WO2006052911A2 - Laforin expression in diagnosis and treatment of cancer and other diseases - Google Patents

Abstract

Methods of characterizing the etiology of a cancer and other human diseases comprising testing at least one cell for the presence of at least one of a) methylation of EPM2A gene; b) reduced expression of EPM2A gene; and c) mutation of EPM2A gene. Methods of treating cancer and other diseases are also provided.

Description

LAFORIN EXPRESSION IN DIAGNOSIS AND TREATMENT OF CANCER AND OTHER DISEASES

[001] Work leading to this invention was funded by NIH grants CA82355, CA69091 , and CA58033. The government has certain rights in this invention.

[002] This application claims priority to U.S. Provisional Application 60/626,266, filed November 8, 2004, the entire disclosure of which is incorporated herein by reference.

[003] This invention relates to diagnosis and treatment of cancer and other diseases. In particular, it relates to diseases that involve abnormal expression of the EPM2A gene, its product, laforin and/or its substrate GSK-3.

[004] In some embodiments, this invention relates to methods of characterizing an etiology of a cancer by testing at least one cancer cell for the presence of at least one of a) methylation of the EPM2A gene; b) reduced expression of the EPM2A gene; and c) mutation of the EPM2A gene. In some embodiments, the invention relates to methods of characterizing an etiology of a cancer by testing at least one cancer cell for the presence of lafora bodies.

[005] In some embodiments, the invention relates to methods of treating a cancer in a person identified as having a cancer associated with methylated EPM2A gene(s), by administering to the person an effective amount of a drug that demethylates DNA. In some embodiments, the invention relates to methods of treating a person identified as having a disease associated with reduced expression or mutation of the EPM2A gene, by activating EPM2A gene(s) in cells of the person's body. In these methods, the disease can be chosen, for example, from Lafora disease and cancer. [006] In some embodiments, the invention relates to methods of treating a person identified as having a disease associated with reduced expression or mutation of the EPM2A gene, by administering laforin to the person. In these methods, the disease can be chosen from, for example, Lafora disease and cancer. In some embodiments, administering the laforin involves administering the laforin protein to the person. In other embodiments, administering the laforin involves administering a laforin-expressing genetic construct to the person.

[007] In other embodiments, the invention involves methods of treating a person identified as having a disease associated with reduced expression or mutation of the EPM2A gene, by administering a genetically engineered GSK3β that cannot be phosphorylated at the Serine 9 position. In some embodiments, the GSK3/? that cannot be phosphorylated at the Serine 9 position includes an amino acid mutation at Serine 9 with Alanine or any other amino acids.

[008] In some embodiments, the invention relates to methods of treating a person identified as having a disease associated with reduced expression or mutation of EPM2A gene, by administering to the person at least one compound that restores laforin's ability to phosphorylate GSK3y?.

[009] The invention also relates to methods of identifying compounds that modify laforin's ability to phosphorylate GSK3/? at Serine 9, wherein the methods include a step of testing laforin's ability to phosphorylate GSK3/? in the presence and absence of the test compounds. In some embodiments, the laforin tested is wild-type laforin and the compounds identified aje either inhibitors or enhancers of laforin's ability to phosphorylate GSK3/?. In other embodiments, the laforin tested is mutated laforin and the compounds identified restore laforin's ability to phosphorylate GSK3β. The invention also provides methods of treating Alzheimer's disease or other diseases with over-activated GSK3 by administering to a person in need of treatment a compound identified according to the aforementioned methods.

[010] Still further, the invention provides methods of determining the type of cancer in a person, by assaying a tissue sample from the person for the presence of at least one of a) methylation of EPM2A gene; b) reduced expression of EPM2A gene; and c) mutation of EPM2A gene. In some embodiments, the person is a blood relative of a person having Lafora disease.

[011] In some embodiments, the invention is directed to methods of reducing the likelihood of cancer or other diseases in a person identified as positive for the presence of at least one of a) reduced expression of EPM2A, b) methylation of EPM2A gene, or c) mutation of EPM2A gene, comprising modifying the person's glucose metabolism. The person's glucose metabolism may be modified through diet modification. In other embodiments, the person's glucose metabolism is modified by administration of a. drug such as 2- deoxyglucose and/or metforin.

[012] In some embodiments, the invention is directed to methods of reducing the likelihood of diseases in a person identified as having over-activated GSK3, such as Alzheimer's disease, by administering drugs that reduce expression of EPM2a gene or its products. One of the compounds can be SiRNA using sequence information from Laforin cDNA.

[013] Additional features and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The features and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[014] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

[015] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiment(s) of the invention and together with the description, serve to explain the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS [016] Figure 1. A transgenic mouse (TG-B) line with high penetrance.

early lethality lymphoma. The transgenes, the re-arranged α and β chain genes

of the T cell receptor, were obtained from a CD8⁺ T cell clone with specificity for SV40 large T antigen. Since its generation, the transgenic mice have been backcrossed to B10.BR background for more than 30 generations. A. The survival distribution function among B10.BR, TG-B and F1 (TG-B x C57BL/6) mice. The incidence of lymphoma is significantly higher among TG-B in comparison to WT (P<0.0001) and F1 (TG-B x B6) mice (PO.0001). F1 (TG-B x B6) mice developed lymphoma at a significant rate in comparison to WT mice (PO.0001). B. Enlargement of spleen (top), thymus (middle) and mesenteric lymph nodes (MLN, bottom) in TG-B mice with tumor (left panels). Organs from a TG-B mouse that have not developed tumor are shown as controls. C. lmmunohistochemical staining with anti-Thyl antibody in spleens from TG-B mouse without tumor (a) and TG-B mouse with tumor (b). H&E sections of the liver (c) and the kidney (d) of the tumor-bearing TG-B mouse. [017] Figure 2. Identification of the integration site of the TCR transqene. A. Cytogenetic analysis of the TCR gene localization by FISH. The

probe used is the constant region of the TCR α chain. The location of the

transgene is indicated by an arrow in the left panel, the positions of chromosomes 10 and 14 are marked in the right panel. The signals in

chromosome 14 reflect the location of the endogenous TCR α loci. B.

Identification of the TCR integration site by PCR-based TOPO^®Walker method

using the TOPO^®Linker primers and the gene specific sequence primers from

both 3' and 5' termini of the TCR g and TCR β transgenes. The primer that

corresponded to the 3' terminus of the TCRα-chain yielded PCR products with

TCR gene sequence. The junction sequence of TCR Ca and chromosome 10

DNA is shown. This is extracted from the sequence of 4 clones. C.

Chromatogram depicting the junctional sequence of TCRα and β constructs. D.

Confirmation of the integration sites by Southern blot. The restriction enzyme map of endogenous and Tg alleles is shown in the left and a Southern blot autograph is shown in the right. E. A portion of the 10A2 region in relation to the integration site is indicated. Note that the integration site is in intron 1 of Epm2a, which encodes for laforin.

[018] Figure 3. Defective expression of laforin in lymphoma. A. Defective expression of EPM2A mRNA in transgenic mice with lymphoma (Tg- Tu), but not in the non-transgenic littermates (Ntg), or in young transgenic mice that have not yet developed lymphoma (Tg). Total RNA was isolated from thymi. The cDNA was prepared by reverse transcription and amplified using primers that amplify the nearly entire coding sequence of Epm2a. B. Absence of the laforin protein expression in Tg-Tu cells, as analyzed by Western blot using a polyclonal anti-laforin antibody. C. Down-regulation of the laforin protein in multiple murine malignancies of T, B cell lineages. The thymocytes from non-transgenic littermates (Ntg) and two transgenic mice with lymphoma (Tg-Tu) were examined as control. The malignant cell lines used were T cell lymphoma EL4, YAC-1 , BW5147 and RMA-S₁ B cell lymphoma A20, RAW8.1 , CH27, and myeloma S194. Data shown are representative of three independent experiments.

[019] Figure 4. Epm2a is hvpermethylated in lymphoma cells. A. Diagram of 5'-CpG island and the location of the 4 primer sets used to amplify the bisulfite treated DNA to examine the methylation status of the Epm2a gene. The 5'-CpG island is 1225 bp in length with 59.2% GC content. The ratio of observed CpG : expected CpG is 0.948. The length of PCR products and the number of CpG di-nucleotides (in parentheses) within each PCR products are labelled. B. Summary of the results of bisulfite sequencing of PCR products with LDM3 which contained 45 CpG di-nucleotides. Fifty seven clones were obtained from six lymphoma mice (clone numbers from each mouse are labelled in parentheses) and 45 clones were obtained from six transgenic littermates without lymphoma (10 each from three mice and 15 clones from a pool of three mouse thymi). Each circle represents the methylation status of a CpG di-nucleotide from 8-10 clones, except the last line of circles from which each circle represents results from 15 clones. Methylated and unmethylated alleles are shown as solid and open circles, respectively. C. 5-AzaC-induced Epm2a expression in lymphoma cell lines, but not in 3 non-lymphoid tumor cell lines. Cells were collected after 48 hr of treatment with or without 5-aza-2'-deoxycytidine. Total RNA was isolated and RT- PCR was done using primers that amplify the entire coding sequence of Epm2a. The cell lines used were T cell lymphoma EL4, YAC-1 and BW5147, mastocytoma P815, colon cancer MC38 and fibrosarcoma Meth A. D. DNA methylation in lymphoma cell lines. Genomic DNA from the cell lines were isolated and subjected to digested or undigested with methylation sensitive enzyme Sacll overnight. The 280 bp promoter was amplified by real-time PCR. To normalize against DNA loss associated with enzyme digestion and purification, the difference between Sacll-treated and untreated samples with 0% methylation (as determined by bisulfate sequencing, mouse No. 11992) was subtracted from all digested samples. DNA from a TG-B thymoma (No. 4012) with 100% methylation was used as positive control.

[020] Figure 5. Laforin suppresses the growth of tumor cells. A. Laforin induces apoptosis in lymphoma cells. V5 tagged Epm2a lentiviral expression construct was transduced into murine T lymphoma cell lines, EL-4, YAC-1 , RMA- S, and BW5147. Forty-eight hours after transduction, the cells were stained with FITC conjugated anti-V5 antibody and 7-amino-actinomycin (7-AAD) to measure DNA contents. Cells were gated for V5 negative (upper panels) and V5 positive cells (Epm2a transduced cells) (lower panels). DNA contents corresponding to 2C and 4C are indicated by arrows. The numbers represent the percentage of cells with DNA contents less than 2C. B. Laforin suppresses the growth of murine T cell lymphoma BW5147. BW5147 cells were transduced with either vector or Epm2a cDNA lentivirus. Polyclonal stable transfectants were injected subcutaneously into the RAG-2(-/-) BALB/c mice (3 x 10⁵ cells/mouse).The growth kinetics are presented. C. Laforin suppresses the growth of P185neu- transformed NIH3T3 cell line B104-1-1. B104-1-1 cells were transfected with either vector or Epm2a cDNA. Three stable clones from each group (Vector or Laforin) were injected into the RAG-2(-/-) BALB/c mice (3x10⁵ cells/mouse). The growth kinetics are presented. Data shown are representative of two independent experiments. D. RNAi silencing of laforin expression in fibroblast cell line EEF8 increases its tumorigenicity. EEF8 were transduced with either Epm2a-siRNA or its control lentivirus as detailed in methods. The insert shows the laforin protein levels of representative clones from each group. The control and silenced clones of EEF8 cells (1x10⁶/mouse) were injected subcutaneously into RAG-2(-/-) mice. The incidence and growth kinetics were recorded over a two-month period. Data shown represent two independent experiments. In combination, tumor was detected in all 8 mice that received the silenced clone, but in none of the 8 mice that received control clone.

