EP1863512A1

EP1863512A1 - Enhancing myc-dependent sensitivity of cancer to dr5 agonists

Info

Publication number: EP1863512A1
Application number: EP06737889A
Authority: EP
Inventors: Marc Nasoff; Quinn L. Deveraux; Kim C. Quon; Sabine Rottman
Original assignee: IRM LLC
Current assignee: IRM LLC
Priority date: 2005-03-24
Filing date: 2006-03-08
Publication date: 2007-12-12
Also published as: JP2008536820A; WO2006104672A1

Abstract

The present application provides methods and compositions for inducing apoptosis in cancer cells by combining DR5 agonists and either GSK3 antagonists or hCDC4 antagonists.

Description

ENHANCING MYC-DEPENDENT SENSITIVITY OF CANCER TO DR5

AGONISTS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S.

Provisional Patent Application Nos. 60/665,646 (filed March 24, 2005) and 60/717,644 (filed September 15, 2005). The disclosures of the priority applications are incorporated herein by reference in their entirety and for all purposes.

BACKGROUND OF THE INVENTION

[0002] The MYC proto-oncogene is a member of the basic helix-loop-helix leucine zipper family of transcription factors (reviewed in (Luscher, B., (2001). Gene 277, 1-14). Believed to bind promoters in as many as 15% of the genes in the human genome (Patel, J.H., Loboda, A.P., Showe, M.K., Showe, L.C. and McMahon, S.B., (2004). Nat Rev Cancer 4, 562-568), MYC plays a role in a diverse array of processes of cancer development, including angiogenesis, genomic instability, inhibition of differentiation, proliferation, immortalization and metabolism (for review, (Oster, S.K., Ho, C.S., Soucie, E.L. and Perm, L.Z., (2002). Adv Cancer Res 84, 81-154)). Paradoxically, under certain conditions, MYC is also capable of sensitizing cells to apoptotic cell death a process normally associated with tumor suppression rather than promotion (Pelengaris, S., Khan, M. and Evan, G., (2002). Nat Rev Cancer 2,

764-776.).

[0003] MYC is estimated to be amplified, translocated, mutated or otherwise deregulated in up to 70% of human tumors (Nilsson, J.A. and Cleveland, J.L., (2003). Oncogene 22, 9007-9021), suggesting that MYC activation may be an obligate step in the majority of human cancers. Transgenic mouse tumorigenesis studies have borne out MYCs key role, demonstrating a function for MYC in tumor initiation, progression and maintenance (Jonkers, J. and Berns, A., (2004) Cancer Cell 6, 535-538). However, despite its clear association with cancer, success in pharmacologically inhibiting its activity has not been forthcoming. [0004] DR5 is a receptor for TRAIL (Tumor necrosis factor related apoptosis- inducing ligand, also known as apo2L, TNFSFlO), an apoptosis-inducing cytokine of the tumor necrosis factor (TISfF) superfamily (reviewed in LeBlanc, H.N. and Ashkenazi, A., (2003) Cell Death Differ 10, 66-75.; Ozoren, N. and El-Deiry, W.S., (2002) Neoplasia 4, 551-557). Recombinant human TRAIL and agonistic antibodies against its two death- inducing receptors, DR4 and DR5, are currently undergoing extensive pre-clinical and clinical testing as cancer therapeutics.

SUMMARY OF THE INVENTION

[0005] In one aspect, the present invention provides methods of inducing apoptosis in a cancer cell. The methods entail contacting a MYC-expressing and DR5 -expressing cancer cell with (i) a DR5 agonist; and (ii) an antagonist of a MYC-interacting gene listed in Table 1 (e.g., GSK3 β) or a hCDC4 antagonist. In some embodiments, the cancer cell is in an animal and the contacting step comprises administering an agonist of a MYC-interacting gene listed in Table 1 (e.g., GSK3 β) or a hCDC4 antagonist to the animal. In some embodiments, the animal is a human. In some embodiments, the animal is not a human. In some embodiments, the contacting step comprises contacting the cell with an agonist of a MYC-interacting gene listed in Table 1 (e.g., GSK3β). In some embodiments, the contacting step comprises contacting the cell with a hCDC4 antagonist. In some embodiments, the DR5 agonist is an antibody. In some embodiments, the DR5 agonist is TRAIL.

[0006] The present invention also provides methods of inducing apoptosis in a cancer cell. In some embodiments, the methods involve contacting a MYC-expressing and DR5- expressing cancer cell with a DR5 agonist; and introducing into the cell an antisense or siRNA that inhibits expression of a MYC-interacting gene listed in Table 1 (e.g., GSK3β) or hCDC4. The present invention also provides compositions comprising a therapeutically- effective dose of (i) a DR5 agonist; and (ii) an agonist of a MYC-interacting gene listed in Table 1 (e.g., GSK3β) or a hCDC4 antagonist. In some embodiments, the DR5 agonist is an antibody. In some embodiments, the DR5 agonist is TRAIL.

[0007] The present invention also provides methods of identifying an agent for inducing apoptosis in cancer cells. In some embodiments, the methods involve (i) contacting one or more agents to a polypeptide comprising a phosphodegron-binding fragment of a hCDC4 polypeptide, wherein the phosphodegron comprises LPTPP (SEQ ID NO:2), wherein the threonine in SEQ ID NO:2 is phosphorylated; and (ii) selecting one or more agents that inhibits the polypeptide binding of the phosphodegron, thereby identifying an agent that induces apoptosis in a cancer cell. In some embodiments, the phosphodegron comprises KKFELLPTPPLSPSRR (SEQ ID NO: 1). In some embodiments, the method further involve (iii) contacting the selected one or more agents to cancer cells that expresses MYC in the presence of a DR5 agonist; and (iv) selecting an agent that induces more apoptosis in the cancer cells than when the cells are contacted with the DR5 agonist in the absence of the agent.

[0008] In some of these methods, the polypeptide is linked to a solid support. In some embodiments, the polypeptide is associated with a first fluorescent label and the phosphodegron is associated with a second fluorescent label and the first and second labels interact to produce a fluorescent signal when the polypeptide binds the phosphodegron, and wherein a reduction of the fluorescent signal in the presence of an agent indicates that the agent inhibits binding of the polypeptide to the phosphodegron. In some embodiments, the first fluorescent label is Europium and the first fluorescent label is linked to an antibody that binds to the polypeptide; and the second fluorescent label is selected from the group consisting of Allophycocyanin (APC) and C-Phycocyanin (CPC).

[0009] The present invention also provides methods of inducing apoptosis in cancer cells in an individual. In some embodiments, the methods entail administering a DR5 agonist to the individual, wherein the cancer cells in the individual are pre-determined to have a mutation in a polynucleotide encoding hCDC4 or glycogen synthase kinase-3β (GSK3β) resulting in a reduced activity of hCDC4 or GSK3β compared to a wildtype hCDC4 or GSK3β polypeptide. In some embodiments, the methods involve determining the hCDC4 or GSK3β genotype of cancer cells in the individual prior to the administering step. In some embodiments, the methods comprise determining the hCDC4 genotype of the cancer cells. In some embodiments, the methods comprise determining the GSK3β genotype of the cancer cells. In some embodiments, the DR5 agonist is selected from the group consisting of an antibody that binds to DR5 and TRAIL.

[0010] The present invention also provides methods of inducing apoptosis in cancer cells in an individual. In some embodiments, the methods comprise administering a DR5 agonist to the individual, wherein the cancer cells in the individual are pre-determined to have a mutation at position T58. In some embodiments, the mutation is T58I or T58A. In some embodiments, the methods comprise determining the MYC genotype of cancer cells in the individual prior to the administering step. BRIEF DESCRIPTION OF THE FIGURES

[0011] Figure 1 illustrates the amino acid sequence of the heavy chain variable region of an anti-DR5 agonist antibody. The complementarity determining regions (CDRs) are boxed. The remaining amino acids are part of the framework region (FR). [0012] Figure 2 illustrates the amino acid sequence of the light chain variable region of the anti-DR5 agonist antibody referred to in Figure 1. The complementarity determining regions (CDRs) are boxed. The remaining amino acids are part of the framework region (FR).

[0013] Figure 3 illustrates the DNA coding sequences for the variable regions displayed in Figures 1 and 2.

DEFINITIONS

[0014] "GSK3 β antagonists" or "hCDC4 antagonists" are compounds that partially

(e.g., at least 5, 10, 20, 50% or more) or totally block stimulation, decrease, prevent, delay activation, inactivate, or desensitizeGSK3β or hCDC4, respectively. Antagonists can include, e.g., antibodies, organic small molecules (e.g., less than 1500 Daltons), etc. [0015] "DR5 agonists" are compounds that stimulate, increase, activate, enhance activation, sensitize or up regulate the activity of DR5. Agonists can include, e.g., antibodies, organic small molecules (e.g., less than 1500 Daltons), etc. Agonists may, but do not necessarily, compete with known DR5 ligands (e.g., TRAIL) for binding to DR5 and induce apoptosis in cancer cells that express MYC and DR5. In some embodiments, the amount of apoptosis induced by a DR5 agonist is at least 5, 10, 20, 50, 100, 150%, 200% or more compared to the apoptosis induced by TRAIL.

[0016] As used herein, the term "cancer" is used to mean a condition in which a cell in a patient's body undergoes abnormal, uncontrolled proliferation. The abnormal cell may proliferate to form a solid tumor, or may proliferate to form a multitude of cells (e.g., leukemia).

