[0001] This application claims priority to U.S. Provisional Pat. App. No.

61/547,671, filed on October 14, 201 1, which is fully incorporated by reference herein for all purposes.

BACKGROUND OF THE INVENTION

[0002] This invention was made with government support under AG21842-02 awarded by the National Institute on Aging, and under DK54687-06 awarded by the National Institute of Diabetes and Digestive and Kidney Diseases. The government has certain rights in the invention.

1. Field of the Invention

[0003] The invention relates to methods of inhibiting tumor cell proliferation by inhibiting FoxMlB activity. Specifically, the invention relates to methods and compositions for inhibiting tumor cell proliferation by inhibiting FoxMlB activity, expression, or nuclear localization in a tumor cell.

2. Background of the Related Art

[0004] The Forkhead box transcription factors have been implicated in regulating cellular longevity and proliferative capacity. Such studies include a finding of increased longevity in C. elegans bearing a mutant daf-2 gene, which encodes the worm homolog of the insulin/Insulin-like Growth Factor 1 (IGFl) receptor (Lin et al, 1997, Science 278: 1319-1322; Ogg et al, 1997, Nature 389: 994-999). Disruption of the daf-2 gene abolishes insulin-mediated activation of the phosphatidylinositol 3- kinase (PI3K) - protein kinase B/Akt (Akt) signal transduction pathway and prevents inhibition of the forkhead transcription factor daf-16 (corresponding to mammalian homologs FoxO l or Fkhr) (Paradis and Ruvkun, 1998, Genes Dev. 12: 2488-2498). Activation of the PI3K/Akt pathway phosphorylates the C-terminus of the Daf-16 (FoxOl; Fkhr) gene product and mediates its nuclear export into the cytoplasm, thus preventing FoxOl transcriptional activation of target genes (Biggs et al, 1999, Proc. Natl. Acad. Sci. USA 96: 7421-7426; Brunei et al, 1999, Cell 96: 857-68; Guo et al,

1999, J. Biol. Chem. 274: 17184-17192).

[0005] More recent studies oiDaf-2 C. elegans mutants have demonstrated that Daf-16 stimulates expression of genes that limit oxidative stress (Barsyte et al, 2001, FASEB J. 15: 627-634; Honda et al, 1999, FASEB J. 13 : 1385-1393; Wolkow et al,

2000, Science 290: 147-150) and that the mammalian FoxOl gene could functionally replace the Daf-16 gene in C. elegans (Lee et al, 2001, Curr. Biol. jj_: 1950-1957). In proliferating mammalian cells, the PI3K/Akt signal transduction pathway is essential for Gl to S-phase progression because it prevents transcriptional activity of the FoxO l and Fox03 proteins, which stimulate expression of the CDK inhibitor p27 ^kipl gene (Medema et al, 2000, Nature 404: 782-787). Moreover, genetic studies in budding yeast demonstrated that forkhead Fkhl and Fkh2 proteins are components of a transcription factor complex that regulates expression of genes critical for progression into mitosis (Hollenhorst et al, 2001, Genes Dev. 15: 2445-2456;

Koranda et al, 2000, Nature 406: 94-98; Kumar et al, 2000, Curr. Biol. 10: 896-906; Pic et al, 2000, EMBO J. 19: 3750-3761).

[0006] Forkhead Box M1B (FoxMlB) transcription factor (also known as Trident and HFH-11B) is a proliferation-specific transcription factor that shares 39% amino acid homology with the HNF-3 winged helix DNA binding domain. The molecule also contains a potent C-terminal transcriptional activation domain that possesses several phosphorylation sites for M-phase specific kinases as well as PEST sequences that mediate rapid protein degradation (Korver et al, 1997, Nucleic Acids Res. 25: 1715-1719; Korver et al, 1997, Genomics 46: 435-442; Yao et al, 1997, J. Biol. Chem. 272: 19827-19836; Ye et al, 1997, Mol. Cell Biol. 17: 1626-1641).

[0007] In situ hybridization studies have shown that FoxMlB is expressed in embryonic liver, intestine, lung, and renal pelvis (Ye et al, 1997, Mol. Cell Biol. 7: 1626-1641). In adult tissue, however, FoxMlB is not expressed in postmitotic, differentiated cells of the liver and lung, although it is expressed in proliferating cells of the thymus, testis, small intestine, and colon (Id). FoxMlB expression is reactivated in the liver prior to hepatocyte DNA replication following regeneration induced by partial hepatectomy (Id).

[0008] FoxMlB is expressed in several tumor-derived epithelial cell lines and its expression is induced by serum prior to the Gi/S transition (Korver et al, 1997 ^' , Nucleic Acids Res. 25: 1715-1719; Korver et al, 1997, Genomics 46: 435-442; Yao et al, 1997, J. Biol. Chem. 272: 19827-19836; Ye et al, 1997, Mol. Cell Biol. 17: 1626- 1641). Consistent with the role of FoxMlB in cell cycle progression, elevated FoxMlB levels are found in numerous actively -proliferating tumor cell lines (Korver et al, 1997, Nucleic Acids Res. 25: 1715-1719; Yao et al, 1997, J. Biol. Chem. 272: 19827-36; Ye et al, 1997, Mol. Cell Biol. 17: 1626-1641). Increased nuclear staining of FoxMlB was also found in human basal cell carcinomas (Teh et al, 2002, Cancer Res. 62: 4773-80), suggesting that FoxMlB is required for cellular proliferation in human cancers.

[0009] These studies and others suggest that FoxMlB plays some role in human cancers. FoxMlB, therefore, would provide an attractive target for anti-cancer therapies because FoxMlB expression typically declines during normal aging (see co- owned U.S. patent application US 2004-0109844 Al, filed August, 28 2003, incorporated by reference herein). Thus, FoxMlB might provide a selective target that is more active in tumor cells than in normal cells, particularly terminally- differentiated, aged or aging normal cells that surround a tumor, allowing tumor cells to be treated while minimizing the deleterious side-effects of such compounds on normal cells.

SUMMARY OF THE INVENTION

[0010] The invention provides methods of inhibiting proliferation of a tumor cell, comprising the step of inhibiting FoxMlB activity in the tumor cell. The methods of the invention can be accomplished by regulating FoxMlB activity through any of the mechanisms as described herein or described in co-owned U.S. Patent Application Serial No. 12/871,560 and co-owned U.S. Patent Nos. 7,799,896 and 7,635,673. The disclosures of U.S. Patent Application Serial No. 12/871,560 and U.S. Patent Nos. 7,799,896 and 7,635,673 are herein incorporated by reference in their entireties.

[0011] In one aspect of the invention, cellular FoxMlB activity is inhibited by causing FoxMlB protein to localize in the tumor cell cytoplasm or to localize to the nucleolus of the tumor cell nucleus and/or preventing or inhibiting translocation of FoxMlB to the cell nucleus. Causing FoxMlB protein to localize in the cytoplasm can be accomplished, for example, by contacting a cell with a compound that causes FoxMlB to translocate from the nucleus to the cytoplasm, or that sequesters FoxMlB in the cytoplasm and prevents FoxMlB from translocating from the cytoplasm to the nucleus. Causing FoxMlB protein to localize in the nucleolus of the nucleus can occur when FoxMlB protein interacts with the tumor suppressor pl9 ^ARF protein or a peptide containing the pl9 ^ARF sequences 26-44 or compounds that mimic pl9 ^ARF function. Such compounds can be identified using screening methods of the invention as described herein.

[0012] In another aspect, FoxMlB activity can be inhibited by contacting a cell, preferably a tumor cell, with a peptide having an amino acid sequence of the pl9 ^ARF tumor suppressor protein as set forth in SEQ ID NO: 10

(rrrrrrrrrKFVRSRRPRTASCALAFVN; referred to herein as the (D-Arg) ₉ -pl9 ^ARF 26- 44 peptide), SEQ ID NO: 11 (KFVRSRRPRTASCALAFVN; referred to herein as the pl9 ^ARF 26-44 peptide), or SEQ ID NO: 12

(KFVRSRRPRTASCALAFVNMLLRLERIL RR; referred to herein as the pl9 ^ARF 26- 55 peptide).

[0013] In another aspect, the invention provides a modified polypeptide that inhibits FoxMlB activity in a tumor cell. In a preferred embodiment, the polypeptide comprises a pl9Arf peptide fragment comprising pl9Arf amino acid residues 26-44 of SEQ ID NO: 16, and an HIV Tat peptide of SEQ ID NO: 17. In another embodiment, the polypeptide comprises a pl9Arf peptide fragment comprising pl9Arf amino acid residues 26-44 of SEQ ID NO: 16, and a nine-D-Arg peptide of SEQ ID NO: 18. In some embodiments, the nine-D-Arg peptide of SEQ ID NO: 18 or the HIV Tat peptide of SEQ ID NO: 17 is covalently linked to the N-terminus of the pl9Arf peptide fragment. In other embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 19.

[0014] In another aspect, the invention provides a modified polypeptide that inhibits FoxMlB activity in a tumor cells wherein the polypeptide is modified at the N-terminus, at the C-terminus, or at both the N terminus and the C terminus. In certain embodiments, the polypeptide is modified by acetylation. In other embodiments, the polypeptide is modified by amidation. In still other embodiments, the polypeptide is modified by both acetylation and amidation.

[0015] The methods of the invention can be used to inhibit growth of any tumor cell that expresses FoxMlB protein or that is derived from a cell that expressed FoxMlB protein. A cell that expressed FoxMlB protein can be, for example, a cell from an aging individual, wherein expression of FoxMlB protein is diminished as a result of aging. In a particular aspect, the methods of the invention can be used to inhibit tumor cell growth in vitro (i.e. under cell culture conditions) or in vivo (i.e. in a live animal). In other aspects, the methods of the invention can be used to inhibit growth of tumor cells that are derived from benign or malignant tumors. In a particular aspect, the tumor cells are of epithelial cell origin, for example, from liver, lung, skin, intestine (small intestine or colon), spleen, prostate, breast, brain, or thymus cells. The tumor cells can also be of mesoderm cell origin, for example, from liver, lung, skin, intestine (small intestine or colon), spleen, prostate, breast, brain, bone marrow or thymus cells.

[0016] The invention also provides methods for inhibiting tumor growth in an animal comprising administering to an animal, bearing at least one tumor cell present in its body, a therapeutically effective amount of a FoxMlB inhibitor for a therapeutically effective period of time. In another aspect, the FoxMlB inhibitor can be a compound that inhibits FoxMlB activity. In yet another aspect, the FoxMlB inhibitor can be a peptide having an amino acid sequence as set forth in SEQ ID NO: 10, SEQ ID NO: 1 1, or SEQ ID NO: 12, for a therapeutically effective period of time. In additional aspects, a combination of peptides that inhibit FoxMlB activity can be administered to the animal. For example, peptides having an amino acid sequence as set forth in SEQ ID NO: 10 can be administered with peptides having an amino acid sequence as set forth in SEQ ID NO: 11 and/or SEQ ID NO: 12. One of skill in the art will recognize that any combination of these peptides can be administered to the animal bearing at least one tumor cell in its body.

[0017] The invention also provides pharmaceutical compositions comprising a peptide having an amino acid sequence as set forth in SEQ ID NO: 10, SEQ ID NO: 1 1, or SEQ ID NO: 12 or therapeutically-effective mixture thereof. In certain aspects, pharmaceutical compositions of the invention are useful for inhibiting tumor cell growth in an animal by inhibiting FoxMlB activity in the tumor cell. In other embodiments, the pharmaceutical composition comprises a modified polypeptide that inhibits FoxMlB activity in a tumor cell.

[0018] Specific preferred embodiments of the invention will become evident from the following more detailed description of certain preferred embodiments and the claims. DETAILED DESCRIPTION OF THE DRAWINGS

[0019] Figures 1A and IB depict a human FoxMlB cDNA comprising a deletion of the terminal 972 nucleotides at the 3 ' end (SEQ ID NO: 1).

[0020] Figure 1C depicts a human FoxMlB protein sequence (SEQ ID NO: 2) encoded by the nucleotide sequence as set forth in SEQ ID NO: 1.

[0021] Figure 2 is a schematic representation of triple-LoxP FoxMlB targeting vector used to generate conditional FoxMlB knockout mice.

[0022] Figures 3A and 3B show RNase protection assays (RPA) with a FoxMlB probe after infection of human hepatoma HepG2 cells with Adenovirus expressing antisense human FoxMlB cDNA (AdFoxMlB AS). [0023] Figures 4A and 4B show R ase protection assays (RPA) with a FoxMlB probe after infection of human osteoblastoma U20s cells with AdFoxMlB AS.

[0024] Figure 5 A shows the FoxMlB amino acid sequence from amino acid residue 582-662 (SEQ ID NO: 8) and the LXLXXL (SEQ ID NO: 3) motif, which extends from amino acid residue 635-662 (SEQ ID NO: 9). All of the Thr or Ser residues in the FoxMlB protein sequence that are potential Cdkl/Cdk2

phosphorylation sites were changed to alanine and the Leu residue at 641 in the LXLXXL (SEQ ID NO: 3) motif was changed to alanine.

[0025] Figure 5B depicts a graph showing that mutation of the Cdkl phosphorylation site at 596 and Leu residue at 641 causes diminished FoxMlB transcriptional activity. Results are expressed as the percent activity with respect to wild-type FoxMlB where CMV-empty served as a control for basal expression levels of the FoxMlB reporter gene. Four separate transfection experiments were performed in triplicate to calculate ±SD.

[0026] Figure 5C shows the results of Western blot analysis with T7 epitope- tagged antibody of U20S cells transiently transfected with CMV-GFP-T7-FoxMlB following immunoprecipitation with a Cdkl or Cdk2 polyclonal antibody. The immunoprecipitated proteins were subjected to Western blot analysis using a monoclonal antibody against the T7 epitope tagged antibody protein. These co- immunoprecipitation studies showed that the Leu residue at 641 was required for association with the Cdk-Cyclin complexes.

