MOLECULAR MODULATORS OF THE WNT/BETA-CATENIN PATHWAY

Abstract

The present invention is directed toward a method of treating a subject for a condition mediated by aberrant Wnt/β-catenin signaling by selecting a subject with a condition mediated by aberrant Wnt/β-catenin signaling and administering to the selected subject at least one compound selected from the group consisting of those set forth in Table 1, Table 2, Table 3, and a pharmaceutically acceptable salt thereof. A method of similarly modulating the Wnt/β-catenin pathway in a subject is also discussed.

Description

FIELD OF THE INVENTION

This invention relates to molecular modulators of the Wnt/β-catenin pathway.

BACKGROUND OF THE INVENTION

Wnt/β-catenin signaling regulates cell fate and proliferation during development, homeostasis, and disease. The canonical Wnt pathway describes a series of events that occur when Wnt proteins bind to cell-surface receptors of the Frizzled family, causing the receptors to activate Dishevelled family proteins and ultimately resulting in a change in the amount of β-catenin that reaches the nucleus. Dishevelled (DSH) is a key component of a membrane-associated Wnt receptor complex which, when activated by Wnt binding Frizzled, inhibits a second complex of proteins that includes axin, GSK-3, and the protein APC. The axin/GSK-3/APC complex normally promotes the proteolytic degradation of the β-catenin intracellular signaling molecule. After this “β-catenin destruction complex” is inhibited, a pool of cytoplasmic β-catenin stabilizes, and some β-catenin is able to enter the nucleus and interact with TCF/LEF family transcription factors to promote specific gene expression.

Numerous diseases and several conditions have been linked to aberrant Wnt/β-catenin signaling (Moon R T, “WNT and Beta-catenin Signaling: Diseases and Therapies,” Nat Rev Gen 5(9):691-701 (2004)). It is also clear that modulation of Wnt/β-catenin signaling may be therapeutic for a variety of other indications including those involving a deficit in stem/progenitor cells. Lithium chloride is currently the only FDA approved small molecule modulator of Wnt/β-catenin signaling. The narrow therapeutic range of lithium combined with the vast number of diseases linked to Wnt/β-catenin signaling begs the discovery of additional small molecule modulators.

The present invention is directed, in part, to identifying small molecule modulators of Wnt/β-catenin signaling.

SUMMARY OF THE INVENTION

One aspect of the present invention is directed toward a method of treating a subject for a condition mediated by aberrant Wnt/β-catenin signaling by selecting a subject with a condition mediated by aberrant Wnt/β-catenin signaling and administering to the selected subject at least one compound selected from the group consisting of those set forth in Table 1, Table 2, Table 3, and a pharmaceutically acceptable salt thereof.

Another aspect of the present invention is directed toward a method of modulating the Wnt/β-catenin pathway in a subject including selecting a subject in need of Wnt/β-catenin pathway modulating and administering to the selected subject at least one compound selected from the group consisting of those set forth in Table 1, Table 2, Table 3, and a pharmaceutically acceptable salt thereof.

Yet another aspect of the present invention is directed toward a method of contacting a cell having aberrant Wnt/β-catenin signaling with at least one compound selected from the group consisting of those set forth in Table 1, Table 2, Table 3, and a pharmaceutically acceptable salt thereof, thereby modulating the Wnt/β-catenin signaling of the cell.

The present invention identifies small molecule modulators of the Wnt/β-catenin pathway. These small molecule modulators are potential therapies for the multiple diseases associated with the Wnt/β-catenin misregulation. Diseases that will be treated with the small molecule modulators of the present invention include, without limitation, cancer (malignant melanoma, colorectal cancer, renal, liver, lung, breast, prostate, ovarian, parathyroid, leukemias, glioma, neuroblastoma, astrocytoma, etc), bone mass diseases, fracture repair, FEVR, diabetes mellitus, cord blood transplants, psychiatric disease (e.g., bipolar depression), neurodegenerative disease (Alzheimer's, ALS), hair loss, diseases linked to loss of stem/progenitor cells, conditions improved by increasing stem/progenitor cell populations, HIV, and tooth agenesis.

The methods of the present invention, by their mechanism of action, further provide an insight in understanding the molecular, cellular, and organismic aspects of Wnt signaling. Characterizing small molecule probes that regulate Wnt signaling have utility as research tools for investigating various aspects in Wnt signaling in vitro and in vivo.

Currently available tools for conditional modulation of the Wnt pathway in humans are generally lacking. The present invention provides for pharmaceutical products that have previously unrecognized capabilities for activating or inhibiting the Wnt pathway. Since some of the compounds can enhance the activity of lithium, a drug used for the treatment of bipolar disorder, these agents may used in combination for effective lowering of required doses.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-G illustrate that nuclear β-catenin predicts improved survival in melanoma patients and correlates with decreased tumor proliferation. FIG. 1A is a graph showing that patients with the highest levels of nuclear β-catenin (upper tertile) exhibit an increased survival probability by Kaplan-Meier analysis compared to patients in the middle and lower tertile. This trend was statistically significant by log-rank test. FIG. 1B is a graph showing metastases separated into those with the highest nuclear β-catenin levels (upper 20%, n=46) and those with lower nuclear β-catenin levels (remaining 80%, n=179). Kaplan-Meier analysis showed a significantly increased survival probability in patients with the highest amount of nuclear β-catenin (Gehan-Breslow-Wilcoxon test). FIG. 1C is a graph showing the subset of patients with available data on tumor depth (Breslow thickness) analyzed by Kaplan-Meier survival curves. Tumors were grouped based on the AJCC tumor staging guidelines for tumor depth into T1 (0-1.00 mm, n=35), T2 (1.01-2.00 mm, n=26), T3 (2.01-4.00 mm, n=32) or T4 (>4.00 mm, n=20). The survival curves exhibited an extremely significant trend by log-rank test. FIG. 1D and FIG. 1E are graphs showing tumors grouped by tumor staging depth evaluated for proliferation (FIG. 1D) and for expression of nuclear β-catenin (FIG. 1E). Bars show the mean and standard deviation for each group, while gray dots represent individual tumors. The horizontal dotted lines represent the mean Ki-67 and nuclear β-catenin seen for all tumors in the array. As expected, increasing tumor depth is associated with increased proliferation. By contrast, levels of nuclear β-catenin decrease with increasing tumor depth, suggesting that activation of Wnt/β-catenin signaling is lost with melanoma progression. The trend for both % Ki-67 and nuclear β-catenin was extremely significant by ANOVA (*p<0.002). FIG. 1F is a histogram showing primary tumors stratified into tertiles based on levels of nuclear β-catenin (see FIG. 5), and the distribution of proliferation as measured by % Ki-67 was assessed in each tertile. Patients with the highest levels of nuclear β-catenin (upper tertile, n=39) showed a lower mean % Ki-67 than patients in the middle tertile (n=39) or the lower tertile (n=40). This trend was extremely significant by ANOVA (*p<0.0001). The histogram illustrates that tumors with the lowest levels of nuclear β-catenin (lower tertile) show a clear shift towards higher proliferation compared to patients with the highest levels of nuclear β-catenin (upper tertile). FIG. 1G is a graph showing normalized levels of nuclear β-catenin in primary tumors plotted against proliferation as measured by % Ki-67, and a Deming regression analysis (diagonal line) reveals an extremely significant inverse correlation between levels of nuclear β-catenin and proliferation as measured by Ki-67 (slope=−1.089+/−0.24).

FIGS. 2A-G illustrate activation of Wnt/β-catenin signaling changes melanoma cell fate. FIG. 2A is a photograph showing B16 cells expressing GFP, WNT3A or WNT5A isolated at equivalent confluency, spun down and photographed in a 96-well plate, demonstrating the marked difference in pigmentation seen in melanoma cells expressing WNT3A. FIG. 2B shows expression of WNT5A was confirmed by immunoblotting of cell lysates. FIG. 2C shows immunofluorescent staining demonstrating increased nuclear β-catenin in B16 cells expressing WNT3A, consistent with activation of the Wnt/β-catenin pathway. FIG. 2D is a histogram showing conditioned media from B16:GFP, B16:WNT3A and B16:WNT5A cells incubated with a human melanoma cell line stably transduced to express firefly luciferase under the control of a TCF-based Wnt/β-catenin-responsive promoter. Media from B16:WNT3A cells activate the reporter, indicating that these cells secrete active WNT3A. FIG. 2E is a histogram showing expression of the Wnt/β-catenin target gene Axin2 measured by quantitative real-time PCR and normalized to Gapdh. Upregulation of Axin2 is seen in WNT3A cells, indicating activation of the Wnt/β-catenin pathway. FIG. 2F is a histogram, showing proliferation of cells expressing GFP, WNT3A, or WNT5A, which was measured by hematocytometer after six days of culture (shaded bars, left y-axis) or by MTT assay after three days of culture (unshaded bars, right y-axis). Bars represent the average and standard deviation of three to six biological replicates. The inhibition of proliferation seen with WNT3A cells is extremely significant by ANOVA with both proliferation assays (*p<0.001). FIG. 2G is a histogram showing cell cycle analysis where cells expressing WNT3A demonstrated a decreased population in S phase and an increased population in G1 compared to cells expressing GFP or WNT5A. Bars indicate the average and standard deviation of three biologic replicates, and the data shown are representative of five individual experiments, each with at least three biologic replicates per condition. The changes observed in % G1 and % S with the WNT3A cells is extremely significant by ANOVA (*p<0.001).

