CA2772049A1 - Wd repeat 79 protein targeted therapy - Google Patents

Abstract

Methods and compositions are disclosed for inhibiting cell proliferation and diagnosing and treating cancer comprising antagonists or inhibitors of the WDR79 protein. Also disclosed are methods for screening compounds for WDR79 inhibitory or antagonist activity.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit under 35 USC 119(e) to United States Provisional Patent Application 61/230,453 filed July 31, 2009, the entire contents of which are incorporated by reference herein.

FIELD OF THE INVENTION

[0002] The present disclosure relates to methods and compositions for treatment or diagnosis of cancer by targeting the WD repeat protein 79.

BACKGROUND OF THE INVENTION

[0003] Normal cell growth is controlled by a balance of positive and negative regulatory factors. Growth-inducing genes that have been well characterized are those encoded by so-called oncogenes, whereas growth-inhibiting genes are generally termed as tumor suppressors. Aberrant behavior of any of these oncogenic or growth suppressive activities may lead to uncontrolled growth of cells, a hallmark of cancer. Cancer, or tumorogenesis, is intimately associated with the genetic activation of oncogenes and/or genetic inactivation of tumor suppressor genes. The discovery and understanding of the molecular mechanisms of, and the balance between, oncogenes and tumor suppressors provides targets for the development of new methods of cancer therapy, prevention and diagnosis.

[0004] The WD-repeat 79 (WDR79; Telomerase Cajal body protein 1, WD repeats-containing protein encoding RNA antisense to p53) protein is a novel member of a family of proteins that function as adaptors in cellular organization and signal transduction. WD
repeat proteins are defined by the presence of four or more repeating units containing a conserved core of approximately 40 amino acids that usually ends with tryptophan-aspartic acid (WD) and belong to a large and fast-expanding conservative protein family. The WD-repeat domains bear a C-terminal tryptophan-aspartic acid dipeptide and fold into 13-propellers mediating protein complex formation. Each WD repeat is part of one of the four or more anti-parallel strands that form one of the blades of these propeller-like structures. The propeller structure provides extensive surface areas serving as a very rigid platform or scaffold for the assembly of stable protein complexes.

[0005] WD repeat proteins have diverse biological functions including, but not limited to, signal transduction, RNA synthesis and processing, chromatin assembly, vesicular trafficking, cytoskeletal assembly, cell cycle control, and apoptosis. WD-repeat proteins are associated with human diseases including, but not limited to, cancer, lissencephaly

6 PCT/US2010/043986 syndrome, Cockayne syndrome, late-onset sensorineural deafness and triple-A
syndrome (Allgrove syndorme). WD proteins associated with cancer include those involved in tumor promotion, such as Rack1 (WD-repeat protein receptor for activated C-kinase), WDRPUH
(WD40 repeat protein up-regulated in hepatocellular carcinoma), endonuclein, and STRAP
(serine-threonine kinase receptor-associated protein) and those associated with tumor suppression including FBW7 (F-box and WD repeat domain-containing 7), RbAp46 (retinoblastoma (Rb) suppressor-associated protein 46) and WDR6.

[0006] WD-repeat 79 is a six-repeat member whose homologs bear high sequence identities in yeast to human. WDR79 is encoded by the WRAP53 gene (WD repeat containing, antisense to TP53). It is a scaRNA-binding protein participating in telomerase assembly and Cajal body formation and its antisense transcript affects DNA
damage-induced p53 response. WDR79 has been implicated in human disease in that estrogen receptor-negative breast cancer is associated with its single nucleotide polymorphism.
WDR79 also contains binding motifs for USP7 (ubiquitin specific peptidase 7, also called herpesvirus-associated ubiquitin-specific protease, Hausp).

[0007] WDR79 regulates p53 and Mdm2. p53 (also known as protein 53 or tumor protein 53), is a transcription factor which in humans is encoded by the TP53 gene. p53 is important in multicellular organisms, where it regulates the cell cycle and thus functions as a tumor suppressor that is involved in preventing cancer. As such, p53 has a role in conserving stability by preventing genome mutation. Mdm2 (encoded for by the murine double minute oncogene) and USP7, the two proteins that act as ubiqitin-specific E3 ligase and hydrolase, respectively, serve as key switches to control p53 activation and stability. In unstressed cells, p53 is a low-level, short-lived protein owing to its ubiquitin-dependent proteosomal degradation. In response to diverse stressors such as DNA damage, hypoxia, oncogenic insults and telomere erosion, p53 is activated and stabilized, leading to cell cycle arrest, senescence or apoptosis. In this reciprocal form of control, while Mdm2 depletes p53 through ubiquitination, USP7 binds Mdm2 or p53 via its N-terminal MATH (meprin and TRAF
homology) domain to remove ubiquitins to stabilize the two proteins. The binding affinity of USP7 is several folds higher for Mdm2 than p53, imparting a competitive differential regulatory mechanism. However, little is known of how USP7 itself is regulated and whether other proteins cooperate with USP7 in these physiological processes.

SUMMARY OF THE INVENTION

[0008] Disclosed herein is the discovery of a molecular interaction between and USP7 and its functional significance in linking the Mdm2-p53 pathway to tumor cell proliferation. WDR79 is up-regulated in tumor cell lines and varies in subcellular distribution, but it markedly shuttles to, and accumulates in, the nuclear compartment upon overexpression. In the nucleus, WDR79 colocalizes and interacts with USP7, a molecular relationship that was shown to occur also in vitro. This event in turn reduces the ubiquitination of Mdm2 and p53, thereby increasing the stability and extending the half-life of the two proteins. The effects of WDR79 depended upon USP7, because knockdown of USP7 results in its attenuation. Given the opposed enzymatic activity of Mdm2 versus USP7, the effect of WDR79 on tumor cell growth was investigated demonstrating that promotes tumor cell proliferation. Taken together, these findings reveal a novel role of WDR79 in tumor cell proliferation and pinpoint a new mechanism by which WDR79 and USP7 functionally interact to modulate the Mdm2-p53 antagonism.

[0009] Disclosed herein are methods for identifying an agent that binds to WDR79 or modulates the interaction between WDR79 and USP7.

[0010] Also disclosed are methods for diagnosing cancer in a subject by assaying a biological sample for WDR79 expression wherein elevation of WDR79 expression above a normal level is indicative of the presence of cancer in the subject.

[0011] The present disclosure also includes methods for modulating deubiquitination and/or stability of p53 and/or MDM2.

[0012] In one embodiment disclosed herein, a method of inhibiting tumor cell proliferation is provided comprising exposing a cell population or a tissue to an effective amount of an antagonist or inhibitor of WD-repeat protein 79 (WDR79).

[0013] In another embodiment, the antagonist or inhibitor is a small molecule, a nucleic acid or an antibody. In another embodiment, the nucleic acid is selected from the group consisting of short interfering RNA (sRNA), short hairpin RNA (shRNA), micro-RNA
(miRNA), double-stranded RNA (dsRNA) or anti-sense molecule.

[0014] In one embodiment disclosed herein, a method of screening for anti-proliferative compounds is provided comprising obtaining potential anti-proliferative compounds and determining the effects of said compounds on the expression of WDR79 in a cell. In another embodiment, the cell is a cancer cell.

[0015] In another embodiment, the anti-proliferative compound is a small molecule, a nucleic acid or an antibody. In another embodiment, the nucleic acid is selected from the group consisting of short interfering RNA (sRNA), short hairpin RNA (shRNA), micro-RNA
(miRNA), double-stranded RNA (dsRNA) or anti-sense molecule.

[0016] In one embodiment, a method of modulating the activity of USP7 is provided comprising modulating the levels of WDR79 in a cell or tissue.

[0017] In another embodiment, the modulating the activity of USP7 comprises increasing the activity of USP7. In another embodiment, the modulating the activity of USP7 comprises decreasing the activity of USP7. In yet another embodiment, the modulating the activity of USP7 comprises modulating the activity or expression of WDR79.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 depicts the structure (FIG. 1A), expression (FIG. 113) and normalized levels (FIG. 1C) (n=3) of WDR79 in normal human epidermal melanocytes (NHEM) and tumor cells. FIG. 1A: The antigenic region (Abl-50), WD-repeats and USP7 motifs (U) are labeled in FIG. 1A. Actin shows protein loading levels in FIG. 1 B.

[0019] FIG. 2 depicts the subcellular location of WDR79 in four cell types (FTIC-WDR79, PI-counter stained nuclei and merged images shown).

[0020] FIG. 3 depicts the accumulation of WDR79 in the nucleus upon overexpression.
Images of p53-null H1299 cells and p53wt U2OS cells are presented in FIG. 3A.
FIG. 3B
depicts expression of WDR79, RCC1 and a-tubulin in cytosol extracts (CE) and nuclear extracts (NE). FIG. 3C depicts histograms of the NE/CE ratios of WDR79.

[0021] FIG. 4 depicts the colocalization of WDR79 and USP7 in the nucleus (FIG. 4A), association in vivo (FIG. 4B) and binding in vitro (FIG. 4C). FIG. 4A depicts the co-localization of WDR79 with USP7 in the nuclear compartment. Shown are WDR79, USP7, WDR79-USP7 merge and differential interference contrast (DIC) images of H1299 and U20S cells. FIG. 4B depicts that WDR79 and USP7 associate in vivo in three different cell types. The WDR79-USP7 complex was precipitated with anti-flag and detected with anti-USP7. FIG. 4C depicts WDR79 binding to the N-terminal MATH domain of USP71_210 in a GST pull-down assay, confirming the direct interaction.

[0022] FIG. 5 depicts that WDR79 stabilizes Mdm2 and p53 and prolongs their half-life by decreasing ubiquitination in a USP7-dependent manner. FIG. 5A depicts Mdm2 and p53 stabilization via WDR79 in all cell types. Actin shows protein loading levels.
FIG. 5B depicts that WDR79 prolongs the half-life of Mdm2 and p53. Cycloheximide (CHX) treatment (0-90 min) is indicated. FIG. 5C depicts that WDR79 reduces ubiquitination of Mdm2 and p53 in the presence of proteasome inhibitor MG132. Mdm2 (left) or p53 (right) immunoprecipitates (IP) were analyzed, with size markers at left. FIG. 5D depicts that USP7 knockdown attenuates the effect of WDR79 on Mdm2 and p53 in U20S cells. Absence or presence of control shRNA, USP7-shRNA or WDR79 plasmid and the antibody-specificity are indicated.