[021] Figure 6. Laforin is a phosphatase of GSK-36 at Ser 9. A. Laforin

selectively prevented PDGF-induced phosphorylation of GSK-3β. Vector or

Iaforin-V5 transfected NIH3T3 cells were stimulated with PDGF for given periods of time. The activation of PDK (anti-phosphorylated Ser241), Akt (anti-

phosphorylated Ser473), GSK-3β (anti-phosphorylated Ser9), FKHR(anti-

phosphorylated Ser256), and AFX(anti-phosphorylated Ser193) were analyzed by Western blot. The amounts of protein loaded were revealed using the total Akt as determined by Western blot. B. Laforin dephosphorylated the phospho-

Ser 9, but not P-tyrosine 216 of GSK-3β and P-tyrosine 279 of GSK-3α. Two

stable laforin transfected clones are presented (D14 and D7). C. Laforin

prevented PDGF induced inactivation of GSK-3β kinase activity. Vector or

laforin-transfected NIH3T3 cells were stimulated with PDGF for 30 minutes and

the cells were lysed and subjected to immunoprecipitation with anti-GSK-3β

antibody. The immunoprecipitates were used for kinase assay to determine the

GSK-3β kinase activity. Recombinant Tau protein and ³²P-γ-ATP were used as substrates. Specific phosphorylation of Tau was determined by ³²P

autoradiograph. The GSK-3β in immunoprecipitates was determined by Western

blot with anti-GSK-3β. The radioactive intensity in each lane was normalized

based on the amounts of GSK-3β in each sample, as determined by Western blot. In order to compare different experiments, we arbitrarily assigned the

activity of the GSK-3β isolated from untreated vector-transfectants as 1.0.

Statistical difference was calculated by student t-tests. Data shown are summary of 3 independent experiments. D. Inhibitory silencing siRNA suppresses endogenous laforin and enhances the PDGF induced phosphorylation of GSK-

3β. Oligonucleotides encoding siRNA against the C-termini of Epm2a was cloned

into pSilencer™ 1.0-1)6 siRNA expression vector. The upper panel shows the efficiency of endogenous Epm2a suppression in three stable clones C1 , C2 and

C3 by RT-PCR. The activation of Akt (anti-phosphorylated Ser473) and GSK-3β (anti-phosphorylated Ser 9) from vector or siRNA C3 transfected NIH3T3 cells

were analyzed by Western blot. GSK-3β was the protein loading control. E.

Physical association between GSK-3β and laforin. Upper panel shows

association of transfected laforin with GSK3β. Vector or laforin-transfected

NIH3T3 cells were stimulated with PDGF for 30 min and cell lysates were

immunoprecipitated with either control IgGI or anti-GSK-3β monoclonal antibody.

The precipitates were then blotted with either anti-GSK-3β or anti-V5 antibodies

to detect the V5-tagged laforin protein. The lower panel shows association

between GSK-3β and Laforin in untransfected NIH 3T3 cells. As in the upper

panel, except that the cells were untransfected and the Laforin were probed with

anti-Laforin antibody. Laforin dephosphorylates GSK-3β in vitro., F. Laforin dephosphorylates GSK-3β but not BAD in vitro.. Recombinant GSK-3β or BAD

was phosphorylated by constitutively activated Akt in the presence of ATP. Wild

type or mutant laforin proteins (0.5 or 1.0 μg/sample) were added to the mixture

and after the phosphorylation is complete. The phosphatase activity was

determined by Western blot, using either anti-phospho-Ser9 GSK-3β or anti- phospho-BAD (S112) antibodies. Data shown represent at least three independent experiments.

[022] Figure 7. Laforin modulates Wnt signaling pathway. A. Vector (V), laforin (D2, D7, D14) or mutant (C265S)-transfected NIH3T3 cells were

treated with 0.5 μM PMA with or without recombinant Wnt-3A. At 3 hours after

stimulation, the cell lysates were analyzed for phosphorylation of GSK-3β at Ser

9 by Western blot. Protein loading is indicated by blotting with anti-GSK-3β

antibody. The nuclear fractions were isolated to determine the amounts of β-

catenin. Protein loadings in the nuclear fractions were indicated by anti-SP1 blot. B. TCF reporter assay. pTOPFLASH or pFOPFLASH TCF luciferase reporter vector was co-transfected with pRL-SV40 Ranilla control luciferase reporter plasmid into Vector (V), laforin (D14) or mutant laforin (C265S) stably transfected NIH3T3 cells. Twenty four hours after transfection, the cells were stimulated with Wnt-3A conditional medium or control medium overnight. Cell lysate supematants were evaluated for luciferase activity by Dual-luciferase reporter assay system. The results are shown after normalized to corresponding Renilla

reporter value. C. Silencing siRNA increased the GSK-3β-phosphorylation and β-

catenin nuclear accumulation by Wnt-3A. Vector (V) or C-terminal silencing

siRNA (C3) transfected NIH3T3 cells were cultured in the presence of 0.5 μM

PMA with or without recombinant Wnt-3A. Phospho-Ser 9 GSK-3β in cell lysate and β-catenin in nuclear fractions were analyzed by Western blot. D. Laforin

expression (B-D1) in tumor cell line B104-1-1 inhibited the GSK-3β-

phosphorylation and β-catenin nuclear accumulation induced by Wnt-3A. E.

Increased GSK-3β-phosphorylation and β-catenin nuclear accumulation in the

thymocytes from transgenic mice with lymphoma (Tg-Tu), but not the normal mice (Ntg) and the transgenic mice yet to develop lymphoma (Tg). Freshly isolated thymi were lysed and nuclear and cytoplasmic proteins were analyzed

for the presence of P-GSK-3β (Ser 9), and nuclear β-catenin. Data shown

represent three independent experiments.

[023] Figure S1. Over-expression of genes adjacent to TCR transqene integration site does not lead to tumorigenesis. a. Genomic organization of Epm2a and the only two other genes within 300 Kb of the integration site on chromosome 10. The cDNA designations are based on Celera database, b. Expression of the two adjacent genes in non-transgenic littermates (Ntg), transgenic mice without tumor (Tg) and transgenic mice that developed tumor (Tg-Tu), as determined by RNase protection assay, c. Summary of observation period of 8 transgenic founder mice that express mCG16603 under the control of the proximal lck promoter to induce high expression in the thymus.

[024] Figure S2. Over-expression of laforin selectively prevented

PDGF-induced phosphorylation of GSK-3β. Vector or Iaforin-V5 transfected

NIH3T3 cells were stimulated with PDGF for given periods of time. The

activation of Akt (anti-phosphorylated Ser473) and GSK-3β (anti-phosphorylated

Ser9) were analyzed by Western blot. Amounts of protein loaded were revealed using the total Akt as determined by Western blot. Summaries of data from three independent experiments presented as the means +/- S. D. of the relative amounts of phosphorylated proteins at 30 minutes after PDGF-treatment. The films were scanned using CanoScan LiDE 30 (Canon USA Inc, Salt Lake City, UT), and the intensity of the band was determined using SigmaScan Pro 4 for

windows (SPSS Inc., Chicago, IL). The signals of phosphorylated Akt or GSK-3β

were divided by the signals of total Akt or GSK-3β. These ratios were then

normalized over those from PDGF-treated vector-transfectants (1.0). The statistical significance was determined by student t-test.

[025] Figure S3. Dose-dependence and specificity of siRNA effect. A. Two different knock down construct resulted in different degrees of laforin knock¬

down (upper panel) and biological effect on GSK-3β phosphorylation at Ser 9

(Lower panel). B. Human EPM2A cDNA restored inhibition of GSK-3β

phosphorylation in laforin-knock down cells. Human EPM2A cDNA was transduced by lentivirus. It had two base-pair mismatches in the C-terminal silencing sequence of mouse Epm2a. Note that the anti-laforin antibody was cross-reactive with both mouse and human Laforin.

[026] Fig. S4. A preliminary study identifying a 333 bp fragment with most methylated CpG. Top diagram illustrates the CpG contents of the 5' region of the mouse EPM2a gene, with the CpG island shaded. The numbers underneath the black/white are those of the methylated clones over the number of clones analyzed. The average numbers of methylated per clone are given in the parentheses. The sequence of the 5' region of the gene is provided in the bottom. The sequence in exon 1 are shown in upper cases, with remaining sequence in lower case.

[027] Fig. S5. The CpG island of human EPM2a gene. Top diagram illustrates the CpG contents of the 5' region of the mouse EPM2a gene, with the CpG island shaded. The sequence of the 5^J region of the gene is provided in the bottom. The sequence in exon 1 are shown in upper cases, with remaining sequence in lower case.

[028] Fig. S6. Treatment of 2-dβ prolong survival of TqB mice. TgB mice (20 mice/group) between 2-4 month of age were treated with either PBS or 2-dG (20 mg/kg) every 3 days for 6 weeks. After 4 weeks of interval, all mice were again treated for another cycle. Mice that were moribund were sacrificed, and the presence of lymphoma was confirmed by necropsy.

DESCRIPTION OF THE EMBODIMENTS

[029] Reference will now be made in detail to the present embodiments of the invention.

[030] In some embodiments, this invention relates to methods of characterizing an etiology of a cancer by testing at least one cancer cell for the presence of at least one of a) methylation of the EPM2A gene; b) reduced expression of the EPM2A gene; and c) mutation of the EPM2A gene. The invention also provides methods of determining an increased likelihood of cancer in a person, by assaying a tissue sample from the person for the presence of at least one of a) methylation of EPM2A gene; b) reduced expression of EPM2A gene; and c) mutation of EPM2A gene.

[031] Methylation of the EPM2A gene can be tested using the bisulfite sequencing method, which generally involves converting non-methylated cytosines into thymines, while methylated cytosines remain unchanged. The putative CpG island of the human EPM2a gene is illustrated in Fig. S5. The modified DNA can then be analyzed using a variety of methods, examples of which are known in the art. Examples of methods for determining the level of DNA expression are also well known in the art, and include but are not limited to, assaying mRNA or protein. Finally, mutation of the EPM2A gene can be determined with a variety of well known methods, including but not limited to, those involving T4 endonuclease VII, which recognizes structural differences in heteroduplex DNA, rather than the sequence itself. Many readily available assays can be used in accordance with this aspect of the invention.

[032] In some embodiments, the invention relates to methods of characterizing an etiology of a cancer by testing at least one cancer cell for the presence of lafora bodies. These stored polysaccharide bodies are pale, well- defined masses, round or kidney-shaped, that generally displace the nuclei and reside primarily in periportal hepatocytes. They are weakly positive with diastase- PAS, stain with methenamine silver and colloidal iron stains, and, by electron microscopy, are composed of a nonmembrane-bound combination of interwoven fibrils (6 to 10 nm diameter), aggregates of smooth endoplasmic reticulum, and granular material with glycogen rosettes. Other methods of testing for these bodies include those described in Chapter 19 of Theory and Practice of Histotechnoloqy, Sheehan and Hrapchak, eds., (The CV. Mosby Company, 1980), the entire disclosure of which is incorporated herein by reference.

