKAP 12, AKAP3, AKAP9, AKTl, AKT2, ALS2CR2, ALS2CR7, BLK, BMPRlA, BMX, CAMK2B, CAMKIINalpha, CARKL, CDC42BPB, CDK3, CDK5R2, CDKL3, CHK, CIB3, CLK3, CSNKlAl, CSNKlD, DAPK3, DGKE, DOKl, DYRKlB, DYRK4, EGFR, EPHB6, ERK8, FLTl, HIPKl, HKl, HRI, IKBKE, ILK, ITK, ITPKA, KIP2, LAK, MAK, MAP2K1IP1 , MAP3K3, MAP3K7IP1, MAP4K2, MAP4K4, MAPK8IP3, MAPKAPl, MAPKAPK2, MAPKAPK3, MARKl, MARK3, MAST2, MK-STYX, NAGK, NEK8, NEK9, NIPA, NME3, NTRKl, NTRK3, NUCKS, PAK4, PFKFBl, PFKFB4, PFKM, PHKGl, PIK3CD, PIP5K1C, PIP5K2A, PIP5K2B, PKNbeta, PLKl, PRKABl, PRKACA, PRKACG, PRKAG3, PRKAR2A, PRKCABP, PRKCE, PRKCQ, PRKX, PTK6, PXK, RI0K3, RIPKl, ROCK2, ROR2, SMGl, STKl 6,

STK22C, STK22D, STK25, STK29, STK35, STK38L, TKl, TK2, and TNNI3K. In a preferred embodiment of the above method, the least one kinase is selected from the group of ACVR2B, CAMK2B, CHK, CSNKlAl, DGKE, IKBKE, MAP4K4, PIP5K2A, PRKACG, PRKCQ, PTK6, and ROR2. In this method, an increase in the kinase activity of the kinase or kinases, as compared to the level in a reference sample, is a diagnostic indicator of a malignant proliferative disorder or a propensity to develop a malignant proliferative disorder. In another aspect the invention features a method for identifying a gene necessary for anchorage-independent growth. This method includies contacting cells expressing a gene selected from IGF-IR, ErbB2, and phosphatidylinositol 3 '-kinase alpha and also expressing a class of mRNA, each member having a known sequence, with one or more siRNAs specific to each sequence of the class and measuring the ability to survive under anchorage-independent conditions of the cells after the contacting. In this method, a reduction in the ability identifies an mRNA as essential to anchorage-independent survival.

In another aspect the invention features a method for identifying a therapeutic compound for treating a proliferative disease (e.g., cancer). This method includes contacting cells (e.g., cells expressing a gene selected from IGF-IR, ErbB2, and phosphatidylinositol 3 '-kinase alpha) with a candidate compound and measuring the ability to survive under anchorage-independent conditions of the cells after the contacting. In this method a reduction in the ability to survive identifies a candidate compound as inhibiting survival under the anchorage-independent conditions. In any of the forgoing aspects the cell may be a suspect cell.

By "Candidate Kinases" is meant the kinases set forth in Table IA. Exemplary Genebank accession numbers corresponding to the sequences of Candidate Kinases are also set forth in Table IA. By Candidate Kinases is also meant a nucleic acid with at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% percent sequence identity to any of the kinases listed in Table IA as determined by the NCBI BLAST program having the relevant kinase biological phenotype. Additionally and alternatively, a Candidate Kinase is defined as a nucleic acid that hybridizes under high stringency conditions to a nucleic acid of a kinase listed in Table IA having the relevant biological phenotype. Table IA Candidate Kinases

Table IB Candidate Kinases-siRNA sequences

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; Sequence Analysis Primer, Gribskov and Devereux, Eds., M. Stockton Press, New York, 1991 ; and Carillo and Lipman, SIAMJ. Applied Math 48:1073, 1988.

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 from websites such as the NCBI website (http://www. ncbi.nlm.nih.gov/BLAST/unfinishedgenome.html) or the TIGR website (http://www. tigr.org/cgi-bin/BlastSearch/blast.cgi). These software programs match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. 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.

By "hybridize" is meant pair to form a double-stranded complex containing complementary paired nucleobase sequences, or portions thereof, under various conditions of stringency (see, e.g., Wahl. and Berger, Methods Enzymol 152:399, 1987; Kimmel, Methods Enzymol 152:507, 1987).

By "hybridizes under high stringency conditions" is meant under conditions of stringent salt concentration, stringent temperature, or in the presence of formamide. For example, 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. In a more preferred embodiment, hybridization will occur at 37 °C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/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 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art. For most applications, washing steps that follow hybridization will also vary in stringency. 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. For example, 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 ⁰C, and most preferably of at least about 68 °C. In a preferred embodiment, wash steps will occur at 25 ⁰C in 30 mM NaCl, 3 tnM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, 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 ⁰C 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. Nat. Acad Sci. U.S.A. 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). Preferably, hybridization occurs under physiological conditions. Typically, complementary nucleobases hybridize via hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

By a "suspect cell" is meant a cell that has been isolated (e.g., a biopsy) from a tissue (e.g., breast, epithelium, or subepithelium) that may contain a neoplasia. Examples of suspect cells are cells with irregular margins and cells capable of anchorage-independent growth.

By "kinase activity" is meant the activity whereby an enzyme phosphorylates a substrate (e.g., a protein, lipid, or carbohydrate substrate). This activity is meant to include 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.

By 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 (e.g., siRNA molecules or shRNA molecules), antibodies (e.g., an antibody which binds to and/or decreases the activity of a Candidate Kinase), and components or combinations thereof. Examples of compounds include the RNAi molecules listed in Table IB and any art-known antibody that binds or decreases the activity of a Candidate Kinase. Any of compound as herein defined (e.g., RNAi molecule or antibody) may be used as a therapeutic agent for the treatment of a proliferative disorder (e.g., cancer).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a series of micrographs and diagrams showing 3D models of distinct breast tumor phenotypes.

Figure 2 is a diagram of a 3D culture model of mammary epithelial acini.

Figure 3 is a diagram and a series of immuno fluorescent micrographs showing the apico-basal cell polarity and lumen formation within epithelial acini.