[0023] FIG. 6 depicts that WDR79 stable expression promotes tumor cell proliferation.
FIG. 6A depicts three different cell types transfected with Flag-WDR79 or a control vector followed by G418 selection. FIG. 6B depicts a model pathway from WDR79-USP7 interaction to growth promotion in which the comparative effects on Mdm2 and p53 are arrow-indicated. WDR79 lies upstream of USP7 and regulates USP7 to promote cell proliferation by balancing Mdm2-p53 antagonism.

[0024] FIG. 7 depicts the evolutionary conservation of WDR79 homolog. FIG. 7A
depicts the alignment of the amino acid sequences of WDR79 proteins from human (Hs_WDR79, SEQ ID NO:1), mouse (Mm_WDR79; SEQ ID NO:2) and zebrafish (Dr WDR79; SEQ ID NO:3). Identical amino acid residues are highlighted in light gray.
Similar residues are highlighted in dark gray. Dashes denote gaps in the sequence alignment. The six genuine WD40 repeats (E value < le-12) are labeled with stars and the 7th putative WD40 repeat with # symbols. The USP7-binding motifs are each indicated by a horizontal bar. No nuclear localization signal was detected. The predicted conserved nuclear export signal (N ES) is underlined. FIG. 7B depicts the phylogenetic tree of the 16 WDR79 homologs from 16 representative species (in abbreviated taxonomic names and retrieved from GenBank). The number at the node indicates the bootstrap value. The scale bar at the bottom denotes 0.2 substitutions per residue.

[0025] FIG. 8 depicts that transient expression of WDR79 markedly enhances its nuclear translocation and accumulation. Human WDR79 full-length cDNA was subcloned in pEGFP-C1 vector and transfected into four different cell types for 48 hours.
Shown from left to right are images of GFP-WDR79, DIC and their merge. In all instances WDR79 is predominantly located in the nuclear compartment.

[0026] FIG. 9 depicts that the nuclear translocation of WDR79 is driven by itself and is independent of whether or not the expression plasmid carries an epitope tag.
Human WDR79 full-length cDNA was subcloned into pCMV-Script vector lacking the epitope tag sequence. This tag-free WDR79 construct was transiently expressed in A375 and cells and the WDR79 protein was detected with dye-conjugated anti-WDR79 antibody.
Shown from left to right are images of FTIC-WDR79, DIC and their merge.

[0027] FIG. 10 depicts the effects of WDR79 on cell cycle progression in A375 cells.
DETAILED DESCRIPTION OF THE INVENTION

[0028] Disclosed herein is a new molecular process in which WDR79 interacts with USP7 to modulate the Mdm2-p53 pathway and promote tumor cell proliferation.
Disclosed herein is that WDR79 is up-regulated in tumor cell lines and, upon overexpression, accumulates in the nuclear compartment where it co-localizes with USP7, forming a complex via their direct physical interaction. This event in turn increases the stability and prolongs the half-life of Mdm2 and p53 via decreasing ubiquitination. These studies further reveal that the effects of WDR79 are dependent upon USP7 and manifest a net outcome as promoting tumor cell proliferation. Accordingly, these findings identify WDR79 as a critical factor acting upstream, whereby it binds and potentiates USP7 to modulate the Mdm2-p53 antagonism.

[0029] Also disclosed is a model that accounts for the function of WDR79 via regulation. WDR79 moves to and accumulates in the nuclear compartment where it interacts with USP7 via direct physical association. This molecular process activates USP7 and potentiates its ability to deubiquitinate Mdm2 and p53, thereby stabilizing both Mdm2 and p53 and extending their half-life. Given the opposite functions between p53 and Mdm2, the WDR79-USP7 interaction may render USP7 more active toward Mdm2 than p53, a dynamic interplay ultimately resulting in cell proliferation (FIG. 6B). This view reinforces the importance of relative effects on the Mdm2-p53 pathway in terms of their antagonism and links the complexity of additional factors to this central and competitive regulation. The role of the WDR79-USP7 interaction in tumor cell proliferation is a novel finding.

[0030] Included within the scope of the present disclosure are insertion, deletion or conservative amino acid substitution variants of WDR79. As used herein, a conservative variant refers to alterations in the amino acid sequence that do not adversely affect the biological functions of the protein. A substitution, insertion or deletion is said to adversely affect the protein when the altered sequence prevents or disrupts a biological function associated with the protein. For example, the overall charge, structure or hydrophobic/hydrophilic properties of the protein, in certain instances, may be altered without adversely affecting a biological activity. Accordingly, the amino acid sequence can be altered, for example to render the peptide more hydrophobic or hydrophilic, without adversely affecting the biological activities of the protein.

[0031] Ordinarily, the allelic variants, the conservative substitution variants, and the members of the protein family, will have an amino acid sequence having at least about 50%, 60%, 70% or 75% amino acid sequence identity with WDR79, more preferably at least about 80-90%, even more preferably at least about 92-94%, and most preferably at least about 95%, 98% or 99% sequence identity. Identity or homology with respect to such sequences is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with WDR79, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity (see section B for the relevant parameters).
Fusion proteins, or N-terminal, C-terminal or internal extensions, deletions, or insertions into the peptide sequence shall not be construed as affecting homology.

[0032] Thus, the proteins disclosed herein include molecules having the amino acid sequence of WDR79; fragments thereof having a consecutive sequence of at least about 3, 4, 5, 6, 10, 15, 20, 25, 30, 35 or more amino acid residues of these proteins;
amino acid sequence variants wherein one or more amino acid residues has been inserted N-or C-terminal to, or within, the disclosed coding sequence; and amino acid sequence variants of the disclosed sequence, or their fragments as defined above, that have been substituted by at least one residue. Such fragments, also referred to as peptides or polypeptides, may contain antigenic regions, functional regions of the protein identified as regions of the amino acid sequence which correspond to known protein domains, as well as regions of pronounced hydrophilicity. The regions are all easily identifiable by using commonly available protein sequence analysis software such as MacVector (Oxford Molecular).

[0033] Contemplated variants further include those containing predetermined mutations by, e.g., homologous recombination, site-directed or PCR mutagenesis, and the corresponding proteins of other animal species, including but not limited to rabbit, mouse, rat, porcine, bovine, ovine, equine and non-human primate species, and the alleles or other naturally occurring variants of the family of proteins; and derivatives wherein the protein has been covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid (for example a detectable moiety such as an enzyme or radioisotope).

[0034] The present disclosure further provides compositions comprising a protein or polypeptide of WDR79 and a diluent. Suitable diluents can be aqueous or non-aqueous solvents or a combination thereof, and can comprise additional components, for example water-soluble salts or glycerol, that contribute to the stability, solubility, activity, and/or storage of the protein or polypeptide.

[0035] As described below, WDR79 proteins can be used: (1) to identify agents which modulate the level of or at least one activity of a protein, (2) to identify binding partners for a protein, (3) as an antigen to raise polyclonal or monoclonal antibodies, (4) as a therapeutic agent or target for cancer and (5) as a diagnostic agent or marker of cancer and other diseases associated with WDR79.

[0036] The present disclosure further provides nucleic acid molecules that encode the protein of WDR79 and the related proteins herein described, preferably in isolated form. As used herein, "nucleic acid" is defined as RNA or DNA that encodes a protein or peptide as defined above, is complementary to a nucleic acid sequence encoding such peptides, hybridizes to the nucleic acid of such proteins or peptides and remains stably bound to it under appropriate stringency conditions, encodes a polypeptide sharing at least about 50%, 60%, 70% or 75%, preferably at least about 80-90%, more preferably at least about 92-94%, and most preferably at least about 95%, 98%, 99% or more identity with the peptide sequence of WDR79 or exhibits at least 50%, 60%, 70% or 75%, preferably at least about 80-90%, more preferably at least about 92-94%, and even more preferably at least about 95%, 98%, 99% or more nucleotide sequence identity over the open reading frames of the WRAP53 gene (GenBank accession numbers AK001247 [SEQ ID NO:4] and DQ431240 [SEQ ID NO:5]).

[0037] The present disclosure further includes isolated nucleic acid molecules that specifically hybridize to the complement of the sequence of WRAP53, particularly molecules that specifically hybridize over the open reading frames. Such molecules that specifically hybridize to the complement of the sequence of WRAP53 typically do so under stringent hybridization conditions.

[0038] Specifically contemplated are genomic DNA, cDNA, mRNA, siRNA and antisense molecules, as well as nucleic acids based on alternative backbones or including alternative bases, whether derived from natural sources or synthesized. Such hybridizing or complementary nucleic acids, however, are defined further as being novel and unobvious over any prior art nucleic acid including that which encodes, hybridizes under appropriate stringency conditions, or is complementary to nucleic acid encoding a protein according to the present disclosure.

[0039] Homology or identity at the nucleotide or amino acid sequence level is determined by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx which are tailored for sequence similarity searching. The approach used by the BLAST program is to first consider similar segments, with and without gaps, between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance. The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter (low complexity) are at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix, recommended for query sequences over 85 nucleotides or amino acids in length.

[0040] For blastn, the scoring matrix is set by the ratios of M (i.e., the reward score for a pair of matching residues) to N (i.e., the penalty score for mismatching residues), wherein the default values for M and N are 5 and -4, respectively. Four blastn parameters were adjusted as follows: Q=10 (gap creation penalty); R=10 (gap extension penalty); wink=1 (generates word hits at every winks" position along the query); and gapw-16 (sets the window width within which gapped alignments are generated). The equivalent Blastp parameter settings were Q=9; R=2; wink=1; and gapw=32. A Bestfit comparison between sequences, available in the GCG package version 10.0, uses DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty) and the equivalent settings in protein comparisons are GAP=8 and LEN=2.