[0017] For purposes of the invention, the term "cancer cell" refers to any cell that proliferates abnormally, including, without limitation, pancreatic, colon, breast, prostate, renal, lung, ovarian, gastric, esophageal, hepatocellular, or head and neck cancer cells, melanoma cells, leukemia cells, and multiple myeloma cells. In some embodiments, the cancer cell is grown in cell culture, including primary cultures and immortalized cell lines. In some other embodiments, the cancer cell is in an animal, preferably a mammal. As used herein, the term "mammal" includes, without limitation rats, mice, dogs, pigs, rabbits, non- human primates, and humans.

[0018] As used herein, "phosphodegron" refers to a phosphorylated polypeptide.

[0019] "Nucleic acid" or "polynucleotide" refers to deoxyribonucleotides (e.g., DNA) or ribonucleotides (e.g., RNA) and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). [0020] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et ah, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). [0021] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

[0022] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ- carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an .alpha, carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0023] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (e.g., a polypeptide of the invention), which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. [0024] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same sequences. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. The invention provides polypeptides comprising a sequence substantially identical to the polypeptides exemplified in Figure 1 and Figure 2). Optionally, the identity exists over a region that is at least about 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length.

[0025] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. [0026] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. AppL Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. MoI. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sd. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Generics Software Package, Generics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0027] Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al (1990) J. MoI. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl, Acad. ScL USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. [0028] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sd. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

DETAILED DESCRIPTION OF THE INVENTION Induction ofapoptosis in cells

[0029] The present invention is based, in part, on the surprising discovery that inhibition of proteins encoded by a number of MYC-interacting genes such as GSK3β (listed in Table 1) or hCDC4 sensitizes cancer cells to induction of DR5 agonist-induced apoptosis. Accordingly, some embodiments of the invention provide for inliibiting these MYC- interacting genes (e.g., GSK3β) or hCDC4 in combination with triggering the DR5 receptor. [0030] The cells in which apoptosis is induced are typically MYC-expressing cancer cells or other cells that are sensitive to DR5 agonists. The cells in which apoptosis is induced can be, in vivo or in vitro. In some embodiments, the methods of the invention are performed ex vivo, i.e., a cell sample is removed from an individual, apoptosis is induced in cancer cells from the sample, and the treated cells are inserted back into the individual.

DR5 agonists

[0031] Any DR5 agonist can be used in accordance with the present invention.

Exemplary DR5 agonists include, e.g., anti-DR5 agonist antibodies and TRAIL. See, e.g., Griffith TS, et al, Curr Opin Immunol. 10(5):559-63 (1998).

[0032] Any anti-DR5 antibody agonist can be used according to the methods of the invention. DR5 (also referred to as Death Receptor 5) is a receptor of the ligand TRAIL. See, e.g., Pan et al., Science 277:815-8 (1997); Sheridan, et al, Science 277:818-21 3 (1997); Walczak et al, EMBOJ. 16:5386-974 (1997). Anti-DR5 antibodies have been described previously in, e.g., PCT WO 01/83560 (antibody TRA-8; ATCC PTA-1428) and PCT WO

02/079377.

[0033] In addition, anti-DR5 agonist antibody agonists are described herein. The variable regions of the heavy and light chains of an exemplary anti-DR5 antibody agonist are provided in Figure 1 and Figure 2. See α/so, PCT Patent Publication WO 2004/050895. In some embodiments, the anti-DR5 agonist antibodies used in the present invention compete with an antibody with the variable regions described in Figure 1 and Figure 2 for binding to

DR5. In some embodiments, the DR5 antibody agonists used in the invention have CDRs that are substantially identical to the CDRs exemplified in Figure 1 and Figure 2. Exemplary anti-DR5 antibodies include those with the specificity of an antibody comprising the light and/or heavy chain variable region sequences displayed in Figure 1 and Figure 2.

[0034] Any type of antibody agonist may be used according to the methods of the invention. Generally, the antibodies used are monoclonal antibodies. Monoclonal antibodies can be generated by any method known in the art (e.g., using hybridomas, recombinant expression and/or phage display).

[0035] The antibodies of the invention need not be cross-linked or otherwise treated prior to administration. However, in some embodiments, the antibodies of the invention are cross-linked. Cross-linking (e.g., using hetero- or homo-bifunctional chemical cross-linkers) is well known in the art. Alternatively, stable multivalent Fabs (e.g., trimers or tetramers, etc.) can be administered. See, e.g., PCT WO 99/27964.

[0036] In numerous embodiments, the anti-DR5 antibodies of the invention do not bind to other polypeptides. In some embodiments, the ant-DR5 antibodies do not bind any other receptor in the TNF receptor family (e.g., TNFR2, TNFR3, OX40, CD40, FAS, DcR3,

CD27, CD30, CD137, DR4, DcRl, DcR2, RANK, OPG, DR3, TR2, NGFR, TNFRl, and

TACl). In some embodiments, the ant-DR5 antibodies do not bind to DR4, DTRl, DTR2 or

OPG.

[0037] The anti-DR5 agonist antibodies of the invention can be extremely potent. For example, in some embodiments, in a standard subcutaneous tumor ablation assay, the antibodies of the invention can reduce tumor size by 50% at a concentration of 1 or less mg/kg body weight (and in some embodiments, 0.50 mg/kg, 0.05 mg/kg, or 0.01 mg/kg or less) when administered to an animal 3 times a week for two weeks and ablate tumors completely when ten times that amount is used.

[0038] In some cases, the anti-DR5 antibodies of the invention are designed to lack or have a reduced antibody-dependent cellular cytotoxicity (ADCC). For example, in some embodiments, the antibodies of the invention comprise an IgG-I, IgG-2, IgG-2A, IgG 3 or IgG-4 Fc region.

Humanized Antibodies

[0039] In some embodiments, the antibody used according to the present invention is a chimeric (e.g., mouse/human) antibody made up of regions from a non-human anti-DR5 antibody agonist together with regions of human antibodies. For example, a chimeric H chain can comprise the antigen binding region of the heavy chain variable region (e.g., the sequence displayed in Figure 1 and Figure 2) of the non-human antibody linked to at least a portion of a human heavy chain constant region. This humanized or chimeric heavy chain may be combined with a chimeric L chain that comprises the antigen binding region of the light chain variable region (e.g., the sequence displayed in Figure 1 and Figure 2) of the non- human antibody linked to at least a portion of the human light chain constant region. In some embodiments, the heavy chain constant region can be an IgM or IgA antibody. [0040] The chimeric antibodies of the invention may be monovalent, divalent, or polyvalent immunoglobulins. For example, a monovalent chimeric antibody is a dimer (HL) formed by a chimeric H chain associated through disulfide bridges with a chimeric L chain, as noted above. A divalent chimeric antibody is a tetramer (H₂ L₂) formed by two HL dimers associated through at least one disulfide bridge. A polyvalent chimeric antibody is based on an aggregation of chains.

[0041] The nucleotide and amino acid sequences of the variable region of an exemplary anti-DR5 antibody agonist are provided in Figure 3. The DNA sequences of the antibodies of the invention can be identified, isolated, cloned, and transferred to a prokaryotic or eukaryotic cell for expression by procedures well-known in the art. Such procedures are generally described in Sambrook et al, supra, as well as CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel et al. , eds., 1989). Expression vectors and host cells suitable for expression of recombinant antibodies and humanized antibodies in particular, are well known in the art. The following references are representative of methods and vectors suitable for expression of recombinant immunoglobulins which may be utilized in carrying out the present invention: Weidle et al, Gene, 51: 21-29 (1987); Dorai et al., J. Immunol, 13(12):4232-4241 (1987); De Waele et al, Eur. J. Biochem., 176:287-295 (1988); Colcher et al, Cancer Res., 49:1738-1745 (1989); Wood et al., J. Immunol, 145(a):3011-3016 (1990); Bulens et al, Eur. J. Biochem., 195:235-242 (1991); Beggington et al, Biol. Technology, 10:169 (1992); King etal., Biochem. J, 281:317-323 (1992); Page et al., Biol. Technology, 2:64 (1991); King et al, Biochem. J., 290:723-729 (1993); Chaudary et al, Nature, 339:394- 397 (1989); Jones et al, Nature, 321. -522-525 (1986); Morrison and Oi, Adv. Immunol, 44:65-92 (1988); Benhar et al, Proc. Natl. Acad. Sd. USA, 91:12051-12055 (1994); Singer et al.,J. Immunol, 150:2844-2857 (1993); Cooto etal., Hybridoma, 13(3):215-219 (1994); Queen et al, Proc. Natl. Acad. Set USA, 86:10029-10033 (1989); Caron etal, Cancer Res., 32:6761-6767 (1992); Cotoma et al, J. Immunol Metk, 152:89-109 (1992). Moreover, vectors suitable for expression of recombinant antibodies are commercially available. [0042] Host cells capable of expressing functional immunoglobulins include, e.g., mammalian cells such as Chinese Hamster Ovary (CHO) cells; COS cells; myeloma cells, such as NSO and SP2/0 cells; bacteria such as Escherichia coli; yeast cells such as Saccharomyces cerevisiae; and other host cells.

Single Chain Antibodies

[0043] In some embodiments, the antibodies of the invention are single chain antibodies (for example, comprising one or more sequence in Figure 1 and Figure 2). Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al, Methods in Enzymology 203:46-88 (1991); Shu et al, Proc. Natl Acad. ScL USA 90:7995-7999 (1993); and Skerra et al, Science 240:1038-1040 (1988).

Human Antibodies

[0044] In some embodiments, human antibodies are used according to the present invention. Human antibodies can be made by a variety of methods known in the art including by using phage display methods using antibody libraries derived from human immunoglobulin sequences. See, e.g., Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995), U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741. [0045] In some embodiments, the antibodies of the present invention are generated using phage display. For example, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. Such phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds DR5 can be selected or identified with DR5, e.g., using labeled DR5. Phage used in these methods are typically filamentous phage including fd and Ml 3 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Bririkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al, J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al, Gene 187:9-18 (1997); Burton et al, Advances in Immunology 57: 191- 280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.

