CA2781300A1 - Methods and compositions for treating hedgehog-associated cancers - Google Patents

Abstract

Provided herein are methods and compositions for treating or preventing a hedgehog-associated cancer (e.g., a hedgehog ligand-dependent cancer cell growth chosen from a neuroendocrine cancer, a sarcoma (e.g., a musculoskeletal sarcoma, such as chondrosarcoma and osteosarcoma)), a head and neck cancer, or a lung cancer by administering to a subject a hedgehog inhibitor, alone or combination with another anticancer agent (e.g., paclitaxel or a paclitaxel agent, a tyrosine kinase inhibitor (e.g., receptor tyrosine kinase (RTK) inhibitor) or an mTOR inhibitor).

Description

Attorney Docket No. I2041-7000WO/3020PCT
METHODS AND COMPOSITIONS FOR TREATING

HEDGEHOG-ASSOCIATED CANCERS
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. Application Serial No.
12/762,008, filed April 16, 2010. This application also claims the benefit of priority to U.S. Provisional Application Serial No. 61/263,184, filed November 20, 2009;
U.S.
Provisional Application Serial No. 61/294,029, filed January 11, 2010; U.S.
Provisional Application Serial No. 61/324,934, filed April 16, 2010; U.S. Provisional Application Serial No. 61/327,373, filed April 23, 2010; U.S. Provisional Application Serial No.
61/331,365, filed May 4, 2010; U.S. Provisional Application Serial No.
61/351,082, filed June 3, 2010, and U.S. Serial No. 61/386,763, filed September 27, 2010. The contents of all of the aforesaid applications are hereby incorporated by reference in their entirety.

BACKGROUND
Hedgehog signaling plays a role in many stages of development, especially in formation of left-right symmetry. Loss or reduction of hedgehog signaling leads to multiple developmental deficits and malformations, one of the most striking of which is cyclopia.
Many cancers and proliferative conditions have been shown to depend on the hedgehog pathway. The growth of such cells and survival can be affected by treatment with the compounds disclosed herein. It has been reported that activating hedgehog pathway mutations occur in sporadic basal cell carcinoma (Xie et al. (1998) Nature 391:
90-2) and primitive neuroectodermal tumors of the central nervous system (Reifenberger et al. (1998) Cancer Res 58: 1798-803). Uncontrolled activation of the hedgehog pathway has also been shown in numerous cancer types such as GI tract cancers including pancreatic, esophageal, gastric cancer (Berman et al. (2003) Nature 425: 846-51, Thayer et al. (2003) Nature 425: 851-56) lung cancer (Watkins et al. (2003) Nature 422: 313-317, prostate cancer (Karhadkar et al (2004) Nature 431: 707-12, Sheng et al.
(2004) Molecular Cancer 3: 29-42, Fan et al. (2004) Endocrinology 145: 3961-70), breast cancer Attorney Docket No. I2041-7000WO/3020PCT
(Kubo et al. (2004) Cancer Research 64: 6071-74, Lewis et al. (2004) Journal of Mammary Gland Biology and Neoplasia 2: 165-181) and hepatocellular cancer (Sicklick et al. (2005) ASCO conference, Mohini et al. (2005) AACR conference).
The need still exists for identifying new cancer therapies, in particular new uses for hedgehog inhibitors, alone or in combination with other therapeutic agents, for treatment of cancers that are responsive to hedgehog modulation.

SUMMARY
The invention discloses, at least in part, that a hedgehog (Hh) inhibitor, as a single agent or in combination with other anti-cancer agents, can reduce hedgehog-associated cancer cell growth. In one embodiment, Applicants have discovered that administration of a hedgehog inhibitor, alone or in combination with a tyrosine kinase inhibitor (e.g., sunitinib), reduced neuroendocrine cancer growth and/or tumor progression in vivo. It was further discovered that administration of the hedgehog inhibitor reduced the expression of hedgehog-dependent markers (e.g., genes) in stroma surrounding neuroendocrine cancers, while no significant effect was detected on hedgehog dependent markers (e.g., genes) in the neuroendocrine tumor cells.
In other embodiments, administration of a hedgehog inhibitor, alone or in combination with an mTOR inhibitor or other anti-cancer agents (e.g., one or more of:
doxorubicin, cisplatin, ifosfamide, or methotrexate (e.g., high dose methotrexate)) reduced the growth and/or tumor progression of musculoskeletal sarcomas, such as chondrosarcomas and osteosarcomas. Applicants have discovered that administration of the hedgehog inhibitor reduced a signaling pathway in the tumor cells and the surrounding stroma, thus supporting a direct signaling mechanism between the hedgehog ligand and the tumor, as well as an indirect effect on the tumor via the surrounding stroma.
In yet other embodiments, administration of a hedgehog inhibitor in combination with paclitaxel or a paclitaxel agent reduces the growth and/or tumor progression of a pancreatic cancer, e.g., metastatic pancreatic cancer, to a greater extent than administration of each agent alone. The combination of the hedgehog inhibitor and paclitaxel or paclitaxel agent can further include gemcitabine (e.g., GEMZAR
). It is Attorney Docket No. I2041-7000WO/3020PCT
believed that administration of the hedgehog inhibitor results in enhancement of the delivery of the paclitaxel or the paclitaxel agent and/or gemcitabine, when used in combination, compared to the use of the paclitaxel or paclitaxel agent (and/or gemcitabine) without the hedgehog inhibitor. In one embodiment, the subject is a patient with metastastatic pancreatic cancer. In another embodiment, the subject is a patient with pancreatic or metastastatic pancreatic cancer who has received no prior cancer treatment (e.g., no prior exposure to other anti-cancer agent, surgical or radiation procedure for, e.g., no prior cancer treatment for their disease).
In yet another embodiment, administration of a hedgehog inhibitor in combination with a VEGF (Vascular Endothelial Growth Factor) antagonist, e.g., an anti-VEGF
antibody (e.g., bevacizumab), reduces the growth and/or tumor progression of a pancreatic cancer to a greater extend than administration of either agent alone.
In other embodiments, administration of a hedgehog inhibitor in combination with a tyrosine kinase inhibitor (e.g., a receptor tyrosine kinase (RTK) inhibitor, such as an EGFR inhibitor) reduces the growth and/or tumor progression of a head and neck cancer and/or lung cancer (e.g., non-small cell lung cancer). In one embodiment, the hedgehog inhibitor extends the relapse free survival of a subject who is undergoing, or has been previously treated with, another anti-cancer agent (e.g., a tyrosine kinase inhibitor). In one embodiment, the tyrosine kinase inhibitor is geftinib or cetuximab. For example, the hedgehog inhibitor reduces or inhibits tumor re-growth of a hedgehog-associated cancer after therapy with a tyrosine kinase inhibitor is less effective or ineffective (e.g., a subject having a relapse after therapy with, or a tumor developing resistance to, a tyrosine kinase inhibitor). In one embodiment, the subject is a patient with lung cancer (e.g., non-small cell lung cancer) who relapses after geftinib therapy. In another embodiment, the subject is a patient with head and neck squamous cell carcinoma (HNSCC) who is undergoing or has undergone therapy with a tyrosine kinase inhibitor (e.g., an EGFR tyrosine kinase inhibitor such as cetuximab).
Thus, methods and compositions for treating or preventing a hedgehog-associated cancer (e.g., a hedgehog ligand-dependent cancer cell growth chosen from a neuroendocrine cancer, a sarcoma (e.g., a musculoskeletal or soft-tissue sarcoma, such as chondrosarcoma, osteosarcoma, synovial sarcoma or liposarcoma), a head and neck Attorney Docket No. I2041-7000WO/3020PCT
cancer, or a lung cancer by administering to a subject a hedgehog inhibitor, alone or combination with another anti-cancer agent (e.g., paclitaxel or a paclitaxel agent, a tyrosine kinase inhibitor (e.g., receptor tyrosine kinase (RTK) inhibitor) or an mTOR
inhibitor) are disclosed.
Accordingly, in one aspect, the invention features a method of reducing or inhibiting growth of a tumor or cancer, e.g., a hedgehog-associated tumor or cancer, in a subject. The invention also features a method of treating a subject having, or at risk of having, a tumor or cancer, e.g., a hedgehog-associated cancer or tumor. The method includes administering to the subject a hedgehog inhibitor, e.g., one or more hedgehog inhibitors as described herein, in an amount sufficient to reduce or inhibit the tumor cell growth, and/or treat or prevent the cancer or tumor, in the subject. In certain embodiments, the hedgehog inhibitor is administered as a single agent, or in combination with other anti-cancer agents (e.g., in combination with a paclitaxel or a paclitaxel agent, a tyrosine kinase inhibitor (e.g., receptor tyrosine kinase (RTK) inhibitor), an mTOR
inhibitoror a VEGF inhibitor). In certain embodiments, the hedgehog-associated tumor or cancer is a hedgehog ligand-dependent cancer cell growth chosen from one or more of a neuroendocrine cancer, a sarcoma (e.g., a musculoskeletal or soft-tissue sarcoma, such as chondrosarcoma, osteosarcoma, synovial sarcoma or liposarcoma), a pancreatic cancer, a head and neck cancer, prostate cancer, ovarian cancer, or a lung cancer (e.g., a small cell or a non-small cell lung cancer).
In another aspect, the invention features a method of treating a hedgehog-associated cancer or tumor, in a subject in need of hedgehog inhibition. The method includes administering to the subject a first anti-cancer agent and a second anti-cancer agent, in an amount sufficient to treat the cancer or tumor, wherein the first anti-cancer agent is a hedgehog inhibitor. In certain embodiments, the hedgehog-associated cancer or tumor and the second anti-cancer agent are each chosen from: (i) the hedgehog-associated cancer or tumor is a sarcoma and the second anti-cancer agent is chosen from one or more of. mTOR inhibitor, doxorubicin, cisplatin, ifosfamide, or methotrexate; (ii) the hedgehog-associated cancer or tumor is a neuroendocrine cancer and the second anti-cancer agent is a tyrosine kinase inhibitor; (iii) the hedgehog-associated cancer or tumor is a head and neck squamous cell cancer and the second anti-cancer agent is a tyrosine Attorney Docket No. I2041-7000WO/3020PCT

kinase inhibitor; (iv) the hedgehog-associated cancer or tumor is a pancreatic cancer and the second anti-cancer agent is a paclitaxel agent; (v) the hedgehog-associated cancer or tumor is a pancreatic cancer and the second anti-cancer agent is a VEGF
inhibitor; or (vi) the hedgehog-associated cancer or tumor is a lung cancer and the second anti-cancer agent is a tyrosine kinase inhibitor is chosen from sunitinib, erlotinib, gefitinib, or sorafenib. Unless explicitly noted otherwise, the use herein of the term "first," "second,"
or "third" agent is not intended to imply a particular order of administration. It is intended to clarify the different classes of agents used.
In another aspect, the invention features a method of reducing or preventing a relapse in a subject having a tumor or cancer, e.g., a hedgehog-associated tumor or cancer. The method includes administering to the subject a hedgehog inhibitor, e.g., one or more hedgehog inhibitors as described herein, in an amount sufficient to reduce or inhibit the tumor or cancer re-growth or relapse, in the subject. In certain embodiments, the hedgehog inhibitor is administered as a single agent, or in combination with other anti-cancer agents (e.g., in combination with a paclitaxel or a paclitaxel agent, a tyrosine kinase inhibitor (e.g., receptor tyrosine kinase (RTK) inhibitor) or an mTOR
inhibitor).
In certain embodiments, the hedgehog-associated tumor or cancer is a hedgehog ligand-dependent cancer cell growth chosen from one or more of a neuroendocrine cancer, a sarcoma (e.g., a musculoskeletal sarcoma, such as chondrosarcoma and osteosarcoma, or soft-tissue sarcoma, such as synovial sarcoma or liposarcoma), a pancreatic cancer, a head and neck cancer, or a lung cancer (e.g., a small cell or a non-small cell lung cancer).
In certain embodiments, the subject is a patient who is undergoing cancer therapy (e.g., treatment with other anti-cancer agents, surgery and/or radiation). In certain embodiments, the subject is a patient who has undergone cancer therapy (e.g., treatment with other anti-cancer agents, surgery and/or radiation). In one embodiment, the relapse reduced or prevented occurs after tyrosine kinase inhibitor therapy, e.g., a subject that has undergone or is undergoing therapy with a tyrosine kinase inhibitor therapy.
In some embodiments, the cancer is a lung cancer (e.g., a non-small cell lung cancer) or a head and neck squamous cell cancer.
In those subjects treated with the methods of the invention, treatment can include, but is not limited to, inhibiting tumor growth; reducing tumor mass or volume;
reducing Attorney Docket No. I2041-7000WO/3020PCT
size or number of metastatic lesions; inhibiting the development of new metastatic lesions; reducing one or more of non-invasive tumor volume, metabolism;
prolonged survival; prolonged progression-free survival; prolonged time to progression;
and/or enhanced quality of life.
In one embodiment, the hedgehog inhibitor reduces or inhibits a hedgehog signaling pathway. For example, the hedgehog inhibitor reduces or inhibits the activity of a hedgehog receptor, e.g., Smoothened. In one embodiment, the hedgehog inhibitor reduces or inhibits the binding of a hedgehog ligand to a hedgehog receptor, e.g., Patched. In one embodiment, the hedgehog inhibitor is a Smoothened inhibitor.
In other embodiments, the hedgehog inhibitor targets a hedgehog ligand-dependent cancer or tumor, e.g., targets one or more of the tumor cell, the tumor micro environment, or other residual diseases that is responsive to a hedgehog ligand (e.g., a target tumor cell, a target tumor microenviroment, and/or a target residual disease as shown in Figure 19). In some embodiments, the hedgehog inhibitor targets the tumor micro environment of a hedgehog ligand-dependent cancer or tumor (e.g., a desmoplastic tumor, such as pancreatic cancer and/or neurodendocrine tumors) thereby causing one or more of. (i) depleting or reducing desmoplastic stroma and/or the stroma support provided to the tumor; (ii) increasing the vascularity of the tumor; or (iii) rendering the tumor more accessible to chemotherapy. In such embodiments, the hedgehog inhibitor can decrease fibrosis, thus leading to improved drug delivery and/or survival.
In other embodiments, the hedgehog inhibitor targets a hedgehog ligand-independent cancer or tumor, e.g., a cancer or tumor having a genetic mutation in a hedgehog receptor (e.g., a Patched mutant tumor). Exemplary hedgehog ligand-independent cancers or tumors include, but are not limited to, basal cell carcinoma (e.g., advanced basal cell carcinoma) and medulloblastoma.
In certain embodiments, the tumor or cancer, e.g., the hedgehog-associated cancer or tumor, treated includes, but is not limited to, a solid tumor, a soft tissue tumor (e.g., a heme malignancy), and a metastatic lesion, e.g., a metastatic lesion of any of the cancers disclosed herein.
In one embodiment, the tumor or cancer, e.g., the hedgehog-associated cancer or tumor, treated with the hedgehog inhibitor is a sarcoma, e.g., a bone or soft tissue Attorney Docket No. I2041-7000WO/3020PCT