[0041] "Stringent conditions" are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCI/0.0015 M sodium citrate/0.1% SDS at 50 C, or (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.19% Ficoll/0.1 %
polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM
sodium citrate at 42 C. Another example is hybridization in 50% formamide, 5xSSC (0.75 M
NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5xDenhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42 C, with washes at 42 C in 0.2xSSC and 0.1% SDS.
A skilled artisan can readily determine and vary the stringency conditions appropriately to obtain a clear and detectable hybridization signal. Preferred molecules are those that hybridize under the above conditions to the complement of the sequence of WRAP53 and which encode a functional or full-length protein. Even more preferred hybridizing molecules are those that hybridize under the above conditions to the complement strand of the open reading frame of the sequence of WRAP53.

[0042] As used herein, a nucleic acid molecule is said to be "isolated" when the nucleic acid molecule is substantially separated from contaminant nucleic acid molecules encoding other polypeptides.

[0043] The present disclosure further provides fragments of the disclosed nucleic acid molecules. As used herein, a fragment of a nucleic acid molecule refers to a small portion of the coding or non-coding sequence. The size of the fragment will be determined by the intended use. For example, if the fragment is chosen so as to encode an active portion of the protein, the fragment will need to be large enough to encode the functional region(s) of the protein. For instance, fragments which encode peptides corresponding to predicted antigenic regions may be prepared. If the fragment is to be used as a nucleic acid probe or PCR
primer, then the fragment length is chosen so as to obtain a relatively small number of false positives during probing/priming.

[0044] Fragments of the nucleic acid molecules of the present disclosure (i.e., synthetic oligonucleotides) that are used as probes or specific primers for the polymerase chain reaction (PCR), or to synthesize gene sequences encoding proteins, can easily be synthesized by chemical techniques or using automated synthesis methods. In addition, larger DNA segments can readily be prepared by well known methods, such as synthesis of a group of oligonucleotides that define various modular segments of the gene, followed by ligation of oligonucleotides to build the complete modified gene.

[0045] The nucleic acid molecules of the present disclosure may further be modified so as to contain a detectable label for diagnostic and probe purposes. A variety of such labels are known in the art and can readily be employed with the encoding molecules herein described. Suitable labels include, but are not limited to, biotin, radiolabeled or fluorescently labeled nucleotides and the like. A skilled artisan can readily employ any such label to obtain labeled variants of the nucleic acid molecules.

[0046] As described above, the identification and characterization of the nucleic acid molecule having the sequence of WDR79/WRAP53 allows a skilled artisan to isolate nucleic acid molecules that encode other members of the protein family in addition to the sequences herein described. Further, the presently disclosed nucleic acid molecules allow a skilled artisan to isolate nucleic acid molecules that encode other members of the family of proteins in addition to the WDR79 proteins

[0047] For instance, a skilled artisan can readily use the amino acid sequence of WDR79 to generate antibody probes to screen expression libraries prepared from appropriate cells. Typically, polyclonal antiserum from mammals such as rabbits immunized with the purified protein (as described below) or monoclonal antibodies can be used to probe a mammalian cDNA or genomic expression library to obtain the appropriate coding sequence for other members of the protein family. The cloned cDNA sequence can be expressed as a fusion protein, expressed directly using its own control sequences, or expressed by constructions using control sequences appropriate to the particular host used for expression of the enzyme.

[0048] Alternatively, a portion of the coding sequence herein described can be synthesized and used as a probe to retrieve DNA encoding a member of the protein family from any mammalian organism. Oligomers containing approximately 18-20 nucleotides (encoding about a 6-7 amino acid stretch) are prepared and used to screen genomic DNA or cDNA libraries to obtain hybridization under stringent conditions or conditions of sufficient stringency to eliminate an undue level of false positives.

[0049] Additionally, pairs of oligonucleotide primers can be prepared for use in PCR to selectively clone an encoding nucleic acid molecule. A PCR
denature/anneal/extend cycle for using such PCR primers is well known in the art and can readily be adapted for use in isolating other encoding nucleic acid molecules.

[0050] Nucleic acid molecules encoding proteins related to WDR79 may also be identified in existing genomic or other sequence information using any available computational method, including but not limited to: PSI-BLAST; PHI-BLAST, 3D-PSSM; and other computational analysis methods.

[0051] The present disclosure further provides recombinant DNA molecules (rDNAs) that contain a coding sequence. As used herein, a rDNA molecule is a DNA
molecule that has been subjected to molecular manipulation in situ. Methods for generating rDNA
molecules are well known in the art. In exemplary rDNA molecules, a coding DNA
sequence is operably linked to expression control sequences and/or vector sequences.

[0052] The choice of vector and/or expression control sequences to which one of the disclosed protein family encoding sequences is operably linked depends directly, as is well known in the art, on the functional properties desired, e.g., protein expression, and the host cell to be transformed. A vector is at least capable of directing the replication or insertion into the host chromosome, and preferably also expression, of the structural gene included in the rDNA molecule.

[0053] Expression control elements that are used for regulating the expression of an operably linked protein encoding sequence are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, and other regulatory elements. Preferably, the inducible promoter is readily controlled, such as being responsive to a nutrient in the host cell's medium.

[0054] In one embodiment, the vector containing a coding nucleic acid molecule will include a prokaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
Such replicons are well known in the art. In addition, vectors that include a prokaryotic replicon may also include a gene whose expression confers a detectable marker such as a drug resistance.
Typical bacterial drug resistance genes are those that confer resistance to ampicillin, kanamycin, chloramphenicol or tetracycline.

[0055] Vectors that include a prokaryotic replicon can further include a prokaryotic or bacteriophage promoter capable of directing the expression (transcription and translation) of the coding gene sequences in a bacterial host cell, such as E coli. A promoter is an expression control element formed by a DNA sequence that permits binding of RNA

polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment. Typical of such vector plasmids are pUC8, pUC9, pBR322 and pBR329 available from BioRad Laboratories, (Richmond, CA), pPL and pKK223 available from Pharmacia (Piscataway, NJ).

[0056] Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can also be used to form rDNA molecules that contain a coding sequence. Eukaryotic cell expression vectors, including viral vectors, are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired DNA
segment. Typical of such vectors are pSVL and pKSV-10, pBPV-1/pML2d, pTDT1 (ATCC, #31255), pCDM8, and the like eukaryotic expression vectors. Vectors may be modified to include tissue specific promoters if needed.

[0057] Eukaryotic cell expression vectors used to construct the rDNA molecules may further include a selectable marker that is effective in a eukaryotic cell, preferably a drug resistance selection marker. One drug resistance marker is the gene whose expression results in neomycin resistance, i.e., the neomycin phosphotransferase (neo) gene.
Alternatively, the selectable marker can be present on a separate plasmid, and the two vectors are introduced by co-transfection of the host cell, and selected by culturing in the appropriate drug for the selectable marker.

[0058] The present disclosure further provides host cells transformed with a nucleic acid molecule that encodes the WDR79 or related protein. The host cell can be either prokaryotic or eukaryotic. Eukaryotic cells useful for expression of a protein are not limited, so long as the cell line is compatible with cell culture methods and compatible with the propagation of the expression vector and expression of the gene product.
Exemplary eukaryotic host cells include, but are not limited to, yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human cell line.
Exemplary eukaryotic host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells (NIH/3T3) available from the ATCC as CRL 1658, baby hamster kidney cells (BHK), and the like eukaryotic tissue culture cell lines.

[0059] Any prokaryotic host can be used to express a rDNA molecule encoding a disclosed protein. An exemplary prokaryotic host is E. coli.

[0060] Transformation of appropriate cell hosts with an rDNA molecule of the present disclosure is accomplished by well known methods that typically depend on the type of vector used and host system employed. With regard to transformation of prokaryotic host cells, electroporation and salt treatment methods are typically employed. With regard to transformation of vertebrate cells with vectors containing rDNAs, electroporation, cationic lipid or salt treatment methods are typically employed.

[0061] Successfully transformed cells, i.e., cells that contain a rDNA
molecule, can be identified by well known techniques including the selection for a selectable marker. For example, cells resulting from the introduction of an rDNA can be cloned to produce single colonies. Cells from those colonies can be harvested, lysed and their DNA
content examined for the presence of the rDNA or the proteins produced from the cell assayed via an immunological method.

[0062] Further provided herein are methods for producing a WDR79 protein using nucleic acid molecules herein described. In general terms, the production of a recombinant form of a protein typically involves the following steps:

[0063] First, a nucleic acid molecule is obtained that encodes a protein of WDR79, such as a nucleic acid molecule comprising, consisting essentially of, or consisting of, WRAP53. If the encoding sequence is uninterrupted by introns, as are these open-reading-frames, it is directly suitable for expression in any host.

[0064] The nucleic acid molecule is then preferably placed in operable linkage with suitable control sequences, as described above, to form an expression unit containing the protein open reading frame. The expression unit is used to transform a suitable host and the transformed host is cultured under conditions that allow the production of the recombinant protein. Optionally the recombinant protein is isolated from the medium or from the cells;
recovery and purification of the protein may not be necessary in some instances where some impurities may be tolerated.

[0065] Each of the foregoing steps can be done in a variety of ways. For example, the desired coding sequences may be obtained from genomic fragments and used directly in appropriate hosts. The construction of expression vectors that are operable in a variety of hosts is accomplished using appropriate replicons and control sequences, as set forth above. The control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene and were discussed in detail earlier. Suitable restriction sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors. A skilled artisan can readily adapt any host/expression system known in the art for use with nucleic acid molecules to produce recombinant protein.

[0066] Another embodiment provides methods for identifying agents that modulate the expression of a nucleic acid encoding a WDR79 protein. Such assays may utilize any available means of monitoring for changes in the expression level of the nucleic acids. As used herein, an agent is said to modulate the expression of a nucleic acid if it is capable of up- or down-regulating expression of the nucleic acid in a cell.