[0027] Figure 5D shows the results of a kinase assay of U20S cells transiently transfected with CMV GFP-FoxMlB (lanel), CMV-GFP-FoxMlB T585A (lane 2), CMV GFP-FoxMlB T596A (lane 3), CMV GFP-FoxMlB L641A (lane 4), or CMV GFP-FoxMlBS657A (lane 5). [0028] Figure 5E shows diminished in vivo phosphorylation of the FoxMlB T596A Cdk mutant and FoxMlB L641A mutant proteins by the Cdk-Cyclin protein complexes. U20S cells were transiently transfected with either CMV T7-FoxMlB, CMV T7-FoxMlB T596A or FoxMlB L641A, and transfected cells were then serum starved for 48 hours. The cells were then incubated in the presence or absence of serum for 12 or 18 hours, the cells harvested and protein extracts prepared. Protein extracts were immunoprecipitated (IP) with an antibody specific for the T7 epitope and then subjected to Western blot analysis with MPM2 monoclonal antibody that recognizes phosphorylated Cdk sites. Western blot analysis with T7 antibody demonstrated equal amounts of FoxMlB protein in all the lanes. Relative intensity of MPM2 signal was determined and FoxMlB levels from cells not stimulated with serum was set at one.

[0029] Figure 6A is a schematic diagram depicting inhibition of Cdkl kinase activity by either Mytl phosphorylation, dominant-negative (DN) Cdkl or the Cdkl inhibitor Alsterpaullone.

[0030] Figure 6B is a schematic diagram depicting stimulation of Cdkl activity by Cdc25B and Cdc25C dephosphorylation.

[0031] Figure 6C is a graph demonstrating that inhibition of Cdkl activity diminished FoxMlB transcriptional activity in cotransfection assays. U20S TetR cells were transiently co-transfected with the reporter 6X-FoxMlB-TATA-

Luciferase and CMV-TO-FoxMlB (500 ng) alone or with increasing amounts of either CMV-DN-Cdkl, Cdkl pharmacological inhibitor Alsterpaullone or CMV- Mytl. Results are expressed as the percent activity with respect to wild-type FoxMlB using four separate transfection experiments were performed in triplicate to calculate ±SD. [0032] Figure 6D is a graph demonstrating that activation of Cdkl activity by dephosphorylation with either Cdc25B or Cdc25C stimulated FoxMlB

transcriptional activity, which was potentiated by increased CBP levels.

[0033] Figures 7A-H show nuclear localization of GFP-FoxMlB fusion protein following treatment with either pharmacological kinase inhibitors or dominant negative kinases. U20S cells were transiently transfected with CMV GFP-FoxMlB with the indicated pharmacological kinase inhibitors (B-D) or dominant-negative kinase expression vectors (E-H). Cells in panel (A) were untreated.

[0034] Figure 8A is a graph demonstrating that inhibition of CBP histone acetyl transferase activity by E1A decreased the FoxMlB transcriptional activity. U20S cells were transiently co-transfected with the reporter 6X-FoxMlB-TATA- Luciferase and CMV- FoxMlB alone or in different combinations with CBP and El A expression vectors.

[0035] Figure 8B shows the results of Western blot analysis of cell lysates after immunoprecipitation with a monoclonal antibody that recognized CBP. U20S cells were transiently transfected with CBP and either CMV WT GFP-FoxMlB (lanes 1- 2), CMV GFP-FoxMlB L641A (lanes 3-4), CMV GFP-FoxMlB S657A (lanes 5-6), or mock transfected (lanes 7-8). The first lane of each set contains 1/10 of the input protein extract (50ug) and the second lane contains the immunoprecipitated (IP) protein extracts.

[0036] Figure 9A shows a schematic diagram depicting the

Ras/MEK/MAPK/p90Rsk/Myt 1 and PI3K/PDKl/p90Rsk/Mytl pathways, which prevent Mytl phosphorylation mediated inhibition of Cdkl activity. Also shown is the action of DN-RasN17, the MEK1/2 inhibitor U0126, PI3K inhibitor Ly294002, DN-Akt and Akt pharmacological kinase inhibitor and DN-p90Rsk. [0037] Figure 9B shows the results of Western blot analysis with GFP antibody of protein extracts from U20S cells transiently transfected with CMV GFP-FoxMlB plasmid with either CMV DN-p90Rsk or CMV DN-RasN17 or 50 μΜ of U0126, 50 μΜ of PI3K inhibitor Ly294002 or 25 μΜ of Akt inhibitor.

[0038] Figure 9C is a graph demonstrating that inhibition of

Ras/MEK/MAPK/p90Rsk and PI3K/PDKl/p90Rsk pathways resulted in diminished FoxMlB transcriptional activity. U20S TetR cells were transiently co-transfected with the reporter 6X-FoxMlB-TATA-Luciferase and CMV-TO-FoxMlB (500 ng) with CMV-DN-p90Rsk, CMV-DN-Ras or DN-AKT or with 50 μΜ of either U0126 or Ly294002 alone or together or with 25 μΜ of Akt inhibitor. Four separate transfection experiments were performed in triplicate to calculate ±SD.

[0039] Figures 10A-B show fluorescent micrographs of TU EL assay (100 X) demonstrated similar apoptosis levels in Alb-Cre Foxmlb -I- and Foxmlb fl/fl control after 23 weeks of DEN/PB exposure.

[0040] Figure IOC shows a graph of the number of apoptotic cells (TUNEL positive) per 1000 hepatocytes (±SD) in non-tumor regions of livers from male Foxmlb fl/fl or Alb-Cre Foxmlb -I- mice after either 0, 6, 23, or 33 weeks of DEN/PB exposure.

[0041] Figures 10D-G show high power magnification of hepatocytes in which the nuclei were counterstained with DAPI (630 X; D-E) or visualized by Laser Confocal microscopy (F-G; bar indicates 2 μιη). A centromere-specific mouse fluorescent in situ hybridization (FISH) probe was used to show that Alb-Cre Foxmlb -I- hepatocyte nuclei possessed an increase in the number of hybridizing

chromosomes compared to control hepatocyte nuclei at 23 weeks of DEN/PB treatment. [0042] Figure 1 OH is a graph of the mean number of DAPI stained hepatocyte nuclei per 200X field (±SD) in non-tumor regions of livers from male Foxmlb fl/fl or Alb-Cre Foxmlb -I- mice either untreated or after 6, 23, or 33 weeks of DEN/PB exposure. The mean number (±SD) of TU EL or DAPI positive hepatocyte nuclei per 1000 cells or 200X field was calculated by counting the number of positive hepatocyte nuclei using 5 different liver sections from 3 male mice at the indicated times of DEN/PB exposure.

[0043] Figure 1 1A-H shows immunohistochemically stained liver sections from Foxmlb fl/fl and Alb-Cre Foxmlb -I- mice either untreated or treated with DEN/PB for either 6, 23 or 33 weeks stained for nuclear expression of FoxMlB protein.

Abundant nuclear staining of FoxMlB protein was induced as early as 6 weeks after DEN/PB exposure in Foxmlb fl/fl hepatocytes surrounding the periportal vein (PV, C), but not in hepatocytes near the central vein (CV). High levels of nuclear FoxMlB protein persisted in hyper-proliferative hepatic adenomas and HCC (C and E, margins of tumor indicated by arrows). As expected, nuclear staining of Foxmlb protein was not found in Alb-Cre Foxmlb -I- hepatocytes at any of the time points following DEN/PB treatment (B, D, F and H). Abbreviations are PV, portal vein and CV, central vein. Magnifications are 200X.

[0044] Figure 12A-I shows that Alb-Cre Foxmlb -I- livers exhibit normal expression of GST-pi and CAR following DEN/PB treatment. Alb-Cre Foxmlb -/- and Foxmlb fl/fl livers isolated from male mice after 23 weeks of DEN/PB exposure were immunohistochemically stained with antibody specific to Glutathionine-S- transferase placental isoform (GST-pi). Both Alb-Cre Foxmlb -I- and Foxmlb fl/fl hepatocytes were strongly immunostained for GST-pi after 23 weeks of DEN/PB treatment (C-F) but its expression was not detected in untreated control Foxmlb fl/fl mouse liver (A-B). Western blot analysis with liver protein extracts demonstrated that hepatic expression of GST-pi protein was induced as early as 6 weeks following DEN/PB treatment and that its hepatic expression continued following 23 weeks of DEN/PB exposure (G). Normal hepatocyte nuclear levels of the CAR nuclear receptor were found in male Alb-Cre Foxmlb -I- mice following DEN/PB treatment (H-I). Magnifications: A, C, E is 50X; B, D, F, H, I is 200X.

[0045] Figures 13A-B show p27 ^Kipl immunohistochemical staining of liver sections from untreated Alb-Cre Foxmlb -I- and Foxmlb fl/fl mice.

[0046] Figures 13C-J show immunohistochemical staining of liver sections from Alb-Cre Foxmlb -I- and Foxmlb fl/fl male mice after either untreated or after 6, 23, or 33 weeks of DEN/PB exposure to examine hepatocyte nuclear expression of p27 ^&pl protein. In Figure 13E and G, the margins of hepatic adenoma (Ad) or hepatocellular Carcinoma (HCC) are indicated by arrows. Magnification: A-J is 200X.

[0047] Figure 13K shows immunohistochemical staining of p27 ^&pl protein in female Alb-Cre Foxmlb -I- mice hepatocytes after 50 weeks DEN/PB treatment.

[0048] Figure 13L shows immunohistochemical staining of p27 ^Kipl protein in male Alb-Cre Foxmlb -I- mice hepatocytes after 50 weeks of DEN/PB.

[0049] Figures 13M-N show graphs of percent p27 ^&pl positive hepatocyte nuclei per 200X field liver section during tumor progression. Number of hepatocyte nuclei per 200X section was determined by DAPI staining.

[0050] Figure 14A shows results from Western blot analysis of p27 ^&pl , Cdc25B or Cdc25C protein expression in liver protein extracts isolated from either untreated or DEN/PB treated mice. Expression levels of Cdk2 were used as a loading control. [0051] Figure 14B is a drawing depicting the FoxMlB winged helix DNA binding domain (WHD), the C-terminal transcriptional activation domain (TAD), and the FoxMlB LXL motif (639-641) that recruits either the Cdk2-Cyclin E/A (S-phase) or Cdkl-Cyclin B (G2 phase) complexes.

[0052] Figure 14C shows co-immunoprecipitation (Co-IP) assays with protein extracts prepared from U20S cells that were transiently transfected CMV p27 ^&pl and with CMV expression vectors containing either WT GFP-FoxMlB or GFP-Foxmlb L641A mutant protein that fail to recruit the Cdk-Cyclin complexes. Also shown is a control lane containing 1/10 of the extract used in the Co-IP experiment.

[0053] Figure 14D shows that p27 ^&pl protein inhibited FoxMlB transcriptional activity in cotransfection assays. Transfections were performed twice in triplicate and used to calculate percent WT FoxMlB transcriptional levels (±SD).

[0054] Figure 15 A shows Western Blot analysis, blotting with a p 19 ^ARF (p 19) antibody, of liver extracts prepared from two mice following either no treatment or 6, 23 and 33 weeks of DEN/PB exposure. Expression levels of Cdk2 were used as a loading control.

[0055] Figure 15B shows co-immunoprecipitation (Co-IP) assays performed with liver protein extracts prepared from Foxmlb fl/fl and Alb-Cre Foxmlb -I- mice following either 6 or 23 weeks of DEN/PB treatment. The protein extracts were first immunoprecipitated with p 19 antibody and then analyzed by Western blot analysis with a mouse FoxMlB antibody.

[0056] Figure 15C is a drawing depicting functional domains of the FoxMlB and pl9 ^ARF tumor suppressor proteins. Schematically shown is the FoxMlB winged helix DNA binding domain (WHD), the C-terminal transcriptional activation domain (TAD) and the C-terminal region (688-748) required for pl9 ^ARF (pl9) binding. Schematically shown are the l9 nucleolar localization sequence ( rLS) and the pi 9 Mdm2 and FoxMlB binding sites.

[0057] Figure 15D shows co-IP assays with protein extracts prepared from U20S cells that were transiently transfected with CMV green fluorescent protein (GFP)- FoxMlB fusion protein and with pi 9 expression vectors. These included expression vectors containing either WT pi 9 protein or N-terminal deletion mutants of the p 19 protein (ΔΙ-14, Δ15-25, Δ26-37, Δ26-37 + Δ1-14) that were fused with an hemagglutinin (HA) epitope tag. The pl9 protein was immunoprecipitated from transfected protein extracts with HA antibody followed by Western blot analysis with a monoclonal antibody specific to the GFP protein to detect the GFP-FoxMlB fusion protein.

[0058] Figure 15E shows co-IP assays with protein extracts prepared from U20S cells that were transiently transfected with CMV GFP-FoxMlB fusion protein and expression vector containing V5 epitope tagged pi 9 ^ARF 26-44 or pl9 ^ARF 26-55 sequences. The pl9 protein was immunoprecipitated from transfected protein extracts with V5 epitope antibody followed by Western blot analysis with GFP monoclonal antibody.

[0059] Figure 15F shows that the p 19 protein inhibits FoxMlB transcriptional activity in cotransfection assays.

[0060] Figure 16A-D shows immunostaining of U20S cells transfected with HA- pl9ARF and GFP- FoxMlB expression vectors demonstrating that the HA tagged pl9 was able to target nuclear fluorescence of WT GFP-Foxmlb fusion protein (D) to the nucleolus (B, C). [0061] Figures 16E-I shows nucleolar targeting of GFP- FoxMlB WT protein in cotransfections with CMV expression vectors containing mutant pi 9 ^ARF proteins (Δ1- 14, Δ15-25, 26-44 or 26-55) that were still able to associate with FoxMlB protein.

[0062] Figure 161 shows nucleolar fluorescence of CMV GFP- pl9 ^ARF 26-44.

[0063] Figure 16J shows nuclear fluorescence of CMV WT GFP- FoxMlB and expression vector containing mutant pl9 ^ARF A26-37 protein that failed to interact with FoxMlB.

[0064] Figure 16K shows transfection of CMV WT pl9 expression vector was unable to elicit nucleolar targeting of GFP- FoxMlB 1-688 protein, which failed to bind to p 19 protein.

[0065] Figure 16L shows that treatment of U20S cells for three days with the TRITC fluorescently tagged (D-Arg) ₉ -pl9 ^ARF 26-44 peptide demonstrated that this pl9 ^ARF peptide was transduced into the cell and was localized to the nucleolus.

[0066] Figure 17A is a graph showing that the (D-Arg) ₉ -p 19 ^ARF 26-44 peptide was an effective inhibitor of FoxMlB transcriptional activity.