FIGS. 3A-E illustrate that elevation of melanocyte differentiation markers by WNT3A corresponds with decreased tumor growth and metastasis in vivo. FIG. 3A is a heatmap of whole genome expression profiles of WNT3A or WNT5A cell lines compared to gene expression in GFP cells, which served as the reference sample. Three biologic replicates were analyzed for each cell line. The heatmap illustrates the differences between the most significant regulated genes in WNT3A cells compared to WNT5A cells by unpaired t-test. Genes that were among the most significantly regulated in WNT3A cells are listed with normalized fold-change (log 2) compared to GFP cells shown in parentheses. The most significantly regulated genes include known Wnt/β-catenin targets, genes involved in melanocyte and neural crest differentiation, and genes implicated in melanoma prognosis or therapeutics. FIG. 3B is a histogram showing several genes selected for validation using real-time quantitative PCR (qPCR), including genes implicated in melanocyte differentiation (Met, Kit, Sox9, Mitf, Si/Gp100), melanoma biology (Trpm1, Kit, Mme, Mlze), and genes that are known Wnt target genes (Axin2, Met, Sox9). Genes that were upregulated in WNT3A cells by transcriptional profiling are all upregulated by qPCR, while genes that are downregulated in WNT3A cells on the array (Mlze, Mme) are also downregulated by qPCR. Genes upregulated in WNT3A cells are universally downregulated in the WNT5A cells, providing evidence that WNT5A can antagonize transcription of Wnt/β-catenin gene targets in melanoma cells, even in the absence of WNT3A. Data are expressed as log 2-transformed fold-change compared to B16:GFP cells, and are representative of three or more experiments with similar results. FIG. 3C is a histogram showing gene changes induced by WNT3A inhibited upon treatment with β-catenin siRNA (20 nM) compared to control siRNA (20 nM). Data are expressed as log 2-transformed fold-change in cells treated with β-catenin siRNA compared to control siRNA. FIG. 3D is a graph showing tumor explants demonstrating that B16 cells expressing WNT3A form smaller tumors than cells expressing GFP or WNT5A. Data are expressed as the mean and standard deviation from four mice for each tested cell line. The experiment shown is representative of four independent experiments with the same result, all involving at least four mice for each cell line tested. The decrease in tumor size with WNT3A was highly significant by ANOVA at 14 days post-implantation (*p=0.004). FIG. 3E is a plot showing metastases to the popliteal sentinel lymph node bed evaluated by Firefly luciferase assay, demonstrating significantly decreased metastases in tumors expressing WNT3A.

FIGS. 4A-H illustrate a high-throughput screen for therapeutic activators of Wnt/β-catenin signaling. FIG. 4A is a schematic diagram showing the design of the high-throughput pharmacologic screen for Wnt activators and compounds that synergized in combination with WNT3A in murine HT-22 cells, using a Wnt/β-catenin-responsive luciferase reporter. The screen of FDA-approved compounds, some with multiple representations by different formulations or concentrations, is shown with a heatmap. Top compounds with the greatest percent change of activity with the growth media (GM) were then re-sorted based on the percent change with the WNT3A (W3a) stimulus, resulting in a final list of compounds that could activate in the absence and presence of WNT3A. Riluzole represented 2 of the top 6 compounds that fulfilled both screening requirements. FIG. 4B is a graph showing that in a secondary screen using HEK293T cells, riluzole activated expression of firefly luciferase under the transcriptional control of the Axin2 promoter, both on its own as well as in synergy with WNT3A conditioned media. Note that activation of the reporter by 10 μM riluzole was similar to activation by WNT3A alone. FIG. 4C is a graph showing that in B16 cells, riluzole enhances the transcription of endogenous Axin2, Si/Gp100, and Kit in the presence of WNT3A conditioned media, demonstrating dose-dependent synergy. FIG. 4D is a histogram showing that B16 cells were treated in culture for 72 hours with either 10 mM lithium chloride (with a control of 10 mM sodium chloride) or 10 μM riluzole (with a vehicle control of DMSO). Riluzole demonstrates upregulation of melanocytic genes, similar to WNT3A. By comparison, lithium upregulates a more limited set of melanocytic genes. Data are expressed as log 2-transformed fold-change compared to control. The data shown are the averages of three biologic replicates. FIG. 4E is a photograph of B16 cells treated for 2 passages with riluzole (10 μM) were compared to control B16 cells at equivalent confluency, demonstrating increased pigmentation with riluzole treatment. FIG. 4F is a histogram showing that B16 cells treated for three days with 10 μM riluzole exhibit decreased proliferation by MTT assay, which was extremely significant by two-tailed t-test (*p<0.0001) in this representative experiment. FIG. 4G is a graph showing B16 cells injected into footpads of C57BL/6 mice, and treatment with riluzole was initiated one week post-injection. No significant difference was seen in tumor size after 21 days. FIG. 4H is a plot showing sentinel lymph nodes in the popliteal fossa adjacent to the injected foot assayed for the presence of metastases as measured by Firefly luciferase. Bars represent the mean and standard deviation of 9 mice for each group, and indicate that tumors from mice treated with riluzole exhibited significantly decreased metastasis compared to control mice with no treatment (unpaired two-tailed t-test).

FIGS. 5 A-D illustrate figures related to tumor microarray analysis. FIG. 5A is a histogram depicting the distribution of nuclear β-catenin staining in the cohort of primary tumors. The bar below shows the cut-offs for the three tertiles used for analysis of survival in FIG. 1. FIG. 5B is a histogram depicting survival analysis in metastases. The upper 20% was selected based on both the population distribution and the absolute levels of nuclear-catenin, which correspond roughly with the upper tertile of the population. FIG. 5C is a plot showing levels of nuclear β-catenin compared in primary tumors and metastases/recurrences, showing a decrease in nuclear β-catenin in metastases/recurrences that approximated statistical significance using an unpaired two-tailed t-test. This data supports the hypothesis that Wnt/β-catenin signaling is lost with melanoma progression. FIG. 5D is a plot comparing % Ki-67 with another marker of proliferation, % PCNA. Deming regression analysis gave an extremely significant correlation, with a slope of 1.04 suggesting that proliferation was robustly measured by % Ki-67.

FIGS. 6A-D illustrate Wnt expression in the context of human melanoma. FIG. 6A is a table showing data from the NCBI Gene Expression Omnibus used to evaluate the expression of Wnt isoforms in benign nevi and melanoma tumors (see also Barrett et al., Nucleic Acids Res. D760-5 (2007), which is hereby incorporated by reference in its entirety). The datasets used include GDS1375 (Talantov et al., Clin. Cancer Res. 11(20):7234-42 (2005), which is hereby incorporated by reference in its entirety) and GDS1989 (Smith et al., Cancer Biol. Ther. 4(9):1018-29 (2005), which is hereby incorporated by reference in its entirety). The primary expression data is shown, and the above table summarizes the data from these two datasets. The data summarization is based on the reported ‘detection call’ of the Affymetrix data used for all three datasets, and the scale indicates the percentage of samples with ‘present’ calls on the expression of the different Wnt isoforms. In the primary data presented above, ‘absent’ calls are faded out. Scoring was as follows: 0 calls were ‘absent’ in all samples; + represents up to 25% of specimens have expression; ++ represents 25-50% of specimens have expression; +++ represents 50-75% of specimens have expression; ++++ represents 75-100% of specimens have expression. Few Wnt isoforms are expressed by melanoma tumors based on this transcriptional profiling, and only wnt3, wnt4, wnt5a and wnt6 were detected in melanomas from both gene datasets. FIG. 6B and FIG. 6C are histograms showing the human melanoma cell lines Mel375 (FIG. 6B) and UACC 1273 (FIG. 6C) were transduced with lentiviral constructs for encoding either GFP or WNT3A. Cells were counted after 3-7 days by hematocytometer, and the panels above are representative of multiple experiments with similar results. The bars represent the average and standard deviation from three biologic replicates. P-values for two-tailed t-tests were statistically significant (*p<0.05). Expression of WNT3A also led to a consistent and reproducible decrease in proliferation by MTT assay. No consistent effect on proliferation was seen with expression of WNT5A, again similar to the B16 cell lines. FIG. 6D is a histogram showing human melanoma cell lines cultured for 3-7 days in the presence of either 10 mM sodium chloride or 10 mM lithium chloride. Proliferation was measured by hematocytometer or MTT assay, and normalized to growth observed in the samples cultured in 10 mM sodium chloride. Lithium chloride inhibited proliferation in all human melanoma cell lines tested.