sarcoma (e.g., a synovial sarcoma, a liposarcoma, a musculoskeletal sarcoma, such as bone and cartilage sarcoma, chondrosarcoma and osteosarcoma). In one embodiment, the hedgehog inhibitor alone or combination with a second agent (e.g., an mTOR
inhibitor and/or other anti-cancer agents (e.g., one or more of. doxorubicin, cisplatin, ifosfamide, or methotrexate (e.g., high dose methotrexate))) reduces or inhibits local or metastatic sarcoma invasion. In one embodiment, the hedgehog inhibitor, alone or combination with the mTOR inhibitor, treats or prevents a chondrosarcoma. In other embodiments, the hedgehog inhibitor alone or combination with the mTOR inhibitor treats or prevents an osteosarcoma, e.g., a relapsed or refractory osteosarcoma.
In other embodiments, the tumor or cancer, e.g., the hedgehog-associated cancer or tumor, treated with the hedgehog inhibitor is a neuroendocrine cancer or tumor. In one embodiment, the cancer or tumor treated is a neuroendocrine cancer chosen from one or more of, e.g., a neuroendocrine cancer of the pancreas (e.g., a pancreatic endocrine tumor), lung, appendix, duodenum, ileum, rectum, small intestine; or a neuroendocrine cancer from the adrenal medulla, the pituitary, the parathyroids, thyroid endocrine islets, pancreatic endocrine islets, or dispersed endocrine cells in the respiratory or gastrointestinal tract. In other embodiments, the cancer or tumor treated is a carcinoid tumor, e.g., a functional or a non-functional carcinoid neuroendocrine cancer.
In certain embodiments, the hedgehog inhibitor is administered in combination with a tyrosine kinase inhibitor (e.g., one or more of a receptor tyrosine inhibitor (RTK), e.g., sunitinib) in an amount sufficient to treat or prevent the neuroendocrine tumor. In one embodiment, the hedgehog inhibitor and the tyrosine kinase inhibitor are administered concurrently. In other embodiments, the hedgehog inhibitor and the tyrosine kinase inhibitor are administered sequentially. For example, the hedgehog inhibitor can be administered before initiating treatment with, or after ceasing treatment with, the tyrosine kinase inhibitor. Administration of the hedgehog inhibitor and the tyrosine kinase inhibitor can overlap in part with each other, and either of which can be continued as a single agent after cessation of treatment with the other.
In yet another embodiment, the tumor or cancer, e.g., the hedgehog-associated cancer or tumor, is a pancreatic cancer and is treated with a combination of a hedgehog inhibitor and paclitaxel or a paclitaxel agent (e.g., a paclitaxel formulation such as Attorney Docket No. I2041-7000WO/3020PCT
TAXOL ), an albumin-stabilized nanoparticle paclitaxel formulation (e.g., ABRAXANE ) or a liposomal paclitaxel formulation, e.g., Endo Tag 1). In certain embodiments, the hedgehog inhibitor is administered concurrently with the paclitaxel or the paclitaxel agent. In other embodiments, the hedgehog inhibitor and the paclitaxel or the paclitaxel agent are administered sequentially. For example, the hedgehog inhibitor can be administered before initiating treatment with, or after ceasing treatment with, the paclitaxel or the paclitaxel agent. In yet other embodiments, administration of the hedgehog inhibitor overlaps with the treatment with the paclitaxel or the paclitaxel agent, and continues after treatment with the paclitaxel or the paclitaxel agent has ceased. In other embodiments, the hedgehog inhibitor and the paclitaxel are administered in combination with an additional therapeutic agent (e.g., a third anti-cancer agent chosen from gemcitabine, cisplatin, epirubicin, 5-fluorouracil, leucovorin, oxaplatin, a VEGF
antagonist, e.g., an anti-VEGF antibody (e.g., bevacizumab), or a combination thereof).
In certain embodiments, the hedgehog inhibitor, the paclitaxel agent and the third anti-cancer agent are administered concurrently, sequentially, or in a partially overlapping schedule. In other embodiments, the third anti-cancer agent is administered prior to initiating treatment with, or after ceasing treatment with, the hedgehog inhibitor and the paclitaxel or the paclitaxel agent. In one embodiment, the combination of the hedgehog inhibitor and paclitaxel or paclitaxel agent can further include gemcitabine (e.g., GEMZAR ). In one embodiment, the subject is a patient with metastastatic pancreatic cancer. In another embodiment, the subject is a patient with pancreatic or metastastatic pancreatic cancer who has received no prior cancer treatment (e.g., no prior exposure to other anti-cancer agent, surgical or radiation procedure for, e.g., no prior cancer treatment for their disease, e.g., their metastatic disease).
In other embodiments, the tumor or cancer, e.g., the hedgehog-associated cancer or tumor, is a pancreatic cancer and is treated with a hedgehog inhibitor, and can further include one or more of. paclitaxel, a paclitaxel agent, a VEGF antagonist, e.g., an anti-VEGF antibody (e.g., bevacizumab), 5-fluorouracil, or oxaplatin.
In other embodiments, the tumor or cancer, e.g., the hedgehog-associated cancer or tumor, is a pancreatic cancer and is treated with a combination of a hedgehog inhibitor Attorney Docket No. I2041-7000WO/3020PCT
and paclitaxel or a paclitaxel agent, and can further include one or more of a VEGF
antagonist, e.g., an anti-VEGF antibody (e.g., bevacizumab), 5-fluorouracil, or oxaplatin.
In other embodiments, the tumor or cancer, e.g., the hedgehog-associated cancer or tumor, treated with the hedgehog inhibitor is a head and neck squamous cell cancer. In certain embodiments, the hedgehog inhibitor is administered in combination with a tyrosine kinase inhibitor (e.g., in combination with one or more of a receptor tyrosine inhibitor (RTK), e.g., an EGFR-tyrosine kinase inhibitor such as an anti-EGFR
antibody (e.g., cetuximab)) to treat or prevent the head and neck squamous cell cancer.
In one embodiment, the hedgehog inhibitor and the tyrosine kinase inhibitor are administered concurrently. In other embodiments, the hedgehog inhibitor and the tyrosine kinase inhibitor are administered sequentially. For example, the hedgehog inhibitor can be administered before initiating treatment with, or after ceasing treatment with, the tyrosine kinase inhibitor. In one embodiment, the administration of the hedgehog inhibitor overlaps with the treatment with the tyrosine kinase inhibitor, and continues after treatment with the tyrosine kinase inhibitor has ceased.
In yet another embodiment, the tumor or cancer, e.g., the hedgehog-associated cancer or tumor, treated with the hedgehog inhibitor is a lung cancer, e.g., a small or a non-small lung cancer. In one embodiment, the lung cancer is non-small lung cancer. In certain embodiments, the hedgehog inhibitor is administered in combination with a tyrosine kinase inhibitor (e.g., in combination with one or more of a receptor tyrosine inhibitor (RTK), e.g., gefitinib or a VEGF inhibitor) to treat or prevent the lung cancer.
In one embodiment, the lung cancer is non-small cell lung cancer and the hedghehog inhibitor is administered in combination with a VEGF inhibitor (e.g., an anti-VEGF
antibody such as bevacizumab) in combination with carboplatin and/or paclitaxel or a paclitaxel agent. In one embodiment, the hedgehog inhibitor and the tyrosine kinase or other agent inhibitor are administered concurrently. In other embodiments, the hedgehog inhibitor and the tyrosine kinase inhibitor or other agent are administered sequentially.
For example, the hedgehog inhibitor can be administered before initiating treatment with, or after ceasing treatment with, the tyrosine kinase inhibitor. In one embodiment, the administration of the hedgehog inhibitor overlaps with the treatment with the tyrosine Attorney Docket No. I2041-7000WO/3020PCT
kinase inhibitor or other agent, and continues after treatment with the tyrosine kinase inhibitor or other agent has ceased.
In certain embodiments, the tyrosine kinase inhibitor is a receptor tyrosine kinase inhibitor. In one embodiment, the tyrosine kinase inhibitor is chosen from sunitinib, erlotinib, gefitinib, or sorafenib. In certain embodiment, the tyrosine kinase inhibitor is an EGFR-tyrosine kinase inhibitor. In certain embodiments, the EGFR-tyrosine kinase inhibitor is a small molecule EGFR-tyrosine kinase inhibitor. In one embodiment, the small molecule EGFR-tyrosine kinase inhibitor is chosen from one or more of erlotinib, gefitinib, icotinib, lapatinib, neratinib, vandetanib, BIBW 2992 or XL-647. In other embodiments, the small molecule EGFR-tyrosine kinase inhibitor is gefitinib or erlotinib.
In certain embodiments, the small molecule EGFR-tyrosine kinase inhibitor is gefitinib.
In other embodiments, the small molecule EGFR-tyrosine kinase inhibitor is erlotinib. In certain embodiments, the EGFR-tyrosine kinase inhibitor is a monoclonal antibody. In certain embodiments, the monoclonal antibody is chosen from cetuximab, panitumumab, zalutumumab, nimotuzumab necitumumab or matuzumab. In one embodiment, the monoclonal antibody is cetuximab.
The methods and compositions of the invention can optionally be used in combination with other therapeutic modalities, e.g., one or more anti-cancer agents, and/or in combination with surgical and/or radiation procedures as described herein.
Additional embodiments or features of the present invention are as follows:
In one embodiment, the subject treated is a mammal, e.g., a primate, typically a human (e.g., a patient having, or at risk of, a cancer or tumor as described herein). In another embodiment, the subject treated is in need of hedgehog inhibition (e.g., has been evaluated to show elevated hedgehog levels). In certain embodiment, the subject is a human having, or at risk of having, a hedgehog-associated tumor or cancer. In one embodiment, the subject is a human having, or at risk of having, a hedgehog ligand-independent tumor or cancer. In another embodiment, the subject is a human having, or at risk of having, a hedgehog ligand-dependent cancer or tumor. In one embodiment, the subject is a human having, or at risk of having, a hedgehog ligand-dependent cancer or tumor chosen from one or more of a neuroendocrine cancer, a sarcoma (e.g., a Attorney Docket No. I2041-7000WO/3020PCT
musculoskeletal sarcoma, such as chondrosarcoma and osteosarcoma), a pancreatic cancer, a head and neck cancer, or a lung cancer (e.g., a small cell or a non-small cell lung cancer). In one embodiment, the subject is a patient suffering from multiple endocrine neoplasia type 1. In other embodiments, the subject is a patient suffering from a chondrosarcoma, or an osteosarcoma, e.g., a relapsed or refractory osteosarcoma.
In another embodiment, the subject is in need of, or being considered for, treatment with a hedgehog inhibitor, alone or in combination, with any of the anti-cancer agents disclosed herein. The subject can be one at risk of having the disorder, e.g., a subject having a relative afflicted with the disorder, or a subject having a genetic trait associated with risk for the disorder. In one embodiment, the subject can be symptomatic or asymptomatic. In certain embodiments, the subject harbors an alteration in an EGFR
gene or gene product. In certain embodiments, the subject is a patient who is undergoing cancer therapy (e.g., other anti-cancer agents, surgery and/or radiation). In certain embodiments, the subject is a patient who has undergone cancer therapy (e.g., other anti-cancer agents, surgery and/or radiation). In one embodiment, the subject has been treated with a tyrosine kinase inhibitor (e.g., sunitinib, cetuximab or geftinib). In certain embodiments, the subject has developed a partial or complete resistance to a previous anti-cancer treatment, e.g., the subject does not respond well to treatment with a tyrosine kinase inhibitor (e.g., sunitinib, cetuximab or geftinib).
In one embodiment, the subject is a patient with a metastatic cancer, e.g., metastastatic pancreatic cancer. In another embodiment, the subject is a patient with a cancer or a metastatic cancer (e.g., pancreatic or metastastatic pancreatic cancer) who has received no prior cancer treatment (e.g., no prior exposure to other anti-cancer agent, surgical or radiation procedure for, e.g., no prior cancer treatment for their disease, e.g., their metastatic disease).
In one embodiment, the hedgehog inhibitor used in the methods or compositions of the invention is a compound of formula I:

Attorney Docket No. I2041-7000WO/3020PCT
H

O NH
~H
H

_Pg,N"
O H

or a pharmaceutically acceptable salt thereof. An example of a pharmaceutically acceptable salt of the compound of formula I is the hydrochloride salt.
In some embodiments, the hedgehog inhibitor is administered as a pharmaceutical composition comprising the hedgehog inhibitor, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
In certain embodiments, the hedgehog inhibitor is administered, or is present in the composition, e.g., the pharmaceutical composition.
The hedgehog inhibitors described herein can be administered to the subject systemically (e.g., orally, parenterally, subcutaneously, intravenously, rectally, intramuscularly, intraperitoneally, intranasally, transdermally, or by inhalation or intracavitary installation). Typically, the hedgehog inhibitors are administered orally.
In one embodiment, the hedgehog inhibitor is IPI-926. IPI-926 can be administered orally in a daily schedule at a dose of about 20 mg to 200 mg, typically about 50 to 150 mg, 75 to 140 mg, and more typically 120 to 130 mg, alone or in combination with a second agent as described herein.
The methods and compositions of the invention can, optionally, be used in combination with other therapeutic modalities, e.g., one or more additional anti-cancer agents, and/or in combination with surgical and/or radiation procedures. In other embodiments, the methods and compositions of the invention are used in combination with surgical and/or radiation procedures. Any combination of the hedgehog inhibitor and other therapeutic modalities can be used. For example, the hedgehog inhibitor and the other therapeutic modalities can be administered during periods of active disorder, or during a period of remission or less active disorder. The hedgehog inhibitor and other therapeutic modalities can be administered before treatment, concurrently with treatment, Attorney Docket No. I2041-7000WO/3020PCT

post-treatment, or during remission of the disorder. In one embodiment, the anti-cancer agent is administered simultaneously or sequentially with the hedgehog inhibitor.
In other embodiments, the hedgehog inhibitor and the anti-cancer agent are administered as separate compositions, e.g., pharmaceutical compositions. In other embodiments, the hedgehog inhibitor and the anti-cancer agent are administered separately, but via the same route (e.g., both orally or both intravenously).
In still other instances, the hedgehog inhibitor and the anti-cancer agent are administered in the same composition, e.g., pharmaceutical composition.
In some embodiments, the hedgehog inhibitor is a first line treatment for the cancer, e.g., the hedgehog-associated cancer or tumor, i.e., it is used in a subject who has not been previously administered another drug intended to treat the cancer.
In other embodiments, the hedgehog inhibitor is a second line treatment for the cancer, e.g., hedgehog-associated cancer or tumor, i.e., it is used in a subject who has been previously administered another drug intended to treat the cancer.
In other embodiments, the hedgehog inhibitor is a third or fourth line treatment for the cancer, e.g., the hedgehog-associated cancer or tumor, i.e., it is used in a subject who has been previously administered two or three other drugs intended to treat the cancer.
In other embodiments, the hedgehog inhibitor is administered as neoadjuvant therapy, i.e., prior to another treatment.
In other embodiments, the hedgehog inhibitor is administered as adjuvant therapy, i.e., a treatment in addition to a primary therapy.
In some embodiments, the hedgehog inhibitor is administered to a subject prior to, or following surgical excision/removal of the cancer, e.g., the hedgehog-associated cancer or tumor.
In some embodiments, the hedgehog inhibitor is administered to a subject before, during, and/or after radiation treatment of the cancer, e.g., the hedgehog-associated cancer or tumor.
In some embodiments, the hedgehog inhibitor is administered to a subject, e.g., a cancer patient who is undergoing or has undergone cancer therapy (e.g., treatment with another anti-cancer agent, radiation therapy and/or surgery). In other embodiments, the Attorney Docket No. I2041-7000WO/3020PCT

hedgehog inhibitor is administered concurrently with the cancer therapy. In instances of concurrent administration, the hedgehog inhibitor can continue to be administered after the cancer therapy has ceased. In other embodiments, the hedgehog inhibitor is administered sequentially with the cancer therapy. For example, the hedgehog inhibitor can be administered before initiating treatment with, or after ceasing treatment with, the cancer therapy. In one embodiment, the administration of the hedgehog inhibitor overlaps with the cancer therapy, and continues after the cancer therapy has ceased. In one embodiment, the hedgehog inhibitor is administered after cancer therapy has ceased (i.e., with no period of overlap with the cancer treatment). For example, the cancer therapy and the hedgehog inhibitor can be administered concurrently, sequentially, or as a combination of concurrent administration followed by monotherapy with either the anti-cancer agent, or the hedgehog inhibitor.
In one embodiment, the method includes administering the hedgehog inhibitor as a first therapeutic agent, followed by administration of a cancer therapy (e.g., treatment with a second therapeutic agent (e.g., another anti-cancer agent), radiation therapy and/or surgery). In another embodiment, the method includes administering a cancer therapy first (e.g., treatment with a first therapeutic agent (e.g., another anti-cancer agent), radiation therapy and/or surgery), followed by administering the hedgehog inhibitor as a second therapeutic agent.
In yet other embodiments, the method includes administering the hedgehog inhibitor in combination with a second, third or more additional therapeutic agents (e.g., anti-cancer agents as described herein). For example, the hedgehog inhibitor and another anti-cancer agent (e.g., a second anti-cancer agent chosen from a tyrosine kinase inhibitor, paclitaxel or a paclitaxel agent, an mTOR inhibitor) are administered in combination with yet another therapeutic agent (e.g., a third anti-cancer agent chosen from gemcitabine, cisplatin, epirubicin, 5-fluorouracil, a VEGF antagonist (e.g., an anti-VEGF antibody (bevacizumab), leucovorin, oxaplatin, or a combination thereof).
In one embodiment, the hedgehog inhibitor, the second and third anti-cancer agents are administered concurrently. In other embodiments, the third anti-cancer agent is administered prior to initiating treatment with, or after ceasing treatment with, the hedgehog inhibitor and the second anti-cancer agents. Any order and combination of the Attorney Docket No. I2041-7000WO/3020PCT

administration of the hedgehog inhibitor, with a second, third or more anti-cancer agent is within the scope of the present invention.
In other embodiments, one or more hedgehog inhibitors (e.g., one or more hedgehog inhibitors are described herein) are administered in combination. In one embodiment, the hedgehog inhibitors are administed concurrently. In another embodiment the inhibitors are administered sequentially. For example, a combination of e.g., IPI-926 and GDC-0449 can be administered concurrently or sequentially.
In one embodiment, GDC-0449 is administered first, followed, with or without a period of overlap, by administration of IPI-926. In another embodiment, IPI-926 is administered first, followed, with or without a period of overlap, by administration of GDC-0449.
In one embodiment, the anti-cancer agent used in combination with the hedgehog inhibitor is a cytotoxic or a cytostatic agent. Exemplary cytotoxic agents include antimicrotubule agents, topoisomerase inhibitors (e.g., irinotecan), or taxanes (e.g., docetaxel), antimetabolites, mitotic inhibitors, alkylating agents, intercalating agents, agents capable of interfering with a signal transduction pathway, agents that promote apoptosis and radiation. In yet other embodiments, the methods can be used in combination with immunodulatory agents, e.g., IL-1, 2, 4, 6, or 12, or interferon alpha or gamma, or immune cell growth factors such as GM-CSF. In one embodiment, the anti-cancer agent is a topoisomerase inhibitor, e.g., irinotecan.
In other embodiments, the anti-cancer agent used in combination with the hedgehog inhibitor is paclitaxel or a paclitaxel agent, a tyrosine kinase inhibitor (e.g., a receptor tyrosine kinase (RTK) inhibitor, e.g., an EGFR inhibitor, gefitinib, sunitinib) or an mTOR inhibitor.
In certain embodiments, the anti-cancer agent is a tyrosine kinase inhibitor (e.g., a receptor tyrosine kinase (RTK) inhibitor). Thus, certain embodiments, the methods of the invention include administering to the subject in need of treatment, or at risk of having the cancer, a hedgehog inhibitor as described herein, in combination with a tyrosine kinase inhibitor (e.g., a receptor tyrosine kinase (RTK) inhibitor) in an amount effective to reduce or treat the cancer or tumor, e.g., the hedgehog ligand dependent cancer or tumor described herein. In one embodiment, the tyrosine kinase inhibitor include, but is not limited to, an epidermal growth factor (EGF) pathway inhibitor (e.g., Attorney Docket No. I2041-7000WO/3020PCT

an epidermal growth factor receptor (EGFR) inhibitor), a vascular endothelial growth factor (VEGF) pathway inhibitor (e.g., a vascular endothelial growth factor receptor (VEGFR) inhibitor (e.g., a VEGFR-1 inhibitor, a VEGFR-2 inhibitor, a VEGFR-3 inhibitor)), a platelet derived growth factor (PDGF) pathway inhibitor (e.g., a platelet derived growth factor receptor (PDGFR) inhibitor (e.g., a PDGFR-13 inhibitor)), a RAF-1 inhibitor, a KIT inhibitor and a RET inhibitor. In some embodiments, the anti-cancer agent used in combination with the hedgehog inhibitor is selected from the group consisting of. axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTIN
TM
AZD2171), dasatinib (SPRYCEL , BMS-354825), erlotinib (TARCEVA ), gefitinib (IRESSA ), imatinib (Gleevec , CGP57148B, STI-571), lapatinib (TYKERB , TYVERB ), lestaurtinib (CEP-701), neratinib (HKI-272), nilotinib (TASIGNA ), semaxanib (semaxinib, SU5416), sunitinib (SUTENT , SU11248), toceranib (PALLADIA ), vandetanib (ZACTIMA , ZD6474), vatalanib (PTK787, PTK/ZK), trastuzumab (HERCEPTIN ), bevacizumab (AVASTIN ), rituximab (RITUXAN ), cetuximab (ERBITUX ), panitumumab (VECTIBIX ), ranibizumab (Lucentis ), nilotinib (TASIGNA ), sorafenib (NEXAVAR ), alemtuzumab (CAMPATH ), gemtuzumab ozogamicin (MYLOTARG ), ENMD-2076, PCI-32765, AC220, dovitinib lactate (TK1258, CHIR-258), BIBW 2992 (TOVOKTM), SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120 (VARGATEF ), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154, CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, and/or XL228. Selected tyrosine kinase inhibitors are chosen from sunitinib, erlotinib, gefitinib, or sorafenib. In certain embodiments, the tyrosine kinase inhibitor is chosen from cetuximab, bevacizumab, panitumumab, zalutumumab, nimotuzumab necitumumab or matuzumab. In certain embodiments, the monoclonal antibody is cetuximab.
In one embodiment, the hedgehog inhibitor is administered in combination with an mTOR inhibitor, e.g., one or more mTOR inhibitors chosen from one or more of rapamycin, temsirolimus (TORISEL ), everolimus (RAD001, AFINITOR ), ridaforolimus, AP23573, AZD8055, BEZ235, BGT226, XL765, PF-4691502, GDC0980, SF1126, OSI-027, GSK1059615, KU-0063794, WYE-354, INK128, temsirolimus (CCI-779), Palomid 529 (P529), PF-04691502, or PKI-587. In one embodiment, the mTOR

