WO2006116503A2 - Methods and compositions for modulating wnt signaling pathway - Google Patents

Abstract

This invention provides novel Wnt signaling pathway regulators. The invention also provides methods of using the Wnt signaling pathway regulators to screen for compounds that modulate Wnt signaling pathway. The methods comprise first screening test compounds for modulators of a Wnt signaling pathway regulator disclosed herein, and then further screening the identified modulating agents for ability to modulate Wnt signaling pathway. The invention further provides methods for modulating Wnt signaling pathway and pharmaceutical compositions for treating diseases and conditions (e.g., tumors) associated with abnormal Wnt signaling activities.

Description

METHODS AND COMPOSITIONS FOR MODULATING WNT SIGNALING PATHWAY

CROSS-REFERENCE TO RELATED APPLICATIONS

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

U.S. Provisional Patent Application No. 60/675,301, filed April 26, 2005. The disclosure of the priority application is incorporated herein by reference in its entirety and for all purposes.

FIELD OF THE INVENTION

[0002] The present invention generally relates to modulation of Wnt signaling pathways. More particularly, the invention pertains to novel Wnt signaling pathway regulators, and to methods of using such regulators to identify novel compounds that modulate Wnt signaling pathways.

BACKGROUND OF THE INVENTION

[0003] Wnt signaling pathways play important roles in cell proliferation, differentiation, adhesion, motility, polarity and morphogenetic movements (Cadigan et al., Genes Dev. 11:3286-3305, 1997; and Polakis, Genes Dev. 14:1837-1851, 2000). Many of the Wnt ligands activate the so-called canonical pathway which signals by regulating the cytoplasmic abundance of β-catenin. In the absence of Wnt signaling, the cytoplasmic β- catenin is actively targeted for degradation through interaction with a protein complex comprised of APC, CKl, Axin, protein phosphatase 2A (PP2A), and glycogen synthase kinase 3β (GSK-3β). In this complex, β-catenin is phosphorylatedby CKl and then by GSK-3 β. Phosphorylation of β-catenin results in its ubiquitination mediated by β-Trcp, and subsequent degradation through a proteasome-dependent pathway. [0004] Activation of Wnt signaling leads to inactivation of GSK-3 β through DvI and Frat. This results in accumulation and nuclear translocation of β-catenin. β-catenin transduces Wnt signals by associating with T cell factor (TCF) and lymphoid enhancer factor (LEF) transcription factors. The binding of β-catenin to TCF/LEF-1 is required for the former to transactivate target genes such as c-myc, c-jun, fra-1 , and cyclin Dl . In the absence of Wnt signaling, β-catenin level in the cells is low and nuclear TCF/β-catenin complex formation is prevented. This leads to target gene suppression by TCF/LEF which acts as transcriptional repressor.

[0005] There is a need in the art for better means for modulating Wnt signaling pathway, and for treating disease and conditions due to abnormal Wnt signaling (e.g., tumorigenesis). The present invention addresses this and other needs.

SUMMARY OF THE INVENTION

[0006] In one aspect, the invention provides methods for identifying agents that modulate Wnt signaling pathway. The methods involve assaying a biological activity of a Wnt signaling pathway regulator identified by the present inventors (one encoded by a polynucleotide listed in Tables 1-6) in the presence of test compounds to identify one or more modulating agents that modulate the biological activity of the regulator. This is followed by testing the modulating agents for ability to modulate Wnt signaling pathway. Some of the methods employ a positive Wnt signaling pathway regulator that is encoded by a polynucleotide in Tables 1, 3, and 5. Some other methods use a negative Wnt signaling pathway regulator that is encoded by a polynucleotide in Tables 2, 4, and 6. In some of the methods, the identified modulating agents stimulate the biological activity of the Wnt signaling pathway regulator. In some other methods, the identified modulating agents inhibit the biological activity of the Wnt signaling pathway regulator. [0007] In some of the screening methods, the further testing on the modulating agents involves examining the modulating agents for ability to modulate expression of a gene responsive to β-catenin signaling. In some of these methods, the gene responsive to β-catenin signaling encodes a component of the Wnt signaling pathway downstream of β- catenin or a target gene of Wnt signaling pathway. In some of the screening methods of the invention, the modulating agents are further tested for ability to modulate expression of a reporter gene under the control of a promoter containing a TCF/LEF binding site. In some other methods, the modulating agents were tested for ability to modulate proliferation of a cell in response to Wnt signaling.

[0008] In some methods, the biological activity assayed is expression of a gene encoding the Wnt signaling pathway regulator. In some other methods, the employed Wnt signaling pathway regulator is an enzyme, e.g., a kinase. In these methods, the biological activity assayed can be an enzymatic activity of the Wnt signaling pathway regulator. [0009] In a related aspect, the invention provides methods for identifying modulators of Wnt signaling pathway. The methods entail (a) contacting test agents with a Wnt signaling pathway regulator identified by the present inventors (one encoded by a polynucleotide listed in Tables 1-6); (b) identifying one or more modulating agents which modulate a biological activity of the Wnt signaling pathway regulator; and (c) screening the modulating agents for ability to regulate expression of a gene from a promoter that is responsive to β-catenin signaling.

[0010] Some of the methods use a positive Wnt signaling pathway regulator that is encoded by a polynucleotide in Tables 1, 3, and 5. Some other methods employ a negative Wnt signaling pathway regulator that is encoded by a polynucleotide in Tables 2, 4, and 6. The modulating agents identified in the screening can either stimulate or inhibit the biological activity of the Wnt signaling pathway regulator. In some methods, the promoter responsive to β-catenin signaling contains a TCE/LEF binding site. In some methods, the Wnt signaling pathway regulator employed in the screening is an enzyme (e.g., a kinase), and the biological activity assayed is its enzymatic activity. [0011] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 shows that NME2 inhibits migration of breast cancer MDA-

MB-231 cells.

[0013] Figure 2 shows that overexpression of NME2 in Xenopus embryos result in impaired dual axis formation. DETAILED DESCRIPTION

I. Overview

[0014] The invention is predicated in part on the discoveries by the present inventors of molecules that positively or negatively regulate Wnt signaling pathway. As detailed in the Examples below, the present inventors carried out genome-wide siRNA (loss-of-function) and cDNA (gain-of-function) screening for cellular regulators of Wnt signaling pathway. As detailed in the Examples, the screening employed a transfection grade T-cell factor reporter (TOPFlash) under control of nuclear β-catenin to reveal the level of Wnt signaling. In order to identify both activators and inhibitors of Wnt signaling pathways, the basal level of Wnt signaling is increased by stimulating with condition medium collected from stably transfected L-wnt3 A cells (ATCC). In addition to some known Wnt components (which validate the authenticity of the screens), a number of other genes that activate or inhibit Wnt signaling were identified from the screening. These genes (shown in Tables 1-6) and their encoded polypeptides are termed herein "Wnt signaling pathway regulators." One such regulator, NME2, was chosen for further studies. As detailed in the Examples below, it was found that regulating NME2 expression level changed migration behavior of cultured cancer cells. Further, it was also observed that overexpression of NME2 can lead to developmental defect in whole organism. [0015] The Wnt signaling pathway regulators identified by the present inventors provide novel targets which can be used to screen for compounds that modulate the Wnt signaling pathway. Compounds identified from the screening can be used to treat diseases and conditions associated with or mediated by defective Wnt signaling, e.g., cancer and neurodegenerative diseases. In addition, Wnt ligand and signaling have been reported to regulate stem cell proliferation and differentiation in several systems. The identified modulators of Wnt signaling pathways can also be used for generating desired cells for cell based therapy of various diseases. For example, they can be employed in expanding hematopoietic stem cells for chemotherapy and differentiating neural stem cells for treatment of neurodegenerative diseases.

