Inhibitors of atypical protein kinase c and their use in treating hedgehog pathway-dependent cancers

Abstract

To provide methods for treating hedgehog pathway-dependent cancers, where aspects of the methods include inhibition of hedgehog pathway-dependent cancer growth, proliferation or metastasis that is promoted by hedgehog pathway signaling.SOLUTION: The invention provides a method of treating a hedgehog pathway-dependent cancer using an inhibitor of atypical protein kinase C iota. The method comprises administering to the subject a therapeutically effective amount of a composition comprising CRT0422839 or CRT0364436 or a pharmaceutically acceptable salt thereof.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Contract AR054780 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

[0002] FIELD OF THEINVENTION The present invention relates to therapeutic methods for treating hedgehog pathway dependent cancers and disorders. In particular, the present invention relates to methods of treating hedgehog pathway dependent cancers and disorders using inhibitors of atypical protein kinase C (aPKC) iota. [Background technology]

[0003] 2. Background of the Invention The Hedgehog (Hh) signaling pathway plays a key role in development and tumorigenesis in all metazoa. Three Hh genes have been identified in mammals: Sonic hedgehog (SHh), Desert hedgehog (DHh), and Indian hedgehog (IHh). These proteins are secreted proteins that act in opposition to the receptor Patched (Ptch1 or Ptch2 in humans). Ptch acts, in part, by antagonizing the activity of Smoothened (Smo), a G protein-coupled receptor that activates the transcription factor Gli. Binding of Shh to Ptch relieves Ptch-mediated inhibition of Smo, allowing Smo to promote Gli-dependent transcription. During development, Hh-induced Smo activity promotes proliferation, migration, and differentiation of progenitor cells to pattern organ development. However, dysregulation of Hh pathway signaling, for example by inactivating mutations in Ptch or activating mutations in Smo, is associated with cancer (Toftgard, R. Hedgehog signaling in cancer. Cell Mol. Life Sci., 57: 1720-1731 (2000)). Induction of Hh target genes is required for tumor growth and maintenance in tumor epithelium, and Hh pathway signaling is involved in tumor metastasis of many epithelial tumors. For example, high levels of Hh pathway signaling are required for the development and spread of basal cell carcinoma (BCC).

[0004] There is a need for better methods for treating cancers and disorders associated with the Hh pathway. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Toftgard, R. Hedgehog signaling in cancer. Cell Mol. Life Sci., 57: 1720-1731 (2000) Summary of the Invention

[0006] Provided is a method for treating Hedgehog pathway-dependent cancer.Aspects of the method include inhibiting the growth, proliferation and / or metastasis of Hedgehog pathway-dependent cancer that is promoted by Hedgehog pathway signaling.In particular, disclosed is a method for treating Hedgehog pathway-dependent cancer with an inhibitor of aPKC iota.

[0007] In one aspect, a method of treating a subject with a Hedgehog pathway dependent cancer is provided, the method comprising administering to the subject a composition comprising a therapeutically effective amount of CRT0422839 or CRT0364436, or a pharma- ceutically acceptable salt thereof.

[0008] In certain embodiments, the cancer comprises a constitutively active Hedgehog pathway. In one embodiment, the cancer is basal cell carcinoma (BCC). In some embodiments, the cancer is metastatic.

[0009] Multiple cycles of treatment can be administered to the subject over a period of time.For example, treatment is administered to the subject for at least 3 months, at least 6 months, at least 9 months, at least 12 months, or longer.Preferably, multiple cycles of treatment are administered to the subject for a period of time sufficient to cause at least partial tumor response, more preferably complete tumor response.In certain embodiments, the composition comprising CRT0422839 or CRT0364436 or its pharmacologic acceptable salt is administered according to a daily dosing regimen or intermittently.

[0010] In certain aspects, the method further comprises administering an additional anti-cancer therapy, such as, but not limited to, surgery, chemotherapy, radiation therapy, immunotherapy, biological therapy, or a combination thereof.

[0011] In certain embodiments, the composition to be administered further comprises a pharma- ceutically acceptable excipient.

[0012] In certain embodiments, the method further comprises administering a histone deacetylase (HDAC) inhibitor in combination with CRT0422839 or CRT0364436 or its pharmaceutically acceptable salt.Exemplary HDAC inhibitors include hydroxamic acids, such as vorinostat, belinostat, panobinostat, gibinostat, dacinostat (LAQ824), trichostatin A; sesquiterpene lactones, such as parthenolide; cyclic tetrapeptides, such as trapoxin B; depsipeptides, such as romidepsin; and benzamides, such as entinostat (MS-275), tacedinaline (CI994), mocetinostat, etc. In one embodiment, the HDAC inhibitor is vorinostat.

[0013] In certain embodiments, the subject is a mammal, such as a human or non-human primate, a rodent, a farm animal, or a pet.

[0014] In certain embodiments, CRT0422839 or CRT0364436, or a pharma- ceutically acceptable salt thereof, is administered in an amount sufficient to reduce the viability of Hedgehog pathway-dependent cancerous cells in the subject.

[0015] In certain embodiments, CRT0422839 or CRT0364436, or a pharma- ceutically acceptable salt thereof, is administered in an amount sufficient to reduce production of Gli1 mRNA in hedgehog pathway-dependent cancerous cells in the subject.

[0016] In certain embodiments, CRT0422839 or CRT0364436, or a pharma- ceutically acceptable salt thereof, is administered in an amount sufficient to reduce Hedgehog pathway-dependent cancerous cell growth and cell proliferation in a subject.

[0017] In another aspect, a method of inhibiting the growth or proliferation of a Hedgehog pathway-dependent cancerous cell is provided, the method comprising contacting the Hedgehog pathway-dependent cancerous cell with an effective amount of CRT0422839 or CRT0364436, or a pharma- ceutical acceptable salt thereof.

[0018] In certain embodiments, the Hedgehog pathway dependent cancerous cells are BCC cells.

[0019] In certain aspects, the hedgehog pathway dependent cancerous cells contain a constitutively active hedgehog pathway.

[0020] In certain aspects, the Hedgehog pathway dependent cancerous cells are in vivo or in vitro.

[0021] In certain aspects, the Hedgehog pathway-dependent cancerous cell is a cancerous cell of a mammal (eg, a human or non-human primate, a rodent, a livestock animal, or a pet).

[0022] In certain embodiments, the method further comprises contacting the Hedgehog pathway dependent cancerous cell with an HDAC inhibitor.

[0023] In another aspect, a composition is provided that comprises CRT0422839 or CRT0364436 or a pharma- ceutically acceptable salt thereof for use in treating Hedgehog pathway dependent cancer.In some embodiments, the composition further comprises a HDAC inhibitor.In one embodiment, the HDAC inhibitor is vorinostat.