FIGS. 7A-F illustrate inhibitors of GSK3 activate Wnt/β-catenin signaling and inhibit proliferation of B16 melanoma cells. FIG. 7A and FIG. 7B are photographs showing immunofluorescent staining of β-catenin demonstrates increased nuclear β-catenin in B16 cells treated with 10 mM lithium chloride or 1 μM BIO compared to control cells treated with 10 mM sodium chloride or DMSO, respectively, consistent with activation of the Wnt/β-catenin pathway by lithium and BIO. FIG. 7C and FIG. 7D are histograms showing quantitative PCR demonstrates increased Axin2 levels in B16 cells treated with 10 mM lithium chloride or 1 μM BIO compared to control cells, also consistent with activation of the Wnt/β-catenin pathway by both drugs. FIG. 7E and FIG. 7F are histograms showing representative MTT proliferation assays and demonstrate the decreased proliferation seen in B16 cells treated with 10 mM lithium chloride or 1 μM BIO compared to control cells. Bars represent the mean and standard deviation of three to six biologic replicates. The difference is extremely significant by unpaired two-tailed t-test (p<0.001).

FIGS. 8A-C illustrate microarray analysis of B16 cells expressing WNT3A and WNT5A. FIG. 8A and FIG. 8B are Venn diagrams which compare the genes upregulated and downregulated in B16 cells expressing WNT3A or WNT5A compared to control B16 cells expressing GFP, which served as the reference for Agilent whole mouse genome two-channel arrays. Very few genes were regulated by WNT5A compared to WNT3A, consistent with previous results in human melanoma cells. FIG. 8C shows B16 melanoma cells transfected for 72 hours with either control siRNA or siRNA targeting murine β-catenin were analyzed by immunoblotting to assess knockdown of β-catenin protein. The siRNA sequences (SEQ ID NOs: 1-3) tested are on the right. It was found that siRNA #2 and #3 produced marked knockdown of β-catenin protein and for the validation of microarray target genes presented in FIG. 3. Cells were transfected with a pool consisting of 10 nM of siRNA #2 and #3 to minimize off-target effects of each individual siRNA.

FIG. 9 illustrates a model for differentiation therapy using Wnt/β-catenin activators in melanoma. This is a schematic diagram depicting a model of melanoma arising through transformation of differentiated melanocytes and nevus (mole) cells or from melanocytic progenitor cells, taking into account that clinical melanomas arise both from established melanocytic lesions and also de novo (Barnhill et al., Pathology of Melanocytic Nevi and Malignant Melanoma (2004), which is hereby incorporated by reference in its entirety). Based readouts of differentiation such as gene expression profiles, previous studies have found that melanoma progression appears to correlate with the loss of expression of melanocytic markers. Additionally, this model also incorporates the concept of cancer stem cells (or tumor initiating cells) in melanoma (Hendrix et al., Nat. Rev. Cancer 7:246 (2007), which is hereby incorporated by reference in its entirety), which give rise to highly proliferative bulk tumor cells, and are themselves highly resistant to conventional chemotherapy in the context of melanoma and other cancer stem cell models. Based on the finding that WNT3A is one of only three factors needed to generate functional melanocytes from embryonic stem cells (Fang et al., Stem Cells 24:1668 (2006), which is hereby incorporated by reference in its entirety), as well as the well-described requirement for Wnt/β-catenin signaling in melanocyte development from animal models (Dorsky et al., Nature 396:370 (1998), which is hereby incorporated by reference in its entirety), the leveraging of this pathway to force cell fate changes in melanoma offers an attractive choice for therapeutic manipulation. The findings herein, as well as other supporting published results (Bachmann et al., Clin. Cancer Res. 11:8606 (2005); Kageshita et al., Br. J. Dermatol. 145:210 (2001), which are hereby incorporated by reference in their entirety) documenting the loss of β-catenin with melanoma progression and decreased survival are depicted below the model. The present data suggest that using activators of Wnt/β-catenin signaling in melanoma can force differentiation in both bulk tumor cells and cancer stem cells to promote cell fates associated with less aggressive tumors through the reactivation of melanocyte-associated transcriptional programs that are downregulated or lost during normal melanoma progression. The goal of differentiation therapy using Wnt/β-catenin activators would be to elicit changes in tumor cell properties through reprogramming of cell, generating tumors that are less aggressive, less proliferative, or potentially more susceptible to currently available melanoma therapies. The availability of several previously FDA-approved activators of Wnt/β-catenin signaling, including riluzole, can facilitate the rapid testing of this therapeutic approach in clinical trials.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention is directed toward a method of treating a subject for a condition mediated by aberrant Wnt/β-catenin signaling by selecting a subject with a condition mediated by aberrant Wnt/β-catenin signaling and administering to the selected subject at least one compound selected from the group consisting of those set forth in Table 1, Table 2, Table 3, and a pharmaceutically acceptable salt thereof.

In a preferred embodiment of this and other aspects described herein, the subject is human.

In another embodiment of this and other aspects described herein, the compound is selected from the group consisting of flunarizine, alendronate, SNDX-275, vorinostat, isotretinoin, etoposide, virginiamycin, amoxapine, riluzole, mercaptopurine, milrinone, clofazimine, melphalan, and thioguanine.

The condition which can be treated in accordance with this aspect of the present invention can be any one of the following: cancer (malignant melanoma, colorectal cancer, renal, liver, lung, breast, prostate, ovarian, parathyroid, leukemias, glioma, neuroblastoma, astrocytoma, etc), bone mass diseases, fracture repair, FEVR, diabetes mellitus, cord blood transplants, psychiatric disease (e.g., bipolar depression), neurodegenerative disease (Alzheimer's, ALS), hair loss, diseases linked to loss of stem/progenitor cells, conditions improved by increasing stem/progenitor cell populations, HIV, and tooth agenesis.

The compound of the present invention can activate or inhibit the Wnt/β-catenin pathway.

Another aspect of the present invention is directed toward a method of modulating the Wnt/β-catenin pathway in a subject including selecting a subject in need of a Wnt/β-catenin pathway modulating and administering to the selected subject at least one compound selected from the group consisting of those set forth in Table 1, Table 2, Table 3, and a pharmaceutically acceptable salt thereof.

Yet another aspect of the present invention is directed toward a method of contacting a cell having aberrant Wnt/β-catenin signaling with at least one compound (e.g., 1, 2, 3, 4, 5 or more compounds) selected from the group consisting of those set forth in Table 1, Table 2, Table 3, and a pharmaceutically acceptable salt thereof, thereby modulating the Wnt/β-catenin signaling of the cell.

The Wnt signaling pathway is essential in many biological processes. During embryogenesis this pathway is evolutionary conserved and controls many events. At the cellular level the Wnt pathway regulates morphology, proliferation, motility, and cell fate. Also during tumorigenesis the Wnt signaling pathway has a central role and inappropriate activation of this pathway are observed in several human cancers (Spink et al., “Structural Basis of the Axin-adenomatous Polyposis Coli Interaction,” EMBO J, 19(20):2270-2279 (2000), which is hereby incorporated by reference in its entirety). β-catenin is a protein which is known to be a key mediator of Wnt signaling (McCrea et al., “A Homolog of the Armadillo Protein in Drosophila (plakoglobin) Associated with E-cadherin,” Science 254(5036):1359-1361 (1991); Gumbiner “Signal Transduction of Beta-catenin,” Curr. Opin. Cell. Biol. 7(5):634-640 (1995) which are hereby incorporated in their entirety). The primary structure of β-catenin comprises an amino-terminal domain of approximately 130 amino acids, a central region of 12 imperfect repeats of 42 amino acids known as arm repeats (since they show homology with the repeats found in Arm protein of Drosophila), and a carboxy-terminal domain of 110 amino acids. The amino-terminus of β-catenin is important for regulating its stability whereas the carboxy-terminal works as a transcriptional activator domain (Willert et al., “Beta-catenin: a Key Mediator of Wnt Signaling,” Curr. Opin. Genet. Dev. 8(1):95-102 (1998) which is hereby incorporated in its entirety). β-catenin activity can be controlled by a large number of binding partners that will affect the stability and localization of the β-catenin. The compounds of the present invention (Tables 1, 2, 3, and 4) provide such an interaction with the β-catenin.

For purposes of clarity, the following terms shall be understood to have the following meanings. All other terms used herein have the same meaning as commonly understood by one of ordinary skills in the art.

The term “alkyl” means an aliphatic hydrocarbon group which may be straight or branched having about 1 to about 10 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, 3-pentyl, hexyl, heptyl, octyl, nonyl, and decyl.