[0016] The following sections provide guidance for making and using the compositions of the invention, and for carrying out the methods of the invention.

II. Definitions [0017] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Oxford Dictionary of Biochemistry arid Molecular Biology, Smith et al. (eds.), Oxford University Press (revised ed., 2000); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3 ^rd ed., 2002); and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (Eds.), Oxford University Press (4^th ed., 2000). In addition, the following definitions are provided to assist the reader in the practice of the invention. [0018] The term "agent" or "test agent" includes any substance, molecule, element, compound, entity, or a combination thereof. It includes, but is not limited to, e.g., protein, polypeptide, small organic molecule, polysaccharide, polynucleotide, and the like. It can be a natural product, a synthetic compound, or a chemical compound, or a combination of two or more substances. Unless otherwise specified, the terms "agent", "substance", and "compound" can be used interchangeably.

[0019] The term "analog" is used herein to refer to a molecule that structurally resembles a reference molecule but which has been modified in a targeted and controlled manner, by replacing a specific substituent of the reference molecule with an alternate substituent. Compared to the reference molecule, an analog would be expected, by one skilled in the art, to exhibit the same, similar, or improved utility. Synthesis and screening of analogs, to identify variants of known compounds having improved traits (such as higher binding affinity for a target molecule) is an approach that is well known in pharmaceutical chemistry.

[0020] As used herein, "contacting" has its normal meaning and refers to combining two or more molecules (e.g., a test agent and a polypeptide) or combining molecules and cells (e.g., a test agent and a cell). Contacting can occur in vitro, e.g., combining two or more agents or combining a test agent and a cell or a cell lysate in a test tube or other container. Contacting can also occur in a cell or in situ, e.g., contacting two polypeptides in a cell by coexpression in the cell of recombinant polynucleotides encoding the two polypeptides, or in a cell lysate. [0021] A "heterologous sequence" or a "heterologous polynucleotide," as used herein, is one that originates from a source foreign to the particular host cell, or, if from the same source, is modified from its original form. Thus, a heterologous polynucleotide in a host cell includes a polynucleotide that, although being endogenous to the particular host cell, has been modified. Modification of the heterologous sequence can occur, e.g., by treating the polynucleotide with a restriction enzyme to generate a polynucleotide fragment that is capable of being operably linked to the promoter. Techniques such as site-directed mutagenesis are also useful for modifying a heterologous polynucleotide.

[0022] The term "homologous" when referring to proteins and/or protein sequences indicates that they are derived, naturally or artificially, from a common ancestral protein or protein sequence. Similarly, nucleic acids and/or nucleic acid sequences are homologous when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Homology is generally inferred from sequence similarity between two or more nucleic acids or proteins (or sequences thereof). The precise percentage of similarity between sequences that is useful in establishing homology varies with the nucleic acid and protein at issue, but as little as 25% sequence similarity is routinely used to establish homology. Higher levels of sequence similarity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more can also be used to establish homology.

[0023] A "host cell," as used herein, refers to a prokaryotic or eukaryotic cell into which a heterologous polynucleotide (e.g., an expression vector) is to be introduced. The heterologous polynucleotide can be introduced into the host cell by any means, e.g., transfection, electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, and/or the like.

[0024] The term "Wnt signaling pathway regulator" or "regulator of Wnt signaling pathway" refers a gene or its encoded polypeptide which can modulate Wnt/β- catenin signaling activities. Thus, it encompasses "Wnt signaling pathway-modulating genes" and "Wnt signaling pathway-modulating polypeptides." As shown in Tables 1-6, it includes both agonizing regulators and antagonizing regulators of the Wnt/β-catenin signaling pathway. [0025] The term "sequence identity" in the context of two nucleic acid sequences or amino acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. A "comparison window" refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are aligned optimally. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482; by the alignment algorithm of Needleman and Wunsch (1970) J. MoI. Biol. 48:443; by the search for similarity method of Pearson and Lipman (1988) Proc. Nat. Acad. Sci U.S.A. 85:2444; by computerized implementations of these algorithms (including, but not limited to CLUSTAL in the PC/Gene program by Intelligentics, Mountain View, CA; and GAP, BESTFIT, BLAST, FASTA, or TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., U.S.A.). Alignment can also be performed by inspection and manual alignment.

[0026] A "substantially identical" nucleic acid or amino acid sequence refers to a nucleic acid or amino acid sequence which has at least 90% sequence identity to a reference sequence using the programs described above (e.g., BLAST) using standard parameters. The sequence identity is preferably at least 95%, more preferably at least 98%, and most preferably at least 99%. Preferably, the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues. In a most preferred embodiment, the sequences are substantially identical over the entire length of the coding regions.

[0027] The term "modulate" with respect to a biological activity of a reference protein or its fragment refers to a change in the expression level or other biological activities of the protein. For example, modulation may cause an increase or a decrease in expression level of the reference protein, eri2ymatic modification (e.g., phosphorylation) of the protein, binding characteristics (e.g., binding to a target polynucleotide), or any other biological, functional, or immunological properties of the reference protein. The change in activity can arise from, for example, an increase or decrease in expression of one or more genes that encode the reference protein, the stability of an mRNA that encodes the protein, translation efficiency, or from a change in other biological activities of the reference protein. The change can also be due to the activity of another molecule that modulates the reference protein (e.g., a kinase which phosphorylates the reference protein). Modulation of a reference protein can be up-regulation (i.e., activation or stimulation) or down- regulation (i.e. inhibition or suppression). The mode of action of a modulator of the reference protein can be direct, e.g., through binding to the protein or to genes encoding the protein, or indirect, e.g., through binding to and/or modifying (e.g., enzymatically) another molecule which otherwise modulates the reference protein.

[0028] The term "subject" includes mammals, especially humans. It also encompasses other non-human animals such as cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys. These subjects are all amenable to treatment with the Wnt-modulating compounds that can be identified in accordance with the present invention.

[0029] A "variant" of a reference molecule refers to a molecule substantially similar in structure and biological activity to either the entire reference molecule, or to a fragment thereof. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if the composition or secondary, tertiary, or quaternary structure of one of the molecules is not identical to that found in the other, or if the sequence of amino acid residues is not identical.

III. Screening for Novel Modulators of Wnt signaling pathways - General Scheme [0030] The Wnt signaling pathway regulators identified by the present inventors provide novel targets to screen for modulators of Wnt signaling pathways. Various biochemical and molecular biology techniques or assays well known in the art can be employed to practice the present invention. Such techniques are described in, e.g., Handbook of Drug Screening, Seethala et al. (eds.), Marcel Dekker (1^st ed., 2001); High Throughput Screening: Methods and Protocols (Methods in Molecular Biology, 190), Janzen (ed.), Humana Press (1^st ed., 2002); Current Protocols in Immunology, Coligan et al. (Ed.), John Wiley & Sons Inc (2002); Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (3^rd ed., 2001); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003).