[0024] In another aspect, a composition is provided that comprises CRT0422839 or CRT0364436 or a pharma- ceutically acceptable salt thereof for use in treating basal cell carcinoma.In some embodiments, the composition further comprises a HDAC inhibitor.In one embodiment, the HDAC inhibitor is vorinostat. [The present invention 1001] A method for treating a subject with a hedgehog pathway dependent cancer, comprising administering to the subject a composition comprising a therapeutically effective amount of CRT0422839 or CRT0364436, or a pharma- ceutically acceptable salt thereof. [The present invention 1002] The method of claim 1001, wherein the cancer is basal cell carcinoma (BCC). [The present invention 1003] The method of any one of claims 1001 to 1002, wherein said cancer comprises a constitutively active Hedgehog pathway. [The present invention 1004] The method according to any one of claims 1001 to 1003, wherein the cancer is metastatic. [The present invention 1005] The method of any of claims 1001-1004, wherein multiple cycles of treatment are administered to the subject for a period of time sufficient to result in at least a partial tumor response. [The present invention 1006] The method of claim 1005, wherein said period is at least 6 months. [The present invention 1007] The method of claim 1006, wherein said period is at least 12 months. [The present invention 1008] The method of any of claims 1005-1007, wherein multiple cycles of treatment are administered to the subject for a period of time sufficient to result in a complete tumor response. [The present invention 1009] The method of any of claims 1005 to 1008, wherein the composition is administered according to a daily dosing regimen or intermittently. [The present invention 1010] The method of any one of claims 1001 to 1009, further comprising the step of administering an additional anticancer therapy. [The present invention 1011] The method of claim 1010, wherein said additional anti-cancer therapy is surgery, chemotherapy, radiation therapy, immunotherapy, biological therapy, or a combination thereof. [The present invention 1012] The method of any one of claims 1001 to 1011, wherein the composition further comprises a pharma- ceutically acceptable additive. [The present invention 1013] The method of any of claims 1001 to 1012, further comprising the step of administering a histone deacetylase (HDAC) inhibitor in combination with CRT0422839 or CRT0364436 or a pharma- ceutically acceptable salt thereof. [The present invention 1014] The method of claim 1013, wherein said HDAC inhibitor is vorinostat. [The present invention 1015] The method according to any one of claims 1001 to 1014, wherein the subject is a mammal. [The present invention 1016] The method of claim 10, wherein the subject is a human. [The present invention 1017] The method of any of claims 1001 to 1016, wherein CRT0422839 or CRT0364436, or a pharma- ceutically acceptable salt thereof, is administered in an amount sufficient to reduce the viability of Hedgehog pathway-dependent cancerous cells in the subject. [The present invention 1018] Any of the methods of claims 1001 to 1017, wherein CRT0422839 or CRT0364436, or a pharma- ceutically acceptable salt thereof, is administered in an amount sufficient to reduce production of Gli1 mRNA in Hedgehog pathway-dependent cancerous cells in the subject. [The present invention 1019] The method of any of claims 1001-1018, wherein CRT0422839 or CRT0364436, or a pharma- ceutically acceptable salt thereof, is administered in an amount sufficient to reduce the growth and cell proliferation of Hedgehog pathway-dependent cancerous cells in the subject. [The present invention 1020] A method for inhibiting the growth or proliferation of hedgehog pathway-dependent cancerous cells, comprising contacting the hedgehog pathway-dependent cancerous cells with an effective amount of CRT0422839 or CRT0364436, or a pharma- ceutically acceptable salt thereof. [The present invention 1021] The method of claim 1020, wherein said Hedgehog pathway dependent cancerous cells are basal cell carcinoma (BCC) cells. [The present invention 1022] The method of any one of claims 1020 to 1021, wherein said hedgehog pathway dependent cancerous cells comprise a constitutively active hedgehog pathway. [The present invention 1023] The method of any one of claims 1020 to 1022, wherein the Hedgehog pathway dependent cancerous cell is in vivo or in vitro. [The present invention 1024] The method according to any one of claims 1020 to 1023, wherein the Hedgehog pathway dependent cancerous cells are human cancerous cells. [The present invention 1025] The method of any of claims 1020 to 1024, further comprising contacting the Hedgehog pathway dependent cancer cell with a deacetylase (HDAC) inhibitor. [The present invention 1026] The method of claim 10, wherein said HDAC inhibitor is vorinostat. [The present invention 1027] A composition comprising CRT0422839 or CRT0364436, or a pharma- ceutically acceptable salt thereof, for use in treating a hedgehog pathway dependent cancer. [The present invention 1028] The composition of the present invention 1027, wherein the cancer is basal cell carcinoma (BCC). [The present invention 1029] The composition of any one of claims 1027 to 1028, further comprising a histone deacetylase (HDAC) inhibitor. [The present invention 1030] The composition of the present invention, wherein said HDAC inhibitor is vorinostat. [Brief description of the drawings]

[0025] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings, in which: It is emphasized that, according to common practice, the various features of the drawings are not to scale. On the contrary, dimensions of the various features have been arbitrarily expanded or reduced for clarity. The drawings include the following figures: [Figure 1A]We show that atypical protein kinase iota (aPKCι) inhibitors modulate BCC cell viability. The effects of compounds CRT0422839 and CRT0364436 on BCC viability were tested in a mouse BCC cell line (BSC1) and compared to results using a known peptide inhibitor, PSI, previously described in Mirza et al. (JCI Insight (2017) 2(21):e97071). Shown is the viability of BSCI cells following treatment with the aPKC inhibitors PSI (Figure 1A), CRT0329868 (Figure 1B), CRT0364436 (Figure 1C), or CRT0422839 (Figure 1D) at concentrations of 1 μm and 10 μm. [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 1D] See legend to Figure 1A. [Diagram 2] Figure 2 shows the effect of treatment of a mouse BCC cell line (BSC1) with PSI, CRT0329868, CRT0422839, or CRT0364436 on the expression levels of Gli 1 mRNA, a marker of Shh pathway output, which was measured to further demonstrate inhibition of BCC growth and Shh pathway signaling. Results are shown after treatment with 1 μM and 10 μM concentrations of the compounds for 6 hours (Figure 2A) or 24 hours (Figure 2B). [Figure 3A] The chemical structure and properties of the aPKCι inhibitor CRT0364436 / TEV-44229 are shown. [Figure 3B] The chemical structure and properties of the aPKCι inhibitor CRT0422839 / TEV-47448 are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Detailed Description of the Invention Before the present methods and compositions are described, it is to be understood that this invention is not limited to the particular methods or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, and the scope of the present invention will be limited only by the appended claims.

[0027] Where a range of numerical values ​​is provided, it is understood that each intervening value, to the nearest tenth of the lower limit, between the upper and lower limits of that range, unless the context clearly dictates otherwise, is also specifically disclosed. Each smaller range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may be independently included or excluded in the range; each range in which either or both of the limits in the smaller range are included or not included in the range is also encompassed within the invention, subject to the specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the invention.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Although any method and material similar or equivalent to those described herein can be used to carry out or test this invention, some possible and preferred methods and materials are described here.All publications mentioned herein are incorporated by reference to disclose and describe the method and / or material in relation to which the publication is cited.It is understood that this disclosure takes precedence over any disclosure of the incorporated publication to the extent that there is a discrepancy.

[0029] As will be apparent to one of skill in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has distinct components and features which may be readily separated from, or combined with, the features of any of the other aspects without departing from the scope or spirit of the invention. A recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0030] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells, a reference to "the inhibitor" includes a reference to one or more inhibitors and equivalents thereof known to those skilled in the art, e.g., compounds or drugs, and so forth.

[0031] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0032] The term "about," particularly with respect to a given amount, is meant to encompass a deviation of plus or minus 5%.

[0033] The terms "tumor", "cancer" and "neoplasm" are used interchangeably and refer to a cell or cell population whose growth, proliferation or survival is greater than that of a corresponding normal cell, e.g., a cell proliferation disorder, a hyperproliferative disorder or a differentiation disorder. Generally, the growth is uncontrolled. The term "malignant" refers to invasion into nearby tissues. The term "metastasis" or secondary, recurrent or recurrent tumor, cancer or neoplasm refers to the spread or dissemination of a tumor, cancer or neoplasm to another site, location or region within a subject, which site, location or region is different from the primary tumor or cancer. Neoplasms, tumors and cancers include benign, malignant, metastatic and non-metastatic types, and include neoplasm, tumor or cancer stage (I, II, III, IV or V) or grade (G1, G2, G3, etc.), or neoplasm, tumor, cancer or metastasis that is progressing, worsening, stabilized or in remission. In particular, the terms "tumor," "cancer," and "neoplasm" include carcinomas such as squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, and small cell carcinoma.

[0034] The term "hedgehog pathway dependent cancer" includes cancers that depend on activation of the hedgehog pathway or that are associated with aberrant activation of the hedgehog pathway, including, but not limited to, basal cell carcinoma, medulloblastoma, rhabdomyosarcoma, small cell lung cancer, retinoblastoma, gastric and upper gastrointestinal cancer, osteosarcoma, pancreatic cancer, breast cancer, colon cancer, ovarian cancer, brain cancer, mammary gland cancer, thyroid cancer, and prostate cancer.

[0035] By "antitumor activity" is intended the slowing of cell proliferation, and therefore the slowing of the growth rate of existing tumors or tumors that develop during treatment, and / or the destruction of existing neoplastic (tumor) cells or newly formed neoplastic cells, and therefore the overall size of tumors during treatment. Such activity can be evaluated using animal models, such as xenograft models of human renal cell carcinoma. For example, see Pulkkanen et al., In Vivo (2000) 14:393-400 and Everitt et al., Toxicol. Lett. (1995) 82-83:621-625 for descriptions of animal models.

[0036] By "therapeutically effective dose or amount" of an aPKC iota inhibitor (e.g., CRT0422839 or CRT0364436) is intended an amount that results in a positive therapeutic response, such as anti-tumor activity, when the aPKC iota inhibitor is administered, or when a histone deacetylase inhibitor (e.g., vorinostat) is further administered in combination with the aPKC iota inhibitor as described herein. Additionally, an "effective amount" of an aPKC iota inhibitor can inhibit the growth, proliferation, and / or metastasis of Hedgehog pathway-dependent cancerous cells, and / or reduce the production of Gli 1 mRNA in Hedgehog pathway-dependent cancerous cells, and / or reduce the viability of Hedgehog pathway-dependent cancerous cells.

[0037] The term "tumor response" as used herein means the reduction or disappearance of all measurable lesions. The criteria for tumor response are based on WHO reporting criteria [WHO Offset Publication, 48-World Health Organization, Geneva, Switzerland, (1979)]. Ideally, all lesions that are measurable in one or two dimensions should be measured at each evaluation. In the presence of multiple lesions in an organ, such measurements may not be possible, and in such situations, up to six representative lesions should be selected, if possible.

[0038] As used herein, the term "complete response" (CR) means the complete disappearance of all clinically detectable malignancy as determined by two assessments at least four weeks apart.