[0067] Figure 17B is a Western blot analysis showing that the CMV-TETO GFP- Foxmlb U20S clone C3 cell line displayed Doxycycline inducible expression of the GFP- FoxMlB fusion protein.

[0068] Figure 17C-H shows results of colony formation assays wherein the (D- Arg) ₉ -pl9 ^ARF 26-44 peptide significantly diminished the ability of induced GFP- FoxMlB to stimulate colony formation of the U20S clone C3 cells on soft agar. Doxycycline induced FoxMlB-GFP expression stimulated anchorage-independent growth in the U20S clone C3 cell line (F-G) as assessed by propagation for two weeks on soft agar while the (D-Arg)9-pl9 ^ARF 26-44 peptide significantly inhibited colony formation of U20S cells on soft agar (E and H). [0069] Figure 171 shows a graph depicting quantitation of FoxMlB induced formation of U20S cell colonies on soft agar treated or not treated with the (D-Arg) ₉ - pl9 ^ARF 26-44 peptide. The number of U20S colonies of the indicated treatments were counted in 4 to 5 different 100X fields and determined the mean number of cell colonies (±SD).

[0070] Figures 18A and 18B show graphs depicting quantitation of

deoxynucleotidyl transferase dUTP nick end labeling ("TUNEL") positive cells treated with either WT -blocked, WT-unblocked or mutant-unblocked (D-Arg) ₉ - pl9 ^ARF 26-44 peptide. The cells treated with WT-blocked (D-Arg) ₉ -pl9 ^ARF 26-44 peptides had a significantly higher number of TUNEL positive cells compared to cells treated with WT-unblocked and mutant-blocked (D-Arg) ₉ -pl9 ^ARF 26-44 peptides. In Fig. 18A WT-unblocked (D-Arg) ₉ -pl9 ^ARF 26-44 peptides showed some activity compared to the mutant-blocked peptides at doses higher than 30 μΜ (statistically significant at 30 and 40 μΜ). The experiments underlying Figures 18A (experiment 1) and 18B (experiment 2) were performed using the same protocol on separate dates.

[0071] Figures 19A and 19B show graphs depicting quantitation of TUNEL positive cells following treatment using various concentrations of the WT-blocked (D- ArgVpl^ 26-44 peptides. The EC50 (D-Arg^-pl^ 26-44 peptides for WT-blocked was 30.08 μΜ and 30.73 μΜ for Figures 19A and 19B, respectively). The experiments underlying Figures 19A (experiment 1) and 19B (experiment 2) were performed using the same protocol on separate dates.

[0072] Figure 20A shows photographs of tumor nodules isolated from ALb- HRasV12 mice treated with PBS, mutant ARF-peptide or ARF-peptide for three weeks. [0073] Figure 20B is a graph showing the quantification of tumor nodules from ALb-HRasV12 mice treated with PBS, mutant ARF-peptide or ARF-peptide for three weeks. The ARF-peptide treated mice showed a reduction of tumor nodules.

[0074] Figure 20C is a graph showing the percentage of CD45- CD90 + cells from ALb-HRasV12 mice treated with PBS, mutant ARF-peptide or ARF-peptide for three weeks. The results indicate that there was a considerable reduction in the CD45- CD90 + cells in the ARF-peptide treated mice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0075] Conventional techniques well known to those with skill in the art were used for recombinant DNA production, oligonucleotide synthesis, and tissue culture and cell transformation (e.g., electroporation, lipofection) procedures. Enzymatic reactions and purification techniques were performed according to manufacturers' specifications or as commonly accomplished in the art or as described herein. The techniques and procedures were generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al, 2001, MOLECULAR CLONING: A LABORATORY MANUAL, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference for any purpose. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, molecular biology, genetic engineering, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0076] Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

Definitions

[0077] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0078] The term "isolated protein" referred to herein means a protein encoded by a nucleic acid including, inter alia, genomic DNA, cDNA, recombinant DNA, recombinant RNA, or nucleic acid of synthetic origin or some combination thereof, which (1) is free of at least some proteins with which it would normally be found, (2) is essentially free of other proteins from the same source, e.g., from the same cell or species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is naturally found when isolated from the source cell, (5) is not linked (by covalent or noncovalent interaction) to all or a portion of a polypeptide to which the "isolated protein" is linked in nature, (6) is operatively linked (by covalent or noncovalent interaction) to a polypeptide with which it is not linked in nature, or (7) does not occur in nature. Preferably, the isolated protein is substantially free from other contaminating proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic or research use.

[0079] The terms "polypeptide" or "protein" is used herein to refer to native proteins, that is, proteins produced by naturally-occurring and specifically non- recombinant cells, or by genetically-engineered or recombinant cells, and comprise molecules having the amino acid sequence of the native protein, or sequences that have deletions, additions, and/or substitutions of one or more amino acids of the native sequence. The terms "polypeptide" and "protein" specifically encompass FoxMlB protein, or species thereof that have deletions, additions, and/or substitutions of one or more amino acids of FoxMlB having at least one functional property of the FoxMlB protein. In addition, the terms "polypeptide" and "protein" specifically encompass peptides that can inhibit FoxMlB activity, including the (D-Arg)g- pl9ARF 26-44 peptide (SEQ ID NO: 10; rrrrrrrrrKFVRSRRPRTASCALAFVN), the pl9 ^ARF 26-44 peptide (SEQ ID NO: 1 1; KFVRSRRPRTASCALAFVN), and the pl9 ^ARF 26-55 peptide (SEQ ID NO: 12;

KFVRSRRPRTASCALAFVNMLLRLERILRR), or species thereof that have deletions, additions, and/or substitutions of one or more amino acids of SEQ ID NO: 10, SEQ ID NO: 1 1, or SEQ ID NO: 12 having the ability to inhibit FoxMlB activity. The term "naturally-occurring" as used herein refers to an object that can be found in nature, for example, a polypeptide or polynucleotide sequence that is present in an organism (including a virus) that can be isolated from a source in nature and which has not been intentionally modified by man. The term "naturally occurring" or "native" when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, and the like, refers to materials which are found in nature and are not manipulated by man. Similarly, "recombinant," "non-naturally occurring" or "non-native" as used herein refers to a material that is not found in nature or that has been structurally modified or synthesized by man.

[0080] As used herein, the twenty conventional amino acids and their

abbreviations follow conventional usage. See IMMUNOLOGY-A SYNTHESIS, 2nd Edition, (E. S. Golub and D. R. Gren, Eds.), 1991, Sinauer Associates,

Sunderland, Mass., which is incorporated herein by reference for any purpose.

[0081] In a specific embodiment, the invention provides a polypeptide that inhibits FoxMlB activity in a tumor cells wherein the polypeptide is modified at the N-terminus, at the C-terminus, or at both the N terminus and the C terminus. In order to remove electric charge from polypeptide ends, the polypeptides can be modified by N-terminal acetylation and/or C-terminal amidation. In some embodiments, the modifications can help the polypeptide mimic uncharged natural peptides. In other embodiments, the modified ends are blocked against synthetase activities. In other embodiments, the modified polypeptide has the amino acid sequence of SEQ ID

NO: 19. Other known modifications to the N and C termini of a polypeptide can also be used according to the invention. In another embodiment, the N and/or C termini of the polypeptide are modified such that polypeptide is less likely or more likely to cyclize. Cyclization of polypeptides has been shown to affect the structural rigidity of the polypeptide. In one embodiment, a linker is provided to facilitate the cyclization of the polypeptide

[0082] In some embodiments, the polypeptide is modified by amidation. Many bioactive peptides have carboxyl terminal alpha-amide residues. Presence of the alpha-amide can be critical for biological activity. Amidation of peptides can enhance activity of certain polypeptides. Polypeptide amidation is known to one of ordinary skill in the art. Many of the precursor proteins to amidated peptides contain the amino acid sequence -X-Gly -Basic-Basic- where X is the residue that becomes amidated in the mature peptide and the basic residues can be lysine or arginine. Briefly, in a first reaction step the glycine is oxidized to form alpha-hydroxy-glycine. The oxidized glycine cleaves into the C-terminally amidated peptide and an N-glyoxylated peptide. Typically the resulting sequence is -X-NH ₂ . Any combination of these recognized sequences is contemplated by the invention. [0083] In other embodiments, the polypeptide is modified by acetylation.

Acetylation occurs when a polypeptide is modified by the attachment of at least one acetyl group, generally at the N-terminus. The acetylation reaction is known to one of ordinary skill in the art, and can be performed, for example, using an acidic anhydride. In some embodiments, the acetylated peptides can serve as optimized enzyme substrates.

[0084] Peptide analogs are commonly used in the pharmaceutical industry as non- peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics" or "peptidomimetics." (See Fauchere, 1986, Adv. Drug Res. 15: 29; Veber and Freidinger, 1985, TINS p392; and Evans et al, 1987, J. Med. Chem. 30: 1229, which are incorporated herein by reference for any purpose.) Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce a similar therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biochemical property or pharmacological activity), but have one or more peptide linkages optionally replaced by a linkage such as:— CH ₂ NH— , -CH ₂ S-, -CH ₂ -CH ₂ -, -CH=CH- (cis and trans), -COCH ₂ -, - CH(OH)CH ₂ — , and— CH ₂ SO-, by methods well known in the art. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) may be used in certain embodiments to generate more stable peptides. In addition, conformationally- constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch, 1992, Ann. Rev. Biochem. 61_: 387), incorporated herein by reference for any purpose); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.

[0085] The term "polynucleotide" as used herein means a polymeric form of nucleotides that are at least 10 bases in length. In certain embodiments, the bases may be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.

[0086] In one embodiment, the invention provides methods for inhibiting proliferation of a tumor cell comprising the step of inhibiting FoxMlB activity in the tumor cell. Several methods of inhibiting FoxMlB activity can be used to accomplish the methods of the invention. For example, FoxMlB activity in a cell can be inhibited by causing FoxMlB protein to localize in the cytoplasm, rather than in the nucleus. Causing FoxMlB to localize in the cytoplasm can be accomplished, for example, by contacting a cell with a compound that causes FoxMlB to translocate from the nucleus to the cytoplasm, or that sequesters FoxMlB in the cytoplasm and prevents FoxMlB from translocating from the cytoplasm to the nucleus.

[0087] In another embodiment, the inhibitor comprises a polypeptide. In another aspect, the invention provides a modified polypeptide that inhibits FoxMlB activity in a tumor cell. In a preferred embodiment, the polypeptide is isolated.

[0088] In certain embodiments, the polypeptide is a chimeric protein. In other embodiments, the polypeptide comprises a viral protein or a fragment thereof. In one embodiment, the polypeptide comprises the HIV Tat peptide. In another embodiment, the polypeptide comprises the HIV Tat peptide of SEQ ID NO: 17. In another embodiment, the inhibitor comprises a nine-D-Arg peptide of SEQ ID NO: 18. In another embodiment, the inhibitor comprises a pl9Arf peptide fragment comprising pl9Arf amino acid residues 26-44 of SEQ ID NO: 16. In a preferred embodiment, the polypeptide comprises (1) a pl9Arf peptide fragment comprising pi 9Arf amino acid residues 26-44 of SEQ ID NO: 16, and (2) an HIV Tat peptide of SEQ ID NO: 17. In another embodiment, the polypeptide comprises (1) a pl9Arf peptide fragment comprising pl9Arf amino acid residues 26-44 of SEQ ID NO: 16, and (2) a nine-D- Arg peptide of SEQ ID NO: 18 that is covalently linked to the N-terminus of the pl9Arf peptide fragment. In other embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 19. Amino acid sequences are provided in Table 5.

[0089] The polypeptide can be modified at the N-terminus, at the C-terminus or at both the N terminus and the C terminus. Modifications can comprise acetylation, amidation, or any of the other known modifications known in the art and as described. In yet another embodiment, the inhibitor comprises an isolated modified polypeptide that inhibits FoxMlB activity in a tumor cell, said polypeptide comprising (1) a pl9Arf peptide fragment comprising pl9Arf amino acid residues 26-44 of SEQ ID NO: 16, and (2) an HIV Tat peptide of SEQ ID NO: 17 or a nine-D-Arg peptide of SEQ ID NO: 18 that is covalently linked to the N-terminus of the pi 9Arf peptide fragment, wherein the polypeptide is modified at the N-terminus, at the C-terminus or at both the N terminus and the C terminus.

[0090] In one embodiment of the invention, an effective inhibitor of FoxMlB activity causes at least about 50% reduction in FoxMlB activity. Preferably, an effective inhibitor of FoxMlB activity causes at least about 80% reduction in FoxMlB activity. Most preferably, an inhibitor of FoxMlB activity causes at least about 90% reduction in FoxMlB activity.

[0091] Assaying for nuclear localization and expression of FoxMlB protein can be accomplished by any method known the art. For example, immunohistochemistry using detectably-labeled primary anti-FoxMlB antibodies, or unlabeled primary anti- FoxMlB and detectably-labeled secondary antibodies (for example, labeled with fluorescent markers, such as fluorescein isothiocyanate, FITC), can be used to visualize FoxMlB protein localization, inter alia, by fluorescence microscopy.

Alternative labels, such as radioactive, enzymatic and hapten labels, are within the scope of this invention.

[0092] As used herein, the terms "label" or "labeled" refers to incorporation of a detectable marker, e.g., by incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotin moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). In certain embodiments, the label or marker can also be therapeutic. Various methods of labeling polypeptides and glycoproteins can be used that are known in the art. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, "Tc, ^{U 1} ln, ¹²⁵ I, ¹³¹ I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotin, and predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In certain embodiments, labels are attached by spacer arms of various lengths (such as -(CH ₂ ) _n -, n = 1-50, more preferably 1-20) to reduce steric hindrance. [0093] In certain embodiments, the invention provides a method of inhibiting tumor growth in an animal comprising inhibiting FoxMlB activity in a tumor cell in the animal, for example, by administering to the animal, which has at least one tumor cell present in its body, a therapeutically effective amount of a compound that inhibits FoxMlB activity.

