BACKGROUND

Hedgehog (Hh) signaling is an evolutionarily conserved pathway essential for axial patterning and cell fate determination in development. Hedgehog signaling is known to regulate a diverse range of biological processes, such as cellular proliferation, differentiation, and organ formation in a tissue specific and dose dependent manner. Normally, Hedgehog signaling is strictly controlled during cellular proliferation, differentiation and embryonic pattern formation. However, aberrant activity of the Hedgehog signaling pathway, due to mutations that constitutively activate the pathway, for instance, can have pathological consequences.

In mammals, Hh signaling is driven by three ligands, Sonic hedgehog (SHH), Indian hedgehog (IHH), and Desert hedgehog (DHH). In the absence of ligand, the receptor Patched (PTCH) constitutively inhibits the G-coupled-like receptor Smoothened (SMO). SMO is required for canonical pathway activation and is the molecular target of small molecule Hh pathway inhibitors in development as cancer therapeutics. In the presence of ligand, PTCH is inhibited, relieving inhibition of SMO which then moves to the tip of the primary cilia, a single, immotile membrane-bound organelle that coordinates Hh signaling through trafficking of key signaling proteins along a microtubule core. Active SMO blocks the constitutive processing of the GLI proteins, a family of three zinc finger transcription factors (GLI1, 2 and 3). Unprocessed GLI proteins are transcriptionally active, and in turn initiate the expression of Hh target genes, including GLI1.

Human genetics, cancer genomics and genetic mouse models have established a role for aberrant Hh signaling in cancer. For example, loss-of-function mutations of Patched are found in Gorlin's syndrome; and gain-of-function mutations of SMO and GLI are linked to basal cell carcinoma (BCC, the most common form of skin cancer) and glioblastoma. As another example, constitutive activation of Hedgehog has been found to promote tumorigenesis in BCC, medulloblastoma (the most common childhood brain tumor), rhabdomyosarcoma, pancreatic cancer, small cell lung cancer, prostate cancer and breast cancer. Hedgehog signaling is also implicated in the metastasis of prostate cancer.

Cancers driven by inactivating mutations in PTCH or oncogenic mutations in SMO (e.g. medulloblastoma, basal cell carcinoma) exhibit ligand independent pathway activation, and can be successfully treated with SMO antagonists in the clinic. In these settings, malignant transformation is associated with the expression of GLZ-dependent transcription of genes such as GLI1, MYC and CCND1.

Alternatively, tumors such as small cell lung cancer (SCLC) are thought to depend on autocrine ligand-dependent Hh signaling for progression and self-renewal. Despite the mechanistic and therapeutic implications of these observations, there is no genetic biomarker that can predict which cancers expressing Hh ligands are dependent on autocrine signaling.

Itraconazole, a systemic antifungal, is a potent antagonist of the Hedgehog (Hh) signaling pathway that acts by a mechanism distinct from its inhibitory effect on fungal sterol biosynthesis. Systemically administered itraconazole, like other Hh pathway antagonists, can suppress Hh pathway activity and the growth of medulloblastoma in a mouse allograft model and does so at serum levels comparable to those in patients undergoing antifungal therapy. Mechanistically, itraconazole appears to act on the essential Hh pathway component Smoothened (SMO) by a mechanism distinct from that of cyclopamine and other known SMO antagonists, and prevents the ciliary accumulation of SMO normally caused by Hh stimulation.

Despite the therapeutic effects of Hedgehog inhibitors such as itraconazole, it can also have serious side effects, such as elevated alanine aminotransferase levels, a risk of developing congestive heart failure, and liver failure, which can be fatal. Therefore, there is a need for methods that select populations of patients that would respond favourably to the therapeutic effects of Hedgehog inhibitors, such as itraconazole, to minimize exposure of unresponsive or little-responsive patients to the Hedgehog inhibitors ( e.g ., itraconazole). There is also a need for methods that select populations of patients that do not have ligand-independent Hh signalling that would respond favourably to the therapeutic effects of Hedgehog inhibitors, such as itraconazole, to minimize exposure of unresponsive or little-responsive patients to the Hedgehog inhibitors (e.g., itraconazole). This disclosure fulfils these needs and provides further advantages.

SUMMARY

This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

In one aspect, the present disclosure features a method of treating cancer, including administering a therapeutically effective amount of a Hedgehog inhibitor to a subject in need thereof, wherein the subject is selected by: (a) obtaining a tumor biopsy sample from the subject, and (b) assessing expression of a hedgehog ligand, identifying the presence of ciliated cancerous cells in the tumor biopsy sample, identifying genetic mutations in TP 53 and/or RBI, identifying genetic mutations in hedgehog signaling components (e.g., PTCH1, SMO, SUFU, isochromosome 9, CDKN2A, and/or MDM2 ); and (c) selecting the subject when the subject expresses a hedgehog ligand, has ciliated cancerous cells in the tumor biopsy sample, has genetic mutations in TP53 and/or RBI, has genetic mutations in hedgehog signaling components (e.g., PTCH1, SMO, SUFU, isochromosome 9, CDKN2A, and/or MDM2).

In one aspect, the present disclosure features a method of treating cancer, including administering a therapeutically effective amount of itraconazole to a subject in need thereof, wherein the subject is selected by: (a) obtaining a tumor biopsy sample from the subject, and (b) assessing expression of a hedgehog ligand, identifying the presence of ciliated cancerous cells in the tumor biopsy sample, identifying genetic mutations in TP 53 and/or RBI, identifying genetic mutations in hedgehog signaling components (e.g., PTCH1, SMO, SUFU, isochromosome 9, CDKN2A, and/or MDM2 ); and (c) selecting the subject when the subject expresses a hedgehog ligand, has ciliated cancerous cells in the tumor biopsy sample, has genetic mutations in TP53 and/or RBI, has genetic mutations in hedgehog signaling components (e.g., PTCH1, SMO, SUFU, isochromosome 9, CDKN2A, and/or MDM2).

In another aspect, the present disclosure features a method of treating cancer, including administering a therapeutically effective amount of a Hedgehog inhibitor (e.g., itraconazole) to a subject in need thereof, wherein the subject is selected by: identifying activated hedgehog signaling in a tumor biopsy sample from the subject; and if hedgehog signaling is activated in the tumor biopsy sample, determining the presence of ciliated cancerous cells in the tumor biopsy sample; and selecting the subject for treatment with the Hedgehog inhibitor (e.g., itraconazole) when hedgehog signaling is activated and when the tumor biopsy sample is ciliated.

In yet another aspect, the present disclosure features a method of treating basal cell carcinoma, medulloblastoma, or rhabdomyosarcoma, including administering a therapeutically effective amount of a Hedgehog inhibitor (e.g., itraconazole) to a subject in need thereof, wherein the subject is selected by: identifying a gain-of-function gene mutation in sonic hedgehog signaling components in a tumor biopsy sample from the subject; and if a gain-of-function gene mutations in hedgehog signaling components is present in the tumor biopsy sample, selecting the subject for treatment with the Hedgehog inhibitor (e.g., itraconazole).

In a further aspect, the present disclosure features a method of treating cancer, including administering a therapeutically effective amount of a Hedgehog inhibitor (e.g., itraconazole) to a subject in need thereof, wherein the subject is selected by: (a) identifying activated sonic hedgehog signaling in a tumor biopsy sample from the subject, and if sonic hedgehog signaling is activated in the tumor biopsy sample, determining the presence of ciliated cancerous cells in the tumor biopsy sample; and (b) determining genomic mutations of TP 53, RBI, or both TP 53 and RBI; (c) selecting the subject for treatment with the Hedgehog inhibitor ( e.g ., itraconazole) when sonic hedgehog signaling is activated, the tumor biopsy sample is ciliated, and at least one of TP53 and RBI is genomically mutated as determined by targeted sequencing.

In another aspect, the present disclosure features a method for treating cancer, including determining the percentage of ciliated cancerous cells in tumor biopsy samples obtained from a cancer patient population, wherein the greater the percentage of ciliated cancerous cells in a given tumor biopsy sample, the more responsive the corresponding patient would be to treatment with a Hedgehog inhibitor (e.g. , itraconazole) ; and selecting patients having tumor biopsy samples having greater percentages of ciliated cancerous cells for treatment with the Hedgehog inhibitor (e.g., itraconazole).

Various (enumerated) embodiments of the present invention are now described. It will be recognized that features specified in each embodiment may be combined with other specified features to provide further embodiments of the present disclosure.

Embodiment 1: A method for preventing or treating cancer, preferably osteosarcoma or small cell lung cancer (SCLC) or a condition associated with same in an individual comprising providing a therapeutically effective amount of a Hedgehog inhibitor, preferably a SMO inhibitor, more preferably itraconazole in an individual, wherein the individual has a cancer cell having a mutation, preferably a loss of function mutation in a gene selected from the group consisting of: TP53 and RBI, more preferably a loss of function mutation in TP53 and a loss of function mutation in RB 1 , or wherein the individual has a cancer cell having a reduced production or no production of a protein or RNA encoded by a gene selected from said group.

Embodiment 2: A composition comprising a therapeutically effective amount of a Hedgehog inhibitor, preferably a SMO inhibitor, more preferably itraconazole for use in preventing or treating cancer, preferably osteosarcoma or small cell lung cancer (SCLC) or a condition associated with same in an individual, wherein the individual has a cancer cell having a mutation, preferably a loss of function mutation in a gene selected from the group consisting of: TP53 and RBI, more preferably a loss of function mutation in TP53 and a loss of function mutation in RB 1 , or wherein the individual has a cancer cell having a reduced production or no production of a protein or RNA encoded by a gene selected from said group.

Embodiment 3: A use of a composition comprising a Hedgehog inhibitor, preferably a SMO inhibitor, more preferably itraconazole in the manufacture of a medicament for preventing or treating cancer, preferably osteosarcoma or small cell lung cancer (SCLC) or a condition associated with same in an individual, wherein the individual has a cancer cell having a mutation, preferably a loss of function mutation in a gene selected from the group consisting of: TP53 and RBI, more preferably a loss of function mutation in TP53 and a loss of function mutation in RBI, or wherein the individual has a cancer cell having a reduced production or no production of a protein or RNA encoded by a gene selected from said group.

Embodiment 4: A Hedgehog inhibitor, preferably a SMO inhibitor, more preferably itraconazole when used for preventing or treating cancer, preferably osteosarcoma or small cell lung cancer (SCLC) or a condition associated with same in an individual, wherein the individual has a cancer cell having a mutation, preferably a loss of function mutation in a gene selected from the group consisting of: TP53 and RBI, more preferably a loss of function mutation in TP53 and a loss of function mutation in RB 1 , or wherein the individual has a cancer cell having a reduced production or no production of a protein or RNA encoded by a gene selected from said group.

Embodiment 5: A method for preventing or treating cancer, preferably osteosarcoma or small cell lung cancer (SCLC) or condition associated with same in an individual comprising:

- assessing or having assessed, a sample comprising a cell, preferably a cancer cell obtained from an individual for whom cancer is to be prevented or treated to determine if a cell in the sample has a mutation, preferably a loss of function mutation in a gene selected from the group consisting of: TP53 and RBI; more preferably a loss of function mutation in TP53 and a loss of function mutation in RBI, and

- where the individual has a cell having a mutation, preferably a loss of function mutation in a gene selected from the group consisting of: TP53 and RBI, o providing a Hedgehog inhibitor, preferably a SMO inhibitor, more preferably itraconazole to the individual, thereby preventing or treating cancer or condition associated with same in the individual.

Embodiment 6: A method for determining the likelihood of an individual presenting an anti-cancer response, preferably an anti - osteosarcoma or anti - small cell lung cancer (SCLC) response, to administration of a Hedgehog inhibitor, preferably a SMO inhibitor, more preferably itraconazole comprising:

- assessing or having assessed, a sample, preferably a sample of cancer cells obtained from an individual for whom the likelihood of presenting an anti-cancer response to administration of a Hedgehog inhibitor, preferably a SMO inhibitor, more preferably itraconazole is to be determined to determine whether a cancer cell in the sample has a mutation, preferably a loss of function mutation in a gene selected from the group consisting of: TP53 and RBI, more preferably a loss of function mutation in TP53 and a loss of function mutation in RB 1 ;

- determining that the individual has a higher likelihood of presenting an anti -cancer response to administration of a Hedgehog inhibitor, preferably a SMO inhibitor, more preferably itraconazole where a cancer cell in the sample has a mutation, preferably a loss of function mutation in a gene selected from the group consisting of: TP53 and RBI;

- determining that the individual has a lower likelihood of presenting an anti-cancer response to administration of a Hedgehog inhibitor, preferably a SMO inhibitor, more preferably itraconazole where a cancer cell in the sample does not have a mutation, preferably a loss of function mutation in a gene selected from the group consisting of: TP53 and RBI; thereby determining the likelihood of an individual presenting an anti-cancer response to administration of a Hedgehog inhibitor, preferably a SMO inhibitor, more preferably itraconazole.