Figure 4 is a series of photomicrographs showing the distinct biological activities of cells expressing specific oncogenes (cyclin D, ErbB2/HER2, CSF-IR, and IGF-IR) in 3D culture.

Figure 5 is a photomicrograph showing the phenotype of control cells and a photomicrograph showing the invasion phenotype of cells expressing ErbB2/TGFβ.

Figure 6 is a list of facts implicating a role for IGF-I expression in breast cancer.

Figure 7 A is a Western blot showing the levels of phopsho-IGFR and IGFR protein in cells overexpressing IGFR and treated with IGF-I compared to control cells that do not overexpress IGFR and/or are not treated with IGF-I.

Figure 7B is a series of photomicrographs showing the cell growth observed in cells overexpressing IGFR and treated with IGF-I compared to control cells that overexpress IGFR and are not treated with IGF-I, and cells that do not overexpress IGFR and are treated with IGF-I .

Figure 7C is a series of photomicrographs showing the growth morphology of cells which either overexpress IGF-IR and/or are treated with IGF-I.

Figure 7D is a series of immunofluorescent micrographs showing the expression of Ki67 and Caspase 3 in cells overexpressing IGF-IR and treated with IGF-I .

Figure 8 is a list of oncogenes that enhance survival of cells in the luminal space. Figure 9 is a diagram of the siRNA screen used to identify molecules critical for protection from anoikis.

Figure 10 is a flow chart showing the automated transfection protocol.

Figure 11 is a diagram of the analysis of siRNA screen data. Figure 12 is a diagram of the result of the primary siRNA screen in IGF-IR cells and the result of a repeated siRNA screen in IGF-IR cells.

Figure 13 is a diagram of the result of the primary siRNA screen in IGF-IR cells and the result of the counterscreen in MGF-IOA cells cultured in Matrigel.

Figure 14 is a list of RNAi screen hits that retested in a second RNAi screen. Figure 15 is a graph showing the fold change in viability observed for specific

RNAi molecules in the IGFlR and MCFlOA siRNA screens.

Figure 16 is a Northern blot showing the levels of Candidate Kinase expression.

Figure 17A is a immunofluoresecent micrograph showing the amplification of PTK6 in a breast rumor.

Figure 17B is a Kaplan-Meier curve showing the effect of PTK6 on (breast cancer) disease- free survival.

Figure 18A is graph of the RNA expression analysis of PTK6 in various human breast tumor subsets. Figure 18B is a Western blot showing the levels of PTK6 protein in different breast cancer cell lines.

Figure 19A is a graph showing PTK6 siRNA reverses IGF-I and Matri gel- induced survival of MCF-7 cells grown in suspension.

Figure 19B is a Western blot showing the amount of PTK6 and cleaved PARP present in cells transfected with PTK6 siRNA or control vector (Luc) in the presence or absence of IGF- 1.

Figure 2OA is a graph showing the cell survival of MCF-7 cells expressing control siRNA (Luc siRNA) or PTK6 siRNA, when grown in suspension.

Figure 2OB is a Western blot of PARP cleavage and PTK6 levels in MCF-7 cells expressing control siRNA (Luc) or PTK6 siRNA, when grown in suspension.

Figure 21 A is a graph showing the relative cell death of IGF-IR cells expressing different PTK6 siRNA molecules. Figure 21 B is a Western blot showing PARP cleavage in IGF-IR cells expressing different PTK6 siRNA molecules.

Figure 21 C is a graph showing the relative cell death of IGF-IR cells (attached or in suspension) expressing LKO siRNA molecules or PTK6 siRNA molecules. Figure 21 D is a graph showing the PARP cleavage in IGF-IR cells expressing

LKO siRNA molecules or PTK6 siRNA molecules.

Figure 22 A is a graph of the percentage cell death in MCF-7 cells expressing control siRNA (Luc siRNA) or PTK6 siRNA, when cultured in the presence of absence of IGF-I . Figure 22B is a Western blot showing the levels of cleaved PARP and PTK6 in MCF-7 cells expressing control siRNA (Luc siRNA) or PTK6 siRNA, when cultured in the presence or absence of IGF-I .

Figure 22C is a graph of the relative cell death in ZR751 cells expressing control siRNA (Luc siRNA) or PTK6 siRNA at 48 hours and 72 hours after transfection.

Figure 22D is a Western blot showing the levels of cleaved PARP and PTK6 in ZR751 cells expressing control siRNA (Luc siRNA) or PTK6 siRNA at 48 h or 72 h after transfection.

Figure 23 A is a Western blot showing the level of PTK6 expression in IGF-IR cells expressing control siRNA (Luc) or PTK6 siRNAs following growth in suspension for 24 hours.

Figure 23B is a Western blot showing the level of PARP cleavage in IGF-IR cells expressing control siRNA (Luc) or PTK6 siRNAs following growth in suspension for 24 hours. Figure 24 is a phase contrast image of cell growth in 3D Matrigel cultures.

The cells overexpress IGF-IR and express either a control vector or a PTK6 shRNA vector.

Figure 25 A is a Western blot showing the expression of PTK6 protein in IGF- IR cells expressing PTK6 shRNA. Figure 25B is a series of photomicrographs showing the cell morphology of

IGF-IR cells following transfection with a control vector or PTK6 shRNA vector. Figure 26 is a graph showing the amount of cell death in cells transfected with a PTK6 siRNA vector or control vector (Luc siRNA) in mammary epithelial cells expressing the Neu oncogene.

Figure 27 is a Western blot showing the level of PRKCQ (PKCΘ) expression in lysates from different breast cancer cell lines.

Figure 28 is a graph showing the percent cell death in control MCFlOA cells and MCFlOA cells overexpressing PRKCQ (PKCΘ) following culture in suspension for 48 hours.

Figure 29 is a graph of cell migration of control MCF-IOA cells and MCF- 1OA cells overexpressing PRKCQ (PKCΘ) in the presence or absence of 0.5 ng/mL EGF.

Figure 30 is a diagram of lysate array analysis of candidate siRNA-tranfected cells.