[0094] In certain embodiments, the invention provides pharmaceutical compositions comprising a therapeutically effective amount of a compound that inhibits FoxMlB expression, nuclear localization or expression and or nuclear localization in mammalian cells together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant. In other embodiments, the invention provides pharmaceutical compositions that comprise a therapeutically effective amount of a compound that inhibits FoxMlB expression in mammalian cells and also induces FoxMlB protein to translocate into the cytoplasm from the nucleus of tumor cells together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant. Such compounds can be identified in screening methods of the invention. The invention further provides pharmaceutical compositions comprising a peptide having an amino acid sequence as set forth in SEQ ID NO: 10, SEQ ID NO: 1 1, or SEQ ID NO: 12.

[0095] The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.

[0096] The term "pharmaceutical composition" as used herein refers to a composition comprising a pharmaceutically acceptable carrier, excipient, or diluent and a chemical compound, peptide, or composition as described herein that is capable of inducing a desired therapeutic effect when properly administered to a patient. [0097] The term "therapeutically effective amount" refers to the amount of growth hormone or a pharmaceutical composition of the invention or a compound identified in a screening method of the invention determined to produce a therapeutic response in a mammal. Such therapeutically effective amounts are readily ascertained by one of ordinary skill in the art and using methods as described herein.

[0098] As used herein, "substantially pure" means an object species that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition). In certain embodiments, a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis or on a weight or number basis) of all macromolecular species present. In certain embodiments, a substantially pure composition will comprise more than about 80%, 85%, 90%, 95%, or 99% of all macromolar species present in the composition. In certain embodiments, the object species is purified to essential homogeneity (wherein contaminating species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.

[0099] The term "patient" includes human and animal subjects.

[00100] As used herein, the terms "tumor growth" and "tumor cell proliferation" are used to refer to the growth of a tumor cell. The term "tumor cell" as used herein refers to a cell that is neoplastic. A tumor cell can be benign, i.e. one that does not form metastases and does not invade and destroy adjacent normal tissue, or malignant, i.e. one that invades surrounding tissues, is capable of producing metastases, may recur after attempted removal, and is likely to cause death of the host. Preferably a tumor cell that is subjected to a method of the invention is an epithelial-derived tumor cell, such as a tumor cell derived from skin cells, lung cells, intestinal epithelial cells, colon epithelial cells, testes cells, breast cells, prostate cells, brain cells, bone marrow cells, blood lymphocytes, ovary cells or thymus cells.

[00101] Acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. The pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta- cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose or dextrins);

proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as

polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20 and polysorbate 80, Triton, trimethamine, lecithin, cholesterol, or tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol, or sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants. See, for example, REMINGTON'S

PHARMACEUTICAL SCIENCES, 18 ^th Edition, (A.R. Gennaro, ed.), 1990, Mack

Publishing Company.

[00102] Optimal pharmaceutical compositions can be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, Id. Such compositions may influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of the antibodies of the invention.

[00103] The primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Pharmaceutical compositions can comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute therefor. Pharmaceutical compositions of the invention may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (REMINGTON'S PHARMACEUTICAL SCIENCES, Id.) in the form of a lyophilized cake or an aqueous solution. Further, the FoxM IB-inhibiting product may be formulated as a lyophilizate using appropriate excipients such as sucrose. [00104] Formulation components are present in concentrations that are acceptable to the site of administration. Buffers are advantageously used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.

[00105] The pharmaceutical compositions of the invention can be delivered parenterally. When parenteral administration is contemplated, the therapeutic compositions for use in this invention may be in the form of a pyrogen- free, parenterally acceptable aqueous solution comprising the desired compound identified in a screening method of the invention in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which the compound identified in a screening method of the invention is formulated as a sterile, isotonic solution, appropriately preserved. Preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that may provide controlled or sustained release of the product which may then be delivered via a depot injection. Formulation with hyaluronic acid has the effect of promoting sustained duration in the circulation. Implantable drug delivery devices may be used to introduce the desired molecule.

[00106] The compositions may be formulated for inhalation. In these

embodiments, a compound identified in a screening method of the invention or a FoxMlB inhibitor disclosed herein is formulated as a dry powder for inhalation, or inhalation solutions may also be formulated with a propellant for aerosol delivery, such as by nebulization. Pulmonary administration is further described in PCT Application No. PCT/US94/001875, which describes pulmonary delivery of chemically modified proteins and is incorporated by reference. [00107] The pharmaceutical compositions of the invention can be delivered through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art. A FoxMlB inhibitor disclosed herein or compounds of the invention that are administered in this fashion may be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. A capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized.

Additional agents can be included to facilitate absorption of the FoxMlB inhibitor disclosed herein or compound identified in a screening method of the invention.

Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed.

[00108] A pharmaceutical composition may involve an effective quantity of a FoxMlB inhibitor disclosed herein or a compound in a mixture with non-toxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another appropriate vehicle, solutions may be prepared in unit-dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.

[00109] Additional pharmaceutical compositions are evident to those skilled in the art, including formulations involving a FoxMlB inhibitor disclosed herein or compounds of the invention in sustained- or controlled-delivery formulations.

Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See, for example, PCT

Application No. PCT/US93/00829, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. Sustained- release preparations may include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules, polyesters, hydrogels, polylactides (U.S. 3,773,919 and EP 058,481), copolymers of L-glutamic acid and gamma ethyl-L- glutamate (Sidman et al, 1983, Biopolymers 22: 547-556), poly (2 -hydroxy ethyl- methacrylate) (Langer et al, 1981, J. Biomed. Mater. Res. 15: 167-277) and Langer, 1982, Chem. Tech. 12: 98-105), ethylene vinyl acetate (Langer et al, id.) or poly-D(-)- 3-hydroxybutyric acid (EP 133,988). Sustained release compositions may also include liposomes, which can be prepared by any of several methods known in the art. See e.g., Eppstein et al, 1985, Proc. Natl. Acad. Sci. USA 82: 3688-3692; EP

036,676; EP 088,046 and EP 143,949.

[00110] The pharmaceutical composition to be used for in vivo administration typically is sterile. In certain embodiments, this may be accomplished by filtration through sterile filtration membranes. In certain embodiments, where the composition is lyophilized, sterilization using this method may be conducted either prior to or following lyophilization and reconstitution. In certain embodiments, the composition for parenteral administration may be stored in lyophilized form or in a solution. In certain embodiments, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[00111] Once the pharmaceutical composition of the invention has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.

[00112] The present invention is directed to kits for producing a single-dose administration unit. Kits according to the invention may each contain both a first container having a dried protein compound identified in a screening method of the invention and a second container having an aqueous formulation, including for example single and multi-chambered pre-filled syringes (e.g., liquid syringes, lyosyringes or needle-free syringes).

[00113] The effective amount of a pharmaceutical composition of the invention to be employed therapeutically will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment, according to certain embodiments, will thus vary depending, in part, upon the molecule delivered, the indication for which the pharmaceutical composition is being used, the route of administration, and the size (body weight, body surface or organ size) and/or condition (the age and general health) of the patient. A clinician may titer the dosage and modify the route of administration to obtain the optimal therapeutic effect. Typical dosages range from about 0.1 g/kg to up to about 100 mg/kg or more, depending on the factors mentioned above. In certain embodiments, the dosage may range from 0.1 g/kg up to about 100 mg/kg; or 1 |^g/kg up to about 100 mg/kg; or 5 g/kg up to about 100 mg/kg.

[00114] The dosing frequency will depend upon the pharmacokinetic parameters of a FoxMlB inhibitor disclosed herein in the formulation. For example, a clinician administers the composition until a dosage is reached that achieves the desired effect. The composition may therefore be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages may be ascertained through use of appropriate dose-response data.

[00115] Administration routes for the pharmaceutical compositions of the invention include orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices. The pharmaceutical compositions may be administered by bolus injection or continuously by infusion, or by implantation device. The pharmaceutical composition also can be administered locally via implantation of a membrane, sponge or another appropriate material onto which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration.

[00116] In certain embodiments, it may be desirable to use a FoxMlB inhibitor disclosed herein or pharmaceutical compositions thereof in an ex vivo manner. In such instances, cells, tissues or organs that have been removed from the patient are exposed to pharmaceutical compositions of the invention after which the cells, tissues and/or organs are subsequently implanted back into the patient.

[00117] Pharmaceutical compositions of the invention can be administered alone or in combination with other therapeutic agents, in particular, in combination with other cancer therapy agents. Such agents generally include radiation therapy or chemotherapy. Chemotherapy, for example, can involve treatment with one or more of the following agents: anthracyclines, taxol, tamoxifene, doxorubicin, 5- fluorouracil, and other drugs known to one skilled in the art.

[00118] The following Examples are provided for the purposes of illustration and are not intended to limit the scope of the present invention. The present invention is not to be limited in scope by the exemplified embodiments, which are intended as illustrations of individual aspects of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

EXAMPLES

Example 1

Generation of Conditional FoxMlB Knockout Mice

[00119] FoxMlB knockout mice die immediately after birth. Therefore, to examine the role of FoxMlB in adult tissues, conditional FoxMlB knockout mice were generated using a triple-LoxP FoxMlB targeting vector to create a "Floxed" FoxMlB targeted locus (see Figure 2 for a schematic diagram of the vector). Cre recombinase-mediated deletion of the FoxMl genomic sequences spanning the two LoxP sites removes the entire winged helix DNA binding domain and the C-terminal transcriptional activation domain, thereby preventing expression of functional FoxMl isoforms. Following standard electroporation and culture of mouse embryonic stem (ES) cells to select for homologous recombination (G418 and gangcyclovir), homologous recombinants were identified by Southern blotting of ES cell genomic DNA. [00120] Mouse blastocysts were injected with the ES cells comprising the "Floxed" (fl/+) FoxMlB targeted allele, and chimeric mice with germ line transmission were selected. Viable mice homozygous for the "Floxed" (fl/fl) FoxMlB targeted allele were generated in this manner. Mice either homozygous (fl/fl) or heterozygous (fl/+) for the FoxMlB (fl) allele were verified by PCR amplification of mouse genomic DNA with primers that flanked the LoxP site. Breeding the albumin promoter Cre recombinase transgene into the FoxMlB (fl/fl) mouse genetic background allowed hepatocyte deletion of the FoxMlB locus within six weeks after birth, which was verified by Southern blot using liver genomic DNA.

Example 2

TTR-FoxMlB transgenic livers display increased size of hepatic preneoplastic and neoplastic nodules

[00121] To investigate the influence of increased FoxMlB expression on liver tumor formation, wild type (WT) and TTR-FoxMlB transgenic (TG) CD-I mice were treated for 23 weeks with diethylnitrosamine (DEN)/Phenobarbital (PB) liver tumor induction (Goldfarb et ah, 1983, Environ. Health Perspect. 50: 149-161 ; Russell et ah, 1996, Mo Carcinog. 15: 183-189; Slagle e/ a/., 1996, Mo/. Carcinog. 15:261-269; Tamano et ah, 1994, Carcinogenesis L5: 1791-1798). Transgenic CD-I mice were generated using the -3 kb transthyretin (TTR) promoter to constitutively express the FoxMlB transgene (SEQ ID NO: 1 as shown in Figure 1) in hepatocytes as described (Ye et ah, 1999, Moh Cell Biol, 19: 8570-8580). At 14 days postnatal of age 17 WT and TTR-FoxMlB TG CD-I mice received a single IP injection of 5 μg of DEN/g body weight (10 μΐ/g body weight of 0.05% solution of DEN in water). At 4 weeks of age, mice were placed on water containing 0.05% of PB for 21 weeks. The mice were sacrificed at 25 weeks of age, the livers were fixed in paraformaldehyde, paraffin embedded, sectioned and then H&E stained and examined for tumors. The TTR- FoxMlB TG livers exhibited larger preneoplastic and neoplastic nodules (Table 1 ; greater than 200 μιη in size) and hepatocyte proliferation was stimulated in these hepatic nodules as determined by immunohistochemical staining for Ki67 antigen. However, increased FoxMlB levels did not increase the number of hepatic tumor nodules, suggesting that FoxMlB enhanced the growth of hepatic tumors but did not stimulate tumor initiation.

Table 1

[00122] Table 1 shows the mean number ± (s.e.m.) of hepatic preneoplastic or neoplastic nodules (adenomas) per cm ³ within the range of sizes shown (n= 17 for each genotype). As shown in column 2 (a) and 3 (b), values are significantly different from control mice based on the Student's t-test P=0.019 and P=0.0027, respectively.

Example 3

Infection of proliferating human cell lines with Adenovirus expressing antisense human FoxMlB cDNA inhibits expression of endogenous FoxMlB mRNA

[00123] Proliferating human hepatoma HepG2 cells were infected with an increasing amounts of plaque forming units (PFU) per cell of either an adenovirus expressing antisense human FoxMlB cDNA (Figure 3A, AdFoxMlB AS) or Adenovirus expressing bacterial LacZ gene (Figure 3B, AdLacZ) and total RNA was isolated 20 hours following post infection. Expression of human FoxMlB mRNA was measured using an RNase protection assay (RPA) with a FoxMlB probe as described previously (Ye et al, 1999, Mol. Cell. Biol. 19:8570-8580; Ye et al, 1997, Mol. Cell Biol. 17: 1626-1641). These RPA studies demonstrated that AdFoxMlB AS infection at 30 pfu per cell is sufficient to inhibit endogenous FoxMlB expression (Figure 3 A), but AdLacZ control infections did not influence FoxMlB expression (Figure 3B). Furthermore, AdFoxMlB infection of human osteoblastoma U20S cells was sufficient to prevent FoxMlB expression in this human tumor cell line as well (Figure 4). Taken together infection of cells with AdFoxMlB AS is an effective means by which to inhibit FoxMlB expression in tumor cell lines.