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inhibitor inhibits TORCI and TORC2. Examples of TORCI and TORC2 dual inhibitors include, e.g., OSI-027, XL765, Palomid 529, and INK128. The hedgehog inhibitor can be administered via the same or a different route than the mTOR inhibitor. In one embodiment, the mTOR inhibitor is administered systemically, e.g., orally, subcutaneously, or intravenously.
In other embodiments, the hedgehog inhibitor is administered with an inhibitor of insulin-like growth factor receptor (IGF-1R). Exemplary IGF-1R inhibitors include, but are not limited to, small molecule IGF-1R antagonists (e.g., GSK1904529A), antibody antagonists, IGF-1R peptide antagonists, or anti-sense or other nucleic acid antagonists.
In yet another embodiment, the hedgehog inhibitor is administered in combination with an ALK kinase inhibitor(s). Exemplary ALK inhibitors include TAE-684 (also referred to herein as "NVP-TAE694"), PF02341066 (also referred to herein as "crizotinib" or "1066"), and AP26113. Additional examples of ALK kinase inhibitors are described in example 3-39 of WO 2005016894 by Garcia-Echeverria C, et at.
In some embodiments, the hedgehog inhibitor is administered in combination with folfirinox. Folfirinox comprises oxaliplatin 85 mg/m2 and irinotecan 180 mg/m2 plus leucovorin 400 mg/m2 followed by bolus fluorouracil (5-FU) 400 mg/m2 on day 1, then 5-FU 2,400 mg/m2 as a 46-hour continuous infusion.
In some embodiments, the hedgehog inhibitor is administered in combination with a P13K inhibitor. In one embodiment, the P13K inhibitor is an inhibitor of delta and gamma isoforms of P13K. In another embodiment, the P13K inhibitor is a dual inhibitor of P13K and mTOR. Exemplary P13K inhibitors that can be used in combination with the hedgehog inhibitor, include but are not limited to, GSK 2126458, GDC-0980, GDC-0941, Sanofi XL147, XL756, XL147, PF-46915032, BKM 120, CAL-101, CAL 263, SF1126, PX-886, and a dual P13K inhibitor (e.g., Novartis BEZ235). In one embodiment, the P13K inhibitor is an isoquinolinone. In one embodiment, the inhibitor is INKI 197 or a derivative thereof. In other embodiments, the P13K
inhibitor is INKI 117 or a derivative thereof.
In some embodiments, the hedgehog inhibitor is administered in combination with a BRAF inhibitor, e.g., GSK2118436, RG7204, PLX4032, GDC-0879, PLX4720, and sorafenib tosylate (Bay 43-9006).

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In some embodiments, the hedgehog inhibitor is administered in combination with a MEK inhibitor, e.g., ARRY-142886, GSKI 120212, RDEA436, RDEA119/BAY
869766, AS703026, AZD6244 (selumetinib), BIX 02188, BIX 02189, CI-1040 (PD184352), PD0325901, PD98059, and U0126.
In some embodiments, the hedgehog inhibitor is administered in combination with a JAK2 inhibitor, e.g., CEP-701, NCB 18424, CP-690550 (tasocitinib).
In other embodiments, the hedgehog inhibitor is administered in combination with a vascular disrupting agent (e.g., DMXAA, vadimezan).
The aforesaid combinations can be used to treat any of the cancers and metastic growths described herein. The combinations described herein can be administered in any order. Unless explicitly noted otherwise, the use herein of the term "first,"
"second," or "third" agent is not intended to imply a particular order of administration.
It is intended to clarify the different classes of agents used.
In yet other embodiments, the hedgehog inhibitor, alone or combination with the anti-cancer agent is administered in a therapeutically effective amount, e.g., at a predetermined dosage schedule.
In certain embodiments, wherein the hedgehog inhibitor is used in combination with a tyrosine kinase inhibitor, the method includes administering the hedgehog inhibitor and/or the tyrosine kinase inhibitor at sub-cytotoxic levels. In some embodiments, the tyrosine kinase inhibitor is administered to a subject at a dose (e.g., oral dose) of at least about 10 mg, about 25 mg, about 37.5 mg, about 50 mg, about 70 mg, about 87.5 mg, about 100 mg, about 125 mg, or about 150 mg per day. In some embodiments, the tyrosine kinase inhibitor is administered to a subject at a dose (e.g., oral dose) of about 37.5 mg, about 50 mg, or about 87.5 mg per day. In some embodiments, the tyrosine kinase inhibitor is administered to a subject at a dose (e.g., oral dose) of about 50 mg per day. In some embodiments, the tyrosine kinase inhibitor is administered to a subject once, twice, three, or more times per day. In some embodiments, the tyrosine kinase inhibitor is administered to a subject once per day. In some embodiments, the tyrosine kinase inhibitor is administered to a subject daily for about one, two, three, four or more weeks. In some embodiments, the tyrosine kinase inhibitor is administered to a subject daily for about four weeks.

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For example, in embodiments where sunitinib is the tyrosine kinase inhibitor administered in combination with the hedgehog inhibitor, it can be administered at a dose of about 50 mg daily; less than 50 mg daily, e.g., 37.5 mg daily; or greater than 50 mg daily, e.g., 62.5 mg daily. In one embodiment, the tyrosine kinase inhibitor (e.g., sunitinib) is administered daily for one, two, three, four or five weeks, followed by one, two, or three weeks without administration. In certain embodiments, sunitinib is administered orally.
The methods of the invention can further include the step of evaluating a sample from the tumor, the cancer cell or the subject, e.g., to detect the presence or absence of an alteration in an EGFR gene or gene product. The method can be used to identify or select a tumor, a cancer cell, or a subject (e.g., a subject having a cancer or tumor, or at risk for developing a cancer or tumor) as having a likelihood (e.g., increased or decreased likelihood), to respond to a treatment comprising an EGFR inhibitor in combination with a hedgehog inhibitor. Exemplary alterations in an EGFR gene or gene product that can be evaluated and/or treated, include but are not limited to, an EGFR exon deletion (e.g., EGFR exon 19 deletion, E746-A750 deletion), and/or exon mutation (e.g., an L858R/T790M EGFR mutation). Other exemplary alterations include, but are not limited to, EGFR D770 N771>AGG; EGFR D770 N77l insG; EGFR D770 N77l insG;
EGFR D770 N771insN: EGFR E709A; EGFR E709G: EGFR 709H; EGFR E709K:
EGFR E709V; EGFR E746 A750de1: EGFR E746 A750de1, T75 IA;
EGFR E746 A750de1, V ins; EGFR E746 T751 del, I ins; EGFR E746 T751 del, S752A; EGFR E746 T751del, S752D; EGFR E746 T751 del, V ins; EGFR G719A;
EGFR G719C; EGFR G719S: EGFR H773 V774insH; EGFR H773 V774insNPH;
EGFR H773 V774insPH; EGFR H773>NPY; EGFR L747 E749de1;
EGFR L747 E749de1, A750P; EGFR L747 S752de1: EGFR L747 S752de1, P753S;
EGFR_L747_S752de1, Q ins; EGFR_L747_T750de1, P ins; EGFR L747_T751de1;
EGFR_L858R; EGFR L861Q; EGFR M766 A767insA1; EGFR_P772_H773insV;
EGFR S752 1759de1; EGFR S7681; EGFR T790M: EGFR V769 D770insASV;
EGFR V769 D770insASV, and EGFR V774 C775insHV.
The methods of the invention can further include the step of monitoring the subject, e.g., for a change (e.g., an increase or decrease) in one or more of.
tumor size;

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hedgehog levels or signaling; stromal activation; levels of one or more cancer markers;
the rate of appearance of new lesions, e.g., in a bone scan; one or more of non-invasively tumor volume, metabolism, hypoxia evolution and/or tumor bone synthesis; the appearance of new disease-related symptoms; the size of soft tissue mass, e.g., a decreased or stabilization; quality of life, e.g., amount of disease associated pain, e.g., bone pain; histological analysis, e.g., synthesis of cartilage, lobular pattern, and/or the presence or absence of mitotic cells; tumor aggressivity, vascularization of primary tumor, metastatic spread; tumor size and location can be visualized using multimodal imaging techniques (e.g18F-FDG PET, 18FNa, MRI 18FMISO scintigraphies); or any other parameter related to clinical outcome. The subject can be monitored in one or more of the following periods: prior to beginning of treatment; during the treatment;
or after one or more elements of the treatment have been administered. Monitoring can be used to evaluate the need for further treatment with the same hedgehog inhibitor, alone or in combination with, the same anti-cancer agent, or for additional treatment with additional agents. Generally, a decrease in one or more of the parameters described above is indicative of the improved condition of the subject, although with serum hemoglobin levels, an increase can be associated with the improved condition of the subject.
The methods of the invention can further include the step of analyzing a nucleic acid or protein from the subject, e.g., analyzing the genotype of the subject.
In one embodiment, a hedgehog protein, or a nucleic acid encoding a hedgehog ligand and/or an upstream or downstream component(s) of the hedgehog signaling, e.g., a receptor, activator or inhibitor of hedgehog, is analyzed. The elevated hedgehog ligand can be detected in blood, urine, circulating tumor cells, a tumor biopsy or a bone marrow biopsy.
The elevated hedgehog ligand can also be detected by systemic administration of a labeled form of an antibody to a hedgehog ligand followed by imaging. The analysis can be used, e.g., to evaluate the suitability of, or to choose between alternative treatments, e.g., a particular dosage, mode of delivery, time of delivery, inclusion of adjunctive therapy, e.g., administration in combination with a second agent, or generally to determine the subject's probable drug response phenotype or genotype. The nucleic acid or protein can be analyzed at any stage of treatment, but preferably, prior to administration of the hedgehog inhibitor and/or anti-cancer agent, to thereby determine Attorney Docket No. I2041-7000WO/3020PCT

appropriate dosage(s) and treatment regimen(s) of the hedgehog inhibitor (e.g., amount per treatment or frequency of treatments) for prophylactic or therapeutic treatment of the subject.
In certain embodiments, the methods of the invention further include the step of detecting elevated hedgehog ligand in the subject, prior to, or after, administering a hedgehog inhibitor to the patient. The elevated hedgehog ligand can be detected in blood, urine, circulating tumor cells, a tumor biopsy or a bone marrow biopsy. The elevated hedgehog ligand can also be detected by systemic administration of a labeled form of an antibody to a hedgehog ligand followed by imaging. The step of detecting elevated hedgehog ligand can include the steps of measuring hedgehog ligand in the patient prior to administration of the other cancer therapy, measuring hedgehog ligand in the patient after administration of the other cancer therapy, and determining if the amount of hedgehog ligand after administration of the other chemotherapy is greater than the amount of hedgehog ligand before administration of the other chemotherapy. The other cancer therapy can be, for example, a chemotherapeutic or radiation therapy.
In another aspect, the method further includes the step of identifying one or more anti-cancer agents that elevate hedgehog ligand expression in a tumor (e.g., a neuroendocrine cancer or sarcoma), and administering a therapeutically effective amount of the one or more anti-cancer agents that elevate hedgehog ligand expression in the tumor and a therapeutically effective amount of a hedgehog inhibitor. The step of identifying the anti-cancer agent that elevate hedgehog expression can include the steps of exposing cells from the tumor to one or more anti-cancer agents in vitro and measuring hedgehog ligand in the cells.
In another aspect, the invention features a composition, e.g., a pharmaceutical composition, that includes one or more hedgehog inhibitors, e.g., a hedgehog inhibitor as described herein, and one or more anti-cancer agents (e.g., an anti-cancer agent as disclosed herein). The composition can further include a pharmaceutically-acceptable carrier or excipient.
In another aspect, the invention features a composition for use, or the use, of a hedgehog inhibitor, alone or in combination with an anti-cancer agent described herein (e.g., a paclitaxel or a paclitaxel agent, tyrosine kinase inhibitor, mTOR
inhibitor, and/or Attorney Docket No. I2041-7000WO/3020PCT

IGF-1R antagonist) for the treatment of a cancer or tumor, e.g., a hedgehog associated cancer or tumor described herein.
In another aspect, the invention features therapeutic kits that include the hedgehog inhibitor, alone or in combination with an anti-cancer agent described herein (e.g., a paclitaxel or a paclitaxel agent, a tyrosine kinase inhibitor, mTOR inhibitor, and/or IGF-1R antagonist), and instructions for use the treatment of cancer, e.g., a hedgehog associated cancer or tumor described herein.
All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.
Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DEFINITIONS
Definitions of specific functional groups and chemical terms are described in detail below. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in, for example, Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH
Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.
Certain compounds of the present invention can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, i.e., stereoisomers (enantiomers, diastereomers, cis-trans isomers, E/Z isomers, etc.). Thus, inventive compounds and pharmaceutical compositions thereof can be in the form of an individual enantiomer, diastereomer or other geometric isomer, or can be in the form of a mixture of stereoisomers. Enantiomers, diastereomers and other geometric isomers can be isolated from mixtures (including racemic mixtures) by any method known to those skilled in the Attorney Docket No. I2041-7000WO/3020PCT

art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts or prepared by asymmetric syntheses; see, for example, Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, S.H., et al., Tetrahedron 33:2725 (1977); Eliel, E.L.
Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, S.H. Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972).
Carbon atoms, unless otherwise specified, can optionally be substituted with one or more substituents. The number of substituents is typically limited by the number of available valences on the carbon atom, and can be substituted by replacement of one or more of the hydrogen atoms that would be available on the unsubstituted group.
Suitable substituents are known in the art and include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, alkoxy, aryl, aryloxy, arylthio, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, heterocyclyl, halo, azido, hydroxyl, thio, alkthiooxy, amino, nitro, nitrile, imino, amido, carboxylic acid, aldehyde, carbonyl, ester, silyl, alkylthio, haloalkyl (e.g., perfluoroalkyl such as -CF3), =0, =S, and the like.
When a range of values is listed, it is intended to encompass each value and sub-range within the range. For example, an alkyl group containing 1-6 carbon atoms (Ci_6 alkyl) is intended to encompass, C1, C2, C31 C41 C51 C61 C1-6, C2_6, C3_6, C4-6, C5_6, C1-51 C2-5, C3-5, 04_5, C1_4, C2_4, C3_4, CI-3, C2-3, and CI-2 alkyl.
The term "alkyl," as used herein, refers to saturated, straight- or branched-chain hydrocarbon radical containing between one and thirty carbon atoms. In certain embodiments, the alkyl group contains 1-20 carbon atoms. Alkyl groups, unless otherwise specified, can optionally be substituted with one or more substituents. In certain embodiments, the alkyl group contains 1-10 carbon atoms. In certain embodiments, the alkyl group contains 1-6 carbon atoms. In certain embodiments, the alkyl group contains 1-5 carbon atoms. In certain embodiments, the alkyl group contains 1-4 carbon atoms. In certain embodiments, the alkyl group contains 1-3 carbon atoms.
In certain embodiments, the alkyl group contains 1-2 carbon atoms. In certain embodiments, the alkyl group contains 1 carbon atom. Examples of alkyl radicals include, but are not limited to, methyl, ethyl, n propyl, isopropyl, n-butyl, iso-butyl, Attorney Docket No. I2041-7000WO/3020PCT