[0039] As used herein, the term "partial response" (PR) means a 50% or greater reduction from baseline in the sum of the products of the longest perpendicular diameters of all measurable disease without evaluable disease progression and without evidence of new lesions, as determined by at least two consecutive assessments at least 4 weeks apart. Assessments must demonstrate a partial reduction in the size of lytic lesions, remineralization of lytic lesions, or a reduction in density of blastic lesions. Transient inflammation at sites of metastatic disease is not uncommon. Individual lesions that appear to increase in size do not necessarily disqualify a PR unless the increase is documented on two consecutive measurements taken at least 28 days apart.

[0040] A "pharmaceutically acceptable excipient or carrier" refers to an optional excipient that may be included in a composition that does not cause significant adverse toxicological effects in a patient.

[0041] "Pharmaceutically acceptable salt" includes, but is not limited to, amino acid salts, salts prepared with inorganic acids, such as chloride salts, sulfate salts, phosphate salts, diphosphate salts, bromides, and nitrates, or salts prepared from any of the corresponding inorganic acid forms, such as hydrochlorides, or salts prepared with organic acids, such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, paratoluenesulfonate, pamoate, salicylate, and stearate, as well as estolate, gluceptate, and lactobionate.Similarly, salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).

[0042] As used herein, the terms "treatment", "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in the sense of preventing, completely or partially, a disease or its symptoms, and / or therapeutic, in the sense of curing, partially or completely, a disease and / or adverse effects resulting from the disease. "Treatment", as used herein, covers any treatment of a disease in a mammal, particularly a human, and includes: (a) increasing survival time; (b) reducing the risk of death from the disease; (c) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (d) inhibiting the disease, i.e., arresting its onset / progression (e.g., slowing the rate of disease progression), and (e) palliating the disease, i.e., causing regression of the disease.

[0043] "Substantially purified" generally refers to the isolation of a substance (e.g., a compound, molecule, drug) such that it constitutes the majority of the sample in which it is present. Typically, in a sample, the substantially purified component constitutes 50% of the sample, preferably 80%-85%, and more preferably 90-95%.

[0044] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, prognosis, treatment, or therapy is desired. A therapeutic "mammal" refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, and the like. In some cases, the methods of the invention are utilized in laboratory animals, in veterinary applications, and in the development of animal models of disease, including, but not limited to, rodents such as mice, rats, and hamsters, primates, and transgenic animals.

[0045] method A method for treating Hedgehog pathway-dependent cancer with an inhibitor of aPKC iota is disclosed. In some embodiments, an inhibitor of aPKC iota is used in combination with an inhibitor of HDAC1. Without being bound to a particular theory, aPKC phosphorylates GLI1 transcription factor, resulting in chromatin association of GLI1 and activation of gene transcription, which leads to activation of Hedgehog pathway. The activity of GLI1 is controlled by regulating its nuclear transport. That is, GLI1 moves between the nuclear lamina, where it is inactive, and the nucleoplasm, where it is active. The mechanism by which aPKC iota activates GLI1 appears to involve HDAC1 being recruited to GLI1, which is activated by HDAC1-mediated deacetylation. Therefore, not only an inhibitor of aPKC iota, but also an inhibitor of HDAC1 may be useful for treating Hedgehog pathway-dependent cancer.

[0046] It will be appreciated that although the methods of the present invention are directed to the treatment of existing tumors, the methods may also be useful in preventing further tumor proliferation and growth that arise during treatment.

[0047] Atypical protein kinase C iota inhibitors As described above, the method of the present invention includes administering an inhibitor of aPKC iota. Exemplary inhibitors of aPKC iota include CRT0422839 (TEV-47448) and CRT0364436 (TEV-44229), or pharma- ceutically acceptable salts thereof. CRT0422839 has the chemical formula: TIFF2025038028000002.tif3128 and CRT0364436 have the chemical formula: I have TIFF2025038028000003.tif7128.

[0048] Such aPKC iota inhibitors have antitumor activity in the treatment of hedgehog pathway dependent cancer.In particular, these aPKC iota inhibitors can inhibit the growth, proliferation and metastasis of hedgehog pathway dependent cancer cells, reduce the production of Gli 1 mRNA in hedgehog pathway dependent cancer cells, and reduce the survival of hedgehog pathway dependent cancer cells (see examples).

[0049] HDAC1 inhibitors In certain embodiments, combination therapy is performed with aPKC iota inhibitor and HDAC1 inhibitor.Exemplary HDAC1 inhibitors include hydroxamic acids, such as vorinostat, belinostat, panobinostat, gibinostat, dacinostat (LAQ824), trichostatin A; sesquiterpene lactones, such as parthenolide; cyclic tetrapeptides, such as trapoxin B; depsipeptides, such as romidepsin; and benzamides, such as entinostat (MS-275), tacedinaline (CI994), mocetinostat.In some embodiments, an inhibitor that selectively inhibits HDAC1 without affecting other classes of HDACs, such as parthenolide, is used.

[0050] Inhibition of the Hedgehog signaling pathway As described herein, aPKC iota inhibitor can be used alone or in combination with HDAC inhibitor to inhibit the signal transduction of Hh pathway.Inhibited means that the activity of the pathway is reduced, suppressed, decreased, attenuated or antagonized.For example, in cells (e.g., cancerous cells) in which Hh pathway is overactive or constitutively active, for example, in cells that contain activating mutations of Smo gene or inactivating mutations of Ptch or SUFU gene, or in cells that overexpress pathway activators such as SHH / IHH ligand, SMO or GLI1 / 2, it may be desirable to use aPKC iota inhibitor and / or HDAC inhibitor to inhibit the signal transduction of Hh pathway.Other mutations that promote abnormal activation of Hedgehog signal transduction pathway are well known and can be easily identified by those skilled in the art.For mutations associated with aberrant activation of Hedgehog signaling, in particular mutations involved in cancer, see, for example:Pellegrini et al. (2017) Int. J. Mol. Sci. 18(11) pii: E2485;Bao et al. (2018) Mol. Nutr. Food Res. 62(1);Levanat et al. (2017) Curr. Pharm. Des. 23(1):73-94;Laukkanen et al. (2016) Anticancer Agents Med. Chem. 16(3):309-317;Suzman et al. (2015) Cancers (Basel). 7(4):1983-1993;Holikova et al. (2004) Int. J. Dermatol. 43(12):865-869;Wetmore (2003) Curr. Opin. Genet. Dev. 13(1):34-42; Bale (2002) Annu. Rev. Genomics Hum. Genet. 3:47-65; Lacour et al. (2002) Br. J. Dermatol. 146 Suppl 61:17-19; Wicking et al. (2001) Cancer Lett. 173(1):1-7; and Daya-Grosjean et al. (2005) Cancer Lett. 225(2):181-192; which are incorporated herein by reference.

[0051] In carrying out the methods of the invention, an aPKC iota inhibitor, alone or in combination with an HDAC inhibitor, is provided to a cell in an effective amount, i.e., an amount effective to inhibit Hh pathway signaling. Biochemically speaking, an effective amount or dose of an aPKC iota inhibitor and / or HDAC inhibitor is an amount sufficient to inhibit Hh pathway signaling in a cell by 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 200% or more, or 500% or more. In other words, the activity of the Hh signaling pathway in cells contacted with an effective amount or dose of an aPKC iota inhibitor or HDAC inhibitor will be about 70% or less, about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 10% or less, about 5% or less, or about 0%, i.e., negligible, of the activity observed in cells not contacted with an effective amount / dose of an aPKC iota inhibitor and / or HDAC inhibitor. In other words, Hh pathway signaling will be altered by about 0.5-fold or more, 1-fold or more, 2-fold or more, 5-fold or more, 8-fold or more, or 10-fold or more.

[0052] The amount of inhibition of cellular activity by aPKC iota inhibitors or HDAC inhibitors can be examined by several methods known to those skilled in the art of molecular biology. For example, the amount of phosphorylated transcription factor Gli in cells can be measured by Western blotting; the amount of Gli binding to a DNA target sequence can be measured by electrophoretic mobility assay (EMSA); the expression level of transcription factors normally activated by Hh signaling, such as ptch1, ptch2, hhip1, nhk2, rab34, etc., can be measured, for example, by measuring the RNA or protein levels of genes that are transcriptional targets of Gli, or by transfecting / infecting cells with a nucleic acid vector containing a Gli-responsive promoter operably linked to a reporter protein, such as luciferase, EGFP, and qualitatively or quantitatively measuring the amount of reporter protein produced. In this way, the inhibitory effect of aPKC iota inhibitors or HDAC inhibitors can be confirmed.