Example 4

Generation of FoxMlB expression plasmids and luciferase reporter plasmid

[00124] The CMV-FoxMlB expression plasmid was generated by PCR amplification of the CMV Human FoxMlB expression plasmid (Ye et al, 1997, Mol. Cell Biol. 17: 1626-1641) with 5' EcoRl T-epitope tagged FoxMlB primer:

5'- gcggaattcaccatggctagcatgactggtggacagcaaatgggtTGGCAGAACTCTGTG TCTGAG (SEQ ID NO: 4) and a 3' antisense primer that hybridized to the CMV expression vector SV-40 poly A region: 5'-gtttgtccaattatgtca (SEQ ID NO: 5). The resulting 3.3 KB FoxMlB PCR product was digested with EcoRl and Hindlll, generating the 2.5 KB EcoRI-Hindlll T7 tagged FoxMlB cDNA fragment and removing 800 nucleotides from the 3' untranslated region. This FoxMlB cDNA fragment was subsequently cloned in the corresponding sites in the CMV expression vector (Pani et al, 1992, Mol. Cell Biol. 12:3723-373245). [00125] A CMV pEGFP-FoxMlB expression plasmid was generated by liberating a 2.5 KB EcoRI-Hindlll fragment from the CMV FoxMlB expression vector. The Hindlll site was made blunt by T4 polymerase fill in reaction and then the FoxMlB cDNA fragment was cloned into EcoRI-Smal sites of the pEGFP-C2 expression plasmid (Clontech). The CMV tetracycline operator (CMV-TO) FoxMlB expression plasmid was generated by excising an EcoRI-BamHI fragment from pEGFP-FoxMlB expression plasmid. The BamHI site was made blunt by a T4 polymerase reaction and then the FoxMlB cDNA fragment was cloned into EcoRI and EcoRV sites of the pCDNA4-TO expression plasmid (T-Rex system, Invitrogen). [00126] A 6X FoxMlB/FoxA TATA-Luciferase utilized 6 copies of the

FoxMlB/FoxA binding site (TTTGTTTGTTTG; SEQ ID NO: 6) from the cdx-2 promoter region driving expression of the CMV-TATA box luciferase reporter gene as described previously (Rausa et al, 2003, Mol. Cell. Biol. 23:437-449; Samadani et al, 1996, Mol. Cell. Biol. 16:6273-6284; Ye et al, 1997, Mol. Cell Biol. 17: 1626- 1641).

Example 5

FoxMlB-dependent transcription requires the 596 Cdk phosphorylation site and binding of Cdkl/Cdk2 proteins through the FoxMlB LXLXXL sequence

[00127] Previous transfection studies demonstrated that the FoxMlB

transcriptional activation domain was contained within the carboxyl-terminal 365 to 748 amino acid residues (Ye et. al, 1997. Mol. Cell. Biol. 17: 1626-1641). Searching the FoxMlB C-terminal sequence for Cdkl/2 consensus phosphorylation sites X- pS/T-P-X-R/K revealed three potential Cdkl/2 sites at residues 585, 596 and 657 in the FoxMlB protein (Figure 5 A). In order to assess the transcriptional function of these potential FoxMlB Cdkl/2 sites, site-directed mutagenesis was used to alter either Thr or Ser residue to an Ala residue to prevent their Cdk phosphorylation in vivo. Transient transfection assays with 6X FoxMlB TATA-luciferase reporter and CMV vectors expressing either WT or Cdkl/2 mutant FoxMlB protein revealed that mutation of Cdkl/2 sites at either 585 or 657 resulted in only a marginal decrease (20% to 30%) in FoxMlB transcriptional activity (Figure 5B). In contrast, mutation of the FoxMlB 596 Thr residue (FoxMlB T596A) caused an 80% decrease in transcriptional activity, suggesting that this particular Cdkl/2 phosphorylation site plays an important role in FoxM IB-dependent transcription (Figure 5B). Moreover, FoxMlB was unable to activate expression of the TATA-luciferase control reporter in cotransfection assays, demonstrating that the multimerized FoxMlB binding sites were required for FoxMlB-dependent transcriptional activation (Figure 5B).

[00128] To identify FoxMlB sequences involved in the interaction with Cdk proteins, site-directed mutagenesis was used to convert the Leu 641 residue to an Ala residue thereby disrupting the FoxMlB LXL (639-641) motif shown in Figure 5A, which has been shown to bind to Cdk-Cyclin proteins as efficiently as the Cyclin- binding Cy (RXL) motif (Takeda et al, 2001, J Biol Chem 276: 1993-1997:

Wohlschlegel et ah, 2001, Mol Cell Biol 21 :4868-4874). Transient transfection assays demonstrated that FoxMlB L641A mutant protein displayed an 80% reduction in transcriptional activity (Figure 5B). Furthermore, increasing amounts of the CMV FoxMlB L641A expression vector inhibited transcriptional activity of the WT

FoxMlB protein in cotransfection assays, suggesting that the CMV FoxMlB L641A mutant protein functioned as a dominant negative inhibitor. Moreover, both GFP-T7- FoxMlB L641A and GFP-T7-FoxMlB T596A mutant proteins are retained in the nucleus (Fig. 4A-C), indicating that their diminished transcriptional activity was not due to inhibition of nuclear localization. [00129] To determine whether the FoxMlB T596A or FoxMlB L641A mutant proteins exhibited diminished protein association with either the Cdkl or Cdk2 protein, co-immunoprecipitation (Co-IP) experiments were performed with protein extracts prepared from U20S cells transfected with either CMV T7-FoxMlB WT or mutant expression constructs (Figure 5C). The transfected U20S cell extracts were Co-IP with either Cdkl or Cdk2 antibody and then FoxMlB protein was visualized by Western blot analysis with the T7 epitope Tag monoclonal antibody. These studies demonstrated that CMV T7-FoxMlB L641A mutant protein was unable to interact with either Cdkl or Cdk2 proteins, whereas the FoxMlB mutant proteins disrupted in each of the Cdkl phosphorylation sites could efficiently associate with the Cdk proteins (Figure 5C). These results suggested that retention of the second Leu residue within the LXL sequence was essential for interaction between FoxMlB and Cdk proteins, and that FoxMlB binding of either Cdkl or Cdk2 Cyclin protein complexes was required for its transcriptional activity.

[00130] To examine whether the Cdkl -Cyclin B complex phosphorylates the

FoxMlB protein, Co-immunoprecipitation (Co-IP) Cdkl in vitro kinase assays were performed with ³² P labeled γ-ΑΤΡ. Protein extracts prepared from U20S cells transfected with either CMV GFP-T7-FoxMlB WT or GFP-T7-FoxMlB Cdk mutant expression vectors were co-immunoprecipitated with Cdk- 1 antibody and were then used for radioactive Cdkl in vitro kinase assay. The proteins phosphorylated in the Co-IP Cdkl in vitro kinase reaction were resolved on SDS-PAGE and visualized by autoradiography. Consistent with reduced transcriptional activity, the Cdkl Co-IP kinase assay demonstrated that GFP-T7-FoxMlB T596A mutation exhibited reduced phosphorylation by the Cdkl protein, whereas Cdkl phosphorylated the GFP-T7- FoxMlB T585A and GFP-T7-FoxMlB S657A proteins to levels found with the GFP- T7-FoxMlB WT protein (Figure 5D). As expected, the GFP-T7-FoxMlB L641A mutant protein failed to interact efficiently with Cdkl protein (Figure 5C) and therefore only low levels of FoxMlB L641A mutant protein were available for Cdkl phosphorylation in the Co-IP Cdkl kinase assay (Figure 5D). [00131] To examine Cdk phosphorylation in vivo, protein extracts were prepared from serum stimulated U20S cells transfected with either CMV T7-FoxMlB WT, CMV T7-FoxMlB T596A or CMV FoxMlB L641A expression constructs. These protein extracts were IP with the T7 antibody and then Western blot analysis with the MPM2 monoclonal antibody was used to determine Cdk phosphorylation in vivo. These results demonstrated that Cdk phosphorylation of T7-FoxMlB WT protein was increased following serum stimulation and that the FoxMlB Thr 596 residue was required for phosphorylation by the Cdk-Cyclin complexes in vivo (Figure 5E). Furthermore, in vivo Cdk phosphorylation of the T7-FoxMlB L641A mutant protein was significantly reduced (Figure 5E), suggesting that recruitment of the Cdk-Cyclin complex by the FoxMlB LXL sequence was critical for its efficient Cdk phosphorylation in vivo.

Example 6

FoxMlB-dependent transcription is stimulated by increased Cdkl activity and CBP co-activator levels

[00132] CMV-FoxMlB and the 6X FoxMlB TATA luciferase constructs were co- transfected with increasing amounts of CMV-DN-Cdkl or cells were treated with increasing concentration of the pharmacological Cdkl inhibitor Alsterpaullone (Figure 6A) to demonstrate that Cdkl activity is necessary for FoxMlB transcriptional activity. Inhibiting Cdkl activity with either dominant negative (DN) Cdkl or a pharmacologically active concentration of Alsterpaullone (1 μΜ) caused an 80% to 90% reduction in FoxMlB transcriptional activity (Figure 6C). Neither DN- Cdkl nor Alsterpaullone (1 μΜ) altered nuclear localization of transfected CMV GFP-FoxMlB protein (Figure 7A, B and E), suggesting that inhibiting Cdkl activity alone diminished FoxMlB-dependent transcription. Furthermore, co-transfection of CMV WT-Mytl kinase, which negatively regulates Cdkl activity through phosphorylation, resulted in a 64% reduction in FoxMlB transcriptional activity (Figure 6C). Consistent with these findings, stimulation of Cdkl activity by co- transfection of either CMV Cdc25B or Cdc25C phosphatases enhanced FoxMlB transcriptional activity by 3.4-fold and 1.7-fold, respectfully (Figure 6B and 6D). Furthermore, co-transfection of CMV Cdc25B and CMV CBP together significantly augmented CBP-mediated stimulation of FoxMlB transcriptional activity from 1.4- fold to 6.2-fold increase (Figure 6D). Taken together, these results provided evidence that Cdkl activity was required to stimulate FoxMlB transcriptional activity.

Example 7

FoxMlB transcriptional activity involves recruitment of CBP through phosphorylation of the FoxMlB 596 Cdkl site

[00133] Co-transfection assays were performed with CMV-CBP or CMV- Adeno virus El A alone or in combination to determine if FoxMlB transcriptional activity required the CBP co-activator protein. Co-transfection of CMV-CBP stimulated FoxMlB transcriptional activity by 50%, whereas inhibition of CBP function with E1A resulted in a 75% reduction in FoxMlB transcriptional activity (Figure 8A). These studies suggested that recruitment of the p300/CBP family of coactivator proteins was essential for FoxMlB transcriptional activation. [00134] U20S cells were transiently transfected with CMV-CBP and either CMV GFP-FoxMlB, CMV GFP-FoxMlB comprising an L641A mutation, or CMV GFP- FoxMlB comprising an T596A mutation to determine if the critical FoxMlB 596 Cdkl phosphorylation site was required for recruitment of CBP. Protein extracts were prepared 48 hours after transfection, and then used for immunoprecipitation with CBP antibody followed by Western blot analysis with GFP monoclonal antibody. These co-IP experiments demonstrated that both WT and FoxMlB L641A mutant proteins could efficiently interacted with the CBP protein (Figure 8B). In contrast, disruption of the FoxMlB Cdkl phosphorylation site at Thr residue 596 significantly diminished FoxMlB's ability to associate with the CBP protein (Figure 8B). Taken together these results showed that FoxMlB phosphorylation by Cdkl-Cyclin B complex was required for recruitment of the p300/CBP coactivator protein, serving as a mechanism for proliferation-specific stimulation of FoxMlB transcriptional activity.

Example 8

Blocking the Ras-MAPK and PBK-PDKl pathways diminished FoxMlB transcriptional activity, but inhibiting Akt did not influence FoxMlB-dependent transcription

[00135] The role of the MAPK and PI3K pathways in regulating FoxMlB activity was examined using FoxMlB transcription assays performed in U20S cells that were either treated with the pharmacological MEK1/2 inhibitor U0126 or PI3K inhibitor Ly294002, or co-transfected with CMV DN-Ras 17 expression vector (Figure 9A). These transfection studies demonstrated that inhibition of MEK1/2, PI3K or Ras caused a 70 to 80% reduction in FoxMlB-dependent transcription (Figure 9C), a finding consistent with the important roles of Ras/MAPK and PI3K/PDK1 pathways in Cdkl-Cyclin B activation. In contrast, blocking the Akt pathway with either CMV DN-Akt or the Akt pharmacological kinase inhibitor did not significantly alter

FoxMlB transcriptional activity (Figure 9C). Furthermore, combining the MEK1/2 (U0126) and PI3K (Ly294002) inhibitors resulted in a 90% reduction in FoxM IB- dependent transcription demonstrating the importance of the Ras/MAPK and PI3K/PDK1 pathway in regulating FoxM IB transcriptional activity (Figure 9C). Co- transfection of CMV DN-p90Rsk (Figure 9A) resulted in a 56% reduction in FoxM IB transcriptional activity (Figure 9C), which was similar to the transcriptional reductions found with CMV WT-Mytl (Figure 6C). Addition of the Ras/MEKl/2 or PI3K/Akt pathway inhibitors did not diminish expression (Figure 9B) or nuclear localization of GFP-FoxMlB protein (Figure 7C, D, G and H), suggesting that these inhibitors caused decreases in FoxM IB transcriptional activity. However, co- transfection of DN-p90Rsk resulted in redistribution of a portion of GFP-FoxMlB fluorescence to the periphery of the nucleus (Figure 7F), suggesting that p90Rsk signaling may influence FoxMlB nuclear localization. Taken together, these studies demonstrated that FoxMlB transcriptional activity required Cdkl-Cyclin Bl activation, which was mediated by growth factor stimulation of the Ras/MAPK and PI3K/PDK1 signaling cascades.

Example 9

Alb-Cre Foxmlb -I- livers fail to develop hepatic adenomas or hepatocellular carcinomas after DEN/PB treatment

[00136] A well-established Diethylnitrosamine (DEN)/Phenobarbital (PB) liver tumor induction protocol (see Tamano et ah, 1994, Carcinogenesis 15: 1791-1798; Sargent et ah, 1996, Cancer Res. 56:2985-91; Kalinina et ah, 2003, Oncogene 22:6266-6276) was used to determine whether Foxmlb was required for proliferative expansion during mouse liver tumor formation. A single intraperitoneal (IP) injection of the tumor initiator Diethylnitrosamine (DEN) was given at 14 days postnatally to the entire mouse litter containing both Foxmlb fl/fl (control) and Alb-Cre Foxmlb -/- (experimental) pups. Two weeks later, the mice were placed on drinking water containing 0.05% of the liver tumor promoter Phenobarbital (PB) for the duration of the liver tumor induction experiment.

[00137] Eight control Foxmlb fl/fl mice and 11 experimental Alb-Cre Foxmlb -/- mice were sacrificed at 23 weeks of DEN/PB exposure and seven control Foxmlb fl/fl and 13 experimental Alb-Cre Foxmlb -I- mice were sacrificed at 33 weeks following DEN/PB treatment (Table 2).