Generating agonist antibodies

[0046] Agonist antibodies can be identified by generating anti-DR5 antibodies and then testing each antibody for the ability trigger DR5 mediated events, e.g., inducing apoptosis in a cancer cell. A variety of assays known in the art can be used to detect induction of apoptosis.

[0047] In one assay, DOHH-2 or Jurkat cells are contacted with a candidate antibody agonist and then monitored for viability as a function of antibody concentration. Reduced cell viability (e.g., caused by increased apoptosis) with increased antibody concentration indicates that the antibody is an agonist. Cell viability can be assayed by adding Alamar blue, which fluoresces in the presence of living, but not dead, cells. As described in PCT Patent Publication No. WO 2004/050895, agonist antibodies can be identified by screening hybridomas raised against DR5 and then screening the hybridoma supernatant for the ability to induce apoptosis in DOHH-2 or Jurkat cells. Appropriate positive and negative controls can be used to confirm the results. For example, a cell line that does not go through DR5- mediated TRAIL induced apoptosis should not go through apoptosis in response to a candidate anti-DR5 agonist.

antagonists of MYC-interacting genes [0048] Any molecule that inhibits activity or expression of a polypeptide encoded by the MYC-interacting genes (e.g., GSK3β) show in Table 1 can be used in combination with a DR5 agonist to induce apoptosis as described herein. Using GSKβ as an example, antagonists can include, e.g., antibodies that bind to GSK3β and inhibit its activity as well as small molecule inhibitors. A wide variety of GSK3β antagonists are known in part due the role of GSK3 in diabetes. In some embodiments, the GSK3β antagonists do not significantly antagonize other kinases, including other kinases such as GSK3α. One exemplary GSK3β antagonist is LiCl. In some embodiments, the GSK3β antagonist is not LiCl. Exemplary GSK3β antagonists are described in, e.g., WO 00/38675 and U.S. Patent Publications 2005/0026946, 2005/0004201, 2005/0004202, 2004/0266815, 2004/0209878, 2004/0192718, 2004/0192698, 2004/0186119, 2004/0186113, 2004/0162234, 2004/0138273, 2004/0106574, 2004/0092535, 2004/0082581, 2004/0077699, 2004/0077642, 2004/0059113, 2004/0054180, 2004/0006095, 2004/0006094, 2003/0225085, 2003/0212079, 2003/0194750, 2003/0105075, 2003/0078280, 2003/0060629, 2003/0055097, 2002/0198219, 2002/0160478, 2002/0151574, 2002/0147146, 2001/0052137 and U.S. Patent No. 6,608,063.

[0049] Exemplary GSK3β inhibitors include those depicted in formulas 1 and 2. See, e.g., WO04/043467 & WO 01/072745 for descriptions of the molecules and methods for their production.

[0050] Additional agents that inhibit GSK3 β can be identified by any method known in the art. For example, compounds may be screened in the following manner for their ability to inhibit GSIG β using a standard coupled enzyme assay (e.g., Fox et ah, Protein Sd 7:2249 (1998)) or other assays used to determine kinase activity.

[0051] hCDC4 antagonists

[0052] Any hCDC4 antagonists can be used according to the methods of the invention. Antagonists can include, e.g., antibody antagonists or other organic small molecule inhibitors.

[0053] hCDC4 acts as part of the skp-cullin-Fbox (SCF) complex, which functions as an E3 ligase — facilitating the attachment of ubiquitin to cellular substrates, which can target them for degradation by the 26S proteasome. See, e.g., Cardozo and Pagano, MoI. Cell. Biol. 5:739-751 (2004). The function of hCDC4 within the SCF complex is substrate recognition. hCDC4 contains an Fbox motif in its N-terminal region that binds to Skpl, thereby anchoring hCDC4 to the SCF complex. A flexible linker between the Fbox and the WD40 repeats in hCDC4 allows the circular D -propeller motif, formed by the WD40 repeats, to recognize appropriate substrates, thereby recruiting these substrates to the SCF for ubiquitination. The hCDC4 circular D -propeller motif recognizes a phosphodegron motif within the targeted substrate, which in the case of MYC is KKFELLPTPPLSPSRR (SEQ ID NO: 1), where the threonine (T) is phosphorylated by GSK3 D . A minimal phsophodegron sequence an L*TPXX consensus, which in the case of MYC is LPTPP. Thus, GSK3 D phophorylates MYC allowing recognition by Fbw7, thereby recruiting MYC to the SCF complex where it is ubiquitinated and subsequently degraded by the 26S proteasome. [0054] In view of the mechanism of action of hCDC4, a number of screening assays can be performed. In some embodiments, the active ligase complex can be contacted with candidate antagonists and then tested for a reduction in ligase activity. Ligase activity can be measured directly or indirectly (e.g., by measuring changes in ubiquitination of a target protein or ubiquitin-mediated degradation of the protein target.

[0055] Alternatively, assays that measure interference in binding of hCDC4 or a phosphodegron-binding fragment thereof to a phosphodegron (i.e., a phosphorylated polypeptide comprising the LPTPP sequence from MYC) in the presence of a candidate antagonist can be used to identify hCDC4 antagonists. In general, WD40 repeats of hCDC4 are involved in phosphodegron binding. See, .e.g., Nash et ah, Nature 414(6863):498-9 (2001). Accordingly, phosphodegron-binding fragments of hCDC4 will generally comprise at least some one of the WD40 repeats of hCDC4 (e.g., WD40 repeat #3, located at amino acids 533-572 of FBW7U, amino acids 482-521 of FBW7D and amino acids 377-416 of FBW7D). In some embodiments, the phosphodegron-binding fragment comprises all of the WD40 repeats of hCDC4 (e.g., amino acids 467-735 of FBW7D, amino acids 416-684 of FBW7D and amino acids 311-579 of FBW7D The phosphodegron-binding fragment can be expressed and used in the assays of the invention as a fusion protein with a heterologous amino acid sequence. hCDC4-phosphodegron binding can be performed by any methods known in the art for measuring protein-protein binding. In some embodiments, the methods comprise labeling the hCDC4 polypeptide, or an active fragment thereof, with a first label and labeling the phosphodegron with a second label, wherein proximity of the two labels (e.g., when hCDC4 binds the phosphodegron) results in a detectable signal that does not occur when the two labels are not in proximity. Examples of labels that create a signal when in proximity include the pairing of Europium with either Allophycocyanin (APC) or C- Phycocyanin (CPC). Labels can be linked directly to the hCDC4 polypeptide and the phosphodegron or indirectly. Indirect linkages include, e.g., linking the label to an antibody that binds to either the hCDC4 polypeptide and the phosphodegron, or a fusion of one of them with a common fusion partner such as glutathione-S-transferase (GST). Alternatively, various binding pairs such as biotin/strepavidin can be used to link the label. For example, in some embodiments, one of the binding members is biotinylated and labeled by contacting the biotinylated member with streptavidin linked to the label. Signal for the labels can be detected using, e.g., standard fluorescence detectors. Inhibition of binding in the presence of a candidate inhibitor can be detected by a reduction of signal from the proximity labels. siRNA inhibition of expression of hCDC4 or the MYC-interacting genes

[0056] In some embodiments of the invention, small polynucleotides that interfere with expression can be used to inhibit expression of hCDC4 or the MYC-interacting genes (e.g., GSK3D) show in Table 1 in combination with administration of a DR5 agonist. In some embodiments, the polynucleotides comprise sequences that are in the sense, antisense, or both orientations of at least a fragment of the desired target mRNA. [0057] Antisense technology is well known. Generally, nucleic acids sequences that are at least substantially complementary to at least a portion (e.g., at least 5, 10, 20, or more contiguous nucleotides) in an mRNA target (e.g., mRNA encoding hCDC4 or GSK3 D) are generated in, or administered to, a cell in which it is desired to inhibit expression. Examples of GSK3D antisense molecules have been described in, e.g., U.S. Patent No. 6,323,029. [0058] siRNAs that inhibit expression of GSK3 D or hCDC4 can also be administered in combination with a DR5 agonist. "siRNA" refers to small interfering RNAs, that are capable of causing interference and can cause post-transcriptional silencing of specific genes in cells, for example, mammalian cells (including human cells) and in the body, for example, mammalian bodies (including humans). The phenomenon of RNA interference is described and discussed in Bass, Nature 411 : 428-29 (2001); Elbahir et at, Nature 411 : 494-98 (2001); and Fire etal, Nature 391: 806-11 (1998); and WO 01/75164, where methods of making interfering RNA also are discussed. siRNAs can include hairpin loops comprising self- complementary sequences or double stranded sequences. The siRNAs based upon the sequences and nucleic acids encoding the gene products disclosed herein typically have fewer than 100 base pairs and can be, e.g., about 30 bps or shorter, and can be made by approaches known in the art, including the use of complementary DNA strands or synthetic approaches. The siRNAs are capable of causing interference and can cause post-transcriptional silencing of specific genes in cells, for example, mammalian cells (including human cells) and in the body, for example, mammalian bodies (including, or optionally excluding, humans). Exemplary siRNAs according to the invention could have up to 29 bps, 25 bps, 22 bps, 21 bps, 20 bps, 15 bps, 10 bps, 5 bps or any integer thereabout or therebetween. Tools for designing optimal inhibitory siRNAs include that available from DNAengine Inc. (Seattle, WA) and Ambion, Inc. (Austin, TX).