The term “alkenyl” means an aliphatic hydrocarbon group containing At least one carbon-carbon double bond and which may be straight or branched having about 2 to about 10 carbon atoms in the chain. Preferred alkenyl groups have 2 to about 4 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl, or propyl are attached to a linear alkenyl chain. Exemplary alkenyl groups include, without limitation, ethenyl, propenyl, n-butenyl, i-butenyl, prenyl, and isoprenyl.

As used herein, “cycloalkyl” refers to a non-aromatic saturated or unsaturated mono- or polycyclic ring system which may contain 3 to 6 carbon atoms; and which may include at least one double bond. Exemplary cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, anti-bicyclopropane, or syn-bicyclopropane.

The term “alkoxy” means an alkyl-O—, alkenyl-O—, or alkynyl-O— group wherein the alkyl, alkenyl, or alkynyl group is described above. Exemplary alkoxy groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, pentoxy, and hexoxy.

The term “alkanoyl” refers to a radical of the formula R^aC(O)— where R^a is an alkyl or cycloalkyl radical as defined above. The alkyl radical and the cycloalkyl radical may be optionally substituted as defined above.

As used herein, “aryl” refers to aromatic monocyclic or polycyclic ring system containing from 6 to 19 carbon atoms, where the ring system may be optionally substituted. Aryl groups of the present invention include, but are not limited to, groups such as phenyl, naphthyl, azulenyl, phenanthrenyl, anthracenyl, fluorenyl, pyrenyl, triphenylenyl, chrysenyl, and naphthacenyl.

The term “aryloxy” means an aryl-O—, where aryl is described as above. Exemplary aryloxy groups include phenoxy and naphthoxy.

The term “arylalkyl” refers to a radical of the formula —R^aR^b where R^a is an alkyl radical as defined above and R^b is an aryl radical as defined above. The alkyl radical and the cycloalkyl radical may be optionally substituted as defined above.

The term “arylalkoxy” refers to a radical of the formula —O—R^aR^b where R^a is an alkyl or cycloalkyl radical as defined above and R^b is an aryl radical as defined above. The alkyl radical and the cycloalkyl radical may be optionally substituted as defined above.

As used herein, “heteroaryl” refers to an aromatic ring radical which consists of carbon atoms and from one to five heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. For purposes of this invention the heteroarayl may be a monocyclic or polycyclic ring system; and the nitrogen, carbon, and sulfur atoms in the heteroaryl ring may be optionally oxidized; the nitrogen may optionally be quaternized. Examples of heteroaryl groups include, without limitation, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, furyl, thiophenyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, thienopyrrolyl, furopyrrolyl, indolyl, azaindolyl, isoindolyl, indolinyl, indolizinyl, indazolyl, benzimidazolyl, imidazopyridinyl, benzotriazolyl, benzoxazolyl, benzoxadiazolyl, benzothiazolyl, pyrazolopyridinyl, triazolopyridinyl, thienopyridinyl, benzothiadiazolyl, benzofuyl, benzothiophenyl, quinolinyl, isoquinolinyl, tetrahydroquinolyl, tetrahydroisoquinolyl, cinnolinyl, quinazolinyl, quinolizilinyl, phthalazinyl, benzotriazinyl, chromenyl, naphthyridinyl, acrydinyl, phenanzinyl, phenothiazinyl, phenoxazinyl, pteridinyl, and purinyl.

As used herein, the term “monocyclic” indicates a molecular structure having one ring.

As used herein, the term “polycyclic” indicates a molecular structure having two or more rings, including, but not limited to, fused, bridged, or spiro rings.

The term “substituted” or “substitution” of an atom means that one or more hydrogen on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency is not exceeded. “Unsubstituted” atoms bear all of the hydrogen atoms dictated by their valency. When a substituent is oxo (i.e., ═O), then 2 hydrogens on the atom are replaced. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds; by “stable compound” or “stable structure” is meant a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. Exemplary substitutents include, without limitation, oxo, thio (i.e. ═S), nitro, cyano, halo, OH, NH₂, C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₆ cycloalkyl, C₄-C₇ cycloalkylalkyl, monocyclic aryl, monocyclic hetereoaryl, polycyclic aryl, and polycyclic heteroaryl.

TABLE 1
Activators/Synergizers
Family I  wherein Ar is a member selected from the group consisting of phenyl and fluorophenyl, preferably para-fluorophenyl; provided that at least one of said Ar groups is fluorophenyl.
Family II wherein n is an integer from 3 to 5
Family III
Family IV wherein each of R1 and R2 are independently the same as or different from each other; when R1 and R2 are the same, each is a substituted or unsubstituted cycloalkylamino, pyridineamino, piperidino, 9-purine-6-amine, or thiazoleamino group; when R1 and R2 are different, R1═R3—N—R4, wherein each of R3 and R4 are independently the same as or different from each other and are a hydrogen atom, a hydroxyl group, a substituted or unsubstituted, branched or unbranched alkyl, alkenyl, cycloalkyl, aryl, alkyloxy, aryloxy, arylalkyloxy, or pyridine group, or R3 and R4 bond together to form a piperidine group and R2 is a hydroxylamino, hydroxyl, amino, alkylamino, or alkyloxy group; and n is an integer from about 4 to about 8.
Family V

An example of a suitable compound of Family I is flunarizine, which has the following structure:

An example of a suitable compound of Family II is alendronate, which has the following structure:

An example of a suitable compound of Family III is SNDX-275, which has the following structure:

An example of a suitable compound of Family IV is vorinostat, which has the following structure:

An example of a suitable compound of Family V is isotretinoin, which has the following structure:

TABLE 2
Inhibitors
Family VI   wherein R2 is H and R1 is selected from the group consisting of C1-10)alkyl, (C2-10)alkenyl, (C5-6)cycloalkyl, 2-furyl, 2-thienyl, (C6-10)aryl, and (C7-14)aralkyl; or R1 and R2 are each (C1-10)alkyl; or R1, R2 and the carbon to which they are attached together represent C5-6)cycloalkyl; one of R3 and R4 is H and the other is selected from the group consisting of (C1-5)alkanoyl and benzoyl; or R3 and R4 are the same and are selected from the group consisting of (C1-5) alkanoyl and benzoyl: R5 is H or a phosphate group.
Family VII  wherein: R1 is selected from the group consisting of: (1) hydrogen (2) —COR2, wherein R2 is selected from the group consisting of: (a) C1-4 alkyl, (b) benzyl, and (c) phenyl, (3) —CONHR3, wherein R3 is selected from the group consisting of: (a) C1-4 alkyl, (b) benzyl, unsubstituted or substituted with —CH3 or —NO2, (c) phenyl, unsubstituted or substituted with —CH3 or —NO2, (d) naphthyl, unsubstituted or substituted with —CH3 or —NO2, X is oxo, (H, OH) or (H, —OCOR4), wherein R4 is independently selected from the definitions of R2, and the symbol of a line and a dashed line is a single bond or a double bond; with the proviso that if X is oxo or (H, OH), the symbol of a line and a dashed line is a single bond; and the further proviso that if X is oxo, R1 is other than hydrogen.
Family VIII wherein each X is, independently, H, Cl, F, Br, I, CH3, CF3, OH, OCH3, CH2CH3, or OCH2CH3; B is independently H, Cl, F, Br, I, CX3, CH2CH3, OCX3, or OCX2CX3; and D is CH2, O, NH, S(O)0-2.

An example of a suitable compound of Family VI is etoposide, which has the following structure:

An example of a suitable compound of Family VII is virginiamycin, which has the following structure:

An example of a suitable compound of Family VIII is amoxapine, which has the following structure:

TABLE 3
Other Compounds
Compound 1: ascomycin
Compound 2: azelaic acid
Compound 3: carbenicillin
Compound 4: stearoylcarnitine
Compound 5: clotrimazole
Compound 6: fluvastatin sodium salt
Compound 7: lorcainide
Compound 8: mianserin
Compound 9: oxyphenbutazone
Compound 10: riluzole hydrochloride
Compound 11: salsolinol hydrobromide
Compound 12: 5-aza-2′- deoxycytidine
Compound 13: 6-mercaptopurine
Compound 14: all-trans- retinoic acid
Compound 15: azathioprine
Compound 16: doxycycline
Compound 17: famprofazone
Compound 18: idoxuridine
Compound 20: metixene hydrochloride
Compound 21: taxol
Compound 22: thioguanine
Compound 23: milrinone
Compound 24: Melphalan
Compound 25: 9-cis retinoic acid
Compound 26: Lovastatin
Compound 27: Israpidine

Compounds of the present invention can be administered to a subject at risk for a condition mediated by aberrant Wnt/β-catenin signaling, a subject that is diagnosed with a condition mediated by aberrant Wnt/β-catenin signaling, or a subject already afflicted with a condition mediated by aberrant Wnt/β-catenin signaling.