[0031] Typically, test agents are first assayed for their ability to modulate a biological activity of a Wnt signaling pathway regulator encoded by the polynucleotides shown in Tables 1-6 ("the first assay step"). Modulating agents thus identified are then subject to further screening for ability to modulate Wnt signaling, typically in the presence of the Wnt signaling pathway regulator ("the second testing step"). Depending on the Wnt signaling pathway regulator employed in the method, modulation of different biological activities of the Wnt signaling pathway regulator can be assayed in the first step. For example, a test agent can be assayed for binding to the Wnt signaling pathway regulator. The test agent can be assayed for activity to modulate expression of the Wnt signaling pathway regulator, e.g., transcription or translation. The test agent can also be assayed for activities in modulating the cellular level or stability of the Wnt signaling pathway regulator, e.g., post-translational modification or proteolysis.

[0032] If the Wnt signaling pathway regulator has a known biological or enzymatic function (e.g., kinase activity or protease activity), the biological activity monitored in the first screening step can also be the specific biochemical or enzymatic activity of the Wnt signaling pathway regulator. These include kinases (e.g., LOC283458, CDK5, IRAKI, CAMKlG, FLTl, DGKA, JAK2, MAP2K4, and PCTKl), proteases (e.g., USP9Y, USP14 USP8, and SENPl), phosphatases (e.g., PTP4A2, INPP5D, DUSP16, PTPRD, FBP2, and PPPlCA), or other enzymes shown in Tables 1-6. Any of these molecules can be employed in the first screening step. Methods for assaying the enzymatic activities of these molecules are well known and routinely practiced in the art. For any Wnt pathway regulator, the substrate to be used in the screening can be a molecule known to be enzymatically modified by the enzyme (e.g., a kinase), or a molecule that can be easily identified from candidate substrates for a given class of enzymes. For example, many kinase substrates are available in the art. See, e.g., www.emdbiosciences.com; and www.proteinkinase.de. In addition, a suitable substrate of a kinase can be screened for in high throughput format. For example, substrates of a kinase can be identified using the Kinase-Glo® luminescent kinase assay (Promega) or other kinase substrate screening kits (e.g., developed by Cell Signaling Technology, Beverly, Massachusetts).

[0033] In an exemplary embodiment, the Wnt signaling pathway regulator employed in the screening is the IRAKI kinase, and test agents are first screened for ability to modulate the kinase's activity in autophosphorylation or phosphorylation of a substrate. Autophosphorylation of IRAKI can be examined as described in the literature, e.g., Maschera et al., Biochem J. 339:227-31, 1999. Kinase activity of IRAKI can also be examined using other substrates known to be phosphorylated by IRAKI, e.g., Pellino2. Pellino 2 phosphorylation by IRAKI can be assayed using methods known in the art, e.g., Strelow et al., FEBS Lett. 547:157-61, 2003. Similarly, activities of the other enzymes (e.g., the other kinases) shown in Tables 1-6 can also be examined using methods that are well known in the art.

[0034] As noted above, the Wnt signaling pathway regulators or polypeptides include both positive and negative regulators of Wnt signaling pathway. Therefore, test agents can be screened for ability to either up-regulate or down-regulate a biological activity of the Wnt signaling pathway regulator in the first assay step. Once test agents that modulate the Wnt signaling pathway regulator are identified, they are typically further tested for ability to modulate Wnt signaling. This further testing step is often needed to confirm that their modulatory effect on the Wnt signaling pathway regulator would indeed lead to regulation of Wnt signaling activities. For example, a test agent which inhibits a biological activity of a positive Wnt signaling pathway regulator (e.g., one shown in Tables 1, 3, and 5) needs to be further tested in order to confirm that such modulation can result in suppressed or reduced Wnt signaling activities. Similarly, a test agent which modulates (e.g., stimulating) a biological activity of a negative Wnt signaling pathway regulator (e.g., one shown in Tables 2, 4, and 6) can also be further tested to confirm that it can lead to down-regulation of Wnt signaling activities.

[0035] In both the first assaying step and the second testing step, either an intact

Wnt signaling pathway regulator, or a fragment thereof, may be employed. Molecules with sequences that are substantially identical to that of the Wnt signaling pathway regulators can also be employed. Analogs or functional derivatives of the Wnt signaling pathway regulator could similarly be used in the screening. The fragments or analogs that can be employed in these assays usually retain one or more of the biological activities of the Wnt signaling pathway regulator (e.g., kinase activity if the Wnt signaling pathway regulator employed in the first assaying step is a kinase). Fusion proteins containing such fragments or analogs can also be used for the screening of test agents. Functional derivatives of a Wnt signaling pathway regulator usually have amino acid deletions and/or insertions and/or substitutions while maintaining one or more of the bioactivities and therefore can also be used in practicing the screening methods of the present invention. A functional derivative can be prepared from a Wnt signaling pathway regulator by proteolytic cleavage followed by conventional purification procedures known to those skilled in the art. Alternatively, the functional derivative can be produced by recombinant DNA technology by expressing only fragments of a Wnt signaling pathway regulator that retain one or more of their bioactivities.

IV. Test Compounds

[0036] Test agents or compounds that can be screened with methods of the present invention include polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycines, oligocarbamates, polypeptides, saccharides, fatty acids, steroids, purines, pyrrolidines, derivatives, structural analogs or combinations thereof. Some test agents are synthetic molecules, and others natural molecules.

[0037] Test agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. Combinatorial libraries can be produced for many types of compound that can be synthesized in a step-by-step fashion. Large combinatorial libraries of compounds can be constructed by the encoded synthetic libraries (ESL) method described in WO 95/12608, WO 93/06121, WO 94/08051, WO 95/35503 and WO 95/30642. Peptide libraries can also be generated by phage display methods (see, e.g., Devlin, WO 91/18980). Libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts can be obtained from commercial sources or collected in the field. Known pharmacological agents can be subject to directed or random chemical modifications, such as acylation, alkylation, esterifϊcation, amidification to produce structural analogs.

[0038] The test agents can be naturally occurring proteins or their fragments.

Such test agents can be obtained from a natural source, e.g., a cell or tissue lysate. Libraries of polypeptide agents can also be prepared, e.g., from a cDNA library commercially available or generated with routine methods. The test agents can also be peptides, e.g., peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred. The peptides can be digests of naturally occurring proteins, random peptides, or "biased" random peptides. In some methods, the test agents are polypeptides or proteins. The test agents can also be nucleic acids. Nucleic acid test agents can be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids. For example, digests of prokaryotic or eukaryotic genomes can be similarly used as described above for proteins.

[0039] In some preferred methods, the test agents are small molecule organic compounds, e.g., chemical compounds with a molecular weight of not more than about 1,000 or not more than about 500. Preferably, high throughput assays are adapted and used to screen for such small molecules. In some methods, combinatorial libraries of small molecule test agents as described above can be readily employed to screen for small molecule compound modulators of Wnt signaling. A number of assays are available for such screening, e.g., as described in Schultz (1998) Bioorg Med Chem Lett 8:2409-2414; Weller (1997) MoI Divers. 3:61-70; Fernandes (1998) Curr Opin Chem Biol 2:597-603; and Sittampalam (1997) Curr Opin Chem Biol 1:384-91.