[033] The invention also relates to methods of identifying compounds that modify laforin's ability to phosphorylate GSK3/? at Serine 9, wherein the methods include a step of testing laforin's ability to phosphorylate GSK3/? in the presence and absence of the test compounds. In some embodiments, the laforin tested is wild-type laforin and the compounds identified are inhibitors of laforin's ability to phosphorylate GSK3/?. In other embodiments, the laforin tested is mutated laforin and the compounds identified restore laforin's ability to phosphorylate GSK3/?. These assays can be performed in a variety of ways, examples of which are well known in the art. The invention also provides methods of treating Alzheimer's disease by administering to a person in need of treatment a compound identified according to the aforementioned methods.

[034] The invention also relates to methods of treating cancer in a person identified as having a cancer associated with methylated EPM2A gene(s), by administering to the person an effective amount of a drug that demethylates DNA. In some embodiments, the invention relates to methods of treating a person identified as having a disease associated with reduced expression or mutation of the EPM2A gene, by activating EPM2A geπ e(s) in cells of the person's body. In some embodiments, the invention relates to methods of treating a person identified as having a disease associated with reduced expression or mutation of the EPM2A gene, by administering laforin to the person. In some embodiments, the invention involves methods of treating a person identified as having a disease associated with reduced expression or mutation of the EPM2A gene, by administering a genetically engineered GSK3/? that cannot be phosphorylated at the Serine 9 position. In some embodiments, the invention relates to methods of treating a person identified as having a disease associated with reduced expression or mutation of EPM2A gene, by administering to the person at least one compound that restores laforin's ability to phosphorylate GSK3/?.

[035] Methods for targeting vectors to cancer cells are described in Nakanishi T, Tamai I, Takaki A, Tsuji A. (2000) Cancer cell-targeted drug delivery utilizing oligopeptide transport activity. Int. J. Cancer. 88 : 274-280, and Poul MA, Becerril B, Nielsen UB, Morisson P, Marks JD. (2000) Selection of tumor-specific internalizing human antibodies from phage libraries. J. MoI. Biol. 301 : 1149-1161 , both of which are incorporated herein in their entirety. Methods for delivering isolated oligonucleotides and polynucleotides to cells, including the nucleus of cells, are described in Lebedeva I, Benimetskaya L, Stein CA, Vilenchik M. (2000) Cellular delivery of antisense oligonucleotides. Eur. J. Pharm. Biopharm. 50: 101-119. Review., and Fisher KD, Ulbrich K, Subr V, Ward CM, Mautner V, Blakey D, Seymour LW. (2000) A versatile system for receptor-mediated gene delivery permits increased entry of DNA into target cells, enhanced delivery to the nucleus and elevated rates of transgene expression. Gene. Ther. 7: 1337-1343.

[036] In some embodiments, an expression construct comprising the oligonucleotide for treatment may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They can form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat (1991) Targeting of liposomes to hepatocytes. Targeted Diagn. Ther 4: 87-103). Also contemplated are lipofectamine-DNA complexes.

[037] A variety of methods exist for introducing proteins and polypeptides into cells. Such methods include, but are not limited to, "protein transduction" or "protein therapy" as described in publications by Nagahara et al. (Nagahara, et al., 1998, Nat Med, 4:1449-52.) and in publications from the laboratory of Dowdy (Nagahara, et al., 1998, Nat Med, 4:1449-52.; Schwarze, et al., 1999, Science, 285:1569-72.; Vocero-Akbani, et al., 2000, Methods Enzymol, 322:508-21 ; Ho, et al., 2001 , Cancer Res, 61 :474-7.; Vocero-Akbani, et al., 2001 , Methods Enzymol, 332:36-49; Snyder and Dowdy, 2001 , Curr Opin MoI Ther, 3:147-52.; Becker-Hapak, et al., 2001 , Methods, 24:247-56.), publications which are incorporated herein by reference.

[038] In some embodiments, an eleven-amino-acid sequence, the "protein transduction domain" (PTD), from the human immunodeficiency virus TAT protein (Green and Loewenstein, 1988, Cell, 55:1179-88.; Frankel and Pabo, 1988, Cell, 55:1189-93.) is fused to the protein to be delivered, e.g., laforin. The purified protein can then be put in contact with the surface of the tumor cells and the cells take up the protein, which functions to inhibit or suppress growth of that cell. In the case where it is desired to introduce the laforin protein containing the fused PTD into cells comprising a tumor in a human or animal, the protein can be administered to the human by a variety of methods. In some embodiments, the protein is administered by intratumoral or intralesional injection. Methods of administration are provided in more detail below.

[039] Laforin proteins that are fused to PTD can be made by fusing the DNA sequence encoding the laforin protein with the DNA sequence encoding the PTD. The resulting laforin-PTD fusion gene can be incorporated into a vector, for example a plasmid or viral vector, that facilitates introduction of the fusion gene into a organism and expression of the gene at high levels in the organism such that large amounts of the fusion protein are made therein. One such organism in which the vector containing the fusion gene can be expressed is a bacterium, such as Escherichia coli. Other organisms are also commonly used by those skilled in the art. After the fusion protein is expressed at a high level in any of these organisms, the fusion protein is purified from the organism using protein purification techniques well known to those skilled in the art.

[040] The methods of the invention can be practiced orally, parenterally (IV, IM, depot-lM, SQ, and depot-SQ), sublingually, intranasally (inhalation), intrathecal^, topically, or rectally. Dosage forms known to those of skill in the art are suitable for delivery of the compounds employed in the methods of the invention.

[041] Compositions are provided that contain therapeutically effective amounts of the compounds employed in the methods of the invention. The compounds can be formulated into suitable pharmaceutical preparations such as tablets, capsules, or elixirs for oral administration or in sterile solutions or suspensions for parenteral administration. The compounds can be formulated into pharmaceutical compositions using techniques and procedures well known in the art.

[042] About 0.1 to 1000 mg of a therapeutic compound or mixture of compounds employed in the methods of the invention, or a physiologically acceptable salt or ester is compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, etc., in a unit dosage form as called for by accepted pharmaceutical practice. The amount of active substance in those compositions or preparations is such that a suitable dosage in the range indicated is obtained. The compositions can be formulated in a unit dosage form, each dosage containing from about 1 to about 500 mg, or about 10 to about 100 mg of the active ingredient. The term "unit dosage from" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.

[043] To prepare compositions, one or more compounds employed in the methods of the invention are mixed with a suitable pharmaceutically acceptable carrier. Upon mixing or addition of the compound(s), the resulting mixture may be a solution, suspension, emulsion, or the like. Liposomal suspensions may also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient for lessening or ameliorating at least one symptom of the disease, disorder, or condition treated and may be empirically determined.

[044] Pharmaceutical carriers or vehicles suitable for administration of the compounds in the methods provided herein include any such carriers suitable for the particular mode of administration. In addition, the active materials can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, or have another action. The compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.

[045] Where the compounds exhibit insufficient solubility, methods for solubilizing may be used. Such methods are known and include, but are not limited to, using co-solvents such as dimethylsulfoxide (DMSO), using surfactants such as TWEEN, and dissolution in aqueous sodium bicarbonate. Derivatives of the compounds, such as salts or prodrugs, may also be used in formulating effective pharmaceutical compositions. As noted above, the compounds - be it polynucleotides or polypeptides - can be complexed or tagged with targeting moieties to improve their delivery.

[046] The concentration of the compound is effective for delivery of an amount upon administration that lessens or ameliorates at least one symptom of the disorder for which the compound is administered. Typically, the compositions are formulated for single dosage administration.

[047] The compounds employed in the methods of the invention may be prepared with carriers that protect them against rapid elimination from the body, such as time-release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, microencapsulated delivery systems. The active compound can be included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated. The therapeutically effective concentration may be determined empirically by testing the compounds in known in vitro and in vivo model systems for the treated disorder.

[048] The compounds and compositions for use in the methods of the invention can be enclosed in multiple or single dose containers. The enclosed compounds and compositions can be provided in kits, for example, including component parts that can be assembled for use. For example, a compound in lyophilized form and a suitable diluent may be provided as separated components for combination prior to use. A kit may include a compound and a second therapeutic agent for co-administration. The compound and second therapeutic agent may be provided as separate component parts. A kit may include a plurality of containers, each container holding one or more unit dose of the inventive compound employed in the method of the invention. The containers can be adapted for the desired mode of administration, including, but not limited to tablets, gel capsules, sustained-release capsules, and the like for oral administration; depot products, pre-filled syringes, ampoules, vials, and the like for parenteral administration; and patches, medipads, creams, and the like for topical administration.

[049] The concentration of active compound in the drug composition will depend on absorption, inactivation, and excretion rates of the active compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.

[050] The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

[051] If oral administration is desired, the compound can be provided in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition may also be formulated in combination with an antacid or other such ingredient.

[052] Oral compositions can include an inert diluent or an edible carrier and may be compressed into tablets or enclosed in gelatin capsules. For the purpose of oral therapeutic administration, the active compound or compounds can be incorporated with excipients and used in the form of tablets, capsules, or troches. Pharmaceutically compatible binding agents and adjuvant materials can be included as part of the composition.

[053] The tablets, pills, capsules, troches, and the like can contain any of the following ingredients or compounds of a similar nature: a binder such as, but not limited to, gum tragacanth, acacia, corn starch, or gelatin; an excipient such as microcrystalline cellulose, starch, or lactose; a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate; a glidant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and a flavoring agent such as peppermint, methyl salicylate, or fruit flavoring.

[054] When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials, which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. The compounds can also be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings, and flavors.

[055] The active materials can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action. The compounds can be used, for example, in combination with an antitumor agent, a hormone, a steroid, or a retinoid. The antitumor agent may be one of numerous chemotherapy agents such as an demethylating agent, an antimetabolite, a hormonal agent, an antibiotic, colchicine, a vinca alkaloid, L- asparaginase, procarbazine, hydroxyurea, mitotane, nitrosoureas, or an imidazole carboxamide.

[056] Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent such as water for injection, saline solution, fixed oil, a naturally occurring vegetable oil such as sesame oil, coconut oil, peanut oil, cottonseed oil, and the like, or a synthetic fatty vehicle such as ethyl oleate, and the like, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvent; antimicrobial agents such as benzyl alcohol and methyl parabens; antioxidants such as ascorbic acid and sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, and phosphates; and agents for the adjustment of tonicity such as sodium chloride and dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass, plastic, or other suitable material. Buffers, preservatives, antioxidants, and the like can be incorporated as required.

[057] Where administered intravenously, suitable carriers include, but are not limited to, physiological saline, phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, polypropyleneglycol, and mixtures thereof. Liposomal suspensions including tissue-targeted liposomes may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known in the art.

[058] The compounds may be prepared with carriers that protect the compound against rapid elimination from the body, such as time-release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and the like. Methods for preparation of such formulations are known to those skilled in the art.

[059] Compounds employed in the methods of the invention may be administered enterally or parenterally. When administered orally, compounds employed in the methods of the invention can be administered in usual dosage forms for oral administration as is well known to those skilled in the art. These dosage forms include the usual solid unit dosage forms of tablets and capsules as well as liquid dosage forms such as solutions, suspensions, and elixirs. When the solid dosage forms are used, they can be of the sustained release type so that the compounds employed in the methods of the invention need to be administered only once or twice daily.