[0059] One RNAi technique employs genetic constructs within which sense and anti- sense sequences are placed in regions flanking an intron sequence in proper splicing orientation with donor and acceptor splicing sites. Alternatively, spacer sequences of various lengths may be employed to separate self-complementary regions of sequence in the construct. During processing of the gene construct transcript, intron sequences are spliced- out, allowing sense and anti-sense sequences, as well as splice junction sequences, to bind forming double-stranded RNA. Select ribonucleases then bind to and cleave the double- stranded RNA, thereby initiating the cascade of events leading to degradation of specific mRNA gene sequences, and silencing specific genes.

[0060] While polynucleotides that inhibit expression can include naturally occurring nucleotides, the present invention comprehends other oligomeric nucleic acid compounds, including but not limited to oligonucleotide mimetics such as are described below. The inhibitory polynucleotides of the invention (e.g., antisense, sense or a combination thereof) can comprise, e.g., from about 8 to about 30 or more nucleobases (i.e. from about 8 to about 30 or more linked nucleosides). For example inhibitory oligonucleotides can comprise from about 12 to about 25 nucleobases. Specific examples of inhibitory polynucleotides useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages.

[0061] The inhibitory polynucleotides of the invention can be combined any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the polynucleotide.

[0062] Antisense or siRNAs can also be delivered to a cell as part of an expression cassette. In some embodiments, the expression cassette is delivered using a viral vector, e.g., an adenoviral or lentiviral vector.

Screening for agonists or antagonists

[0063] DR5 agonists, GSK3β antagonists and/or hCDC4 antagonists can be identified by many art-recognized methods.

[0064] As some molecules that bind a target may activate or inhibit the activity of that target, in some embodiments, binding assays are performed as a preliminary screen. Binding assays usually involve contacting a target protein or active or inactive fragment thereof with one or more test agents and allowing sufficient time for the protein and test agents to form a binding complex. Any binding complexes formed can be detected using any of a number of established analytical techniques. Protein-protein binding assays include, but are not limited to, methods that measure co-precipitation or co-migration on non-denaturing SDS- polyacrylamide gels, and co-migration on Western blots {see, e.g., Bennet, J.P. and Yamamura, H.I. (1985) "Neurotransmitter, Hormone or Drug Receptor Binding Methods," in Neurotransmitter Receptor Binding (Yamamura, H. I, et al., eds.), pp. 61-89. Other binding assays involve the use of mass spectrometry or NMR techniques to identify molecules bound to a polypeptide of the invention or displacement of labeled substrates. The polypeptides of the invention utilized in such assays can be naturally expressed, cloned or synthesized. In addition, mammalian or yeast two-hybrid approaches (see, e.g., Bartel, PX. et. al. Methods EnzymoU 254:241 (1995)) can be used to identify polypeptides or other molecules that interact or bind when expressed together in a host cell or to identify molecules that interfere with such binding (e.g., binding of hCDC4 to a phosphodegron) .

[0065] In addition, mammalian or yeast two-hybrid approaches (see, e.g. , Bartel, P.L. et. al. Methods Enzymol, 254:241 (1995)) can be used to identify polypeptides or other molecules that interact or bind when expressed together in a host cell. [0066] Agents can also be directly selected for their ability to activate (i.e., act as an agonist in the case of DR5) or antagonize (in the cases of GSK3β and hCDC4) the activity of a particular target protein. Exemplary activity assays for DR5, GSK3β and hCDC4 are described herein. However, it will be understood that the particular activity assays described herein are merely examples and any assay designed to measure activity of a target, either directly or indirectly, can be used in the present invention.

[0067] As described herein, the inventors have determined that inhibit either GSK3β or hCDC4, while activating DR5, results in greater induction of apoptosis in certain cells (e.g., cancer cells, and particularly MYC-expressing cells) than DR5 activation alone. Thus in some embodiments, a known DR5 agonist is contacted to cells (e.g., MYC-expressing cells) in the presence or one or more candidate GSK3β or hCDC4 antagonist and an increase in apoptosis in the cells is detected compared to DR5 agonist treatment alone. Similarly, apoptosis assays can be used to identify DR5 agonists by contacting cells (e.g., MYC- expressing cells) with candidate DR5 agonists and detecting an increase in apoptosis. Further experiments may be performed to determine whether the DR5 receptor mediates the apoptosis.

[0068] A variety of assays for determining cell viability or apoptosis are well known in the art. Such methods include light microscopy for determining the presence of one or more morphological characteristics of apoptosis, such as condensed or rounded morphology, shrinking and blebbing of the cytoplasm, preservation of structure of cellular organelles including mitochondria, and condensation and margination of chromatin. Apoptosis can also be measured using terminal deoxytransferase-mediated (TdT) dUTP biotin nick end-labeling (TUNEL) (Gavriel et al, J. Cell Biol. 119:493 (1992); Gorczyca et al, Int. J Oncol. 1 :639 (1992)). APOPTAG (ONCOR, Inc.; Gaithersburg Md.), PhiPhiLux® (Oncolmmunin, Inc.) and the "Homogeneous Caspases Assay" (Roche Molecular Biochemicals) are commercially available kits for identification of apoptotic cells. In addition, apoptosis can be assayed by detecting nucleosomal DNA fragments using agarose gel electrophoresis (Gong et ah, Anal. Biochem. 218:314 (1994)). Apoptotic or anti-apoptotic activity also can be detected and quantified by determining an altered mitochondrial to nuclear DNA ratio as described in Tepper et al., Anal. Biochem. 203:127 (1992) and Tepper and Studzinski, J. Cell Biochem. 52:352 (1993). One skilled in the art understands that these, or other assays for apoptotic or anti-apoptotic activity, can be performed using routine methodology. Viability screens can be conducted in a high throughput format using, for example, alamar blue. Alternatively, commercially available kits measuring caspase activity can be used to run similar screens aimed at apoptosis detection.

[0069] The agents tested in the methods of the invention can be any small chemical compound, or a biological entity, such as a protein, sugar, nucleic acid, lipid, or combination thereof. Alternatively, test agents will be small organic molecules or peptides. Essentially any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds that can be dissolved in aqueous or organic (especially DMSO-based) solutions are used. The assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microliter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma- Aldrich (St. Louis, MO), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland) and the like. Generally, the compounds to be tested are present in the range from 1 pM to 100 mM. [0070] In some embodiments, high throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (potential modulator compounds). Such "combinatorial chemical libraries" or "ligand libraries" are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics. [0071] A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. [0072] Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010,175, ¥urka, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et αl, Nature 354:84-88 (1991)). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al, Proc. Nat. Acad. Set USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al, J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al, J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho et al, Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleic acid libraries (see Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S. Patent 5,539,083), antibody libraries (see, e.g., Vaughn et al, Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al. , Science, 21 A: 1520-1522 (1996) and U.S. Patent 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent 5,569,588; thiazolidinones and metathiazanones, U.S. Patent 5,549,974; pyrrolidines, U.S. Patents 5,525,735 and 5,519,134; morpholino compounds, U.S. Patent 5,506,337; benzodiazepines, 5,288,514, and the like).

[0073] Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433 A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA). In addition, numerous combinatorial libraries are themselves commercially available {see, e.g., ComGenex, Princeton, N.J., Tripos, Inc., St. Louis, MO, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.). [0074] Agents that are initially identified by any of the foregoing screening methods can be further tested to validate the apparent activity. Modulators that are selected for further study can be tested for an effect on cancer, tumor formation, tumor inhibition, metastases or other cancer-related phenotypes.

[0075] For example, the effect of the compound can be assessed in animals. Standard mouse tumor models include the use of SCID mice in which cancer cells are implanted and which form a tumor. In some embodiments, candidate agonists or antagonists, in combination or alone can be administered to animals to determine an effect on the tumor. [0076] In a high throughput assay of the invention, it is possible to screen several thousands or more of different potential modulators in a single day. In particular, each well of a microliter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microliter plate can assay about 100 {e.g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100 to about 1500 different compounds. It is possible to assay several different plates per day; assay screens for up to about 6,000-20,000, 100,000 or more different compounds are possible using the integrated systems of the invention. In addition, microfluidic approaches to reagent manipulation can be used.

Pharmaceutical compositions

[0077] The antibodies and agents of the invention can be administered directly to the mammalian subject for treatment, e.g., of hyperproliferative disorders including cancer such as, but not limited to: carcinomas, gliomas, mesotheliomas, melanomas, lymphomas, leukemias, adenocarcinomas, breast cancer, ovarian cancer, cervical cancer, glioblastoma, leukemia, lymphoma, prostate cancer, and Burkitt's lymphoma, head and neck cancer, colon cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, cancer of the esophagus, stomach cancer, pancreatic cancer, hepatobiliary cancer, cancer of the gallbladder, cancer of the small intestine, rectal cancer, kidney cancer, bladder cancer, prostate cancer, penile cancer, urethral cancer, testicular cancer, cervical cancer, vaginal cancer, uterine cancer, ovarian cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine cancer, carcinoid cancer, bone cancer, skin cancer, retinoblastomas, multiple myelomas, Hodgkin's lymphoma, and non-Hodgkin's lymphoma {see, CANCER.-PRINCIPLES AND PRACTICE (DeVita, V.T. et al. eds 1997) for additional cancers). [0078] Administration of the compositions of the present invention can be by any of the routes normally used for introducing a chemotherapeutic compound into ultimate contact with the tissue to be treated. The antibodies and agents are administered in any suitable manner, optionally with pharmaceutically acceptable carriers. Suitable methods of administering such antibodies and agents are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.

[0079] Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 17th ed. 1985)).

[0080] The antibodies and agents, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. [0081] Formulations suitable for administration include aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered, for example, by orally, topically, intravenously, intraperitoneally, intravesically or intrathecally. Optionally, the compositions are administered nasally. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials. Solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. The modulators can also be administered as part a of prepared food or drug. The compounds of the present invention can also be used effectively in combination with one or more additional active agents (e.g., chemotherapeutics) depending on the desired therapy or effect.