[0040] Libraries of test agents to be screened with the claimed methods can also be generated based on structural studies of the Wnt signaling pathway regulators discussed above or their fragments. Such structural studies allow the identification of test agents that are more likely to bind to the Wnt signaling pathway regulators. The three-dimensional structures of the Wnt signaling pathway regulators can be studied in a number of ways, e.g., crystal structure and molecular modeling. Methods of studying protein structures using x-ray crystallography are well known in the literature. See Physical Bio-chemistry, Van Holde, K. E. (Prentice-Hall, New Jersey 1971), pp. 221-239, and Physical Chemistry with Applications to the Life Sciences, D. Eisenberg & D. C. Crothers (Benjamin Cummings, Menlo Park 1979). Computer modeling of Wnt signaling pathway regulators' structures provides another means for designing test agents to screen for modulators of Wnt signaling pathways. Methods of molecular modeling have been described in the literature, e.g., U.S. Patent No. 5,612,894 entitled "System and method for molecular modeling utilizing a sensitivity factor," and U.S. Patent No. 5,583,973 entitled "Molecular modeling method and system." In addition, protein structures can also be determined by neutron diffraction and nuclear magnetic resonance (NMR). See, e.g., Physical Chemistry, 4th Ed. Moore, W. J. (Prentice-Hall, New Jersey 1972), and NMR of Proteins and Nucleic Acids, K. Wuthrich (Wiley-Interscience, New York 1986).

[0041] Modulators of the present invention also include antibodies that specifically bind to a Wnt signaling pathway regulator in Tables 1-6. Such antibodies can be monoclonal or polyclonal. Such antibodies can be generated using methods well known in the art. For example, the production of non-human monoclonal antibodies, e.g., murine or rat, can be accomplished by, for example, immunizing the animal with a Wnt signaling pathway regulator in Tables 1-6 or its fragment (See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor New York). Such an immunogen can be obtained from a natural source, by peptides synthesis or by recombinant expression.

[0042] Humanized forms of mouse antibodies can be generated by linking the

CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques. See Queen et al., Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989) and WO 90/07861. Human antibodies can be obtained using phage-display methods. See, e.g., Dower et al., WO 91/17271; McCafferty et al., WO 92/01047. In these methods, libraries of phage are produced in which members display different antibodies on their outer surfaces. Antibodies are usually displayed as Fv or Fab fragments. Phage displaying antibodies with a desired specificity are selected by affinity enrichment to a Wnt signaling pathway regulator in Tables 1-6.

[0043] Human antibodies against a Wnt signaling pathway regulator can also be produced from non-human transgenic mammals having transgenes encoding at least a segment of the human immunoglobulin locus and an inactivated endogenous immunoglobulin locus. See, e.g., Lonberg et al., WO93/12227 (1993); Kucherlapati, WO 91/10741 (1991). Human antibodies can be selected by competitive binding experiments, or otherwise, to have the same epitope specificity as a particular mouse antibody. Such antibodies are particularly likely to share the useful functional properties of the mouse antibodies. Human polyclonal antibodies can also be provided in the form of serum from humans immunized with an immunogenic agent. Optionally, such polyclonal antibodies can be concentrated by affinity purification using a Wnt signaling pathway regulator or its fragment. V. Screening for Modulators of Wnt Signaling Pathway Regulators

[0044] Typically, test agents are first screened for ability to modulate a biological activity of a Wnt signaling pathway regulator identified by the present inventors. A number of assay systems can be employed in this screening step. The screening can utilize an in vitro assay system or a cell-based assay system. In this screening step, test agents can be screened for binding to a Wnt signaling pathway regulator, altering expression level of the Wnt signaling pathway regulator, or modulating other biological activities (e.g., enzymatic activities) of the Wnt signaling pathway regulator.

1. modulating binding activities of Wnt signaling pathway regulators

[0045] In some methods, binding of a test agent to a Wnt signaling pathway regulator is determined in the first screening step. Binding of test agents to a Wnt signaling pathway regulator can be assayed by a number of methods including e.g., labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.), and the like. See, e.g., U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168; and also Bevan et al., Trends in Biotechnology 13:115-122, 1995; Ecker et al., Bio/Technology 13:351-360, 1995; and Hodgson, Bio/Technology 10:973-980, 1992. The test agent can be identified by detecting a direct binding to the Wnt signaling pathway regulator, e.g., co- immunoprecipitation with the Wnt signaling pathway regulator by an antibody directed to the Wnt signaling pathway regulator. The test agent can also be identified by detecting a signal that indicates that the agent binds to the Wnt signaling pathway regulator, e.g., fluorescence quenching or FRET.

[0046] Competition assays provide a suitable format for identifying test agents that specifically bind to a Wnt signaling pathway regulator. In such formats, test agents are screened in competition with a compound already known to bind to the Wnt signaling pathway regulator. The known binding compound can be a synthetic compound. It can also be an antibody, which specifically recognizes the Wnt signaling pathway regulator, e.g., a monoclonal antibody directed against the Wnt signaling pathway regulator. If the test agent inhibits binding of the compound known to bind the Wnt signaling pathway regulator, then the test agent also binds the Wnt signaling pathway regulator.

[0047] Numerous types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al., Methods in Enzymology 9:242-253, 1983); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614-3619, 1986); solid phase direct labeled assay, solid phase direct labeled sandwich assay (see, Harlow and Lane, "Antibodies, A Laboratory Manual," Cold Spring Harbor Press, 3^rd ed., 2000); solid phase direct label RIA using ¹²⁵I label (see Morel et al., MoI. Immunol. 25(1):7-15, 1988); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546-552, 1990); and direct labeled RIA (Moldenhauer et al., Scand. J. Immunol. 32:77-82, 1990). Typically, such an assay involves the use of purified polypeptide bound to a solid surface or cells bearing either of these, an unlabelled test agent and a labeled reference compound. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test agent. Usually the test agent is present in excess. Modulating agents identified by competition assay include agents binding to the same epitope as the reference compound and agents binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference compound for steric hindrance to occur. Usually, when a competing agent is present in excess, it will inhibit specific binding of a reference compound to a common target polypeptide by at least 50 or 75%.

[0048] The screening assays can be either in insoluble or soluble formats. One example of the insoluble assays is to immobilize a Wnt signaling pathway regulator or its fragment onto a solid phase matrix. The solid phase matrix is then put in contact with test agents, for an interval sufficient to allow the test agents to bind. After washing away any unbound material from the solid phase matrix, the presence of the agent bound to the solid phase allows identification of the agent. The methods can further include the step of eluting the bound agent from the solid phase matrix, thereby isolating the agent. Alternatively, other than immobilizing the cellular regulator, the test agents are bound to the solid matrix and the Wnt signaling pathway regulator is then added.

[0049] Soluble assays include some of the combinatory libraries screening methods described above. Under the soluble assay formats, neither the test agents nor the Wnt signaling pathway regulator are bound to a solid support. Binding of a Wnt signaling pathway regulator or fragment thereof to a test agent can be determined by, e.g., changes in fluorescence of either the Wnt signaling pathway regulator or the test agents, or both. Fluorescence may be intrinsic or conferred by labeling either component with a fluorophor.