Table 2. Number of tumors per cm ² liver tissue after 23 or 33 weeks of

DEN/PB treatment

l# Mice: Number of mice (male or female) analyzed for liver tumors after either 23 or 33 weeks of Diethylnitrosamine (DEN)/Phenobarbital (PB) treatment. ² Number of liver tumors per cm ² liver tissue ± SD (adenomas or hepatocellular carcinomas greater than 0.1 mm in size) determined from Hematoxylin and Eosin stained liver sections obtained from four different mouse liver lobes.

[00138] Livers were harvested from male Foxmlb fl/fl and Alb-Cre Foxmlb -/- mice after 6 weeks of DEN/PB exposure to provide an early time point during liver tumor promotion. Liver sections were histologically stained with Hematoxylin and Eosin (H&E) and hepatocyte DNA replication was determined by immunofluorescent detection of BrdU that had been administered in drinking water 4 days before sacrificing the mice following the procedure described in Ledda-Columbano et ah, 2002, Hepatology 36: 1098-1105. After 23 weeks of DEN/PB treatment, H&E stained liver sections from Foxmlb fl/fl male mice revealed numerous hepatic adenomas with abundant BrdU labeling (Table 2). Highly proliferative hepatocellular carcinomas (HCC) with abundant BrdU labeling were visible in liver sections from each of the male control Foxmlb fl/fl mice following 33 weeks of DEN/PB exposure (Table 2). Furthermore, significant numbers of hyper-proliferative adenomas were found in liver sections from female and male Foxmlb fl/fl mice after 33 weeks of DEN/PB treatment (Table 2). No hepatic adenomas or HCC were detected in male or female Alb-Cre Foxmlb -I- mice at either 23 or 33 weeks following DEN/PB exposure (Table 2). At 6, 23 and 33 weeks following DEN/PB treatment, low levels of BrdU incorporation were found in Foxmlb deficient hepatocytes, which was approximately 30% of BrdU labeling levels found in Foxmlb fl/fl hepatocytes of non-tumor regions following DEN/PB exposure.

[00139] In addition, rabbit polyclonal antibodies specific to cc-fetoprotein (AFP) (Dako Corp., Carpinteria, CA) proteins were used for immunohistochemical detection of 5 μιη liver sections using methods described previously (Ye et al, 1997, Mol Cell Biol 17: 1626-1641; Ye et al, 1999, Mol. Cell. Biol. 19:8570-8580; Wang et al, 2002, J. Biol. Chem. 277:44310-44316). AFP and BrdU positive immunofluorescent cells were detected in the Foxmlb fl/fl HCC liver tumors induced by DEN/PB exposure, which identified proliferating AFP-positive hepatocellular carcinoma cells. Fetal hepatocytes express abundant levels of (AFP), its hepatic expression is extinguished postnatally, but AFP expression is reactivated in HCC (Kunnath and Locker, 1983, Embo J2:317-324; Chen et al, 1997 ^' , Crit Rev Eukaryot Gene Expr 7: 1 1-41). Thus, these studies suggested that Foxmlb is required for proliferative expansion during tumor development of hepatic adenomas and HCC. [00140] Together, these experiments demonstrated that male Alb-Cre Foxmlb -I- mice were resistant to developing HCC in response to 33 weeks of DEN/PB exposure, a treatment sufficient to induce multiple HCC tumors in male Foxmlb fl/fl mice (Table 2). [00141] Furthermore, control Foxmlb fl/fl and experimental Alb-Cre Foxmlb -/- mice were treated with DEN/PB for 50 weeks to determine whether Foxmlb deficient livers were resistant to a prolonged hepatic tumor induction protocol. After 50 weeks of DEN/PB exposure, all nine female Alb-Cre Foxmlb -I- mice were devoid of any liver tumors, whereas HCC tumors were found in all four control female livers with one additional control female mouse dying prematurely. Following 50 weeks of DEN/PB exposure, no liver tumors were found in two out of the four male Alb-Cre Foxmlb -I- mice, while one male mouse exhibited hepatic adenomas and the last male mouse displayed HCC tumors that were negative for Foxmlb protein staining. These studies indicated that following prolonged DEN/PB tumor promotion hepatic tumors were found in a subset of the male Alb-Cre Foxmlb -I- livers, suggesting that they developed secondary mutations that allowed tumor formation bypassing the block in Foxmlb -I- hepatocyte proliferation.

Example 10 Alb-Cre Foxmlb -I- male mouse hepatocytes exhibited no elevation in apoptosis and increased hypertrophy in response to DEN/PB treatment

[00142] TUNEL staining of liver sections from DEN/PB treated mice was used to determine whether increased apoptosis contributed to the failure of male Alb-Cre Foxmlb -I- mice to develop liver tumors in response to 33 weeks of DEN/PB treatment. The TUNEL assay was performed using the ApoTag Red in situ apoptosis detection kit from Intergen (Purchase, NY) according to the manufacturer's recommendations. No difference was found in hepatocyte apoptosis between Alb-Cre Foxmlb -I- and Foxmlb fl/fl mice after 6, 23, or 33 weeks of DEN/PB exposure (Figure lOA-C). These results suggested that the absence of liver tumors in Foxmlb - I- mice following DEN/PB exposure was not due to an increase in hepatocyte apoptosis.

[00143] Hypertrophy of the Alb-Cre Foxmlb -I- hepatocytes was significantly increased compared to that of control hepatocytes (non-tumor liver regions) at 23 weeks of DEN/PB exposure (Figure 10D-E). A centromere-specific FISH probe purchased from Vysis Inc. (Downers Grove, IL) was used to hybridize paraffin embedded liver sections according to manufacturer's protocol, demonstrating that Alb-Cre Foxmlb -I- hepatocyte nuclei possessed an increase in hybridizing chromosomes compared to control hepatocyte nuclei at 23 weeks of DEN/PB treatment (Figure 10F-G). To quantitate this increase in size, the number of DAPI stained hepatocyte nuclei were counted (per 200X field) in Foxmlb fl/fl and Alb-Cre Foxmlb -I- liver sections and the data for each of the time points following DEN/PB exposure was plotted (Figure 10H). The mean number (±SD) of DAPI positive hepatocyte nuclei per 1000 cells or 200X field by counting the number of positive hepatocyte nuclei using 5 different 200X liver sections from 3 distinct male mice at the indicated times of DEN/PB exposure or untreated. After 23 or 33 weeks of DEN/PB exposure, half the number of hepatocyte nuclei per 200X field was found in Foxmlb -I- livers compared to Foxmlb fl/fl control liver (Figure 10H). The data suggested that Foxmlb deficient hepatocytes undergo greater hypertrophy and become more polyploid than Foxmlb fl/fl control hepatocytes at 23 and 33 weeks of DEN/PB treatment. These results suggested that Alb-Cre Foxmlb -I- hepatocytes exhibited low levels of DNA replication with a significant reduction in mitosis as was previously found in Foxmlb deficient hepatocytes during liver regeneration and development (Korver et al, 1998, Nucleic Acids Res 25: 1715-1719; Wang et al, 2002, Proc Natl Acad Sci USA 99: 16881-16886). Moreover, Alb-Cre Foxmlb -/- hepatocytes displayed normal serum levels of albumin, bilirubin and glucose after 33 weeks of DEN/PB exposure indicating that their livers functioned normally.

Example 11

Hepatocyte expression of nuclear Foxmlb protein increases prior to tumor formation and continues during tumor progression

[00144] Immunohistochemical staining of liver sections with an antibody specific to Foxmlb protein (Ye et al., 1997, Mol Cell Biol Π _. -Λ626-1641; Ye et al., 1999, Mol. Cell. Biol. 19:8570-8580; Wang et al, 2002, J. Biol. Chem. 277:44310-44316) demonstrated that untreated hepatocyte nuclei displayed no significant expression of the Foxmlb protein (Figure 1 1A-B). Abundant nuclear staining of Foxmlb protein was detected in periportal Foxmlb fl/fl hepatocytes as early as 6 weeks of DEN/PB (Figure 1 1C), yet these hepatocytes failed to exhibit abundant BrdU incorporation levels. High levels of nuclear FoxMlB protein persisted in hyper-proliferative liver adenomas and HCC at 23 weeks and 33 weeks following DEN/PB exposure (Figure 1 IE and G). As expected, nuclear staining of FoxMlB protein was not found in Alb- Cre Foxmlb -I- hepatocytes at any of the time points following DEN/PB treatment (Figure 1 ID, F and H), confirming that the Alb-Cre transgene protein efficiently deleted the Foxmlb floxed targeted allele in hepatocytes (Wang et al, 2002, Proc Natl Acad Sci USA 99: 16881-16886). These studies demonstrated that hepatocyte nuclear levels of FoxMlB were induced in control hepatocytes prior to tumor formation following DEN/PB treatment and that this nuclear expression persisted in hepatic adenomas and HCC. Example 12

Alb-Cre Foxmlb -I- livers exhibit normal expression of GST-pi and CAR following DEN/PB treatment

[00145] Glutathionine-S-transferase placental isoform (GST-pi) is an early marker for "altered enzyme foci" in response to DEN/PB exposure (Hatayama et al , 1993,

Carcinogenesis 14:537-538). Rabbit polyclonal antibodies specific to GST-pi (Dako

Corp., Carpinteria, CA) proteins were used for immunohistochemical detection of 5 μιη liver sections using methods described previously (Ye et al, 1997, Mol Cell Biol 17: 1626- 1641 ; Ye et al, 1999, Mol. Cell. Biol. 19:8570-8580; Wang et al , 2002, J. Biol. Chem. 277:44310-443 16). GST-pi expression was not detected in liver sections of untreated control mice (Figure 12A-B), but both Alb-Cre Foxmlb -I- and Foxmlb fl/fl hepatocytes were strongly immunostained for GST-pi after 23 weeks of DEN/PB treatment (Figure 12C-F). Western blot analysis demonstrated that hepatic expression of GST-pi protein was induced as early as 6 weeks following DEN/PB treatment in both Alb-Cre Foxmlb -I- and Foxmlb fl/fl livers with continued expression after 23 weeks of DEN/PB exposure (Figure 12G). Phenobarbital (PB) stimulates nuclear translocation of the constitutive androstane receptor (CAR) nuclear receptor (Chawla et al , 2001 , Science 294: 1866- 1870). No difference in nuclear staining of the CAR receptor was found between Foxmlb fl/fl and Alb-Cre Foxmlb -I- hepatocytes following DEN/PB treatment (Figure 12H-I), indicating that the Foxmlb deficient hepatocytes were still responsive to the PB tumor promoter. Taken together, the data suggest that Alb-Cre Foxmlb -I- livers responded normally to DEN/PB tumor induction and expressed the "altered enzyme foci" GST-pi marker, but that they failed to undergo the proliferation required for tumor progression. Example 13

Persistent nuclear accumulation of the Cdk inhibitor γιΠ^ ¹ protein and diminished Cdc25B expression in Alb-Cre Foxmlb -I- livers follows DEN/PB exposure

[00146] Liver regeneration studies demonstrated that increased expression of Foxmlb protein was associated with reduced hepatocyte nuclear levels of the Cdk inhibitor ρ27 ^βρ1 protein (Wang et al, 2002, Proc Natl Acad Sci USA 99: 16881 - 16886; Wang et al., 2002, J. Biol. Chem. 277:44310-44316; Krupczak-Hollis et al., 2003, Hepatology 38: 1552-1562). Consistent with these findings, persistent nuclear accumulation of hepatocyte p27 ^Kipl protein was found only in Alb-Cre Foxmlb -I- liver sections at 36 hours after partial hepatectomy (PHx; Figure 13A-B). Nuclear expression of p27 ^&pl protein was examined in mouse liver sections from untreated and DEN/PB treated mice using immunohistochemical staining. Rabbit polyclonal antibodies specific to p27 ^kipl (Cell Signaling, Beverly, MA) proteins were used for immunohistochemical detection of 5 μιη liver sections using methods described previously (Ye et al, 1997, Mol Cell Biol 17: 1626-1641 ; Ye et al, 1999, Mol. Cell. Biol. 19:8570-8580; Wang et al, 2002, J. Biol. Chem. TTTAAi ^' 10-44316). Similar hepatocyte levels of nuclear p27 ^&pl protein were found in untreated Alb-Cre Foxmlb -I- and Foxmlb fl/fl mice (Figure 13C-D), a finding consistent with abundant nuclear expression of the Cdk inhibitor p27 ^&pl protein in quiescent hepatocytes (Kwon et al, 2002, J Biol Chem 277:41417-41422). Hepatocyte nuclear staining of ρ27 ^βρ1 protein was significantly diminished in Foxmlb fl/fl hepatocytes beginning at 6 weeks and continuing through 33 weeks after DEN/PB treatment (Figure 13E, G, I and M). [00147] Furthermore, nuclear expression of p27 ^Kipl protein was undetectable in hepatic tumor cells at all time points following DEN/PB treatment (Figure 13G and I). In contrast, hepatocyte nuclear staining of p27 ^&pl protein was sustained in Alb-Cre Foxmlb -I- mice at 6, 23 and 33 weeks after DEN/PB exposure (Figure 13F, H, J and M). After 50 weeks DEN/PB treatment, nuclear staining of p27 ^Kipl protein was sustained in Female Alb-Cre Foxmlb -I- mouse hepatocytes (Figure 13K and N) and these livers were resistant to development of adenomas and HCC. In contrast, male Alb-Cre Foxmlb -I- mouse hepatocytes exhibited nearly undetectable nuclear staining of p27 ^&pl protein after 50 weeks of DEN/PB exposure (Figure 13L and N) and was associated with 50% of the male Alb-Cre Foxmlb -I- mice developing liver tumors. These results suggested that an increase in liver tumor incidence in male mice following prolonged response to DEN/PB treatment was associated with loss of hepatocyte nuclear levels of p27 ^&pl protein.

[00148] Diminished hepatocyte DNA replication in regenerating Alb-Cre Foxmlb - I- livers was associated with increased nuclear levels of the Cdk inhibitor p21 ^Cipl protein (Wang et al, 2002, Proc Natl Acad Sci US A 99: 16881-16886).