[0082] The dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial response in the subject over time. The dose will be determined by the efficacy of the particular modulators employed and the condition of the subject, as well as the body weight or surface area of the area to be treated. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular compound or vector in a particular subject. Administration can be accomplished via single or divided doses. [0083] The DR5 agonist and the GSK3 β antagonist or hCDC4 antagonist can be administered together in a mixture or each can be administered separately. The DR5 agonist and the GSK3β antagonist or hCDC4 antagonist can, but need not, be administered concurrently.

Use of DRS agonists in GSK3β- or hCDC4- individuals

[0084] In view of the observation of a synergistic effect of inhibition of either GSK3 β orhCDC4 and induction of apoptosis with DR5 agonists, some embodiments of the invention involve detecting the genotype of GSK3β or hCDC4 in cancer cells from an individual and then administering a DR5 agonist to the individual if the individual has cancer cells that lack functional GSK3β or hCDC4 alleles.

[0085] Any mutation reducing the activity of GSK3 β or hCDC4 is indicative of an individual who can be selected for treatment with a DR5 agonist. Thus, nucleotide mutations, resulting in changes in the coding sequence (i.e., resulting in introduction of a different amino at a particular position, introduction of a stop codon in the coding sequence, deletions or inversions, etc.) of GSK3 β or hCDC4 will affect the activity of these proteins. Without intending to limit the invention for use in a particular type of cancer, mutations in hCDC4 have been previously observed in breast and ovarian cancers. See, e.g., Cardozo & Pagano, MoL Cell. Biol 5:739-751 (2004).

[0086] A number of diagnostic tests can be used to determine the genotype of GSK3 β or hCDC4 in cancer cells from an individual. Typically, a biopsy comprising cancer cells within an individual is obtained. The GSK3β or hCDC4 gene sequences can then be analyzed to determine if they comprise any mutations that inhibit their activity. In some embodiments, the chromosomes of the cancer cells will have gone through deletions or translocations, effectively eliminating the genes(s). Alternatively, point mutations or small deletions can indicate functional changes in the resulting encoded proteins. Any nucleotide change that results in coding for a different amino acid than is known to exist at that position in naturally-occurring active GSK3β or hCDC4 proteins is considered a mutation that inhibits the activity of GSK3β or hCDC4, respectively unless there is evidence to the contrary. [0087] Methods for detecting mutations in GSK3β or hCDC4 include, but are not limited to, nucleotide sequencing and/or use of polynucleotide amplification (e.g., PCR) and probe hybridization techniques that allow for detection of sequence alterations in the probe target, e.g., using hybridization conditions that does not allow for significant hybridization if the target gene includes a nucleotide mis-match.

[0088] In other embodiments, individuals are screened for the presence of alleles of

MYC that comprise an alteration at the wildtype T58 position. In these embodiments, at least a portion of the MYC allele flanking the T58 position is isolated and sequences or otherwise detected. Amino acids encoded at that position other than threonine, or the absence of an amino acid at that position, as indicated by amino acid alignment, indicate that the cancer cells are particularly susceptible to treatment with DR5 agonists. Although any change at the T58 position in MYC can induce increased sensitivity to DR5 agonists, in some embodiments, the mutations are selected from T58A or T58I. Cancers comprising mutations at T58 in MYC have been described previously and include Burkitt's lymphoma. See, e.g., Chang et al. , MoI. Cell. Biol. 20(12):4309-4319 (2000). Those of skill in the art will appreciate, however, that any cancer comprising T58 mutations will be more sensitive to DR5 agonist-induced apoptosis induction. Thus the methods of the invention provide for identification of individuals with mutations at the T58 position of MYC and administering DR5 agonists to individuals with such mutations.

EXAMPLES

[0089] The Examples herein are intended to exemplify, not limit, the present invention.

[0090] The MYC proto-oncogene is frequently deregulated in human cancers. We screened a kinase-directed library of small inhibitory RNAs (siRNAs) to uncover genes that display synthetic lethal interactions with MYC. We identified a number of genes as potentiators of apoptosis specifically in MYC-overexpressing cells, which are shown in Table 1. Among the screen hits, we further examined the role of GSKβ in regulating MYC- dependent sensitivity to DR5 death receptor agonists. We found that siRNA-mediated silencing of GSK3β, but not the related GSK3α, prevents phosphorylation of MYC on threonine residue 58, thereby inhibiting recognition of MYC by the E3 ubiquitin ligase component hCDC4 (referred to in this Example as "hFBW7"). Attenuating the GSK3β- FBW7 axis stabilizes MYC, and results in upregulation of surface levels of the TRAIL receptor DR5, potentiation of DR5-induced apoptosis in vitro, and enhancement CDC4 (referred to in this Example as Fbw7) of DR5 agonist treatment efficacy in vivo in a tumor xenograft model. These results identify GSK3β and FB W7, as well as the other MYC- interacting genes listed in Table 1, as cancer therapeutic targets. The data also indicate MYC as a useful substrate in the GSK3β survival-signaling pathway, and demonstrate paradoxically, that MYC-overexpressing tumors are treatable by agents that increase MYC oncogene function.

Table 1. hits of the loss of function screen - ranked with decreasing viability ratio

[0091] High-throughput cellular phenotypic screening methodologies based on gene silencing by small inhibitory RNAs (siRNAs) potentially provide a way of systematically identifying novel synthetic lethal interactions in mammalian cells (reviewed in (Deveraux, Q.L., Aza-Blanc, P., Wagner, K. W., Bauerschlag, D., Cooke, M.P. and Hampton, G.M., (2003) Semin Cancer Biol 13, 293-300; Paddison, PJ. and Harmon, GJ., (2002) Cancer Cell 2, 17-23; Willingham, A.T., Deveraux, Q.L., Hampton, G.M. and P., A.-B., (2004) Oncogene 23, 8392-8400). Here, we employed a focused library of siRNAs directed against the superfamily of human kinases to identify genes whose inactivation is synthetically lethal with MYC overexpression. Among the genes identified was glycogen synthase kinase 3 beta (GSK3β), a gene with a critical but poorly characterized role in suppressing death receptor- induced apoptosis (Hoeflich, K.P., Luo, J., Rubie, E.A., Tsao, M.S., Jin, O. and Woodgett, J.R., (2000) Nature 406, 86-90.; Jope, R.S. and Johnson, G. V., (2004). Trends Biochem Sci 29, 95-102.; Liao, X., Zhang, L., Thrasher, J.B., Du, J. and Li, B., (2003). MoI Cancer Thar 2, 1215-1222). We show that GSK3β's ability to phosphorylate and target MYC for recognition by the E3 ubiquitin ligase component FBW7 (also known as FBXW7, hCDC4, AGO, and SELlO) underlies its synthetic lethal interaction with MYC. These results identify GSK3β and FB W7 as cancer therapeutic targets, and demonstrate a novel, counterintuitive approach toward cancer therapy: that of increasing rather than decreasing oncogene function.

RESULTS

Screening of a kinase-directed siRNA library identifies a synthetic lethal interaction between knockdown of a number of genes and MYC overexpression

[0092] To identify genes whose knockdown is synthetically lethal with MYC overexpression, we screened an arrayed library of 624 siRNAs (Aza-Blanc, P., Cooper, C.L., Wagner, K., Batalov, S., Deveraux, Q.L. and Cooke, M.P., (2003) MoI Cell 12(3):627-37) for the ability to differentially potentiate DR5-mediated apoptosis in an isogenic pair of cell lines differing only in their level of MYC expression. The siRNA library, arrayed into 384 well cell culture plates, was reverse-transfected into either the immortalized but non-transformed kidney epithelial cell HAlE, which expresses low endogenous MYC levels, or its transformed derivative HAlE-MYC that ectopically expresses MYC from an integrated retroviral provirus (Wang, Y., Engels, I.H., Knee, D.A., Nasoff, M., Deveraux, Q.L. and Quon, K.C., (2004) Cancer Cell 5:501-512.). To increase the sensitivity to apoptosis, the siRNA-transfected cells were treated with a suboptimal dose of agonistic monoclonal antibodies specific for DR5 (DR5-A) (Ren, Y.G., Wagner, K.W., Knee, D.A., Aza-Blanc, P., Nasoff, M. and Deveraux, Q.L., (2004) MoI Biol Cell 15, 5064-5074.; Wang, Y., Engels, I.H., Knee, D.A., Nasoff, M., Deveraux, Q.L. and Quon, K.C., (2004) Cancer Cell 5, 501- 512) for 24 h prior to assaying for cell viability. Of the 624 individual siRNAs tested, 14 sensitized the MYC expressing cells > 2 fold over their non-MYC expressing counterparts. As shown in Table 1, genes targeted by these siRNAs and their encoded protein products are termed herein MYC-interacting genes and MYC-interacting polypeptides, respectively. The gene targeted by one of these siRNAs, GSK3β, was selected for further study. [0093] To confirm that siRNA-mediated knockdown of GSK3 β specifically potentiated apoptosis in MYC-expressing cells, we repeated the cell viability assays using multiple non-overlapping siRNAs directed against GSK3β as well as the related isoform GSK3β, across a range of DR5-A concentrations. At all DR5-A concentrations tested, each of the siRNAs targeting GSK3β (2 individual siRNAs, and a "smartpool" pool of 4 siRNAs) sensitized the MYC overexpressing HAlE-MYC cells to the action of DR5-A, while have little or no effect on the isogenic parental cell line. siRNAs against the GSK3β isoform sensitized neither HAlE nor HAlE-MYC cells, despite reducing target protein expression to a similar extent as the GSK3β siRNAs.