[0050] In some binding assays, either the Wnt signaling pathway regulator, the test agent, or a third molecule (e.g., an antibody against the Wnt signaling pathway regulator) can be provided as labeled entities, i.e., covalently attached or linked to a detectable label or group, or cross-linkable group, to facilitate identification, detection and quantification of the polypeptide in a given situation. These detectable groups can comprise a detectable polypeptide group, e.g., an assayable enzyme or antibody epitope. Alternatively, the detectable group can be selected from a variety of other detectable groups or labels, such as radiolabels (e.g., ¹²⁵1, ³²P, ³⁵S) or a chemiluminescent or fluorescent group. Similarly, the detectable group can be a substrate, cofactor, inhibitor or affinity ligand. 2. modulating other activities ofWnt signaling pathway regulators

[0051] Binding of a test agent to a Wnt signaling pathway regulator provides an indication that the agent can be a modulator of the Wnt signaling pathway regulator. It also suggests that the agent may modulate Wnt signaling pathway by acting on the Wnt signaling pathway regulator. Thus, a test agent that binds to a Wnt signaling pathway regulator can be tested for ability to modulate a Wnt signaling activity (i.e., in the second testing step outlined above). Alternatively, a test agent that binds to a Wnt signaling pathway regulator can be further examined to determine whether it indeed modulates a biological activity (e.g., an enzymatic activity) of the Wnt signaling pathway regulator. The existence, nature, and extent of such modulation can be tested with an activity assay. More often, such activity assays can be used independently to identify test agents that modulate activities of a Wnt signaling pathway regulator (i.e., without first assaying their ability to bind to the Wnt signaling pathway regulator).

[0052] In general, the methods involve adding a test agent to a sample containing a Wnt signaling pathway regulator in the presence or absence of other molecules or reagents which are necessary to test a biological activity of the Wnt signaling pathway regulator (e.g., enzymatic activity if the Wnt signaling pathway regulator is an enzyme), and determining an alteration in the biological activity of the Wnt signaling pathway regulator. Methods for monitoring various enzymatic activities are well known in the art. For example, for Wnt signaling pathway regulator NME2 (listed in Table 6), its enzymatic activities can be assessed by the methods described in Hippe et al., J Biol Chem.278:7227-33, 2003. Briefly, NME2 encodes the B isoform of nucleoside diphosphate kinase (NDPK). NDPK exists as a hexamer composed of the A isoform and (encoded by NMEl) and the B isoform. Modulation of NME2 enzymatic activity can be examined by monitoring NDPK catalyzed formation of nucleotides from [³H]GDP. In addition, many other assays for monitoring protein kinase activities are also described in the art. These include assays reported in, e.g., Chedid et al., J. Immunol. 147: 867-73, 1991; Kontny et al., Eur J Pharmacol. 227: 333-8, 1992; Wang et al., Oncogene 13: 2639- 47, 1996; Murakami et al., Oncogene 14: 2435-44, 1997; Pyrzynska et al., J. Neurochem.74: 42-51, 2000; Berry et al., Biochem Pharmacol. 62: 581-91, 2001; Cai et al., Chin Med J (Engl). 114: 248-52, 2001. Any of these methods may be employed and modified to assay modulatory effect of a test agent on a Wnt signaling pathway regulator that is a kinase, e.g., LOC283458, CDK5, IRAKI, CAMKlG, FLTl, DGKA, JAK2, MAP2K4, or PCTKl.

[0053] In addition to assays for screening agents that modulate enzymatic or other biological activities of a Wnt signaling pathway regulator, the activity assays also encompass in vitro screening and in vivo screening for alterations in expression level of the Wnt signaling pathway regulator (e.g., NME2 expression level). These assays can be performed using methods well known and routinely practiced in the art, e.g., Samrbook et al., supra; and Brent et al., supra. In some embodiments, endogenous levels of a Wnt signaling pathway regulator can be directly monitored in cells normally expressing it. In some embodiments, expression or cellular level of a Wnt signaling pathway regulator can be examined in an expression system using cloned cDNA or genomic sequence encoding the Wnt signaling pathway regulator.

[0054] Alternatively, modulation of expression of a Wnt signaling pathway regulator can be examined in a cell-based system by transient or stable transfection of an expression vector into cultured cell lines. Assay vectors bearing transcription regulatory sequences (e.g., promoter) of a Wnt signaling pathway regulator operably linked to reporter genes can be transfected into any mammalian host cell line for assays of promoter activity. Constructs containing a transcription regulatory element of the Wnt signaling pathway regulator that is operably linked to a reporter gene can be prepared using only routinely practiced techniques and methods of molecular biology (see, e.g., Sambrook et al. and Brent et al., supra). General methods of cell culture, transfection, and reporter gene assay have been described in the art, e.g., Brent et al, supra; and Transfection Guide, Promega Corporation, Madison, WI (1998). Any readily transfectable mammalian cell line may be used to assay expression of a reporter gene, e.g., CHO, COS, HCTl 16, HEK293, MCF-7, and HepG2 can be employed.

[0055] When inserted into the appropriate host cell, the transcription regulatory element (e.g., promoter) in the expression vector induces transcription of the reporter gene by host RNA polymerases. Reporter genes typically encode polypeptides with an easily assayable en2ymatic activity that is naturally absent from the host cell. Typical reporter polypeptides for eukaryotic promoters include, e.g., chloramphenicol acetyltransferase (CAT), firefly or Renilla luciferase, beta-galactosidase, beta-glucuronidase, alkaline phosphatase, and green fluorescent protein (GFP).

VI. Testing Modulating Agents for Regulators ofWnt Signaling Pathway

[0056] Once modulating compounds have been identified to bind to a Wnt signaling pathway regulator and/or to modulate a biological activity (including expression level) of the Wnt signaling pathway regulator, they can be further tested for ability to modulate Wnt signaling pathways. Typically, this screening step involves monitoring effects of the modulating compounds on a Wnt signaling related activity. The screening is performed in the presence of the Wnt signaling pathway regulator on which the modulating compounds act. Preferably, this screening step is performed in vivo using cells that endogenously express the Wnt signaling pathway regulator. As a control, effect of the modulating compounds on a cell that does not express the Wnt signaling pathway regulator may also be examined. The Wnt signaling pathway regulator against which the modulating agents are identified in the first screening step can be either expressed endogenously by the cell or expressed from second expression vector. For example, if the Wnt signaling pathway regulator (e.g., encoded by a mouse gene) used in the first screening step is not endogenously expressed by the cell line (e.g., a human cell line), a second vector expressing the polypeptide can be introduced into the cell. By comparing a Wnt signaling related activity in the presence or absence of a test compound, regulatory activities of the compounds on Wnt signaling pathway can be identified.

[0057] Regulation of Wnt signaling activities by the modulating compounds can be examined with a number of methods known to the skilled artisans. Any Wnt signaling related activities can be assayed in the screening. In some methods, the screening monitors expression of a gene that is regulated by the Wnt signaling pathway. For example, the screening can monitor any regulatory effect of the compounds on the expression levels of key Wnt pathway members or other proteins that are responsive to Wnt signaling. For example, the screening can examine the modulating compounds for ability to regulate cellular levels of β-catenin in response to Wnt signaling. This can be performed using methods known in the art, e.g., Wright et al., Biochem Biophys Res Commun. 263:384-8, 1999; and Holman et al. (J. Biol. Chem. 277:34727-34735, 2002). In addition to β-catenin, the screening can also monitor effect of the compounds on the expression of other genes that are responsive to Wnt signaling. Examples include genes up-regulated by Wnt and β-catenin signaling such as c-myc, c-jun, fra-1, and cyclin Dl.