[0053] In clinical terms, the effective dose of aPKC iota inhibitor or HDAC inhibitor is the dose that, when administered for a suitable period, usually at least about 1 week, sometimes about 2 weeks or more, up to about 4 weeks, 8 weeks or more, demonstrates a change in symptoms associated with the undesirable activity of Hh signaling pathway.For example, the effective dose of aPKC iota inhibitor or HDAC inhibitor is the dose that, when administered for a suitable period, usually at least about 1 week, sometimes about 2 weeks or more, up to about 4 weeks, 8 weeks or more, slows, stops or reverses the growth and metastasis of tumors in patients suffering from cancer.Those skilled in the art will understand that an initial dose can be administered for such a period, and then a maintenance dose, optionally with a reduced dose, can be administered.

[0054] Calculating the effective amount or effective dose of the aPKC iota inhibitor or HDAC inhibitor to be administered is within the skill of the art and would be routine for the artisan. Of course, the final amount to be administered will depend on a variety of factors, including the route of administration, the nature of the disease or condition to be treated, and patient-specific factors. A competent clinician will be able to determine the effective amount of the therapeutic agent to be administered to a patient to stop or reverse the progression of the condition, as necessary. LD 50 Using animal data and other information available about the drug, the clinician can determine the maximum safe dose for an individual depending on the route of administration. For example, a dose administered intravenously may be higher than a dose administered intrathecally or topically, given the greater volume of bodily fluid into which the therapeutic composition is administered. Similarly, compositions that are rapidly cleared from the body may be administered at higher or repeated doses to maintain therapeutic concentrations. With ordinary skill, a competent clinician would be able to optimize the dosage of a particular therapeutic agent during the course of routine clinical trials.

[0055] The methods of the invention can be used to inhibit Hh pathway signaling, and thus cellular activities associated with Hh pathway signaling, in cells in vitro and in vivo. For example, cells in which Hh pathway signaling is undesirable, such as cancerous cells in which unregulated Hh pathway signaling promotes proliferation or metastasis, can be contacted with an aPKC iota inhibitor and / or an HDAC inhibitor. Cells can be from any mammalian species, such as mouse, rodent, canine, feline, equine, bovine, ovine, primate, human, etc.

[0056] When the method of the present invention is carried out in vitro, the cells may be derived from an established cell line or may be primary cells; herein, "primary cells", "primary cell lines" and "primary cultures" are used interchangeably and refer to cells and cell cultures derived from a subject that are grown in vitro for a limited number of passages (i.e., splitting) of the culture. For example, a primary culture is a culture that may have been passaged 0, 1, 2, 4, 5, 10, or 15 times, but not enough times to enter a critical stage. Typically, the primary cell lines of the present invention are maintained in vitro for less than 10 passages.

[0057] If the cells are primary cells, they can be harvested from an individual by any convenient method. For example, cells, such as blood cells, e.g., leukocytes, can be harvested by apheresis, leukapheresis, density gradient separation, and the like. As another example, cells, such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, nervous system tissue, and the like, can be harvested by biopsy. A suitable solution can be used to disperse or suspend the harvested cells. Such solutions are generally balanced salt solutions, e.g., saline, PBS, Hank's balanced salt solution, and the like, conveniently supplemented with fetal bovine serum or other naturally occurring factors, and generally in combination with a low concentration of an acceptable buffer, typically 5-25 mM. Convenient buffers include HEPES, phosphate buffer, lactate buffer, and the like. The cells can be used immediately or frozen for long periods and thawed and reused. In such cases, the cells are typically frozen in 10% DMSO, 50% serum, 40% buffered media, or other solution as commonly used in the art for preserving cells at freezing temperatures, and thawed by methods commonly known in the art for thawing frozen cultured cells.

[0058] The aPKC iota inhibitor or HDAC inhibitor can be dissolved in a solvent such as water, alcohols, or DMSO, DMF, and diluted with water or an appropriate buffer before being provided to the cells.

[0059] To modulate Hh pathway signaling, the aPKC iota inhibitor or HDAC inhibitor can be provided to the cells for about 30 minutes to about 24 hours, e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, or any other period of about 30 minutes to about 24 hours, which can be repeated about every day to about every 4 days, e.g., every 1.5 days, every 2 days, every 3 days, or any other frequency of about every day to about every 4 days. The agent can be provided to the subject's cells one or more times, e.g., 1, 2, 3, or 4 or more times; following each contact event, the cells can be incubated with the agent for a period of time, e.g., 16 to 24 hours, after which the medium is replaced with fresh medium and the cells are further cultured.

[0060] The step of contacting the cells with the aPKC iota inhibitor or HDAC inhibitor can be carried out in any medium and under any culture conditions that promote cell survival. For example, the cells can be suspended in any convenient suitable nutrient medium, such as Iscove's modified DMEM or RPMI 1640, supplemented with fetal bovine serum or heat-inactivated goat serum (about 5-10%), L-glutamine, thiols, particularly 2-mercaptoethanol, and antibiotics, such as penicillin and streptomycin. The culture medium may contain growth factors to which the cells respond. Growth factors, as defined herein, are molecules that can promote cell survival, growth, and / or differentiation in culture or in intact tissues through specific action on transmembrane receptors. Growth factors include polypeptide factors and non-polypeptide factors. Conditions that promote cell survival are typically conditions that allow non-homologous end joining and homologous recombination.

[0061] Cancerous cells of interest for research and treatment in this application include precancerous (e.g., benign), malignant, premetastatic, metastatic, and nonmetastatic cells, where the cancerous phenotype is driven by Hh pathway signaling. In other words, Hh pathway signaling (and often unregulated Hh pathway signaling) predisposes an individual's cells to cancer or induces or enhances an individual's symptoms of cancer, such as tumor growth and metastasis. In many such instances, Hh pathway signaling is elevated in tumor cells compared to the level of signaling observed in healthy cells, e.g., 2-fold or more, 3-fold or more, 4-fold or more, 6-fold or more, 8-fold or more, 10-fold or more, 20-fold or more, or 50-fold or more compared to the amount of Hh pathway signaling in healthy cells. The level of Hh signaling can be measured by any convenient method, as known in the art or described herein.

[0062] In some applications, aPKC iota inhibitors or HDAC inhibitors are used to regulate the signal transduction of the Hh pathway in vivo, for example, to inhibit tumor growth or metastasis to treat cancer. In these in vivo embodiments, aPKC iota inhibitors or HDAC inhibitors are administered directly to individuals. aPKC iota modulating agents can be administered by any of a number of methods well known in the art, as described below.

[0063] formulation aPKC iota inhibitors (e.g., CRT0422839, CRT0364436) or HDAC inhibitors can be incorporated into various formulations. More specifically, aPKC iota inhibitors or HDAC inhibitors can be formulated into pharmaceutical compositions by combining with suitable pharmaceutically acceptable carriers or diluents. A pharmaceutical formulation is a composition that includes one or more aPKC iota inhibitors and / or HDAC inhibitors in a pharmaceutically acceptable vehicle. A "pharmaceutically acceptable vehicle" can be a vehicle approved by federal or state regulatory agencies or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in mammals, such as humans. The term "vehicle" refers to a diluent, adjuvant, excipient, or carrier used in formulating the compounds of the present invention for administration to mammals. Such pharmaceutical vehicles can be lipids, such as liposomes, such as liposomal dendrimers; liquids, such as water and oils, such as those of petroleum, animal, vegetable, synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, saline; gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. In addition, auxiliary agents, stabilizers, thickeners, lubricants, and colorants may be used. The pharmaceutical compositions can be formulated into solid, semi-solid, liquid, gaseous formulations, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Thus, administration of aPKC iota inhibitors and / or HDAC inhibitors can be achieved in a variety of ways, including transdermal, intradermal, oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, etc. administration. The active agent may be systemic following administration or may be localized by the use of topical administration, intramural administration, or the use of an implant that acts to retain the active dose at the site of implantation. The active agent may be formulated for immediate activation or for sustained release.

[0064] In some diseases, particularly those of the central nervous system, drugs may need to be formulated to cross the blood-brain barrier (BBB). One strategy for drug delivery through the blood-brain barrier (BBB) ​​involves disruption of the BBB by osmotic means such as mannitol or leukotrienes, or biochemically by using vasoactive substances such as bradykinin. The possibility of using BBB opening to target specific drugs to brain tumors is also an option. If the therapeutic composition of the present invention is administered by intravascular injection, a BBB disrupting agent can be co-administered with the therapeutic composition of the present invention. Other strategies for crossing the BBB may involve the use of endogenous transport systems, including caveolin-1-mediated transcytosis, carrier-mediated transporters such as glucose and amino acid carriers, insulin or transferrin receptor-mediated transcytosis, and active diffusion transporters such as p-glycoprotein. Active transport moieties can also be conjugated to therapeutic compounds for use in the present invention to facilitate transport across the endothelial wall of blood vessels. Alternatively, drug delivery of a therapeutic agent behind the BBB can be by local delivery, e.g., intrathecal delivery, e.g., via an Ommaya reservoir (see, e.g., U.S. Patent Nos. 5,222,982 and 5,385,582; incorporated herein by reference); by bolus injection, e.g., with a syringe, intravitreal or intracranially; by continuous infusion, e.g., by cannulation, e.g., using convection (see, e.g., U.S. Application Nos. 20070254842; incorporated herein by reference); or by implantation of a device to which the agent is reversibly affixed (see, e.g., U.S. Application Nos. 20080081064 and 20090196903; incorporated herein by reference).