[0067] In one assay format, cell lines that contain reporter gene fusions between nucleotides from within the open reading frame defined by WDR79/WRAP53 and/or the 5' and/or 3' regulatory elements and any assayable fusion partner may be prepared. Numerous assayable fusion partners are known and readily available including the firefly luciferase gene and the gene encoding chloramphenicol acetyltransferase. Cell lines containing the reporter gene fusions are then exposed to the agent to be tested under appropriate conditions and time. Differential expression of the reporter gene between samples exposed to the agent and control samples identifies agents which modulate the expression of a nucleic acid of the WDR79/WRAP53 gene.

[0068] Additional assay formats may be used to monitor the ability of the agent to modulate the expression of a nucleic acid encoding a WDR79 protein. For instance, mRNA
expression may be monitored directly by hybridization to the nucleic acids.
Cell lines are exposed to the agent to be tested under appropriate conditions and time and total RNA or mRNA is isolated by standard procedures.

[0069] In one embodiment, the cells are derived from human tissue, for instance, biopsy tissue or cultured cells from patients with cancer. Cell lines from tissue such as, but not limited to, breast, colon, lung, ovary, prostate, stomach, intestine, liver, skin, muscle, kidney, bladder, brain, bone, etc. may be used. Alternatively, other available cells or cell lines may be used.

[0070] Probes to detect differences in RNA expression levels between cells exposed to the agent and control cells may be prepared from the nucleic acids related to WDR79 or WRAP53. It is preferable, but not necessary, to design probes which hybridize only with target nucleic acids under conditions of high stringency. Only highly complementary nucleic acid hybrids form under conditions of high stringency. Accordingly, the stringency of the assay conditions determines the amount of complementarity which should exist between two nucleic acid strands in order to form a hybrid. Stringency should be chosen to maximize the difference in stability between the probe:target hybrid and probe: non-target hybrids.

[0071] Probes may be designed from the nucleic acids through methods known in the art. For instance, the G+C content of the probe and the probe length can affect probe binding to its target sequence. Methods to optimize probe specificity are commonly available.

[0072] Hybridization conditions are modified using known methods as required for each probe. Hybridization of total cellular RNA or RNA enriched for polyA RNA can be accomplished in any available format. For instance, total cellular RNA or RNA
enriched for polyA RNA can be affixed to a solid support and the solid support exposed to at least one probe comprising at least one, or part of one of the sequences under conditions in which the probe will specifically hybridize. Alternatively, nucleic acid fragments comprising at least one, or part of one of the sequences can be affixed to a solid support, such as a silicon chip, porous glass wafer or membrane. The solid support can then be exposed to total cellular RNA or polyA RNA from a sample under conditions in which the affixed sequences will specifically hybridize. Such solid supports and hybridization methods are widely available.
By examining for the ability of a given probe to specifically hybridize to an RNA sample from an untreated cell population and from a cell population exposed to the agent, agents which up- or down-regulate the expression of a nucleic acid encoding the WDR79 protein are identified.

[0073] Hybridization for qualitative and quantitative analysis of mRNAs may also be carried out by using, for example, a RNase Protection Assay. Briefly, an expression vehicle comprising cDNA encoding the gene product and a phage specific DNA dependent RNA
polymerase promoter (e.g., T7, T3 or SP6 RNA polymerase) is linearized at the 3' end of the cDNA molecule, downstream from the phage promoter, wherein such a linearized molecule is subsequently used as a template for synthesis of a labeled antisense transcript of the cDNA by in vitro transcription. The labeled transcript is then hybridized to a mixture of isolated RNA (i.e., total or fractionated mRNA) by incubation at 45 C
overnight in a buffer comprising 80% formamide, 40 mM Pipes, pH 6.4, 0.4 M NaCl and 1 mM EDTA. The resulting hybrids are then digested in a buffer comprising 40 .tg/ml ribonuclease A and 2 g/ml ribonuclease. After deactivation and extraction of extraneous proteins, the samples are loaded onto urea/polyacrylamide gels for analysis.

[0074] In another assay, to identify agents which affect the expression of the instant gene products, cells or cell lines are first identified which express the gene products physiologically. Cells and/or cell lines so identified would be expected to comprise the necessary cellular machinery such that the fidelity of modulation of the transcriptional apparatus is maintained with regard to exogenous contact of agent with appropriate surface transduction mechanisms and/or the cytosolic cascades. Further, such cells or cell lines would be transduced or transfected with an expression vehicle (e.g., a plasmid or viral vector) construct comprising an operable non-translated 5' promoter-containing end of the structural gene encoding the instant gene products fused to one or more antigenic fragments, which are peculiar to the instant gene products, wherein the fragments are under the transcriptional control of the promoter and are expressed as polypeptides whose molecular weight can be distinguished from the naturally occurring polypeptides or may further comprise an immunologically distinct tag or other detectable marker.
Such a process is well known in the art

[0075] Cells or cell lines transduced or transfected as outlined above are then contacted with agents under appropriate conditions. For example, the agent in a pharmaceutically acceptable excipient is contacted with cells in an aqueous physiological buffer such as phosphate buffered saline (PBS) at physiological pH, Eagles balanced salt solution (BSS) at physiological pH, PBS or BSS comprising serum or conditioned media comprising PBS or BSS and/or serum incubated at 37 C. The conditions may be modulated as deemed necessary by one of skill in the art. Subsequent to contacting the cells with the agent, said cells will be disrupted and the polypeptides of the lysate are fractionated such that a polypeptide fraction is pooled and contacted with an antibody to be further processed by immunological assay (e.g., ELISA, immunoprecipitation or Western blot). The pool of proteins isolated from the "agent-contacted" sample will be compared with a control sample where only the excipient is contacted with the cells and an increase or decrease in the immunologically generated signal from the "agent-contacted" sample compared to the control will be used to distinguish the effectiveness of the agent.

[0076] Another embodiment provides methods for identifying agents that modulate the level or at least one activity of a protein, such as the protein having the amino acid sequence of WDR79. Such methods or assays may utilize any means of monitoring or detecting the desired activity and are particularly useful for identifying agents that treat cancer.

[0077] In one format, the relative amounts of a protein between a cell population that has been exposed to the agent to be tested, compared to an un-exposed control cell population may be assayed. In this format, probes such as specific antibodies are used to monitor the differential expression of the protein in the different cell populations. Cell lines or populations are exposed to the agent to be tested under appropriate conditions and time.
Cellular lysates may be prepared from the exposed cell line or population and a control, unexposed cell line or population. The cellular lysates are then analyzed with the probe.

[0078] Antibody probes are prepared by immunizing suitable mammalian hosts in appropriate immunization protocols using the peptides, polypeptides or proteins disclosed herein if they are of sufficient length, or, if desired, or if required to enhance immunogenicity, conjugated to suitable carriers. Methods for preparing immunogenic conjugates with carriers such as BSA, KLH, or other carrier proteins are well known in the art. In some circumstances, direct conjugation using, for example, carbodiimide reagents may be effective; in other instances linking reagents such as those supplied by Pierce Chemical Co.
(Rockford, IL), may be desirable to provide accessibility to the hapten. The hapten peptides can be extended at either the amino or carboxy terminus with a cysteine residue or interspersed with cysteine residues, for example, to facilitate linking to a carrier.
Administration of the immunogens is conducted generally by injection over a suitable time period and with use of suitable adjuvants, as is generally understood in the art. During the immunization schedule, titers of antibodies are taken to determine adequacy of antibody formation.

[0079] While the polyclonal antisera produced in this way may be satisfactory for some applications, for pharmaceutical compositions, use of monoclonal preparations is preferred.
Immortalized cell lines which secrete the desired monoclonal antibodies may be prepared using the standard methods or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known. The immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is the peptide hapten, polypeptide or protein. When the appropriate immortalized cell culture secreting the desired antibody is identified, the cells can be cultured either in vitro or by production in ascites fluid.

[0080] The desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Fragments of the monoclonal antibodies or the polyclonal antisera which contain the immunologically significant (antigen-binding) portion can be used as antagonists, as well as the intact antibodies. Use of immunologically reactive (antigen-binding) antibody fragments, such as the Fab, Fab', or F(ab')2 fragments is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin.

[0081] The antibodies or antigen-binding fragments may also be produced, using current technology, by recombinant means. Antibody regions that bind specifically to the desired regions of the protein can also be produced in the context of chimeras with multiple species origin, such as humanized antibodies.

[0082] Agents that are assayed in the above method can be randomly selected or rationally selected or designed. As used herein, an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences involved in the association of a protein alone or with its associated substrates, binding partners, etc. An example of randomly selected agents is the use a chemical library or a peptide combinatorial library, or a growth broth of an organism.

[0083] As used herein, an agent is said to be rationally selected or designed when the agent is chosen on a nonrandom basis which takes into account the sequence of the target site and/or its conformation in connection with the agent's action. Agents can be rationally selected or rationally designed by utilizing the peptide sequences that make up these sites.
For example, a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to or a derivative of any functional consensus site.

[0084] The agents of the present disclosure can be, as examples, peptides, small molecules, vitamin derivatives, as well as carbohydrates. Dominant negative proteins, DNAs encoding these proteins, antibodies to these proteins, peptide fragments of these proteins or mimics of these proteins may be introduced into cells to affect function.
"Mimic" used herein refers to the modification of a region or several regions of a peptide molecule to provide a structure chemically different from the parent peptide but topographically and functionally similar to the parent peptide. A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present disclosure.

[0085] The peptide agents disclosed herein can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art.
In addition, the DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. The production using solid phase peptide synthesis is necessitated if non-gene-encoded amino acids are to be included.

[0086] Another class of agents disclosed herein are antibodies immunoreactive with critical positions of WDR79proteins or peptides. Antibody agents are obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of the protein intended to be targeted by the antibodies.

[0087] As provided herein, the proteins and nucleic acids related to WDR79 and WRAP53 are differentially expressed between normal and cancerous cells. Agents that up-or down-regulate or modulate the expression of the protein or at least one activity of the protein, such as agonists or antagonists, may be used to modulate biological and pathologic processes associated with the protein's function and activity. This includes agents identified employing homologues and analogues.