[0058] In some embodiments, instead of directly assaying expression levels of

Wnt pathway components or target genes, the screening can involve measuring expression of a reporter gene under the control of a transcription regulatory element (e.g., an enhancer or a promoter element) that is responsive to Wnt signaling. For example, as detailed in the Example below, the screening can monitor in HEK 293T host cells expression of a luciferase reporter gene in a Wnt-responsive reporter construct, TOPFlash. In addition to the reporter gene, this reporter construct contains a promoter under the control of multimeric TCF/LEF binding site which is active only in the presence of β-catenin. Therefore, this reporter system allows monitoring of the level of β-catenin and Wnt signaling activities. Similar assay systems have also been described in the art. For example, Hocevar et al. (EMBO J. 22:3084-3094, 2003) employed the TOPFlash reporter construct to show that expression of the adaptor molecule Disabled-2 (Dab2) in NIH-3T3 cells abrogated Wnt-3A mediated stimulation of TOPFlash activity. Jansson et al. (Proc Natl Acad Sci USA 102:1460-5, 2005) described a reporter assay by which β-catenin induced upregulation of luciferase expression driven by PPAR-responsive elements is detected in HEK293 cells. Sampson et al. (EMBO J. 20: 4500-4511, 2001) employed reporter assays to show that HMG-box repressor HBPl inhibits promoter activities of several Wnt pathway components (e.g., Wnt, β-catenin, GSK3 and LEF/TCF) or targets of the Wnt pathway (e.g., cyclin Dl and c-myc).

[0059] Any of these assays systems may be employed and modified in the present invention to screen the modulating compounds for ability to regulate Wnt signaling. Other than HEK293T cells, any readily transfectable mammalian cell line may be used to in the in vitro reporter assay systems. Examples include HEK 293, CHO, COS, HepG2, NIH 3T3, HCTl 16, Caco 2, HUVEC, MCF-7, MCF-IOA, CCD-I8C0, and NOV- 31 cell lines. In addition to luciferase gene, other reporter genes that can be employed in the reporter construct include, e.g., chloramphenicol acetyltransferase (CAT), beta- galactosidase, beta-glucuronidase, alkaline phosphatase, and green fluorescent protein (GFP). General methods of cell culture, transfection, and reporter gene assay have been described in the art, e.g., Brent et al., supra; and Transfection Guide, Promega Corporation, Madison, WI (1998).

[0060] Instead of employing an in vitro reporter assay as described above, some other screening methods examine the modulating compounds for ability to regulate other cellular processes or activities regulated by Wnt signaling pathway. In these methods, the compounds modulating a Wnt signaling pathway regulator can be further screened for activity in regulating a Wnt-mediated activity such as cell proliferation or differentiation. For example, Wright et al. (Biochem Biophys Res Commun. 263:384-8, 1999) described a proliferation assay to study proliferation of primary endothelial cells in response to Wntl expression. In Wright et al., a Wntl -expressing construct was rransfected into mouse brain microvascular endothelial cells. Following incubation and expression of Wntl in the cells, proliferation of the cells was measured using a MTT assay. Similarly, Castelo-Branco et al. (Proc Natl Acad Sci USA. 100:12747-52, 2003) assessed the role of Wnt signaling in the development of ventral midbrain dopaminergic (DA) neurons. Using in vitro cultured precursor neuron cells from rats, they showed that Wntl, Wnt3a and Wnt5a can enhance development of mature DA neurons from ventral midbrain precursors. Another in vitro culture system is described in Viti et al., J Neurosci. 23:5919-27, 2003. Here, explants of dorsolateral embryonic mouse cortex were used to examine the effects of Wnts on progenitor maturation in the cortex. Proliferation of progenitor cells that express Wnt exogenously was assessed by staining for proliferating cell nuclear antigen (PCNA) or by incorporation of bromodeoxyuridine (BrdU).

[0061] In still some other methods, other than in vitro systems, the compounds are examined for ability to modulate a Wnt signaling related activity via an in vivo system, e.g., with a whole animal model. For example, as illustrated in the Examples below, a Xenopus system can be used to further screen the compounds for ability to modulate Wnt signaling activities. During early Xenopus development, Wnt antagonism plays an essential role in head specification. Inhibition of Wnt signaling can result in large heads and forebrains. Conversely, an up-regulation of Wnt signaling leads to microcephalic embryos. To screen for compounds that modulate Wnt signaling, Xenopus embryos can be treated with a compound that modulate a Wnt signaling pathway regulator identified in the first screening step. For example, the embryo can be administered with the compound at a stage 10.5 (gastrulation stage) for, e.g., 24 hours. The treated embryo is then allowed to develop, e.g., until stage 40 (tadpole stage). Development of the embryo is then examined using techniques well known in the art and compared to an embryo that has not been treated with the compound or that has been treated with the vehicle. A Wnt- modulating compound is identified if treatment with a compound leads to significant phenotypic changes in the embryo development. For example, the phenotypic changes can be substantial head defects ranging from significantly diminished heads and complete loss of eyes to reduced eyes and heads while the posterior structures of tadpoles remained intact.

[0062] Similar animal models are described in the art which can be employed in the present invention to screening for Wnt-modulating compounds. For example, a Xenopus system is described in Yamada et al. (Genes Cells. 8:677-84, 2003). Using Xenopus embryos, this animal model examines effect of β-catenin signaling on ectopic axis formation in Xenopus embryos. Another example is provided in Suksaweang et al. (Dev Biol. 266:109-22, 2004) which employed chicken liver to show that β-catenin/Wnt signaling plays a role in regulating growth zone activities during liver development. Any of these in vivo systems can be employed and/or modified in the present invention to screen the identified compounds for ability to modulate Wnt signaling. This additional screening could confirm that the compounds, by regulating a Wnt signaling pathway regulator, can indeed lead to modulation of Wnt signaling activities.

VII. Therapeutic Applications

[0063] The present invention provides novel methods and compositions for modulating Wnt signaling pathways and for treating diseases and conditions associated with or mediated by deregulated Wnt signaling activities. Wnt signaling pathway is known to be involved in the regulation of cell growth, oncogenesis and apoptosis. For example, deregulation of Wnt signaling pathway has been implicated in tumorigenesis, such as colorectal cancers, β-catenin regulates transcription of oncogenes such as c-Myc and cyclin Dl, as well as initiation of tumor metastasis. Wnt ligand and signaling have also been shown to regulate stem cell proliferation and differentiation in several systems. The Wnt-modulating compounds that can be identified in accordance with the present invention provide new approaches to treat various clinical conditions or disease states that are linked to abnormal Wnt signaling, e.g., cancers. In addition, they are also useful for preventing or modulating the development of such diseases or disorders in a subject suspected of being, or known to be, prone to such diseases or disorders.