[0065] For inclusion in a pharmaceutical product, the aPKC iota inhibitor and / or HDAC inhibitor can be obtained from a suitable commercial source. As a general proposition, the total pharmacologic effective amount of the aPKC iota inhibitor and / or HDAC inhibitor administered parenterally will be within a range that can be determined by a dose-response curve.

[0066] The aPKC iota inhibitor-based therapeutic agent, with or without HDAC inhibitor, i.e., the formulation used for therapeutic administration, can be sterile. Sterility is easily achieved by filtration through a sterile filtration membrane (e.g., 0.2 μm membrane). The therapeutic composition is generally placed in a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle. The composition containing the aPKC iota inhibitor and / or HDAC inhibitor can be stored in a single-dose or multi-dose container, for example, a sealed ampule or vial, as an aqueous solution or as a lyophilized formulation for extemporaneous preparation. An example of a lyophilized formulation is filling a 10 mL vial with 5 mL of a sterile-filtered 1% (w / v) aqueous solution of the compound, and lyophilizing the resulting mixture. An infusion solution is obtained by extemporaneously preparing the lyophilized compound with bacteriostatic water for injection. Alternatively, the aPKC iota inhibitor and / or HDAC inhibitor can be formulated into a lotion for topical administration.

[0067] The pharmaceutical composition may contain pharma- ceutically acceptable non-toxic carriers or diluents depending on the desired formulation, and these carriers or diluents are defined as vehicles commonly used to formulate pharmaceutical compositions for administration to animals or humans. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may contain other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, excipients, etc. The composition may also contain additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents, surfactants, etc.

[0068] The composition may also include any of a variety of stabilizing agents, such as, for example, antioxidants. When the pharmaceutical composition includes a polypeptide, the polypeptide may be complexed with a variety of known compounds that enhance the in vivo stability of the polypeptide or otherwise enhance its pharmacological properties (e.g., increase the half-life of the polypeptide, reduce its toxicity, improve solubility or uptake). Examples of such modifying or complexing agents include sulfate, gluconate, citrate, and phosphate. The nucleic acids or polypeptides of the composition may also be complexed with molecules that enhance their in vivo properties. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.

[0069] Further guidance regarding suitable formulations for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief review of methods for drug delivery, see Langer, Science 249:1527-1533 (1990).

[0070] The pharmaceutical composition can be administered for prophylactic and / or therapeutic treatment. The toxicity and therapeutic effect of the active ingredient can be determined according to standard pharmaceutical procedures, such as determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) in cell cultures and / or experimental animals. The dose ratio between toxicity and therapeutic effect is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Therapeutic agents that exhibit large therapeutic indices are preferred.

[0071] Data obtained from cell culture and / or animal studies can be used in formulating a range of human dosages. The dosage of the active ingredient generally falls within a range of circulating concentrations that include the ED50 with low toxicity. The dosage can vary within this range depending on the dosage form and route of administration used.

[0072] The components used in formulating pharmaceutical compositions are preferably of high purity and substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, typically at least analytical grade, and more typically at least pharmaceutical grade). Furthermore, compositions intended for in vivo use are usually sterile. Where a given compound must be synthesized prior to use, the resulting product is usually substantially free of potentially toxic substances, particularly endotoxins, that may be present during the synthesis or purification process. Compositions for parenteral administration are also sterile, substantially isotonic, and manufactured under GMP conditions.

[0073] The aPKC iota inhibitor and / or HDAC inhibitor may be provided in addition to other drugs.For example, in the method of treating cancer promoted by the signal transduction of Hh pathway, the aPKC iota inhibitor and / or HDAC inhibitor can be co-administered with other known cancer therapeutics.

[0074] Administration Hedgehog pathway-dependent cancers that can be treated according to the methods described herein include, but are not limited to, cancers that depend on activation of the Hedgehog pathway or are associated with aberrant or constitutive activation of the Hedgehog pathway, such as: basal cell carcinoma, medulloblastoma, rhabdomyosarcoma, small cell lung cancer, retinoblastoma, gastric and upper gastrointestinal cancer, osteosarcoma, pancreatic cancer, breast cancer, colon cancer, ovarian cancer, brain tumors, mammary gland cancer, thyroid cancer, and prostate cancer.

[0075] At least one therapeutically effective dose of an aPKC iota inhibitor (e.g., CRT0422839 or CRT0364436), either alone or in combination with an HDAC inhibitor, and / or optionally other anti-cancer agents, is administered. By "therapeutically effective dose or amount" of each of these agents is intended an amount that, when administered, results in a positive therapeutic response in terms of treating an individual against a Hedgehog pathway-dependent cancer. Of particular interest are the amounts of these agents that result in an anti-tumor effect, as defined herein. By "positive therapeutic response" is intended an individual undergoing treatment according to the present invention shows improvement in one or more symptoms of the Hedgehog pathway-dependent cancer for which the individual is undergoing treatment.

[0076] Thus, for example, a "positive therapeutic response" would be an improvement in the disease associated with the treatment (e.g., treatment with an aPKC iota inhibitor, or combination therapy with an aPKC iota inhibitor and an HDAC inhibitor and / or optionally other anti-cancer agents) and / or an improvement in one or more symptoms of the disease associated with the treatment. Thus, for example, a positive therapeutic response would refer to one or more of the following improvements in the disease: (1) reduction in tumor size; (2) reduction in the number of cancer cells; (3) inhibition (i.e., slowing to some extent, preferably stopping) of tumor growth; (4) inhibition (i.e., slowing to some extent, preferably stopping) of cancer cell invasion into peripheral organs; (5) inhibition (i.e., slowing to some extent, preferably stopping) of tumor metastasis; and (6) some alleviation of one or more symptoms associated with cancer.

[0077] Such treatment responses can be further characterized in terms of the degree of improvement. Thus, for example, an improvement can be characterized as a complete response. "Complete response" means disappearance of all symptoms and signs of all measurable or evaluable disease as confirmed by repeated physical, laboratory, nuclear and radiological examinations (i.e., computed tomography (CT) and / or magnetic resonance imaging (MRI)) or other non-invasive techniques for all initial abnormalities or sites that were positive at the time of entry into the study. Alternatively, disease improvement may be classified as a partial response. By "partial response" it is intended that the sum of the products of the perpendicular diameters of all measurable lesions is reduced by more than 50% when compared to the pretreatment measurement (partial response does not apply to patients with only evaluable responses).

[0078] In certain embodiments, multiple therapeutically effective doses of aPKC iota inhibitor, either alone or in combination with HDAC inhibitor, and optionally other anti-cancer drugs, are administered according to a daily dosing regimen or intermittently.For example, therapeutically effective doses are administered 1 day per week, 2 days per week, 3 days per week, 4 days per week, or 5 days per week, etc.By "intermittent" administration, it is intended that therapeutically effective doses are administered, for example, every other day, every 2 days, every 3 days, etc.For example, in some embodiments, aPKC iota inhibitor, either alone or in combination with HDAC inhibitor, and / or optionally other anti-cancer drugs, are administered twice or thrice per week for an extended period of time, for example, for 1, 2, 3, 4, 5, 6, 7, 8...10...15...24 weeks, etc. "Twice weekly" or "twice weekly" is intended to mean that two therapeutically effective doses of the agent in question are administered to the subject within seven days, starting from the first day of the first week of administration, with a minimum of 72 hours and a maximum of 96 hours between doses. "Three times weekly" or "three times weekly" is intended to mean that three therapeutically effective doses are administered to the subject within seven days, with a minimum of 48 hours and a maximum of 72 hours between doses. In the present invention, this type of administration is referred to as "intermittent" therapy. According to the method of the present invention, the subject can receive intermittent therapy (i.e., a therapeutically effective amount is administered twice weekly or three times weekly) for one or more cycles of one week or more until a desired therapeutic response is achieved. The agent can be administered by any acceptable route of administration, as described herein below.

[0079] In certain embodiments, a combination therapy of an aPKC iota inhibitor, an HDAC inhibitor, and optionally other anti-cancer agents is administered. The aPKC iota inhibitor can be administered before the HDAC inhibitor, simultaneously with the HDAC inhibitor, or after the HDAC inhibitor. When provided simultaneously with the HDAC inhibitor, the aPKC iota inhibitor can be provided in the same composition or in a different composition. Thus, the two agents can be presented to an individual by concurrent therapy. By "concurrent therapy" is intended administration to a human subject such that the therapeutic effect of the combination of substances is provided to the human subject undergoing treatment. For example, concurrent therapy can be achieved by administering a pharmaceutical composition comprising at least one therapeutically effective dose of an aPKC iota inhibitor and a pharmaceutical composition comprising at least one therapeutically effective dose of at least one HDAC inhibitor according to a specific dosing regimen. Similarly, the aPKC iota inhibitor and / or HDAC inhibitor, and optionally other anti-cancer agents, can be administered in at least one therapeutic dose. The administration of the separate pharmaceutical compositions can occur at the same time (i.e., simultaneously) or at different times (i.e., sequentially, in any order, on the same day or on different days), so long as the therapeutic effect of the combination of these agents is provided to the subject being treated.