sec-butyl, sec-pentyl, iso-pentyl, tert-butyl, n-pentyl, neopentyl, n hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, and the like.
The term "alkenyl," as used herein, denotes a straight- or branched-chain hydrocarbon radical having at least one carbon-carbon double bond by the removal of a single hydrogen atom, and containing between two and thirty carbon atoms.
Alkenyl groups, unless otherwise specified, can optionally be substituted with one or more substituents. In certain embodiments, the alkenyl group contains 2-20 carbon atoms. In certain embodiments, the alkenyl group contains 2-10 carbon atoms. In certain embodiments, the alkenyl group contains 2-6 carbon atoms. In certain embodiments, the alkenyl group contains 2-5 carbon atoms. In certain embodiments, the alkenyl group contains 2-4 carbon atoms. In certain embodiment, the alkenyl group contains 2-carbon atoms. In certain embodiments, the alkenyl group contains 2 carbon atoms.
Alkenyl groups include, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-l-yl, and the like.
The term "alkynyl," as used herein, denotes a straight- or branched-chain hydrocarbon radical having at least one carbon-carbon triple bond by the removal of a single hydrogen atom, and containing between two and thirty carbon atoms.
Alkynyl groups, unless otherwise specified, can optionally be substituted with one or more substituents. In certain embodiments, the alkynyl group contains 2-20 carbon atoms. In certain embodiments, the alkynyl group contains 2-10 carbon atoms. In certain embodiments, the alkynyl group contains 2-6 carbon atoms. In certain embodiments, the alkynyl group contains 2-5 carbon atoms. In certain embodiments, the alkynyl group contains 2-4 carbon atoms. In certain embodiments, the alkynyl group contains carbon atoms. In certain embodiments, the alkynyl group contains 2 carbon atoms.
Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), l propynyl, and the like.
The terms "cycloalkyl", used alone or as part of a larger moiety, refer to a saturated monocyclic or bicyclic hydrocarbon ring system having from 3-15 carbon ring members. Cycloalkyl groups, unless otherwise specified, can optionally be substituted with one or more substituents. In certain embodiments, cycloalkyl groups contain 3-10 carbon ring members. In certain embodiments, cycloalkyl groups contain 3-9 carbon Attorney Docket No. I2041-7000WO/3020PCT
ring members. In certain embodiments, cycloalkyl groups contain 3-8 carbon ring members. In certain embodiments, cycloalkyl groups contain 3-7 carbon ring members.
In certain embodiments, cycloalkyl groups contain 3-6 carbon ring members. In certain embodiments, cycloalkyl groups contain 3-5 carbon ring members. Cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. The term "cycloalkyl" also includes saturated hydrocarbon ring systems that are fused to one or more aryl or heteroaryl rings, such as decahydronaphthyl or tetrahydronaphthyl, where the point of attachment is on the saturated hydrocarbon ring.
The term "aryl" used alone or as part of a larger moiety (as in "aralkyl"), refers to an aromatic monocyclic and bicyclic hydrocarbon ring system having a total of carbon ring members. Aryl groups, unless otherwise specified, can optionally be substituted with one or more substituents. In certain embodiments of the present invention, "aryl" refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, anthrancyl and the like, which can bear one or more substituents. Also included within the scope of the term "aryl", as it is used herein, is a group in which an aryl ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl or tetrahydronaphthalyl, and the like, where the point of attachment is on the aryl ring.
The term "aralkyl" refers to an alkyl group, as defined herein, substituted by aryl group, as defined herein, wherein the point of attachment is on the alkyl group.
The term "heteroatom" refers to boron, phosphorus, selenium, nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of abasic nitrogen.
The terms "heteroaryl" used alone or as part of a larger moiety, e.g., "heteroaralkyl", refer to an aromatic monocyclic or bicyclic hydrocarbon ring system having 5-10 ring atoms wherein the ring atoms comprise, in addition to carbon atoms, from one to five heteroatoms. Heteroaryl groups, unless otherwise specified, can optionally be substituted with one or more substituents. When used in reference to a ring atom of a heteroaryl group, the term "nitrogen" includes a substituted nitrogen.
Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, Attorney Docket No. I2041-7000WO/3020PCT

pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. The terms "heteroaryl" and "heteroar-", as used herein, also include groups in which a heteroaryl ring is fused to one or more aryl, cycloalkyl or heterocycloalkyl rings, wherein the point of attachment is on the heteroaryl ring.
Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, and tetrahydroisoquinolinyl.
The term "heteroaralkyl" refers to an alkyl group, as defined herein, substituted by a heteroaryl group, as defined herein, wherein the point of attachment is on the alkyl group.
As used herein, the terms "heterocycloalkyl" or "heterocyclyl" refer to a stable non-aromatic 5-7 membered monocyclic hydrocarbon or stable non-aromatic 7-10 membered bicyclic hydrocarbon that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more heteroatoms. Heterocycloalkyl or heterocyclyl groups, unless otherwise specified, can optionally be substituted with one or more substituents. When used in reference to a ring atom of a heterocycloalkyl group, the term "nitrogen" includes a substituted nitrogen. The point of attachment of a heterocycloalkyl group can be at any of its heteroatom or carbon ring atoms that results in a stable structure. Examples of heterocycloalkyl groups include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. "Heterocycloalkyl" also include groups in which the heterocycloalkyl ring is fused to one or more aryl, heteroaryl or cycloalkyl rings, such as indolinyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, where the radical or point of attachment is on the heterocycloalkyl ring.
The term "unsaturated", as used herein, means that a moiety has one or more double or triple bonds.

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As used herein, the term "partially unsaturated" refers to a ring moiety that includes at least one double or triple bond. The term "partially unsaturated"
is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aromatic groups, such as aryl or heteroaryl moieties, as defined herein.
The term "diradical" as used herein refers to an alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, and heteroaralkyl groups, as described herein, wherein 2 hydrogen atoms are removed to form a divalent moiety.
Diradicals are typically end with a suffix of "-ene". For example, alkyl diradicals are referred to as alkylenes (for example: . , , and -(CR'2)X wherein R' is hydrogen or other substituent and x is 1, 2, 3, 4, 5 or 6); alkenyl diradicals are referred to as "alkenylenes' ; alkynyl diradicals are referred to as "alkynylenes"; aryl and aralkyl diradicals are referred to as "arylenes" and "aralkylenes", respectively (for example:
); heteroaryl and heteroaralkyl diradicals are referred to as O
"heteroarylenes" and "heteroaralkylenes", respectively (for example: );
cycloalkyl diradicals are referred to as "cycloalkylenes"; heterocycloalkyl diradicals are referred to as "heterocycloalkylenes"; and the like.
The terms "halo", "halogen" and "halide" as used herein refer to an atom selected from fluorine (fluoro, F), chlorine (chloro, Cl), bromine (bromo, Br), and iodine (iodo, I).
As used herein, the term "haloalkyl" refers to an alkyl group, as described herein, wherein one or more of the hydrogen atoms of the alkyl group is replaced with one or more halogen atoms. In certain embodiments, the haloalkyl group is a perhaloalkyl group, that is, having all of the hydrogen atoms of the alkyl group replaced with halogens (e.g., such as the perfluoroalkyl group -CF3).
As used herein, the term "azido" refers to the group -N3.
As used herein, the term "nitrile" refers to the group -CN.
As used herein, the term "nitro" refers to the group -NO2.
As used herein, the term "hydroxyl" or "hydroxy" refers to the group -OH.
As used herein, the term "thiol" or "thio" refers to the group -SH.

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As used herein, the term "carboxylic acid" refers to the group -CO2H.
As used herein, the term "aldehyde" refers to the group -CHO.
As used herein, the term "alkoxy" refers to the group -OR', wherein R' is an alkyl, alkenyl or alkynyl group, as defined herein.
As used herein, the term "aryloxy" refers to the group -OR', wherein each R' is an aryl or heteroaryl group, as defined herein.
As used herein, the term "alkthiooxy" refers to the group -SR', wherein each R' is, independently, a carbon moiety, such as, for example, an alkyl, alkenyl, or alkynyl group, as defined herein.
As used herein, the term "arylthio" refers to the group -SR', wherein each R' is an aryl or heteroaryl group, as defined herein.
As used herein, the term "amino" refers to the group -NR'2, wherein each R' is, independently, hydrogen, a carbon moiety, such as, for example, an alkyl, alkenyl, alkynyl, aryl or heteroaryl group, as defined herein, or two R' groups together with the nitrogen atom to which they are bound form a 5-8 membered ring.
As used herein, the term "carbonyl" refers to the group -C(=O)R', wherein R' is, independently, a carbon moiety, such as, for example, an alkyl, alkenyl, alkynyl, aryl or heteroaryl group, as defined herein.
As used herein, the term "ester" refers to the group -C(=O)OR' or -OC(=O)R' wherein each R' is, independently, a carbon moiety, such as, for example, an alkyl, alkenyl, alkynyl, aryl or heteroaryl group, as defined herein.
As used herein, the term "amide" or "amido" refers to the group -C(=O)N(R')2 or - NR'C(=O)R' wherein each R' is, independently, hydrogen or a carbon moiety, such as, for example, an alkyl, alkenyl, alkynyl, aryl or heteroaryl group, as defined herein, or two R' groups together with the nitrogen atom to which they are bound form a 5-8 membered ring.
The term "sulfonamido" or "sulfonamide" refers to the group N(R')SO2R' or -SO2N(R')2, wherein each R' is, independently, hydrogen or a carbon moiety, such as, for example, an alkyl, alkenyl, alkynyl, aryl or heteroaryl group, as defined herein, or two R' groups together with the nitrogen atom to which they are bound form a 5-8 membered ring.

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The term "sulfamido" or "sulfamide" refers to the group -NR'SO2N(R')2, wherein each R' is, independently, hydrogen or a carbon moiety, such as, for example, an alkyl, alkenyl, alkynyl, aryl or heteroaryl group, as defined herein, or two R' groups together with the nitrogen atom to which they are bound form a 5-8 membered ring.
As used herein, the term "imide" or "imido" refers to the group -C(=NR')N(R')2 or -NR'C(=NR')R' wherein each R' is, independently,hydrogen or a carbon moiety, such as, for example, an alkyl, alkenyl, alkynyl, aryl or heteroaryl group, as defined herein, or wherein two R' groups together with the nitrogen atom to which they are bound form a 5-8 membered ring.
As used herein "silyl" refers to the group -SiR' wherein R' is a carbon moiety, such as, for example, an alkyl, alkenyl, alkynyl, aryl or heteroaryl group.
In some cases, the hedgehog inhibitor can contain one or more basic functional groups (e.g., such as an amino group), and thus is capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term "pharmaceutically acceptable salts" in these instances refers to the relatively non-toxic, inorganic and organic acid addition salts. These salts can be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately treating the compound in its free base form with a suitable acid. Examples of pharmaceutically acceptable, nontoxic acid addition salts from inorganic acids include, but are not limited to, hydrochloric, hydrobromic, phosphoric, sulfuric, nitric and perchloric acid or from organic acids include, but are not limited to, acetic, adipic, alginic, ascorbic, aspartic, 2-acetoxybenzoic, benzenesulfonic, benzoic, bisulfonic, boric, butyric, camphoric, camphorsulfonic, citric, cyclopentanepropionic, digluconic, dodecylsulfonic, ethanesulfonic, 1,2-ethanedisulfonic, formic, fumaric, glucoheptonic, glycerophosphonic, gluconic, hemisulfonic, heptanoic, hexanoic, hydroiodic, 2 hydroxyethanesulfonic, hydroxymaleic, isothionic, lactobionic, lactic, lauric, lauryl sulfonic, malic, maleic, malonic, methanesulfonic, 2-naphthalenesulfonic, napthylic, nicotinic, oleic, oxalic, palmitic, pamoic, pectinic, persulfonic, 3 phenylpropionic, picric, pivalic, propionic, phenylacetic, stearic, succinic, salicyclic, sulfanilic, tartaric, thiocyanic, p-toluenesulfonic, undecanoic, and valeric acid addition salts, and the like. In other cases, the hedgehog inhibitor can contain one or more acidic functional groups, and thus is Attorney Docket No. I2041-7000WO/3020PCT

capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" in these instances refers to the relatively non-toxic, inorganic and organic base addition salts. These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately treating the compound in its free acid form with a suitable base.
Examples of suitable bases include, but are not limited to, metal hydroxides, metal carbonates or metal bicarbonates, wherein the metal is an alkali or alkaline earth metal such as lithium, sodium, potassium, calcium, magnesium, or aluminum. Suitable bases can also include ammonia or organic primary, secondary or tertiary amines.
Representative organic amines useful for the formation of base addition salts include, for example, ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like (see, e.g., Berge et al., supra).
The term "solvate" refers to a compound of the present invention having either a stoichiometric or non-stoichiometric amount of a solvent associated with the compound.
The solvent can be water (i.e., a hydrate), and each molecule of inhibitor can be associated with one or more molecules of water (e.g., monohydrate, dihydrate, trihydrate, etc.). The solvent can also be an alcohol (e.g., methanol, ethanol, propanol, isopropanol, etc.), a glycol (e.g., propylene glycol), an ether (e.g., diethyl ether), an ester (e.g., ethyl acetate), or any other suitable solvent. The hedgehog inhibitor can also exist as a mixed solvate (i.e., associated with two or more different solvents).
The term "sugar" as used herein refers to a natural or an unnatural monosaccharide, disaccharide or oligosaccharide comprising one or more pyranose or furanose rings. The sugar can be covalently bonded to the steroidal alkaloid of the present invention through an ether linkage or through an alkyl linkage. In certain embodiments the saccharide moiety can be covalently bonded to a steroidal alkaloid of the present invention at an anomeric center of a saccharide ring. Sugars can include, but are not limited to ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, glucose, and trehalose.
As used herein, the articles "a" and "an" refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.

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The term "or" is used herein to mean, and is used interchangeably with, the term "and/or", unless context clearly indicates otherwise.
"About" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values.

DESCRIPTION OF THE FIGURES
Figure 1 is a graph depicting the change in tumor volume over time for BxPC-3 pancreatic tumor xenografts treated with vehicle and IPI-926.
Figure 2A is a graph depicting human Gli-1 levels in BxPC-3 pancreatic tumor xenografts treated with vehicle and IPI-926.
Figure 2B is a graph depicting murine Gli-1 levels in BxPC-3 pancreatic tumor xenografts treated with vehicle and IPI-926.
Figure 3 is a graph depicting the change in tumor volume over time for BxPC-3 pancreatic tumor xenografts treated with vehicle, IPI-926, gemcitabine, and a combination of IPI-926 and gemcitabine.
Figure 4 is a graph depicting the change in tumor volume over time for MiaPaCa pancreatic tumor xenografts treated with vehicle, IPI-926, gemcitabine, and a combination of IPI-926 and gemcitabine.
Figure 5 is a graph depicting the change in tumor volume over time for LX22 small cell lung cancer tumor xenografts treated with vehicle, IPI-926, etoposide/carboplatin, and a combination of IPI-926 and etoposide/carboplatin.
Figure 6 is a graph depicting the change in tumor volume over time for LX22 small cell lung cancer tumor xenografts treated with vehicle, IPI-926, etoposide/carboplatin followed by vehicle, and etoposide/carboplatin followed by IPI-926.
Figure 7A is a graph depicting murine Indian hedgehog levels in LX22 small cell lung cancer tumor xenografts that were treated with etoposide/carboplatin followed by vehicle or IPI-926.

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Figure 7B is a graph depicting human Indian hedgehog levels in LX22 small cell lung cancer tumor xenografts that were treated with etoposide/carboplatin followed by vehicle or IPI-926.
Figure 8A is a graph depicting murine Gli-1 expression levels in LX22 small cell lung cancer tumor xenografts that were treated with etoposide/carboplatin followed by vehicle or IPI-926.
Figure 8B is a graph depicting human Gli-1 expression levels in LX22 small cell lung cancer tumor xenografts that were treated with etoposide/carboplatin followed by vehicle or IPI-926.
Figure 9A is a graph depicting the change in murine hedgehog ligand expression levels in UMUC-3 bladder cancer tumor xenografts treated with gemcitabine as compared to naive UMUC-3 bladder cancer tumor xenografts.
Figure 9B is a graph depicting the change in human hedgehog ligand expression levels in UMUC-3 bladder cancer tumor xenografts treated with gemcitabine as compared to naive UMUC-3 bladder cancer tumor xenografts.
Figure 10 is a graph depicting the change in human Sonic, Indian and Desert Hedgehog ligand expression in UMUC-3 bladder cancer tumor cells treated with doxorubicin as compared to naive UMUC-3 bladder cancer tumor cells.
Figure 11 is a graph depicting the change in human Sonic and Indian Hedgehog ligand expression in A2780 ovarian cancer tumor cells treated with carboplatin or docetaxel as compared to naive A2780 ovarian cancer tumor cells.
Figure 12 is a graph depicting the change in human Sonic and Indian Hedgehog ligand expression in IGROV-1 ovarian cancer tumor cells treated with carboplatin or docetaxel as compared to naive IGROV-1 ovarian cancer tumor cells.
Figure 13 is a graph depicting the change in human Sonic and Indian Hedgehog ligand expression in H82 small cell lung cancer tumor cells treated with carboplatin or docetaxel as compared to naive H82 small cell lung cancer tumor cells.
Figure 14 is a graph depicting the change in Sonic Hedgehog ligand expression in UMUC-3 bladder cancer tumor cells exposed to hypoxic conditions as compared to UMUC-3 bladder cancer tumor cells exposed to normoxic conditions.

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Figures 15A-15G are bar graphs depicting the detection of human Sonic Hedgehog (SHH) expression in benign or tumor tissues from appendix (A), duodenum (B), ileum (C), pancreas (D), rectum (E), small intestine (F), or lung (G), by immunohistochemistry. N/A: spot missing/staining un-interpretable; -:
negative; -/+:
overall light staining; +: 5-25%; ++: 25-50%; +++: 50-75%; ++++: 75-100%.
Figures 16A-16B are graphs depicting murine (A) or human (B) Gli-1mRNA
levels in Bon-1 pancreatic neuroendocrine cancer (NET) xenografts treated with ( *p<0.005).
Figures 16C-16D are bar graphs depicting the mRNA levels of human Sonic Hedgehog (SHH) (A) or Indian Hedgehog (IHH) (B) in Bon-1 pancreatic neuroendocrine cancer (NET) xenografts treated with IPI-926.
Figure 17 is an image (I Ox Obj.) depicting the expression of human Sonic Hedgehog (SHH) in Bon-1 pancreatic neuroendocrine cancer (NET) xenografts detected by immunostaining.
Figure 18 is a graph depicting the change in tumor volume over time for Bon-1 pancreatic neuroendocrine cancer (NET) xenografts treated with vehicle, IPI-926, sunitinib, or a combination of IPI-926 and sunitinib.
Figure 19 is a summary of the cancers that can be treated with hedgehog inhibitors, e.g., IPI-926.
Figure 20 is a panel of photograps depicting the detection and histologic characterization of the rat chondrosarcoma model. Panel (A) depicts an MRI
image of tumor lobules at the graft site and in the surrounding muscles (arrows) 11 days after tumor transplantation. Panel (B) shows the histology of the typical pattern of chondrosarcoma with lobules separated by fascia. Panel (C) shows the presence of cartilage and mitotic cells classified in this model as grade II
chondrosarcoma.
Figures 21A-21D is a panel of bar graphs showing the effects of IPI-926 in decreasing Hh signaling in tumor and stromal cells of osteosarcoma xenograft models.
Figures 21A-21B show a decrease in PTCH1 and Glil mRNA expression in tumor cells from Xenograft A and B aninals treated with IPI-926 compared to controls.
Similar decreases in PTCH1 and Glil mRNA expression is detected in stromal cells treated with IPI-926 compared to controls (Figures 21C-21D).