[0088] As used herein, a subject can be any mammal, so long as the mammal is in need of modulation of a pathological or biological process mediated by a protein or nucleic acid disclosed herein. The term "mammal" is defined as an individual belonging to the class Mammalia.

[0089] Pathological processes refer to a category of biological processes which produce a deleterious effect. For example, expression of a protein may be associated with cell growth or hyperplasia. As used herein, an agent is said to modulate a pathological process when the agent reduces the degree or severity of the process. For instance, cancer may be prevented or disease progression modulated by the administration of agents which up- or down-regulate or modulate in some way the expression or at least one activity of a protein disclosed herein.

[0090] The agents can be provided alone, or in combination with other agents that modulate a particular pathological process. For example, an agent can be administered in combination with other known drugs. As used herein, two agents are said to be administered in combination when the two agents are administered simultaneously or are administered independently in a fashion such that the agents will act at the same time or in some way potentiate each other's activity, or both contribute to amelioration of the disease process.

[0091] The agents can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

[0092] The present disclosure further provides compositions containing one or more agents which modulate expression or at least one activity of a protein or gene. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typical dosages comprise 0.1 to 100 g/kg body wt.
The preferred dosages comprise 0.1 to 10 g/kg body wt. The most preferred dosages comprise 0.1 to 1 g/kg body wt.

[0093] In addition to the pharmacologically active agent, the compositions disclosed herein may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action. Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts. In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered.
Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension and include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for delivery into the cell.

[0094] The pharmaceutical formulation for systemic administration may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient.

[0095] Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.

[0096] In practicing the methods disclosed herein, the compositions may be used alone or in combination, or in combination with other therapeutic or diagnostic agents. In certain embodiments, the compositions may be coadministered along with other compositions typically prescribed for these conditions according to generally accepted medical practice.
The compositions can be utilized in vivo, ordinarily in mammals, such as humans, sheep, horses, cattle, pigs, dogs, cats, rats and mice, or in vitro.

[0097] Another embodiment provides methods for isolating and identifying binding partners of WDR79 proteins. In general, a disclosed protein is mixed with a potential binding partner or an extract or fraction of a cell under conditions that allow the association of potential binding partners with the protein. After mixing, peptides, polypeptides, proteins or other molecules that have become associated with a protein are separated from the mixture.
The binding partner that bound to the protein can then be removed and further analyzed. To identify and isolate a binding partner, the entire protein, for instance a protein comprising the entire amino acid sequence of the protein can be used. Alternatively, a fragment of the protein can be used.

[0098] As used herein, a cellular extract refers to a preparation or fraction which is made from a lysed or disrupted cell. The preferred source of cellular extracts will be cells derived from human tumors or transformed cells, for instance, biopsy tissue or tissue culture cells from carcinomas. Alternatively, cellular extracts may be prepared from normal tissue or available cell lines.

[0099] A variety of methods can be used to obtain an extract of a cell. Cells can be disrupted using either physical or chemical disruption methods. Examples of physical disruption methods include, but are not limited to, sonication and mechanical shearing.

Examples of chemical lysis methods include, but are not limited to, detergent lysis and enzyme lysis. A skilled artisan can readily adapt methods for preparing cellular extracts in order to obtain extracts for use in the present methods.

[0100] Once an extract of a cell is prepared, the extract is mixed with the protein under conditions in which association of the protein with the binding partner can occur. A variety of conditions can be used, the most preferred being conditions that closely resemble conditions found in the cytoplasm of a human cell. Features such as osmolarity, pH, temperature, and the concentration of cellular extract used, can be varied to optimize the association of the protein with the binding partner.

[0101] After mixing under appropriate conditions, the bound complex is separated from the mixture. A variety of techniques can be utilized to separate the mixture.
For example, antibodies specific to a protein can be used to immunoprecipitate the binding partner complex. Alternatively, standard chemical separation techniques such as chromatography and density/sediment centrifugation can be used. After removal of non-associated cellular constituents found in the extract, the binding partner can be dissociated from the complex using conventional methods. For example, dissociation can be accomplished by altering the salt concentration or pH of the mixture.

[0102] To aid in separating associated binding partner pairs from the mixed extract, the protein can be immobilized on a solid support. For example, the protein can be attached to a nitrocellulose matrix or acrylic beads. Attachment of the protein to a solid support aids in separating peptide/binding partner pairs from other constituents found in the extract. The identified binding partners can be either a single protein or a complex made up of two or more proteins. Alternatively, binding partners may be identified using a Far-Western assay or identified through the use of epitope tagged proteins or GST fusion proteins.

[0103] Alternatively, the nucleic acid molecules disclosed herein can be used in a yeast two-hybrid system or other in vivo protein-protein detection system. The yeast two-hybrid system has been used to identify other protein partner pairs and can readily be adapted to employ the nucleic acid molecules herein described.

[0104] Once isolated, the binding partners of the disclosed proteins, and homologues and analogues thereof, obtained using the above described methods can be used for a variety of purposes. The binding partners can be used to generate antibodies that bind to the binding partner using techniques known in the art. Antibodies that bind the binding partner can be used to assay the activity of the protein, as a therapeutic agent to modulate a biological or pathological process mediated by the protein, or to purify the binding partner.
These uses are described in detail below.

[0105] Another embodiment provides methods for identifying agents that reduce or block the association of a protein with a binding partner. Specifically, a protein is mixed with a binding partner in the presence and absence of an agent to be tested. After mixing under conditions that allow association of the proteins, the two mixtures are analyzed and compared to determine if the agent reduced or blocked the association of the protein with the binding partner. Agents that block or reduce the association of the protein with the binding partner will be identified as decreasing the amount of association present in the sample containing the tested agent.

[0106] As used herein, an agent is said to reduce or block the association between a protein and a binding partner when the presence of the agent decreases the extent to which, or prevents, the binding partner from becoming associated with the protein.
One class of agents will reduce or block the association by binding to the binding partner while another class of agents will reduce or block the association by binding to the protein.

[0107] The binding partner used in the above assay can either be an isolated and fully characterized protein or can be a partially characterized protein that binds to the protein or a binding partner that has been identified as being present in a cellular extract. It will be apparent to one of ordinary skill in the art that so long as the binding partner has been characterized by an identifiable property, e.g., molecular weight, the present assay can be used.

[0108] Agents that are assayed in the above method can be randomly selected or rationally selected or designed. As used herein, an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences involved in the association of the protein with the binding partner. An example of randomly selected agents is the use of a chemical library or a peptide combinatorial library, or a growth broth of an organism.

[0109] As used herein, an agent is said to be rationally selected or designed when the agent is chosen on a nonrandom basis which takes into account the sequence of the target site and/or its conformation in connection with the agent's action. Agents can be rationally selected or rationally designed by utilizing the peptide sequences that make up the contact sites of the binding partner with the protein. For example, a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to the contact site of the protein on the binding partner. Such an agent will reduce or block the association of the protein with the binding partner by binding to the binding partner.

[0110] The agents can be, as examples, peptides, small molecules, vitamin derivatives, as well as carbohydrates. A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents.

[0111] One class of agents are peptide agents whose amino acid sequences are chosen based on the amino acid sequence of the WDR79 protein. The peptide agents can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art. In addition, the DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. The production using solid phase peptide synthesis is necessitated if non-gene encoded amino acids are to be included.

[0112] Another class of agents are antibodies immunoreactive with critical positions of the protein or the binding partner. As described above, antibodies are obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of the protein or the binding partner, intended to be targeted by the antibodies. Critical regions include the contact sites involved in the association of the protein with the binding partner.

[0113] As discussed below, the important minimal sequence of residues involved in activity of the disclosed proteins define a functional linear domain that can be effectively used as a bait for two hybrid screening and identification of potential associated molecules.
Use of such fragments will significantly increase the specificity of the screening as opposed to using the full-length molecule and is therefore preferred. Similarly, this linear sequence can be also used as an affinity matrix also to isolate binding proteins using a biochemical affinity purification strategy.

[0114] Further disclosed herein are compositions containing one or more agents that block association of a WDR79 protein with a binding partner. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typical dosages comprise 0.1 to 100 .tg/kg body wt, 0.1 to 10 .tg/kg body wt or 0.1 to 1 .tg/kg body wt.

[0115] The present disclosure further encompasses rational drug design and combinatorial chemistry. Those of skill will recognize appropriate methods to utilize and exploit aspects of the present proteins in identifying compounds which can be developed for cancer treatment. Rational drug design involving polypeptides requires identifying and defining a first peptide with which the designed drug is to interact, and using the first target peptide to define the requirements for a second peptide. With such requirements defined, one can find or prepare an appropriate peptide or non-peptide that meets all or substantially all of the defined requirements. Thus, one goal of rational drug design is to produce structural or functional analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g., agonists, antagonists, null compounds) in order to fashion drugs that are, for example, more or less potent forms of the ligand.
Combinatorial chemistry is the science of synthesizing and testing compounds for bioactivity en masse, instead of one by one, the aim being to discover drugs and materials more quickly and inexpensively than was formerly possible. Rational drug design and combinatorial chemistry have become more intimately related in recent years due to the development of approaches in computer-aided protein modeling and drug discovery.

[0116] The use of molecular modeling as a tool for rational drug design and combinatorial chemistry has dramatically increased due to the advent of computer graphics.
Not only is it possible to view molecules on computer screens in three dimensions but it is also possible to examine the interactions of macromolecules such as enzymes and receptors and rationally designed derivative molecules to test. A vast amount of user-friendly software and hardware is now available and virtually all pharmaceutical companies have computer modeling groups devoted to rational drug design.

[0117] In another embodiment, genetic therapy can be used as a means for modulating biological and pathologic processes associated with the protein's function and activity. This comprises inserting into a cancerous cell a gene construct encoding a protein comprising all or at least a portion of the sequences of WDR79, or alternatively a gene construct comprising all or a portion of the non-coding region of WRAP53, operably linked to a promoter or enhancer element such that expression of said protein causes suppression of said cancer and wherein said promoter or enhancer element is a promoter or enhancer element modulating said gene construct.