[060] The oral dosage forms can be administered to the patient 1 , 2, 3, or 4 times daily. The compounds employed in the methods of the invention can be administered either three or fewer times, or even once or twice daily. Hence, the compounds employed in the methods of the invention be administered in oral dosage form. Whatever oral dosage form is used, they can be designed so as to protect the compounds employed in the methods of the invention from the acidic environment of the stomach. Enteric coated tablets are well known to those skilled in the art. In addition, capsules filled with small spheres each coated to protect from the acidic stomach, are also well known to those skilled in the art.

[061] The compounds and methods can be used to inhibit neoplastic cell proliferation in an animal. The methods comprise administering to an animal having at least one neoplastic cell present in its body a therapeutically effective amount of at least one of the compounds, in compositions as described above. The animal can be a mammal, including a domesticated mammal. The animal can be a human.

[062] The term "neoplastic cell" is used to denote a cell that shows aberrant cell growth. The aberrant cell growth of a neoplastic cell includes increased cell growth. A neoplastic cell may be, for example, a hyperplastic cell, a cell that shows a lack of contact inhibition of growth in vitro, a benign tumor cell that is incapable of metastasis in vivo, or a cancer cell that is capable of metastases in vivo and that may recur after attempted removal. The term "tumorigenesis" is used to denote the induction of cell proliferation that leads to the development of a neoplastic growth.

[063] The terms "therapeutically effective amount" and "therapeutically effective period of time" are used to denote treatments at dosages and for periods effective to reduce neoplastic cell growth. As noted above, such administration can be parenteral, oral, sublingual, transdermal, topical, intranasal, or intrarectal. When administered systemically, the therapeutic composition can be administered at a sufficient dosage to attain a blood level of the compounds of from about 0.1 μM to about 100 mM. For localized administration, much lower concentrations than this can be effective, and much higher concentrations may be tolerated. One of skill in the art will appreciate that such therapeutic effect resulting in a lower effective concentration of the compound may vary considerably depending on the tissue, organ, or the particular animal or patient to be treated according to the invention. It is also understood that while a patient may be started at one dose, that dose may be varied over time as the patient's condition changes.

[064] Described in other terms, the methods of the invention can be used in the treatment of cell proliferative diseases or conditions. The term "cell proliferative disease or condition" is meant to refer to any condition characterized by aberrant cell growth, preferably abnormally increased cellular proliferation. Examples of such cell proliferative diseases or conditions include, but are not limited to, cancer, restenosis, and psoriasis. Cancers treatable according to the invention include, but are not limited to, prostate cancer, lung cancer, acute leukemia, multiple myeloma, bladder carcinoma, renal carcinoma, breast carcinoma, colorectal carcinoma, neuroblastoma, or melanoma.

[065] It should be apparent to one skilled in the art that the exact dosage and frequency of administration will depend on the particular compounds employed in the methods of the invention administered, the particular condition being treated, the severity of the condition being treated, the age, weight, general physical condition of the particular patient, and other medication the individual may be taking as is well known to administering physicians who are skilled in this art. [066] EXAMPLES

[067] Experimental Procedures

[068] Antibodies and recombinant proteins. Antibodies against the

following proteins were used in this study: Akt, lκBα, β-catenin, phospho-Akt (Ser

473), phospho-GSK-3β (Ser 9), phospho-FKHR (Ser 256), phospho-PDK1 (Ser

241 ), phospho-GSK-3α/β(Ser 21/9) (above antibodies are from Cell Signaling,

Beverly, MA), GSK-3β (BD Pharmingen/Transduction Labs, San Diego, CA), V5

and anti-V5-HRP (Invitrogen Corp., Carlsbad, CA), p50, p65, Sp1 , (Santa Cruz

Biotechnology, Inc. Santa Cruz, CA), and phospho-GSK-3α/β (pY 279/216)

(Biosource International, Camarillo, CA). Other reagents were recombinant GSK-

3β fusion protein and Akt1/PKB protein kinase (Cell Signaling), recombinant Tau

protein (PanVera LLC, Madison, Wl), human platelet-derived growth factor

(PDGF), phobol myristate acetate (PMA), and β-actin monoclonal antibody

(Sigma, St. Louis, MO) and fast flow protein G Sepharose (Amersham, Sunnyvale, CA).

[069] Identification of transgene integration site. The fluorescence in situ hybridization (FISH) was performed by SeeDNA Biotech Inc. (Windsor,

Ontario, Canada) using a 1.4 kb DNA fragment of TCR α chain constant region

as the probe. The integration site of TCR transgene in chromosome 10 was

identified using the TOPO^®WaIker Kit (Invitrogen) according to the protocol

provided. Briefly, TCR transgenic mouse genomic DNA was digested by restrictive enzyme Pst\ and dephosphorylated to allow ligation with the

TOPO^®Linker (Shuman, 1994). Primer extension using Taq polymerase and TCR

specific primer 1 was performed to create double strand DNA that contained 3' A-

overhangs. Dephosphorylated A-tailed DNA was ligated with TOPO^®Linker and further amplified by PCR. The PCR products were cloned into a TA-TOPO cloning vector (Invitrogen) and sequenced. The sequences were searched with the Celera Discovery System (Rockville, MD).

[070] Bisulfite-PCR and sequencing. The methylation status of Epm2a was determined by bisulfite-PCR and sequencing. Bisulfite treatment was carried out as described previously (Clark et al., 1994). PCR was carried out with bisulfite-treated DNA as templates with 4 pairs of primers, LDM1.F. 5'- AGAGGTTTATAAAGGATTAAA TGTTGG, LDM1. R. 5'- TAAAATCAAAATTCCCAAAC (278bp); LDM2.F. 5'- GTTTGGGAATTTTGATTTTAC, LDM2.R. 5'- TAACCCGAATCCTAAAAACCTAAC (124bp); LDM3.F. 5'- GTTAGGTTTTTAGGATTYGGGTTAT, LDM3.R. 5'-CAATACCTTCCCAA TACAACTC (333bp); LDM4.F. δ'-GAGTTGTATTGGGAAGGTATTG, LDM4.R. 5'- AACTCTCCRAAATAAACCTAAAACC (222bp). The PCR products were purified by Gel Extraction kit (Qiagen), subcloned into the TA-TOPO vector system (Invitrogen) and sequenced by an ABI/PE 3700 capillary sequencer. 8-10 clones were sequenced for each lymphoma or normal thymus.

[071] 5-Aza-2'-deoxycitidine treatment. Cells were seeded at a density of 6 x 10⁶ cells/10-cm plate and 5-Aza-dC (Sigma, St. Louis, MO) was added to different cell lines at maximal tolerable doses based on a 24-hour viability test (1 μM for P815, 2 μM for EL-4, RAW8.1 and S194, 3 μM for BW5147, CH27 and J558, 5 μM for RMA-S and A20, 10 μM for Yac-1 , Meth A and Ag8.653, 20 μM for MC38 ). After 24 hrs, the medium was changed, and the cells were treated with fresh 5-Aza-dC for an additional 24 hrs. Two days after 5- Aza-dC treatment, the cells were harvested for RT-PCR analysis on EPM2A expression. Genomic DNA from three Epm2a high cell lines (P815, Meth A and MC38) and three Epm2a low lymphoma cell lines (EL-4, BW5147 and YAC-1) were subjected to methylation-sensitive Sac Il digestion. Digested or undigested genomic DNA were used as templates for real time PCR.

[072] Anti-laforin polyclonal antibody. The antibody was produced by Genemed Synthesis, Inc. (San Francisco, CA). Rabbits were immunized with the synthetic peptide of 16 amino acid residues (YKFLQREPGGELHWEG, residues 85-100 in laforin protein, Accession No. AAD26336) coupled with keyhole limpet hemocyanin (KLH) in complete Freund's adjuvant. The antiserum was purified using peptide conjugated Affigel column.

[073] Epm2a siRNA constructs. Oligonucleotides encoding siRNA directed against Epm2a at C terminal region of 934 to 954 nucleotides (5'- AAGGTGCAGTACTTCATCATG-3') (C) or polyA region of 1212 to 1233 nucleotides (δ'-AAGGAAAACTGCATGCCACAT-S¹) (E) were cloned into pSilencer 1.0-U6 siRNA expression vector (Ambion, Austin, TX) to generate C- siRNA according to manufacturer's protocol. NIH3T3 cells were co-transfected with C- or E-siRNA vector and pcDNA4/V5-His (Invitrogen) using Fugene 6 and

selected by Zeocin at a concentration of 250 μg/ml. Individual clones were

analyzed for Epm2a expression by RT-PCR.

[074] Preparation of lentiviral stocks and viral transduction. Full length cDNAs of both EPM2A and Emp2a were subcloned into pIenti/V5-D-TOPO vector (Invitrogen). Lentiviral construct for silencing Epm2a and its corresponding control construct were made by replacing CMV promoter in plenti/V5-D-TOPO vector with Epm2a-siRNA expressing cassette in C-siRNA construct and with siRNA-empty corresponding cassette in pSilence1.0-U6, respectively. Viral particles were produced in 293FT cells according to manufacturer's protocol (Invitrogen). T lymphoma cell lines at cell number 3 x 10⁵ in a 6-well plate were

transduced by the concentrated virus at 1 :50 dilution in the presence of 6 μg/ml

polybrene for 16 hours. The fresh medium was replaced and continued to culture for another 24 hours before flow cytometry analysis.

[075] GSK-3β kinase assay. The in vitro kinase assay measuring the

kinase activity of GSK-3β was performed according to a previously described

protocol (Song et al., 2002).

[076] Phosphatase assay. Fusion GSK-3β protein was phosphorylated

in vitro with active Akt according to manufacturer's protocol (Cell Signalling). The Epm2a full length cDNA was cloned into the BamH\ and Xho\ sites of pGEX-6P- 1(Amersham) to create GST-Epm2a fusion protein construct and transformed into E.coli strain BL21. The soluble GST-fused protein was purified by glutathione-agarose beads, and eluted with 5OmM reduced glutathione. Given

concentrations of the laforin protein was mixed with the phosphorylated GSK-3β

in phosphatase assay buffer as described previously (Taylor et al., 1997).

[077] Cell fractionation and /?-catenin analysis. The recombinant mouse Wnt-3A was purchased from R&D Systems (Minneapolis, MN). Vector-, Epm2a- or siRNA-transfectants of NIH3T3 or B104-1-1 cells were plated for 16 hours. After an additional 2-4 hours of culture in serum-free medium, the transfectants were treated with or without recombinant mouse Wnt-3A protein

(100 -200 ng/ml) in DMEM medium containing 0.5μM of PMA for 3 hours. The

cells were either directly lysed for the detection of p-GSK-3β levels in total lysate

or homogenized and fractioned for the detection of β-catenin levels in the nuclei. The total cell lysates and nuclear fractions were separated in 12% SDS-PAGE and probed with different antibodies after transfer to nitrocellulose membranes.

[078] TCF reporter assay. NIH3T3 transfectants in a 24-well plate

were transfected with 0.2 μg of pTOPFLASH or pFOPFLASH TCF luciferase

reporter constructs (Cell Signalling) plus 0.05 μg of pRL-SV40 renilla luciferase

control reporter. 24 hours later, after an additional 2-4 hours of culture in serum- free medium, the transfectants were treated with Wnt-3A conditional medium or L-cell control medium for 16 hr. The luciferase activity was determined by the Dual-luciferase Reporter Assay System (Promega, Madison, Wl). The results were shown after normalized to corresponding Renilla reporter value.