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Figures 22A-22D is a panel of bar graphs showing the effects of IPI-926 in proliferation and apoptosis in osteosarcoma xenograft models. Figures 22A and show a decrease in proliferation of tumor cells detected by the percentage of cells showing Ki-67 staining in two different animals in response to IPI-926 compared to controls. Figures 22B and 22D show an increase in apoptosis detected by Tunel Staving in response to IPI-926 compared to controls.
Figure 23 is a panel of bar graphs showing the inhibition of Hh pathway markers in tumor cells treated with IPI-926, compared to the vehicle control.
Figure 24 is a bar graph showing a comparison of IPI-926-treated primary chondrosarcoma xenografts to other chemotherapies.
Figure 25 is a bar graph summarizing the effects in human Glil modulation in primary chondrosarcoma xenograft models treated with multiple inhibitors.
Figure 26 depicts the change in tumor volume over time for L3.6p1 pancreatic tumor xenografts treated with vehicle, ABRAXANE , and a combination of ABRAXANE and IPI-926.
Figure 27A shows images of phospho histone 3 (PH3) staining on the L3.6p1 tumor model comparing vehicle, ABRAXANE , and a combination of IPI-926 and ABRAXANE treated tumors.
Figure 27B quantitates the % PH3 positive neoplastic nuclei per stained tumor section in Figure 27A.
Figure 28A shows the change in tumor growth over time for ASPC-1 tumor-bearing Ncr nude mice treated with vehicle, IPI-926, ABRAXANE , and a combination of IPI-926 and ABRAXANE .
Figure 28B shows the change in tumor growth over time for ASPC-1 tumor-bearing Ncr nude mice treated with vehicle, IPI926, paclitaxel, and a combination of IPI-926 and paclitaxel.
Figure 29A shows the change in tumor volume over time for L3.6p1 tumor bearing mice treated with Vehicle, IPI-926 alone, Abraxane +/- IPI-926, Gemzar +/-IPI-926, ABRAXANE + Gemzar and ABRAXANE + Gemzar + IPI-926.

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Figure 29B shows the change in tumor volume over time for L3.6p1 tumor bearing mice treated with Vehicle, IPI-926 alone, ABRAXANE alone, and the combination of ABRAXANE and IPI-926.
Figure 29C compares survival of L3.6p1 tumor bearing mice being treated with Vehicle, IPI-926 alone, ABRAXANE +/- IPI-926, Gemzar +/- IPI-926, ABRAXANE + Gemzar , and ABRAXANE + Gemzar + IPI-926.
Figure 29D compares survival of L3.6p1 tumor bearing mice being treated with Vehicle, IPI-926 alone, Abraxane alone and the combination of ABRAXANE and IPI-926.
Figure 30A depicts contrast enhanced ultrasound images showing tumor perfusion in vehicle treated animals.
Figure 30B depicts contrast enhanced ultrasound images showing tumor perfusion in IPI-926 treated animals.
Figure 30C depicts quantitation of contrast enhanced ultrasound images showing tumor perfusion in vehicle treated animals.
Figure 30D depicts quantitation of contrast enhanced ultrasound images showing tumor perfusion in IPI-926 treated animals.
Figure 31 depicts the amount of Gli-1 inhibition in excised IPI-926 treated tumors of L3.6p1 pancreatic cell lines and ASPC-1 pancreatic cell lines versus control.
Figure 32 is a graph depicting therapeutic testing of IPI-926 and cetuximab (ERBITUX ) in direct patient tumor model (DPTM) of head and neck squamous cell cancer (HNSCC). Passage 3 CUHN004 tumors were implanted subcutaneously into nude mice and allocated to Control (vehicle), ERBITUX (ERBITUX 40 mg/kg/day by IP
injection 2/week for 5 weeks), IPI-926 (IPI-926 at 40 mg/kg/day PO for 5 weeks), and Combination (ERBITUX + IPI-926 for 5 weeks). N = 10 per group. Following 5 weeks of treatment, tumor bearing animals were observed for re-growth for 2 additional months. Tumor volumes were measured as indicated and are expressed as normalized to baseline (Day 1 prior to treatment).
Figure 33 is a graph depicting that IPI-926 delays re-growth in non-small cell cancer NCI-H1650 xenograft model post gefitinib therapy. NCI-H1650 were grown subcutaneously in nude mice. Tumor bearing mice were administered gefitinib (40 Attorney Docket No. I2041-7000WO/3020PCT

mg/kg, p.o) for 7 days then followed-by (fb) IPI-926 (40 mg/kg, p.o) every other day.
H 1650 sensitivity (regression) to gefitinib in vivo was followed by a 65%
inhibition (p<0.02) of tumor re-growth with IPI-926 treatment.
Figure 34 is a graph depicting that IPI-926 delays tumor re-growth in non-small cell cancer HCC827 xenograft model post gefitinib therapy. HCC827 cells were grown subcutaneously in nude mice. Gefitinib was administered (10 mg/kg, p.o) for 3 days then followed-by (fb) IPI-926 (40 mg/kg, p.o) every other day. A 70% inhibition (p<0.03) of tumor re-growth post regression with gefitinib was observed with IPI-926 treatment.
Figure 35 is a graph showing that tumor human hedgehog ligands IHh and DHh are upregulated in the non-small cell cancer NCI-H1650 xenograft model post gefitinib treatment.
Figure 36 is a graph showing that IPI-926 inhibits the up-regulation of stromal cell Gli I and Gli2 in the non-small cell cancer NCI-H1650 xenograft model post gefitinib treatment. Murine Gli l is up-regulated (p<O.05) post therapy compared to vehicle treated tumor, and down modulated (p<0.0001) with IPI-926 treatment. Murine Gli2 is up-regulated (p<0.01) post target therapy when compared to vehicle, and down modulated (p<0.03) with IPI-926 treatment.
Figure 37 is a linear graph showing the effects of IPI-926, Avastin , or the combination of IPI-926 and Avastin in BXPC3 tumor bearing mice.

DETAILED DESCRIPTION
Malignant activation of the Hedgehog (Hh) pathway is associated with multiple tumor types and can promote the growth of certain cancers via at least three modes: Hh ligand-dependent signaling between tumor cells, Hh ligand-dependent signaling between tumor cells and their microenvironment, and ligand-independent signaling caused by mutations in the Hh receptors Patched or Smoothened. Figure 19 provides a summary of the cancers that can be treated with hedgehog inhibitors, such as IPI-926. For example, hedgehog inhibitors can target the tumors directly. In other embodiments, Hh inhibitors can target the tumor microenvironment of ligand dependent cancers (e.g., desmoplastic tumors, such as pancreatic cancer and/or neurodendocrine tumors). In such embodiments, hedgehog inhibitors can decrease fibrosis, thus leading to improved drug delivery and/or Attorney Docket No. I2041-7000WO/3020PCT

survival. In other embodiments, hedgehog inhibitors can target ligand-dependent residual disease. In yet other embodiments, hedgehog inhibitor can target ligand-independent cancers.
In certain cancers (such as pancreatic, ovarian, prostate, colorectal and small cell lung cancers), Hh ligands are believed to act via a paracrine role whereby cancer cells produce the Hh ligand that activates the Hh pathway in the surrounding stromal cell micro environment. In other cancers, Hh ligands can signal a tumor or cancer cell directly, as opposed to through the surrounding stromal tissue. Exemplary tumors and cancer cells that are believed to be activated directly by Hh ligands include chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), multiple myeloma, small cell lung cancer, chondrosarcoma and osteosarcoma.
Other classes of cancers are believed to be Hh ligand-independent as they involve genetic mutations in the Hh receptors Patched or Smoothened. Examples of such cancers include, but are not limited to, Gorlin's Syndrome, basal cell carcinoma (BCC) and medulloblastoma.
IPI-926 is a potent and selective Smoothened (Smo) inhibitor currently in clinical trials in solid tumors and metastatic pancreatic cancer. Smo is believed to play an important role in the malignant activation of the hedgehog pathway in both Hh ligand dependent and ligand independent cancers. Thus, IPI-926 is believed to disrupt the malignant activation of both Hh ligand dependent and ligand independent cancers. For example, IPI-926 is believed to inhibit pancreatic cancer by inhibiting Smo within the stroma. Inhibition of Smo within the tumor micro environment is believed to deplete the desmoplastic stroma, increasing the vascularity of the tumor and rendering it more accessible to chemotherapy. Consistent with this model, IPI-926, blocks Hh signaling in tumor-associated stromal cells -but not in the cancer cells- of several pancreatic xenograft models, resulting in reduced growth of the xenografts. This leads to a depletion of stromal tissue. Studies in a Kras, p53 model of pancreatic cancer demonstrated that IPI-926 decreased the desmoplastic stroma and enabled chemotherapy to access the tumor cells, leading to decreased incidence of tumor metastases and an increase in median survival. Improved survival of pancreatic cancer models is seen when IPI-926 is combined with gemcitabine.

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In one embodiment, Applicants show that administration of a hedgehog inhibitor, alone or in combination with a tyrosine kinase inhibitor (in this case, sunitinib), reduced pancreatic neuroendocrine cancer cell growth in vivo (Examples 14-15).
Expression of Sonic Hedgehog (SHH) ligand was detected in neuroendocine tumors of various organs, e.g., pancreas, duodenum, lung, rectum, and small intestine (Example 13). It was further discovered that administration of the hedgehog inhibitor reduced expression of Hh-dependent genes in the stroma surrounding neuroendocrine cancers, while no significant reduction of Hh dependent genes in the neuroendocrine tumor was detected, thus supporting a paracrine signaling mechanism between the hedgehog-secreting tumors and hedgehog signaling pathway in the surrounding stroma (Example 14). In other embodiments, the hedgehog inhibitor reduced the activity of a hedgehog receptor, e.g., Smoothened and/or Patched, in a tumor microenvironment, thereby causing one or more of. (i) depleting or reducing desmoplastic stroma; (ii) increasing the vascularity of the tumor; or (iii) rendering the tumor more accessible to chemotherapy. Thus, methods and compositions for treating or preventing a cancer (e.g., a neuroendocrine cancer) by administering to a subject a hedgehog inhibitor, alone or combination with a tyrosine kinase inhibitor (e.g., a receptor tyrosine kinase (RTK) inhibitor) are disclosed.
In other embodiments, preclinical studies using inhibitors of hedgehog signaling in chondrosarcoma and osteosarcoma cell lines provided evidence for the potency of Hh-inhibitors as future agents for musculoskeletal sarcoma treatment (see Example 16).
Inhibiting Hh pathway is believed to have antitumor and anti-stromal activity, and can be used to limit or prevent sarcoma invasion (local and metastatic). Previous studies established that inhibiting mTOR pathway had a strong antitumor activity towards sarcoma such as chondrosarcoma (see e.g., Brown, R. E. (2004) Annals of Clinical &
Laboratory Science 34:397-399; Chan, S. (2004) Br J Cancer 91(8):1420-4; Geryk-Hall, M. et al. (2009) Curr Oncol Rep. 11 (6):446-53). Thus, administration of a hedgehog inhibitor, alone or in combination with an mTOR inhibitor, is expected to reduce the growth and/or tumor progression of musculoskeletal or soft-tissue sarcomas, such as chondrosarcomas, synovial sarcoma, liposarcoma, and osteosarcomas.
In yet other embodiments, administration of a hedgehog inhibitor in combination with paclitaxel or a paclitaxel agent reduces the growth and/or tumor progression of a Attorney Docket No. I2041-7000WO/3020PCT

pancreatic cancer to a greater extent than administration of each agent alone (Examples 17-19). This combination can additionally include gemcitabine and/or a VEF
inhibitor (e.g., bevacizumab). Without wishing to be bound by theory, it is believed that administration of the hedgehog inhibitor results in enhancement of the delivery of the paclitaxel or the paclitaxel agent when used in combination, compared to the use of the paclitaxel agent alone.
In other embodiments, administration of a hedgehog inhibitor in combination with a tyrosine kinase inhibitor (e.g., a receptor tyrosine kinase (RTK) inhibitor, such as an EGFR inhibitor) reduces the growth and/or tumor progression of a hedgehog-associated cancer or tumor (e.g., a head and neck cancer and/or lung cancer (e.g., non-small cell lung cancer)) (Examples 22-25). In one embodiment, the hedgehog inhibitor extends the relapse free survival of a subject who is undergoing, or has been previously treated with, an anti-cancer agent (e.g., a tyrosine kinase inhibitor). In one embodiment, the tyrosine kinase inhibitor is geftinib or cetuximab. For example, the hedgehog inhibitor reduces or inhibits tumor re-growth of a hedgehog-associated cancer after therapy with a tyrosine kinase inhibitor is less effective or ineffective (e.g., a subject having a relapse after therapy with a tyrosine kinase inhibitor). In one embodiment, the subject is a patient with lung cancer (e.g., non-small cell lung cancer) who relapses after geftinib therapy. In another embodiment, the subject is a patient with head and neck squamous cell carcinoma (HNSCC) who is undergoing or has undergone therapy with a tyrosine kinase inhibitor (e.g., an EGFR tyrosine kinase inhibitor such as cetuximab).
Accordingly, methods and compositions for treating or preventing a hedgehog-associated cancer (e.g., a hedgehog ligand-dependent cancer cell growth chosen from a neuroendocrine cancer, a sarcoma (e.g., a musculoskeletal sarcoma, such as chondrosarcoma and osteosarcoma)), a head and neck cancer, or a lung cancer by administering to a subject a hedgehog inhibitor, alone or combination with another anti-cancer agent (e.g., paclitaxel or a paclitaxel agent, a tyrosine kinase inhibitor (e.g., receptor tyrosine kinase (RTK) inhibitor) or an mTOR inhibitor) are disclosed.
Various aspects of the invention are described in further detail below.
Additional definitions are set out throughout the specification.

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Hedgehog Inhibitors Suitable hedgehog inhibitors include, for example, those 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, and U.S. Patent Application Publication No. 2008/0293755, the entire disclosures of which are incorporated by reference herein. Examples of other suitable hedgehog inhibitors include those described in U.S. Patent Application Publication Nos. US 2002/000693 1, 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 2003/088970, 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 are described in Yauch, R. L. et al.
(2009) Science 326: 572-574 Sciencexpress: 1-3 (10.1126/science. 1179386); Rudin, C.
et al.
(2009) New England J of Medicine 361-366 (10.1056/nejmaO902903).
For example, the hedgehog inhibitor can be a compound having the following structure:

Me H N
Me Me O H
Me H
C H H

or a pharmaceutically acceptable salt thereof, wherein R' is H, alkyl, -OR, amino, sulfonamido, sulfamido, -OC(O)R5, - N(R5)C(O)R5, or a sugar;
R2 is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, nitrile, or heterocycloalkyl;
or RI and R2 taken together form =O, =S, =N(OR), =N(R), =N(NR2), or =C(R)2;

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R3 is H, alkyl, alkenyl, or alkynyl;
R4 is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, haloalkyl, -OR, -C(O)R5, -C02R5, -S02R5, -C(O)N(R5)(R5), -[C(R)z]q Rs, -[(W)-N(R)C(O)]gR5, -[( W)-C(O)]gR5, -[(W)-C(0)0]gR5, -[(W)-OC(O)]gR5, -[(W)-S02]gR5, -[(W)-N(R5)SO2]gR5, -[(W)-C(O)N(R5)]gR5, -[(W)-O]gR5, -[(W)-N(R)]gR5, -W-NR3+X- or -[(W)-S]gR5;
each W is independently for each occurrence a diradical;
each q is independently for each occurrence 1, 2, 3, 4, 5, or 6;
X- is a halide;
each R5 is independently for each occurrence H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl or -[C(R)2]p-R6;
or any two occurrences of R5 on the same substituent can be taken together to form a 4-8 membered optionally substituted ring which contains 0-3 heteroatoms selected from N, 0, S, and P;
pis 0-6;
each R6 is independently hydroxyl, -N(R)COR, -N(R)C(O)OR, -N(R)S02(R), -C(O)N(R)2, -OC(O)N(R)(R), -SO2 N(R)(R), -N(R)(R), -000R, -C(O)N(OH)(R), -OS(O)20R, -S(O)20R, -OP(O)(OR)(OR), -NP(O)(OR)(OR), or -P(O)(OR)(OR);
provided that when R2, R3 are H and R4 is hydroxyl; R' can not be hydroxyl;
provided that when R2, R3, and R4 are H ; Ri can not be hydroxyl; and provided that when R2, R3, and R4 are H ; Ri can not be sugar.
Examples of compounds include:

Attorney Docket No. I2041-7000WO/3020PCT
H Me Me H N Me H N
Me Me Me Me Me H/ H Me H/ 'H

H H H H

H H

H H
Me H N Me H N
Me Me Me Me O 'H
Me H O CV
H H H
Me 2N\"
H H
Me Me H N Me H N
Me Me Me Me Me H / 'H Me H / 'H

H H Fi H
O O
H H

Attorney Docket No. I2041-7000WO/3020PCT
-N

Me H Me, HN
Me O Me Me -H , O
o Me H % Me H H
Me H H Ph H
O
H H
Ph -N H-N
Me, H N Me H N
Me Me Me Me Me H O ,H Me O H

H H H H
O O
H H
Ph H-N

O Me H
Me H N
Me Me Me O N O
\- ~
Me H / ,H Me H Me H H

H H H Ph H H

Attorney Docket No. I2041-7000WO/3020PCT
H
Me H N
Me Me / O
Me H H
H H
MeO
H

H
Me H N
Me Me Me H

IO H H
H
H H
Me H N Me H N
Me Me Me Me Me H O H Me H O

O H H H H
AN H H H

H
Me H N
Me Me 0 CO H H

AN \,.