Exemplary conditions which can be treated in accordance with these aspects of the present invention, include, but are not limited to, cancer (malignant melanoma, colorectal cancer, renal, liver, lung, breast, prostate, ovarian, parathyroid, leukemias, glioma, neuroblastoma, astrocytoma, etc), bone mass diseases, fracture repair, FEVR, diabetes mellitus, cord blood transplants, psychiatric disease (e.g., bipolar depression), neurodegenerative disease (Alzheimer's, ALS), hair loss, diseases linked to loss of stem/progenitor cells, conditions improved by increasing stem/progenitor cell populations, HIV, and tooth agenesis.

The compounds of the present invention can be administered orally, parenterally, for example, subcutaneously, intravascularly, intraarterially, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by inhalation, or by application to mucous membranes, such as, that of the nose, throat, and bronchial tubes. The compounds may be administered alone or with suitable pharmaceutical carriers, and can be in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions.

One of ordinary skill in the art would be able to use routine methods in order to determine the appropriate route of administration and the correct dosage of an effective amount of a cell-based composition for methods of the present invention. It would also be known to those having ordinary skill in the art to recognize that in certain therapies, multiple administrations of pharmaceutical compositions of the invention will be required to effect therapy. For example a composition may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a span of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5, years, 10 years, or more.

Compounds of the present invention and compositions and compositions comprising the same are often administered, in an effective amount. As used herein, the term “effective amount” includes an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

A “therapeutically effective amount” of a compound or composition of the invention, may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of a compound or combination of compounds to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of one or more compounds are outweighed by the therapeutically beneficial effects.

A “prophylactically effective amount” refers to an amount of a compound or combination of compounds effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, a prophylactic dose is used in subjects prior to or at an earlier stage of disease; thus, the prophylactically effective amount is less than the therapeutically effective amount.

The active compounds of the present invention may be orally administered, for example, with an inert diluent, or with an assimilable edible carrier, or they may be enclosed in hard or soft shell capsules, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. For oral therapeutic administration, these active compounds may be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compound in these compositions may, of course, be varied and may conveniently be between about 2% to about 60% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.

The amount of the active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the ingredient which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.1 percent to about 99 percent of active ingredient, about 1 percent to about 90 percent of active ingredient, about 10 percent to about 80 percent of active ingredient, about 25 percent to about 75 percent of active ingredient, about 30 percent to about 70 percent of active ingredient, about 40 percent to about 60 percent of active ingredient, or about 50 percent of active ingredient.

In one embodiment, the amount of active ingredient in a single dosage to produce a therapeutic effect is about 0.1% active ingredient, about 1% active ingredient, about 5% active ingredient, about 10% active ingredient, about 15% active ingredient, about 20% active ingredient, about 25% active ingredient, about 30% active ingredient, about 35% active ingredient, about 40% active ingredient, about 45% active ingredient, about 50% active ingredient, about 55% active ingredient, about 60% active ingredient, about 65% active ingredient, about 70% active ingredient, about 75% active ingredient, about 80% active ingredient, about 85% active ingredient, about 90% active ingredient, or about 95% active ingredient or more.

An effective dose will generally depend upon the factors described above. Generally, oral, intravenous, intracerebroventricular, and subcutaneous doses of the adjunct therapies used in combination with a cell-based composition in various embodiments of this invention, will range from about 0.000001 to about 1000 mg per kilogram, about 0.000005 to about 950 mg per kilogram, about 0.00001 to about 850 mg per kilogram, about 0.00005 to about 750 mg per kilogram, about 0.0001 to about 500 mg per kilogram, about 0.0005 to about 250 mg per kilogram, about 0.001 to about 100 mg per kilogram, about 0.001 to about 50 mg per kilogram, about 0.001 to about 25 mg per kilogram, about 0.001 to about 10 mg per kilogram, about 0.001 to about 1 mg per kilogram, about 0.005 to about 100 mg per kilogram, about 0.005 to about 50 mg per kilogram, about 0.005 to about 25 mg per kilogram, about 0.005 to about 10 mg per kilogram, about 0.005 to about 1 mg per kilogram, about 0.01 to about 100 mg per kilogram, about 0.01 to about 50 mg per kilogram, about 0.01 to about 25 mg per kilogram, about 0.01 to about 10 mg per kilogram, about 0.01 to about 1 mg per kilogram, about 0.05 to about 50 mg per kilogram, about 0.05 to about 25 mg per kilogram, about 0.05 to about 10 mg per kilogram, about 0.05 to about 1 mg per kilogram, about 0.1 to about 25 mg per kilogram, about 0.1 to about 10 mg per kilogram, about 0.1 to about 1 mg per kilogram, and about 0.1 to about 0.5 mg per kilogram of body weight per day.

Preferred compositions according to the present invention are prepared so that an oral dosage unit contains between about 1 and 250 mg of active compound.

The tablets, capsules, and the like may also contain a binder such as gum tragacanth, acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose, or saccharin. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a fatty oil.

Various other materials may be present as coatings or to modify the physical form of the dosage unit. For instance, tablets may be coated with shellac, sugar, or both. A syrup may contain, in addition to active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and flavoring such as cherry or orange flavor.

These active compounds may also be administered parenterally. Solutions or suspensions of these active compounds can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols such as, propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

The compounds of the present invention may also be administered directly to the airways in the form of an aerosol. For use as aerosols, the compounds of the present invention in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The materials of the present invention also may be administered in a non-pressurized form such as in a nebulizer or atomizer.

The compounds of the present invention may also be administered directly to the airways in the form of a dry powder. For use as a dry powder, the compounds of the present invention may be administered by use of an inhaler. Exemplary inhalers include metered dose inhalers and dry powdered inhalers. A metered dose inhaler or “MDI” is a pressure resistant canister or container filled with a product such as a pharmaceutical composition dissolved in a liquefied propellant or micronized particles suspended in a liquefied propellant. The correct dosage of the composition is delivered to the patient. A dry powder inhaler is a system operable with a source of pressurized air to produce dry powder particles of a pharmaceutical composition that is compacted into a very small volume. For inhalation, the system has a plurality of chambers or blisters each containing a single dose of the pharmaceutical composition and a select element for releasing a single dose.

Suitable powder compositions include, by way of illustration, powdered preparations of the active ingredients thoroughly intermixed with lactose or other inert powders acceptable for intrabronchial administration. The powder compositions can be administered via an aerosol dispenser or encased in a breakable capsule which may be inserted by the patient into a device that punctures the capsule and blows the powder out in a steady stream suitable for inhalation. The compositions can include propellants, surfactants and co-solvents and may be filled into conventional aerosol containers that are closed by a suitable metering valve.

EXAMPLES

Example 1

Cell Lines

B16 murine melanoma cells expressing firefly luciferase were used as the parental line for experiments described herein (Murakami et al., Cancer Res. 62:7328 (2002), which is hereby incorporated by reference in its entirety). Human melanoma UACC1273 and M92047 cell lines are as described in Bittner et al., Nature 406:536 (2000), which is hereby incorporated by reference in its entirety). The human melanoma cell lines Mel375, A2058, Mel 29.6 and Mel501 were obtained from Fred Hutchinson Cancer Research Institute; Seattle, Wash. The murine cell line HT22, a subclone of the HT4 hippocampal cell line, was obtained from The Salk Institute for Biological Studies. Sequences for human WNT3A and WNT5A were amplified by polymerase chain reaction (PCR) and cloned into third generation lentiviral vectors derived from backbone vectors (Dull et al., J. Virol. 72:8463 (1998), which is hereby incorporated by reference in its entirety). These lentiviral vectors contained an EF 1-alpha promoter driving a bi-cistronic message encoding human Wnt isoforms plus GFP. Cells were sorted by fluorescence activated cell sorting (FACS) for GFP expression, with the goal of obtaining cells with approximately equivalent levels of GFP expression.

Example 2

Cell Culture

B16 murine melanoma cells were cultured in Dulbeccos modified Eagle's media (DMEM) supplemented with 2% Fetal Bovine Serum, and 1% antibiotic/antimycotic (Invitrogen; Grand Island, N.Y.) (Murakami et al., Cancer Res. 62:7328 (2002), which is hereby incorporated by reference in its entirety). The human melanoma lines Mel375, M92047, A2058, Mel 29.6, Mel501 and Mel526 were cultured in DMEM supplemented with 2% FBS and 1% antibiotic/antimycotic. UACC1273 cells were cultured in RPMI (Invitrogen; Grand Island, N.Y.) supplemented with 2% FBS and 1% antibiotic/antimycotic. All cell lines were cultured in the presence of 0.02% Plasmocin (InvivoGen; San Diego, Calif.). Synthetic siRNAs (Invitrogen; Grand Island, N.Y.) were transfected into cultured cells at a final concentration of 20 nM using Lipofectamine 2000 (Invitrogen; Grand Island, N.Y.). HT22 cells were cultured in DMEM supplemented with 10% FBS and 1% antibiotic/antimycotic. Sequences for β-catenin siRNA are described in FIG. 8.