Embodiment 7: A method for selecting an individual for treatment of cancer, preferably osteosarcoma or small cell lung cancer (SCLC) with a Hedgehog inhibitor, preferably a SMO inhibitor, more preferably itraconazole comprising the step of:

- assessing or having assessed a cell of the individual, preferably a cancer cell to determine if the cell has a mutation, preferably a loss of function mutation in a gene selected from the group consisting of: TP53 and RBI, more preferably a loss of function mutation in TP53 and a loss of function mutation in RBI;

- selecting the individual for treatment of cancer with a Hedgehog inhibitor, preferably a SMO inhibitor, more preferably itraconazole where the individual has a mutation, preferably a loss of function mutation in a gene selected from the group consisting of: TP53 and RBI; thereby selecting an individual for treatment of cancer with a Hedgehog inhibitor, preferably a SMO inhibitor, more preferably itraconazole.

Embodiment 8: A method for administration of a Hedgehog inhibitor, preferably a SMO inhibitor, more preferably itraconazole to an individual requiring therapy for cancer, preferably osteosarcoma or small cell lung cancer (SCLC) comprising the step of:

- assessing or having assessed a cell of the individual, preferably a cancer cell to determine if the cell has a mutation, preferably a loss of function mutation in a gene selected from the group consisting of: TP53 and RBI, more preferably a loss of function mutation in TP53 and a loss of function mutation in RBI; optionally

- administering a therapeutically effective amount of a Hedgehog inhibitor, preferably a SMO inhibitor, more preferably itraconazole to the individual for therapy of cancer. Embodiment 9: A kit comprising:

- a probe for assessing for presence or absence of a mutation, preferably a loss of function mutation in a gene selected from the group consisting of TP53 and RB 1 ; or

- a probe for assessing for reduced production or no production of a protein or RNA encoded by a gene selected from said group;

- written instructions for use of the kit in an enumerated embodiment described above.

The above enumerated embodiments may comprise the step of determining whether the cell of the individual has reduced production, or no production of a protein or RNA encoded by a gene selected from the group consisting of: TP53 and RBI, wherein reduced production or no production of a protein or RNA encoded by a gene selected from said group determines that the cell has a loss of function mutation in the gene, thereby determining whether the cell has a loss of function mutation in a gene of said group.

In the above enumerated embodiments, it is preferred that an individual to whom a Hedgehog inhibitor, preferably a SMO inhibitor, more preferably itraconazole is administered does not have a loss of function mutation in PTCH or a gain of function in SMO, as for example as is observed in a condition such as Gorlin’s syndrome. The PTCH and/or SMO genotype or phenotype of an individual may be known prior to the implementation of an above enumerated method, or may be determined as an embodiment of an above enumerated method.

In the above enumerated embodiments, it is preferred that a cancer cell for treatment with a Hedgehog inhibitor, preferably a SMO inhibitor, more preferably itraconazole has a phenotype including a higher than normal abundance of primary cilia. This phenotype of a cancer cell of an individual may be known prior to the implementation of an above enumerated method, or may be determined as an embodiment of an above enumerated method.

In the above enumerated embodiments, it is preferred that a cancer cell for treatment with a Hedgehog inhibitor, preferably a SMO inhibitor, more preferably itraconazole has a phenotype including autocrine expression of a Hedgehog ligand, for example Sonic Hedgehog, Indian Hedgehog or Desert Hedgehog. This phenotype of a cancer cell of an individual may be known prior to the implementation of an above enumerated method, or may be determined as an embodiment of an above enumerated method.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of this disclosure will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings.

FIGURE 1 is a schematic of model depicting the effect of Trp53 (p53) and Rbl (Rb) on Hedgehog (Hh) signaling. Hh, Smo and Gli are positive Hh pathway regulators, whereas Ptch is a negative Hh pathway regulator.

FIGURES 2A-2E are a series of graphs showing inhibition of Hedgehog signaling using SUBA-Itraconazole in Trp53 and Rbl mutant mouse embryonic fibroblasts (MEFs) and mouse osteosarcoma cells.

FIGURE 2A is a graph of GUI expression in p53Rb KO MEFs treated with PBS control, 1 mg/ml rhSHH, or rhSHH and 2pg/ml and 5pg/ml SUBA-Itraconazole for 24 hours, normalized to the expression of b2 -microglobulin n = 4, mean + SEM. ^**** p < 0.0001, two-way ANOVA / Tukey’s test.

FIGURE 2B is a graph of GUI expression in WT MEFs treated with PBS control, 1 mg/ml rhSHH, or rhSHH and 2pg/ml and 5pg/ml SUBA-Itraconazole for 24 hours, normalized to the expression of b2 -micro globulin. n = 4, mean + SEM. ^**** p < 0.0001, two-way ANOVA / Tukey’s test.

FIGURE 2C is a graph of GUI expression in mouse osteosarcoma cell line D12M treated with PBS control, 1 mg/ml rhSHH, or rhSHH and 2pg/ml and 5pg/ml SUBA-Itraconazole for 24 hours, normalized to the expression of b2 -micro globulin n = 4, mean + SEM. ^**** p < 0.0001, two- way ANOVA / Tukey’s test.

FIGURE 2D is a graph of GUI expression in mouse osteosarcoma cell line mOS25 treated with PBS control, 1 mg/ml rhSHH, or rhSHH and 2pg/ml and 5pg/ml SUBA-Itraconazole for 24 hours, normalized to the expression of b2 -micro globulin. n = 4, mean + SEM. ^**** p < 0.0001, two- way ANOVA / Tukey’s test.

FIGURE 2E is a graph of GUI expression in mouse osteosarcoma cell line mOS 18 treated with PBS control, 1 mg/ml rhSHH, or rhSHH and 2pg/ml and 5pg/ml SUBA-Itraconazole for 24 hours, normalized to the expression of b2 -micro globulin. n = 4, mean + SEM. ^**** p < 0.0001, two- way ANOVA / Tukey’s test.

FIGURES 3A-3C are a series of graphs showing inhibition of Smoothened signaling using SUBA-Itraconazole in human and mouse osteosarcoma in vivo.

FIGURE 3A shows graphs of tumour volume and Kaplan-Meier analysis of survival in human osteosarcoma xenografts MG63 treated with vehicle control or lOOmg/kg SUBA- Itraconazole. P < 0.0001, Log-rank (Mantel-Cox) test.

FIGURE 3B shows graphs of tumour volume and Kaplan-Meier analysis of survival in p53Rb KO mouse osteosarcoma allografts (D12M) treated with vehicle control or lOOmg/kg SUBA-Itraconazole. P < 0.0001, Log-rank (Mantel-Cox) test.

FIGURE 3C shows graphs of tumour volume and Kaplan-Meier analysis of survival in induced mouse osteosarcoma allografts (mOS18) treated with vehicle control or lOOmg/kg SUBA-Itraconazole. P < 0.0001, Log-rank (Mantel-Cox) test. DETAILED DESCRIPTION

The present disclosure features a method of treating cancer, including administering a therapeutically effective amount of a Hedgehog inhibitor ( e.g ., itraconazole) to a subject in need thereof. In some embodiments, the subject may be selected according to a set of criteria including expression of Hh ligand, the presence of primary ciliated cancerous cells (also referred to herein as ciliated cancerous cells), gene mutations in hedgehog signaling components, tumor suppressor TP53 somatic gene mutations, and/or tumor suppressor RBI somatic gene mutations, such that the subject would be responsive to treatment with the Hedgehog inhibitor (e.g., itraconazole). Definitions

Various terms that will be used throughout this specification have meanings that will be well understood by a skilled practitioner. However, for ease of reference, some of these terms will now be defined.

As used herein, the term “condition associated” with cancer refers to one or more symptoms or co-morbidities that associate with the relevant cancer.

The terms “treating” or “treatment” as used herein, refer to both direct treatment of a subject by a medical professional (e.g., by administering a therapeutic agent to the subject), or indirect treatment, effected, by at least one party, (e.g., a medical doctor, a nurse, a pharmacist, or a pharmaceutical sales representative) by providing instructions, in any form, that (i) instruct a subject to self-treat according to a claimed method (e.g., self-administer a drug) or (ii) instruct a third party to treat a subject according to a claimed method. Also encompassed within the meaning of the term “treating” or “treatment” are prevention of relapse or reduction of the disease to be treated, e.g., by administering a therapeutic at a sufficiently early phase of disease to prevent or slow its progression.

The terms "effective amount" or "therapeutically effective amount," as used herein, refer to a sufficient amount of an administered agent (i.e., a hedgehog inhibitor) which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.

As used herein, the term “drug” refers to a compound having beneficial prophylactic and/or therapeutic properties when administered to, for example, humans.

As used herein, the term “a solid dispersion” refers to a system in solid state including at least two components, wherein one component is dispersed more or less evenly throughout the other component or components. In particular, and with reference to a widely accepted definition from the early 1970's, “solid dispersions” are the dispersion of one or more active ingredients in an inert carrier or matrix at solid state, prepared by the melting, solvent, or melting-solvent methods.

As used herein, the term “matrix” refers to a medium that surrounds the Hedgehog inhibitor ( e.g ., itraconazole) particles.

As used herein, the term “in vivo” refers to in the living body of an animal such as human, whereas the term “in vitro” refers to outside the body and in an artificial environment.

Reference will also be made to the pharmacokinetic parameter AUC. This is a widely accepted parameter determined from the graphical presentation of actual or theoretical plasma profiles (concentration vs time), and represents the area under the curve (AUC) of such a profile.

As will be explained below, it will be appreciated that the pharmaceutical composition can include other components within it, such as disintegrants, diluents, fillers and the like.

As used herein, the term about, unless stated to the contrary, refers to +/- 10%, more preferably +/- 5%, of the designated value.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

"Itraconazole” is a common name for a triazole antifungal compound, the specific chemical structure and IUPAC name of which are well known in the art. It is available commercially (see Merck Index Reg. No. 5262 (12 ^th ed. 1996) and US 4,267,179). As used herein, "itraconazole" includes not only the chemical compound (free base form, also referred to as "free itraconazole"), but also all optical isomers, such as enantiomers, diastereomers, meso compounds, and the like, as well as pharmaceutically acceptable salts, solvates, and prodrugs (such as esters) thereof.

Patient population selection and cancer treatment methods

In some embodiments, the hedgehog signaling-ligand dependent cancer is, for example, osteosarcoma, prostate cancer, basal cell carcinoma, colorectal cancer, glioblastoma, medulloblastoma, rhabdomyosarcoma, pancreatic cancer, small cell lung cancer, and/or breast cancer.

The present disclosure features, inter alia, presents a method of treating cancer, including administering a therapeutically effective amount of a Hedgehog inhibitor ( e.g ., itraconazole) to a subject in need thereof. The subject may be responsive to cancer therapy using the Hedgehog inhibitor (e.g., itraconazole) when the subject expresses hedgehog ligand, has ciliated cancerous cells in the tumor biopsy sample, has gene mutations in hedgehog signaling components (e.g., PTCH1, SMO, SUFJJ, isochromosome 9, CDKN2A, and/or MDM2, in the tumor as a whole), and/or has genetic mutations in TP53 and/or RBI (e.g., in the tumor as a whole). The subject can be selected by: identifying genetic mutations in TP 53 and/or RBI. The gene mutation in TP 53, and/or RBI is preferably a loss-of-function mutation. These characteristics, individually or in any combination, are indicative of cancer cell hypersensitivity to the hedgehog ligand and thereby the likely response of the cancer to the Hedgehog inhibitor ( e.g ., itraconazole) treatment. For example, the subject can be responsive to cancer therapy using the Hedgehog inhibitor (e.g., itraconazole) when the subject expresses a hedgehog ligand, has ciliated cancerous cells in the tumor biopsy sample, and/or has genetic mutations in TP53 and/or RBI, and can be selected by: assessing the expression of hedgehog (e.g., SHH) ligand, identifying the presence and/or percentage of primary cilia, and/or identifying genetic mutations in TP 53 and/or RBI.

The Examples below demonstrate the effectiveness of itraconazole, for instance, in the treatment of cancers that include expression of Hh ligand, the presence of primary ciliated cancerous cells, gene mutations in hedgehog signaling components, tumor suppressor TP53 somatic gene mutations, and/or tumor suppressor RBI somatic gene mutations. A person of ordinary skill in the art would readily recognize that the effects of itraconazole, for instance, can be readily extrapolated to Hedgehog inhibitors in general.

In some embodiments, the subject is selected by obtaining a tumor biopsy sample from the subject; and (a) determining the presence and/or percentage of ciliated cancerous cells in the tumor biopsy sample, (b) identifying gene mutations in hedgehog signaling components (e.g., PTCH1, SMO, SUFU, isochromosome 9, CDKN2A, and/or MDM2), (c) assessing the expression of a hedgehog signaling ligand (e.g., in the tumor as a whole), and/or (d) identifying mutations in TP 53 and/or RBI (e.g., in the tumor as a whole).