DETAILED DESCRIPTION OF THE INVENTION

In the post-genomic era, researchers can use the tools of molecular biology to identify specific proteins with roles in cancer cell growth and/or survival and then develop chemical compounds (drugs) that disrupt function of these proteins, with the idea that these drugs (e.g., siRNA molecules) may be used as cancer therapies. One of the challenges of the approach is to first identify appropriate protein targets for specific cancer types. An ideal target protein might be one that is essential for viability in a cancer cell, but not essential for viability in healthy, non-cancerous cells in the same tissue or organ. For example, a protein involved in anchorage- independent survival of neoplastic cells would be a desirable target because disruption of its activity would not disrupt the anchorage-dependent growth of a normal cell The invention features a method of identifying pharmaceutical compounds useful for the disruption of the anchorage-independent survival of neoplastic cells. In one aspect, the invention features the identification of genes necessary for anchorage- independent survival of anchorage-independent cells. In another aspect, the invention features the method of identifying pharmaceutical compounds which modulate the activity of one or more of the genes necessary for anchorage-independent cell survival (e.g., ACVR2B, CAMK2B, CHK, CSNKlAl, DGKE, IKBKE, MAP4K4, PIP5K2A, PRKACG, PRKCQ, PTK6, and ROR2). I. Identification of genes necessary for anchorage-independent growth

One of the most exciting discoveries of recent years is RNA interference or RNAi (Fire et al., 1998; Bernstein et al., 2001; Elbashir et al., 2001; Hammond et al., 2001 ; Ketting et al, 2001). 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). The RISC complex unwinds the duplex, uses one strand of the duplex for recognition of homologous sequences in mRNA, and then cleaves the mRNA. The outcome is the lowering of specific mRNA levels and the subsequent reduction of specific protein levels. Two methods are available to generate siRNAs. In one method, one can 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. The shRNA is recognized by a cellular enzyme called Dicer 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. (Exemplary methods are set forth in Berns et al., 2004; Paddison et al., 2004; Zheng et al., 2004; Kittler et al., 2005; Mackeigan et al., 2005; and Pelkmans et al., 2005). In the present invention, 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 can be used in these screens. The RNAi molecules 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 molecules 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.

The kinome

Kinases are important for reasons including: (1) their established roles in many important cellular 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.

The methods of the invention are applicable to all kinases, including the lipid, nucleotide, and carbohydrate kinases. Non-protein kinases, like the lipid kinases, also help control cell metabolism. Any splice isoform of a kinase may be assayed.

The methods of the invention are also applicable to other proteins (e.g., transcription factors), which may be involved in anchorage-independent survival.

siRNA screen

Normal epithelial cells undergo apoptosis (programmed cell death) when detached from extracellular matrix and cultured in suspension. This specific type of apoptosis has been referred to as anoikis. Many oncogenes have been demonstrated to protect cells from anoikis. This activity of oncogenes is believed to contribute to tumorigenesis by allowing tumor cells to survive when they locate to sites outside of their normal "niches" (e.g., after invasion into their microenvironment or metastasis to other tissues). We have developed a high throughput siRNA screen to identify key signaling molecules involved in anoikis-resistance mediated by oncogenes (e.g., the IGF-I receptor (IGF-IR), ErbB2 (e.g., Genbank Accession Nos. NM_001005862, and NM_004448), and PIK3CA (e.g., Genbank Accession No. NM_006218, and the mutant variants E545K and H 1047R). Using this screen, we have identified multiple siRNAs that prevent IGF-IR mediated, anchorage-independent survival. The genes targeted by these siRNAs represent potential targets for therapeutic intervention in cancer because the inhibition of these proteins may prevent the survival of tumors cells outside their natural tissue environments. Cells overexpressing IGF-IR are detached from matrix, transfected with a library of small interfering RNA oligonucleotides (siRNAs) that target cellular kinases and then cultured in suspension for 72 hours. Cell viability is monitored, for example, using the Alamar Blue assay, which measures cellular reducing equivalents, a reflection of cellular metabolic activity. The reduction of Alamar Blue causes the dye to change in color from a deep blue to a pinkish color; a change that can easily be quantitated fluorimetrically. siRNAs that result in compromised cell viability are identified for additional validation and evaluation. This assay has identified multiple siRNAs that reproducibly impair IGF-IR driven viability in suspension without significant toxicity to normal epithelial cells attached to matrix (see, Example 2 below). The kinases targeted by these siRNAs represent attractive candidates for therapeutic intervention in proliferative diseases.

This screen can also be used to test compounds for their ability to disrupt anchorage-independent growth. In this embodiment, cells overexpressing a gene involved in anchorage-independent growth (e.g., IGF-IR, ErbB2, and PIK3CA) are tested for anchorage-independent cell viability, as described above, in the presence of candidate therapeutic compounds.

II. Screens for pharmaceutical compounds

Screening assays to identify compounds that modulate the expression or activity of Candidate Kinases are carried out by standard methods. The screening methods may involve high-throughput techniques. In addition, these screening techniques may be carried out in cultured cells or in organisms such as worms, flies, yeast, or mammals. Screening in these organisms may include the use of polynucleotides homologous to Candidate Kinases.

Any number of methods are available for carrying out such screening assays. According to one approach, candidate compounds are added at varying concentrations to the culture medium of cells expressing a polynucleotide coding for a Candidate Kinase. Gene expression is then measured. There are several methods to assay for gene expression known in the art. Some examples include the preparation of RNA from samples and the use of the RNA for Northern blotting, PCR-based amplification (e.g., RT-PCR), or RNAse protection assays. (Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience, New York, 1997). The level of gene expression in the presence of the candidate compound is compared to the level measured in a control culture medium lacking the candidate molecule. A compound which promotes a change (e.g., a decrease) in the expression of a Candidate Kinase is considered useful in the invention; such a molecule may be used, for example, as a therapeutic for a proliferative disorder (e.g., cancer).

While a candidate compound may be identified through modulation of any one of the Candidate Kinases, particularly promising compounds would modulate several, or many (e.g., at least 1, 2, 3, 4, 5, 6, 7, or 8) of the Candidate Kinases (e.g., ACVR2B, CAMK2B, CHK, CSNKlAl, DGKE, IKBKE, MAP4K4, PIP5K2A, PRKACG, PRKCQ, PTK6, and ROR2). It is well known in the art that the gene expression of a large number of genes can be measured using a nucleotide microarray. Compounds which modulate Candidate Kinases could be identified by comparing the expression profile of Candidate Kinases from cells treated with a candidate compound compared to a control (e.g., untreated) sample. Promising candidates would likely result in decreased expression of Candidate Kinases.