Example 3

Conditioned Media and Measurement of Wnt Pathway Activation Using a Reporter Assay

Conditioned media was collected from sub-confluent melanoma cell lines, and this media was tested for its ability to activate Wnt/β-catenin signaling in UACC1273 cells stably transduced with a previously described Wnt/β-catenin-responsive firefly luciferase reporter and a constitutive Renilla luciferase gene used for normalization (Major et al., Science 316:1043 (2007), which is hereby incorporated by reference in its entirety). Conditioned media from B16 melanoma cells was spun down to clear cell debris and then incubated with reporter cells overnight. Activation of the Wnt/β-catenin reporter was measured using a dual luciferase reporter (DLR) assay kit (Promega; Madison, Wis.).

Example 4

RNA Purification from B16 Melanoma Cells and PCR Analysis

Cells were cultured for approximately 72 hours until they reached 80-90% confluency. RNA was purified using the RNeasy kit using the manufacturer's protocol (Qiagen; Maryland, Md.). cDNA was synthesized using Superscript Reverse Transcriptase (Invitrogen; Grand Island, N.Y.). Light Cycler FastStart DNA Master SYBR Green1 (Roche; Mannheim, Germany) was used for real-time PCR as previously described (Major et al., Science 316:1043 (2007), which is hereby incorporated by reference in its entirety). Quantitative PCR results presented in the manuscript are representative of experiments performed on a minimum of three biologic replicates.

Example 5

In Vivo Tumor Inoculation and Measurements of Lymph Node Metastasis

Footpad injections of transduced B16 melanoma cells and measurement of popliteal lymph node and lung metastasis was performed as previously described (Murakami et al., Cancer Res. 62:7328 (2002), which is hereby incorporated by reference in its entirety). For the experiments using riluzole (Matrix Scientific; Columbia, S.C.), mice were dosed orally according to previously published protocols (Namkoong et al., Cancer Res. 67:2298 (2007); Fumagalli et al., Exp. Neurol. 198:114 (2006), which are hereby incorporated by reference in their entirety). Mice received approximately 200 g/day (˜10 mg/kg/day) of riluzole dosed in their drinking water, since this dose was estimated to achieve serum levels comparable to that obtained in humans receiving 1-2 mg/kg/day (Namkoong et al., Cancer Res. 67:2298 (2007); Fumagalli et al., Exp. Neurol. 198:114 (2006), which are hereby incorporated by reference in their entirety). Riluzole dosing was initiated 7 days after tumor inoculation, and mice were sacrificed at 28 days post-inoculation for sentinel lymph node metastases studies. All animal studies were performed using IACUC protocols approved by institutional review boards.

Example 6

Cell Proliferation Assays

For cell counts by hematocytometer, cells were seeded at a uniform density (usually between 10,000 to 25,000 cells per well) in a 12 or 24 well tissue culture plate in the appropriate media. At the end of 3-7 days, cells were trypsinized, resuspended in the appropriate media and counted. Dead cells were identified by 0.4% Trypan Blue stain and excluded from hematocytometer measurements. Cell proliferation experiments were performed with a minimum of six biologic replicates. Similar results were observed for all cell lines using the MTT assay (ATCC; Manassas, Va.), performed according to manufacturer's protocol. For relative cell proliferation assays of B16: GFP cells incubated with lithium chloride or sodium chloride, cell proliferation was measured by luciferase assay. Cell cycle analysis was performed using DAPI-staining and flow cytometry. The Ki-67 rabbit monoclonal antibody was purchased from ThermoFisher (catalog no. RM-9106).

Example 7

Immunohistochemistry and Immunoblotting Studies

A polyclonal rabbit anti-β-catenin antibody was used for detection of β-catenin (1:1000 dilution for immunoblot, 1:200 dilution for immunohistochemistry). Cells were grown on 18 mm glass coverslips, for 48-72 hours, fixed using 4% paraformaldahyde, permeabilized using 0.25% Triton X-100, and then blocked with 10% goat serum. Goat anti-rabbit Alexa Fluor-568 antibody (Molecular Probes; Eugene, Oreg.) was diluted 1:1000. Cells were counterstained for nucleic acid with DAPI (Molecular Probes; Eugene, Oreg.). Paraffin-embedded nevus sections were stained using an antibody dilution of 1:200. Cellular lysates were obtained by lysing cells on plate with a 0.1% NP-40 based buffer and analyzed by NuPage 4-12% gradient gels (Invitrogen; Grand Island, N.Y.). The WNT5A antibody was obtained from Cell Signaling Technologies (Danvers, Mass.).

Example 8

Tumor Microarrays

Tumor microarrays were assembled at the Yale Tissue Microarray Facility. Staining and scoring of tissue microarrays was performed using automated quantification (AQUA) as previously described (Camp et al., Nat. Med. 8:1323 (2002), which is hereby incorporated by reference in its entirety). Statistical analysis, including Kaplan-Meier survival probabilities, ANOVA, and t-tests, was performed using the GraphPad Prism software package (GraphPad Software; La Jolla, Calif.).

Example 9

cDNA Microarrays

Agilent whole mouse genome array analysis was performed through the microarray core facility at the Huntsman Cancer Institute (Salt Lake City, Utah). Data analysis, including the t-test (Pan, Bioinformatics 18:546 (2002), which is hereby incorporated by reference in its entirety) was performed using the TM4 microarray software suite, which is freely available online (Saeed et al., Biotechniques 34:374 (2003), which is hereby incorporated by reference in its entirety). Two-channel hybridizations were performed with labeled cDNA isolated from three biologic replicates each for cells expressing either WNT3A or WNT5A, using cDNA from GFP-expressing cells as the reference sample. These studies revealed gene sets regulated in both WNT3A and WNT5A cells (FIG. 8), which were then filtered to obtain the top 10% of most variant genes in the WNT3A and WNT5A datasets. Subsequently, an unpaired two-tailed t-test analysis was used to identify genes that were significantly different between the most variant genes in the WNT3A and WNT5A replicate samples, using an arbitrary p-value of p<0.04 as a cut-off. The rationale for further comparing the regulated genes in WNT3A cells to those in WNT5A cells was based on the finding that WNT5A did not have significant phenotypic effects (pigmentation, proliferation or cell cycle), and this subsequent comparison allowed identification of potentially important genes regulated by WNT3A that might be missed by setting arbitrary cut-off values for significant genes (i.e. 2-fold upregulated or 50% downregulated).

Example 10

High Throughput Small Molecule Screen

Compounds were dissolved in dimethylsulphoxide (DMSO). For the primary screen, performed in duplicate, HT22 cells stably expressing the beta-catenin activated reporter (BAR) were cultured in growth medium (DMEM/10% FBS/1% antibiotic). 3000 cells per well were transferred to 384-well clear bottom plates (Nalgene Nunc; Rochester, N.Y.) in 30 μL of growth medium. The following day, 100 nL of compound and 10 μL of either growth media or WNT3A conditioned media (E.C.₅₀ dose) was transferred to the cells. The next day each well was imaged using transmitted light with the ImageXpress Micro (Molecular devices; Sunnyvale, Calif.) followed by the addition of 10 μL of Steady-Glo (Promega; Madison, Wis.) as per the manufacture's instructions, and luminescence measurement on an EnVision Multilabel plate reader (PerkinElmer; Waltham, Mass.). Viability was scored by analyzing the ImageXpress images. As described in detail in Seiler et al. (Seiler et al., Nucleic Acids Res. 36:D351 (2008), which is hereby incorporated by reference in its entirety), each compound well received an algebraically signed Z-score corresponding to the number of standard deviations it fell above or below the mean of a well-defined mock-treatment distribution of DMSO controls. Z-score normalized data from the growth media stimulus group were sorted by average percent change. The fold-increase over the background of DMSO controls for each treatment was also calculated. Top compounds with the greatest percent change of activity with the growth media were then resorted based on the percent change with the WNT3A stimulus.

Biological activity of the screened compounds are set forth in Table 4. These compounds represent FDA approved compounds and drugs or known bioactive molecules. RKO (human colorectal carcinoma red line) and HT22 (mouse hyppocampal line) cell lines were screened. The lines were tested without a stimulus, in presence of an E.C.₅₀ dose of Wnt 3A condition media and in presence of a subthreshold dose of LiCl. Results of the screening are set forth below.

TABLE 4
Biological Activity of the High Throughput Small Molecules Screen.