[0094] Similarly, the commonly employed GSK3 inhibitor lithium chloride (LiCl) sensitized HAlE-MYC, but not HAlE cells, to DR5-A in a concentration dependent manner. Substitution of potassium chloride (KCl) for LiCl to control for changes in osmolality did not significantly influence DR5-A sensitivity in either cell line. Two additional commercially available small molecule GSK3 inhibitors (6-bromoindirubin-3-oxime (Meijer, L., Skaltsounis, A.L., Magiatis, P., Polychronopoulos, P., Knockaert, M., Leost, M., Ryan, X.P., Vonica, C.A., Brivanlou, A., Dajani, R., Crovace, C, Tarricone, C, Musacchio, A., Roe, S.M., Pearl, L. and Greengard, P., (2003). Chem Biol 10, 1255-1266),and AR-A014418 (Bhat, R., Xue, Y., Berg, S., Hellberg, S., Ormo, M., Nilsson, Y., Radesater, A.C., Jerning, E., Markgren, P.O., Borgegard, T., Nylof, M., Gimenez-Cassina, A., Hernandez, F., Lucas, JJ., Diaz-Nido, J. and Avila, J., (2003) J Biol Chem 278, 45937-45945) gave similar results to those for LiCl. While none of these chemical inhibitors distinguish between the α and β GSK3 isoforms, these data, when combined with the siRNA results presented above, provide strong evidence that reducing or inhibiting the GSK3β isoform potentiates DR5-mediated apoptosis specifically in MYC overexpressing cells.

GSK3β and FBW7-dependent phosphorylation and degradation of MYC determine the sensitivity of MYC expressing cells to DR5-A-mediated apoptosis

[0095] The synthetic lethal relationship between GSK3 β and MYC suggested that

GSK3β might act through MYC to influence cell survival and apoptosis. Recently, GSK3 was shown to regulate MYC protein stability by phosphorylating MYC at threonine residue 58 (T58) (Gregory, M.A., Qi, Y. and Hann, S.R., (2003) J Biol Chem 278, 51606-51612). Phosphorylation at this site creates a recognition site termed a Cdc4 phosphodegron or CPD that allows MYC to be recognized and targeted for degradation by FBW7, an F-box containing component of the SCF (Skp-Cullin-F-box) ubiquitin ligase complex (Welcker, M., Orian, A., Jin, J., Grim, J.A., Harper, J. W., Eisenman, R.N. and Clurman, B.E., (2004) Proc Natl Acad Sd USA 101, 9085-9090.; Yada, M., Hatakeyama, S., Kamura, T., Nishiyama, M., Tsunematsu, R., Imaki, H., Ishida, N., Okumura, F., Nakayama, K. and Nakayama, K.I., (2004) EMBO J23, 2116-2125). To determine if phosphorylation of MYC at T58 and subsequent FBW7-dependent degradation play a role in the control of cell survival by GSK3β, we tested whether suppression of FBW7 function would mimic depletion of GSK3β. siRNAs against FB W7 and GSK3β increased MYC protein levels and enhanced sensitivity to DR5-A in HAlE-MYC, but not in HAlE cells, suggesting that GSK3β and FBW7 act in a common pathway regulating MYC-dependent apoptosis. Notably, siRNA mediated silencing of GSK3β and FB W7 protein expression was similar in HAlE-MYC and HAlE cells, indicating the lack of sensitization in HAlE cells did not result from failure to downregulate GSK3β or FBW7. Although both GSK3β and FBW7 siRNAs enhanced MYC protein levels in HAlE-MYC cells, only siRNAs against GSK3β prevented MYC T58 phosporylation indicating that FB W7 function downstream of GSK3β-dependent MYC T58 phosporylation — consistent with previous observations (Welcker, M., Orian, A., Jin, J., Grim, J.A., Harper, J. W., Eisenman, R.N. and Clurman, B.E., (2004) Proc Natl Acad Sci USA 101, 9085-9090; Yada, M., Hatakeyama, S., Kamura, T., Nishiyama, M., Tsunematsu, R., Imaki, H., Ishida, N., Okumura, F., Nakayama, K. and Nakayama, K.I., (2004) EMBO J23, 2116- 2125).

[0096] In HAlE cells where retrovirally introduced MYC was mutated to alanine at

T58 (MYCT58A), such that it could no longer be phosphorylated by GSK3β and therefore not recognized by FBW7(Welcker, M., Orian, A., Jin, J., Grim, J.A., Harper, J.W., Eisenman, R.N. and Clurman, B.E., (2004) Proc Natl Acad Sci USA 101, 9085-9090; Yada, M., Hatakeyama, S., Kamura, T., Nishiyama, M., Tsunematsu, R., Imaki, H., Ishida, N., Okumura, F., Nakayama, K. and Nakayama, K.I., (2004). EMBO J 23, 2116-2125), neither GSK3β nor FB W7 siRNAs could further sensitize cells to DR5-A, in striking contrast to similar experiments performed with wild-type (wt) MYC. These results indicate that MYC is phosphorylated at T58 in order for siRNAs against either GSK3βor FBW7 to additionally sensitize to apoptosis. Notably, the MYCT58A allele was significantly more potent in sensitizing HAlE cells to DR5-A-induced apoptosis than was wt MYC, sensitizing to a similar extent as the combination of wt MYC plus siRNA against GSK3β or FB W7. Taken together, these data strongly argue for a model in which inactivation of GSK3β enhances sensitivity to DR5 agonists by preventing phosphorylation of MYC at T58, thereby preventing FBW7-dependent MYC recognition and degradation.

Stabilization of MYC by inactivation of the GSK3β-FBW7 CPD recognition module increases DR5 receptor levels and stimulates Caspase-8 processing

[0097] Previously, we showed that ectopic expression of MYC results in the upregulation of DR5 receptor on the cell surface and subsequent sensitization to apoptosis induced by DR5 agonists (Wang, Y., Engels, I.H., Knee, D.A., Nasoff, M., Deveraux, Q.L. and Quon, K.C., (2004) Cancer Cell 5, 501-512). Thus, we asked if the additional stabilization of expressed MYC, caused by FB W7 or GSK3β depletion, could further influence surface DR5 receptor levels. Flow cytometric analysis of HAlE-MYC cells transfected with various siRNAs showed that both GSK3β and FBW7-depleted cells increased DR5 receptors relative to cells transfected with control siRNAs. In contrast, an siRNA targeting DR5 decreased cell surface DR5 expression, confirming the specificity of the reagents employed in these assays. Notably, increased DR5 receptor levels in GSK3β or FBW7-depleted cells were associated with increased activation and proteolytic processing of Caspase-8, thus correlating increased receptor levels to increased functional apoptotic signaling. GSK3β and FB W7 siRNAs had no detectable influence on DR5 receptors in HAlE cells, suggesting that disrupting the GSK3β/FBW7-dependent MYC degradation process in cells with low MYC expression, e.g. non-transformed cells, is not sufficient to enhance DR5 cell surface expression and subsequent sensitization to DR5 agonists.

Disruption of the GSK3β-FBW7 module controlling MYC stability in human tumor- derived cells upregulates DR5 surface receptor levels and enhances DR5-A-induced apoptosis

[0098] To determine if our results could be recapitulated in human tumor-derived material with high endogenous MYC levels, we employed the human colon carcinoma cell line, HCTl 16. Previously, we showed that this cell line, which overexpresses MYC (Wang, Y., Engels, IJH., Knee, D.A., Nasoff, M., Deveraux, Q.L. and Quon, K.C., (2004) Cancer Cell 5, 501-512) but is wild-type (wt) for FBW7 (Rajagopalan, H., Jallepalli, P.V., Rago, C, Velculescu, V.E., Kinzler, K.W., Vogelstein, B. and Lengauer, C, (2004) Nature 428, 77- 81), undergoes apoptosis when treated with DR5-A, and that this sensitivity requires endogenous MYC function (Wang, Y., Engels, LH., Knee, D.A., Nasoff, M., Deveraux, Q.L. and Quon, K.C., (2004) Cancer Cell 5, 501-512). Like HAlE-MYC cells, HCTl 16 could be sensitized to DR5-A by siRNAs against GSK3β and FBW7, but not GSK3β. Importantly, these siRNAs did not sensitize to an agonistic antibody against the DR4 TRAIL receptor, highlighting the role of DR5 in GSK3β and FBW7-mediated sensitization. Furthermore, derivatives of HCTl 16, in which one or both copies of FBW7 were disrupted by homologous recombination (FB W7 +/- and FBW7 -/- respectively, (Rajagopalan, H., Jallepalli, P.V., Rago, C, Velculescu, V.E., Kinzler, K.W., Vogelstein, B. and Lengauer, C, (2004) Nature 428, 77-81) were similarly hypersensitive to DR5-A when compared to the siGSK3β and siFBW7 parental HCTl 16 line. Interestingly, both heterozygous and homozygous disruption of FBW7 strongly enhanced apoptosis by DR5-A, consistent with the observation that FB W7 can act as a haploinsufficient tumor suppressor gene (Mao, J.H., Perez-Losada, L, Wu, D., Delrosario, R., Tsunematsu, R., Nakayama, K.I., Brown, K., Bryson, S. and Balmain, A., (2004) Nature 432, 775-779), although the effect was more dramatic in the homozygotes. FBW7 gene silencing by siRNA's or genetic deletion was confirmed by western blot or PCR analysis.