One aspect of this invention is a microarray containing nucleic acid molecules which hybridize nucleic acids substantially identical to Candidate Kinases or fragments thereof. These microarrays would be useful for identifying compounds which effect the expression multiple Candidate Kinases (e.g., ACVR2B, CAMK2B, CHK, CSNKlAl, DGKE, IKBKE, MAP4K4, PIP5K2A, PRKACG, PRKCQ, PTK6, and ROR2). Preferably, the microarray would contain nucleic acid molecules that hybridize nucleic acids substantially identical to all of the Candidate Kinases or fragments thereof. Yet another feature of the invention is the method of analyzing data from previously conducted microarray experiments, where the microarray based candidate drug screen contains Candidate Kinases (e.g., ACVR2B, CAMK2B, CHK, CSNKlAl, DGKE, IKBKE, MAP4K4, PIP5K2A, PRKACG, PRKCQ, PTK6, and ROR2), to identify candidate compounds. A feature of this aspect of the invention is that large portions of the experimentation has already been completed and is available in the art.

If desired, the effect of candidate compounds may, in the alternative, be measured at the level of polypeptide production using the same general approach and standard immunological techniques, such as Western blotting or immunoprecipitation with an antibody specific for Candidate Kinases (e.g., ACVR2B, CAMK2B, CHK, CSNKlAl , DGKE, IKBKE, MAP4K4, PIP5K2A, PRKACG, PRKCQ, PTK6, and RO R2). For example, immunoassays may be used to detect or monitor the expression of Candidate Kinases. Polyclonal or monoclonal antibodies which are capable of binding to such a polypeptide may be used in any standard immunoassay format (e.g., ELISA, Western blot, RIA assay, or protein microarray) to measure the level of Candidate Kinases. A compound which promotes a change (e.g., a decrease) in the expression of Candidate Kinases is considered particularly useful. Again, such a molecule may be used, for example, as a therapeutic for a proliferative disorder (e.g., cancer). Alternatively, or in addition, candidate compounds may be screened for those that specifically bind to and modulate the activity of Candidate Kinases. The efficacy of such a candidate compound is dependent upon its ability to interact with the polypeptide. Such an interaction can be readily assayed using any number of standard binding techniques and functional assays (e.g., those described in Ausubel et al., supra). For example, a candidate compound may be tested in vitro for interaction and binding with a Candidate Kinase and its ability to modulate a Candidate Kinase's activity may be assayed by any standard assays (e.g., in vitro kinase assays).

In one particular embodiment, a candidate compound that binds to Candidate Kinases may be identified using a chromatography-based technique. For example, a recombinant Candidate Kinase may be purified by standard techniques from cells engineered to express a Candidate Kinase and may be immobilized on a column. A solution of candidate compounds is then passed through the column, and a compound specific for the Candidate Kinase is identified on the basis of its ability to bind to the polypeptide and be immobilized on the column. To isolate the compound, the column is washed to remove non-specifically bound molecules, and the compound of interest is then released from the column and collected. Compounds isolated by this method (or any other appropriate method) may, if desired, be further purified (e.g., by high performance liquid chromatography). Compounds isolated by this approach may also be used, for example, as therapeutics to treat a proliferative disorder (e.g., cancer). Compounds which are identified as binding to a Candidate Kinase with an affinity constant less than or equal to 10 mM and/or mediate a decrease (e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or at least 95% decrease) in the activity of the Candidate Kinase are considered particularly useful in the invention.

.' Potential antagonists of Candidate Kinases can also be identified using assays that detect the enzymatic activity of the kinases. Assays (e.g., high throughput assays) to measure the enzymatic activity of kinases are well known in the art (see, for example, Figure 30).

Potential antagonists include organic molecules, peptides, peptide mimetics, polypeptides, and antibodies that bind to a Candidate Kinase, or a polynucleotide encoding a Candidate Kinase and thereby increase or decrease its activity. Potential antagonists include small molecules that bind to and occupy the binding sites of a Candidate Kinase. Other potential antagonists include antisense molecules.

Polynucleotide sequences coding for Candidate Kinases may also be used in the discovery and development of compounds to treat proliferative disorders (e.g., cancer). Candidate Kinases (e.g., ACVR2B, CAMK2B, CHK, CSNKlAl, DGKE, IKBKE, MAP4K4, PIP5K2A, PRKACG, PRKCQ, PTK6, and ROR2), upon expression, can be used as a target for the screening of drugs. Additionally, the polynucleotide sequences encoding the amino terminal regions of the encoded polypeptide, Shine-Delgarno sequence, or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest. Polynucleotides encoding fragments of a Candidate Kinase may, for example, be expressed such that RNA interference takes place, thereby reducing expression or activity of a Candidate Kinase. Examples of sequences useful for inhibiting the expression of Candidate Kinases are set forth in Table IB. The antagonists of the invention may be employed, for instance, to treat a variety of proliferative disorders (e.g., cancer).

Optionally, compounds identified in any of the above-described assays may be confirmed as useful in delaying or ameliorating proliferative disorders (e.g., at least a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% delay in the onset of a proliferative disorder, reduction in cancer cell number, tumor size, or cell migration, or prevention of metastasis) in either standard tissue culture methods or animal models and, if successful, may be used as therapeutics for treating proliferative disorders. Small molecules provide useful candidate therapeutics. Preferably, such molecules have a molecular weight below 2,000 daltons, more preferably between 300 and 1,000 daltons, and most preferably between 400 and 700 daltons. It is preferred that these small molecules are organic molecules.

Test Compounds and Extracts

In general, compounds capable of treating a proliferative disorder are identified from large libraries of both natural product or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal- based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and polynucleotide-based (e.g., siRNA and shRNA) compounds. Synthetic compound libraries are commercially available. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available. In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.

In addition, those skilled in the art of drug discovery and development readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their activity in treating proliferative disorders should be employed whenever possible.