H H

Attorney Docket No. I2041-7000WO/3020PCT
H
Me H N
Me Me CO H H

MeO"
H

HN
H

ee Me H N
Me Me Me Me Me HO H

H
Me2N\' O
H H
H
Me H N
Me Me /
Me H
H H
HO"
H

H
Me H N
Me Me O
'1-~
Me H H
O H H

H

Attorney Docket No. I2041-7000WO/3020PCT
H
Me H N
Me Me Me H

~O H H

H

H
Me H N
Me Me O
Me H H

H
HO"N
H

H
Me H N
Me Me Me H
O H H
H

MeN
H
Me H N
Me Me Me H N
Me Me o ;H o 0e ', Me H Me H
H
O H H H H

O
H H

Attorney Docket No. I2041-7000WO/3020PCT
Ph ~O
H HN
Me H N
Me Me Me H N
O Me Me Me H H j9:0H
H
MeON H

H
H H
O NH O NH
"'H "'H
H H

, Ph, O H H N H H

H H
O NH O NH
,H ~H
H H
\ H H
'I . N N,N H
O H H

H H
O NH O N
H
,,H
H H N ' H H

N C N N, ~ H H

Attorney Docket No. I2041-7000WO/3020PCT
H

H O NH
O "H
,4) H H
NH H
~H

N,N
O NP

O McO2C
H H
O NH O

H H , H H H
O H H H H
N O
H H H

H H
O N O N
'H ,O 'H 'SO2Me H H
H H
O H and O H Z

and pharmaceutically acceptable salts thereof.
One example of a suitable hedgehog inhibitor for the methods of the current invention is the compound of formula I:

H
O NH
'H
H

H Fi 0H ` H

I

Attorney Docket No. I2041-7000WO/3020PCT
or a pharmaceutically acceptable salt thereof. An example of a pharmaceutically acceptable salt is a hydrochloride salt of the compound of formula I (also referred to herein as "Compound 42" or "IPI-926").
IPI-926 is believed to disrupt malignant activation of the hedgehog pathway in both Hh ligand dependent and Hh ligand independent cancers. IPI-926 shows an EC50 of 7-15 nM in C3H10 cells, and inhibits Smo binding with an IC50 of 1-2 nM. IPI-926 can be administered orally either once a day or continuously; it has a half-life of 20-40 hours after a single dose.
Hedgehog inhibitors useful in the current invention can contain a basic functional group, such as amino or alkylamino, and are thus capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable acids. The term "pharmaceutically-acceptable salts" in this respect, refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately treating the compound in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed during subsequent purification.
Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, besylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like (see, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).
The pharmaceutically acceptable salts of the present invention include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g., from non-toxic organic or inorganic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, benzenesulfonic, ethane disulfonic, oxalic, isothionic, and the like.

Attorney Docket No. I2041-7000WO/3020PCT

In other cases, the compounds of the present invention can contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases. The term "pharmaceutically-acceptable salts" in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately treating the compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like (see, for example, Berge et at., supra).

Pharmaceutical Compositions To practice the methods of the invention, the hedgehog inhibitor and/or the chemotherapeutic agent can be delivered in the form of pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of one or more hedgehog inhibitors and/or one or more chemotherapeutic formulated together with one or more pharmaceutically acceptable excipients. In some instances, the hedgehog inhibitor and the chemotherapeutic agent are administered in separate pharmaceutical compositions and can (e.g., because of different physical and/or chemical characteristics) be administered by different routes (e.g., one therapeutic is administered orally, while the other is administered intravenously). In other instances, the hedgehog inhibitor and the chemotherapeutic can be administered separately, but via the same route (e.g., both orally or both intravenously). In still other instances, the hedgehog inhibitor and the chemotherapeutic can be administered in the same pharmaceutical composition.
Pharmaceutical compositions can be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those Attorney Docket No. I2041-7000WO/3020PCT

targeted for buccal, sublingual, and systemic absorption), capsules, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin;
intravaginally or intrarectally, for example, as a pessary, cream or foam;
sublingually;
ocularly; transdermally; pulmonarily; or nasally.
Examples of suitable aqueous and nonaqueous carriers which can be employed in pharmaceutical compositions include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents, dispersing agents, lubricants, and/or antioxidants. Prevention of the action of microorganisms upon the compounds of the present invention can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It can also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
Methods of preparing these formulations or compositions include the step of bringing into association the hedgehog inhibitor and/or the chemotherapeutic with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
The hedgehog inhibitors and the chemotherapeutics of the present invention can be given per se or as a pharmaceutical composition containing, for example, about 0.1 to Attorney Docket No. I2041-7000WO/3020PCT

99%, or about 10 to 50%, or about 10 to 40%, or about 10 to 30%, or about 10 to 20%, or about 10 to 15% of active ingredient in combination with a pharmaceutically acceptable carrier. Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
The selected dosage level will depend upon a variety of factors including, for example, the activity of the particular compound employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
In general, a suitable daily dose of a hedgehog inhibitor and/or a chemotherapeutic will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, oral, intravenous and subcutaneous doses of the compounds of the present invention for a patient, when used for the indicated effects, will range from about 0.0001 mg to about 100 mg per day, or about 0.001 mg to about mg per day, or about 0.01 mg to about 100 mg per day, or about 0.1 mg to about 100 mg per day, or about 0.0001 mg to about 500 mg per day, or about 0.001 mg to about 500 mg per day, or about 0.01 mg to about 500 mg per day, or about 0.1 mg to about 500 mg per day.
The subject receiving this treatment is any animal in need, including primates, in particular humans, equines, cattle, swine, sheep, poultry, dogs, cats, mice and rats.
The compounds can be administered daily, every other day, three times a week, twice a week, weekly, or bi-weekly. The dosing schedule can include a "drug holiday,"
i.e., the drug can be administered for two weeks on, one week off, or three weeks on, one week off, or four weeks on, one week off, etc., or continuously, without a drug holiday.
The compounds can be administered orally, intravenously, intraperitoneally, topically, Attorney Docket No. I2041-7000WO/3020PCT
transdermally, intramuscularly, subcutaneously, intranasally, sublingually, or by any other route.
Since the hedgehog inhibitors are administered in combination with other treatments (such as additional chemotherapeutics, radiation or surgery) the doses of each agent or therapy can be lower than the corresponding dose for single-agent therapy. The dose for single-agent therapy can range from, for example, about 0.0001 to about 200 mg, or about 0.001 to about 100 mg, or about 0.01 to about 100 mg, or about 0.1 to about 100 mg, or about 1 to about 50 mg per kilogram of body weight per day. The determination of the mode of administration and the correct dosage is well within the knowledge of the skilled clinician.

Therapeutic Methods In one aspect, the invention relates to a method of treating cancer by administering to a patient a hedgehog inhibitor, alone or in combination with a second therapeutic agent, e.g., an anti-cancer agent (e.g., a receptor tyrosine kinase inhibitor, paclitaxel or a paclitaxel agent, an mTOR inhibitor, and/or an IGF-1R
antagonist).
As used herein, and unless otherwise specified, the terms "treat," "treating"
and "treatment" contemplate an action that occurs while a patient is suffering from cancer, which reduces the severity of the cancer, or retards or slows the progression of the cancer.
As used herein, unless otherwise specified, the terms "prevent," "preventing"
and "prevention" contemplate an action that occurs before a patient begins to suffer from the regrowth of the cancer and/or which inhibits or reduces the severity of the cancer.
As used herein, and unless otherwise specified, the terms "manage," "managing"
and "management" encompass preventing the recurrence of the cancer in a patient who has already suffered from the cancer, and/or lengthening the time that a patient who has suffered from the cancer remains in remission. The terms encompass modulating the threshold, development and/or duration of the cancer, or changing the way that a patient responds to the cancer.
As used herein, and unless otherwise specified, a "therapeutically effective amount" of a compound is an amount sufficient to provide a therapeutic benefit in the Attorney Docket No. I2041-7000WO/3020PCT

treatment or management of the cancer, or to delay or minimize one or more symptoms associated with the cancer. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapeutic agents, which provides a therapeutic benefit in the treatment or management of the cancer.
The term "therapeutically effective amount" can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the cancer, or enhances the therapeutic efficacy of another therapeutic agent.

As used herein, and unless otherwise specified, a "prophylactically effective amount" of a compound is an amount sufficient to prevent regrowth of the cancer, or one or more symptoms associated with the cancer, or prevent its recurrence. A
prophylactically effective amount of a compound means an amount of the compound, alone or in combination with other therapeutic agents, which provides a prophylactic benefit in the prevention of the cancer. The term "prophylactically effective amount" can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.
As used herein, "cancer" and "tumor" are synonymous terms.
As used herein, "cancer therapy" and "cancer treatment" are synonymous terms.
As used herein, "chemotherapy" and "chemotherapeutic" and "chemotherapeutic agent" are synonymous terms.
As used herein, the term "patient" or "subject" refers to an animal, typically a human (i.e., a male or female of any age group, e.g., a pediatric patient (e.g, infant, child, adolescent) or adult patient (e.g., young adult, middle-aged adult or senior adult) or other mammal, such as a primate (e.g., cynomolgus monkey, rhesus monkey);
commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and/or dogs; and/or birds, including commercially relevant birds such as chickens, ducks, geese, and/or turkeys, that will be or has been the object of treatment, observation, and/or experiment.
When the term is used in conjunction with administration of a compound or drug, then the patient has been the object of treatment, observation, and/or administration of the compound or drug.

Attorney Docket No. I2041-7000WO/3020PCT
In some embodiments, the hedgehog inhibitor is a first line treatment for the cancer, i.e., it is used in a subject who has not been previously administered another drug intended to treat the cancer.
In other embodiments, the hedgehog inhibitor is a second line treatment for the cancer, i.e., it is used in a subject who has been previously administered another drug intended to treat the cancer.
In other embodiments, the hedgehog inhibitor is a third or fourth line treatment for the cancer, i.e., it is used in a subject who has been previously administered two or three other drugs intended to treat the cancer.
In some embodiments, a hedgehog inhibitor is administered to a subject following surgical excision/removal of the cancer.
In some embodiments, a hedgehog inhibitor is administered to a subject before, during, and/or after radiation treatment of the cancer.
In other embodiments, the hedgehog inhibitor is administered as neoadjuvant therapy, i.e., prior to another treatment.
In other embodiments, the hedgehog inhibitor is administered as adjuvant therapy, i.e., a treatment in addition to primary therapy.
In certain embodiments, the methods include administration of a first therapeutic agent and a second therapeutic agent, wherein the second therapeutic agent is a hedgehog inhibitor. The two agents can be administered concurrently (i.e., essentially at the same time, or within the same treatment) or sequentially (i.e., one immediately following the other, or alternatively, with a gap in between administration of the two). In some embodiments, the hedgehog inhibitor is administered sequentially (i.e., after the first therapeutic). The first therapeutic agent can be a chemotherapeutic agent, or multiple chemotherapeutic agents administered sequentially or in combination.
In another aspect, the invention relates to a method of treating cancer including the steps of administering to a patient a first therapeutic agent, then administering the first therapeutic agent in combination with a second therapeutic agent, wherein the second therapeutic agent is a hedgehog inhibitor.
In another aspect, the invention relates to a method of treating a condition mediated by the hedgehog pathway by administering to a patient a first therapeutic agent Attorney Docket No. I2041-7000WO/3020PCT
and a second therapeutic agent, wherein the second therapeutic agent is a hedgehog inhibitor. The two agents can be administered concurrently (i.e., essentially at the same time, or within the same treatment) or sequentially (i.e., one immediately following the other, or alternatively, with a gap in between administration of the two). In some embodiments, the hedgehog inhibitor is administered sequentially (i.e., after the first therapeutic). The first therapeutic agent can be a chemotherapeutic agent.
In another aspect, the invention relates to a method of treating a condition mediated by the hedgehog pathway including the steps of administering to a patient a first therapeutic agent, then administering the first therapeutic agent in combination with a second therapeutic agent, wherein the second therapeutic agent is a hedgehog inhibitor.
The invention also relates to methods of extending relapse free survival in a cancer patient who is undergoing or has undergone cancer therapy (for example, treatment with a chemotherapeutic (including small molecules and biotherapeutics, e.g., antibodies), radiation therapy, surgery, RNAi therapy and/or antisense therapy) by administering a therapeutically effective amount of a hedgehog inhibitor to the patient.
"Relapse free survival", as understood by those skilled in the art, is the length of time following a specific point of cancer treatment during which there is no clinically-defined relapse in the cancer. In some embodiments, the hedgehog inhibitor is administered concurrently with the cancer therapy. In instances of concurrent administration, the hedgehog inhibitor can continue to be administered after the cancer therapy has ceased.
In other embodiments, the hedgehog inhibitor is administered after cancer therapy has ceased (i.e., with no period of overlap with the cancer treatment). The hedgehog inhibitor can be administered immediately after cancer therapy has ceased, or there can be a gap in time (e.g., up to about a day, a week, a month, six months, or a year) between the end of cancer therapy and the administration of the hedgehog inhibitor. Treatment with the hedgehog inhibitor can continue for as long as relapse-free survival is maintained (e.g., up to about a day, a week, a month, six months, a year, two years, three years, four years, five years, or longer).
In one aspect, the invention relates to a method of extending relapse free survival in a cancer patient who had previously undergone cancer therapy (for example, treatment with a chemotherapeutic (including small molecules and biotherapeutics, e.g., antibodies), Attorney Docket No. I2041-7000WO/3020PCT

radiation therapy, surgery, RNAi therapy and/or antisense therapy) by administering a therapeutically effective amount of a hedgehog inhibitor to the patient after the cancer therapy has ceased. The hedgehog inhibitor can be administered immediately after cancer therapy has ceased, or there can be a gap in time (e.g., up to about a day, a week, a month, six months, or a year) between the end of cancer therapy and the administration of the hedgehog inhibitor.
Hedgehog inhibitors, e.g., IPI-926, described in PCT publications WO
2008083252 and WO 2008083248, both of which are incorporated herein by reference, have been shown to inhibit in vitro growth of human cell lines derived from patients with pancreatic cancer, medulloblastoma, lung cancer, multiple myeloma, acute lymphocytic leukemia, myelodysplatic syndrome, non-Hodgkin's type lymphoma, Hodgkin's disease and lymphocygtic leukemia.
Hedgehog inhibitors, e.g., IPI-926, have also shown tumor growth inhibition in a number of preclinical in vivo models, such as medulloblastoma (Pink et al., American Association for Cancer Research, 1588, 2008; Villavicencia et al. American Association for Cancer Research, 2009); small cell lung cancer (Travaglione et al., American Association for Cancer Research, 4611, 2008; Peacock et al., American Association for Cancer Research, 2009); and ovarian cancer (Growdon et al, Society of Gynecologic Oncologists Annual Meeting on Women's Cancer, 2009).
Additionally, hedgehog inhibitors, e.g., IPI-926, have demonstrated rapid and sustained Hedgehog pathway inhibition in stromal cells, a downstream mediator of Hedgehog signaling, after single administration in a model of human pancreatic cancer (Traviglione et al., EORTC-NCI-AACR Symposium on "Molecular Targets and Cancer Therapeutics" 2008).
Inhibition of the hedgehog pathway has also been shown to reduce or inhibit the growth of a variety of cancers, such as acute lymphocytic leukemia (ALL) (Ji et al., Journal of Biological Chemistry (2007) 282:37370-37377); basal cell carcinoma (Xie et al., Nature (1998) 391:90-92; Williams et al., PNAS (2003) 100:4616-4621; Bale and Yu (2001) Human Molecular Genetics (2001) 10:757-762); biliary cancer (Berman et al., Nature (2003) 425:846-85 1; WO 2005/013800); brain cancer and glioma (Clement et al., Current Biology (2007) 17:1-8; Ehtesham et al., Ongogene (2007) 1-10); bladder cancer;

Attorney Docket No. I2041-7000WO/3020PCT
breast cancer (Kubo et al., Cancer Research (2004) 64:6071-6074; Lewis et al., J.
Mammary Gland Biology and Neoplasia (2004) 2:165-181); chondrosarcoma (Wunder et al., Lancet Oncology (2007) 513-524); chronic lymphocytic leukemia (CLL) (Hedge et al., Mol. Cancer Res. (2008) 6:1928-1936); chronic myeloid leukemia (CML) (Dierks et al., Cancer Cell (2008) 14:238-249); colon cancer (Yang and Hinds, BMC
Developmental Biology (2007) 7:6); esophageal cancer (Berman et al., Nature (2003) 425:846-851; WO 2005/013800); gastric cancer (Berman et al., Nature (2003) 425:846-851; Ma et al., Carcinogenesis (2005) 26:1698-1705; WO 2005/013800; Shiotani et al., J. Gastroenterol. Hepatol. (2008) S 161-S 166; Ohta et al., Cancer Research (2005) 65:10822-10829; Ma et al., World J. Gastroenterol (2006) 12:3965-3969);
gastrointestinal stromal tumor (GIST) (Yoshizaki et al., World J.
Gastroenterol (2006) 12:5687-5691); hepatocellular cancer (Sicklick et al., Carcinogenesis (2006) 27:748-757;
Patil et al., Cancer Biology & Therapy (2006) 5:111-117); kidney cancer (Cutcliffe et al., Human Cancer Biology (2005) 11:7986-7994); lung cancer (Watkins et al., Nature (2003) 422:313-317); medulloblastoma (Berman et al., Science (2002) 297:1559-1561;
Pietsch et al. Cancer Research (1997) 57:2085-208 8); melanoma (Stecca et al., PNAS
(2007) 104:5895-5900; Geng et al., Angiogenesis (2007) 10:259-267); multiple myeloma (Peacock et al., PNAS USA (2007) 104:4048-4053; Dierks et al., Nature Medicine (2007) 13:944-951); neuroectodermal tumors (Reifenberger et al., Cancer Research (1998) 58:1798-1803); non-Hodgkin's type lymphoma (NHL) (Dierks et al., Nature Medicine (2007) 13:944-951; Lindemann, Cancer Research (2008) 68:961-964); osteosarcoma (Warzecha et al., J. Chemother. (2007) 19:554-561); ovarian cancer (Steg et al., J.
Molecular Diagnostics (2006) 8:76-83); pancreatic cancer (Thayer et al., Nature (2003) 425:851-856; Berman et al., Nature (2003) 425:846-851; WO 2005/013800);
prostate cancer (Karhadkar et al., Nature (2004) 431:707-712; Sheng et al., Molecular Cancer (2004) 3:29-42; Fan et al., Endocrinology (2004) 145:3961-3970); and testicular cancer (Dormeyer et al., J. Proteome Res. (2008) 7:2936-2951).
Examples of conditions that can be treated include lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), pancreatic cancer, bladder cancer, ovarian cancer, breast cancer, colon cancer, multiple myeloma, acute myelogenous leukemia Attorney Docket No. I2041-7000WO/3020PCT

(AML), chronic myelogenous leukemia (CML), neuroendocrine cancer, and a sarcoma (e.g., a musculoskeletal sarcoma, such as chondrosarcoma and osteosarcoma).
A more comprehensive list of proliferative disorders and cancers that can be treated using the methods disclosed herein include, for example, lung cancer (including small cell lung cancer and non small cell lung cancer), other cancers of the pulmonary system, medulloblastoma and other brain cancers, pancreatic cancer, basal cell carcinoma, breast cancer, prostate cancer and other genitourinary cancers, gastrointestinal stromal tumor (GIST) and other cancers of the gastrointestinal tract, colon cancer, colorectal cancer, ovarian cancer, cancers of the hematopoietic system (including multiple myeloma, acute lymphocytic leukemia, acute myelocytic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, and myelodysplastic syndrome), polycythemia Vera, Waldenstrom's macroglobulinemia, heavy chain disease, soft-tissue sarcomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, melanoma, and other skin cancers, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, stadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, bladder carcinoma, and other genitourinary cancers, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, endometrial cancer, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, hepatocellular carcinoma, thyroid cancer, gastric cancer, esophageal cancer, head and neck cancer, small cell cancers, essential thrombocythemia, agnogenic myeloid metaplasia, hypereosinophilic syndrome, systemic mastocytosis, familiar hypereosinophilia, chronic eosinophilic leukemia, thyroid cancer, neuroendocrine cancers, and carcinoid tumors.