[0064] Typically, a subject in need of treatment is administered with a pharmaceutical composition comprising one or more of the Wnt-modulating compounds described above. The compounds can be directly administered under sterile conditions to the subject to be treated. They can be administered alone or as the active ingredient of a pharmaceutical composition. Some applications employ small molecule Wnt-modulating compounds that can be identified in accordance with the above described screening methods. Some other applications employ antagonist or agonist antibodies which specifically recognize one or more of the Wnt signaling pathway regulators identified herein. Wnt signaling modulators that can be used in therapeutic applications of the invention further include agents that specifically modulate expression or cellular levels of the Wnt signaling pathway regulators. Such compounds include, e.g., nucleic acid modulators such as short interfering RNA (siRNA), microRNA (miRNA), and synthetic hairpin RNA (shRNA), anti-sense nucleic acid, or complementary DNA (cDNA). For example, as illustrated in the Examples below, a siRNA that specifically targets NME2 can be used to inhibit tumor metastasis.

[0065] Therapeutic composition of the present invention can be combined with or used in association with other therapeutic agents. For example, a subject with cancer may be treated concurrently with conventional chemotherapeutic agents, particularly those used for tumor and cancer treatment. Examples of such chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis- chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6- mercaptopurine, 6-thioguanine, cytarabine (CA), 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5- fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, trimetrexate, teniposide, cisplatin and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., pp. 1206-1228, Berkow et al., eds., Rahay, NJ., 1987).

[0066] Pharmaceutical compositions of the present invention typically comprise at least one active ingredient together with one or more acceptable carriers thereof. Pharmaceutically carriers enhance or stabilize the composition, or to facilitate preparation of the composition. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered {e.g., nucleic acid, protein, modulatory compounds or transduced cell), as well as by the particular method used to administer the composition. They should also be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the subject. This carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral, sublingual, rectal, nasal, or parenteral. For example, the Wnt- modulating compound can be complexed with carrier proteins such as ovalbumin or serum albumin prior to their administration in order to enhance stability or pharmacological properties.

[0067] The pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, and the like. The concentration of therapeutically active compound in the formulation may vary from about 0.1 100% by weight. Therapeutic formulations are prepared by any methods well known in the art of pharmacy. The therapeutic formulations can be delivered by any effective means which could be used for treatment. See, e.g., Goodman & Gilman's The Pharmacological Bases of Therapeutics, Hardman et al., eds., McGraw-Hill Professional (10^th ed., 2001); Remington: The Science and Practice of Pharmacy, Gennaro, ed., Lippincott Williams & Wilkins (20^th ed., 2003); and Pharmaceutical Dosage Forms and Drug Delivery Systems, Ansel et al. (eds.), Lippincott Williams & Wilkins (7^th ed., 1999). [0068] The therapeutic formulations can conveniently be presented in unit dosage form and administered in a suitable therapeutic dose. A suitable therapeutic dose can be determined by any of the well known methods such as clinical studies on mammalian species to determine maximum tolerable dose and on normal human subjects to determine safe dosage. Except under certain circumstances when higher dosages may be required, the preferred dosage of a Wnt-modulating agent of the present invention usually lies within the range of from about 0.001 to about 1000 mg, more usually from about 0.01 to about 500 mg per day. The preferred dosage and mode of administration of a Wnt-modulating agent can vary for different subjects, depending upon factors that can be individually reviewed by the treating physician, such as the condition or conditions to be treated, the choice of composition to be administered, including the particular Wnt- modulating agent, the age, weight, and response of the individual subject, the severity of the subject's symptoms, and the chosen route of administration. As a general rule, the quantity of a Wnt-modulating agent administered is the smallest dosage which effectively and reliably prevents or minimizes the conditions of the subjects. Therefore, the above dosage ranges are intended to provide general guidance and support for the teachings herein, but are not intended to limit the scope of the invention.

EXAMPLES

[0069] The following examples are provided to further illustrate, but not to limit the present invention.

EXAMPLES

[0070] The following examples are provided to illustrate, but not to limit the present invention.

Example 1. Identification of Novel Regulators of Wnt Signaling Pathway

[0071] We performed genome-wide gain-of-function (by overexpressing cDNAs) and loss-of-function (by knocking-down expression of genes through synthetic siRNAs or shDNAs) screens. A number of novel Wnt activators and inhibitors were identified from the screens, in addition to several known Wnt components (which validate the authenticity of the screens). A transfection grade T-cell factor reporter (TOPFlash) under the control of nuclear β-catenin is used to reveal the level of Wnt signaling. In order to obtain information on both up-regulation and down-regulation, the basal level of Wnt signaling is increased by stimulating with condition medium collected from stably transfected L-wnt3A cells (ATCC).

Gain-of-function Screen

[0072] An arrayed and annotated cDNA library in a mammalian expression vector was used for gain-of-function screen. The library, consisting of approximately 20,000 full-length human cDNAs was spotted in 384 well plates such that each well contained an individual cDNA with known identity. In a semi-automated process, cDNAs were incubated with a non-liposomal transfection reagent (Fugeneό, Roche Applied Science, Indianapolis, IN) and TOPFlash (Upstate, Lake Placid, NY) vector. This vector contains two sets of three copies of the TCF binding site upstream of the Thymidine Kinase (TK) minimal promoter and luciferase open reading frame. Human kidney derived HEK 293T were then introduced into each well to complete the transfection procedure. After 24 hours of incubation at 37 C and 5% CO2, equal volumes L-wnt3A condition medium was added as a stimulant. The cells were incubated for another 2 days. Bright-glo reagent (Promega, Madison, WI) was then added to each well and relative luminescence was quantitated using an Acquest (LJL Biosystems, Sunnyvale, CA) plate reader. The assay was run in duplicates and the plate data were normalized to a mean value and compared across the library (20,000 wells). As respectively shown in Tables 1 and 2, cDNAs with mean activity values > or < 3 standard deviations from the whole experimental mean were selected from the library, and were amplified and isolated utilizing commercially available DNA isolation reagents (Qiagen, Germany). These samples were reconfirmed utilizing the methods outlined above. Other than the novel Wnt pathway regulators listed in the tables, several known Wnt modulators were also uncovered in the screen. Examples include β-catenin (CTNNBl and CTNNAl), GSK3A, WNT5A, GSK3B, cullin 1, CSNKlD, CSNKlE, JUP, FBXWlB₅ and dishevelled 2. This validates the authenticity of the screen. Loss-of-function Screens

[0073] a) PCR-based siRNA library (Zheng et al., Proc. Natl. Acad. Sci.

USA, 101: 135-140, 2004): Using siRNAs generated by PCR which target a library of different genes, effect of loss-of-function of these genes on Wnt signaling was examined. This screen was performed in the same way as that described above for the cDNA library screen. As shown in Tables 3 and 4, a number of novel regulators of Wnt signaling pathway were identified. Several known modulators of Wnt signaling pathway were also among the hits identified from the screening, e.g., PPP2R4, FZD4, LRP6, and β-catenin.

[0074] b) Synthetic siRNA library: This screen was similarly performed as that described above except that Lipofectamine 2000 (Invitrogen) was used instead of Fugeneβ (Roche). Around 75 Wnt regulators (both novel and known) were identified. The novel Wnt up-regulators and down-regulators are listed in Tables 5 and 6, respectively.