When a crude extract is found to have an activity that modulates Candidate Kinase expression or activity, or a binding activity, further fractionation of the positive lead extract is necessary to isolate chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the characterization and identification of a chemical entity within the crude extract having activity that may be useful in treating a proliferative disorder. Methods of fractionation and purification of such heterogenous extracts are known in the art. If desired, compounds shown to be useful agents for the treatment of a proliferative disorder are chemically modified according to methods known in the art.

Compounds capable of treating a proliferative disorder (e.g., cancer) may also include siRNA molecules (e.g., any of the RNA molecules listed in Table IB) or antibodies that bind or decrease the activity of a Candidate Kinase (e.g., art-known antibodies specific for ACVR2B, CAMK2B, CHK, CSNKlAl, DGKE, IKBKE,

MAP4K4, PIP5K2A, PRKACG, PRKCQ, PTK6, and ROR2). Any of the compounds identified in the disclosed screening methods may be administered to a subject (e.g., a human) to delay or ameliorate a proliferative disorder (e.g., at least a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% delay in the onset of a proliferative disorder, reduction in cancer cell number, tumor size, or cell migration, or prevention of metastasis).

III. Diagnostic methods

The genes identified in this screen represent candidate targets for therapeutic intervention in cancer, especially tumors in which the IGF-IR or PIK3CA pathways are activated. The latter can be identified by examining for known activating mutations in the PIK3CA gene or of other alterations in the PIK3CA pathway (e.g., loss of ftinction of PTEN, a phosphatase that prevents accumulation of the PIK3CA product and inactivates this pathway).

The present invention provides assays useful in the diagnosis of proliferative disorders such as cancer, based on the discovery that the proteins listed in Table IA (i.e., Candidate Kinases) are involved in anchorage-independent survival.

Accordingly, diagnosis of proliferative disorders can be performed by measuring the level of expression or activity of the Candidate Kinases (e.g., ACVR2B, CAMK2B, CHK, CSNKlAl, DGKE, IKBKE, MAP4K4, PIP5K2A, PRKACG, PRKCQ, PTK6, and ROR2), individually or in combination, in a sample taken from a subject. This level of expression or activity can then be compared to a control sample, for example, a sample (e.g., a cell) taken from a control subject, and a decrease in the specified proteins relative to the control is taken as diagnostic of a proliferative disorder, or a risk of or propensity to develop a proliferative disorder (e.g., cancer) or a malignant proliferative disorder. Analysis of levels of the mRNA or polypeptides of the Candidate Kinases, or activity of the polypeptides, may be used as the basis for screening the subject sample (e.g., a blood or tissue sample). The nucleic acid and amino acid sequences are known in the art and correspond to the Genebank Accession Numbers set forth in Table IA. Methods for screening mRNA levels include any of those standard in the art, for example, the use of Northern blotting, quantitative rtPCR, or microarrays. Methods for screening polypeptide levels may include immunological techniques standard in the art (e.g., Western blot or ELISA), or may be performed using chromatographic or other protein purification techniques. In another embodiment, the activity (e.g., kinase activity) of any of the Candidate Kinases may be measured, where a decrease or increase in the activity of a Candidate Kinase relative to sample taken from a control subject is diagnostic of the proliferative disorder (e.g., cancer).

IV. Database Development

The methods described herein may also be employed to generate a database containing the identity of proteins whose down-regulation produces a particular result, either desirable or undesirable. V. Experimental Results Example 1: Tissue Culture Models of Breast Cancer Cells

Breast cancer tumors have several distinct growth phenotypes (Figure 1). A three-dimensional tissue culture model of breast cancer tumors has been developed that utilizes the growth of MCF-IOA cells (Figure 2). The three-dimensional tissue culture model results in the formation of structures similar to the epithelial acini present in mammary tissue. Epithelial acini have specific apical/basal cell polarity and the inner cells normally undergo apoptosis (Figure 3).

Expression of several different oncogenes has been shown to confer distinct growth morphology to cells in three-dimensional tissue culture models (Figure 4). For example, the expression of ErbB2/TGFβ has been shown to induce a cellular invasion phenotype on cultured cells (Figure 5) and has been implicated for a role in breast cancer.

In addition to ErbB2/TGFβ, IGF-I has been implicated for a role breast cancer (Figure 6). Hyperstimulation of the IGF-I receptor (IGF-IR) in mammary epithelial cells results in epithelial cell morphogenesis (Figure 7).

Cells within the luminal space of epithelial acini normally undergo anoikis. However, the oncogenes ErbB2, IGF-IR, and CSF-IR have been shown to prevent anchorage-independent growth or anoikis (Figure 8). We have developed a high throughput siRNA screen to identify key signaling molecules involved in anoikis- resistance mediated by oncogenes (e.g., IGF-IR, ErbB2, and PIK3CA).

Example 2: High throughput siRNA screen for viability in suspension

In this screen, IGF-IR overexpressing MCF-IOA cells were detached from matrix by trypsinization, and incubated in suspension in 96 well plates coated with Polyhema to prevent their attachment (Figure 9). The cells in each well were transfected with a 25 nM of a single siRNA molecule that target one of the 646 kinases that are part of the Qiagen Kinase siRNA library (Figure 10). There are two siRNAs that target each kinase (thus, a total of 1292 siRNAs were tested). The cells were then cultured for an additional 72 hours (see the protocol set forth below).

Cell viability was assessed using a colorimetric assay employing Alamar Blue (resazurin) dye that measures the reducing capacity of cells. Alamar blue changes from a blue to a pink and highly fluorescent molecule when reduced and can be measured either colorimetrically or fluorimetrically. It is believed that the extent of reduction is dependent on the metabolic activity of the cells (likely to be by oxygen consumption through metabolism). There is a direct correlation between the reduction of Alamar Blue in the growth media and the quantity/proliferation of living organisms. Unlike other reporters of cellular reducing potential, Alamar Blue is nontoxic and doesn't require fixation. The screen is performed in quadruplicate and fold- changes in Alamar Blue were calculated relative to cells transfected with a negative control siRNA (Luciferase siRNA) (Figure 11). The screen was repeated in quadruplicate at least three times to generate a list of candidate "hits" for further validation. siRNAs targeting 104 kinases scored in this screen (Figure 12).