Attorney Docket No. I2041-7000WO/3020PCT

In certain embodiments, the cancer is selected from bladder cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, endometrial cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, lymphoma, leukemia, meduloblastoma, melanoma, multiple myeloma, neuroendocrine cancer, osteosarcoma, ovarian cancer, pancreatic cancer and prostate cancer.
In certain embodiments, the cancer is lung cancer. In certain embodiments, the lung cancer is small cell lung cancer (SCLC). In certain embodiments, the lung cancer is non-small cell lung cancer (NSCLC).
In certain embodiments, the cancer is colorectal cancer.
In certain embodiments, the cancer is neuroendocrine cancer.
Neuroendocrine cancers (also known as gastroenteropancreatic tumors or gastroenteropancreatic neuroendocrine cancers), are cancers derived from cells at the interface between the endocrine (hormonal) system and the nervous system. The majority of neuroendocrine cancers fall into two categories: carcinoids and pancreatic endocrine tumors (also known as endocrine pancreatic tumors or islet cell tumors). In addition to the two main categories, other forms of neuroendocrine cancers exist, including neuroendocrine lung tumors, which arise from the respiratory rather than the gastro-entero-pancreatic system. Neuroendocrine cancers can originate from endocrine glands such as the adrenal medulla, the pituitary, and the parathyroids, as well as endocrine islets within the thyroid or the pancreas, and dispersed endocrine cells in the respiratory and gastrointestinal tract. The total incidence of neuroendocrine cancers in the United States is about 9,000 new cases per year.
For example, the cancer treated can be a neuroendocrine cancer chosen from one or more of, e.g., a neuroendocrine cancer of the pancreas, lung, appendix, duodenum, ileum, rectum or small intestine. In other embodiments, the neuroendocrine cancer is chosen from one or more of. a pancreatic endocrine tumor; a neuroendocrine lung tumor;
or a neuroendocrine cancer from the adrenal medulla, the pituitary, the parathyroids, thyroid endocrine islets, pancreatic endocrine islets, or dispersed endocrine cells in the respiratory or gastrointestinal tract.
Pancreatic endocrine tumors can secrete biologically active peptides (e.g., hormones) that can cause various symptoms in a subject. Such tumors are referred to Attorney Docket No. I2041-7000WO/3020PCT

functional or secretory tumors. Functional tumors can be classified by the hormone most strongly secreted. Examples of functional pancreatic endocrine tumors include gastrinoma (producing excessive gastrin and causing Zollinger-Ellison Syndrome), insulinoma (producing excessive insulin), glucagonoma (producing excessive glucagon), vasoactive intestinal peptideoma (VIPoma, producing excessive vasoactive intestinal peptide), PPoma (producing excessive pancreatic polypeptide), somatostatinoma (producing excessive somatostatin), watery diarrhea hypokalemia-achlorhydria (WDHA), CRHoma (producing excessive corticotropin-releasing hormonse), calcitoninoma (producing excessive calcitonin), GHRHoma (producing excessive growth-hormone-releasing hormone), neurotensinoma (producing excessive neurotensin), ACTHoma (producing excessive adrenocorticotropic hormone), GRFoma (producing excessive growth hormone-releasing factor), and parathyroid hormone-related peptide tumor. In some instances, pancreatic endocrine tumors can arise in subjects who have multiple endocrine neoplasia type 1 (MEN1); such tumors often occur in the pituitary gland or pancreatic islet cells. Pancreatic endocrine tumors that do not secrete peptides (e.g., hormones) are called nonfunctional (or nonsecretory or nonfunctional) tumors. In one embodiment, the cancer treated is a pancreatic ductal adenocarcinoma.
In other embodiments, the cancer treated is a carcinoid tumor, e.g., a carcinoid neuroendocrine cancer. Carcinoid tumors tend to grow more slowly than pancreatic endocrine tumors. A carcinoid tumor can produce biologically active molecules such as serotonin, a biogenic molecule that causes a specific set of symptoms called carcinoid syndrome. Carcinoid tumors that produce biologically active molecules are often referred to as functional carcinoid tumors, while those that do not are referred to as nonfunctional carcinoid tumors. In some embodiments, the neuroendocrine cancer is a functional carcinoid tumor (e.g., a carcinoid tumor that can produce biologically active molecules such as serotonin). In other embodiments, the neuroendocrine cancer is a non-functional carcinoid tumor. In certain embodiments, the carcinoid tumor is a tumor from the thymus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon), rectal, pancreatic, appendix, ovarian or testicular carcinoid.

Attorney Docket No. I2041-7000WO/3020PCT

Carcinoid tumors can be further classified depending on the point of origin, such as lung, thymus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon), rectum, pancreas, appendix, ovaries and testes.
In some embodiments, the neuroendocrine cancer is a carcinoid tumor. In other embodiments, the neuroendocrine cancer is a pancreatic endocrine tumor. In still other embodiments, the neuroendocrine cancer is a neuroendocrine lung tumor. In certain embodiments, the neuroendocrine cancers originate from the adrenal medulla, the pituitary, the parathyroids, thyroid endocrine islets, pancreatic endocrine islets, or dispersed endocrine cells in the respiratory or gastrointestinal tract.
Further examples of neuroendocrine cancers that can be treated include, but are not limited to, medullary carcinoma of the thyroid, Merkel cell cancer (trabecular cancer), small-cell lung cancer (SCLC), large-cell neuroendocrine carcinoma (of the lung), extrapulmonary small cell carcinomas (ESCC or EPSCC), neuroendocrine carcinoma of the cervix, Multiple Endocrine Neoplasia type 1 (MEN-1 or MEN 1), Multiple Endocrine Neoplasia type 2 (MEN-2 or MEN2), neurofibromatosis type 1, tuberous sclerosis, von Hippel-Lindau (VHL) disease, neuroblastoma, pheochromocytoma (phaeochromocytoma), paraganglioma, neuroendocrine cancer of the anterior pituitary, and/or Carney's complex.
In other embodiments, the cancer or tumor treated is a sarcoma, e.g., a musculoskeletal sarcoma (e.g., bone and cartilage sarcoma). Exemplary musculoskeletal sarcomas, include but are not limited to, osteosarcoma (e.g., conventional osteogenic sarcoma), chondrosarcoma (e.g., conventional chondrosarcoma), Ewing sarcoma, dedifferentiated chondrosarcoma, parosteal osteogenic sarcoma, periosteal osteogenic sarcoma, mesenchymal chondrosarcoma, giant cell tumor of bone, adamantinoma, chordoma and other sarcomas that typically occur in soft tissue in adults that can also occur in bone, such as malignant fibrous histiocytoma (MFH [also termed high grade undifferentiated pleomorphic sarcoma or HGUPS]), fibrosarcoma, leiomyosarcoma, and angiosarcoma, among others. Each one is described in more detail herein below.
Examples of relatively common bone and cartilage sarcoma subtypes, include but are not limited to osteosarcoma (e.g., conventional osteogenic sarcoma), chondrosarcoma Attorney Docket No. I2041-7000WO/3020PCT
(e.g., conventional chondrosarcoma), Ewing sarcoma, and dedifferentiated chondrosarcoma.
Osteogenic sarcoma (also called osteosarcoma) is the most common tumor of bone. Approximately 800-1000 cases of osteogenic sarcoma are seen in the United States each year. A second peak of incidence of osteosarcoma occurs in the 8th decade of life, typically associated with Paget disease of bone. Osteosarcoma typically affects adolescents, and generally affects bones around the knee joint, though any bone of the body can be affected. Treatment typically involves chemotherapy and surgery to try to achieve the best cure rate. Standard drugs that are used include doxorubicin and cisplatin in adults, and the same two drugs with high-dose methotrexate in children, adolescents, or young adults. The use of ifosfamide remains controversial. Recurrences typically occur in the lungs. This is one situation where surgery can be curative;
resection of lung metastases from a primary osteosarcoma is a standard of care when there is a small number of lung nodules that can be removed safely, and can be associated with a 30-35%
cure rate. Osteosarcomas occur commonly in familial syndromes associated with sarcoma, such as Li-Fraumeni syndrome (involving a mutation in the p53 gene), retinoblastoma (involving a mutation in the Rb gene), and Rothmund-Thomson syndrome.
Conventional chondrosarcoma can be a difficult tumor to treat. It often arises in older patients, and often in the pelvis. As a result, people with multiple medical diagnoses are put in the position of requiring a very large operation with a high risk of post-operative complications, with subsequent loss of function. For chondrosarcomas that arise in other sites, surgery can be less morbid and represents the standard of care. People with metastatic disease often times do not respond well to chemotherapy. Grade chondrosarcomas nearly never metastasize, Grade 2 chondrosarcomas have only a 15% risk of metastasis, and grade 3 chondrosarcomas have a two-thirds or higher risk of metastasis. As a result, some people with grade 3 chondrosarcomas will be given adjuvant chemotherapy. A version of chondrosarcoma called clear cell chondrosarcoma has an intermediate risk of metastasis, but treatment is typically surgery alone.
Ewing sarcoma is the third most common sarcoma of bone, and second most common in children. The same tumor occurs in the soft tissue of adults more than it occurs in bone. We estimate there are fewer than 500 cases a year in the United States.

Attorney Docket No. I2041-7000WO/3020PCT

Without chemotherapy the cure rate is at best 10%, but with chemotherapy a cure rate of up to 75% in children and 50-60% in adults is seen. Surgery and radiation are also commonly used as treatment for the primary tumor in order to try to achieve the highest cure rate possible. Ewing sarcomas can appear in any site of the body. Then they recur, it is most commonly in the lungs and bones.
Dedifferentiated chondrosarcoma is a more aggressive version of chondrosarcoma, typically occurring in adolescence and in people over age 60. It shows features of both chondrosarcoma and of elements of a less differentiated tumor, such as MFH
(malignant fibrous histiocytoma), which does not show even a hint of relatedness to the chondrosarcoma. This version of chondrosarcoma has a high risk of recurrence, even greater than that of grade 3 conventional chondrosarcoma (described above).
Less common bone and cartilage sarcoma subtypes include parosteal osteogenic sarcoma, periosteal osteogenic sarcoma, mesenchymal chondrosarcoma, giant cell tumor of bone, adamantinoma, and chordoma.
Parosteal osteosarcoma is a low grade osteosarcoma of bone that grows from the surface of the bone without lifting off the surface connective tissue of bone, called periosteum. It occurs by far most commonly along the posterior, distal femur in the 3rd decade of life. Treatment for this rare form of osteosarcoma is usually surgery alone, although if there are aggressive features such as dedifferentiation or a high grade component seen, chemotherapy is also often given.
Periosteal osteosarcoma is a low grade osteosarcoma of bone that grows from the surface of the bone and lifts off the surface connective tissue of bone, called periosteum, and is also associated with new bone formation in the area of the lifted periosteum. It typically occurs between ages of 10 and 30. Treatment for this rare form of osteosarcoma is usually surgery alone. It is not clear if chemotherapy is helpful for this type of osteosarcoma, though it is often given if the tumor appears more aggressive than usual.
Mesenchymal chondrosarcoma is a rare bone tumor which shows a mixture of aggressive small round blue cells mixed with more typical lower grande chondrosarcoma.
They usually affect people between ages of 15 and 30, and have a high risk of recurrence.
The benefit of chemotherapy is not known, though chemotherapy is often used.
The Attorney Docket No. I2041-7000WO/3020PCT

typical chemotherapy drugs that are used in the adjuvant setting (or metastatic setting, for that matter) are the drugs used for Ewing sarcoma and similar sarcomas.
Giant cell tumor of bone is a tumor of bone that typically occurs between ages and 40, and has a unique appearance under the microscope. It occurs in the area of the knee and lower spine, typically. It is treated by scraping out the tumor and treating the tumor cavity with cement (which heats up and also destroys tumor as a result) or with liquid nitrogen (freezing and thawing the tumor in place, often killing remaining cells). A
bone graft is often used to try to reconstruct the area as well. In some cases, the tumor can be removed as one piece without damaging other tissues, and in these cases a bone graft can be performed as well. Conventional giant cell tumors have a risk of recurrence where they start, and have a low but real chance of metastasis to the lungs. Giant cell tumors must be differentiated from aneurysmal bone cysts, Adamantinoma is a very rare tumor of cells that are associated with bone formation that are can be similar to the cells responsible for forming teeth.
The latter cells can form cancers of the lower jaw more than the upper jaw, termed ameloblastoma.
Adamantinoma nearly always affects the tibia, and is treated with surgery.
Rare cases can travel elsewhere in the body, at which point chemotherapy is used to try and increase lifespan.
Chordoma is a tumor that appears very similar to the cells that fetal cells that formed the spine during development, the notochord. The relationship to development of the bone while the fetus is growing in the uterus is hard to understand, since tumors of this sort typically only arise in people over age 50. It typically occurs at the base of the skull, or in the sacrum (the very base of the spine in the pelvis). Given these locations, surgical removal is often times not possible. This is one tumor that can respond to radiation, with an intent to cure even tumors that are not surgically removable, and is an ideal type of tumor with which to try proton beam radiation. For tumors that recur, there are hints that imatinib (Gleevec ) can be of some use, and only infrequently are responses seen to other chemotherapy drugs.
Other sarcomas that typically occur in soft tissue in adults that can also occur in bone. These include malignant fibrous histiocytoma (MFH [also termed high grade Attorney Docket No. I2041-7000WO/3020PCT

undifferentiated pleomorphic sarcoma or HGUPS]), fibrosarcoma, leiomyosarcoma, and angiosarcoma, among others.
Certain methods of the current invention can be especially effective in treating cancers that respond well to existing chemotherapies, but suffer from a high relapse rate.
In these instances, treatment with the hedgehog inhibitor can increase the relapse-free survival time or rate of the patient. Examples of such cancers include lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), bladder cancer, ovarian cancer, breast cancer, colon cancer, multiple myeloma, acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), neuroendocrine cancer, and sarcomas.
The invention also encompasses the use of a chemotherapeutic agent and a hedgehog inhibitor for preparation of one or more medicaments for use in a method of extending relapse free survival in a cancer patient. The invention also relates to the use of a hedgehog inhibitor in the preparation of a medicament for use in a method of extending relapse free survival in a cancer patient who had previously been treated with a chemotherapeutic. The invention also encompasses the use of a hedgehog inhibitor in the preparation of a medicament for use in a method of treating pancreatic cancer patient.
It has been discovered that multiple tumor types exhibit up-regulation of Hh ligands post chemotherapy (see Examples 11 and 12 herein) and in response to other stress, such as hypoxia (see Example 12). The type of Hh ligand that is up-regulated (i.e., Sonic, Indian and/or Desert) and the degree of up-regulation vary depending upon the tumor type and the chemotherapeutic agent. Without wishing to be bound to any theory, these results suggest that stress (including chemotherapy) induces Hedgehog ligand production in tumor cells as a protective or survival mechanism. The results further suggest that up-regulation of tumor-derived Hh ligand post-chemotherapy can confer upon the surviving cell population a dependency upon the Hh pathway that is important for tumor recurrence, and thus can be susceptible to Hh pathway inhibition.
Thus, an aspect of the invention is a method of treating cancer by determining whether expression of one or more hedgehog ligands has increased during or after chemotherapy, then administering a hedgehog inhibitor. Ligand expression can be measured by detection of a soluble form of the ligand in peripheral blood and/or urine (e.g., by an ELISA assay or radioimmunoassay), in circulating tumor cells (e.g., by a Attorney Docket No. I2041-7000WO/3020PCT

fluorescence-activated cell sorting (FACS) assay, an immunohistochemisty assay, or a reverse transcription polymerase chain reaction (RT-PCR) assay), or in tumor or bone marrow biopsies (e.g., by an immunohistochemistry assay, a RT-PCR assay, or by in situ hybridization). Detection of hedgehog ligand in a given patient tumor could also be assessed in vivo, by systemic administration of a labeled form of an antibody to a hedgehog ligand followed by imaging, similar to detection of PSMA in prostate cancer patients (Bander, NH Nat Clin Pract Urol 2006; 3:216-225). Expression levels in a patient can be measured at least at two time-points to determine of ligand induction has occurred. For example, hedgehog ligand expression can be measured pre- and post-chemotherapy, pre-chemotherapy and at one or more time-points while chemotherapy is ongoing, or at two or more different time-points while chemotherapy is ongoing. If a hedgehog ligand is found to be up-regulated, a hedgehog inhibitor can be administered.
Thus, measurement of hedgehog ligand induction in the patient can determine whether the patient receives a hedgehog pathway inhibitor in combination with or following other chemotherapy.
Another aspect of the invention relates to a method of treating cancer in a patient by identifying one or more chemotherapeutics that elevate hedgehog ligand expression in the cancer tumor, and administering one or more of the chemotherapeutics that elevate hedgehog ligand expression and a hedgehog inhibitor. To determine which chemotherapeutics elevate hedgehog expression, tumor cells can be removed from a patient prior to therapy and exposed to a panel of chemotherapeutics ex vivo and assayed to measure changes in hedgehog ligand expression (see, e.g., Am. J. Obstet.
Gynecol.
Nov. 2003, 189(5):1301-7; J. Neurooncol., Feb. 2004, 66(3):365-75). A
chemotherapeutic that causes an increase in one or more hedgehog ligands is then administered to the patient. A chemotherapeutic that causes an increase in one or more hedgehog ligands can be administered alone or in combination with one or more different chemotherapeutics that can or can not cause an increase in one or more hedgehog ligands.
The hedgehog inhibitor and chemotherapeutic can be administered concurrently (i.e., essentially at the same time, or within the same treatment) or sequentially (i.e., one immediately following the other, or alternatively, with a gap in between administration of the two). Treatment with the hedgehog inhibitor can continue after treatment with the Attorney Docket No. I2041-7000WO/3020PCT

chemotherapeutic ceases. Thus, the chemotherapeutic is chosen based upon its ability to up-regulate hedgehog ligand expression (which, in turn, renders the tumors dependent upon the hedgehog pathway), which can make the tumor susceptible to treatment with a hedgehog inhibitor.
Another aspect of the invention relates to a method of treating cancer in a patient by identifying an alteration in an EGFR gene or gene product. The alteration of the EGFR gene or gene product includes, but is not limited to, cytogenetic abnormalities, non-reciprocal translocations, rearrangements, intra-chromosomal inversions, mutations, point mutations, deletions, changes in gene copy number, mutations in a transcript, and changes in expression of a gene or gene product. In certain embodiments, the mutation in a transcript is an mRNA mutation, rRNA mutation or tRNA mutation. In certain embodiments, the expression level, structure (e.g., post-translational modifications, such as phosphorylation) and/or activity of one or more oncogenic polypeptides is evaluated.
In related embodiments, the expression level, structure, and/or activity of one or more mutant oncogenic isoforms, e.g., isoforms arising from one or more of alternative splicing, frameshifting, translational and/or post-translational events, of various proto-oncogene expression products in a cell, e.g., a hyperproliferative cell (e.g., a cancerous or tumor cell) are detected.
Examples of EGFR mutations are described in e.g., Couzin J., (2004) Science 305:1222-1223; Fukuoka, M. et al., (2003) J. Clin. Oncol. 21:2237-46; Lynch et al., (2004) NEJM 350(21):2129-2139; Paez et al. (2004) Science 304:1497-1500; Pao, W. et al. Proc Nat/ Acad Sci USA. (2004) 101(36):13306-11; Gazdar A. F. et al., Trends Mol Med. (2004) 10(10):481-6; Huang S. F. et al. (2004) C/in Cancer Res.
10(24):8195-203;
Couzin J. Science (2004) 305(5688):1222-3; Sordella R. et al. (2004) 305(5687):1163-7;
Kosaka T. et al. (2004) Cancer Res. 64(24):8919-23; Marchetti A. et al. J C/in Onco/.
(2005) 23(4):857-65; Tokumo M. et al. (2005) C/in Cancer Res. 11(3):1167-1173;
Han S. W. et al. (2005) J C/in Onco/. 23(11):2493-501; Mitsudomi T. et al. (2005) J C/in Onco/. 23(11):2513-20; Shigematsu H. et al. JNat/ Cancer Inst. 97(5):339-46;
Kim K. S.
et al., (2005) C/in Cancer Res. 11(6):2244-51; Cappuzzo F. et al. (2005) JNat/
Cancer Inst. 97(9):643-55; Cortes-Funes H. et al. Ann Onco/. (2005) 16(7):1081-6;
Sasaki H. et al. (2005) C/in Cancer Res. 11(8):2924-9; Chou T. Y. et al., (2005) C/in Cancer Res.