Immunostaining of liver sections demonstrated that nuclear expression of p21 ^Qpl protein in Alb-Cre Foxmlb -I- and Foxmlb fl/fl hepatocytes was similar and restricted to hepatocytes surrounding the central vein after 6, 23 or 33 weeks of DEN/PB treatment. The similar expression pattern of nuclear p21 ^Cipl protein in hepatocytes of DEN/PB treated mice suggested that elevated p21 ^Qpl protein levels were unlikely to be involved in suppressing tumor formation in Alb-Cre Foxmlb -I- livers.

[00149] Western blot analysis revealed similar levels of total p27 ^Kipl protein in Foxmlb fl/fl and Alb-Cre Foxmlb -I- liver extracts at 6, 23 or 33 weeks following DEN/PB exposure (Figure 14A). These results suggested that Foxmlb deficiency resulted in sustained hepatocyte levels of nuclear p27 ^&pl protein after DEN/PB treatment without changing total expression of the p27 ^Kipl protein. [00150] The Western blot was then stripped and probed sequentially with antibodies specific to the Cdk-activating Cdc25B or Cdc25C phosphatases (Santa Cruz Biotech) at a concentration of 1 : 1000. Foxmlb fl/fl control livers exhibited a transient increase in expression of the M-phase promoting Cdc25B phosphatase protein at 6 weeks after DEN/PB exposure, whereas hepatic levels of Cdc25B protein were significantly diminished in Alb-Cre Foxmlb -I- livers (Figure 14A). Similar levels of Cdc25C protein are found in liver extracts from Alb-Cre Foxmlb -I- and Foxmlb fl/fl mice after 6 weeks of DEN/PB treatment (Figure 14A). However, diminished hepatic expression of Cdc25B and Cdc25C proteins is observed at either 23 or 33 weeks after DEN/PB exposure (Figure 14A). Taken together, the data suggested that decreased proliferation in Alb-Cre Foxmlb -I- hepatocytes was likely due to sustained nuclear levels of Cdk inhibitor p27 ^Kipl protein and diminished expression of the Cdkl -activator Cdc25B.

Example 14

The Cdk inhibitor ρΠ^ ¹ protein associates with FoxMlB through the Cdk- Cyclin complexes and inhibits its transcriptional activity

[00151] FoxMlB transcriptional activity requires an LXL Cdk docking site (639- 641) that recruits either the Cdk2-Cyclin E/A (S-phase) or Cdkl-Cyclin B (G2 phase) complexes to the FoxMlB transcriptional activation domain, which is required for efficient phosphorylation of the FoxMlB Cdk 596 site (Major et al, 2004, Mol. Cell. Biol. 24:2649-2661). Retention of this Foxmlb Cdk site at Thr 596 residue was found to be essential for transcriptional activity by mediating phosphorylation dependent recruitment of the CREB Binding Protein (CBP) histone acetyltransferase (Major et al, 2004, Mol. Cell. Biol. 24:2649-2661). [00152] Protein extracts were prepared from U20S cells that were transiently transfected with the CMV p27 ^Kipl and CMV expression constructs containing either WT GFP-Foxmlb or the GFP- FoxMlB L641A mutant that failed to interact with the Cdk-Cyclin complexes (Major et al, 2004, Mol Cell Biol 24:2649-2661). These U20S cell transfected lysates were immunoprecipitated (IP) with the p27 ^Kipl antibody (Cell Signaling, Beverly, MA; 1 : 1000) followed by Western blot analysis with GFP antibody. These Co-IP experiments demonstrated that the p27 ^Kipl protein associated with the WT FoxMlB protein, whereas p27 ^Kipl was unable to bind to the GFP- FoxMlB L641A mutant protein (Figure 14C). These results suggested that the p27 ^&pl protein associated with the Cdk-Cyclin complexes, which are recruited by the FoxMlB transcriptional activation domain through the LXL Cdk docking motif (Figure 14B).

[00153] In addition, U20S cells were transiently transfected with the 6X FoxMlB -TATA-luciferase reporter plasmid (Rausa et al, 2003, Mol Cell Biol 20:8264-8282; Major et al, 2004, Mol Cell Biol 24:2649-2661) with the CMV WT FoxMlB and p27 ^&pl expression vectors to determine whether the p27 ^&pl protein could inhibit Foxmlb transcriptional activity. Transfected cells were harvested at 48 hours after transfection and processed for dual luciferase assays to determine FoxMlB transcriptional activity. Cotransfection of p27 ^Kipl expression vector caused a significant reduction in FoxMlB transcriptional activity (Figure 14D). This finding was consistent with the ability of the p27 ^Kipl protein to inhibit kinase activity of the Cdk-Cyclin complexes (Polyak et al, 1994, Genes Dev 8:9-22; Zerfass-Thome et al, 1997, Mol Cell Biol 17:407-415) required for FoxMlB transcriptional activity through Cdk phosphorylation dependent recruitment of the CBP coactivator protein (Major et al, 2004, Mol Cell Biol 24:2649-2661). Example 15

Endogenous pl9 tumor suppressor associates with FoxMlB protein in liver extracts prepared from mice following 6 weeks of DEN/PB exposure

[00154] Hepatic expression of pi 9 protein in livers from mice exposed to DEN/PB was examined by Western blot analysis. For Western blotting, 100 μg of total protein extracts prepared from liver following the procedure in Rausa et al, 2000, Mol Cell Biol 20:8264-8282) were separated on SDS-PAGE and transferred to PVDF membrane (BioRAD). Rabbit antibodies specific to pl9 ^ARF (AB80; GeneTex, San Antonio, TX; 1 :750) proteins were used as primary antibody. The primary antibody signals were amplified by HRP-conjugated secondary antibodies (Bio-Rad, Hercules, CA), and detected with Enhanced Chemiluminescence Plus (ECL-plus, Amersham Pharmacia Biotech, Piscataway, NJ).

[00155] Western Blot analysis demonstrated that hepatic expression of pl9 protein was induced at 6 weeks after DEN/PB exposure, but liver expression of pi 9 was significantly diminished by 23 weeks following DEN/PB exposure (Figure 15 A), a finding consistent with those obtained with other tumors (Sherr and McCormick, 2002, Cancer Cell 2: 103-112).

[00156] Co-immunoprecipitation (Co-IP) assays were performed with liver protein extracts prepared from Foxmlb fl/fl and Alb-Cre Foxmlb -I- mice following either 6 or 23 weeks of DEN/PB treatment (Figure 15B) to determine whether the pl9 tumor suppressor protein associated with the FoxMlB protein. For Co-IP experiments, 500 μg of protein extract prepared from DEN/PB treated liver were immunoprecipitated with pl9 ^ARF antibody (AB80; GeneTex, San Antonio, TX; 2 μg) followed by Western Blot analysis with mouse antibody FoxMlB protein (1 :5000). The signals from the primary antibody were amplified by HRP conjugated anti-mouse IgG (Bio-Rad, Hercules, CA), and detected with Enhanced Chemiluminescence Plus (ECL-plus, Amersham Pharmacia Biotech, Piscataway, NJ). As a positive control, Co-IP experiments were performed with protein extracts prepared from mouse embryo fibroblasts (MEFs) that were cultured in vitro for 12 passages to induce endogenous protein expression of the pl9 tumor suppressor (Kamijo et ah, 1997, Cell 91:649- 659). These Co-IP studies demonstrated efficient association between endogenous FoxMlB and p 19 proteins in extracts prepared from either Foxmlb fl/fl livers at 6 weeks of DEN/PB exposure or late passage MEFs, but not with liver extracts from Alb-Cre Foxmlb -I- mice (Figure 15B). Negative controls showed that Foxmlb protein failed to Co-IP with pl9 in protein extracts prepared from Foxmlb fl/fl livers at 23 weeks of DEN/PB treatment, which no longer expressed the pi 9 protein but continued to express Foxmlb protein (Figure 15B and 11C).

Example 16

FoxMlB and pl9 cotransfection assays and synthesis of (D-Arg)9-pl9 ^ARF 26-44 peptide

[00157] Human osteosarcoma U20S cells were maintained in DMEM

supplemented with 10% fetal calf serum, IX Pen/Strep and IX L-Glutamine (Gibco). For transient transfection, U20S cells were plated in six-well plates and transfected using Fugene 6 reagent (Roche) according to the manufacturer's protocol. Cells were transfected with 500 ng of CMV WT FoxMlB 1-748 alone or with CMV expression vectors containing either WT T7-pl9 ^ARF or N-terminal mutant T7-pl9 ^ARF protein (Δ1- 14, Δ15-25, Δ26-37, or Δ26-37 + Δ1-14) or V5-TAT-pl9 ^ARF 26-44 or V5-TAT- pl9 ^ARF 26-55 sequences and with 1.5 μg of a 6X FoxMlB TATA-Luciferase reporter. Ten nanograms of CMV-Renilla luciferase reporter plasmid were included as an internal control to normalize transfection efficiency. Cotransfection assays were also performed with 500 ng of CMV FoxMlB 1-688 and 6X FoxMlB TATA-Luciferase reporter and 10 ng of CMV-Renilla internal control. Twenty- four hours post- transfection, cells were prepared for dual luciferase assays (Promega). Luciferase activity was determined as percent of wild type FoxMlB activity following normalization to Renilla activity. Experiments were performed at least four times in triplicate and mean ± SD determined.

[00158] The Sigma-Genosys company (The Woodlands, TX) synthesized a (D- Arg) ₉ -pl9ARF 26-44 peptide (rrrrrrrrrKFVRSRRPRTASCALAFVN; SEQ ID NO: 10) containing nine D-Arg residues (SEQ ID NO: 14) at the N-terminus, which has been demonstrated to enhance cellular uptake of polypeptides (Wender et ah, 2000, Proc Natl Acad Sci US A 97: 13003-13008). The (D-Arg) ₉ -pl9ARF 26-44 peptide was tagged with a fluorescent Lissamine (TRITC) on the N-terminus and acetylated at the C-terminus and was purified by high-pressure liquid chromatography (Sigma- Genosys). Cotransfection assays were also performed with 500 ng of CMV FoxMlB 1-688, 6X FoxMlB TATA-Luciferase reporter and 10 ng of CMV-Renilla internal control. The transfected U20S cells were treated with 12 μΜ of the pl9 ^ARF rrrrrrrrrKFVRSRRPRTASCALAFVN (SEQ ID NO: 10) peptide for 24 hours and then harvested for dual luciferase assays (Promega) as described above.

[00159] U20S cells were transiently transfected in 2 well chamber slides (Nunc) with CMV GFP- FoxMlB expression constructs in the presence or absence of either CMV WT T7-pl9 ^ARF , CMV HA-pl9 ^ARF , or CMV expression constructs containing either N-terminal mutant T7-pl9 ^ARF proteins (ΔΙ-14, Δ15-25, or Δ26-37) or V5-TAT- pl9 ^ARF proteins (26-44; SEQ ID NO: 1 1, or 26-55; SEQ ID NO: 12). U20S cells were transiently transfected with CMV EGFP expression vector containing the TAT- pl9 ^ARF proteins (26-44; SEQ ID NO: 1 1, or 26-55; SEQ ID NO: 12). Forty-eight hours post transfection, cells were fixed in 4% Para-formaldehyde for 20 minutes at room temperature. GFP fluorescence or immuno-fluorescence with anti-HA antibody following TRITC conjugated secondary antibody was detected using a Zeiss microscope. U20S cells were treated with 12 μΜ of the rrrrrrrrrKFVRSRRPRTASCALAFV (SEQ ID NO: 10) peptide for 24 hours and then analyzed for TRITC fluorescence as described above.

Example 17

Creation of Doxycycline inducible CMV-TETO GFP- FoxMlB U20S cell line and soft agar assays

[00160] The T-REx™-U20S cells were purchased from Invitrogen Life Technologies (Catalog No. R712-07). The T-REx™-U20S cells express the Tet repressor from pCEP4/tefR that was episomally maintained in tissue culture medium containing 10% fetal calf serum and drug selection with 50 μg/ml of Hygromycin B. Tetracycline regulation in the T-REx System was based on the binding of tetracycline to the TET repressor and de-repressing of the CMV-TETO promoter controlling expression of the gene of interest (Yao et al, 1998, Hum Gene Ther 9: 1939-1950). The pCDNA4-TO GFP- FoxMlB expression plasmid provided in the T-REx™ system was generated as described previously (Major et ah, 2004, Mol. Cell. Biol. 24:2649-2661) and transfected T-REx™-U20S cells with linearized pCDNA4-TO GFP-Foxmlb expression plasmid to select clonal Doxycycline inducible GFP- Foxmlb U20S cell lines. CMV-TETO GFP- FoxMlB U20S clones were isolated by selection for three weeks with tissue culture medium containing 50 μg/ml of Hygromycin B and 250 μ^πιΐ of Zeocin. The CMV-TETO GFP-Foxmlb U20S clone C3 cell line was selected for the soft agar assays because it exhibited intermediate expression of the GFP-Foxmlb fusion protein in response to 1 μg/ml of Doxycycline (Sigma D-9891) as determined by Western blot analysis with GFP monoclonal antibody. Wild type U20S cells or CMV-TETO GFP-Foxmlb U20S clone C3 cells were grown in medium with or without 1 μg/ml of Doxycycline for 2 days prior to either adding the (D-Arg)9-pl9 ^ARF 26-44 peptide or left untreated. A concentration of 12 μΜ of p 19 ^ARF peptide (rrrrrrrrrKFVRSRRPRTASCALAFVN; SEQ ID NO: 10) was added to the cells for 24 hours prior to splitting the cells for the soft agar assays using procedures described previously (Conzen et al. 2000, Mol Cell Biol 20:6008-6018). U20S cells (10 ⁵ ) were plated subconfluently in a 6 well plates in 0.7% agarose on a 1.4% agarose bed in the presence or absence of 12 μΜ of the (D- Arg) ₉ -pl9 ^ARF 26-44 peptide and 1 μg/ml of Doxycycline. Every 4 days the tissue culture medium containing 10% fetal calf serum, 12 μΜ of the (D-Arg) ₉ -pl9 ^ARF 26- 44 peptide and 1 μg/ml of Doxycycline was replaced. Controls included growth in medium containing 10% fetal calf serum with or without 1 μg/ml of Doxycycline. U20S cell colonies that were larger than 1 mm in size were scored after two weeks of growth on the soft agar.