The siRNAs that are identified by the primary screen (e.g., those siRNAs set forth in Table IB) were subjected to a secondary screen to evaluate whether they cause a reduction in cell viability in the context of the 'normal', non-transformed parental line of breast epithelial cells (MCF-IOA) from which the IGF-IR expressing cells were derived. To replicate the suspended state of the cells, but avoid cell death due to anoikis, the MCF-IOA cells were detached from tissue culture plates with trypsin and then incubated in suspension in medium containing 2% Matrigel (a commercial preparation containing basement membrane proteins derived from a mouse tumor) (Figure 13). siRNAs targeting 28 of the kinases decreased the viability of the MCF-IOA cells, whereas 76 of the initial 104 kinases scored showed at least a 1.5 fold preferential inhibition of viability of the IGF-IR cells (see Table IB, Table 2A, Table 2B, Figure 14, and Figure 15). The kinases targeted by these siRNAs (i.e., Candidate Kinases) may play a critical role in the viability of human tumor cells.

Table 2A.

28/43 double se uence hits re eated

+ designates genes that retested withonly 1 sequence.

Table 2B.

76/104 sin le se uence hits re eated

* Designates genes that retest at 1.5 fold threshold only; others are above the 1.7 fold change threshold. Analysis of additional siRNAs that target the Candidate Kinases

Because the siRNAs identified in these screens may decrease the viability of the IGF- IR expressing cells due to off-target effects, additional siRNAs that target the Candidate Kinases were used to transfect suspended IGF-IR cells and MCF-IOA cells suspended in Matrigel. The effects of these siRNAs on cell viability was assessed using the methodology described above. Two siRNAs targeting 16 kinases and three siRNAs targeting 10 kinases show preferential inhibition of viability of the IGF-IR cells relative to the MCF-IOA cells (see Table IB and Table 3). Those kinases in which only a single siRNA caused inhibition of IGF-IR viability are eliminated from consideration as Candidate Kinases.

Table 3

Evaluation of additional duplexes for the primary screen "hits"

Analysis of cells transformed by another oncogene

To determine whether the siRNAs that inhibit cells transformed by IGF-IR also inhibit the viability of another oncogene, the siRNAs targeting the Candidate Kinases in cells expressing a mutant form of the protein phosphatidylinositol 3'- kinase alpha (PIK3CA) were examined. This lipid kinase has been shown to be mutated in about 25-30% of human breast tumors and multiple other tumors. We specifically used one mutant variant - PIK3CAE545K- which represents one of two mutants that are found in at least 90% of breast tumors. Expression of this protein in MCF-IOA cells inhibited anoikis (Isakoff et al., 2005). 54 of 56 Candidate Kinases evaluated thus far also inhibited survival of the PIK3CAE545K expressing MCF-I OA cells. These results support the possibility that the viability of tumors cells expressing these mutant variants of PIK3CA will be compromised by inhibition of these kinases.

Analysis of induction of apoptosis Alamar Blue represents a non-specific reporter of cell viability. To determine whether the siRNAs targeting a subset of the Candidate Kinases specifically enhance apoptosis of IGF-IR expressing cells in suspension, caspase 3 activation in siRNA transfected cells was assessed. A high throughput screen for assaying caspase 3 activation has been optimized for detection of apoptosis in suspended cells. This assay utilizes a commercially available reagent, Caspase-Glo from Promega. In this assay, cells are trypsinized, plated onto 96 well plates coated with Polyhema and transfected with three siRNAs targeting Candidate Kinases. The cells are cultured for an additional 72 hours in suspension, after which the Caspase 3/7 substrate is added. Luminscence resulting from Caspase 3/7 substrate cleavage is then assessed.

Protocol for high throughput screen

Cells used in the following protocol are engineered to expressing IGF-IR as described above.

Preparation of polyhema plates: 100 microliters of polyhema (6 mg/mL [Sigma] in 95% ethanol)are dispensed into each well of 96-well white opaque plates (Corning).

Plates are incubated at 37 ⁰C for 4-5 days to ensure complete evaporation of polyhema. Preparation of cells to be transfected:

Cells are trypsinized, counted and resuspended in OPTIMEM (Gibco) at a density of 20,000 cells/80 microliters/well of a 96 well plate.

Preparation of siRNA source plates, oligofectamine plate and transfection mix plates:

1. Take the siRNA source plate (V bottom plate) out of -20 ⁰C, spin at 900 rpm for 30s or less (pulse) and then thaw on ice. Each well contains 50- 100 μL of siRNA. The stock concentration is either 5 μM or 20 μM. 2. Make 7 colored labels-one for each of the 7 round bottom plates and place on 96 well round bottom plates: a. Source plate 1 and Source plate 2 (identical plates) b. Oligofectamine plate c. TFl , TF2, TF3, TF4 (Empty transfection plates) 3. Write out map on the lid of both source plates:

A4, A5, A8, A9 positive controls Dl and El no cell controls Cl 2 and Dl 2 no cell controls Gl 1 and Hl 1 lipid controls C9, F4, El 1 and Fl 1 luciferase controls

4. Pipet 45ml of Optimem into a 50 mL conical tube. Then pipet 30 mL into a plastic reservoir boat.

5. Pipet 30 μL of optimem with multichannel pipette into every edge well (source plate) except the A4,A5, A8, A9 positive control wells. Also add 30 μL to the lipid control Gl 1 and Hl 1.

6. Add 24 μl of Optimem in all of the center wells (source plate) except the Luciferase control wells (C9, F4, El 1 and FI l).

7. Repeat steps 7 and 8 with the second source plate and place both plates aside. 8. Do not add the siRNA until the last step.

9. Prepare the Oligofectamine plate.

In a 15 mL falcon tube combine 1.6 mL Oligofectamine with4.4 mL optimem (6 mL total). Mix and add to another reservoir boat.

Using a mutichannel pipette, aliquot 50 μL into each well of the round bottom oligofectamine plate and then place on ice.