[0118] In the constructs described, expression of said protein can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element. For example, if desired, enhancers known to preferentially direct gene expression in neural cells, T cells, or B cells may be used to direct the expression. The enhancers used could include, without limitation, those that are characterized as tissue or cell specific in their expression. Alternatively, if a genomic clone is used as a therapeutic construct (for example, following its isolation by hybridization with the nucleic acid molecule described above), regulation may be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.

[0119] Insertion of the construct into a cancerous cell is accomplished in vivo, for example using a viral or plasmid vector. Such methods can also be applied to in vitro uses.
Thus, these methods are readily applicable to different forms of gene therapy, either where cells are genetically modified ex vivo and then administered to a host or where the gene modification is conducted in vivo using any of a number of suitable methods involving vectors especially suitable to such therapies.

[0120] Retroviral vectors, adenoviral vectors, adeno-associated viral vectors, or other viral vectors with the appropriate tropism for cells likely to be involved in cancer may be used as a gene transfer delivery system for a therapeutic gene construct. Numerous vectors useful for this purpose are generally known. Retroviral vectors are particularly well developed and have been used in clinical settings. Non-viral approaches may also be employed for the introduction of therapeutic DNA into cells otherwise predicted to undergo cancer. For example, a gene may be introduced into a neuron or a T cell by lipofection, asialorosonucoid polylysine conjugation, or, less preferably, microinjection under surgical conditions.

[0121] Generally, for a protein to exert an effect, the cell that will use or secrete the protein must create it. To create a protein the cell first makes a copy of the protein's gene sequence in the nucleus of the cell. This copy of the gene sequence that encodes for the protein (called messenger RNA or mRNA) leaves the nucleus and is trafficked to a region of the cell containing ribosomes. Ribosomes read the sequence of the mRNA and create the protein for which it encodes. This process of new protein synthesis is known as translation.
A variety of factors affect the rate and efficiency of protein translation.
Among the most significant of these factors is the intrinsic stability of the mRNA itself. If the mRNA is degraded quickly within the cell (such as before it reaches a ribosome), it is unable to serve as a template for new protein translation, thus reducing the cell's ability to create the protein for which it encoded.

[0122] In one embodiment, the agent comprises nucleic acids involving in RNA
interference (RNAi). RNAi is a naturally-occurring mechanism for suppressing gene expression and subsequent protein translation. RNAi suppresses protein translation by either degrading the mRNA before it can be translated or by binding the mRNA
and directly preventing its translation. This technology provides an avenue to suppress the expression and actions of molecules and their receptors within a given population of cells.

[0123] Specifically, RNAi is mediated by double stranded RNA ("dsRNA"), short hairpin RNA ("shRNA") or other nucleic acid molecules with similar characteristics.
These nucleic acid molecules are processed or cut into smaller pieces by cellular enzymes including Dicer and Drosha. The smaller fragments of the nucleic acid molecules can then be taken up by a protein complex (the RISC complex) that mediates degradation of mRNAs. The RISC
complex will degrade mRNA that complementarily base pairs with the nucleic acid molecules it has taken up. In this manner, the mRNA is specifically destroyed, thus preventing the encoded-for protein from being made. Unless otherwise indicated, a particular nucleic acid sequence also encompasses conservatively modified variants thereof and complementary sequences, as well as the sequence explicitly indicated. The nucleic acid molecules can include any type of nucleic acid molecule capable of mediating RNA
interference, such as, without limitation, short interfering nucleic acid (siNA), short hairpin nucleic acid (shNA), short interfering RNA (sRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), and double-stranded RNA (dsRNA). The nucleic acid molecules also include similar DNA
sequences. Further, the nucleic acid and nucleic acid molecules can contain unmodified or modified nucleotides. Modified nucleotides refer to nucleotides which contain a modification in the chemical structure of a nucleotide base, sugar and/or phosphate. Such modifications can be made to improve the stability and/or efficacy of nucleic acid molecules

[0124] The understanding of the mechanism of RNAi allows the creation of nucleic acid molecules with sequences that are homologous to known gene sequences in order to suppress the expression or formation of certain proteins within a cell. In this disclosure, nucleic acid molecules that are homologous to WDR79 mRNA sequences are introduced into cells locally to suppress expression of these proteins. These nucleic acid molecules specifically suppress the expression of amino acid sequences that encode for WDR79.
Suppressing expression of these proteins locally treats cancer in an area without globally inhibiting other systems.

[0125] Note that, as will be understood by one of skill in the art, the nucleic acid molecules include the sequences of the WRAP53 gene, the reverse complement of those sequences and RNA-based sequences including uracils in the place of the listed thymidines.
Thus, any listing of RNAi sequences may be considered target sequences as well as sequences included in the nucleic acid molecules of WRAP53.

[0126] For any of the methods of application described above, the therapeutic nucleic acid construct is preferably applied to the site of the cancer event (for example, by injection).

However, it may also be applied to tissue in the vicinity of the cancer event or to a blood vessel supplying the cells predicted to undergo cancer.

[0127] Transgenic animals containing mutant, knock-out or modified genes corresponding to the cDNA sequence of WRAP53, or the open reading frame encoding the polypeptide sequence of WDR79, or fragments thereof having a consecutive sequence of at least about 3, 4, 5, 6, 10, 15, 20, 25, 30, 35 or more amino acid residues, are also included.
Transgenic animals are genetically modified animals into which recombinant, exogenous or cloned genetic material has been experimentally transferred. Such genetic material is often referred to as a "transgene." The nucleic acid sequence of the transgene may be integrated either at a locus of a genome where that particular nucleic acid sequence is not otherwise normally found or at, the normal locus for the transgene. The transgene may consist of nucleic acid sequences derived from the genome of the same species or of a different species than the species of the target animal.

[0128] In some embodiments, transgenic animals in which all or a portion of a gene is deleted may be constructed. In those cases where the gene contains one or more introns, the entire gene--all exons, introns and the regulatory sequences--may be deleted.
Alternatively, less than the entire gene may be deleted. For example, a single exon and/or intron may be deleted, so as to create an animal expressing a modified version of a protein.

[0129] The term "germ cell line transgenic animal" refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability of the transgenic animal to transfer the genetic information to offspring. If such offspring in fact possess some or all of that alteration or genetic information, then they too are transgenic animals.

[0130] The alteration or genetic information may be foreign to the species of animal to which the recipient belongs, foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene.

[0131] Transgenic animals can be produced by a variety of different methods including transfection, electroporation, microinjection, gene targeting in embryonic stem cells and recombinant viral and retroviral infection.

[0132] The method of introduction of nucleic acid fragments into recombination competent mammalian cells can be by any method which favors co-transformation of multiple nucleic acid molecules. Detailed procedures for producing transgenic animals are readily available to one skilled in the art.

[0133] As the genes and proteins disclosed herein are differentially expressed in cancer tissues compared to non-cancerous tissues, the genes and proteins may be used to diagnose or monitor cancer, to track disease progression, or to differentiate cancerous tissue from non-cancerous tissue samples. One means of diagnosing cancer using the nucleic acid molecules or proteins involves obtaining tissue from living subjects.

[0134] Assays to detect nucleic acid or protein molecules disclosed herein may be in any available format. Typical assays for nucleic acid molecules include hybridization or PCR
based formats. Typical assays for the detection of proteins, polypeptides or peptides include the use of antibody probes in any available format such as in situ binding assays, etc.

[0135] Generally, the diagnostics can be classified according to whether the embodiment is a nucleic acid or protein-based assay. Some diagnostic assays detect mutations or polymorphisms in the nucleic acids or proteins, which contribute to cancerous aberrations. Other diagnostic assays identify and distinguish defects in protein activity by detecting a level of RNA or protein in a tested organism that resembles the level of RNA or protein in a organism suffering from a disease, such as cancer, or by detecting a level of RNA or protein in a tested organism that is different than an organism not suffering from a disease.

[0136] Additionally, the manufacture of kits that incorporate the reagents and methods described in the following embodiments so as to allow for the rapid detection and identification of aberrations in protein activity or level are contemplated.
The diagnostic kits can include a nucleic acid probe or an antibody or combinations thereof, which specifically detect a mutant form of the protein or a nucleic acid probe or an antibody or combinations thereof, which can be used to determine the level of RNA or protein expression of one or more protein. The detection component of these kits will typically be supplied in combination with one or more of the following reagents. A support capable of absorbing or otherwise binding DNA, RNA, or protein will often be supplied. Available supports include membranes of nitrocellulose, nylon or derivatized nylon that can be characterized by bearing an array of positively charged substituents. One or more restriction enzymes, control reagents, buffers, amplification enzymes, and non-human polynucleotides like calf-thymus or salmon-sperm DNA can be supplied in these kits.

[0137] Useful nucleic acid-based diagnostic techniques include, but are not limited to, direct DNA sequencing, gradient gel electrophoresis, Southern Blot analysis, single-stranded confirmation analysis (SSCA), RNAse protection assay, dot blot analysis, nucleic acid amplification, allele-specific PCR and combinations of these approaches. The starting point for these analyses is isolated or purified nucleic acid from a biological sample. It is contemplated that tissue biopsies would provide a good sample source. The nucleic acid is extracted from the sample and can be amplified by a DNA amplification technique such as the Polymerase Chain Reaction (PCR) using primers. Those of skill in the art will readily recognize methods available for confirming the presence of polymorphisms. In addition, any addressable array technology known in the art can be employed with this aspect of the disclosure.

[0138] In preferred protein-based diagnostic, antibodies are attached to a support in an ordered array wherein a plurality of antibodies are attached to distinct regions of the support that do not overlap with each other. Those of skill in the art will readily recognize available assays that are protein-based diagnostics. Proteins are obtained from biological samples and are labeled by conventional approaches (e.g., radioactivity, calorimetrically, or fluorescently). Employing labeled standards of a known concentration of mutant and/or wild-type protein, an investigator can accurately determine the concentration of the protein in a sample and from this information can assess the expression level of the particular form of the protein. Conventional methods in densitometry can also be used to more accurately determine the concentration or expression level of such protein. These approaches are also easily automated using technology known to those of skill in the art of high throughput diagnostic analysis. As detailed above, any addressable array technology known in the art can be employed with this aspect and display the protein arrays on the chips in an attempt to maximize antibody binding patterns and diagnostic information.