In some embodiments, the subject is selected by obtaining a tumor biopsy sample from the subject; (a) determining the presence and/or percentage of ciliated cancerous cells in the tumor biopsy sample, (b) assessing the expression of a hedgehog ligand (e.g., in the tumor as a whole), and/or (c) identifying mutations in TP53 and/or RBI (e.g., in the tumor as a whole); and selecting the subject when the tumor biopsy sample has ciliated cancerous cells in the tumor biopsy sample, expresses a hedgehog ligand, and/or has genetic mutations in TP 53 and/or RBI.

In some embodiments, in addition or as an alternative to having cancer cells having primary cilia, the selected subject has cancerous cells that (i) express a hedgehog signaling ligand; (ii) have gene mutations in hedgehog signaling components (e.g., PTCH1, SMO, SUFU, isochromosome 9, CDKN2A, and/or MDM2 ); and/or (iii) have gene mutations in the tumor suppressor gene TP53 and/or RBI.

In some embodiments, the present disclosure features, inter alia, a method of treating basal cell carcinoma, medulloblastoma, or rhabdomyosarcoma, including administering a therapeutically effective amount of a Hedgehog inhibitor (e.g., itraconazole) to a subject in need thereof. The subject is selected by identifying one or more gain-of-function gene mutations in sonic hedgehog signaling components (e.g., PTCH1, SMO, SUFU, isochromosome 9, CDKN2A, and/or MDM2), and/or by identifying one or more loss-of-function gene mutations in the tumor suppressor genes TP 53 and RBI in a tumor biopsy sample from the subject. If the one or more loss-of-function gene mutations in the tumor suppressor genes TP53 and RBI are present in the tumor biopsy sample, the subject is selected for treatment with the Hedgehog inhibitor (e.g., itraconazole). As will be discussed below, itraconazole is a Hedgehog inhibitor and a representative Hedgehog inhibitor effective treat subjects having one or more gain-of-function gene mutations in sonic hedgehog signaling components (e.g., PTCH1, SMO, SUFU, isochromosome 9, CDKN2A, and/ r MDM2), and/or one or more loss-of-function gene mutations in the tumor suppressor genes TP53 and RBI in a tumor biopsy sample.

As an example, the cancer is a hedgehog signaling ligand-dependent cancer, such as osteosarcoma, prostate cancer, basal cell carcinoma, colorectal cancer, glioblastoma, medulloblastoma, rhabdomyosarcoma, pancreatic cancer, small cell lung cancer, and/or breast cancer. In some embodiments, the present disclosure presents a method of treating cancer, including administering a therapeutically effective amount of a Hedgehog inhibitor ( e.g ., itraconazole) to a subject in need thereof. The subject is selected by identifying activated sonic hedgehog signaling in a tumor biopsy sample from the subject, and if sonic hedgehog signaling is activated in the tumor biopsy sample, assessing the presence and/or percentage of ciliated cancerous cells in a tumor biopsy sample, and determining genomic mutations of TP53, RBI, or both TP 53 and RBI. The subject is then selected for treatment with the Hedgehog inhibitor (e.g., itraconazole) when sonic hedgehog signaling ligand is detected, the tumor biopsy sample expresses primary cilia, and when at least one of TP53 and RBI is genomically mutated as determined by targeted sequencing using known techniques available to the person skilled in the art.

In some embodiments, the percentage of ciliated cancerous cells is 10%, 20%, 30%, 40%, or 50%. The percentage of ciliated cancerous cells is calculated by determining the percentage of ciliated cancerous cells compared to the total number of cells in the tumor biopsy sample. The percentage can be readily determined by a person of ordinary skill in the art based on the present disclosure. In some embodiments, the cancer is a hedgehog signaling-dependent cancer, such as osteosarcoma, prostate cancer, basal cell carcinoma, colorectal cancer, glioblastoma, medulloblastoma, rhabdomyosarcoma, pancreatic cancer, small cell lung cancer, and/or breast cancer.

The Hedgehog inhibitor (e.g., itraconazole) of the methods of the present disclosure can be administered in any of a number of ways, such as by oral administration (e.g., in a solid oral dosage form, such as a tablet or a capsule), or by parenteral administration.

A hedgehog-signaling ligand dependent tumor biopsy sample can express hedgehog (Hh) ligand, for example, when assessed by immunohistochemical staining of the Hh ligand using one or more Hedgehog antibodies (e.g., Shh antibodies). For example, immunohistochemical analysis can be performed on formalin-fixed paraffin-embedded sections using the Vectastain ABC Elite IgG Kits (Vector Laboratories, Burlingame, USA, Cat no: PK-4000) using the following primary antibodies, Shh (1:200, Santa Cruz: sc9024) and Shh (1:200, Abeam: ab53281). Briefly, slides can be dewaxed in triplicate solutions of Histosol and rehydrated in duplicate solutions of 100% ethanol, for 5 minutes each, followed by immersion into 70%, 50% and 25% ethanol for 3 minutes each, and finally washed in lx PBS solution for 3x5 minutes. Antigen retrieval can be performed by heating slides in I OmM Sodium Citrate Buffer at full power until boiling followed by further heating on power setting 1 for 7 minutes. Incubation in solutions described from this point onwards can be carried out in a humid chamber that can include wet paper towels in an airtight container. In between each critical incubation step, 3x5 minute wash steps in lx PBS were completed. Sections can be encircled with a hydrophobic PAP pen and endogenous hydrogen peroxidase activity can be blocked with 1% H2O2 in lx PBS for 15 minutes at room temperature. 1.5 drops of goat serum in 5mL of lx PBS can be applied to sections and incubated at room temperature for 30 minutes, as a blocking step. One of the two sections can be incubated with primary antibody, diluted in 1% BSA/lx PBS, and the other can receive rabbit IgG as a negative control, and incubated overnight at 4°C. The following day sections can be re incubated with 1 drop of secondary biotinylated goat anti-rabbit IgG in lOmL of lx PBS for 30 minutes at room temperature. Sections can then be incubated in ABC Elite solution for 30 minutes at room temperature, followed by the addition of Sigmafast™ H2O2 activated 3,3 diaminobenzidine (DAB) tablets in 5mL of ultrapure H2O, which are visualized under a Nikon DS-Fil H550S brightfield photomicroscope and timed to monitor development. All slides can be kept to approximately the same development time to ensure consistency of staining intensity. Colour development is terminated in ultrapure H2O and sections are counterstained with hematoxylin for 3 seconds and Scott’s Tap Water for 30 seconds. Sections can then be dehydrated in 25%, 50% and 75% ethanol solutions for 3 minutes each and immersed in triplicate solutions of 100% ethanol and duplicate solutions of Histosol for 5 minutes each. Slides can then be mounted onto coverslips with DPX mounting medium, allowed to dry and imaged. These methods can be utilized to determine or assess either autocrine or paracrine expression of Hedgehog ligands by cells, such as cancer cells, especially ciliated cancer cells.

In some embodiments, the tumor biopsy sample express primary cilia. Immunofluorescence staining of primary cilia can be performed on formalin-fixed paraffin- embedded sections using the following primary antibodies, acetylated alpha-tubulin (1 :500, Sigma T7451) and Arll3b (1:500, ProteinTech: 1711-1-AP). Briefly, slides can be dewaxed in triplicate solutions of Histosol and rehydrated in duplicate solutions of 100% ethanol, for 5 minutes each, followed by immersion into 70%, 50% and 25% ethanol for 3 minutes each, and finally washed in lx PBS solution for 3x5 minutes. Antigen retrieval can be performed by heating slides in Sodium Citrate Buffer at full power until boiling followed by further heating on power setting 1 for 7 minutes. Incubation in solutions described from this point onwards can be carried out in a humid chamber consisting of wet paper towels in an airtight container and in between each critical incubation step, 3x5 minute wash steps in lx PBS can be completed. Sections can be encircled with a hydrophobic PAP pen and blocked with blocking buffer (PBS containing 20% of a 30% BSA solution, 7.5% goat serum, 0.0025% Tween-20) for 1 hour at room temperature. One of the two sections can be incubated with primary antibody, diluted in Incubation buffer (PBS containing 10% of a 30% BSA solution, 5% goat serum, 0.003% Tween-20), and the other can receive rabbit IgG as a negative control, and incubated overnight at 4°C. The following day sections can be washed 3 x 5 minutes in PBS and incubated in secondary antibody (Alexa 488 goat anti-mouse: 1:1000; and Alexa 568 goat anti-rabbit: 1:1000; Molecular Probes) and DAPI nuclear stain (1:1000, Sigma) for 1 hour at room temperature. Sections can then be dehydrated in 25%, 50% and 75% ethanol solutions for 3 minutes each and immersed in triplicate solutions of 100% ethanol and duplicate solutions of Histosol for 5 minutes each. Slides can be mounted onto coverslips with DPX mounting medium, allowed to dry and imaged using a confocal microscope. Without wishing to be bound by theory, it is believed that the cilia frequency (percentage of ciliated cells counted in multiple (e.g., 2, 3, 4, 5, or 10) random fields of the tissue sample) is indicative of potential Hedgehog pathway ligand dependent activation and can predict response of a subject (from which the tumor biopsy sample is obtained) to treatment with a Hedgehog inhibitor (e.g., itraconazole). In some embodiments, the higher the percentage of primary cilia in a given tumor biopsy sample, the greater the responsiveness of the subject (from which the tumor biopsy sample is obtained) to treatment with the Hedgehog inhibitor (e.g., itraconazole). Thus, in some embodiments, the present disclosure features a method for treating cancer, including determining the percentage of ciliated cancerous cells in tumor biopsy samples obtained from a cancer patient population, where the greater the percentage of ciliated cancerous cells in a given tumor biopsy sample, the more responsive the corresponding patient would be to treatment with the Hedgehog inhibitor (e.g., itraconazole); and selecting patients having tumor biopsy samples having greater percentages of ciliated cancerous cells for treatment with the Hedgehog inhibitor (e.g., itraconazole). The ciliated cancerous cells can further express hedgehog ligand, have gene mutations in hedgehog signaling components, or express hedgehog ligand and have gene mutations in hedgehog signaling components.

In some embodiments, primary cilia can be identified by immunofluorescence co localization of acetylated alpha-tubulin (AcTub), a marker of the ciliary axonemes microtubules, and Aril 3b, a marker of the ciliary membrane as described above.

In some embodiments, the tumor biopsy sample is treated with a 5 pg/mL itraconazole solution, in vitro, for 24 hours; and measuring GLI1 expression in the itraconazole-treated tumor biopsy sample; the treated sample can have a decrease in GLI1 expression compared to a control tumor biopsy sample that was not treated with itraconazole. The in vitro treatment of the tumor biopsy sample with a 5 pg/mL itraconazole solution can be conducted in the presence of hedgehog signaling ligand stimulation. In some embodiments, the in vitro treatment of the tumor biopsy sample with a 5 pg/mL itraconazole solution is conducted in the absence of hedgehog signaling ligand stimulation, when the tumor biopsy sample has a gain-of-function gene mutations in hedgehog signaling components. The tumor biopsy sample can be subjected to serum starvation before determining the presence and/or percentage of ciliated cancerous cells.

For example, as will be discussed in Example 1 below, using a panel of cancer cell lines with previously reported genotypes, primary cilia frequency can be assessed using immunofluorescence. Cilia can be either undetectable or at low frequency in certain cancer cell lines both with wild type TP53 and RBI status. In contrast, primary cilia can be frequently detected in cell lines with reported TP53 mutations or rearrangements and perturbation of the RB signaling pathway (such as via CDKN2A deletion) under both serum-starved and normal serum conditions. In some embodiments, the localization of SMO to the primary cilia, a necessary event required for signal transduction in hedgehog-dependent cancer cells, can additionally or alternatively be visualized. Under serum- starved culture conditions SMO infrequently (e.g., <20%) co-localized to the primary cilia consistent with low levels of Hh signaling activation in the cells. In response to treatment with Hh ligand, SMO can translocate into the primary cilia at a much increased frequency (e.g., 100%), which would indicate that these cells are responsive to Hh ligand stimulation. In contrast, basal and ligand-stimulated SMO translocation to the primary cilia can be completely abolished by treatment with an SMO inhibitor. Thus, mutations in TP 53 and RBI, or disruption of their pathways, and/or primary cilia frequency can be predictive of likely response to Hh pathway inhibition.

Compositions

In any of the methods above, the Hedgehog inhibitor (e.g., itraconazole) can be a solid dispersion of the Hedgehog inhibitor (e.g., itraconazole) particles in an acid-stable polymeric matrix. The itraconazole particles in the solid dispersion can include microparticles, nanoparticles, or a combination thereof. In vitro dissolution testing of a solid dispersion of the present disclosure can show itraconazole microparticles and/or nanoparticles when tested at a pH in the range of 5.5 to 7.5. Furthermore, when pretreated at acidic pH (namely, when suspended in a dissolution medium at a pH of about 1.2 for a period of about 20 minutes), in vitro dissolution testing of a solid dispersion of itraconazole particles can show microparticles and nanoparticles when subsequently tested at a pH in the range of 5.5 to 7.5. This pre-treatment can be conducted to simulate in vivo conditions.