10. Make positive and negative controls. Take out 2 RNAse free sterile eppendorf tubes.

Luciferase control

361 μL Optimem

19 μL of Luciferase [20 μM] (in -20 ⁰C) Add 30 μL into the Luciferase control wells on the source plates (C9,

F4, El l and FI l).

Positive controls

133 μL Optimem 7 μL PIM siRNA or EGFR siRNA [20 μM] (in -20 ⁰C)

Final concentration is 2 μM.

Add 30 μl into the positive control wells (A4,A5, A8, A9) on the source plates.

1 1. Final step add siRNA to source plates. Carefully peel off foil in the hood.

Pipet up and down, add 6 μL of the 5 μM stock to the center 6 wells of source plate 1 and 2 excluding the luciferase controls with multichannel pipette.

Spin the source plates 1 and 2 at 900 rpm for 30s or less (pulse) and place on ice.

Cover the stock siRNA source plate with foil and seal the edges with 2 layers of parafilm, put back in the -20 ⁰C.

Place everything on ice.

Cells are transfected according to the automated protocol. Cells are incubated at 37 ⁰C for 4-5 hours and then supplemented with

1/3 volume (50 μL) DMEM F12 supplemented with 15% horse serum, 100 μL EGF (100 μg/mL), 250 μL hydrocortisone (1 mg/mL), 50 μL cholera toxin (lmg/ml), and 1.5 mL insulin (10 mg/mL). Cells are incubated for an additional 72 hours.

Alamar Assay (Biosource)

After 72 hours, 10 microliters of Alamar dye is added to each well of the assay plate. The plates are incubated with reagent at 37 ⁰C. Fluorescence measurements are taken at multiple time points (1 hr, 2 hr, 3 hr, 4hr) using a plate reader (excitation 530 nm, emission 590 nm).

Caspase-Glo Assay (Promega) After 72 hours, 120 microliters of Caspase-Glo reagent is added to each well of the assay plate. The plates are incubated with reagent at room temperature. Luminscence measurements are taken at mutiple time points (1 hr, 2 hr) using a plate reader (integration time 0.5 sec).

Genes identified using the above protocol are indicated, along with their Genebank Accession Number, in Table IA.

Example 3: In Vivo Studies of Specific Candidate Kinases

Additional Candidate Kinases of specific interest include ACVR2B, C AMK2B, CHK, CSNK IAl, DGKE, IKBKE, MAP4K4, PIP5K2 A, PRKACG,

PRKCQ, PTK6, and ROR2. The expression of several of the above-listed Candidate Kinases in MCF-IOA cells overexpressing IGF-IR is shown in Figure 16.

Additional studies with PTK6 have been performed and support its role in IGF-lR-induced survival in suspension. PTK6 is a gene which is amplified in some breast tumors (Figure 17A) and high PTK6 expression is correlated with a worse prognosis (Figure 17B). PTK6 is more highly expressed in high-grade estrogen receptor-positive and Her2 -positive breast tumors (Figure 18).

Dowregulation of PTK6 in results in enhanced cell death not only of IGF-IR overexpressing MCF-IOA cells, but also of IGF-I stimulated MCF-7 cells (a breast cancer cell line that express IGF-IR) cultured in suspension and ZR751 cells (Figure 19A, Figure 2OA, Figure 2 IA, Figure 21C, Figure 22 A, and Figure 22C). Detection of cleaved PARP, a biochemical marker of apoptosis, also supports the conclusion that PTK6 downregulation enhances cell death/apoptosis (Figure 19B, Figure 2OB, Figure 21 B, Figure 2 ID, Figure 22B, Figure 22D, and Figure 23B). In addition, PTK6 downregulation using short hairpin RNA vectors resulted in inhibition of IGF-I stimulated, three-dimensional growth in Matrigel cultures (Figure 24 and Figure 25).

Finally, preliminary studies suggest that PTK6 may play a role in anchorage- independent survival mediated by other oncogenes such as Neu/ErbB2/Her2.

Downregulation of PTK6 in MCF-IOA cells overexpressing Neu (NeuN) partially reverses the anoikis resistance observed with Neu overexpression (Figure 26). As simultaneous overexpression of Her2 and PTK6 have been reported in archival breast cancer specimens (Born et al., J. Pathol. 5: 592-596, 2005), this experimental evidence supports a role for PTK6 in Her2 -mediated tumorigenic phenotypes and could be a therapeutic target for this subtype of breast cancer.

Additional studies with PRKCQ have been performed and support its role in IGF-lR-induced survival in suspension. PRKCQ is overexpressed in a subset of breast cancer cell lines, with higher expression in basal, estrogen receptor-negative cell lines (Figure 27). MCFlOA cells overexpressing PRKCQ also exhibit enhanced anchorage-independent survival when cultured in suspension (Figure 28). Lastly, PRKCQ overexpression induces growth factor-independent cell migration in MCF- 1OA cells (Figure 29).

OTHER EMBODIMENTS

The description of the specific embodiments of the invention is presented for the purposes of illustration. It is not intended to be exhaustive or to limit the scope of the invention to the specific forms described herein. Although the invention has been described with reference to several embodiments, it will be understood by one of ordinary skill in the art that various modifications can be made without departing from the spirit and the scope of the invention, as set forth in the claims. All patents, patent applications, and publications referenced herein are hereby incorporated by reference.

Other embodiments are in the claims.

What is claimed is:

Claims

1. A method of identifying a therapeutic compound for treating a proliferative disease, said method comprising the steps of assaying a candidate therapeutic compound for inhibition of the kinase activity of a kinase selected from the group consisting of ACVR2B, CAMK2B, CHK, CSNKlAl, DGKE, IKBKE, MAP4K4, PIP5K2A, PRKACG, PRKCQ, PTK6, and ROR2, wherein the level of inhibition is indicative of the therapeutic efficacy for treating said proliferative disease with said candidate compound.

2. The method of claim 1 , wherein said assaying comprises contacting said candidate therapeutic compound with the kinase protein in vitro and measuring said kinase activity of said kinase protein.

3. The method of claim 1, wherein said assaying comprises contacting said therapeutic compound with a cell capable of transcribing the mRNA encoding said kinase and measuring the level of expression of said mRNA or said kinase encoded by said mRNA.