[0099] Similarly, the colon carcinoma cell line HTl 15, which carries a naturally occurring heterozygous mutation in FBW7, was highly sensitive to DR5-A. Notably, sensitivity to DR5-A could be rescued by stable over-expression of a wt FBW7 cDNA in HTl 15 cells. The mutation present in HTl 15, Cl 153T, is among the most frequently occurring FBW7 mutations found in colon cancer (Rajagopalan, H., Jallepalli, P.V., Rago, C, Velculescu, V.E., Kinzler, K.W., Vogelstein, B. and Lengauer, C, (2004) Nature 428, 77- 81.; Rajagopalan, H. and Lengauer, C, (2004) Cell Cycle 3, 693-694), and mutates a critical arginine residue in the third WD40 repeat of its substrate recognition domain, thereby disrupting its ability to bind substrate (Orlicky, S., Tang, X., Willems, A., Tyers, M. and Sicheri, F., (2003) Cell 112, 243-256.; Rajagopalan, H., Jallepalli, P.V., Rago, C, Velculescu, V.E., Kinzler, K.W., Vogelstein, B. and Lengauer, C, (2004) Nature 428, 77- 81.). [00100] As in HAlE-MYC cells, transfection of siRNAs targeting GSK3β or FBW7 in

HCTl 16 parental cells resulted in increased cell surface DR5 receptor levels. Similar increases in DR5 receptor levels were also observed when FBW7 was disrupted by homologous recombination, either heterozygously or homozygously, with the increase being slightly greater in the homozygous cells. In HCTl 16 FBW7+/- cells, DR5 cell surface expression could be further increased by transfection of siGSK3β or siFBW7. However no further increases in DR5 receptor levels could be observed following introduction of the GSK3β or FBW7 siRNAs into HCTl 16 FBW7-/- cells devoid of functional FBW7. These results demonstrate that GSK3β regulates DR5 receptor levels only in the presence of functional FBW7, and thus provide compelling evidence that GSK3β acts upstream of FBW7 to regulate DR5 receptor levels. Interestingly, DR5 siRNAs enhanced MYC levels in FBW7+/- and FBW7-/- cells, which may indicate that loss of DR5 provides a survival advantage to MYC overexpressing cells.

[00101] Multiple substrates for FBW7 have been identified in addition to MYC, most notably cyclin E (Koepp, D.M., Schaefer, L.K., Ye, X., Keyomarsi, K., Chu, C, Harper, J.W. and Elledge, S.J., (2001) Science 294, 173-177). Like MYC, cyclin E is recognized by FB W7 following phosphorylation on a residue, T380, acted upon by GSK3 (Welcker, M., Singer, J., Loeb, K.R., Grim, J., Bloecher, A., Gurien-West, M., Clurman, B.E. and Roberts, J.M., (2003) MoI Cell 12, 381-392), raising the possibility that the GSK3β-FBW7 CPD recognition module might also act through cyclin E to regulate DR5-A-induced apoptosis. To address their relative roles in GSK3β/FBW7 control of DR5-mediated cell death, we tested the ability of siRNAs against MYC and cyclin E to rescue FB W7 wt, heterozygous and homozygous mutant HCTl 16 cells from DR5-A-induced apoptosis. Whereas the siRNA targeting cyclin E had no significant effect on cell viability, although it efficiently silence cyclin E expression , the MYC siRNAs rescued viability in all three genotypes, as well as in the naturally occurring heterozygous mutant HTl 15. Qualitatively similar rescue in the HCTl 16 series of cell lines was also obtained if MYC function was reduced by stable retroviral expression of a dominant negative MYC allele, MADMYC (Berns, K., Hijmans, E.M. and Bernards, R., (1997) Oncogene 15, 1347-1356) and was accompanied by down- regulation of DR5 from the cell surface. These results indicate that sensitization to DR5 agonists by disruption of FBW7 is mediated MYC activity, and independent of cyclin E function. [00102] Interestingly, whereas siRNA-mediated knockdown of cyclin E had no effect upon DR5-induced apoptosis, it had a strong suppressing effect on the formation of micronuclei - a proxy for chromosomal instability — induced by FBW7 disruption, as previously reported (Rajagopalan, H., Jallepalli, P.V., Rago, C, Velculescu, V.E., Kinzler, K.W., Vogelstein, B. and Lengauer, C, (2004) Nature 428, 77-81; Rajagopalan, H. and Lengauer, C, (2004) Cell Cycle 3, 693-694). In contrast, MYC siRNAs had no influence on micronuclei formation in FBW7 depleted cells. These data, therefore, delineate the function of two critical FBW7 targets, showing that in FB W7 depleted cells, the resulting increase in DR5 sensitivity is dependent upon MYC stabilization, whereas the increase in chromosomal instability is dependent upon cyclin E stabilization.

The GSK3β-FBW7 module as a potential therapeutic target

[00103] The synthetic lethal relationship between MYC overexpression and inactivation of components of the GSK3β-FBW7 module indicates that targeting GSK3 or FBW7 is an effective therapeutic strategy when used in combination with DR5 agonists. To demonstrate this in vivo, we grew HCTl 16 FBW7+/+ and FBW7+/- cells as subcutaneous xenografted tumors in immune-compromised mice. HCTl 16 FB W7 -/- cells grew poorly in vivo, and were thus excluded from the study. When tumors reached a mean volume of approximately 80 mm³, tumor-bearing mice were treated with 100 μg DR5-A every other day for a total of 7 treatments, or with buffer only as a control. Upon completion of the treatment regimen, the FBW7+/- tumors exhibited a significantly greater response to DR5-A, with a mean rumor volume of 88.05 versus 223.27 for the HCTl 16 FBW7+/+ tumors (p=0.00866), indicating that targeting GSK3 or FBW7 is effective as a combination therapy with DR5 agonists.

CONCLUSIONS

[00104] Here we report identifying a number of genes whose inactivation potentiates apoptosis specifically in MYC-overexpressing cells. Among these genes, GSK3β and FB W7 are a pair of proteins involved in ubiquitin-mediated protein degradation. Apart from identifying these genes as potential cancer therapeutic drug targets, our results elucidate an important mechanism of GSK3β and FBW7-mediated cellular survival signaling, and suggest, paradoxically, that MYC-overexpressing tumors might be treatable by agents that increase MYC oncogene function.

[00105] The sensitization to DR5-mediated apoptosis resulting from suppression of the

GSK3β-FBW7 axis has important implications for how DR5 agonists can be employed in the clinic. Mutations at the T58 GSK3β phosphorylation site on MYC are frequently observed in Burkitt's lymphoma (Bhatia, K., Huppi, K., Spangler, G., Siwarski, D., Iyer, R. and Magrath, L, (1993) Nat Genet 5, 56-61.), and have been shown to inhibit its degradation by the proteasome (Gregory, M.A. and Harm, S.R., (2000) MoI Cell Biol 20, 2423-2435), while inactivating FB W7 mutations have been reported in a variety of solid tumor types, including breast, ovarian, endometrial, pancreatic, and colorectal carcinomas (Calhoun, E.S., Jones, J.B., Ashfaq, R., Adsay, V., Baker, SJ., Valentine, V., Hempen, P.M., Hilgers, W., Yeo, C.J., Hruban, R.H. and Kern, S.E., (2003). Am J Pathol 163, 1255-1260; Rajagopalan, H., Jallepalli, P.V., Rago, C, Velculescu, V.E., Kinzler, K. W., Vogelstein, B. and Lengauer, C, (2004) Nature 428, 77-81.; Spruck, C.H., Strohmaier, H., Sangfelt, O., Muller, H.M., Hubalek, M., Muller-Holzner, E., Marth, C, Widschwendter, M. and Reed, S.I., (2002) Cancer Res 62, 4535-4539).

[00106] Small molecule GSK3 inhibitors are currently under development for several indications, including diabetes, neurodegenerative diseases, and bipolar disorder (Cohen, P. and Goedert, M., (2004) Nat Rev Drug Discov 3, 479-487.). Concerns that long-term treatment by such inhibitors might potentiate tumor incidence, through stabilization of GSK3β oncogenic targets such as β-catenin etc., were not substantiated in animal models (Gould, T.D., Gray, N.A. and Manji, H.K., (2003) Pharmacol Res 48, 49-53.) nor do bipolar patients treated with lithium exhibit increased cancer rates (Cohen, Y., Chetrit, A., Cohen, Y., Sirota, P. and Modan, B., (1998) Med Oncol 15, 32-36). Thus, by indirectly blocking MYC degradation, such inhibitors may be effective against MYC-overexpressing cancers, particularly when combined with DR5 agonists or other therapeutics that act at least in part by harnessing TRAIL signaling pathways, such as histone deacetylase inhibitors, interferons, retinoids, and arsenic trioxide (Akay, C. and Gazitt, Y., (2003) Cell Cycle 2, 358-368.; Altucci, L., Rossin, A., Raffelsberger, W., Reitmair, A., Chomienne, C. and Gronemeyer, H., (2001) Nat. Med. 7, 680-686.; Clarke, N., Jimenez-Lara, A.M., Voltz, E. and Gronemeyer, H., (2004) EMBOJTi, 3051-3060; Insinga, A., Monestiroli, S., Ronzoni, S., Gelmetti, V., Marchesi, F., Viale, A., Altucci, L., Nervi, C, Minucci, S. and Pelicci, P.G., (2005) Nat Med 11, 71-76; Nebbioso, A., Clarke, N., Voltz, E., Germain, E., Ambrosino, C, Bontempo, P., Alvarez, R., Schiavone, E.M., Ferrara, F., Bresciani, F., Weisz, A., de Lera, A.R., Gronemeyer, H. and Altucci, L., (2005) Nat Med U, 77-84).