In some embodiments, the itraconazole can be in a composition that forms a suspension in vitro at a pH in the range of 5.5 to 7.5, the suspension having itraconazole particles in the size range of 1 nm to 10 micrometers, with or without a pre-treatment at acidic pH. In some embodiments, the suspension has at least a portion of its itraconazole particles in the size range of 1 nm to 450 nm in vitro at a pH in the range of 5.5 to 7.5, again with or without a pre-treatment at acidic pH.

In some embodiments, when administered to a subject, the solid dispersions of the present disclosure can form a suspension in vivo. The suspension can be a homogeneous dispersion of itraconazole particles, the particles at least being of a size where, in vitro, they diffract light such that the suspension presents as a cloudy suspension. Evidence of the presence of such a cloudy suspension can be seen during in vitro dissolution testing of the solid dispersion present disclosure.

The itraconazole particles in the cloudy suspension in vitro can include particles of a size greater than about 1 nm but less than about 10 micrometers. In some embodiments, the itraconazole particles are present in nanoparticulate form, such as in the range of 1 nm to 450 nm, and/or a portion of the itraconazole particles are present in microparticulate form (such as in the range of 0.45 micrometer to 10 micrometers). The presence of such nanoparticles in vivo can be determined by testing for them in vitro, such as by passing the cloudy suspension through a 450 nm filter and having the suspension remain cloudy. Such nanoparticles can be present regardless of whether the acidic pre-treatment step described above is utilized in the testing procedure. As discussed above, the composition and/or the methods include a solid dispersion of a Hedgehog inhibitor (e.g., itraconazole) particles in an acid-stable polymeric matrix. The polymers useful for forming the solid dispersion of the pharmaceutical composition can have acidic functional groups. For example, the polymers can be polycarboxylic acids. Such polycarboxylic acids can be any polycarboxylic acid which, when in a solid dispersion with the Hedgehog inhibitor (e.g., itraconazole), results in the formation of the above-mentioned suspension, in the preferred pH ranges, and can provide acceptable absorption in the intestines. In some embodiments, the compositions of the present disclosure upon administration forms a suspension at a pH in the range of 4.0 to 8.0 (e.g., 5.5 to 7.5), and can provide acceptable absorption in the intestines. Thus, in some embodiments, the polymeric matrix of the present disclosure dissolves at a pH of 5.5-7.5, and/or does not dissolve at gastric pH.

The polymers of the polymer matrix can be one or more of hydroxypropyl methylcellulose phthalate (hypromellose phthalate, e.g., HP-50), polyvinyl acetate phthalate (PVAP), hydroxypropylmethylcellulose acetate succinate (HPMCAS), alginate, carbomer, carboxymethyl cellulose, methacrylic acid copolymer, shellac, cellulose acetate phthalate (CAP), starch glycolate, polacrylin, methyl cellulose acetate phthalate, hydroxypropylcellulose acetate phthalate, cellulose acetate terephthalate, cellulose acetate isophthalate and/or cellulose acetate trimellitate, including various grades of each polymer such as HPMCAS -LF, HPMCAS -MF and/or HPMCAS -HG.

In some embodiments, the polymer is a polycarboxylic acid such as a hydroxypropyl methylcellulose phthalate (i.e., hypromellose phthalate) such as that available from Shin-Etsu Chemical Industry Co. Ltd as HP-50, HP-55 or HP-55S. However, in some embodiments, alternatives such as the use of an aqueous based enteric polymer, such as the dispersion Eudragit L30D, or enteric polymers dissolved in water with the addition of ammonia or alkaline agents, are useful. In the solid dispersions of the present disclosure, the ratio of itraconazole to polymer can be in the range of from 3:1 to 1:20. In some embodiments, the ratio of itraconazole to polymer can be in the range of 3:1 to 1:5 (e.g., 3:1 to 1:5, 1:1 to 1:3, 1:2, or 1:1.5 (or 2:3)). In certain embodiments, the ratio of itraconazole to polymer can be in the range of 1:1 to 1:3. In some embodiments, the ratio of itraconazole to polymer can be about 1:1.5 (or 2:3).

The compositions of the present disclosure can be in the form of a solid oral dosage form (e.g., capsule, tablet, or pill).

The solid dispersions of itraconazole can be formed by spray-drying techniques, although it will be understood that suitable solid dispersions can be formed by a skilled practitioner utilizing other conventional techniques, such as co-grinding, melt extrusion, freeze drying, rotary evaporation or any solvent removal process.

In some embodiments, the solid dispersion is formed by dispersing or dissolving the itraconazole and the polymer in a suitable solvent, and subsequently spray drying to form the solid dispersion in the form of a powder. Suitable solvents or dispersion media include methylene chloride, chloroform, ethanol, methanol, propan -2-ol, ethyl acetate, acetone, water or mixtures thereof.

Other excipients can then be blended into the powder (with or without milling or grinding) to form a composition suitable for use in dosage forms such as tablets and capsules.

The compositions of the present disclosure can be formulated into pharmaceutical dosage forms including a therapeutically effective amount of the composition. The dosage forms can include a range of traditional excipients such as disintegrants, diluents, fillers, lubricants, glidants, colorants and flavors. For example, suitable disintegrants can include those that have a large coefficient of expansion, and examples can include crosslinked polymers such as crospovidone (crosslinked polyvinylpyrrolidone), croscarmellose (crosslinked sodium carboxymethylcellulose), and sodium starch glycolate. The dosage form can include an inert substance such as a diluent or a filler. A variety of materials can be used as diluents or fillers, and examples can include sucrose, dextrose, mannitol, sorbitol, starch, micro-crystalline cellulose, and others known in the art, and mixtures thereof.

Lubricants and glidants can be employed in the manufacture of certain dosage forms, and can be employed when producing tablets. Examples of lubricants and glidants are hydrogenated vegetable oils, magnesium stearate, stearic acid, sodium lauryl sulfate, magnesium lauryl sulfate, colloidal silica, talc, mixtures thereof, and others known in the art. A preferred lubricant is magnesium stearate, or mixtures of magnesium stearate with colloidal silica. Excipients such as coloring agents and pigments can also be added to dosage forms in accordance with the present invention, and suitable coloring agents and pigments can include titanium dioxide and dyes suitable for food.

Flavors can be chosen from synthetic flavor oils and flavoring aromatics or natural oils, extracts from plants, leaves, flowers, fruits and so forth and combinations thereof. These can include cinnamon oil, oil of wintergreen, peppermint oils, bay oil, anise oil, eucalyptus, thyme oil. Also useful as flavors are vanilla, citrus oil, including lemon, orange, grape, lime and grapefruit, and fruit essences including apple, banana, pear, peach, strawberry, raspberry, cherry, plum, pineapple, apricot and so forth.

The compositions of the present disclosure can be administered with food or without food. The composition of the present disclosure can provide for acceptable absorption of at least the itraconazole in the intestines where the pH is expected to be in the range of 5.5 to 7.5.

With reference to the pharmacokinetic performance of pharmaceutical compositions in accordance with the present disclosure, it will be appreciated that the parameters that are commonly used in the art to describe the in vivo performance of a formulation (or the bioavailability) are C _max (the maximum concentration of the active in the blood) and, as mentioned previously, AUC (area under the curve — a measure of the total amount of drug absorbed by the patient). These are also the parameters used by regulatory agencies around the world to assess bioequivalence of different formulations. For instance, to be considered bioequivalent, the 90% confidence interval for the ratio of the test to reference product (using natural log -transformed data) for Cmax and AUC are within the range of 80 to 125%.

Compositions of practically insoluble drugs, such as itraconazole, are described, for example, in U.S. Patent Nos. 6,881,745 and 8,771,739, and WIPO application No. PCT/AUOO/01592, each of which is herein incorporated by reference in its entirety.

As described in the above enumerated embodiments 1 to 8, the invention provides methods for preventing or treating cancer, compositions for use in treating cancer, methods for determining the likelihood an individual developing an anti -cancer response, or for selecting an individual for treatment of cancer or for administration of a Hedgehog inhibitor. In these embodiments the cancer the subject of treatment may be characterized by autocrine expression or production of a Hedgehog ligand, or the cancer may not express Hedgehog ligand but is exposed to stromal tissue that expresses or produces Hedgehog ligand. The exposure to autocrine or paracrine produced Hedgehog ligand drives ligand dependent Hedgehog signaling in the cancer cell.

As described in the above enumerated embodiments 1 to 8, there is provide methods involving administration or uses of Hedgehog inhibitors. In these embodiments a Hedgehog inhibitor or Hedgehog pathway inhibitor may be selected from the group consisting of Taladegib (LY2940680), sulforaphane, vismodegib, TAK-441, itraconazole, and erismodegib or the group of Hh ligand inhibitors (e.g., 5E1, robotnikinin), or SMO antagonists (e.g., Taladegib (LY2940680), vismodegib, IPI-926, HhAntag), or Gli-processing inhibitors (e.g., HPI-2, HPI-3, HPI-4, arsenic trioxide). In some embodiments, the Hh inhibitor is described and disclosed in U.S. Patent 7,230,004, U.S. Patent Application Publication No. 2008/0293754, U.S. Patent Application Publication No. 2008/0287420, U.S. Patent Application Publication No. 2008/0293755. Additional examples of Hh inhibitors include, but are not limited to, those described in U.S. Patent Application Publication Nos. US 2002/0006931, US 2007/0021493 and US 2007/0060546, and International Application Publication Nos. WO 2001/19800, WO 2001/26644, WO 2001/27135, WO 2001/49279, WO 2001/74344, WO 2003/011219, WO 5WO 2017/165663 PCT/US2017/023839 5 10 15 20 25 302003/088970, WO 2004/020599, WO 2005/013800, WO 2005/033288, WO 2005/032343, WO 2005/042700, WO 2006/028958, WO 2006/050351, WO 2006/078283, WO 2007/054623, WO 2007/059157, WO 2007/120827, WO 2007/131201, WO 2008/070357, WO 2008/110611, WO 2008/112913, and WO 2008/131354. Additional examples of Hh inhibitors include, but are not limited to, BMS833923 (also known as XL139) (see, e.g., Siu L. et al., J. Clin. Oncol. 2010; 28: 15s (suppl; abstr 2501)); LDE-225 (see, e.g., Pan S. et al., ACS Med. Chem. Lett., 2010; 1(3): 130-134); LEQ-506 (see, e.g., National Institute of Health Clinical Trial Identifier No. NCT01106508); PF-04449913 (see, e.g., National Institute of Health Clinical Trial Identifier No. NCT00953758); Hh pathway antagonists disclosed in U.S. Patent Application Publication No. 2010/0286114; SM012-17 (see, e.g., U.S. Patent Application Publication No. 2010/0093625); SANT-1 and SANT-2 (see, e.g., Rominger CM. et al., J. Pharmacol. Exp. Then 2009; 329(3):995-1005; l-piperazinyl-4-arylphthalazines or analogues thereof (see, e.g., Lucas B.S. et al., Bioorg. Med. Chem. Lett. 2010; 20(12):3618-22).

As described in the above enumerated embodiments 1 to 8, mutation at the TP53 and/or RB 1 loci are utilized to select for patients for treatment with a Hedgehog inhibitor. Typically, the mutation is a loss of function mutation i.e. a mutation that causes a gene product encoded by, or transcribed from, or translated from TP53 and/or RB 1 to have abnormal quantity and/or abnormal quality. A loss of function mutation may lead to production of an abnormal amount of a TP53 or RB 1 gene product, for example an abnormally high, preferably an abnormally low amount of gene product, or no gene product at all. A loss of function mutation may lead to production of a gene product that has sub-optimal function, or no function, when compared to the function of wild type or normal gene product. In embodiments herein, a loss of function mutation of TP53 or RBI genes may lead to sub-optimal, reduced or absence of the normal or physiological function of TP53 and RBI in autophagy and re-cycling of primary cilia on cells. Examples of loss of function mutations are described in Baugh E H et al. 2018 Cell Death and Differentiation, 25: 154-160; Knudsen E S et al 2020 Comms. Biol. 3:158.

As described in the above enumerated embodiments 1 to 8, a reduced production or no production of TP53 or RB 1 protein or RNA may be indicative of an individual having cancer that is susceptible to treatment with a Hedgehog inhibitor. The measurement or detection of functional TP53 or RB 1 can be done utilizing methods known to the skilled worker, or utilizing methods exemplified in the examples that follow. These methods enable one to determine whether there is no production of TP53 or RBI in a cancer cell. These methods also enable one to determine whether there is a reduced production or expression of TP53 or RBI in a cancer cell, for example by comparing the measured amount of TP53 or RBI in a cancer cell with the amount of TP53 or RB 1 in a control defining the amount of TP53 or RB 1 in a non-cancerous cell, preferably a sex, age or tissue matched control.

The following examples are included for the purpose of illustrating, not limiting, the described embodiments.

EXAMPLES

Example 1. Investigation of TP53 and RBI in the Regulation of Autophagy, Primary Cilia Formation and Hedgehog Signaling

To date, there is no genetic biomarker that can predict which cancers expressing Hedgehog (Hh) ligands are dependent on autocrine signaling. Using a combination of genetically modified mice, cell lines, developmental and cancer models, evidence is presented herein that inactivation of the tumor suppressor genes TP53 and RBI results in impaired autophagy, aberrant cilia formation and sensitization of the canonical Hh pathway to ligand-dependent signaling. These findings provide insights into the importance of tumor suppressor genes in development, and into the use of Hh inhibitors as cancer therapies by identifying a genetic basis for dependence on ligand- dependent Hh signaling in small cell lung cancer (SCLC) and osteosarcoma and for the treatment of other common cancers in which both TP53 and RBI are inactivated.