Table 1. Positive Wnt signaling pathway regulators identified by cDNA screening

Table 2. Negative Wnt signaling pathway regulators identified by cDNA screening

Table 3. Positive Wnt signaling pathway regulators identified by PCR-based siRNA screening

Table 4. Negative Wnt signaling pathway regulators identified by PCR-based siRNA screenin

Table 5. Positive Wnt signaling pathway regulators identified by synthetic siRNA screenin

Table 6. Negative Wnt signaling pathway regulators identified by synthetic siRNA screenin

Example 2. Further Study and Characterization ofWnt Signaling Modulators

[0075] If overexpression of cDNA of a gene induces activation or inhibitor of the TOPFlash reporter in the above-described screens, then the gene is apparently sufficient for activation or inhibition of the Wnt signaling pathway. On the other hand, if knockdown of a gene through RNAi induces activation or inhibition of the TOPFlash reporter, then the gene appears to be required for activation or inhibition of the Wnt signaling pathway. To further confirm results from the cDNA, siRNA, and shRNA screens, we performed cross examination experiments. Briefly, cDNAs (obtained from Origene and MGC) encoding the hits identified in the loss-of-function screens or siRNAs (synthesized in-house) targeting the hit genes from the gain-of-function screens were transfected into HEK293 cells, using Fugene 6 as the transfection reagent. The cells were then incubated at 37⁰C for 36 h followed by luciferase assay. The hits listed in the Tables above have all been so tested and showed consistent results in at least two different assays. Form these studies, it is clear that these genes are both sufficient and required in the Wnt signaling pathway.

[0076] Wnt signaling has been implicated in cancer metastasis. One hit from the siRNA screen (loss-of-function), NME2, was chosen in follow-up studies to examine effect of modulating Wnt signaling pathway regulators on cancer metastasis. Expression level of NME2 in metastatic breast cancer MDA-MB-231 cells was modulated by overexpressing cDNA encoding NME2 or siRNA targeting NME2 in a transwell migration assay. The anti-sense strand of the NME2-targeting siRNA has a sequence of 5'- AACACCUGAAGCAGCACUACATT- 3' (SEQ ID NO:1). The assay was performed as follows. MDA-MB-231 cells transfected with cDNAs or siRNAs were plated at a density of 5,000 cells/well (in RPMIl 640 supplemented with 0.5% BSA) in the upper chamber of the Transwell filter in a 24-well plate, with medium containing DMEM supplemented with 10% FBS located in the lower chamber. The cells were allowed to migrate for 12 hours. Thereafter, cells remaining on the upper surface of the filter were removed while cells on the lower side were quantified by counting under a microscope. [0077] Results obtained from the transwell migration assay are shown in Figure

1. As indicated in the Figure, gain-of-function by overexpressing NME2-encoding cDNA increased cell migration compared to control cDNA. Consistently, loss-of-function by transfecting NME2-targeting siRNA had the opposite effect on the cellular migration. These results confirmed that NME2 plays a role in regulating cell migration.

[0078] Moreover, through collaboration with Travis Best and Randall T. Moon at the University of Washington (Seattle), in vivo biological and developmental effect of modulating NME2 expression in whole organism was also examined. Ectopic activation of the Wnt/β-catenin signaling pathway leads to duplication of the embryonic axis. This developmental process has been established as an excellent experimental means to test modulation of Wnt signaling in vivo. To examine the function of NME2 in whole organism, mRNA encoding NME2 (1 ng) was injected into Xenopus embryos. mRNA encoding GFP (1 ng) was used as a control. As shown in Figure 2, the results indicate that dual axis formation was greatly inhibited in embryos treated with NME2 mRNA compared to those treated with GFP mRNA. These data indicate that NME2 is a Wnt antagonist in Xenopus development.

***

[0079] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

[0080] All publications, GenBank sequences, ATCC deposits, patents and patent applications cited herein are hereby expressly incorporated by reference in their entirety and for all purposes as if each is individually so denoted.

Claims

WE CLAIM;

1. A method for identifying an agent that modulate Wnt signaling pathway, the method comprising:

(a) screening test agents to identify one or more modulating agents which modulate a biological activity or expression level of a Wnt signaling pathway regulator encoded by a polynucleotide selected from the members listed in Tables 1-6; and

(b) testing the modulating agents for ability to modulate Wnt signaling pathway.

2. The method of claim 1 , wherein the Wnt signaling pathway regulator positively modulates Wnt signaling pathway and is encoded by a polynucleotide selected from the members listed in Tables 1, 3, and 5.

3. The method of claim 1 , wherein the Wnt signaling pathway regulator negatively modulates Wnt signaling pathway and is encoded by a polynucleotide selected from the members listed in Tables 2, 4, and 6.

4. The method of claim 1 , wherein the modulating agents stimulate the biological activity of the Wnt signaling pathway regulator.

5. The method of claim 1 , wherein the modulating agents inhibit the biological activity of the Wnt signaling pathway regulator.

6. The method of claim 1 , wherein (b) comprises testing the modulating agents for ability to modulate expression of a gene responsive to β-catenin signaling.

7. The method of claim 6, wherein the gene encodes a component of the Wnt signaling pathway downstream of β-catenin or a target gene of Wnt signaling pathway.

8. The method of claim 1, wherein (b) comprises testing the modulating agents for ability to modulate expression of a reporter gene under the control of a promoter containing a TCF/LEF binding site.

9. The method of claim 1 , wherein (b) comprises testing the modulating agents for ability to modulate proliferation of a cell responsive to Wnt signaling.

10. The method of claim 1 , wherein the Wnt signaling pathway regulator is an enzyme, and the biological activity assayed is its enzymatic activity.

11. The method of claim 10, wherein the enzyme is a kinase.

12. The method of claim 11 , wherein the Wnt signaling pathway regulator is NME2 with Accession No. NM_002512.

13. The method of claim 1 , wherein the biological activity assayed is expression of a gene encoding the Wnt signaling pathway regulator.

14. A method for identifying a modulator of Wnt signaling pathway, the method comprising:

(a) contacting test compounds with a Wnt signaling pathway regulator encoded by a polynucleotide selected from the members listed in Tables 1-6;

(b) identifying one or more modulating compounds which modulate a biological activity of said Wnt signaling pathway regulator; and

(c) screening the modulating compounds for ability to regulate expression of a gene from a promoter that is responsive to β-catenin signaling; thereby identifying a modulator of Wnt signaling pathway.

15. The method of claim 14, wherein the promoter comprises a TCE/LEF binding site.

16. The method of claim 14, wherein the Wnt signaling pathway regulator positively modulates Wnt signaling pathway and is encoded by a polynucleotide selected from the members listed in Tables 1, 3, and 5.

17. The method of claim 14, wherein the Wnt signaling pathway regulator negatively modulates Wnt signaling pathway and is encoded by a polynucleotide selected from the members listed in Tables 2, 4, and 6.

18. The method of claim 14, wherein the modulating compounds stimulate the biological activity of the Wnt signaling pathway regulator.

19. The method of claim 14, wherein the modulating compounds inhibit the biological activity of the Wnt signaling pathway regulator.

20. The method of claim 14, wherein the Wnt signaling pathway regulator is an enzyme, and the biological activity is its enzymatic activity.

21. The method of claim 20, wherein the enzyme is a kinase.