Material and Methods

Cell lines

[00107] Genetically defined immortalized HAlE cells were obtained from Robert

Weinberg (Whitehead Institute, Cambridge, MA) and grown in Minimum Essential Medium Eagle supplemented with glutamine, penicillin/streptomycin, and 10% FCS. The colon cancer cell line HCTl 16 and its FBW7+/- and FBW7-/- derivatives were obtained from Bert Vogelstein (Johns Hopkins University, Baltimore, MD) and grown in McCoy's 5A medium supplemented as above. The colon cancer cell line HTl 15 were obtained from the European Collection of Cell Cultures and grown in DMEM supplemented as above.

Retroviruses

[00108] Retroviruses were produced by Lipofectamine 2000 (Invitrogen) -mediated transfection into Phoenix-A producer cells (Garry Nolan, Stanford University, Stanford, CA). Retroviral infections were performed by centrifuging target cells at 2700 rpm for 90 min at 25°C with 50% retroviral supernatant containing 2OmM HEPES and 8 μg/ml polybrene. Two rounds of infection¹ were typically performed. Retroviral vectors used were LZRS-IRES- EGFP for MYC, pBABEpuro for MADMYC, and pWZLblast for MYC or MYC-T58A. 1 μg/ml puromycin or blasticidin was used for selection.

siRNA Library Screening

[00109] The siRNA collection was prepared and plated as previously described Aza-

Blanc, P., Cooper, C.L., Wagner, K., Batalov, S., Deveraux, Q.L. and Cooke, M.P., (2003) MoI Cell 12(3):627-37. The siRNA collection contains 624 siRNAs designed to specifically silence each of 380 known and predicted kinases, 100 genes of unknown function, and 144 known genes of interest including genes known to play a role in apoptosis pathways. siRNAs were arrayed in 384-well microtiter plates in duplicate at 8 ng/well. To transfect siRNA libraries into HAlE and HAlE-MYC cells, we used reverse transfections. Briefly, 10 μl of Opti-Mem/Lipofectamine 2000 (125:1) mixture was dispensed into each well to incubate with siRNAs followed by dispensing of 40 μl of cells. 48 hours later, 10 μl of cross-linked DR5-A was added to one set of plates to reach final concentration of 1 μg/ml. Another 24 hours later, cell viability in each well was measured by CellTiter-Glo (Promega, Madison, WI). For each 384-well plate the signal was normalized by dividing each well by the average of 24 wells on the same plate containing siRNAs against Luciferase (siGL3) and multiplying by 100 to obtain normalized viability ("% viability").

Antibodies

[00110] Antibodies for immunoblotting were obtained from the indicated suppliers:

MYC-9E10 (Santa Cruz), T58-phosphoMYC (#9401, Cell Signaling), Caspase-8 (12F5, Biosource), FLIP (NF-6, Axxora), FBW7 (MoBiTec), GSK3 (Stressgen), Cyclin E (HE12, Pharmingen) and DR5A (generated at GNF). For in vitro apoptosis assays, DR5-A was crosslinked by incubating with F(Ab')₂ fragment goat anti-mouse anti-Fc (Jackson ImmunoResearch) for lhr at room temperature at a 1 :3 ratio by weight. Western blot analysis was performed as previously described.

siRNA

[00111] All siRNA smartpools (targeting GSK3 α, GSK3 β, Cyclin E, MYC) were purchased from Dharmacon. Individual siRNAs used were purchased from Qiagen: siGSK3α (GTG ATT GGC AAT GGC TCA T), siGSK3β -1 (GTA TTG CAG GAC AAG AGA T), siGSK3β-S (GC AAA TCA GAG AAA TGA AC), siFBW7-2 (GGG CAA CAA CGA CGC CGA A), siFBW7-3 (AAG GCA CTC TAT GTG CTT TCA), siMYC (CAC GTC TCC ACA CAT CAG CAC AA); control siRNA siGL3 (directed against the luciferase gene from vector pGL3) AAC TTA CGC TGA GTA CTT CGA TT.

[00112] Cells were transfected with 20ng per well in 96-well plates or 600ng per well in 6-well plates using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions and treated with DR5A or prepared for FACS analysis, immunoblotting or semiquantitative RT-PCR 48 hr after transfection.

Semiquantitative RT-PCR [00113] Total RNA was extracted using RNeasy Mini Kit (Qiagen). 1 μg RNA was reverse-transcribed using ThermalScript System (Invitrogen). lμl cDNA was then used for PCR amplification using the following primers: FBW7_forward (AAG TTG GAC CAT GGT TCT GAG), FBW7_reverse (A CAC AGC GGA CTG CTG CAA C), DR5R_forward (ATG GAA CAA CGG GGA CAG AAC), and DR5R_reverse (TTA GGA CAT GGC AGA GTC TGC).

FACS

[00114] Cells were siRNA transfected as above and 48 hr posttransfection were harvested with Accutase, pelleted, incubated with 10 μg/ml DR5A (mouse antibody) in Hank's buffer (supplemented with 0.1% 0.5 M EDTA, 0.1% IM Hepes, 1% PBS and 0.1% sodium azide) for 90 min at 4°C, washed two times, incubated with 1:1000 diluted APC (allophycocyanin crosslinked goat anti-mouse secondary antibody (Molecular Probes) for 30 min, washed again, and analyzed by FACS.

Cell death assays

[00115] Eight to ten thousand cells per well were plated in 96-well plates in the appropriate cell culture medium, incubated with LiCl/ KCl for 8hr or transfected with siRNAs, and treated with DR5A 48 hr post-transfection for an additional 20 hr. Cell viability was measured in triplicate by CellTiterGlo (Promega) according to the manufacturer's instructions.

Rescue experiments

[00116] HTl 15 cells were transfected with pCMV6-FBW7, coding for full-length

FBW7γ, using Fugene 6 transfection reagent (Roche). pBABEpuro was cotransfected at a 1 :4 ratio by weight allowing for selection with 2 μg/ml puromycin. Stable cells were plated in 96-well plates to attach overnight, treated with DR5-A for 20 h and the cell viability assay was performed subsequently. Animal experiments

[00117] 3 x 10⁶ HCTl 16 FBW7+/+ or FBW7+/- cells were implanted by subcutaneous injection into female, 8 week old Balb/c nude mice using a 27g needle. For each group, tumors formed in 8/8 animals and were measured with calipers in 3 dimensions and volumes were calculated using the formula (L x W x H)/2. When tumors reached a mean volume of 80 mm³, mice were rank-ordered by tumor volume with the smallest and largest tumor excluded, and then divided into even-rank and odd-rank groups for IP injection with 100 μg DR5-A or vehicle control (50 mM sodium citrate, pH 7.0; 140 mM NaCl). Mice were treated every other day for a total of seven times. All animal husbandry and experiments were performed in accordance with protocols approved by the IACUC committee of the Genomics Institute of the Novartis Research Foundation.

[00118] Although the invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[00119] All publications, databases, Geribank sequences, patents, and patent applications cited in this specification are herein incorporated by reference as if each was specifically and individually indicated to be incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A method of inducing apoptosis in a cancer cell, the method comprising: contacting a MYC-expressing and DR5 -expressing cancer cell with: (i) a DR5 agonist; and

(ii) a glycogen synthase kinase-3β (GSK3β) antagonist or a hCDC4 antagonist.

2. The method of claim 1, wherein the cancer cell is in an animal and the contacting step comprises administering the agonist (i) and antagonist (ii) to the animal.

3. The method of claim 2, wherein the animal is a human.

4. The method of claim 2, wherein the animal is not a human.

5. The method of claim 1, wherein the contacting step comprises contacting the cell with a GSK3β antagonist.

6. The method of claim 1, wherein the contacting step comprises contacting the cell with a hCDC4 antagonist.

7. The method of claim 1 , wherein the DR5 agonist is an antibody.

8. The method of claim 1 , wherein the DR5 agonist is TRAIL.

9. A composition comprising a therapeutically-effective dose of: (i) a DR5 agonist; and

(ii) a glycogen synthase kinase-3β (GSK3β) antagonist or hCDC4 antagonist.

10. The composition of claim 9, wherein the composition comprises the GSK3β antagonist.

11. The composition of claim 9, wherein the composition comprises the hCDC4 antagonist.

12. The composition of claim 9, wherein the DR5 agonist is an antibody.

13. The composition of claim 9, wherein the DR5 agonist is TRAIL.

14. A method of identifying an agent for inducing apoptosis in cancer cells, the method comprising:

(i) contacting one or more agents to a polypeptide comprising a phosphodegron-binding fragment of a hCDC4 polypeptide, wherein the phosphodegron comprises LPTPP (SEQ ID NO:2), wherein the threonine in SEQ ID NO:2 is phosphorylated;

(ii) selecting one or more agents that inhibits the polypeptide binding of the phosphodegron, thereby identifying an agent that induces apoptosis in a cancer cell.

15. The method of claim 14, wherein the phosphodegron comprises KKFELLPTPPLSPSRR (SEQ ID NO: 1).

16. The method of claim 14, wherein the method further comprises: (iii) contacting the selected one or more agents to cancer cells that expresses MYC in the presence of a DR5 agonist; and

(iv) selecting an agent that induces more apoptosis in the cancer cells than when the cells are contacted with the DR5 agonist in the absence of the agent.

17. The method of claim 14, wherein the polypeptide is linked to a solid support.

18. The method of claim 14, wherein the polypeptide is associated with a first fluorescent label and the phosphodegron is associated with a second fluorescent label and the first and second labels interact to produce a fluorescent signal when the polypeptide binds the phosphodegron, and wherein a reduction of the fluorescent signal in the presence of an agent indicates that the agent inhibits binding of the polypeptide to the phosphodegron.

19. The method of claim 14, wherein the first fluorescent label is

Europium and the first fluorescent label is linked to an antibody that binds to the polypeptide; and the second fluorescent label is selected from the group consisting of Allophycocyanin (APC) and C-Phycocyanin (CPC).