[0139] As discussed above, the presence or detection of a polymorphism in an gene or protein can provide a diagnosis of a cancer or similar malady in an organism.
Additional embodiments include the preparation of diagnostic kits comprising detection components, such as antibodies, specific for a particular polymorphic variant of the gene or protein. The detection component will typically be supplied in combination with one or more of the following reagents. A support capable of absorbing or otherwise binding RNA or protein will often be supplied. One or more enzymes, such as Reverse Transcriptase and/or Taq polymerase, can be furnished in the kit, as can dNTPs, buffers, or non-human polynucleotides like calf-thymus or salmon-sperm DNA. Results from the kit assays can be interpreted by a healthcare provider or a diagnostic laboratory.
Alternatively, diagnostic kits are manufactured and sold to private individuals for self-diagnosis.

[0140] In addition to diagnosing disease according to the presence or absence of a polymorphism, some diseases involving cancer result from skewed levels of protein or gene in particular tissues or aberrant patterns of protein expression. By monitoring the level of expression in various tissues, for example, a diagnosis can be made or a disease state can be identified. Similarly, by determining ratios of the level of expression of various proteins in specific tissues (e.g., patterns of expression) a prognosis of health or disease can be made.
The levels of protein expression in various tissues from healthy individuals, as well as, individuals suffering from cancers are-determined. These values can be recorded in a database and can be compared to values obtained from tested individuals.
Additionally, the ratios or patterns of expression in various tissues from both healthy and diseased individuals are recorded in a database. These analyses are referred to as "disease state profiles" and by comparing one disease state profile (e.g. from a healthy or diseased individual) to a disease state profile from a tested individual, a clinician can rapidly diagnose the presence or absence of disease.

[0141] The nucleic acid and protein-based diagnostic techniques described above can be used to detect the level or amount or ratio of expression of genes or proteins in a tissue.
Through quantitative Northern hybridizations, in situ analysis, immunohistochemistry, ELISA, genechip array technology, PCR, and Western blots, for example, the amount or level of expression of RNA or protein for a particular protein (wild-type or mutant) can be rapidly determined and from this information ratios of expression can be ascertained.
Alternatively, the proteins to be analyzed can be family members that are currently unknown but which are identified based on their possession of one or more of the homology regions described above.

EXAMPLES
Example 1 WDR79 shuttles to the nucleus and binds USP7 to promote tumor cell proliferation

[0142] 1. Materials and Methods

[0143] Bioinformatics analysis. The WDR79 data set contains 16 homologs from species including unicellular eukaryotes and metazoan animals, which were retrieved from GenBank databases via BLAST search using the human WDR79 protein sequence as the query. The amino acid sequences of WDR79 proteins were aligned using the MUSCLE
program and the alignments were manually inspected. The unrooted protein tree of WDR79 was reconstructed using the neighbor-joining method along with bootstrap test.
Conserved domains and functional sequence motifs of WDR79 were identified and analyzed using complementary methods available from various websites.

[0144] Plasmid constructs. The full-length cDNA sequence of human WDR79 was PCR-amplified using the two primers containing Eco RI and Xho I sites, respectively: forward primer, 5'-CGGAATTCATGAAGACTTTGGAGACT-3' (SEQ ID NO:6) and reverse primer, 5'-CGGCTCGAGTTATATCAGCTCACCCAC-3' (SEQ ID NO:7). The PCR product was digested by Eco RI+Xho I and subcloned into pCMV-Tag2B (with the Flag tag) and pCMV-Script (without the Flag tag) vector (Stratagene) to create Flag-tagged and tag-free WDR79 expression plasmids, respectively. The Eco RI+Xho I digest of WDR79 was also subcloned into the Eco RI+Sal I sites of pEGFP-C1 vector (Clontech). USP7 cDNA fragments were PCR-amplified using the primers that have Eco RI and Xho I sites and subcloned into pGEX-4T-1 vector (Amersham Pharmacia Biotech) to generate the glutathione S-transferase (GST)-USP7 expression vectors as GST-USP71_210 and GST-USP7601_102, respectively. For the GST-USP71-210 fusion construct, the following primers were used: forward primer, 5'-CGGAATTCATGAACCACCAGCAGCAG-3' (SEQ ID NO:8) and reverse primer, 5'-CGGCTCGAGTTAGTGCTTCTTTGAATC-3' (SEQ ID NO:9); the two primers used for production of the GST-USP7601-1102 fusion construct were: forward primer, 5'-CGGAATTCATGTCGCTTGCTGAGTTT-3' (SEQ ID NO:10) and reverse primer, 5'-CGGCTCGAGTCAGTTATGGATTTTAAT-3' (SEQ ID NO:11). All the expression plasmids were sequenced to verify the precision of the cloned cDNA sequence and its orientation prior to their use.

[0145] Cell culture and plasmid transfection. A375, SK-Mel-2, Hs294T, HeLa, SK-N-As, LN-18, H1299 and U20S cells were purchased from ATCC and cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS).
Normal human epidermal melanocyte (NHEM) cells were purchased from Lonza and cultured in melanocyte cell basal medium-4 (MGM-4) with the following growth supplements:
CaCl2, BME (beta-mercaptoethanol), rhFGF-B (recombinant human fibroblast growth factor-B), rh-Insulin, hydrocortisone, PMA (phorbol 12-myristate 13-acetate), GA-1000 (gentamicin sulfate and amphotericin-B) and FBS. All cells were incubated at 37 C in a humidified incubator containing 5% CO2. Transfection into different cell types with expression plasmids was done using FuGENE HD (Roche) according to the manufacturer's instructions.

[0146] Colony formation assay. Cells were transfected with blank pCMV-Tag2B
plasmid and pCMV-Tag2B-WDR79 construct for 48 hr and then plated in 60mm dishes (A375 and SK-Mel-2) or in six-well plates (U20S) at 5X104 cells/ml. Cells were cultured in the selection medium (G418, Sigma) as follows: 400 pg/ml for SK-Mel-2, 800 pg/ml for A375 and U20S. After a 2-week selection, cells were fixed with methanol and stained with 0.1 %
crystal violet.

[0147] Western blot analysis. Whole cell lysate was prepared with M-PER lysis buffer supplemented with protease inhibitor cocktail (Pierce). The protein sample were boiled in SDS sample buffer, resolved on SDS-PAGE, and transferred to nitrocellulose membrane (Bio-Rad). The membranes was blocked in 5% nonfat milk for 1 hr at room temperature and incubated with the indicated antibodies overnight at 4 C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz). Antibody detections were performed with the chemiluninescence ECL kit (Pierce). The following antibodies were used for Western blotting: USP7 and WDR79 (Bethyl Laboratories); p53 (DO-1), Mdm2 (SMP14), RCC1, and GST, ubiquitin (Santa Cruz); Flag and R-actin (Sigma),

[0148] In vivo ubiguitination assay. U20S cells were transfected with pCMV-Tag2B
vector and pCMV-Tag2B-WDR79. After 48 hr of transfection, cells were treated with proteasome inhibitor MG132 (20 pM) for 4 hr and then lysated in pre-boiled SDS-lysis buffer (50 mM Tris-HCI pH7.5, 0.5 mM EDTA, 1% SDS (W/V), 1 mM DTT). Lysate was boiled for min and then clarified by centrifugation at 14,000 rpm for 10 min. Supernatant was diluted 10-fold with 0.5% NP-40 buffer in PBS and 1 mM PMSF. After immunoprecipitation with the anti-Mdm2 or anti-p53 antibody, the immunoprecipitates were resolved by 8% SDS-PAGE
gel and transferred onto nitrocellulose membrane. The blot was blocked in 5%
nonfat milk and probed with anti-ubiquitin antibody. Antibodies were detected with chemiluminescence detection reagents according to the manufacturer's instructions.

[0149] Protein half-life assay. U20S cells were cultured on 6-well plates and were transfected separately with control or WDR79 plasmid as indicated. Forty-eight hours after transfection, cells were treated with cycloheximide (100pg/ml) to block protein biosynthesis for the indicated period of time. Protein levels were analyzed by Western blotting.

[0150] Indirect immunofluorescence. Cells were cultured in Lab-Tek chambers and then transfected separately with control or plasmid as indicated for 24 hr.
They were washed three times with PBS and then fixed with 4% paraformaldehyde for 10 min. After fixation, cells were washed twice with PBS, permeabilized with 0.2% Triton X-100 for 10 min and incubated with 5% BSA for 1 hr prior to incubation with the primary antibody.
The cells were then incubated with FITC-conjugated or Texas red-conjugated secondary antibodies (Santa Cruz) for 1 hr. Cells were washed three times with PBS and finally counterstained with PI
(Sigma). The images were acquired with a confocal microscope (Zeiss LSM510).

[0151] Extraction of cytoplasmic and nuclear proteins. Forty-eight hours after transient transfection, cells were gently washed once in PBS and pelleted by centrifugation at 500 x g for 2-3 min. Ice-cold CERI (NE-PERTM, Pierce) was added to each sample. Cells were resuspended by vortexing for 15 sec and then incubated on ice for 10 min. Ice-cold CERII
was then added to each sample followed by repeated vortexing and centrifugation at 14,000 rpm for 5 min. The supernatant (cytoplasmic extract) was collected. The insoluble nuclear pellet was resuspended in NER and incubated on ice for 40 min. The samples were centrifuged at 14,000 rpm for 10 min, and the supernatant (nuclear extract) was collected.
The protein concentrations were measured using BCA protein assay kit (Pierce).

[0152] Co-immunoprecipitation assay. Protein co-immunoprecipitation was carried out using a universal magnetic Co-IP kit (Active Motif). Five hundred micrograms of whole-cell extract was incubated with 3 pg of mouse anti-flag antibody (Sigma) or normal mouse IgG at 4 C for 3 hr. 25 pl of protein G magnetic beads were added to the mixture and incubated for one more hour with rocking. Beads were washed four times with complete Co-IP/wash buffer. The immunopreccipitated proteins were dissolved in 3xSDS sample buffer and boiled for 5 min prior to Western blot analysis as above.