Ligand-dependent activation of Hh signaling in cancer occurs without mutations in canonical pathway genes. In this Example, the combined inactivation of TP53 and RBI is shown to disrupt components of the autophagic machinery necessary for the degradation of primary cilia, an organelle essential for canonical Hh signaling. This in turn is associated with hypersensitivity to Hh ligand signaling. Also shown is a requirement for Hh signaling in small cell lung cancer (SCLC), uniformly characterized by TP53 and RBI pathway deficiency, and osteosarcoma, a malignant bone tumor that frequently carries somatic mutations in both TP 53 and RBI. Despite the loss of these potent tumor suppressors, the data show that loss of autophagy, primary cilia formation and sensitization to Hh ligand are needed to maintain a malignant, poorly differentiated osteoblast phenotype in osteosarcoma. The results provide a mechanistic framework for aberrant Hh signaling in cancers lacking both TP 53 and RBI, which indicate an important role for the Hh signaling pathway in the maintenance of an undifferentiated malignant phenotype.

Results

Genetic inactivation of Trp53 and Rbl enhances Hedgehog ligand responsiveness in vitro

To investigate the relationship between Trp53 and Rbl mutation status and ligand- dependent Hedgehog signaling, mouse embryonic fibroblasts (MEFs) were utilized in which either Trp53 ( p53 KO), Rbl ( Rb KO) or Trp53 and Rbl ( p53Rb KO) were genetically inactivated through a conditional knockout strategy. In each case, these MEF lines expressed the unprocessed 45kDa SHH protein, but low levels of the 19 kDa active fragment, suggesting that autocrine and/or paracrine signaling was active at low levels. However, when treated with exogenous SHH ligand, upregulation of GUI mRNA, a robust transcriptional target of canonical Hh signaling in murine cells (7) was dramatically enhanced in p53 KO, Rb KO, and p53Rb KO MEFs. This response was completely abrogated by the small molecule SMO inhibitor, LDE225 (8), confirming that the induction of GUI is mediated through canonical activation of the Hh pathway. Since primary cilia are required for canonical Hh signaling via SMO (9), whether an increase in cilia formation was associated with hypersensitivity to Hh ligand in the MEF models of the present example was investigated.

Primary cilia can be identified by immunofluorescence co-localization of acetylated alpha- tubulin (AcTUB), a marker of the ciliary axonemes microtubules, and ARL13B, a marker of the ciliary membrane (10). Cilia induction in cultured cells can be achieved by serum starvation, which is thought to trigger cilia formation due to cell cycle arrest (11). Confocal microscopy analysis showed that p53 KO, Rb KO and p53Rb KO MEFs displayed a 2-4-fold increase in the proportion of ciliated cells compared with their wild type counterparts in serum deprived conditions. In p53Rb KO MEFs, knockdown of KIF3A, a kinesin protein essential for the formation of primary cilia (12), led to a reduction of primary cilia formation, and a commensurate attenuation of the GUI mRNA response induced by SHH ligand. More surprising was the observation that in full serum conditions, cilia were readily detected in all three knockout lines. These results suggest that aberrant cilia formation is the most likely explanation for the enhanced sensitivity to ligand- dependent, canonical Hh signaling seen in p53 KO, Rb KO and p53Rb KO MEFs.

Primary cilia are usually observed in post-mitotic cells, since activation of aurora kinase A and polo-like kinase 1 during the S and G2 phase of the cell cycle trigger cilia resorption (13). Therefore, whether changes in the cell cycle could explain the excess cilia formation seen in the knockout MEF models was investigated. Cell cycle analysis of p53 KO and p53Rb KO MEFs revealed an increase in G2M phase regardless of serum conditions, whereas WT and Rb KO MEFs exhibited similar cell cycle profiles. Co-staining for proliferating cell nuclear antigen (PCNA) and ARL13B revealed that primary cilia could be readily detected in p53 KO, Rb KO and p53Rb KO MEFs in S phase, but not in WT controls. These data show that excess cilia formation in knockout MEF lines cannot be explained by arrest in GO or G1 and suggest that deletion of Trp53 and Rbl disrupts the normal relationship between cell cycle, serum starvation and the formation of primary cilia in vitro.

Genetic inactivation of Trp53 and Rbl disrupts autophagy in vitro

Recent evidence suggests that components of the autophagy pathway play an essential role in the constitutive degradation of cilia in replicating cells in both nutrient-rich and starvation conditions (14). Previous work also suggests that both Trp53 and Rbl can influence the expression of autophagy- related genes (15-17). To explore this potential connection, the autophagic flux in the MEF models was examined by measuring changes in the expression of LC3-II and P62, markers of autophagosome formation (14). In the presence of Bafilomycin A1 (Baf-A), an inhibitor of lysosomal acidification, accumulation of LC3-II or P62 can be used as a marker of autophagic flux by comparing expression in 10% serum to serum starved cells (18, 19). As expected, autophagic flux was upregulated in WT MEFs following serum starvation compared to normal serum culture conditions. In contrast, autophagic flux was reduced in p53 KO, Rb KO and p53Rb KO MEFs, consistent with defective autophagy upstream of autophagosome formation. Similar results were obtained with the lysosomal inhibitor chloroquine.

The formation of autophagosomes can also be assessed by quantitative immunofluorescence for co-localisation of LC3, and an associated protein, P62, in discrete puncta in cultured cells treated with serum deprivation and Baf-A (20). The loss of Trp53, Rbl and both Trp53 and Rbl in MEFs results in a progressive reduction in LC3/P62 puncta formation in serum- starved conditions compared to WT MEFs. Taken together, these data indicate that loss of function mutations in both Trp53 and Rbl can inhibit components of the autophagic pathway that lead to the formation of the autophagosome in serum-starved conditions compared to WT MEFs.

Impaired upregulation of autophagy in serum-deprived conditions could be due to an inability of the P53 and RB 1 -deficient cells to sense changes in the surrounding environment. To explore this, the expression of AMP-activated protein kinase (AMPK), a critical cellular energy sensor that is normally activated in response to nutrient deprivation (21), was analyzed in WT, p53 KO, Rb KO and p53Rb KO MEFs. In serum-deprived conditions, activated AMPK (p-AMPK) increased in all genotypes similar to WT MEFs, suggesting no difference in nutrient sensing. An intact nutrient sensing pathway is also supported by the observed changes in primary cilia, PCNA positive cells and cell cycle in response to serum starvation. Furthermore, the AMPK agonist AICAR, failed to restore autophagic flux in p53Rb KO MEFs.

The expression of 84 autophagy -related genes was determined in the MEF models using a custom quantitative RT-PCR array. In serum-deprived conditions, expression of 30% (25/84) of these genes was significantly downregulated in p53 KO, Rb KO and p53Rb KO MEFs, 2 genes were downregulated in a Rbl -specific manner, 6 genes downregulated in a Tr/xT -specific manner and an additional 4 genes were downregulated only in p53Rb KO MEFs. These included downregulation of the critical autophagy genes Atg5, Atg9b, Ctsd, Cxcr4, Dapkl, Pik3cg and Tnf. Knockdown of Atg5, Atg9b, Ctsd or Pik3cg in wild type MEFs resulted in an enhanced GUI mRNA response to treatment with SHH ligand, demonstrating that genes in the autophagic pathway that are regulated by both P53 and RBI can also act to attenuate canonical Hh signalling.

Comparison of significantly down -regulated genes with published P53 ChIP-seq datasets revealed that of the 56 significantly downregulated genes in the p53 KO MEFs, >32% (18/56) are direct transcriptional targets of P53 (17). Moreover, 89% (16/18) of these direct targets are also significantly reduced in p53Rb KO MEFs and include the autophagy core machinery genes, Atg4a, Atg4c, Atg7, Ulkl and Uvrag, and the lysosomal protein encoding gene Ctsd. Additionally, the E2F1 target genes, Maplc3a (encoding FC3), Ulkl, Atg5 and Bnip3 are significantly downregulated in Rb KO and p53Rb KO MEFs (15). Taken together, these data suggest a model in which P53 and RB 1 directly regulate the expression of genes involved in autophagy, which in turn mediates primary cilia formation and an enhanced cellular response to Hh ligand via the canonical signaling pathway (Figure 1).

Restoration of P53 and RBI function normalizes autophagy, Hedgehog signaling and cilia formation in vitro

To further validate the model proposed in Figure 1, whether re-introduction of both P53 and RBI expression could reverse the effects on Hh signaling, primary cilia formation and autophagy described in the p53Rb KO MEF model was investigated. Lentiviral transduction of p53Rb KO MEFs with mouse Trp53 ORF and mouse Rbl ORF clones tagged with GFP and mCherry, respectively, resulted in robust re-expression of P53 and RBI protein, 72 hrs post infection. Assessment of transduction efficiency by fluorescent imaging and FACS demonstrated low transduction of p53-GFP (<10% of cells) and high transduction of Rb-mCherry (>60% of cells). Notably, nearly all p53-GFP transduced cells were also transduced with Rb-mCherry. Attempts to generate stably expressing p53-GFP and Rb- mCherry clones were unsuccessful since transduced cells progressively underwent senescence and died, consistent with the functional roles of P53 and RBI. Accordingly, all subsequent experiments were performed within 72 hrs of transduction.

In p53Rb KO MEFs, re-expression of both P53 and RBI significantly reduced the GUI mRNA response to SHH ligand treatment to levels more consistent with WT MEFs. In keeping with this result, cilia formation was also inhibited. Reintroduction of P53 and RBI also restored the expression of the autophagy genes Atg5, Atg9b and Ctsd in serum-free conditions, and resulted in a corresponding increase in autophagic flux. These results support a direct connection between genes regulated by both P53 and RBI, autophagy, the formation of primary cilia and the canonical response to Hh ligand.

Requirements for primary cilia in murine lung cancer models In keeping with loss of function mutations in both TP53 and RBI seen in human SCLC (24), mice carrying conditional loxP knockout alleles of both Trp53 and Rbl develop SCLC within 9 months following the inhalation of viral vectors expressing Cre recombinase (25). In this model, a genetic requirement for both Smo (5) and Shh (6) supports findings in human SCLC cell lines that ligand- dependent activation of Hh signaling is functionally important in this disease (4). To test whether a connection between loss of Trp53 and Rbl , Hh signaling and primary cilia formation could be established in a clinically relevant cancer model; whether there was a genetic requirement for the essential cilia protein KIF3A in murine SCLC (mSCLC), and in a counterpart lung adenocarcinoma model (mLUAD) driven by conditional activation of the Kras G12D mutant (26) was determined.

Mice homozygous for conditional loxP knockout alleles of both Trp53 and Rbl ( p53Rb KO) were crossed with mice carrying loxP Kif3a knockout alleles (27) in order to generate triple compound homozygotes ( p53Rb Kif3a KO). In p53Rb KO mice, administration of inhaled recombinant adenoviruses expressing Cre recombinase under a CMV promoter (AdCre) (28) resulted in SCLC tumor formation as expected (25). In p53Rb Kϊba KO mice, AdCre treatment resulted in a marked reduction in both the number and size of tumors. The small tumors that were observed in p53Rb Kif3a KO mice expressed very few primary cilia compared to their p53Rb KO counterparts.

This approach was then repeated in a mLUAD model by crossing mice with a conditional activating Kras G12D mutant allele (Kras) with the conditional Kif3a knockout line (Kras Kif3a KO). In both genotypes, treatment with inhaled AdCre resulted in the formation of multiple adenocarcinomas. Few primary cilia were observed in the tumor cells of both genotypes. These results show that mSCLC can be differentiated from mLUAD, not only by the phenotypic connection to the loss of both Trp53 and Rbl, but by a requirement for the formation of primary cilia. The lack of requirement for primary cilia in the mLUAD model extends the importance of the present findings in MEFs and in the developing neural tube to a model of a common, lethal human cancer.

Autophagic flux and primary cilia formation in murine lung cancer models

The importance of Hh signaling in SCLC, but not in LUAD, was first suggested by differential sensitivity to the SMO antagonist cyclopamine, despite the fact the both tumor types express SHH ligand (4). In light of the results showing enhanced sensitivity to Hh ligand on the basis of both Trp53 and Rbl mutations in MEFs, this question was re-examined in cell lines derived from the mSCLC and mLUAD models. In keeping with the results in MEFs, both mSCLC and mLUAD cells expressed the unprocessed, 45 kDa form of SHH in vitro, however the active 19kDa form was expressed at low levels. These results suggest that in vitro, processing of SHH ligand may not be sufficient for optimal autocrine/juxtacrine signaling.