[0080] In certain embodiments, aPKC iota inhibitor is administered for a short period before administration of HDAC inhibitor and continues for a short period after HDAC inhibitor treatment is stopped, so that the level of aPKC iota inhibitor is sufficient in the subject during treatment to inhibit the association of GLI1 and HDAC1 and the activation of GLI1 and Hedgehog signaling pathway.For example, aPKC iota inhibitor can be administered starting one week before administration of the first dose of HDAC inhibitor and continued one week after administration of the final dose of HDAC inhibitor to the subject.

[0081] In other embodiments, the pharmaceutical composition containing the drug, such as aPKC iota inhibitor and / or HDAC inhibitor and / or optionally other anticancer drugs, is a sustained release formulation or a formulation administered using a sustained release device.Such devices are well known in the art, for example, transdermal patches and small implantable pumps, which can deliver drugs continuously at a steady state over time at various doses to achieve sustained release effect using non-sustained release pharmaceutical compositions.

[0082] The pharmaceutical composition comprising aPKC iota inhibitor and / or HDAC inhibitor and optionally other anti-cancer agents can be administered using the same or different administration routes according to medically acceptable methods known in the art. Suitable administration routes include parenteral administration, for example subcutaneous (SC), intraperitoneal (IP), intramuscular (IM), intravenous (IV), or infusion, oral, pulmonary, intranasal, topical, transdermal, intratumoral, and suppository. When the composition is administered by pulmonary delivery, its therapeutically effective amount is adjusted so that the solubility level of the agent, such as aPKC iota inhibitor and / or HDAC inhibitor, in the bloodstream is equivalent to that obtained by the therapeutically effective amount administered parenterally, for example SC, IP, IM, or IV. In some embodiments, the pharmaceutical composition comprising aPKC iota inhibitor and / or HDAC inhibitor and optionally other anti-cancer agents is administered locally to tumors by IM or SC injection, particularly by IM or SC injection. In some embodiments, the aPKC iota inhibitor and / or HDAC inhibitor and optionally other anti-cancer agents are administered locally, for example as a patch or gel.

[0083] In some embodiments, the aPKC iota inhibitor and / or HDAC inhibitor and optionally other anti-cancer agents are administered by infusion or local injection at a rate of, for example, about 50 mg / h to about 400 mg / h, e.g., about 75 mg / h to about 375 mg / h, about 100 mg / h to about 350 mg / h, about 150 mg / h to about 350 mg / h, about 200 mg / h to about 300 mg / h, about 225 mg / h to about 275 mg / h. Exemplary infusion rates are, for example, about 0.5 mg / m 2 / day~about 10mg / m 2 / day, e.g., about 1 mg / m 2 / day ~ approx. 9mg / m 2 / day, about 2mg / m 2 / day~about 8mg / m 2 / day, about 3mg / m 2 / day~about 7mg / m 2 / day, approximately 4mg / m 2 / day~about 6mg / m 2 / day, approximately 4.5mg / m 2 / day~about 5.5mg / m 2 A desired therapeutic dose of 100 mg / day can be achieved. Administration (e.g., by infusion) can be repeated for a desired period of time, e.g., over a period of about 1 day to about 5 days, or once every few days, e.g., once every 5 days, about 1 month, about 2 months, etc. The aPKC iota inhibitor and / or HDAC inhibitor can also be administered prior to, at the time of, or after other therapeutic interventions, such as surgical interventions to remove cancerous cells. The aPKC iota inhibitor and / or HDAC inhibitor can also be administered as part of a combination therapy in which at least one of immunotherapy, chemotherapy, radiation therapy, or biological therapy is administered to the subject.

[0084] Factors that influence the amount of each of the various compositions administered include, but are not limited to, the method of administration, the frequency of administration (daily or intermittent administration, such as twice or three times a week), the particular disease being treated, the severity of the disease, the history of the disease, whether the individual is receiving concurrent therapy with another therapeutic agent, and the age, height, weight, health, and physical condition of the individual being treated.In general, it is preferable to increase the dosage of this agent as the weight of the subject being treated increases.

[0085] The individual doses of the aPKC iota inhibitor and / or HDAC inhibitor and optionally other anti-cancer drugs are usually not less than the amount required to produce a measurable effect in the subject, and can be determined based on the pharmacokinetics and pharmacology for the absorption, distribution, metabolism, excretion ("ADME") of the aPKC iota inhibitor and / or HDAC inhibitor and optionally other anti-cancer drugs and their by-products, and therefore the disposition of the composition in the subject. This includes consideration of the route of administration as well as the dosage, which can be adjusted for topical application (applied directly to the site of action for a primarily local effect), enteral application (applied via the digestive tract for a systemic or local effect when retained in a portion of the digestive tract), or parenteral application (applied via a route other than the digestive tract for a systemic or local effect). For example, administration of an aPKC iota inhibitor (eg, CRT0422839 or CRT0364436) can be local or by injection, eg, intravenous, intramuscular, or intratumoral injection, or a combination thereof.

[0086] The pharmacokinetics of aPKC iota inhibitors and their corresponding biological activity in a subject is usually evaluated against the percentage of aPKC iota inhibitor present in the target of interest.For example, when administered, aPKC iota inhibitors may accumulate with glycoconjugates or other biological targets, which causes the substance to concentrate in cancer cells and cancerous tissues.Therefore, a dosing regimen in which aPKC iota inhibitors are administered to accumulate in the target of interest over time can be part of a strategy to lower individual doses.This can also mean that, for example, the dose of an aPKC iota inhibitor that is cleared more slowly in vivo can be lowered compared to the effective concentration calculated from in vitro assays (e.g., the effective amount in vitro is close to mM concentration, while in vivo is less than mM concentration).

[0087] As an example, an effective amount of a use or dosing regimen may be determined based on the IC50 of a given aPKC iota inhibitor for inhibiting aPKC kinase activity and / or GLI1 activation and / or Hedgehog pathway activation and / or cell proliferation and / or cell migration / invasion. 50 This can be inferred from the IC 50 " refers to the concentration of drug required for 50% inhibition in vitro. Alternatively, an effective amount is the EC50 of a given aPKC iota inhibitor concentration. 50 It can also be inferred from the "EC 50 " refers to the plasma concentration required to obtain 50% of the maximum effect in vivo. In a related embodiment, ED 50 The dosage can also be determined based on the (effective dosage).

[0088] In general, effective doses are calculated using IC 50 Typically, the amount of aPKC iota inhibitor administered is less than 200-fold greater than the calculated IC 50 Less than about 200-fold, less than about 150-fold, less than about 100-fold, and in many embodiments less than about 75-fold, less than about 60-fold, 50-fold, 45-fold, 40-fold, 35-fold, 30-fold, 25-fold, 20-fold, 15-fold, 10-fold, or even less than about 8-fold or about 2-fold of the calculated IC 50 and sometimes calculated IC 50 In other embodiments, the effective amount is about 2-40 times, about 3-30 times, or about 4-20 times the calculated IC 50 and in certain embodiments, the effective amount is equal to the calculated IC 50 It is a larger amount than that.

[0089] Effective doses are usually calculated EC 50 For example, the amount of aPKC iota inhibitor administered should not exceed the calculated EC 50 The effective amount is less than about 100-fold, less than about 50-fold, less than about 40-fold, 35-fold, 30-fold, or less than 25-fold, and in many embodiments less than about 20-fold, less than about 15-fold, or even less than about 10-fold, 9-fold, 9-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold, or 1-fold of the calculated EC50 Approximately 1 to 30 times the calculated EC 50 The effective amount may be about 1 to 20 times, or about 1 to 10 times, the calculated EC 50 It can be the same as the calculated EC 50 It may be more than IC 50 can be calculated by inhibiting aPKC kinase activity and / or GLI1 activation, and / or cell proliferation and / or cell migration / invasion in vitro.

[0090] To achieve efficacy, the level of the aPKC iota inhibitor must be above a certain level for a certain period of time. Efficacy is dose-dependent, and the higher the level of the aPKC iota inhibitor, the greater the antitumor effect. To minimize toxicity, the level of the aPKC iota inhibitor can be maintained below a certain level within and for a certain period of time (a "drug holiday" allows for clearance of the aPKC iota inhibitor). That is, the drug is held below a certain level for a certain period of time before the next dose is administered. The shorter the drug holiday between doses, the greater the toxicity.