[0153] Knockdown analysis. Suresilencing shRNA plasmid for USP7 and negative control shRNA plasmid were purchased from SABiosciences. U20S cells were transfected separately with a negative control vector containing a scrambled sequence (5'-GGAATCTCATTCGATGCATAC-3', SEQ ID NO:12) and with the pre-designed USP7 shRNA vector containing the target sequence (5'-GCAGTGCTGAAGATAATAAAT-3' SEQ
ID
NO:13). After two rounds of shRNA transfection, cells were plated for additional transfection, if necessary.

[0154] Expression of GST-USP7 fusion proteins and pull-down assay. To prepare GST
fusion proteins, Escherichia coli BL21 were transformed with a pGEX-4T-1-USP7 expression vector containing the appropriate insert. Overnight cultures were diluted 1:10 and grown to A600=0.5, then the fusion proteins was induced by the addition of 0.1 mM
isopropyl-(3-D-thiogalactoside (IPTG). Cell pellets were lysed by mild sonication in ice-cold PBS containing 1% Triton X-100 and protease inhibitor cocktail (Pierce). After centrifugation, supernatants were applied to glutathione Sepharose 4B beads (Amersham Pharmacia Biotech) at overnight. The purified proteins were eluted from beads with 50 mM glutathione and were dialyzed overnight against TBS.

[0155] GST pull-down assay was performed by using the Profound Pull-down GST
Protein-protein Interaction kit (Pierce). Purified GST-USP7 fusion proteins and control GST
protein were immobilized on 50 I glutathione resins by incubation for 2 hr at 4 C. U20S
cells transfected with pCMV-Tag2B-WDR79 were lysed in ProFound Lysis Buffer.
After centrifugation, supernatants were applied to immobilized GST fusion protein beads and incubated for 2 hr at 4 C. The beads were washed four times with 500 I wash buffer/each to remove nonspecific proteins. The bead-bound proteins were added with three volumes of SDS sample buffer and boiled for 5 min prior to Western blot analysis.

WO 2011/014816 FPCT/US2010/043986on 19b i42 /-UU 159

[0156] 2. WDR79 is up-regulated in tumor cell lines and exhibits varied subcellular location

[0157] Bioinformatics analysis of WDR79 was conducted to uncover its WD
repeats, sequence motifs including USP7-binding sites and evolutionarily conserved features in different species (FIG.s 1 and 7). Protein expression in various cell types was then characterized. Compared to NHEM, a higher level of WDR79 was observed in melanoma (A375, SK-Mel-2 and Hs294T) and other tumor cells (cervical cancer HeLa cells, neuroblastoma SK-N-As cells, malignant glioma LN-18 cells, lung cancer H1299 cells and osteosarcoma U2OS cells) (FIG. 1). Because subcellular location is a key index of function, the distribution of WDR79 was refined. It was found primarily in the cytoplasm of A375 and U2OS cells, but was in both the cytosol and the nucleus of SK-Mel-2 and H1299 cells (FIG.
2). These results indicate that WDR79 is up-regulated in human tumor cell lines and is resident in both the cytoplasm and the nucleus.

[0158] 3. WDR79 overexpression enhances its nuclear transport and accumulation

[0159] To further delineate its subcellular location, WDR79 was transiently expressed in various cell types. Notably, in all cell types including A375, SK-Mel-2, U2OS and H1299, WDR79 shuttled to the nucleus and accumulated in that compartment (FIG. 3).
This event was driven by WDR79 itself, because the native (tag-free) and GFP- or Flag-tagged forms showed the identical pattern (FIG.s 8 and 9). To verify this relocation, the specific markers, RCC1 and a-tubulin for nuclear and cytosol protein extracts, were analyzed. In H1299 and U2OS cells, nuclear WDR79 was increased about five- and three-fold, respectively, as compared with controls (FIG.s 3B and 3C). In agreement with recent studies, these results support that despite apparently lacking a nuclear localization signal, WDR79 is capable of becoming nuclear-enriched via forced or stimulated expression.

[0160] 4. WDR79 and USP7 colocalize in the nucleus forming a complex in vivo and in vitro.

[0161] As WDR79 retains USP7-binding sites, the relationship of the two proteins in nuclear colocalization and physical association was studied. USP7 and WDR79 were probed with the specific antibodies conjugated to different dyes. The intense staining in the merged images reveals a strong colocalization of the two proteins in the nucleus (FIG. 4A). Notably, the pattern is evident and same in the nuclei of all cell types including A375, SK-Mel-2, H1299 and U2OS cells. In contrast, overexpression of USP7, mainly a nuclear protein, had no effect (not shown). Thus, nuclear transport of WDR79 does not rely on USP7, but is WO 2011/014816 FPCT/US2010/043986on 19b i42 /-UU 159 required for the colocalization with USP7. It was next determined whether colocalization results from an intracellular complex formation by coimmunoprecipitation. As shown, USP7 was found in the WDR79 immunoprecipitates, not in IgG control (FIG. 4B), indicating their intracellular association. To define which region of USP7 participates in WDR79 binding, the protein pull-down assay was performed. GST-USP71-210 and GST-USP7601-1102 constructs were prepared, given their known roles in binding p53, Mdm2 or EB
virus nuclear antigen 1 and in catalysis. As shown, the USP71-210 region that retains the MATH domain, but not the USP7601-1102 catalytic region, bound WDR79 (FIG. 4C).
Taken together, these data indicate that WDR79 and USP may interact directly in vivo and that the MATH domain of USP7 is involved in, and necessary for, this interaction.

[0162] 5. WDR79 increases the stability of both Mdm2 and p53 in a USP7-dependent manner.

[0163] USP7 is a key deubiquitinase involved in the p53-Mdm2 axis and its forced expression results in p53 and Mdm2 stabilization. Thus, it was examined whether WDR79 acts in concert with USP7 to effect this stabilization. Notably, the steady-state level of p53 and Mdm2 was increased in p53 wild-type A375 and U2OS cells, but only that of Mdm2 was increased in p53-null H1299 cells (FIG. 5A). The effect of WDR79 on the half-life of Mdm2, p53 and USP7 was next determined in U2OS cells using cyclohexamide to inhibit protein synthesis. As expected, WDR79 expression prolonged the half-life of Mdm2 and p53, without apparent effect on itself or its partner USP7 (FIG. 5B).

[0164] Mdm2 is a key negative regulator of p53 via its E3 ligase activity and itself subject to autoubiquitination. To explore the mechanism by which WDR79 stabilizes Mdm2 and p53, the influence of WDR79 on p53 and Mdm2 ubiquitination was determined.
Consistent with its role in p53 and Mdm2 stabilization, WDR79 decreased the ubiquitination of the two proteins (FIG. 5C). To prove the dependence of WDR79 on USP7, shRNA-mediated knockdown of USP7 was performed. As shown in U2OS cells, knockdown of USP7 led to a reduction of the steady-state level of p53 and Mdm2 (FIG. 5D), a result consistent with that of the previous study. Moreover, the data revealed that knockdown of USP7 attenuated the role of WDR79 in stabilizing p53 and Mdm2 (FIG. 5D). Thus, acts through USP7 to modulate the steady-state levels of Mdm2 and p53 proteins.

[0165] 6. WDR79 promotes tumor cell proliferation.

[0166] As shown, WDR79 stabilizes Mdm2 and p53, yet the latter two proteins execute opposing functions in various cellular settings. Thus cell growth was analyzed by colony formation assay to establish the functional significance of WDR79 in relaying its signal to WO 2011/014816 FPCT/US2010/043986on 19b i42 /-UU 159 USP7. Importantly, the WDR79 stable clones derived from A375, SK-Mel-2 and U2OS cells all emerged in greater numbers (FIG. 6A), providing evidence for the increased cell proliferation. Given the different affinities of USP7 for Mdm2 and p53, this result suggests that WDR79 binding stimulates the USP7-Mdm2 axis more effectively than the USP7-p53 counterpart, thereby facilitating cell proliferation.

[0167] Furthermore, WDR79 promotes cell cycle progression from G1 to S and G2/M in A375 cells (FIG. 10).

[0168] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0169] The terms "a," "an," "the" and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.
The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0170] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and WO 2011/014816 FPCT/US2010/043986on 19b i41/-UU159 claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0171] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0172] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term "consisting of" excludes any element, step, or ingredient not specified in the claims. The transition term "consisting essentially of"
limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.

[0173] Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications are individually incorporated herein by reference in their entirety.

[0174] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

Claims (11)

What is claimed is:

1. A method of inhibiting tumor cell proliferation comprising exposing a cell population or a tissue to an effective amount of an antagonist or inhibitor of WDR79.

2. The method of claim 1 wherein said antagonist or inhibitor is a small molecule, a nucleic acid or an antibody.

3. The method of claim 2 wherein said nucleic acid is selected from the group consisting of short interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA
(miRNA), double-stranded RNA (dsRNA) or anti-sense molecule.

4. A method of screening for anti-proliferative compounds comprising obtaining potential anti-proliferative compounds and determining the effects of said compounds on the expression of WDR79 in a cell.

5. The method of claim 4 wherein said anti-proliferative compound is a small molecule, a nucleic acid or an antibody.

6. The method of claim 5 wherein said nucleic acid is selected from the group consisting of short interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA
(miRNA), double-stranded RNA (dsRNA) or anti-sense molecule.

7. The method of claim 4 wherein said cell is a cancer cell.

8. A method of modulating the activity of USP7 comprising modulating the levels of WDR79 in a cell or tissue.

9. The method of claim 8 wherein said modulating the activity of USP7 comprises increasing the activity of USP7.

10. The method of claim 8 wherein said modulating the activity of USP7 comprises decreasing the activity of USP7.

11. The method of claim 8 wherein modulating the activity of USP7 comprises modulating the activity or expression of WDR79.