Treatment of both mSCLC and mLUAD cells with exogenous SHH ligand revealed that mSCLC cells deficient in both Trp53 and Rbl exhibited a strong GUI mRNA response, whereas Kras mutant mLUAD cells showed no response. In keeping with this result, primary cilia were not observed in mLUAD cells, in contrast to reports of cilia in both human and murine SCLC (5). Interrogation of autophagic flux in these cell line models were also in keeping with the MEF results. In mSCLC, consistent reductions in flux measured by LC3 and P62 accumulation, and in puncta formation were seen when compared with mLUAD cells. Assessment of autophagic flux in a cell line derived from the more aggressive mLUAD model in which Trp53 is conditionally inactivated on the Kras G12D background ( Kras p53 KO) revealed no change in LC3 flux and an intermediate reduction in P62 flux compared to the Kras mLUAD. No GUI mRNA response to exogenous SHH ligand was observed. Additionally, in contrast to the Kras mLUAD model, primary cilia were detectable in the Kras p53 KO mLUAD model in vitro, and were comparable in vivo, but not to the same degree seen in mSCLC tumours. Consistent with the above results, increased primary cilia frequency and reduced LC3 and P62 flux were observed in TP53 and RBI- deficient human SCLC cell lines, compared to a TP53 and RB 1 wildtype human LUAD cell line. These results suggest that exogenous Hh ligand may be an important biological tool in understanding the capacity of tumor cells to respond to canonical Hh signaling and may explain discrepancies between in vivo and in vitro models in this regard. Further, these data show that response to Hh ligand in the setting of combined mutations in both Trp53 and Rbl can be replicated in a clinically relevant tumor model along with corresponding reductions in autophagic flux and the formation of primary cilia.

Cilia Formation and Ligand-Dependent Hh Signaling in Murine Osteosarcoma

Mutations in both TP53 and RBI occur frequently in osteosarcoma, the most common primary tumor of bone (Kansara et al., 2014 PMID:25319867). To explore the relationship between Trp53 and Rbl, and Hh signaling in osteosarcoma, two distinct mouse models of osteosarcoma were utilized: (i) the Ca45 radiation-induced model ( ⁴⁵ Ca) (Kansara et al., 2009 PMID: 19307728); and (ii) the Trp53 and Rbl conditional genetic model (p53Rb KO) (Walkley et al., 2008 PMID: 18559481) in which Trp53 and Rbl are specifically inactivated in the osteoblast lineage using OsxCre. Cell lines derived from both models express unprocessed Shh protein and low levels of the active amino-terminal peptide. ⁴⁵ Ca cell lines showed a variable mutational status in Trp53, Rbl and Cdkn2a. To determine Hh ligand responsiveness, a panel of ⁴⁵ Ca and p53Rb KO cell lines was treated with Shh ligand. Canonical Hh pathway activation measured by Glil expression in ⁴⁵ Ca cell lines was variable. In contrast, all OsxCre p53Rb KO cell lines were highly responsive. As expected, inhibition of Smo signaling by LDE225 completely abolished ligand mediated Hh pathway activation in all ⁴⁵ Ca and p53Rb KO cell lines. Consistent with published data in MEFs (Cochrane et al., 2020), primary cilia frequency in murine OS cell lines correlated to Hh responsiveness with non-responsive ⁴⁵ Ca cell lines exhibiting few or a complete absence of cilia, even after serum starvation whilst all p53Rb KO osteosarcoma cell lines were highly ciliated under serum free conditions and also displayed abundant cilia in normal serum conditions. One potential explanation for these results comes from data linking Trp53 and Rbl to the regulation of stem cell pathways of direct relevance to osteosarcoma. This evidence includes (i) a role for Trp53 in controlling neural stem cell fate through a Nanog-GLIl regulatory network (Zbinden et ah, 2010 PMID:20581802); (ii) the capacity of Rbl to restrict the expression of pluripotency genes, Sox2, Oct4 and Nanog, independent of its effects on cell cycle regulation (Kareta et ah, 2015 PMID:25467916); and (iii) a role for Sox2 in inducing transcription of Hedgehog acetyltransferase, an enzyme that catalyzes the rate-limiting step in Hh ligand processing (Justilien et ah, 2014 PMID:24525231). Collectively, loss of Trp53 and/or Rbl may cooperate with ligand-driven Hh signaling through the transcriptional control of pluripotency genes. To test this alternative hypothesis, Sox2, Oct4 and Nanog protein expression was determined in a panel of ⁴⁵ Ca and p53Rb KO mouse osteosarcoma cell lines. Oct4 and Nanog were undetectable in all cell lines whereas no observable differences in Sox2 expression was detected that might account for differences in Hh responsiveness. Furthermore, siRNA knockdown of Sox2 in a representative Hh responsive p53Rb KO mouse osteosarcoma cell line, failed to abrogate Hh responsiveness as would be expected if this were the underlying mechanism in these models.

Analysis of autophagy in mOS cell lines also correlated to Hh response with an upregulation of autophagic flux observed in a non-responsive ⁴⁵ Ca cell line whilst responsive ⁴⁵ Ca and p53Rb KO cell lines exhibited diminished or unchanged autophagic flux following serum removal consistent with defective autophagy. These results in mouse osteosarcoma models implicate Trp53 and Rbl in the regulation of Hh responsiveness via autophagy-mediated ciliogenesis. It was therefore hypothesized that the OsxCre p53Rb KO model could serve as a rigorous test for the role of ligand-dependent Hh signaling in cancer.

Smoothened is Required for the Osteosarcoma Phenotype in OsxCre p53Rb KO Mice.

Since canonical Hh signaling through Smo is both ligand and cilia-dependent, whether conditional deletion of Smo in a genetic mouse model would affect the penetrance of the osteosarcoma phenotype was determined. Osterix (Osx) is a transcription factor involved in the specification of early osteoblast fate (Walkley et al., 2008 PMID: 18559481). Accordingly, Cre recombinase expression driven by the Osx locus can be used to trigger deletion in conditional Trp53 and Rbl alleles to induce osteosarcoma (hereafter Osx p53Rb KO mice) (Walkley et ah, 2008 PMID: 18559481). OsxCre p53Rb KO mice developed osteosarcoma with complete penetrance and a median survival of 137.5 days. To inactivate Hh signaling in this model, these animals were crossed with mice carrying a conditional null Smo allele (Long et ah, 2001 PMID: 11748145) to generate OsxCre p53RbSmo KO mice. Genetic inactivation of Smo on the OsxCre p53Rb KO background (OsxCre p53RbSmo KO) almost completely prevented development of osteosarcoma. Strikingly, all OsxCre p53RbSmo KO mice developed small calcified masses, predominantly on the mandible. Pathological assessment revealed features consistent with osteoid osteoma, a benign tumor of the bone. This result suggests that although the loss of two potent tumor suppressors is sufficient to initiate tumor formation, Hh signaling is required to prevent terminal differentiation and complete penetrance of a malignant phenotype.

In keeping with the in vitro data, OsxCre p53Rb KO tumors express Shh and display prominent primary cilia, consistent with p53 and Rb functions regulating ligand responsiveness and primary cilia frequency. In contrast, even though ⁴⁵ Ca tumors do express Shh they exhibit few if any observable primary cilia. These data strongly support a requirement for Hh signaling in a developmentally regulated tumor model driven by combined loss of Trp53 and Rbl that expresses both primary cilia and Shh ligand in vivo.

Aberrant cilia formation, autophagy and Hh pathway expression in human osteosarcoma

Next, the potential importance of Hh signaling in human osteosarcoma was investigated. Immunohistochemical staining of Hh ligand using two independent SHH antibodies on 120 human osteosarcoma samples revealed positive staining in 47% of cases and was significantly enriched in high tumor grade and the undifferentiated subtype. Using an independent 40 sample human osteosarcoma sample set, both SHH expression and primary cilia were assessed. Here, SHH staining was observed in 88% (35/40) of samples and primary cilia detected in 68% (27/40), with 55% expressing both SHH and primary cilia. Additionally, SHH expression was assessed in human osteosarcoma cell lines, U20S and MG63. Whilst the active SHH carboxy-terminal peptide was observed at low levels in whole cell lysates, expression was markedly upregulated in xenograft tumor lysates, and in lysates obtained from the Osx p53Rb KO cell line D12M. This was also confirmed by immunohistochemistry and suggests that in contrast to the in vitro models, the availability of the Shh signaling peptide is greatly enhanced in vivo, thus rendering the cells competent to receive an autocrine or paracrine signal.

Using a panel of human osteosarcoma cell lines with previously reported genotypes, primary cilia frequency was then assessed using immunofluorescence. Cilia were either undetectable or at low frequency in human osteosarcoma cell lines, U20S and SJSA, both with wild type TP53 and RBI status. In contrast, primary cilia were frequently detected in cell lines with reported TP53 mutations or rearrangements and perturbation of the RB signaling pathway via CDKN2A deletion (B143, HOS, MG63) under both serum-starved and normal serum conditions. Despite these findings, a consistent Glil mRNA response could not be detected in any of the human osteosarcoma cell lines listed. As an alternative measure of canonical Hh pathway activation, the localization of SMO to the primary cilia was visualized, a necessary event required for signal transduction (Bangs and Anderson, 2016 PMID:27881449) in B 143 cells. Under serum- starved culture conditions SMO infrequently (<20%) co-localized to the primary cilia consistent with low levels of Hh signaling activation in these cells. In response to treatment with Hh ligand, SMO translocation was observed into the primary cilia at a frequency of 100% indicating that these cells are indeed responsive to Hh ligand stimulation. In contrast, basal and ligand-stimulated SMO translocation to the primary cilia was completely abolished by treatment with the SMO inhibitor

LDE225. To determine if defective autophagy is a feature of ciliated human osteosarcoma cell lines with perturbations in TP53 and RBI signaling, the state of autophagic flux was assessed. As expected, the non-ciliated U20S cell line demonstrated a marked upregulation of autophagic flux in serum free conditions in presence of chloroquine or bafilomycin A. In contrast, the ciliated MG63 cell line failed to upregulate autophagic flux following serum starvation in presence of chloroquine or bafilomycin A. Together, these data show that Hh signaling is a feature of human osteosarcoma and is associated with autophagy-mediated ciliogenesis.

Inhibition of Hh signaling inhibits osteosarcoma growth and drives differentiation

Based on the data in mouse bone development and osteosarcoma, it was hypothesized that inhibition of Smo signaling in established osteosarcoma would result in tumor growth arrest and differentiation. To test whether this effect could be predicted based on Trp53 and Rbl mutation status, in vivo osteosarcoma flank tumor models generated from OsxCre p53Rb KO mice (D12M) and human MG63 (ciliated) and U20S (unciliated) xenograft models was used. Treatment of tumor-bearing mice with the Smo inhibitor LDE225 resulted in reduced tumor growth and significantly improved survival in both the D12M allograft and MG63 xenograft models, but not in mice with U20S tumors.

Histological analysis of tumor sections revealed focal areas of bone deposition in LDE225 treated tumors that was not apparent in vehicle controls and was confirmed by alizarin red staining. To further assess osteoblast differentiation, immunohistochemical staining was performed using the osteoblast progenitor maker, RUNX2, and the terminal osteoblast marker, Osteopontin (OPN). In both D12M and MG63 tumors, LDE225 treatment resulted in an increase in OPN expression consistent with differentiation. Whilst RUNX2 expression was not detected in MG63 xenografts, vehicle treated D12M tumors exhibited strong Runx2 expression that was significantly reduced in the LDE225 treated tumors. U20S xenografts demonstrated no evidence of intratumoral bone deposition or differential expression of RUNX2 and OPN. These results demonstrate that mutations in TP53 and RBI, or disruption of their pathways, and primary cilia frequency is predictive of likely response to Hh pathway inhibition and support the present conclusions that Hh signaling acts to prevent terminal differentiation in the setting of combined TP53 and RBI mutation.

SUBA-Itraconazole inhibits canonical Hedgehog signaling in vitro and in vivo

To test whether canonical Hedgehog signaling could be inhibited using a representative Hedgehog inhibitor, such as a SMO inhibitor SUBA-Itraconazole, the present Example established P53Rb KO MEF and osteosarcoma cell lines were used to evaluate the hypothesis. Without wishing to be bound by theory, it is believed that the results of the present Example is representative of Hedgehog inhibitors. To determine the response to Hh ligand, p53Rbl KO MEFs were treated with Shh ligand. Canonical hedgehog pathway activation measure by Glil expression, p53Rb KO MEFs were highly responsive to Shh ligand (Figure 2A). As expected, Glil response was completely abolished in presence of SUBA-Itraconazole in a dose dependent manner (Figure 2A). In contrast, there was a modest response to Shh ligand in WT MEFs which was completely inhibited by SUBA-Itraconazole (Figure 2B). Highly ciliated osteosarcoma cell lines cell lines D12M and mOS25 showed significant increase in Glil in response to Shh ligand (Figures 2C, 2D), whereas non-ciliated cell line mOS18 showed marginal response to Shh ligand (Figure 2E). In presence of SUBA-Itraconazole, a complete inhibition of Glil was achieved in a dose dependent manner in osteosarcoma (Figures 2C-2E)

To further validate the in vitro observations of Smo signaling inhibition using SUBA- Itraconazole, it was hypothesized that inhibition of Smo signaling in established osteosarcoma would result in tumor growth arrest and differentiation. To test whether this effect could be predicted based on Trp53 and Rbl mutation status, in vivo osteosarcoma flank tumor models generated from human MG63 (ciliated) xenograft, OsxCre p53Rb KO mice (D12M) and ⁴⁵ Ca induced mOS18 (unciliated) allograft models were used. Treatment of tumor-bearing mice with the Smo inhibitor SUBA-Itraconazole resulted in reduced tumor growth and significantly improved survival in both the MG63 xenograft (Figure 3 A) and D12M allograft models (Figure 3B), but not in mice with mOS18 tumors (Figure 3C). Collectively, the data shows mutational status of Trp53 and Rbl predicts response to a Hedgehog inhibitor, SUBA-Itraconazole, inhibition of canonical Hh signaling in vitro and in vivo.