Example 11

Nuclear β-Catenin Correlates with Improved Patient Survival

Using the expression of nuclear β-catenin as a clinical surrogate marker for Wnt/β-catenin activation (Bachmann et al., Clin. Cancer Res. 11:8606 (2005); T. Kageshita et al., Br. J. Dermatol. 145:210 (2001); Maelandsmo et al., Clin. Cancer Res. 9:3383 (2003), which are hereby incorporated by reference in their entirety), a tumor microarray composed of 343 cores (118 primary tumors, 225 metastases) from patient tumors (Camp et al., Nat. Med. 8:1323 (2002), which is hereby incorporated by reference in its entirety) was scored. Survival probabilities for patients were estimated using Kaplan-Meier analysis after stratifying primary tumors into tertiles based on nuclear β-catenin expression (FIG. 5). This analysis reveals that higher expression of nuclear β-catenin in both primary tumors (FIG. 1A) and in metastases and recurrences (FIG. 1B) predicts significantly increased patient survival. Also, levels of nuclear β-catenin are lower in metastases and recurrences compared to primary tumors (FIG. 5). These findings confirm and extend previous reports of improved prognosis with elevated nuclear β-catenin in melanoma (Bachmann et al., Clin. Cancer Res. 11:8606 (2005); T. Kageshita et al., Br. J. Dermatol. 145:210 (2001); Maelandsmo et al., Clin. Cancer Res. 9:3383 (2003), which are hereby incorporated by reference in their entirety).

Example 12

Nuclear β-Catenin is Negatively Correlated with Proliferation

As tumor depth measurements (Breslow thickness) were obtained for 113 primary tumors in the array cohort, this sub-group of patients was analyzed based on the Breslow thickness stratification used as reported (Thompson, J. A., Semin. Oncol. 29:361 (2002), which is hereby incorporated by reference in its entirety). Increasing tumor depth is correlated with a lower probability of survival (FIG. 1C) and with a higher degree of proliferation, which is measured by the percentage of cells expressing Ki-67 (FIG. 1D). By contrast, nuclear β-catenin levels are highest for shallow tumors (T1) and decrease significantly with increased tumor depth (FIG. 1E).

The percentage of tumors staining positive is then analyzed for the cellular proliferative marker Ki-67 (% Ki-67). Strikingly, distribution histograms of % Ki-67 staining in primary tumors stratified by expression of nuclear β-catenin show a statistically significant shift towards increased proliferation (elevated % Ki-67 staining) in the groups with lower nuclear β-catenin (FIG. 1F). It is shown that there is no correlation between expression of α-catenin and % Ki-67 staining, and PCNA is used as an independent marker of proliferation (FIG. 5). Taken together these data demonstrate that elevated nuclear β-catenin is negatively associated with proliferation as measured by either tumor size/depth, or by the markers Ki-67 and PCNA.

Example 13

-Activation of Wnt/β-Catenin Signaling Changes Melanoma Cell Fate

Wnts, which can activate or antagonize β-catenin signaling, were investigated in order to elicite changes in melanoma cells cultured in vitro that might be consistent with the above clinical data. Since melanoma tumors appear to express WNT3A (FIG. 6), which has a pivotal role in the regulation of melanocyte biology (Dorsky et al., Genes Dev. 14:158 (2000); Fang et al., Stem Cells 24:1668 (2006), which are hereby incorporated by reference in their entirety), and they express WNT5A, which is elevated in melanoma metastases (Bittner et al., Nature 406:536 (2000); Weeraratna et al., Oncogene 23:2264 (2004), which are hereby incorporated by reference in their entirety), B16 mouse melanoma cells were transduced with lentivirus constructs encoding WNT3A, WNT5A, or a GFP control.

B16:WNT3A cells exhibit strikingly increased pigmentation compared to GFP or WNT5A cells (FIG. 2A). Scoring cells for nuclear accumulation of β-catenin revealed that only cells expressing WNT3A, and not WNT5A or GFP, exhibit elevated β-catenin (FIG. 2C). As a positive control, it was shown that conditioned media (CM) from B16 cells expressing WNT3A activates a β-catenin-responsive reporter in UACC1273 melanoma cells (FIG. 2D), confirming that these cells were secreting active WNT3A. Also, it was shown that B16 cells expressing WNT3A exhibit marked increases in expression of the β-catenin target gene Axin2 (Jho et al., Mol. Cell. Biol. 22:1172 (2002), which is hereby incorporated by reference in its entirety) compared to B16:GFP cells (FIG. 2E).

In vitro cell proliferation studies using the MTT cell proliferation assay showed that B16 cells expressing WNT3A exhibit decreased proliferation compared to cells expressing GFP or WNT5A (FIG. 2F). This finding was paralleled in human cell lines (FIG. 6). Cell cycle profiles were then compared to the Wnt-transduced melanoma cell lines, and found that cells expressing WNT3A exhibit an increased population in G1, with a decreased population in S phase, compared to control cells (FIG. 2G). Together, these data suggest that WNT3A can induce differentiation of the melanoma cells to a cell fate that is more pigmented and less proliferative.

Example 14

Elevation of Melanocyte Differentiation Markers by WNT3A

Next, a genome-wide transcriptional profiling was performed to gain further insights into the consequences of expression of WNT3A and WNT5A, which revealed that levels of transcripts elevated by WNT3A were actually reduced by WNT5A (FIG. 3B). Among the most highly significant genes elevated by WNT3A (FIG. 3A) are Axin2 (Jho et al., Mol. Cell. Biol. 22:1172 (2002), which is hereby incorporated by reference in its entirety) and Tcf7 (Roose et al., Science 285:1923 (1999), which is hereby incorporated by reference in its entirety), which are direct targets of Wnt/β-catenin signaling; Mme and Mlze, downregulated genes previously linked to melanoma progression (Watabe et al., Jpn. J. Cancer Res. 92:140 (2001); Bilalovic et al., Mod. Pathol. 17:1251 (2004), which are hereby incorporated by reference in their entirety); Mitf, linked to pigment cell fate, and Trpm1, Met, Sox9 and Kit, which are highly expressed during melanocyte and neural crest development (Loftus et al., Proc. Natl. Acad. Sci. USA 96:9277 (1999, which is hereby incorporated by reference in its entirety)). To confirm the array data levels of selected transcripts were measured by quantitative PCR (FIG. 3B). To establish that the effects of WNT3A on gene expression were specific, it was demonstrated that the changes in gene expression were antagonized by β-catenin siRNA (FIG. 3C). The transcriptional profiling thus supports the conclusion, evident from visual examination of cells (FIG. 2A), that WNT3A promotes melanoma cells adopting characteristics of melanocyte differentiation.

Example 15

WNT3A Reduces Melanoma Tumor Size and Metastasis in Mice

While expression of Trpm1 was elevated by WNT3A (FIG. 3B), its expression is usually reduced during melanoma progression. Taken with the observed changes in cell fate and proliferation seen in cells expressing WNT3A, this led to the prediction that cells expressing WNT3A would form less proliferative and less aggressive tumors in vivo. Indeed, implantation of WNT3A-transduced B16 cells into the footpads of C57BL/6 mice, significantly decreased tumor growth compared to B16 cells transduced with GFP or WNT5A (FIG. 3D) and decreased metastases to popliteal lymph nodes (FIG. 3E).

Example 16

A High-Throughput Screen for Therapeutic Activators of Wnt/β-Catenin Signaling

In support of the hypothesis that activation of Wnt/β-catenin inhibits melanoma growth, treatment of B16 cells with the GSK3 inhibitors lithium chloride (LiCl) or 6-bromoindirubin-3′-oxime (BIO) also resulted in decreased proliferation of cultured cells (FIG. 7). Precluding further consideration of either compound in murine therapeutic trials it was found that lithium and BIO exhibit several obstacles relating to both toxicity and, in the case of lithium, to difficulty maintaining adequate serum levels in mice. Consequently, identifying novel activators of Wnt/β-catenin signaling that would have improved therapeutic efficacy and tolerance became a target.

A high-throughput screen of >60% of the FDA-approved panel of biologically active small molecules was performed using a Wnt-responsive luciferase reporter system to identify compounds that could either activate Wnt/β-catenin signaling on their own, or synergize with WNT3A to enhance reporter activation (FIG. 4).

This screen identified the drug riluzole, an aminobenzothiazole, which is FDA-approved for treatment of amyotrophic lateral sclerosis, as a promising candidate drug for activating Wnt/β-catenin signaling (FIG. 4A). Riluzole was chosen because it was represented by two distinct formulations within the top 6 compounds that activated Wnt/β-catenin signaling on their own as well as in synergy with WNT3A. During the secondary validation, riluzole was confirmed as an activator of Wnt/β-catenin signaling in cells expressing a luciferase-based reporter under control of the Axin2 promoter, where activation was seen with riluzole alone and in combination with WNT3A (FIG. 4B). Riluzole could activate endogenous gene targets in the B16 melanoma cells in synergy with WNT3A (FIG. 4C) and on its own (FIG. 4D).