4. The method of claim 1 , wherein said candidate therapeutic compound is assayed for inhibition of at least 2 of said kinases.

5. The method of claim 4, wherein said candidate therapeutic compound is assayed for inhibition of at least 5 of said kinases.

6. The method of claim 1, further comprising the steps of:

(a) contacting a cell with said candidate therapeutic compound;

(b) measuring the ability of said cell to survive under anchorage-independent conditions after said contacting, wherein the level of reduction in said ability is indicative of the therapeutic efficacy for treating said proliferative disease with said candidate compound; and wherein said cell expresses a gene selected from the group consisting of: IGF- IR, ErbB2, and phosphatidylinositol 3 '-kinase alpha.

7. A method of generating a database, said method comprising the steps of:

(a) contacting a cell expressing a class of mRNA, each member having known sequence, with one or more siRNAs specific to each sequence of said class;

(b) measuring the ability of said cell to survive under anchorage-independent conditions of said cells after said contacting, wherein a reduction in said ability identifies an mRNA as essential to anchorage-independent survival; and

(c) creating a record in said database, wherein said record comprises the identity of an mRNA of said class that is essential.

8. The method of claim 7, wherein said record further comprises the identity of the phenotypic effect of reducing the activity of the protein encoded by the mRNA.

9. A database comprising data relating to biological activity of at least two of the kinases selected from the group consisting of ACVR2B, CAMK2B, CHK, CSNKlAl , DGKE, IKBKE, MAP4K4, PIP5K2A, PRKACG, PRKCQ, PTK6, and ROR2.

10. The database of claim 9, further comprising data relating to the biological activity of at least five of the kinases listed.

1 1. The database of claim 9, further comprising data relating to the biological effect of a compound on said at least two of the kinases listed.

12. The database of claim 9, further comprising data relating to at least two biological activities of said at least two of the kinases listed.

13. The database of claim 9, further comprising data on the biological activity of said at least two kinases in at least two types of cells, wherein one type has pathological characteristics and the other does not.

14. The database of claim 9, wherein said biological activity is anchorage- independent survival.

15. A method of diagnosing a subject as having, or having a propensity to develop, a proliferative disorder, said method comprising the steps of:

(a) isolating a cell from said subject; and

(b) measuring the kinase activity of at least one kinase selected from the group consisting of: ACVR2B, CAMK2B, CHK, CSNKlAl, DGKE, IKBKE, MAP4K4, PIP5K2A, PRKACG, PRKCQ, PTK6, and ROR2, wherein an increase in said kinase activity of said kinase or kinases, as compared to the level in a reference sample, is a diagnostic indicator of a proliferative disorder or a propensity to develop a proliferative disorder.

16. The method of claim 15, wherein said cell is a suspect cell.

17. The method of claim 15 comprising measuring said kinase activity of at least two kinases selected from said group.

18. The method of claim 17 comprising measuring said kinase activity of at least five kinases selected from said group.

19. The method of claim 18 comprising measuring said kinase activity of at least seven kinases selected from said group.

20. A method of diagnosing a subject as having, or having a propensity to develop, a malignant proliferative disorder, said method comprising the steps of:

(a) isolating a cell from said subject; and

(b) measuring the kinase activity of at least one kinase selected from the group consisting of: ACVR2B, CAMK2B, CHK, CSNKlAl, DGKE, IKBKE, MAP4K4, PIP5K2A, PRKACG, PRKCQ, PTK6, and ROR2; wherein an increase in said kinase activity of said kinase or kinases, as compared to the level in a reference sample, is a diagnostic indicator of a malignant proliferative disorder or a propensity to develop a malignant proliferative disorder.

21. The method of claim 20, comprising measuring said kinase activity of at least two kinases selected from said group.

22. The method of claim 21, comprising measuring said kinase activity of at least five kinases selected from said group.

23. The method of claim 22, comprising measuring said kinase activity of at least seven kinases selected from said group.

24. A method for identifying a gene necessary for anchorage-independent growth, said method comprising:

(a) contacting a cell expressing a class of mRNA, each member having a known sequence, with one or more siRNAs specific to each sequence of said class; and,

(b) measuring the ability of said cell to survive under anchorage-independent conditions after said contacting, wherein a reduction in said ability identifies an mRNA as essential to anchorage-independent survival; wherein said cell expresses a gene selected from the group consisting of: IGF- IR, ErbB2, and phosphatidylinositol 3 '-kinase alpha.

25. A method for identifying a therapeutic compound for treating a proliferative disease, said method comprising:

(a) contacting a cell with a candidate compound,

(b) measuring the ability of said cell to survive under anchorage-independent conditions of said cells after said contacting, wherein a reduction in said ability identifies a candidate compound as inhibiting survival under said anchorage- independent conditions; wherein said cell expresses a gene selected from the group consisting of: IGF- IR, ErbB2, and phosphatidylinositol 3 '-kinase alpha.

26. The method of any one of claims 1, 15, or 20, wherein said kinase or at least one kinase is ACVR2B.

27. The method of any one of claims 1, 15, or 20, wherein said kinase or at least one kinase is CAMK2B.

28. The method of any one of claims 1, 15, or 20, wherein said kinase or at least one kinase is CHK.

29. The method of any one of claims 1, 15, or 20, wherein said kinase or at least one kinase is CSNKlAl .

30. The method of any one of claims 1, 15, or 20, wherein said kinase or at least one kinase is DGKE.

31. The method of any one of claims 1 , 15, or 20, wherein said kinase or at least one kinase is IKBKE.

32. The method of any one of claims 1, 15, or 20, wherein said kinase or at least one kinase is MAP4K4.

33. The method of any one of claims 1, 15, or 20, wherein said kinase or at least one kinase is PIP5K2A.

34. The method of any one of claims 1, 15, or 20, wherein said kinase or at least one kinase is PRKACG.

35. The method of any one of claims 1, 15, or 20, wherein said kinase or at least one kinase is PRKCQ.

36. The method of any one of claims 1, 15, or 20, wherein said kinase or at least one kinase is PTK6.

37. The method of any one of claims 1 , 15, or 20, wherein said kinase or at least one kinase is ROR2.