METHODS

Animal experiments

B6;129-Gt(ROSA)26Sortml(cre/ERT)Nat/J (EsrCre,(38)), B6.129P2-Trp53tmlBrn/J

(Trp531ox/lox,(39)), B6;129-Rbltm3Tyj/J (Rbllox/lox,(40)), B6.Cg-Tg(Nes-cre)lKln/J (NestinCre,(41)), 129S/Sv-Krastm4Tyj/J (KrasG12D,(25)), B6.129-Kif3atm2Gsnand (Kif3alox/lox,(26)) and Gt(ROSA)26Sortml(Smo/EYFP)Amc/J (SmoM2,(22)) alleles have been described previously described, and were obtained from Jackson Laboratories. All mice were backcrossed onto and maintained on a C57BL/6J background. Inbred C57BL/6J were obtained from the Monash Animal Research Platform. Genotyping was performed using genomic PCR to amplify DNA purified from placental tissue or tail samples according to Jackson Laboratory protocols for each mouse line. Timed matings were used for the generation of MEFs (E13.5) and neural tube analysis (E10.5). Lung tumor models were administered with 5x108 PFU Ad5CMVCre virus (University of Iowa) by intranasal inhalation as described (6, 42). All mice used in this study were housed under SPF conditions with a standard day /night cycle and fed ad libitum. All experiments involving animals were approved in advance by the Animal Ethics Committee at Monash University (Protocols MMCA2012/24, MMCA2015/11, MMCA2015/12, MMCA2015/13, MMCA2015/41) and were carried out in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.

Genetic mouse model of osteosarcoma OsxCre mice were intercrossed with Trp531ox/lox;Rbllox/lox or Trp531ox/lox; Rbllox/lox; Smolox/lox mice to ultimately generate OsxCre; Trp531ox/lox; Rbllox/lox or OsxCre; Trp531ox/lox; Rbllox/lox; Smolox.lox genotypes. Male OsxCre;Trp531ox/lox;Rbllox/lox or OsxCre; Trp531ox/lox; Rbllox/lox; Smolox.lox mice were bred to Trp531ox/lox; Rbllox/lox or Trp531ox/lox; Rbllox/lox; Smolox.lox females.

Male and female OsxCre; Trp531ox/lox;Rbllox/lox, OsxCre; Trp531ox/lox; Rbllox/lox; Smolox.lox and littermate Trp531ox/lox; Rbllox/lox or Trp531ox/lox; Rbllox/lox; Smolox. lox mice were monitored for signs of palpable and progressive tumor development, body weight loss >10% and signs of distress. At the completion of the study, mice were euthanized in a carbon dioxide chamber, imaged using the Feinfocus Y. Cougar Microfocus X-ray Inspection System (YXLON) and tissue was harvested for cell line generation, pathological examination and immunohistochemistry.

MEF Cell lines

EsrCre mice were intercrossed crossed Trp53lox/lox and Rbllox/lox mice to generate the necessary genotypes. Pregnant dams were sacrificed, embryos removed, and the heads, limb, tail and internal organs dissected sterile phosphate buffered saline (PBS, Gibco; 70011044). The remainder of each embryo was then finely minced using a sterile razor blade and placed in a 10cm round petri dish in DMEM (Gibco; 11965092) supplemented with 10% fetal bovine serum (FBS, Gibco; 26140079) lOOU/mL penicillin and 10 mg/mL streptomycin (Gibco; 15140148). Cells were grown until -80% confluency and were then trypsinized, centrifuged, re-suspended in supplemented DMEM media, then counted and passaged. Following genotyping, were treated with 500nM of 40HT for 24 hours to initiate Cre recombination of flox alleles. 40HT was removed and fresh supplemented DMEM media was added the following day. Authenticated NIH-3T3 cells were obtained from ATCC and maintained in DMEM supplemented with 10% FCS, lOOU/mL penicillin, and 10 mg/mL streptomycin. All MEF lines were cultured in humidified 5% C02 /95% air at 370C.

Human osteosarcoma cell lines

Authenticated U20S, SJSA, B 143, HOS and human osteosarcoma cell lines were obtained from ATCC and maintained in DMEM (Gibco, Invitrogen) supplemented 10% FCS, lOOU/mL penicillin, and 10 mg/mL streptomycin in a humidified 5% C02/95% air atmosphere at 37°C.

Mouse osteosarcoma cell lines

Radiation-induced osteosarcoma cell lines were kindly provided the Garvan Institute of Medical Research and maintained in alpha-MEM (Alpha Modification (Alpha MEM) cell culture medium) supplemented with 10% FCS and lOOU/mL penicillin, and 10 mg/mL streptomycin in a humidified 5% C02/95% air atmosphere at 37°C.

Conditional genetic mouse osteosarcoma cell lines were either provided by St. Vincent’s Institute or generated in this study. To generate cell lines, freshly dissected tumor tissue was mechanically dissociated using scalpel blades in sterile PBS. Cell were centrifuged and resuspended in alpha-MEM supplemented with 10% FCS and lOOU/mL penicillin, and 10 mg/mL streptomycin in a humidified 5% C02/95% air atmosphere at 37°C.

Mouse genotyping PCR

Briefly, DNA was extracted by adding 300pL of 50nM NaOH and incubating at 95 °C until dissolved. Samples were vortexed to complete digestion of tissue. 100pL of 0.5M Tris-HCL (pH 8.0) was then added to neutralise each sample. DNA extracted from embryonic tissue was completed in half the volumes of NaOH and Tris-HCL. 2pL of DNA from each tail sample was added to 18pL aliquots of master mix containing 10pL of GoTaq® DNA Polymerase (Promega, M7123), 0.5pL of IOmM forward and reverse primers to detect Cre-recombinase or floxed and wildtype alleles, and 6pL of nuclease free water, to a final volume of 20pL. PCR products were then separated by gel electrophoresis on a 1.5% agarose gel stained with SYBR® Safe DNA gel stain (ThermoFisher, S33102) . A lkb Plus DNA ladder was used to interpret band sizes. Gels were visualised under a Doc-ItTM 210 imaging system (UVP). Exposure was adjusted for optimal visualisation.

Sanger Sequencing

Sanger sequencing of TP53 and RBI in mouse osteosarcoma cell lines was performed on cDNA PCR product using described primers. Briefly, lpg of RNA was reverse transcribed using the Superscript III First Strand DNA synthesis kit (Invitrogen, #18080051) using random OligodT primers according to manufacturer’s instructions. lpF of cDNA from each cell lines was added to 19pL of master mix containing 10pL of GoTaq®, 0.5pL of IOmM forward and reverse primers. PCR products were purified using Wizard PCR and gel Clean-up kit (Promega) and submitted to the MHTP Medical Genomics Facility for sequencing using forward and reverse primers. Sequences were aligned to reference using SeqMan Pro (DNASTAR, Fasergene, Version 8.1.5).

Detection of Cdnk2a deletion in mouse osteosarcoma cell lines was performed on cDNA PCR product using described primers. Briefly, 1 pg of RNA was reverse transcribed using the Superscript III First Strand DNA synthesis kit (Invitrogen, #18080051) using random OligodT primers according to manufacturer’s instructions. 1 pF of cDNA from each cell lines was added to 19 pF of master mix containing 10pF of GoTaq®, 0.5pF of IOmM forward and reverse primers. PCR products were then separated by gel electrophoresis on a 1.5% agarose gel stained with SYBR® Safe DNA gel stain. A lkb Plus DNA ladder was used to interpret band sizes. Gels were visualized under a Doc-ItTM 210 imaging system (UVP). Exposure was adjusted for optimal visualization.

Cell cycle analysis

Cells were grown in 10cm dishes in the presence of serum free or 10% serum for 24 hours (DMEM+ Penstrep). Cells were trypsinized at 70% confluency, washed in PBS and counted. 1x106 cells were resuspended in 300 mΐ of PBS and 700 mΐ of 100% ice cold ethanol added and mixed gently to fix the cells. The fixed cells were stored at -20°C for cell cycle analysis. For analysis, fixed cells were washed twice with PBS and resuspended in 200 mΐ FxCycle PI/RNAse solution (Thermo Fisher, F10797) and run on BD FACS canto II analyser (BD Biosciences). Data was analysed using FloJo V10 software.

FACS analysis

Transduced p53Rb KO MEFs were trypsinized and counted 48 hours post-infection. Cells were resuspended in DMEM and run on the BD FACS canto II analyser for GFP and mCherry. Flow Jo V10 software was used to analyse percentage positive cells.

Hedgehog ligand and inhibitor treatment

Cells were first seeded at 100,000 cells/well on 6 well plates and grown to 80-90% confluence Treatments were added in media containing 0.2% FBS, with either IOmE of PBS per mL of media, 10mE of lug/mF rhSHH (R&D Systems, 1845-SH-100) per mF of media and/or 400nM of FDE-225 (Selleckchem, S2151) and/or 2pg/ml and 5pg/ml SUBA-Itraconazole. After 24 hours of culture, cells were harvested for RNA extraction or for Western blot analysis.

Quantitative real-time PCR

RNA was isolated from MEF cell lines using the QIAGEN RNeasy mini kit (QIAGEN; Cat# 74106), as per manufacturer’s instructions lpg of RNA was reverse transcribed using the Superscript III First Strand DNA synthesis kit (Invitrogen; 18080051) using random OligodT primers according to manufacturer’s instructions. Real-Time qRT-PCR was performed using SYBR Green (Applied Biosystems; 4309155) on a 7900HT Fast-Real Time PCR System (Applied Biosystems) using custom designed primers. Primers were diluted to a final primer concentration of 300nM in nuclease-free water prior to use. Master mix, containing SYBR Green and primer (6mE) and cDNA (4mE) were loaded and mixed into a 384-well plate manually, were samples were run in triplicate. Transcript levels relative to B2m were calculated using the standard curve method. Analysis of autophagy genes was performed using the mouse Autophagy RT2 Profiler PCR Array (QIAGEN; PAMM-084Z). RNA was isolated from C57B1/6 WT, p53 KO, Rb KO and p53Rb KO MEFS using QIAGEN RNeasy mini kit and reverse transcribed using the RT2 First Strand Kit (QIAGEN; 330401) according to manufacturer’s instructions. The cDNA was added to the RT2 Profiler PCR Array in combination with RT2 SYBR Green qPCR Mastermix (QIAGEN; 330529) and analysed on a 7900HT Fast-Real Time PCR System (Applied Biosystems). Ct values were exported into an Excel file and uploaded onto the data analysis web portal at www.qiagen.com/geneglobe for normalization to control reference genes, fold change calculation, statistical analysis and generation of plots. siRNA knockdown

Reverse transfections using SMARTpool: ON-TARGERT plus siRNAs (Dharmacon) targeted against mouse Atg5 (L-064838-00-0005), Atg9b (L-051438-01-005), Ctsd (L-051673-01- 0005), Pik3ca (L-040929-01-0005) were performed using Lipofectamine RNAiMax (Invitrogen; #13778030) and OptiMEM (Gibco; 31985070). Transfection media consisting of 200pL OptiMEM, lpL of Lipofectamine RNAiMax and 1.2pL of 20mM siRNA was made, producing a final siRNA concentration of 20nM. Transfection media was gently mixed and incubated at room temperature for 20 minutes. MEFs were seeded at 50,000 cells/wall on a 6-well plate in lmL of DMEM/10% FCS media or alpha-MEM/10% FCS (no antibiotic). 200pL of transfection media was added drop-wise per well and plates were rocked gently to mix. Transfection master mixes were made up according to the number of corresponding treatment wells. After 24 hours of culture, transfection media was removed and replaced with DMEM+10% FBS media.