[079] Results

[080] Transgene insertion to and hypermethylation of Epm2a gene induce spontaneous lymphoma in TG-B transgenic mice

[081] The TG-B transgenic mice used in this study have rearranged T

cell receptor α and β-chain genes that isolated from a clonal CD8⁺ cytotoxic T cell

line integrated into the genome of the B10.BR (H-2^k) mice (Geiger et al., 1992). Surprisingly, the TG-B mice had a high incidence of lymphoma regardless of sex. The maximal survival time for TG-B mice was 9 months with a median survival time of 6 months (Fig. 1A). The lymphoma in TG-B mice was first seen in lymphoid organs, including the thymus, spleen and lymph nodes (Fig. 1B). As the cancer progressed, the tumor cells infiltrated other organs such as liver, kidney (Fig. 1C), lung, pancreas, intestines and salivary glands (data not shown). Further characterization of malignant cells revealed the immunophenotype as T cell lymphoma (Fig. 1C) at various stages in T cell development among animals (data not shown). [082] To test if the genetic background of mice affects tumor incidence, we produced TG-B x C57BL/6 F1 mice and monitored their tumor incidence. Although T cell development in the F1 mice was normal (Zheng et al., 2002), significant reductions were observed in the F1 mice for both tumor incidence and onset in comparison to the TG-B mice (Fig. 1A). This result demonstrated that the C57BL/6 mice have dominant genetic modifiers for the development of lymphoma.

[083] In order to understand the mechanism of tumorigenesis in these mice, we first determined the integration site of the transgene by fluorescence in situ hybridization (FISH) using a biotin-labelled DNA fragment probe from the

TCR α chain constant region (Heng et al., 1992). DAPI banding was performed,

and a strong signal was identified on one of the two chromosome 10 (Fig. 2A). The strong signal was localized to region 10A2 based on the summary of 10 images (data not shown). Two weaker FISH signals were detected, one on each

copy of chromosome 14, consistent with the location of the endogenous TCRα

genes. Fine mapping of the integration sites was performed by the

TOPO^®Walker method (Shuman, 1994). PCR products using the TOPO^®Linker

primers and the gene specific sequence primers from both 3' and 5' termini of the

TCR α and TCR β transgenes were cloned and sequenced.

[084] Three independent experiments revealed that the TCR α

transgene was inserted into the intron 1 of Epm2a, which encodes laforin, a dual specific protein phosphatase (Fig. 2B)(Minassian et al., 1998; Serratosa et al., 1999b). Further PCR analysis using primers corresponding to the 3' terminus of

the TCRα transgene and the 5' terminus of the TCRβ transgene confirmed that

these genes were co-integrated into chromosome 10 in TG-B mice (Fig. 2C). The integration was confirmed by PCR using primers outside the sequence

identified by the TOPO^®Walker method (data not shown) and by Southern blot

using the sequence adjacent to the integration site as the probe (Fig. 2D).

[085] To determine whether the integration of the TCR transgene inhibited expression of Epm2a gene, we examined the expression of the Epm2a gene in the thymi of nontransgenic (Ntg), transgenic littermates with no tumor (Tg) and the transgenic mice that developed lymphoma (Tg-Tu). Based on RT- PCR amplification, Epm2a was expressed at significant levels in nontransgenic and transgenic littermates. However, its expression in Tg-Tu lymphoma cells was greatly reduced (Fig. 3A). We prepared antibodies against laforin, which is encoded by the Epm2a gene, to analyze laforin protein levels by Western blotting. This showed that the laforin protein was expressed in Tg mice with approximately half of the amount in NTg mice, but was absent in the transgenic mice that developed lymphoma (Tg-Tu) (Fig. 3B). This method also revealed the down-regulation of laforin in the majority of the murine T and B cell lymphoma cell lines that we tested (Fig. 3C). Thus, tumorigenesis was consistently associated with reduced laforin gene expression.

[086] Interestingly, the high tumor incidence and the lack of laforin documented in Fig. 1 A were observed in heterozygous mice. Since the wild type allele was not deleted in the tumor cells lacking laforin expression (data not shown), it may have been silenced by epigenetic mechanisms. The Epm2a gene contains a 1.2 kb 5'-CpG island that spans the promoter region, exon 1 and part of intron 1 (Fig. 4A). To determine if DNA hypermethylation is an epigenetic mechanism for laforin gene down-regulation in TG-B lymphoma, we treated the genomic DNA with bisulfite and sequenced four PCR products covering 102 CpG di-nucleotides within the 5'-CpG island. Our preliminary analyses of the tumor samples indicated that a 333-bp PCR fragment was the most frequently methylated area (Fig. S4). We therefore focused on this region and examined six TG-B mice that developed lymphoma and six TG-B mice that had not developed tumors. Based on the methylation-mediated protection of OT conversion by the sodium bisulfite, we identified the methylated CpG di-nucleotides. As illustrated in Fig. 4B, extensive hypermethylation was found in the Epm2a gene of all six lymphoma samples. In contrast, none of the normal thyrni had methylation in this region. Our survey of multiple mouse lymphoma cell lines revealed that depression in the Epm2a gene was wide-spread among lymphomas (Fig. 3C). Moreover, treatment with methyl-transferase inhibitor 5-aza-2'-deoxycytidine increased Epm2a expression (Fig. 4C and data not shown). To test whether the hyper-methylation of Epm2a promoter was responsible for the repression of the Epm2a gene, we compared three non-lymphoid tumor cell lines with three T lymphoma cell lines for constitutive and 5-aza-2'-deoxycytidine-induced Epm2a expression. At the same time, Epm2a promoter hypermethylation was determined by quantitative real-time PCR based on susceptibility to a methylation-sensitive restriction enzyme. As shown in Fig. 4D, Epm2a was constitutively expressed at high levels in mastocytoma cell line P815, colon cancer cell line MC38 and fibrosarcoma cell line Meth A. Treatment with 5-aza- 2'-deoxycytidine had little effect on Epm2a expression in these three cell lines. Since the methylation-sensitive Sac Il cuts more than 99% of the Epm2a promoter, lack of PCR product indicated that Epm2a gene in these three tumor cell lines was not methylated. In contrast, in three T lym phoma cell lines tested, the Epm2a promoter region is completely resistant to Sac Il digestion. Correspondingly, very little constitutive expression of Eprn2a was found, and 5- aza-2'-deoxycytidine substantially induced Epm2a expression among the three T lymphoma cell lines. These data are consistent with the notion that hypermethylation is responsible for down-regulation of laforin among T lymphomas.

[087] The transgene expression may potentially activate genes in the area. There are two genes within 300kb to the transgene integration site. The proximal gene is 56 kb away from the integration site and encodes a protein that contains an F-box domain (mCG16603), while the other gene is 135 kb from the integration site (mCG140768) (Fig. S1A). We performed RNase protection assay to determine the mRNA expression of these two genes. The expression level of the distal gene (mCG140768) was not affected by transgene or lymphoma development, while the expression of the proximal gene (mCG16603) was up- regulated by the transgene regardless of lymphoma formation (Fig. S1 b). Although the lack of further elevation of the proximal gene was inconsistent with its role in lymphoma development, we tested this possibility by producing 8 independent founders of transgenic mice that express mCG 16603 under the control of a proximal lck promoter, which resulted in high levels of transgene expression in the thymus. None of the mice developed lymphoma during the over more than 1 year period (Fig. S1c). Thus, the lymphoma development in TG-B mice is unlikely due to the abnormal expression of other genes near the integration site.

[088] Tumor suppressive activity of laforin [089] To test the function of the Epm2a gene in tumor growth, we generated a V5 tagged Epm2a lentiviral expression construct and transduced Epm2a cDNA into four murine T lymphoma cell lines that express very low levels of laforin as shown in Fig. 3C (EL-4, YAC-1 , RMA-S, and BW5147). Forty-eight hours after transduction, we immunostained the cells with anti-V5 antibody and 7- amino-actinomycin (7-AAD), a nucleic acid dye widely used to measure DNA contents. Cells were gated for V5 positive (Epm2a transduced cells) and V5 negative cells. As shown in Fig. 5A, the DNA contents in the majority of the V5 negative cells (upper panels) were between DNA content 2C and 4C, which indicated live cells at different stages of cell cycle. However, 50-70% of the Epm2a-\/5 positive cells had DNA contents less than 2C, indicating very active DNA degradation and cell death. This result demonstrates that laforin expression induces apoptosis of tumor cells.

[090] Despite the rapid cell death, a small number of transfected BW5147 cells survived. After a short-term drug selection, we compared vector and Epm2a-transfected tumor cells for their growth in vivo. As shown in Fig. 5B, the EpA772a-transfectants had a substantially reduced growth rate in vivo. To test if the tumor suppression by the Epm2a gene was limited to lymphoma, we transfected the EPM2a cDNA into B104-1-1 , a tumor cell line transformed by P185neι/ oncogene (Schechter et al., 1984). We inoculated the laforin or vector transfected B104-1-1 cell lines into RAG-2(-/-) mice that lacked T and B lymphocytes. Based on the kinetics of tumor growth, it is clear that laforin had a significant suppressive effect on B104-1-1 tumor growth (Fig. 5C).

[091] As a complementary approach, we silenced the expression of the Epm2a gene in a fibroblast cell line and tested the tumorigenecity of the clones that were transfected with either vector control or siRNA. As shown in Fig. 5D, the vector transfected clones failed to grow tumors in the immune deficient host, while all the mice that received the equal number of the Epm2a-s\\enced cells developed tumors. Thus, laforin protein is a potent suppressor of tumor growth in vivo.

[092] Laforin is a phosphatase for GSK-3β

[093] Because laforin suppressed the growth of Her-2/Neu-transformed tumor cells, we explored the possibility that laforin may be involved in PI-3K/Akt signalling pathway. Activation of the platelet-derived growth factor (PDGF) receptor results in the recruitment of PI-3K isoforms to the inner surface of the plasma membrane. This leads to the activation of protein kinase B/Akt(Romashkova and Makarov, 1999). We therefore transfected the laforin cDNA into N1H3T3 cells and evaluated the effect of laforin on PDGF receptor signaling. We first examined phosphorylation of Akt at Ser 473 by 3- phosphoinositide-dependent protein kinase (PDK) (Alessi et al., 1997a). Activation and phosphorylation of PDK was not significantly affected by PDGF in NIH3T3 cells, regardless of the expression of laforin. As a result of PDK recruitment, significant Akt phosphorylation was observed following PDGF stimulation, and again, this was only marginally affected by laforin expression (Fig. 6A and Fig. S2). Interestingly, of the three down-stream targets of Akt

(Datta et al., 1999) that we examined, namely FKHR, AFX and GSK-3β, only

GSK-3β phosphorylation on Ser 9 was affected by over-expression of laforin (Fig.

6A). The effect was highly selective as the phosphorylation of Tyr 216 of GSK-3β

and Tyr 279 of GSK-3α was not affected by laforin despite laforin's intrinsic dual

phosphatase specificity (Fig. 6B). To determine the effect of laforin expression on

GSK-3β activity, we precipitated GSK-3β and performed the in vitro kinase assay

to test its ability to phosphorylate recombinant protein Tau, a known GSK-3β substrate (Cho and Johnson, 2003). As shown in Fig. 6C, PDGF-induced

inhibition of GSK-3β kinase activity was blocked in laforin-transfected cells. This

was consistent with the effect of laforinon Akt-mediated phosphorylation of GSK-

3β at Ser 9.