Example 18

The pl9 ^ARF 26 to 44 sequences are sufficient to associate with and inhibit FoxMlB transcriptional activity

[00161] To identify pl9 ^ARF protein sequences essential for association with

FoxMlB protein, Co-IP assays were performed with protein extracts prepared from transiently transfected U20S cells, which lack endogenous expression of the pl9 ^ARF tumor suppressor protein (Martelli et al, 2001, Proc Natl Acad Sci USA 98:4455- 4460). U20S cells were co-transfected with CMV Green Fluorescent Protein (GFP)- FoxMlB expression vector and CMV expression plasmids containing either WT pl9 protein or N-terminal deletion mutants of the pl9 protein (ΔΙ-14, Δ15-25, Δ26-37, or Δ26-37 + Δ1-14) that were fused to the HA epitope tag (Weber et al, 2000, Mol Cell Biol 20:2517-2528). Protein extracts were incubated with HA antibody to immunoprecipitate (IP) the HA-pl9 ^ARF protein followed by Western blot analysis with a monoclonal antibody specific to GFP protein to detect the GFP-

FoxMlB fusion protein. These Co-IP experiments demonstrated that the N-terminal

25 amino acid residues of the pl9 ^ARF (pi 9) protein were dispensable for association with the GFP- FoxMlB protein (Figure 15C-D). In contrast, the pl9 amino acid residues 26 to 37 were essential for association with the GFP-Foxmlb fusion protein (Figure 15C-D). Furthermore, retention of the C-terminal 60 amino acids from the FoxMlB protein (688-748) was required for pl9 protein binding (Figure 15C-D).

[00162] To identify pi 9 protein sequences that are sufficient for association with FoxMlB protein, Co-IP assays were performed with protein extracts prepared from U20S cells that were transiently transfected with CMV GFP- FoxMlB expression plasmid and the CMV expression vector containing the V5 epitope tagged pl9 ^ARF 26- 44 or pl9 ^ARF 26-55 sequences. At the amino terminus of either the pl9 ^ARF sequences

26 to 44 or 26 to 55, we placed the protein transduction/nuclear localization domain (MGYGRKKRRQRRR; SEQ ID NO: 13) from the HIV-TAT protein (Becker-Hapak et al, 2001, Methods 24:247-256). Protein extracts were incubated with the V5 epitope tag antibody to IP the p 19 protein followed by Western blot analysis with GFP monoclonal antibody to detect the GFP-FoxMlB fusion protein. These Co-IP experiments demonstrated that pi 9 amino acid residues 26-44 were sufficient to associate with the FoxMlB protein (Figure 15E).

[00163] To determine whether formation of the pl9-FoxMlB protein complex could effectively inhibit FoxMlB transcriptional activity, U20S cells were transiently transfected with the 6X Foxmlb-TATA-luciferase reporter plasmid (Rausa et al, 2003, Mol. Cell. Biol. 23 :437-449; Major et al, 2004, Mol. Cell. Biol. 24:2649-2661) and the CMV WT FoxMlB and p 19 expression vectors (Figure 15F). These cotransfection assays demonstrated that both WT pl9 and mutant T7-pl9 ^ARF Al-14, T7-pl9 ^ARF A15-25, V5-TAT-pl9 ^ARF 26-44 and V5-TAT-pl9 ^ARF 26-55 proteins that retained their ability to associate with FoxMlB protein (Figure 15D-E) were able to significantly decrease FoxMlB transcriptional activity (Figure 15F). In contrast, the T7-pl9 ^ARF A26-37 proteins, which no longer associated with the FoxMlB protein (Figure 15D) were unable to significantly reduce FoxMlB transcriptional activity in these cotransfection assays (Figure 15F). Interestingly, deletion of the FoxMlB C- terminal sequences required for association with pl9 protein (Figure 15D; Foxmlb 1- 688) was also found to be essential for FoxMlB transcriptional activity (Figure 15F). These studies demonstrated that FoxMlB transcription factor was a novel inhibitory target for the pl9 ^ARF tumor suppressor, a finding consistent with the important role of FoxMlB in proliferative expansion during liver tumor progression.

Example 19

The pl9 ^ARF tumor suppressor targets FoxMlB protein to the nucleolus

[00164] U20S cell cotransfection studies demonstrated that HA tagged pi 9 was able to target GFP-FoxMlB fusion protein to the nucleolus (Figure 16A-C). While GFP-FoxMlB 1-748 full-length protein exhibited nuclear staining (Figure 16D), nucleolar targeting of GFP-FoxMlB fusion protein was found in cotransfections with expression vectors containing either WT pi 9 or mutant pi 9 proteins that were still able to associate with FoxMlB protein (Figure 16E-F). The GFP-FoxMlB protein was targeted to the nucleolus by expression vectors containing either the V5-TAT- pl9 ^AKt 26-44 or V5-TAT-pl9 ^AKt 26-55 sequences (Figure 16G-H) and these pl9 sequences were also localized to the nucleolus (Figure 161). In contrast, nuclear fluorescence was found with the GFP-FoxMlB WT protein that was transfected with the CMV pl9 ^ARF A26-37 mutant that failed to associate with FoxMlB protein (Figure 16J). Likewise, cotransfection assays with the CMV WT pi 9 and CMV GFP-

FoxMlB 1-688 expression vectors showed nuclear fluorescence of the mutant GFP- Foxmlb 1-688 protein, a finding consistent with this FoxMlB mutant's inability to associate with the pi 9 protein (Figure 16K and 15B). These studies suggested that association between the pl9 tumor suppressor and FoxMlB resulted in targeting FoxMlB to the nucleolus and FoxMlB transcriptional inhibition.

Example 20

(D-Arg) ₉ pl9 26 to 44 peptide significantly reduces both FoxMlB transcriptional activity and Foxmlb induced cell colony formation on soft agar

[00165] The pl9 ^ARF 26-44 peptide containing nine D-Arg residues (SEQ ID NO: 14) at the N-terminus was fluorescently tagged with Lissamine (TRITC) on the N- terminus and acetylated at the C-terminus as described above. Treatment of U20S cells with 12 μΜ of the (D-Arg) ₉ -pl9 ^ARF 26-44 peptide

(rrrrrrrrrKFVRSRRPRTASCALAFVN; SEQ ID NO: 10) for three days demonstrated that this (D-Arg) ₉ -p 19 ^ARF 26-44 peptide was efficiently transduced into all of the cells and that its fluorescence localized to the nucleolus (Figure 16L). Furthermore, exposure of U20S cells with 12 μΜ of the (D-Arg) ₉ -pl9 ^ARF 26-44 peptide for five days caused neither toxicity nor any increases in apoptosis. Furthermore, treatment of U20S cells with 12 μΜ of the (D-Arg) ₉ -pl9 ^ARF 26-44 peptide that were transfected with CMV- FoxMlB expression vector and the 6X FoxMlB-TATA-luciferase plasmid resulted in significant reduction in FoxMlB transcriptional activity (Figure 17A), suggesting that this p 19 peptide was an effective inhibitor of FoxM 1 B transcriptional activity.

[00166] In addition, the tetracycline (TET) regulated T-REx™ System described above was used to conditionally express the GFP- FoxMlB protein in U20S cells to determine whether conditional overexpression of FoxMlB protein could enhance anchorage-independent growth of U20S cells. The CMV-TETO GFP- FoxMlB expression plasmid was transfected into T-REx™-U20S cells (containing TET repressor) and clonal U20S cell lines were selected that were Doxycycline-inducible for GFP- FoxMlB expression. In response to Doxycycline treatment, the CMV- TETO GFP- FoxMlB U20S clone C3 cell line displayed inducible intermediate levels of the GFP- FoxMlB fusion protein (Figure 17B). The U20S clone C3 cell line was selected to examine whether doxycycline induced FoxMlB -GFP expression enhanced anchorage-independent growth as assessed by propagation for two weeks on soft agar (Conzen et al, 2000, Mol Cell Biol 20:6008-6018). The soft agar experiments demonstrated that induced expression of GFP- FoxMlB protein caused a significant increase in anchorage-independent growth as evidenced by increasing the number and size of U20S cell colonies on soft agar (Figure 17G and I) compared to uninduced controls (Figure 17F) or the WT U20S cells (Figure 17C-D).

[00167] The results suggested that the FoxMlB protein displayed oncogenic properties by stimulating anchorage-independent growth of U20S cell colonies on soft agar. In order to determine whether the (D-Arg)9-pl9 ^ARF 26-44 peptide inhibited FoxMlB induced colony formation of U20S cells on soft agar, the Doxycycline induced U20S clone 3 cells were treated with 12 μΜ of the (D-Arg) ₉ -pl9 ^ARF 26-44 peptide one day prior to plating and was added at this concentration of (D-Arg) ₉ - pl9 ^ARF 26-44 peptide in the soft agar and growth medium throughout the duration of the experiment as described above. The results of these soft agar studies demonstrated that the (D-Arg)9-pl9 ^ARF 26-44 peptide significantly diminished the ability of induced GFP- FoxMlB to stimulate colony formation of the U20S clone C3 cells on soft agar (Figure 17H and I). Furthermore, the (D-Arg)9-pl9 ^ARF 26-44 peptide significantly diminished the ability of the parental U20S cells to form colonies on soft agar (Figure 17E and I). Taken together these studies suggested that the (D-Arg) ₉ -pl9 ^ARF 26-44 peptide is an effective inhibitor of both FoxMlB mediated transcriptional activation and FoxMlB induced stimulation in anchorage-independent growth that is required for cellular transformation.

Example 21

WT-Blocked pl9 ^ARr 26-44 peptide induced apoptosis more significantly than WT-Unblocked and Mutant-blocked pl9 ^ARF 26-44 peptide Activity as shown by TUNEL Assay

[00168] Wildtype-blocked ("WT-blocked") (D-Arg) ₉ -pl9 ^ARF 26-44 peptides, wildtype-unblocked ("WT-unblocked") (D-Arg) ₉ -pl9 ^ARF 26-44 peptides and mutant blocked (D-Arg) ₉ -pl9 ^ARF 26-44 peptides were prepared under good laboratory practice ("GLP") conditions and received from Genemed Synthesis, Inc. (San Antonio, TX). Terminals of the WT-blocked and mutant-blocked peptide were blocked by acetylation on N-terminus and by amidation on C-terminus. [00169] TUNEL assay was used to measure apoptosis in S2 cells treated with WT- blocked, mutant-blocked or WT-unblocked (D-Arg) ₉ -pl9 ^ARF 26-44 peptides. (D- Arg) ₉ -pl9 ^ARF 26-44 peptide treatment of cells was performed in 8-well chamber slides for TUNEL staining. On Day 0, 20,000 cells per well were seeded in the 8-well chamber slides. On Day 1, slides were replenished with fresh media containing one of the following concentrations of either WT-blocked, mutant-blocked or WT- unblocked (D-Arg) ₉ -pl9 ^AKt 26-44 peptides: 10, 15, 25, 30, 40 and 70 μΜ for 24 hours. On Day 2, TU EL staining was performed following the manufacture's protocol (ApopTag® Fluorescein In Situ Apoptosis Detection Kit from CHEMICON ^® International, S71 10). The percent of apoptosis of S2 cells (±SD) was measured by 5 counting the number of TUNEL-positive cells (green fluorescence) per 1,000 nuclei as visualized by DAPI (blue fluorescence) counterstaining. The experiment was repeated in a second experiment using the same protocol. Statistical analysis and EC5 ₀ calculation were performed using GraphPad Prism software. [00170] The results demonstrated that WT-blocked (D-Arg) ₉ -pl9 ^ARF 26-44

10 peptides induced significantly higher apoptosis compared to WT-unb locked and mutant-blocked (D-Arg) ₉ -pl9 ^ARF 26-44 peptides. (Figures 18A and 18B, Tables 3 and 4). WT-unblocked (D-Arg) ₉ -pl9 ^ARF 26-44 peptides showed some activity compared to the mutant-blocked peptide at doses higher than 30 μΜ (statistically significant at 30 and 40 μΜ) in Experiment # 1 only. (Figure 18A, Table 3). There

15 was reproducibility of the apoptotic effect of WT-blocked peptide between the two experiments in the TUNEL assay (EC50 for blocked ARF peptide were 30.08 μΜ and

30.73 μΜ for 02/15/12 and 04/1 1/12, respectively) (Figures 19A and 19B).

Table 3: % of TUNEL Positive S2 Cells after Treatment with WT-Blocked, WT- Unblocked and Mutant (D-Arg) ₉ -pl9 ^ARF 26-44 Peptides— Experiment 1

20

* - when compared with mutant-blocked and WT-unblocked

** - when compared with mutant-blocked Table 4: % of TUNEL Positive S2 Cells after Treatment with WT-Blocked, WT- Unblocked and Mutant (D-Arg) ₉ -pl9 ^ARF 26-44 Peptides— Experiment 2

* - when compared with mutant-blocked and WT-unblocked ** - when compared with mutant-blocked

*** - when compared with WT-unblocked

N/A - Not tested

Example 22

ARF-peptide preferentially eliminated liver cancer stem cells (LCSCs)

10 [00171] ALb-HRasV 12 mice were used to demonstrate that ARF pepides as provided herein were capable of preferentially eliminating liver cancer stem cells (LCSCs). ALb-HRasV 12 mice are a transgnic strain that expresses activated Ras in the liver. These mice developed hepatocellular carcinoma (HCC) by 9 months of age. Alb-HRasV12 mice at 9 months of age were injected with either PBS, mutant peptide 15 or ARF-peptide (3 animals per group) at 5mg/kg every day for a period of 3 weeks. The mice were then sacrified one week later and HCC nodules were quantified (Figures 20A and 20B). In addition, single cell suspensions from the 8 different tumor nodules from the three treatment groups were assayed for CD90 /CD45 ^" cells by fluorescence-activated cell sorting (FACS). As shown in Figure 20C, there was a 20 considerable reduction in the number of the CD90 ⁺ /CD45- cells in the ARF-peptide injected samples suggesting that the ARF peptide preferentially eliminates liver cancer stem cells.

[00172] It should be understood that the foregoing disclosure emphasizes certain specific embodiments of the invention and that all modifications or alternatives 5 equivalent thereto are within the spirit and scope of the invention as set forth in the appended claims.

Table 5: Select Amino Acid Sequences