Example 17

Riluzole Mimics WNT3A and Inhibits Melanoma Metastases In Vivo

Similar to WNT3A, activation of Wnt/β-catenin signaling by riluzole correlates with increased pigmentation (FIG. 4E) and decreased proliferation in cultured B16 cells (FIG. 4F). Riluzole was then tested for inhibition of melanoma progression in vivo using B16 tumor explants, as predicted for a compound that activates β-catenin signaling. Striking reductions in metastases were observed in riluzole-treated mice, as measured by the detection of cells in the sentinel popliteal lymph node bed (FIG. 4H). In related experiments, lung metastasis was tested, and again there was a reduction in mice treated with riluzole, (3 out of 9 control mice vs. 0 out of 9 mice treated with riluzole). While previous studies found that riluzole could decrease proliferation of human melanoma xenografts in a mouse model (Namkoong et al., Cancer Res. 67:2298 (2007), which is hereby incorporated by reference in its entirety), no appreciable difference in tumor growth was observable using the single dose of riluzole tested (FIG. 4G). Together, these data provide proof-of-principle for therapeutic activation of the Wnt/β-catenin pathway to treat metastatic melanoma, based on a mechanism whereby β-catenin signaling leads to changes in the differentiation of melanoma cells.

Data in support of a novel “differentiation therapy” for treating melanoma with agents that activate β-catenin signaling are presented in FIG. 9. Mechanistically, it is proposed that activating Wnt/β-catenin signaling forces melanoma cells to adopt a more differentiated cell fate, resembling melanocytes, which are intrinsically less motile, less proliferative, and thus less deadly. The concept that different states of differentiation and pluripotency exist in melanoma is not new, and in fact recent research has focused on markers that may better identify so-called melanoma stem cells, or tumor initiating cells (Zabierowski et al., Cancer Cell 13:185 (2008); Schatton et al., Nature 451:345 (2008); Grichnik et al., J. Invest. Dermatol. 126:142 (2006), which are hereby incorporated by reference in their entirety). The forced differentiation of these tumor initiating populations into cell fates that are either more benign (i.e. slower growing, or less metastatic) or more treatable provides a new approach that could prove beneficial in concert with current therapies based on cytotoxicity or immunomodulation.

Importantly, in a high throughput cell-based reporter screen riluzole was identified as an FDA-approved compound that has the heretofore unexpected ability to activate Wnt/β-catenin signaling. In light of data showing that riluzole also reduces metastasis in mice like WNT3A, and in light of independent data that riluzole inhibits the growth of human melanoma xenografts in mice (Namkoong et al., Cancer Res. 67:2298 (2007), which is hereby incorporated by reference in its entirety), it can be strongly argued that riluzole should be evaluated further as a therapy for melanoma patients with metastatic disease. More broadly, there are other cancers where conventional treatments have also been disappointing, raising the question of whether the modulation of signaling pathways to manipulate cell fate might prove therapeutic in these cancers as well.

For the activation of β-catenin signaling to be considered as a therapy one would need reasonable assurance that enhancing Wnt/β-catenin signaling in melanoma will not have the undesirable consequence of promoting proliferation. In support of the present data, a recent study found that expression of a stabilized β-catenin mutant (β-cat^STA) in mice did not increase proliferation of melanocytic cells, which is entirely consistent with our findings (Delmas et al., Genes Dev. 21:2923 (2007), which is hereby incorporated by reference in its entirety). This study also found that restricting the expression of β-cat^STA to melanocytes did not lead to any melanomas over a 2-year period (Delmas et al., Genes Dev. 21:2923 (2007), which is hereby incorporated by reference in its entirety). Additionally, other published reports (Ballin et al., Br. J. Cancer 48:83 (1983); Penso et al., Mol. Genet. Metab. 78:74 (2003); Kang et al., Arch. Dermatol. Res. 294:426 (2002), which are hereby incorporated by reference in their entirety) confirm the observed inhibition of melanoma cell proliferation upon treatment with lithium chloride, which is a pharmacologic activator of Wnt/β-catenin signaling. Available data therefore strongly suggest that activation of Wnt/β-catenin signaling is not by itself oncogenic in the context of melanoma.

It has been shown that both WNT3A and riluzole promote pigmentation of melanoma cells in vitro. This observation led to the idea of using these activators of Wnt/β-catenin signaling to force the differentiation of melanoma cells or melanoma cancer stem cells. Supporting the notion that melanoma cells can be directed to adopt characteristics of differentiation, it was demonstrated that B16 cells expressing WNT3A or treated with riluzole express elevated levels of markers of melanocyte differentiation.

Expression of WNT3A or treatment with riluzole also leads to elevation in levels of melastatin (Trpm1) and Kit transcripts, confirming a prior report that these genes are elevated by treating human melanoma cells with recombinant WNT3A (Shah et al., J. Invest. Dermatol. in Press (2008), which is hereby incorporated by reference in its entirety). The loss of expression of TRPM1 is known to correlate with poor prognosis and with progression to metastasis (Duncan et al., J. Clin. Oncol. 19:568 (2001); Duncan et al., Cancer Res. 58:1515 (1998), which are hereby incorporated by reference in their entirety), so its elevation by WNT3A further supports the proposed “differentiation therapy”. The observed elevation of Kit by WNT3A is also extremely interesting given that KIT can be pharmacologically targeted by the kinase inhibitors imatinib (Gleevec) and sunitinib (Sutent) (Grimaldi et al., Ann. Oncol. 18(Suppl. 6 vi):31 (2007), which is hereby incorporated by reference in its entirety). Imatinib is already being tested as a melanoma therapy in clinical trial, but to date has not shown significant promise so far for melanoma (Wyman et al., Cancer 106:2005 (2006); Ugurel et al., Br. J. Cancer 92:1398 (2005); Hodi et al., J. Clin. Oncol. 26:2046 (2008), which are hereby incorporated by reference in their entirety). Herein, it is suggested that future studies should consider a combination therapy in which activation of Wnt/β-catenin signaling forces higher expression of Kit, which may increase their sensitivity to imatinib or sunitinib. Given that current therapeutic strategies have proven largely ineffective for metastatic melanoma, a “differentiation therapy” involving riluzole or other activators of β-catenin signaling, used as monotherapy or in combination therapy, may provide a new alternative for treating this disease.

Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Claims

1. A method of treating a subject for a condition mediated by aberramt Wnt/β-catenin signaling, said method comprising: selecting a subject with a condition mediated by aberrant Wnt/β-catenin signaling andadministering to the selected subject at least one compound selected from the group consisting of those set forth in Table 1, Table 2, Table 3, and a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the subject is human.

3. The method of claim 1, wherein at least one compound of Table 1 or a pharmaceutically acceptable salt thereof is administered.

4. The method of claim 1, wherein at least one compound of Table 2 or a pharmaceutically acceptable salt thereof is administered.

5. The method of claim 1, wherein at least one compound of Table 3 or a pharmaceutically acceptable salt thereof is administered.

6. The method of claim 1, wherein the compound is selected from the group consisting of flunarizine, alendronate, SNDX-275, vorinostat, isotretinoin, etoposide, virginiamycin, amoxapine, riluzole, mercaptopurine, milrinone, clofazimine, melphalan, and thioguanine.

7. The method of claim 1, wherein the compound activates the Wnt/β-catenin pathway.

8. The method of claim 1, wherein the compound inhibits the Wnt/β-catenin pathway.

9. The method of claim 1, wherein the condition is selected from the group consisting of cancer, bone mass diseases, fracture repair, FEVR, diabetes mellitus, cord blood transplants, psychiatric disease, neurodegenerative disease, hair loss, diseases linked to loss of stem/progenitor cells, conditions improved by increasing stem/progenitor cell populations, HIV, and tooth agenesis.

10. The method of claim 9, wherein the condition is cancer selected from the group consisting of malignant melanoma, colorectal cancer, renal, liver, lung, breast, prostate, ovarian, parathyroid, leukemias, glioma, neuroblastoma, and astrocytoma.

11. The method of claim 9, wherein the condition is a psychiatric disease in the form of bipolar depression.

12. The method of claim 9, wherein the condition is a neurodegenerative disease selected from the group consisting of Alzheimer's Disease and ALS.

13. A method of modulating the Wnt/β-catenin pathway in a subject comprising: selecting a subject in need of Wnt/β-catenin pathway modulating andadministering to the selected subject at least one compound selected from the group consisting of those set forth in Table 1, Table 2, Table 3, and a pharmaceutically acceptable salt thereof.

14. The method of claim 13, wherein said compound activates the Wnt/β-catenin pathway.

15. The method of claim 13, wherein said compound inhibits the Wnt/β-catenin pathway.

16. The method of claim 13, wherein at least one compound of Table 1 or a pharmaceutically acceptable salt thereof is administered.

17. The method of claim 13, wherein at least one compound of Table 2 or a pharmaceutically acceptable salt thereof is administered.

18. The method of claim 13, wherein at least one compound of Table 3 or a pharmaceutically acceptable salt thereof is administered.

19. The method of claim 13, wherein the compound is selected from the group consisting of flunarizine, alendronate, SNDX-275, vorinostat, isotretinoin, etoposide, virginiamycin, amoxapine, riluzole, mercaptopurine, milrinone, clofazimine, melphalan, and thioguanine.