[094] The selective effect of laforin on the phosphorylation of GSK-3β at

Ser 9 raises the intriguing possibility that laforin may be a missing phosphatase of

GSK-3β. We used three additional assays to substantiate this hypothesis. First,

we generated the inhibitory siRNA expression construct and used this to eliminate the endogenous laforin in transfected NIH3T3 cells. Three C-terminal Epm2a siRNA stable transfectants were tested for transcriptional inhibition. As shown in Fig. 6D, Epm2a mRNA was reduced to levels undetectable by RT-PCR. Elimination of laforin expression had no effect on cellular levels of phospho-Akt,

but as a result of siRNA inhibition of laforin, the phospho-GSK-3β became

detectable even in the absence of PDGF. Furthermore, upon PDGF stimulation,

levels of phospho-GSK-3β in cells treated with siRNA were greater than those

found in vector-transfected control cells. SiRNA specific for another region of the Epm2a gene partially reduced laforin expression and lead to quantitative changes

in GSK-3β phosphorylation (Fig. S3A). Thus, the levels of GSK-3β

phosphorylation at Ser 9 correlated closely with the efficiency of laforin knockdown (Fig. S3A). In addition, the specificity of the knockdown is confirmed, as the biological effect is restored by reintroduction of human laforin, which has two mismatches in the sequence targeted by the siRNA (Fig. S3B). Thus, laforin

controls GSK-3β phosphorylation at Ser 9.

[095] Secondly, we evaluated whether GSK-3β and laforin can physically associate with each other. We treated the vector and laforin- transfected NIH3T3 cells with PDGF and immunoprecipitated GSK-3β protein

with either control IgGI or a monoclonal anti-GSK-3β antibody. The precipitates

were analyzed by Western blot with either anti-GSK-3β antibody or the anti-V5

antibodies that detect the V5-tagged laforin protein. As shown in Fig. 6E, anti-

GSK-3β, but not control IgGI , brought down both GSK-3β and V5-tagged laforin

proteins in the laforin-transfected cells, but not in vector transfected cells. Thus,

GSK-3β and laforin are associated in the NIH3T3 cells. To determine whether

GSK-3β associate with endogenous laforin protein, we precipitated the GSK-3β in

untransfected NIH3T3 cells and probed the precipitates with antibody specific for laforin. As shown in Fig. 6E lower panel, significant amounts of laforin was co-

precipitated with GSK-3β. Thus, laforin associates with GSK-3β under

physiological condition.

[096] As a third approach, we tested whether recombinant laforin can

directly dephosphorylate GSK-3β in vitro. Recombinant GSK-3β was

phosphorylated in vitro by a constitutively active recombinant Akt mutant protein. Recombinant Laforin expressed in bacteria were added. As shown in Fig. 6F,

laforin reduced GSK-3β phosphorylation at Ser 9 in a concentration dependent

manner. As expected, substitution of a mutant laforin protein (C265S) with a catalytically inactive phosphatase domain(Wang et al., 2002) prevented

dephosphorylation of GSK-3β at Ser 9. The specificity of the de-phosphorylation

is confirmed as laforin does not affect phosphorylation of an unrelated AKT substrate, BAD.

[097] Laforin is an important modulator for Wnt signaling

[098] GSK-3β is a major component in the Wnt signalling pathway (He

et al., 1995; Siegfried et al., 1992). To understand how laforin may suppressor tumor growth, we tested whether it may modulate Wnt signalling. Using a commercially available purified product, we found that in the presence of either suboptimal concentration of PMA or serum (data not shown), purified Wnt3A

consistently induced the phosphorylation of GSK-3β at Ser 9 and induced the

nuclear accumulation of β-catenin. The effect of PMA is consistent with the

recent observation that PKC is component of Wnt signaling pathway (Ossipova et al., 2003). Moreover, expression of laforin, but not the C265S mutant, led to

decreased Ser 9 phosphorylation in GSK-3β and decreased nuclear β-catenin

accumulation in response to Wnt costimulation (Fig. 7A). The functional

importance of laforin expression in β-catenin activity was further confirmed by a

TCF reporter assay (Korinek et al., 1997), as shown in Fig. 7B. Conversely, silencing the laforin expression in NIH3T3 cells significantly increased the

phosphorylation of Ser 9 in GSK-3β and the nuclear accumulation of β-catenin upon the Wnt3A costimulation (Fig. 7C).

[099] To examine the relevance of laforin modulation of Wnt pathway in

tumorigenesis, we evaluated GSK-3β phosphorylation and nuclear β-catenin

accumulation in both a laforin transfected tumor cell line and the spontaneous lymphoma developed in a T cell receptor transgenic line TG-B mouse that has a disrupted Epm2a gene. As shown in Fig. 7D, expression of laforin in B104-1-1

cells decreased the Ser 9 phosphorylation of GSK-3β and drastically decreased

the nuclear accumulation of β-catenin, regardless of whether exogenous Wnt3A

was added. As shown in Fig. 7E, in transgenic mice that have one copy of the Epm2a inactivated, the thymocytes have a modest increase of phosphorylated

GSK-3β in the cytosol, although no β-catenin was detected in the nuclei. In the

lymphoma samples that have complete inactivation of the Epm2a gene, a drastic increase of GSK-3β phosphorylation and β-catenin accumulation in the nuclei

were observed. These results demonstrate that laforin modulate Wnt signalling during tumorigenesis.

[0100] Treatment with 2-deoxyl-glucose (2-dG) reduce cancer incidence in the TgB mice

[0101] The connection between Laforin and GSK3β led us to determine whether modification of glucose metabolism will have an impact on cancer incidence in animal with disruption in one allele of the EPM2a gene. We treated the TgB mice with 2-dG, an inhibitor of the AMPK, a key enzyme in glucose metabolism. As shown in Fig. S6, chronic treatment of 2-dG, substantially prolonged survival of the TgB mice. These results suggest that cancer incidence in animal with disrupted EPM2a gene can be modified by drugs that affect glucose metabolism.

[0102] Discussion

[0103] By following a serendipitous observation of spontaneous lymphoma in a line of TCR transgenic mice, we uncovered that laforin, a dual specific phosphatase with unknown functions in signal transduction, is a key

regulator for GSK3β activity, Wnt signaling and tumorigenesis.

[0104] Laforin is a tumor suppressor in the mouse

[0105] We have presented several lines of evidence that support a role for laforin as a tumor suppressor.

[0106] First, we have identified by position cloning that a line of TCR transgenic mice with rapid onset and high penetrance of lymphoma have the transgene inserted in the intron 1 of the Epm2a gene. The insertion contains

both α and β chains of a genomic clone of TCR and thus exceed 100 kb (if one copy)-300 kb (if 3 copies as estimated by intensity of bands in Southern blot, data not shown). Since the heterozygous mice have roughly 50% of laforin protein expression, it is likely that such insertion inactivated the Epm2a locus. The development of lymphoma associates with complete silencing of the laforin gene, which in turn is due to the hypermethylation of the Epm2a gene. Thus, the genetic lesion and epigenetic inactivation of the Epm2a gene during tumorigenesis is reminiscent of typical tumor suppressor gene (Paige, 2003). Second, we have demonstrated that the hypermethylation of Epm2a gene is not unique to the mouse lymphoma model in the TCR transgenic mice. Down- regulation of laforin was observed in a large panel of mouse lymphoma cell lines and correlates with hypermethylation of the locus. Thirdly, we have directly demonstrated that expression of laforin resulted in apoptosis of tumor cells and suppressed the growth of tumors in vivo. Conversely, suppression of Epm2a gene by siRNA increased tumorigenesis of a fibroblast cell line. These results directly demonstrate that laforin is a potent tumor suppressor gene in the mouse. Fourth, as discussed below, the novel function of laforin as a phosphatase for

GSK-3β and modulator for Wnt signaling provide a mechanism by which laforin

suppresses tumor growth.

[0107] Several caveats deserve consideration.

[0108] First, mice with targeted mutation have been reported to survive to 12 months of age, although it has not been examined as to whether the mice are more susceptible to lymphoma (Ganesh et al., 2002). Therefore targeted mutation of Epm2a in the mixed 129Svj x C57BL/6 background (H-2^b) does not seem to form early lethal lymphoma as observed in the TG-B mice. This apparent inconsistency can be reconciled in two ways. The C57BL/6 (H-2^b) mice are less sensitive to lymphoma in B10.BR, as we showed that the offspring of TG-B(BIO. BR, H-2^k) developed lymphoma substantially faster than the C57BL/6 x TG-B F1 mice. In fact, H-2 region has been shown to contain a major modifier for susceptibility to lymphoma (Kamoto et al., 1996). In addition, TCR transgenic mice are immune-deficient because the majority of their T cells are directed to a single CTL epitope and therefore unable to recognize tumor antigen. In combination, these two factors may explain the lack of report on lymphoma in the Epm2a-deficient mice.

[0109] Second, insertion of TCR may cause aberrant expression of adjacent genes. We have found that the expression of one of the immediate neighbors (within 56 kb of Epm2a) is up-regulated, although no effect was observed on another gene 120 kb away. We have directly tested whether over- expression of the neighboring gene is responsible for tumorigenesis by producing transgenic mice that over-express it in the thymus. Our analysis of 8 independent founders revealed no lymphoma over more than one year period. This result ruled out the possibility that over-expression of the neighbouring gene is the primary event leading to cancer development. The tumor suppressor activity of Epm2a has been confirmed by direct transfection and knock-down experiments in which the adjacent genes should not be affected.

[0110] Thirdly, it is of interest to consider whether the EPM2A gene may be a tumor suppressor in human. Lafora's disease due to EPM2A mutations is an extremely rare genetic disease with early lethality. The patients usually die before they reached 20 years of age. It is therefore not possible to evaluate the direct impact of this mutation on cancer incidence. Nevertheless, we have observed high incidence of loss of heterozygosity (LOH) in human cancer (data not shown). In addition, the EPM2A is located in 6q24 of human genome where deletions or LOH were reported in cancers from a large number of different organs including carcinomas of the breast (Devilee et al., 1991 ; Sheng et al., 1996), endometrium (Tibiletti et al., 1997; Tibiletti et al., 1999), prostate (Cooney et al., 1996), lymphomas and leukemias (Gerard et al., 1997; Zhang et al., 1997), and melanomas (Walker et al., 1994).

[0111] Laforin is a phosphatase for GSK-3β

[0112] GSK-3β is a key modulator for several important signal

transduction pathways. Although GSK-3β is inactivated by phosphorylation at the

Ser 9 position, it is unclear if the inactivated enzyme can be reactivated by dephosphorylation, as no specific phosphatase for this enzyme has been identified. Here we showed that laforin, a dual specific phosphatase with

unknown substrate, is a specific phosphatase for GSK-3β. Transfection of laforin

to cells reduced PDGF-induced phosphorylation of Ser 9 of GSK-3β, but not that

of Y216 of GSK-3β, Y279 of GSK-3α and phosphorylation of PDK, AKT and other

AKT substrates such as FKHR and AFX. Moreover, knockdown of laforin in either NIH3T3 cells or in mouse thymus substantially increased phosphorylation

of GSK-3β. In addition, transfection of laforin substantially increased the

enzymatic activity of GSK-3β. These data demonstrated that laforin is a critical

regulator of Ser 9 phosphorylation and enzymatic activity of GSK-3β. Given the

importance of GSK-3β in a variety of biological processes including cellular

metabolism, growth and transformation (Cohen and Frame, 2001 ; Woodgett, 1990), it would be of interest to determine which of these processes are also regulated by laforin. [0113] Laforin as an important modulator of Wnt signaling

[0114] We have taken two approaches to demonstrate that laforin is an important regulator in Wnt signalling. First, we showed that over-expression of

laforin reduced nuclear accumulation of β-catenin in response to Wnt3A.