[0091] In certain embodiments, the method of treating patients with hedgehog pathway dependent cancers includes cycles of treatment with an aPKC iota inhibitor, either alone or in combination with an HDAC inhibitor, and / or optionally other anti-cancer drugs, followed by a drug holiday without administration of the aPKC iota inhibitor and / or HDAC inhibitor to allow the patient to "recover" from the undesirable effects of the aPKC iota inhibitor and / or HDAC inhibitor. Multiple doses of the aPKC iota inhibitor and / or HDAC inhibitor can be administered according to a daily dosing regimen or intermittently, followed by a drug holiday.

[0092] If the subject undergoing treatment according to the above-mentioned dosing regimen shows partial response or relapses after a long period of remission, a subsequent course of treatment may be required to achieve complete remission of the disease.Thus, following a drug-free period from the first treatment period, the subject may undergo one or more additional treatment periods, including administering aPKC iota inhibitor alone or in combination with HDAC inhibitor, and optionally other anti-cancer drugs.Such drug-free periods between treatment periods are referred to herein as break periods.It will be appreciated that the length of the break period depends on the extent of tumor response (i.e., complete vs. partial) achieved in the previous treatment period with these therapeutic agents.

[0093] Application Examples One example of cancer driven by dysregulation of Hh pathway signaling is basal cell carcinoma (BCC). Gli levels are elevated in BCC tumors, and molecular targeted drugs for BCC focus on antagonizing Smo to reduce Gli mRNA. One such example is the plant alkaloid cyclopamine, which inhibits Smo. Model systems (in vitro and in vivo) have shown that cyclopamine effectively inhibits BCC, but clinical applications of cyclopamine have shown severe side effects that preclude its use. Another Smo antagonist that has shown good efficacy in metastatic BCC tumors is vismodegib. Other treatments include surgery, chemotherapy, immunotherapy (e.g., Euphorbia peplus, Imiquimod, Aldara, etc.), and radiation therapy. In some cases, BCCs are resistant to Smo antagonists or cancer cells have become resistant to Smo antagonists because activating mutations in the Hh pathway are downstream or epistatic of Smo, and the methods of the present invention can be applied to such cancers.

[0094] Another example of a disease driven by dysregulation of Hh pathway signaling is Basal Cell Nevus Syndrome (BCNS), also known as Gorlin Syndrome, a rare multisystem disorder characterized by the development of dozens to hundreds of BCCs. Subjects with BCNS inherit a defective copy of PTCH1. With a prevalence of 1 case per 56,000–164,000 population, BCNS is a rare disease with no effective and acceptable treatment. As a result, drugs to treat or prevent BCC tumors are of interest to patients with BCNS.

[0095] The methods and compositions of the present invention are used to treat or prevent BCC in two clinical populations: i) patients with hereditary BCC tumors, such as those with basal cell nevus syndrome; and ii) patients in the general population with sporadic BCC tumors. In the United States, BCC is the most common cancer with 1 million new diagnoses per year. Although BCC is rarely fatal, the high incidence of BCC and frequent recurrence in affected individuals can result in significant morbidity. Currently, the incidence of skin cancer is increasing year by year, and the treatment of skin cancer places a significant burden on the national health service. Currently, there is no effective treatment for preventing BCC, as sunscreens have not been shown to reduce the occurrence of BCC in randomized controlled trials.

[0096] Another example of cancer driven by dysregulation of Hh pathway signaling is medulloblastoma. Medulloblastoma is an aggressive primary brain tumor originating from the cerebellum or posterior fossa. Medulloblastoma is the most common malignant brain tumor, accounting for 14.5% of newly diagnosed cases. Medulloblastoma usually forms near the fourth ventricle, between the brainstem and the cerebellum. Known treatments for medulloblastoma include chemotherapy, such as one or more of lomustine, cisplatin, carboplatin, vincristine or cyclophosphamide, and vismodegib. The method of the present invention can be applied to medulloblastoma that is resistant (or has become resistant) to Smo antagonists.

[0097] Another example of a cancer driven by dysregulation of Hh pathway signaling is rhabdomyosarcoma. Rhabdomyosarcoma is a sarcoma (cancer of connective tissue) in which cancer cells are thought to arise from skeletal muscle precursor cells. It can be present in any anatomical location. Most arise in areas where skeletal muscle is scarce, such as the head, neck, and genitourinary tract. The diagnosis of rhabdomyosarcoma relies on the recognition of differentiation into skeletal muscle cells. The proteins myoD1 and myogenin are transcription factor proteins normally found in developing skeletal muscle cells that disappear when the muscle matures and becomes innervated. Thus, myoD1 and myogenin are not normally found in normal skeletal muscle and serve as useful immunohistochemical markers for rhabdomyosarcoma. Treatment of rhabdomyosarcoma consists of chemotherapy, radiation therapy, and sometimes surgery.

[0098] Hedgehog pathway-dependent cancers of other tissues, such as variants of Hedgehog pathway-dependent cancers of other tissues that are resistant to Smo antagonists, can also be treated with the methods of the invention, including, for example, subtypes of small cell lung cancer, pancreatic cancer, colon cancer, ovarian cancer, and prostate cancer, all of which have been shown to respond to blockers of the Hedgehog pathway.

[0099] kit A kit is provided that includes one or more containers that hold a composition that includes at least one aPKC iota inhibitor (e.g., CRT0422839 or CRT0364436) and / or HDAC inhibitor, and / or optionally one or more other anti-cancer agents for treating Hedgehog pathway-dependent cancer. The composition may be liquid or lyophilized. Suitable containers for the composition include, for example, bottles, vials, syringes, test tubes, etc. The containers can be made of various materials such as glass, plastic, etc. The containers can have a sterile access port (e.g., the containers can be intravenous solution bags or vials with a stopper that can be pierced by a hypodermic needle).

[0100] The kit may further comprise a second container containing a pharma- ceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It may also comprise other materials useful to the end user, such as other pharma- ceutically acceptable formulation solutions, such as buffers, diluents, filters, needles, and syringes or other delivery devices. The delivery device may be pre-filled with the composition.

[0101] The kit can also include a package insert with written instructions on how to use the composition comprising aPKC iota inhibitor and / or HDAC inhibitor for treating subjects with Hedgehog pathway-dependent cancer. The package insert can be an unapproved draft package insert or a package insert approved by the U.S. Food and Drug Administration (FDA) or other regulatory agencies. Alternatively, the instructions can be provided on a computer-readable medium with information recorded thereon, such as a diskette, CD, DVD, flash drive, etc., or the instructions can be provided on a website address that can be used via the Internet to access the information of the removed site. Any convenient means for providing instructions for treating subjects with Hedgehog pathway-dependent cancer can be included in the kit. EXAMPLES

[0102] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as the invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be accounted for. Unless otherwise specified, parts are parts by weight, molecular weight is weight average molecular weight, temperature is degrees Celsius, and pressure is at or near atmospheric.

[0103] General methods in molecular and cellular biochemistry can be found in standard textbooks such as: Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998); the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.

[0104] Example 1 Regulation of the Hedgehog pathway in BCC cell models Introduction Basal cell carcinoma (BCC) requires high levels of Hedgehog (HH) signaling for survival and growth (Chang et al. (2012) Arch. Dermatol. 148(11):1324-1325; Atwood et al. (2015) Cancer Cell 27(3):342-353; Atwood et al. (2013) Nature 494(7438):484-488; Hutchin et al. (2005) Genes Dev. 19(2):214-223). Activation of the HH pathway requires that HH ligands relieve the inhibition of Smoothened (SMO) by binding to Patched-1. This leads to activation of the GLI family of transcription factors, ultimately promoting the transcription of HH target genes, including Gli1 itself. Recently, SMO inhibitors have been FDA approved for the treatment of BCC, but drug resistance has emerged as a major problem (Chang et al., supra; Atwood et al. (2015), supra; Sekulic et al. (2012) N. Engl. J. Med. 366(23):2171-21791). Because BCCs are uniformly dependent on the HH pathway for growth (Atwood et al. (2015), supra), resistant BCCs have evolved to circumvent pharmacological blockade at the SMO level using pathway-specific mutations as well as non-canonical GLI activation mechanisms (Atwood et al. (2015), supra; Atwood et al. (2013), supra).

[0105] Recently, the present inventors have demonstrated that atypical gliomas are a potent mechanism of drug resistance in BCC. We confirmed hyperactivation of TIFF2025038028000004.tif7153 (Atwood et al. (2013), supra). aPKC phosphorylates the zinc finger domain of GLI1, resulting in chromatin association, gene transcription, and HH pathway activation downstream of input from SMO and Patched-1. In addition, GLI promotes aPKC transcription, forming another positive feedback loop involving GLI. Hyperactivation of this non-canonical HH signaling pathway promotes pathway activation and vismodegib escape in advanced BCC (Atwood et al. (2013), supra). Allosteric (Erdogan et al. (2006) J. Biol. Chem. 281(38):28450-28459) or orthosteric (Kjaer et al. (2013) Biochem. J. 451(2):329-342) small molecule inhibitors of aPKC are in development but have not been applied to treat BCC.