Detection of Lc3/p62 puncta

Cells were seeded at 20,000 cells/well on a 24-well plate containing 14mm circular coverslips. At 80% confluence, cells were treated with 50nM Bafilomycin (Baf-A) in the presence or absence of 10% FBS. After 24 hours of treatment, cells were washed twice in lxPBS and fixed in ice-cold 100% methanol for 15 minutes at -20°C. Cells were then washed three times in lxPBS and incubated in blocking buffer (lxPBS, 5% goat serum, 0.3% Triton X-100) for 1 hour at RT. Blocking buffer was then removed and replaced with antibody dilution solution (lxPBS, 1% BSA, 0.3% Triton X-100) containing Lc3 and p62 primary antibodies and incubated overnight at 4°C overnight on a rocker. The following day, primary antibody solution was then removed, coverslips washed three times in lxPBS and DAPI (1:1000, Sigma-Aldrich; D9542-10MG), Alexa-fluor® 594 goat anti-rabbit and Alexa-fluor® 488 anti-mouse was diluted in antibody dilution solution and applied for 60 minutes -light protected. Coverslips were washed twice in lxPBS then mounted using ProLong™ Gold Antifade Mount with DAPI (Thermo; P36931) on Superfrost slides. High resolution images were acquired using the NIS Elements Confocal Imaging (Nikon) software. The number of Lc3 and p62- stained puncta per cell was determined by an automated macro pipeline on FIJI software (Fassina et al. 2012).

Detection of primary cilia

Cells were washed twice in lxPBS and were fixed in 10% buffered formalin for 10 minutes, then washed twice in PBS. Cells were then permeabilized in 0.1% Triton X-100 /lxPBS for 15 minutes. Next, coverslips were then washed twice in lxPBS and incubated in Odyssey blocking buffer (FI- COR Biosciences; 927-4000) for 30 minutes. The rabbit polyclonal antibody, Arll3b was diluted in Odyssey blocking buffer and incubated for 60 minutes. Primary antibody was then removed, coverslips washed twice in PBS, and DAPI (1:1000, Sigma-Aldrich; D9542-10MG), Alexa-fluor® 594 goat anti-rabbit and acetylated tubulin-488 anti mouse was diluted in Odyssey blocking buffer and applied for 60 minutes - light protected. Coverslips were washed twice for a final time in lxPBS and coverslips were mounted using ProFong™ Gold Antifade Mount with DAPI (Thermo; P36931) on Superfrost slides. Coverslips were viewed using an Eclipse T/-E Nikon Cl Inverted Research Confocal Microscope equipped with a 60x oil immersion objective with identical gain, offset and laser power settings. High-resolution images were acquired using the NIS Elements Confocal Imaging (Nikon) software. The percentage of ciliated cells was calculated from the total number of cells counted per field of vision. Five fields of vision per cell line/or treatment group were manually counted on the Nikon Cl Confocal Microscope. Primary cilia frequency represents the mean percentage of ciliated cells ± SEM.

For analysis of primary cilia in cells following lentiviral reintroduction of Trp53 and Rbl, cells were fixed in 4% PFA for 10 mins and processed, imaged and analysed as described above. Co-staining for Trp53-GFP and Rbl-mCherry and primary cilia was performed using chicken polyclonal anti-GFP, mouse monoclonal mCherry and rabbit polyclonal Aril 3b primary antibodies and Alexa-fluor® 488 goat anti-chicken, Alexa-fluor® 594 goat anti-mouse, and Alexa- fluor® 647 goat anti-rabbit antibodies diluted in Odyssey blocking buffer. Untransduced cells within the same experiments were determined by absence of GFP and/or mCherry staining.

Fentiviral transduction

Mouse Fenti ORF clone of Trp53 (mGFP-tagged) was purchased from Origene (MR206086F4). MouseRbl ORF was cloned into pFVX with mCherry tag and ampicillin resistance selection. Both vectors were transfected into Stbl3 competent cells and single clones selected from agar plates and grown in FB broth. Plasmids were purified using the NEB plasmid purification kit. Insert vector plasmid was mixed with psPAX-2 and VSVG plasmid in a ratio of 5:5:1 respectively and transfected into HEK293T cells using Fipofectamine FTX and Plus reagent. Fresh media was added after 6 hours of transfection and viral particles were collected from the transfected HEK293T cells after a 48 hour incubation. The viral particle media was centrifuged to remove dead cell from media and viral parcel media was diluted 1 : 1 with fresh DMEM and 5pg/ml of polybrene added. 100,000 p53Rb KO MEF cells were transduced with 2 ml of viral particle media in a 6 well plate and centrifuged for 30 mins at 2000 rpm. Cells were used after 48 hours of transduction for further analysis. Western blotting Whole cell extracts prepared in ice-cold RIPA lysis buffer with added HaltTM Protease and Phosphatase inhibitor cocktail (Thermo; 78442). Cells were lysed using a sonicator, 2 cycles at level 5 for 30 seconds, on ice and were then clarified by spinning at 14,000 rpm for 15 minutes at 4°C. The supernatant was then placed in a new 1.5mL Eppendorf tube and then stored at -80°C. Protein concentrations were determined using Bio-Rad DCTM Protein assay ready-to-use reagents (Bio-Rad; 5000116) as per manufacturer’s instructions. 30pg of protein from lysates were re suspended in NuPAGE-LDS sample buffer (Invitrogen; NP0007) and NuPAGE reducing agent (Invitrogen; NP0009), incubated at 70°C for 10 minutes, and then subjected to SDS-PAGE. Protein was transferred onto a nitrocellulose membrane (HybondTM -C Super, Amersham Biosciences; RPN2020E) using a Mini Trans-Blot® Cell wet protein transfer system (Bio-Rad; 1703930), followed by immunoblotting. Protein was then detected using the Li-Cor Odyssey Infrared Imaging System version 3.0.1.6. For analysis of autophagy flux, densitometric analysis of the LC3- II and Actin bands were performed using Odyssey Infrared Imaging System software. Briefly, LC3-II bands were normalized to Actin (LC3 II/actin). Autophagy flux was then determined by dividing the normalized value for the Baf-A or CHQ treated lysate by the normalized value for the control treated lysate of the same sample (14, 18). To determine the autophagic flux ratio, the serum-starved autophagic flux value was divided by the 10% serum autophagic flux value.

Mouse lung tumor analysis

This was performed as described (6, 42). Briefly, 9-month old p53Rb KO AdCre and p53Rb Kba KO AdCre mice and 8-week old Kras AdCre and Kras Kϊba KO AdCre mice were euthanized, lungs dissected, washed in PBS and then inflation fixed in 10% buffered formalin saline overnight. After processing, and embedding, blocks were sectioned to the level of the carina, sections stained with hematoxylin and eosin, and then images acquired using the ImageS cope platform (Leica Biosystems). Tumor number and surface area were determined using the ImageScope software package. Mouse lung cancer cell lines p53Rb KO AdCre mouse small cell lung cancer tumors (mSCLC) and Kras AdCre mouse lung adenocarcinoma tumors (mLUAD) were microdissected from lungs at the ethical endpoints of 9 months and 8 weeks, respectively, in a Class II laminar flow cell culture hood. Tumor tissue was mechanically dissociated using sterile scalpel blades in lxPBS in a culture dish. Cells were placed in a 15mL Falcon tube® and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and dissociated tumour was resuspended in RPMI 1640 with 1% FBS + 1% PenStrep (Gibco), for mSCLC, or DMEM with 1% FBS + 1% PenStrep (Gibco), for mLUAD, for continued growth and maintenance.

Osteosarcoma allograft and xenografts

A total of 1x106 p53Rb KO (D12M), MG63, U20S and mOS18 cells were injected into the flanks of 6-8 week old female BALB/c Nude mice. Cells were resuspended in lOOul of 1:1 mixed cell suspension in PBS and Matrigel. Tumor size was measured daily using digital calipers, and volumes were calculated according to the formula: Tumor Volume (mm3) = (Width2 x Length)/2.

Once tumors reached a volume of 200mm3 mice were randomized to receive the Smo inhibitor LDE225 (20mg/kg) or vehicle control (0.5% Methylcellulose/0.5% Tween 80) or smo inhibitor SUBA-Itraconazole (lOOmg/kg) or vehicle control (ORA-Plus). Mice were treated daily by oral gavage until ethical endpoints were reached. Ethical endpoints were defined as a tumor volume of 800mm3 or greater, a body weight loss of >10% or signs of general distress. At the completion of the study, mice were euthanized in a carbon dioxide chamber and tissue was harvested from the flanks for histology and analysis of differentiation markers.

All mice used in this study were housed under specific pathogen free (SPF) conditions with a standard day/night cycle and fed ad libitum. All experiments involving animals were approved in advance by the Animal Ethics Committee at Monash University (Ethics Number: MMCA20 12/24, MMCA2015/11, MMCA2015/12, MMCA2015/13, MMCA2015/41) and were carried out in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.

Histopathology

At the conclusion of all in vivo studies, mice were euthanized in a carbon dioxide chamber, where tissue was harvested from flanks for histopathological or immunohistochemical analysis. Excised tissue was divided into two halves. The first tumor half was snap frozen on dry ice in a 1.5mL cryovial and stored at -80°C. The second tumor half was encased in a histology cassette and then fixed in 10% buffered formalin (Orion Laboratories, Bayswater, Australia, Cat no: BUF01536F) for 24 hours. Formalin was then switched with 70% Ethanol and cassettes were taken to the Monash Histology Facility for processing and sectioning. The tissue was embedded in a paraffin block, with the cut surface down and subsequently cut at a section thickness of 4pm. Sectioning and Hematoxylin and Eosin (H&E) staining was performed by the Monash Histology Facility - Monash Health Translation Precinct (MHTP) node. Pathological analysis of H&E sections was blindly performed by a pathologist.

Immunohistochemistry

Immunohistochemical analysis was performed on paraffin embedded sections using the Vectastain ABC Elite IgG Kits (Vector Laboratories, Burlingame, USA, Cat no: PK-4000) using the following primary antibodies, Shh (1:200, Abeam: ab53281), OPN (1:500, Abeam: ab8448) and RUNX2 (1:300, Abeam: ab23981). Briefly, slides were dewaxed in triplicate solutions of Histosol and rehydrated in duplicate solutions of 100% ethanol, for 5 minutes each, followed by immersion into 70%, 50% and 25% ethanol for 3 minutes each, and finally washed for 3x5 minutes in lxPBS solution. Antigen retrieval was performed in a microwave by heating slides in lOmM Sodium Citrate Buffer at full power until boiling, followed by further heating on power setting 1 for 7 minutes. Incubation in solutions that are described from this point onwards were carried out in a humid chamber consisting of wet paper towels in an airtight container and in between each critical incubation step that is outlined, 3x5 minute wash steps in lxPBS were completed. Sections were encircled with a hydrophobic PAP pen and were blocked for endogenous hydrogen peroxidase activity with 1% H ₂ 0 ₂ in lxPBS for 15 minutes at room temperature. 1.5 drops of goat serum in 5mL of lxPBS was applied to sections and incubated at room temperature for 30 minutes, as a blocking step. One of the two sections were incubated with primary antibody, while the other received Rabbit IgG as a negative control, and slides were incubated overnight at 4°C. The following day sections were incubated with 1 drop of secondary biotinylated goat anti-rabbit IgG in lOmL of 1XPBS for 30 minutes at room temperature. Sections were then incubated in ABC Elite solution for 30 minutes at room temperature, followed by the addition of SigmafastTM H ₂ 0 ₂ activated 3,3 Diaminobenzidine (DAB) tablets in 5mL of milliQ H20, which were visualized under a Nikon DS-Fil H550S bright field photomicroscope and timed to monitor development. All slides were kept to approximately the same development time to ensure consistency of staining intensity.

Color development was terminated in dH ₂ 0 and sections were counterstained with hematoxylin for 3 seconds and Scott’s Tap Water for 30 seconds. Sections were then dehydrated in 25%, 50% and 75% ethanol solutions for 3 minutes each and immersed in triplicate solutions of 100% ethanol and duplicate solutions of Histosol for 5 minutes each. Slides were then mounted onto coverslips with DPX. Quantification of staining was performed using the multiplicative quickscore method by a blinded observer (Detre et ah, 1995).

Alizarin Red Staining

To visualize calcium deposits in osteosarcoma allograft and xenograft tumor tissue, 2g of alizarin red powder was dissolved in lOOmL of distilled water and adjusted to a pH of 4.1 - 4.3 with 0.5% of ammonium hydroxide. Slides were dewaxed in Histosol, hydrated to 70% ethanol and briefly rinsed in distilled water. Alizarin red solution was applied for 30 seconds to sections and excess dye was blotted off. Slides were then dipped in acetone 20 times, then dipped in a 1:1 Acetone-Histosol solution 20 times, then finally cleared in histosol for 5 minutes. Slides were then mounted onto coverslips using DPX.

Statistics

All data were analyzed with GraphPad Prism (version 7) and represented as mean + SEM. A paired-two tail t test was used for two samples with a single variable. A one-way ANOVA followed by a Tukey’s multiple comparison test was used for more than two samples with one variable. Log-rank (Mantel-Cox) test was used for comparison of Kaplan-Meier survival curves. A P value of less than 0.05 was considered statistically significant and is denoted by *<0.05, **<0.01, ***<0.001, ****<0.0001. The number of samples (‘n’) used for calculating statistics is indicated in the Figures or accompanying legends.

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While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

The present application claims priority from US 63/027,255 filed 19 May 2020, the entire contents which are incorporated herein by reference. All publications discussed and/or referenced herein are incorporated herein in their entirety.