Conversely, knockdown of laforin in both cell lines and in transgenic mice

resulted in a dose-dependent increase of β-catenin in the nuclei. Since nuclear

accumulation of β-catenin play a major role in tumorigenesis, modulation of Wnt

signaling is likely to be responsible for laforin-mediated suppression of tumor growth.

[0115] The detailed mechanism by which laforin regulates Wnt signalling is yet to be fully understood. Since we showed that laforin regulates Ser 9

phosphorylation of GSK-3β, a major regulator of β-catenin, the simplest

hypothesis is that laforin modulates Wnt signaling by regulating Ser 9

phosphorylation of GSK-3β and its biological activity. While our data are entirely

consistent with this notion, two groups have questioned whether GSK-3β-

mediated destruction of β-catenin is regulated by phosphorylation of Ser 9. First,

Ding et al. suggested that Wnt signaling is insulated from AKT pathway. This is in part based on their observation that Wnt3A conditional medium did not induce

GSK-3β phosphorylation above the background (Ding et al., 2000). In addition,

Wnt signalling may reduce GSK activity of Ser9>A mutant protein, as suggested

by the immunoprecipitates of the tagged mutant. However, since the GSK-3β

has the tendency to form dimers (Dajani et al., 2003), it remains possible that

endogenous WT GSK-3β were co-precipitated with the tagged mutant. We

demonstrate dramatic phosphorylation of GSK-3β at Ser9 position in response to

purified active Wnt3A (Willert et al., 2003) under PMA co-stimulation, and such phosphorylation is regulated by laforin. These data indicate that PKC rather than Akt signaling enhanced the Wnt signaling, and is consistent with the observation that PKC is a component of Wnt signaling pathway (Ossipova et al., 2003).

Second, genetic reconstitution in Drosophila demonstrated that the GSK-3β

mutant that lacks Ser 9 can still mediate Wnt signalling (Papadopoulou et al., 2004). This later finding would argue that Ser 9 phosphorylation is not required for Wnt signaling in Drosophila development. Interestingly, Drosophila also lacks the Epm2a gene (Ganesh et al., 2001 ). Thus, laforin is a newly added modulator of Wnt signaling during evolution. This addition allows more accurate modulation

of GSK-3β activity through Ser 9 phosphorylation. Our hypothesis provided a

simple explanation for the requirement of priming sites on β-cantenin (Amit et al.,

2002; Liu et al., 2002; Yanagawa et al., 2002) and the prime phosphate pocket in

GSK-3β that binds the phosphorylated priming sites and phosphor-Ser 9 (Dajani

et al., 2001 ; Frame et al., 2001 ).

[0116] Laforin in cancer and other human diseases

[0117] We have identified that Laforin is a key regulator for the function of GSK3. In addition to its involvement in cancer, GSK3 plays a critical role in a wide spectrum of human diseases, including the Alzheimer's disease (Phiel et al., 2003). Since the expression of Laforin is essential for optimal GSK3 function, it may be possible to inhibit GSK3 function by either down regulating the expression of EPM2a or by inhibition of the Laforin phophatase activity.

[0118] CITED DOCUMENTS

[0119] Alessi, D. R., Deak, M., Casamayor, A., Caudwell, F. B., Morrice, N., Norman, D. G., Gaffney, P., Reese, C. B., MacDougall, C. N., Harbison, D., et al. (1997a). 3-Phosphoinositide-dependent protein kinase-1 (PDK1 ): structural and functional homology with the Drosophila DSTPK61 kinase. Curr Biol 7, 776- 789.

[0120] Alessi, D. R., and Downes, C. P. (1998). The role of Pl 3-kinase in insulin action. Biochim Biophys Acta 1436, 151-164.

[0121] Alessi, D. R., James, S. R., Downes, C. P., Holmes, A. B., Gaffney, P. R., Reese, C. B., and Cohen, P. (1997b). Characterization of a 3- phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha. Curr Biol 7, 261-269.

[0122] Amit, S., Hatzubai, A., Birman, Y., Andersen, J. S., Ben-Shushan, E., Mann, M., Ben-Neriah, Y., and Alkalay, I. (2002). Axin-mediated CKI phosphorylation of beta-catenin at Ser 45: a molecular switch for the Wnt pathway. Genes Dev 16, 1066-1076.

[0123] Behrens, J., von Kries, J. P., Kuhl, M., Bruhn, L., Wedlich, D., Grosschedl, R., and Birchmeier, W. (1996). Functional interaction of beta-catenin with the transcription factor LEF-1. Nature 382, 638-642.

[0124] Bhanot, P., Brink, M., Samos, C. H., Hsieh, J. C, Wang, Y., Macke, J. P., Andrew, D., Nathans, J., and Nusse, R. (1996). A new member of the frizzled family from Drosophila functions as a Wingless receptor. Nature 382, 225-230.

[0125] Cho, J. H., and Johnson, G. V. (2003). Glycogen synthase kinase 3beta phosphorylates tau at both primed and unprimed sites. Differential impact on microtubule binding. J Biol Chem 278, 187-193.

[0126] Clark, S. J., Harrison, J., Paul, C. L, and Frommer, M. (1994). High sensitivity mapping of methylated cytosines. Nucleic Acids Res 22, 2990- 2997. [0127] Cohen, P., and Frame, S. (2001). The renaissance of GSK3. Nat Rev MoI Cell Biol 2, 769-776.

[0128] Cooney, K. A., Wetzel, J. C, Consolino, C. M., and Wojno, K. J. (1996). Identification and characterization of proximal 6q deletions in prostate cancer. Cancer Res 56, 4150-4153.

[0129] Cross, D. A., Alessi, D. R., Cohen, P., Andjelkovich, M., and Hemmings, B. A. (1995). Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378, 785-789.

[0130] Dajani, R., Fraser, E., Roe, S. M., Yeo, M., Good, V. M., Thompson, V., Dale, T. C, and Pearl, L. H. (2003). Structural basis for recruitment of glycogen synthase kinase 3beta to the axin-APC scaffold complex. Embo J 22, 494-501.

[0131] Dajani, R., Fraser, E., Roe, S. M., Young, N., Good, V., Dale, T. C, and Pearl, L. H. (2001). Crystal structure of glycogen synthase kinase 3 beta: structural basis for phosphate-primed substrate specificity and autoinhibition. Cell 105, 721-732.

[0132] Datta, S. R., Brunet, A., and Greenberg, M. E. (1999). Cellular survival: a play in three Akts. Genes Dev 13, 2905-2927.

[0133] Devilee, P., van Vliet, M., van Sloun, P., Kuipers Dijkshoom, N., Hermans, J., Pearson, P. L., and Cornelisse, C. J. (1991). Allelotype of human breast carcinoma: a second major site for loss of heterozygosity is on chromosome 6q. Oncogene 6, 1705-1711.

[0134] Doble, B. W., and Woodgett, J. R. (2003). GSK-3: tricks of the trade for a multi-tasking kinase. J Cell Sci 116, 1175-1186. [0135] Frame, S., and Cohen, P. (2001 ). GSK3 takes centre stage more than 20 years after its discovery. Biochem J 359, 1-16.

[0136] Frame, S., Cohen, P., and Biondi, R. M. (2001 ). A common phosphate binding site explains the unique substrate specificity of GSK3 and its inactivation by phosphorylation. MoI Cell 7, 1321-1327.

[0137] Ganesh, S., Agarwala, K. L., Amano, K., Suzuki, T., Delgado- Escueta, A. V., and Yamakawa, K. (2001 ). Regional and developmental expression of Epm2a gene and its evolutionary conservation. Biochem Biophys Res Commun 283, 1046-1053.

[0138] Ganesh, S., Delgado-Escueta, A. V., Sakamoto, T., Avila, M. R., Machado-Salas, J., Hoshii, Y., Akagi, T., Gomi, H., Suzuki, T., Amano, K., et al. (2002). Targeted disruption of the Epm2a gene causes formation of Lafora inclusion bodies, neurodegeneration, ataxia, myoclonus epilepsy and impaired behavioral response in mice. Hum MoI Genet 11 , 1251-1262.

[0139] Geiger, T., Gooding, L. R., and Flavell, R. A. (1992). T-cell responsiveness to an oncogenic peripheral protein and spontaneous autoimmunity in transgenic mice. Proc Natl Acad Sci U S A 89, 2985-2989.

[0140] Gerard, B., Cave, H., Guidal, C, Dastugue, N., Vilmer, E., and Grandchamp, B. (1997). Delineation of a 6 cM commonly deleted region in childhood acute lymphoblastic leukemia on the 6q chromosomal arm. Leukemia 11 , 228-232.

[0141] Hagen, T., Di Daniel, E., Culbert, A. A., and Reith, A. D. (2002). Expression and characterization of GSK-3 mutants and their effect on beta- catenin phosphorylation in intact cells. J Biol Chem 277, 23330-23335. [0142] He, X., Saint-Jeannet, J. P., Wang, Y., Nathans, J., Dawid, I., and Varmus, H. (1997). A member of the Frizzled protein family mediating axis induction by Wnt-5A. Science 275, 1652-1654.

[0143] He, X., Saint-Jeannet, J. P., Woodgett, J. R., Varmus, H. E., and Dawid, I. B. (1995). Glycogen synthase kinase-3 and dorsoventral patterning in Xenopus embryos. Nature 374, 617-622.

[0144] Heng, H. H., Squire, J., and Tsui, L. C. (1992). High-resolution mapping of mammalian genes by in situ hybridization to free chromatin. Proc Natl Acad Sci U S A 89, 9509-9513.

[0145] Ikeda, S., Kishida, S., Yamamoto, H., Murai, H., Koyama, S., and Kikuchi, A. (1998). Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3beta and beta-catenin and promotes GSK-3beta- dependent phosphorylation of beta-catenin. Embo J 17, 1371-1384.

[0146] Kamoto, T., Shisa, H., Pataer, A., Lu, L., Yoshida, O., Yamada, Y., and Hiai, H. (1996). A quantitative trait locus in major histocompatibility complex determining latent period of mouse lymphomas. Jpn J Cancer Res 87, 401- 404.Korinek, V., Barker, N.,

[0147] Morin, P. J., van Wichen, D., de Weger, R., Kinzler, K. W., Vogelstein, B., and Clevers, H. (1997). Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/- colon carcinoma. Science 275, 1784-1787.

[0148] Liu, C, Li, Y., Semenov, M., Han, C, Baeg, G. H., Tan, Y., Zhang, Z., Lin, X., and He, X. (2002). Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell 108, 837-847. [0149] Manoukian, A. S., and Woodgett, J. R. (2002). Role of glycogen synthase kinase-3 in cancer: regulation by Wnts and other signaling pathways. Adv Cancer Res 84, 203-229.