[0106] GLI proteins are further regulated downstream of SMO by acetylation and subsequent deacetylation by p300. Deacetylation of GLI1 / 2 at K518 and K757 by histone deacetylases 1 / 2 (HDAC1 / 2), respectively, is a critical step in the nuclear maturation process of GLI transcription factors, which is required for chromatin association and gene transcription (Canettieri et al. (2010) Nat. Cell Biol. 12(2):132-142). HDAC1 is itself a transcriptional target of GLI, forming a third positive feedback loop of HH signaling. Of particular interest, HDAC inhibition has been proposed for the treatment of many HH-driven cancers (Canettieri et al., supra; Coni et al. (2017) Sci Rep. 7:44079; Zhao et al. (2014) Pharmacol. Res. Perspect. 2(3):e00043; Coni et al. (2013) PLoS One 8(6):e65718). HDAC inhibitors block growth and promote apoptosis by altering histone-DNA complexes and by changing the acetylation status of non-histone proteins (Falkenberg et al. (2014) Nat. Rev. Drug Discov. 13(9):673-691). Vorinostat, a class I / II HDAC inhibitor, is currently FDA approved for the treatment of cutaneous lymphoma (Mann et al. (2007) Clin. Cancer Res. 13(8):2318-2322). Unfortunately, HDAC inhibition has been hampered by its widespread cytotoxicity. De novo drug discovery remains challenging due to a lack of validated targets and costly clinical development (Hoelder et al. (2012) Mol. Oncol. 6(2):155-176).

[0107] Here we show that the aPKC iota inhibitors CRT0422839 and CRT0364436 regulate BCC cell survival and reduce the levels of GLI1 mRNA, consistent with modulation of the GLI pathway.

[0108] result To examine the effect of aPKC iota inhibition on BCC in vitro, mouse BCC cell lines were treated with increasing doses of CRT0422839 or CRT0364436. Treatment with either CRT0422839 or CRT0364436 resulted in a dose-dependent decrease in BCC growth and survival (Figures 1C and 1D). BCC survival was compared to viability upon treatment with the aPKC inhibitors PSI (Figure 1A) and CRT0329868 (Figure 1B), previously described by Mirza et al. (JCI Insight (2017) 2(21) pii: e97071; incorporated herein by reference in its entirety). Furthermore, treatment with either CRT0422839 or CRT0364436 resulted in a dose-dependent decrease in the levels of Gli1 mRNA (Figures 2A and 2B).

[0109] Example 2 Biochemical kinase assay of aPKC iota The ability of compounds to inhibit the kinase activity of aPKCiota is measured in 384-well black, non-binding, flat-bottom assay plates (Corning 3575) using the IMAP FP progressive binding system (Molecular Devices R8127). The assay mixture (final volume = 10 μl) contains 20 mM Tris-HCL (pH 7.5), 150 μM ATP, 10 mM MgCl2, 0.01% Triton X-100, 250 μM EGTA, 1 mM DTT, 15 pM PKCι (EMD Millipore 14-505), 100 nM FAM-PKCε-pseudosubstrate (Molecular Devices RP7548), 0.1% DMSO, and various concentrations of the test compounds CRT0422839 and CRT0364436. Compound dilutions (prepared in 100% DMSO) are added to the assay plate at 100 nl using a BioMek NX pin tool (Beckman Coulter). The enzymatic reaction is initiated by the addition of ATP (MilliporeSigma A7699) and the plate is then incubated for 1 hour in a 25°C incubator. A 20 μl aliquot of IMAP detection reagent (1:400 in 85% 1X binding buffer A and 15% 1X binding buffer B) is added to each well and then incubated for 2 hours at 25°C. FP is then measured on a PerkinElmer Envision 2102 multilabel plate reader (PerkinElmer) using a FP dual mirror, FP480 excitation filter, P-pol 535 and S-pol 535 emission filters. Data analysis is performed using ActivityBase (IDBS). The IC50 value is calculated by plotting the inhibition rate against the log10 of the compound concentration and fitting it to a four-parameter logistic model (top and bottom are set to 100 and 0, respectively) of XLFit 4 (IDBS).

[0110] The above merely describes the principles of the present invention. It will be understood that those skilled in the art can devise various arrangements that embody the principles of the present invention and are within its spirit and scope, even if not explicitly described or shown herein. Furthermore, all examples and conditional statements described herein are intended to be interpreted as being primarily intended to help the reader understand the principles of the present invention and the concepts provided by the inventors to promote the present technology, and not as being limited to such specifically described examples and conditions. Furthermore, all statements herein that describe the principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Moreover, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., elements developed that perform the same function regardless of structure. Thus, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.

Claims

1. A method for treating a subject with a Hedgehog pathway dependent cancer, comprising administering to the subject a composition comprising a therapeutically effective amount of CRT0422839 or CRT0364436, or a pharma- ceutically acceptable salt thereof.

2. 2. The method of claim 1, wherein the cancer is basal cell carcinoma (BCC).

3. 3. The method of claim 1 or 2, wherein the cancer comprises a constitutively active Hedgehog pathway.

4. The method of any one of claims 1 to 3, wherein the cancer is metastatic.

5. 5. The method of any one of claims 1 to 4, wherein multiple cycles of treatment are administered to the subject for a period of time sufficient to result in at least a partial tumor response.

6. 6. The method of claim 5, wherein the period is at least six months.

7. 7. The method of claim 6, wherein the period is at least 12 months.

8. The method of any one of claims 5 to 7, wherein multiple cycles of treatment are administered to the subject for a period of time sufficient to result in a complete tumor response.

9. The method of any one of claims 5 to 8, wherein the composition is administered according to a daily dosing regimen or intermittently.

10. The method of any one of claims 1 to 9, further comprising administering an additional anti-cancer therapy.

11. 11. The method of claim 10, wherein the additional anti-cancer therapy is surgery, chemotherapy, radiation therapy, immunotherapy, biological therapy, or a combination thereof.

12. The method according to any one of claims 1 to 11, wherein the composition further comprises a pharma- ceutically acceptable excipient.

13. 13. The method of any one of claims 1 to 12, further comprising administering a histone deacetylase (HDAC) inhibitor in combination with CRT0422839 or CRT0364436, or a pharma- ceutically acceptable salt thereof.

14. 14. The method of claim 13, wherein the HDAC inhibitor is vorinostat.

15. The method of any one of claims 1 to 14, wherein the subject is a mammal.

16. The method of claim 15, wherein the subject is a human.

17. The method of any one of claims 1-16, wherein CRT0422839 or CRT0364436, or a pharma- ceutically acceptable salt thereof, is administered in an amount sufficient to reduce viability of Hedgehog pathway-dependent cancerous cells in the subject.

18. The method of any one of claims 1-17, wherein CRT0422839 or CRT0364436, or a pharma- ceutically acceptable salt thereof, is administered in an amount sufficient to reduce production of Gli 1 mRNA in Hedgehog pathway-dependent cancerous cells in the subject.

19. The method of any one of claims 1-18, wherein CRT0422839 or CRT0364436, or a pharma- ceutically acceptable salt thereof, is administered in an amount sufficient to reduce Hedgehog pathway-dependent cancerous cell growth and cell proliferation in the subject.

20. A method for inhibiting the growth or proliferation of hedgehog pathway-dependent cancerous cells, comprising contacting the hedgehog pathway-dependent cancerous cells with an effective amount of CRT0422839 or CRT0364436, or a pharma- ceutically acceptable salt thereof.

21. 21. The method of claim 20, wherein the Hedgehog pathway dependent cancerous cell is a basal cell carcinoma (BCC) cell.

22. 22. The method of claim 20 or 21, wherein the Hedgehog pathway dependent cancerous cells contain a constitutively active Hedgehog pathway.

23. The method of any one of claims 20 to 22, wherein the Hedgehog pathway dependent cancerous cell is in vivo or in vitro.

24. The method of any one of claims 20 to 23, wherein the Hedgehog pathway dependent cancerous cell is a human cancerous cell.

25. The method of any one of claims 20 to 24, further comprising contacting the Hedgehog pathway dependent cancerous cells with a deacetylase (HDAC) inhibitor.

26. 26. The method of claim 25, wherein the HDAC inhibitor is vorinostat.

27. A composition comprising CRT0422839 or CRT0364436, or a pharma- ceutically acceptable salt thereof, for use in treating a hedgehog pathway dependent cancer.

28. 28. The composition of claim 27, wherein the cancer is basal cell carcinoma (BCC).

29. 29. The composition of claim 27 or 28, further comprising a histone deacetylase (HDAC) inhibitor.

30. 30. The composition of claim 29, wherein the HDAC inhibitor is vorinostat.

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