[0150] Mertens, F., Johansson, B., Hoglund, M., and Mitelman, F. (1997). Chromosomal imbalance maps of malignant solid tumors: a cytogenetic survey of 3185 neoplasms. Cancer Res 57, 2765-2780.

[0151] Miller, J. R. (2002). The Wnts. Genome Biol 3, REVIEWS3001.

[0152] Minassian, B. A. (2001). Lafora's disease: towards a clinical, pathologic, and molecular synthesis. Pediatr Neurol 25, 21-29.

[0153] Minassian, B. A. (2002). Progressive myoclonus epilepsy with polyglucosan bodies: Lafora disease. Adv Neural 89, 199-210.

[0154] Minassian, B. A., Lee, J. R., Herbrick, J. A., Huizenga, J., Soder, S., Mungall, A. J., Dunham, I., Gardner, R., Fong, C. Y., Carpenter, S., et al. (1998). Mutations in a gene encoding a novel protein tyrosine phosphatase cause progressive myoclonus epilepsy. Nat Genet 20, 171-174.

[0155] Molenaar, M., van de Wetering, M., Oosterwegel, M., Peterson- Maduro, J., Godsave, S., Korinek, V., Roose, J., Destree, O., and Clevers, H. (1996). XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos. Cell 86, 391-399.

[0156] Ossipova, O., Bardeesy, N., DePinho, R. A., and Green, J. B. (2003). LKB1 (XEEK1 ) regulates Wnt signalling in vertebrate development. Nat Cell Biol 5, 889-894.

[0157] Paige, A. J. (2003). Redefining tumour suppressor genes: exceptions to the two-hit hypothesis. Cell MoI Life Sci 60, 2147-2163. [0158] Papadopoulou, D., Bianchi, M. W., and Bourouis, M. (2004). Functional studies of shaggy/glycogen synthase kinase 3 phosphorylation sites in Drosophila melanogaster. MoI Cell Biol 24, 4909-4919.

[0159] Peifer, M., and Polakis, P. (2000). Wnt signaling in oncogenesis and embryogenesis-a look outside the nucleus. Science 287, 1606-1609.

[0160] Phiel ,CJ., Wilson, CA, Lee, V.M., Klein, P.S. (2003) GSK- 3alpha regulates production of Alzheimer's disease amyloid-beta peptides. Nature 423, 435-9.

[0161] Polakis, P. (2000). Wnt signaling and cancer. Genes Dev 14, 1837-1851.

[0162] Reimann, J., Rudolphi, A., Tcherepnev, G., Skov, S., and Claesson, M. H. (1994). TCR/CD3 ligation of a TCR-transgenic T lymphoma blocks its proliferation in vitro but does not affect its growth in vivo. Exp Clin lmmunogenet 11 , 197-208.

[0163] Romashkova, J. A., and Makarov, S. S. (1999). NF-kappaB is a target of AKT in anti-apoptotic PDGF signalling. Nature 401 , 86-90.

[0164] Schechter, A. L., Stern, D. F., Vaidyanathan, L., Decker, S. J., Drebin, J. A., Greene, M. I., and Weinberg, R. A. (1984). The neu oncogene: an erb-B-related gene encoding a 185,000-Mr tumour antigen. Nature 312, 513-516.

[0165] Serratosa, J. M., Gardiner, R. M., Lehesjoki, A. E., Pennacchio, L. A., and Myers, R. M. (1999a). The molecular genetic bases of the progressive myoclonus epilepsies. Adv Neurol 79, 383-398.

[0166] Serratosa, J. M., Gomez-Garre, P., Gallardo, M. E., Anta, B., de Bernabe, D. B., Lindhout, D., Augustijn, P. B., Tassinari, C. A., Malafosse, R. M., Topcu, M., et al. (1999b). A novel protein tyrosine phosphatase gene is mutated in progressive myoclonus epilepsy of the Lafora type (EPM2). Hum MoI Genet 8, 345-352.

[0167] Sheng, Z. M., Marchetti, A., Buttitta, F., Champeme, M. H., Campani, D., Bistocchi, M., Lidereau, R., and Callahan, R. (1996). Multiple regions of chromosome 6q affected by loss of heterozygosity in primary human breast carcinomas. Br J Cancer 73, 144-147.

[0168] Shuman, S. (1994). Novel approach to molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase. J Biol Chem 269, 32678-32684.

[0169] Siegfried, E., Chou, T. B., and Perrimon, N. (1992). wingless signaling acts through zeste-white 3, the Drosophila homolog of glycogen synthase kinase-3, to regulate engrailed and establish cell fate. Cell 71 , 1167- 1179.

[0170] Song, L., De Sarno, P., and Jope, R. S. (2002). Central role of glycogen synthase kinase-3beta in endoplasmic reticulum stress-induced caspase-3 activation. J Biol Chem 277, 44701-44708.

[0171] Taylor, G. S., Liu, Y., Baskerville, C, and Charbonneau, H. (1997). The activity of Cdc14p, an oligomeric dual specificity protein phosphatase from Saccharomyces cerevisiae, is required for cell cycle progression. J Biol Chem 272, 24054-24063.

[0172] Tibiletti, M. G., Bemasconi, B., Taborelli, M., Furlan, D., Fabbri, A., Franchi, M., Taramelli, R., Trubia, M., and Capella, C. (1997). Involvement of chromosome 6 in endometrial cancer. Br J Cancer 75, 1831-1835.

[0173] Tibiletti, M. G., Furlan, D., Taborelli, M., Facco, C, Riva, C, Franchi, M., Cossu, A., Trubia, M., Taramelli, R., and Capella, C. (1999). Microsatellite instability in endometrial cancer: relation to histological subtypes. Gynecol Oncol 73, 247-252.

[0174] Walker, G. J., Palmer, J. M., Walters, M. K., Nancarrow, D. J., Parsons, P. G., and Hayward, N. K. (1994). Simple tandem repeat allelic deletions confirm the preferential loss of distal chromosome 6q in melanoma, lnt J Cancer 58, 203-206.

[0175] Wang, J., Stuckey, J. A., Wishart, M. J., and Dixon, J. E. (2002). A unique carbohydrate binding domain targets the lafora disease phosphatase to glycogen. J Biol Chem 277, 2377-2380.

[0176] Willert, K., Brown, J. D., Danenberg, E., Duncan, A. W., Weissman, I. L., Reya, T., Yates, J. R., 3rd, and Nusse, R. (2003). Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423, 448-452.

[0177] Woodgett, J. R. (1990). Molecular cloning and expression of glycogen synthase kinase-3/factor A. Embo J 9, 2431-2438.

[0178] Woodgett, J. R. (2001). Judging a protein by more than its name: GSK-3. Sci STKE 2001 , RE12.

[0179] Yanagawa, S., Matsuda, Y., Lee, J. S., Matsubayashi, H., Sese, S., Kadowaki, T., and Ishimoto, A. (2002). Casein kinase I phosphorylates the Armadillo protein and induces its degradation in Drosophϊla. Embo J 21 , 1733- 1742.

[0180] Zeng, L., Fagotto, F., Zhang, T., Hsu, W., Vasicek, T. J., Perry, W. L., 3rd, Lee, J. J., Tilghman, S. M., Gumbiner, B. M., and Costantini, F. (1997). The mouse Fused locus encodes Axin, an inhibitor of the Wnt signaling pathway that regulates embryonic axis formation. Cell 90, 181-192. [0181] Zhang, Y., Weber-Matthiesen, K., Siebert, R., Matthiesen, P., and Schlegelberger, B. (1997). Frequent deletions of 6q23-2<4 in B-cell non-Hodgkin's lymphomas detected by fluorescence in situ hybridization. Genes Chromosomes Cancer 18, 310-313.

[0182] Zheng, X., Gao, J. X., Zhang, H., Geiger, T. L., Liu, Y., and Zheng, P. (2002). Clonal deletion of simian virus 40 large T antigen-specific T cells in the transgenic adenocarcinoma of mouse prostate mice: an important role for clonal deletion in shaping the repertoire of T cells specific for antigens overexpressed in solid tumors. J Immunol 169, 4761-4769.

[0183] Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of characterizing an etiology of a cancer comprising testing at least one cancer cell for the presence of at least one of a) methylation of EPM2A gene; b) reduced expression of EPM2A gene; and c) mutation of EPM2A gene.

2. A method of characterizing an etiology of a cancer comprising testing at least one cancer cell for the presence of lafora bodies.

3. A method of treating a cancer in a person identified as having a cancer associated with methylated EPM2A gene, comprising administering to said person an effective amount of a drug that demethylates DNA.

4. A method of treating a person identified as having a disease associated with reduced expression or mutation of EPM2A gene, comprising activating EPM2A genes in cells of the person's body.

5. The method according to claim 4, wherein the disease is chosen from Lafora disease and cancer.

6. A method of treating a person identified as having a disease associated with reduced expression or mutation of EPM2A gene, comprising administering laforin to the cancer cells.

7. The method according to claim 6, wherein the disease is chosen from Lafora disease and cancer.

8. The method according to claim 6, wherein administering the laforin comprises administering laforin protein to the person.

9. The method according to claim 6, wherein administering the laforin comprises administering a laforin-expressing genetic construct to the person. 10. A method of treating a person identified as having a disease associated with reduced expression or mutation of EPM2A gene, comprising administering a genetically engineered GSK3/? that cannot be phosphorylated at the Serine 9 position.

11. The method according to claim 10, wherein the GSK3/? that cannot be phosphorylated at the Serine 9 position includes an amino acid mutation at Serine 9 with Alanine or any other amino acids.

12. A method of treating a person identified as having a disease associated with reduced expression or mutation of EPM2A gene, comprising administering to the person at least one compound that restores laforin's ability to phosphorylate GSK3β.

13. A method of treating a person identified with at least one disease associated with abnormal activation of GSK3, comprising enhancing expression of laforin by delivery of EPM2A gene, or reducing expression of laforin by administration of at least one of anti-sense or small interfering RNA.

14. A method of identifying compounds that modify laforin's ability to phosphorylate GSK3β at Serine 9 comprising testing laforin's ability to phosphorylate GSK3/? in the presence and absence of the test compo unds.

15. The method according to claim 14, wherein the laforin tested is wild-type laforin and the compounds identified are inhibitors of laforin's ability to phosporylate GSK3/?.

16. The method according to claim 14, wherein the laforin tested is mutated laforin and the compounds identified restore laforin's ability to phosphorylate GSK3β. 17. A method of treating Alzheimer's disease com prising administering to a person in need of treatment a compound identified according to the method of claim 15.

18. A method of determining an increased likelihood of cancer, in a person comprising assaying a tissue sample from the perso n for the presence of at least one of a) methylation of EPM2A gene; b) reduced expression of EPM2A gene; and c) mutation of EPM2A gene.

19. The method according to claim 18, wherein the person is a blood relative of a person having Lafora disease.

20. A method of reducing the likelihood of cancer in a person identified as positive for the presence of at least one of a) reduced expression of EPM2A, b) methylation of EPM2A gene, or c) mutation of EPM2A ge ne, comprising modifying the person's glucose metabolism.

21. The method according to claim 20, wherein the person's glucose metabolism is modified through diet modification.

23. The method according to claim 21, wherein the person's glucose metabolism is modified by administration of a drug chosen from 2-deoxyglucose and metforin.