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11 (10):3750-7; Pao W. et al. (2005) PLoS Med. 2(3):e73; Sasaki H. et al.
(2005) IntJ
Cancer. 118(1):180-4; Eberhard D. A. et al. (2005) J Clin Oncol. 23(25):5900-9; Takano T. et al. (2005) JClin Oncol. 23(28):6829-37; Tsao M. S. et al., (2005) NEngl JMed.
353(2):133-44; Mu X. L. et al. (2005) Clin Cancer Res. 11(12):4289-94; Sonobe M. et al.
(2005) Br J Cancer. 93(3):355-63; Taron M. et al. (2005) Clin Cancer Res.
11(16):5878-85; Mukohara T. et al., (2005) JNatl Cancer Inst. 97(16):1185-94; Zhang X. T.
et al.
(2005) Oncol. 16(8):1334-42. Exemplary alterations in an EGFR gene or gene product, include but are not limited to, an EGFR exon deletion (e.g., EGFR exon 19 Deletion), and/or exon mutation (e.g., an L858R/T790M EGFR mutation). Other exemplary alterations include, but are not limited to, EGFR D770 N771>AGG;
EGFR D770 N771 insG; EGFR D770 N771 insG; EGFR D770 N771 insN;
EGFR E709A; EGFR E709G; EGFR 709H; EGFR E709K; EGFR E709V;
EGFR E746 A750del; EGFR E746 A750de1, T751A; EGFR E746 A750del, V ins;
EGFR E746 T751del, I ins; EGFR E746 T751del, S752A; EGFR E746 T751del, S752D; EGFR E746 T751 del, V ins; EGFR G719A; EGFR G719C; EGFR G719S:
EGFR H773 V774insH; EGFR H773 V774insNPH; EGFR H773 V774insPH;
EGFR H773>NPY; EGFR L747 E749de1; EGFR L747 E749de1, A750P;
EGFR_L747_S752de1; EGFR L747_S752de1, P753S; EGFR L747_S752de1, Q ins;
EGFR_L747_T750de1, P ins; EGFR L747_T751del; EGFR L858R; EGFR_L861Q;
EGFR M766 A767insA1; EGFR P772 H773insV; EGFR S752 1759de1; EGFR S7681;
EGFR T790M; EGFR V769 D770insASV; EGFR V769 D770insASV: and EGFR V774 C775insHV.
The alteration can be detected by any method of detection available in the art, including but not limited to, one or more of nucleic acid hybridization assay, amplification-based assays (e.g., polymerase chain reaction (PCR)), PCR-RFLP
assay, real-time PCR, sequencing, screening analysis (including metaphase cytogenetic analysis by standard karyotype methods, FISH, spectral karyotyping or MFISH, comparative genomic hybridization), in situ hybridization, SSP, HPLC or mass-spectrometric genotyping.

Attorney Docket No. I2041-7000WO/3020PCT
Combination Therapy It will be appreciated that the hedgehog inhibitor, as described above and herein, can be administered in combination with one or more additional therapies, e.g., such as radiation therapy, surgery and/or in combination with one or more therapeutic agents, to treat the cancers described herein.
By "in combination with," it is not intended to imply that the therapy or the therapeutic agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of the invention. The pharmaceutical compositions can be administered concurrently with, prior to, or subsequent to, one or more other additional therapies or therapeutic agents.
In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. In will further be appreciated that the additional therapeutic agent utilized in this combination can be administered together in a single composition or administered separately in different compositions. The particular combination to employ in a regimen will take into account compatibility of the inventive pharmaceutical composition with the additional therapeutically active agent and/or the desired therapeutic effect to be achieved.
In general, it is expected that additional therapeutic agents utilized in combination be utilized at levels that do not exceed the levels at which they are utilized individually.
In some embodiments, the levels utilized in combination will be lower than those utilized individually.
In certain embodiments, the cancer treated by the methods described herein can be selected from, for example, medulloblastoma; a sarcoma (e.g., bone or soft-tissue sacoma (e.g., synovial sarcoma, liposarcoma), musculoskeletal sarcoma such as bone and cartilage sarcoma, osteosarcoma, and chondrosarcoma; pancreatic cancer; lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC));
colorectal cancer; ovarian cancer; head and neck squamous cell carcinoma (HNSCC); chronic myelogenous leukemia (CML); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); multiple myeloma, and prostate cancer.
An example of suitable therapeutics for use in combination with the hedgehog inhibitors for treatment of small cell lung cancer includes, but is not limited to, a Attorney Docket No. I2041-7000WO/3020PCT
chemotherapeutic agent, e.g., etoposide, carboplatin, cisplatin, irinotecan, topotecan, gemcitabine, liposomal SN-38, bendamustine, temozolomide, belotecan, NK012, FR901228, flavopiridol); tyrosine kinase inhibitor (e.g., EGFR inhibitor (e.g., erlotinib, gefitinib, cetuximab, panitumumab); multikinase inhibitor (e.g., sorafenib, sunitinib);
VEGF inhibitor (e.g., bevacizumab, vandetanib); cancer vaccine (e.g., GVAX);
Bcl-2 inhibitor (e.g., oblimersen sodium, ABT-263); proteasome inhibitor (e.g., bortezomib (Velcade), NPI-0052), paclitaxel or a paclitaxel agent; docetaxel; IGF-1 receptor inhibitor (e.g., AMG 479); HGF/SF inhibitor (e.g., AMG 102, MK-0646); chloroquine;
Aurora kinase inhibitor (e.g., MLN8237); radioimmunotherapy (e.g., TF2); hedgehog inhibitor (e.g., IPI-493, IPI-504, tanespimycin, STA-9090); mTOR inhibitor (e.g., everolimus);
Ep-CAM-/CD3-bispecific antibody (e.g., MTI10); CK-2 inhibitor (e.g., CX-4945);
HDAC inhibitor (e.g., belinostat); SMO antagonist (e.g., BMS 833923);
amrubicin, peptide cancer vaccine, and radiation therapy (e.g., intensity-modulated radiation therapy (IMRT), hypofractionated radiotherapy, hypoxia-guided radiotherapy), surgery, and combinations thereof.
An example of suitable therapeutics for use in combination with the hedgehog inhibitors for treatment of non-small cell lung cancer includes, but is not limited to, a chemotherapeutic agent, e.g., vinorelbine, cisplatin, docetaxel, pemetrexed disodium, etoposide, gemcitabine, carboplatin, liposomal SN-38, TLK286, temozolomide, topotecan, pemetrexed disodium, azacitidine, irinotecan, tegafur-gimeracil-oteracil potassium, sapacitabine); tyrosine kinase inhibitor (e.g., EGFR inhibitor (e.g., erlotinib, gefitinib, cetuximab, panitumumab, necitumumab, PF-00299804, nimotuzumab, R05083945), MET inhibitor (e.g., PF-02341066, ARQ 197), PI3K kinase inhibitor (e.g., XL147, GDC-0941), Raf/MEK dual kinase inhibitor (e.g., R05126766), PI3K/mTOR
dual kinase inhibitor (e.g., XL765), SRC inhibitor (e.g., dasatinib), dual inhibitor (e.g., BIBW 2992, GSK1363089, ZD6474, AZD0530, AG-013736, lapatinib, MEHD7945A, linifanib), multikinase inhibitor (e.g., sorafenib, sunitinib, pazopanib, AMG
706, XL 184, MGCD265, BMS-690514, R935788), VEGF inhibitor (e.g., endostar, endostatin, bevacizumab, cediranib, BIBF 1120, axitinib, tivozanib, AZD2171), cancer vaccine (e.g., BLP25 liposome vaccine, GVAX, recombinant DNA and adenovirus expressing L523S
protein), Bcl-2 inhibitor (e.g., oblimersen sodium), proteasome inhibitor (e.g., bortezomib, Attorney Docket No. I2041-7000WO/3020PCT
carfilzomib, NPI-0052, MLN9708), paclitaxel or a paclitaxel agent, docetaxel, receptor inhibitor (e.g., cixutumumab, MK-0646, OSI 906, CP-751,871, BIIB022), hydroxychloroquine, hedgehog inhibitor (e.g., IPI-493, IPI-504, tanespimycin, STA-9090, AUY922, XL888), mTOR inhibitor (e.g., everolimus, temsirolimus, ridaforolimus), Ep-CAM-/CD3-bispecific antibody (e.g., MTI10), CK-2 inhibitor (e.g., CX-4945), HDAC
inhibitor (e.g., MS 275, LBH589, vorinostat, valproic acid, FR901228), DHFR
inhibitor (e.g., pralatrexate), retinoid (e.g., bexarotene, tretinoin), antibody-drug conjugate (e.g., SGN- 15), bisphosphonate (e.g., zoledronic acid), cancer vaccine (e.g., belagenpumatucel-L), low molecular weight heparin (LMWH) (e.g., tinzaparin, enoxaparin), GSK1572932A, melatonin, talactoferrin, dimesna, topoisomerase inhibitor (e.g., amrubicin, etoposide, karenitecin), nelfinavir, cilengitide, ErbB3 inhibitor (e.g., MM-121, U3-1287), survivin inhibitor (e.g., YM155, LY2181308), eribulin mesylate, COX-inhibitor (e.g., celecoxib), pegfilgrastim, Polo-like kinase 1 inhibitor (e.g., BI 6727), TRAIL receptor 2 (TR-2) agonist (e.g., CS-1008), CNGRC peptide-TNF alpha conjugate, dichloroacetate (DCA), HGF inhibitor (e.g., SCH 900105), SAR240550, PPAR-gamma agonist (e.g., CS-7017), gamma-secretase inhibitor (e.g., R04929097), epigenetic therapy (e.g., 5-azacitidine), nitroglycerin, MEK inhibitor (e.g., AZD6244), cyclin-dependent kinase inhibitor (e.g., UCN-01), cholesterol-Fusl, antitubulin agent (e.g., E7389), farnesyl-OH-transferase inhibitor (e.g., lonafarnib), immunotoxin (e.g., BB-10901, SSI
(dsFv) PE38), fondaparinux, vascular-disrupting agent (e.g., AVE8062), PD-Ll inhibitor (e.g., MDX-1105, MDX-1106), beta-glucan, NGR-hTNF, EMD 521873, MEK inhibitor (e.g., GSKI 120212), epothilone analog (e.g., ixabepilone), kinesin-spindle inhibitor (e.g., 4SC-205), telomere targeting agent (e.g., KML-001), P70 pathway inhibitor (e.g., LY2584702), AKT inhibitor (e.g., MK-2206), angiogenesis inhibitor (e.g., lenalidomide), Notch signaling inhibitor (e.g., OMP-21M18), radiation therapy, surgery, and combinations thereof.
An example of suitable therapeutics for use in combination with the hedgehog inhibitors for treatment of colorectal cancer includes, but is not limited to, 5-Fluorouracil (5FU-TS inhibitor); Irinotecan (Topo I poison); Oxaliplatin (DNA adducts), monoclonal antibodies against EGFR, e.g., Erbitux and Vectabix, FOLFOX: 5-Fluorouracil +
Leucovorin +Oxaliplatin; FOLFIRI: 5-Fluorouracil + Leucovorin +Irinotecan, VEGF

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inhibitor (e.g., anti-VEGF antibody) alone or in combination with 5FU, and a combination thereof.
An example of suitable therapeutics for use in combination with the hedgehog inhibitors for treatment of medulloblastoma includes, but is not limited to, a chemotherapeutic agent (e.g., lomustine, cisplatin, carboplatin, vincristine, and cyclophosphamide), radiation therapy, surgery, and a combination thereof.
An example of suitable therapeutics for use in combination with the hedgehog inhibitors for treatment of chondrosarcoma includes, but is not limited to, a chemotherapeutic agent (e.g., one or more of. doxorubicin, cisplatin, ifosfamide, or methotrexate (e.g., high dose methotrexate), trabectedin, triparanol, or DAPT), mTOR
inhibitors, NOTCH inhibitors (e.g., gamma secretase inhibitors (e.g., R0499097), radiation therapy (e.g., proton therapy), surgery, and a combination thereof.
Additional agents that can be used in combination with the hedgehog inhibitors include other anti-cancer agents used for sarcoma treatment.
An example of suitable therapeutics for use in combination with the hedgehog inhibitors for treatment of osteosarcoma includes, but is not limited to, a chemotherapeutic agent (e.g., one or more of. doxorubicin, cisplatin, methotrexate (e.g., high dose methotrexate) (e.g., alone or in combination with leucovorin rescue), gemcitabine, docetaxel, adriamycin, ifosfamide (e.g., alone or in combination with mesna), BCG (Bacillus Calmette-Guerin), etoposide, muramyl tri-peptite (MTP)), radiation therapy, surgery, and a combination thereof. In one embodiment, the hedgehog inhibitor is used I combination with gemcitabine and docetaxel. Additional agents that can be used in combination with the hedgehog inhibitors include other anti-cancer agents used for sarcoma treatment.
An example of suitable therapeutics for use in combination with the hedgehog inhibitors for treatment of pancreatic cancer includes, but is not limited to, a chemotherapeutic agent, e.g., paclitaxel or a paclitaxel agent (e.g., a paclitaxel formulation such as TAXOL ), an albumin-stabilized nanoparticle paclitaxel formulation (e.g., ABRAXANE ) or a liposomal paclitaxel formulation);
gemcitabine (e.g., gemcitabine alone or in combination with AXP107-11); other chemotherapeutic agents such as oxaliplatin, 5-fluorouracil, capecitabine, rubitecan, epirubicin Attorney Docket No. I2041-7000WO/3020PCT
hydrochloride, NC-6004, cisplatin, docetaxel (e.g., TAXOTERE ), mitomycin C, ifosfamide; interferon; tyrosine kinase inhibitor (e.g., EGFR inhibitor (e.g., erlotinib, panitumumab, cetuximab, nimotuzumab); HER2/neu receptor inhibitor (e.g., trastuzumab); dual kinase inhibitor (e.g., bosutinib, saracatinib, lapatinib, vandetanib);
multikinase inhibitor (e.g., sorafenib, sunitinib, XL184, pazopanib); VEGF
inhibitor (e.g., bevacizumab, AV-95 1, brivanib); radioimmunotherapy (e.g., XR303); cancer vaccine (e.g., GVAX, survivin peptide); COX-2 inhibitor (e.g., celecoxib); IGF-1 receptor inhibitor (e.g., AMG 479, MK-0646); mTOR inhibitor (e.g., everolimus, temsirolimus);
IL-6 inhibitor (e.g., CNTO 328); cyclin-dependent kinase inhibitor (e.g., P276-00, UCN-01); Altered Energy Metabolism-Directed (AEMD) compound (e.g., CPI-613); HDAC
inhibitor (e.g., vorinostat); TRAIL receptor 2 (TR-2) agonist (e.g., conatumumab); MEK
inhibitor (e.g., AS703026, selumetinib, GSKI 120212); Raf/MEK dual kinase inhibitor (e.g., R05126766); Notch signaling inhibitor (e.g., MK0752); monoclonal antibody-antibody fusion protein (e.g., L191L2); curcumin; hedgehog inhibitor (e.g., IPI-493, IPI-504, tanespimycin, STA-9090); rIL-2;, denileukin diftitox; topoisomerase 1 inhibitor (e.g., irinotecan, PEP02); statin (e.g., simvastatin); Factor VIIa inhibitor (e.g., PCI-27483); AKT inhibitor (e.g., RX-0201); hypoxia-activated prodrug (e.g., TH-302);
metformin hydrochloride, gamma-secretase inhibitor (e.g., R04929097);
ribonucleotide reductase inhibitor (e.g., 3-AP); immunotoxin (e.g., HuC242-DM4); PARP
inhibitor (e.g., KU-0059436, veliparib); CTLA-4 inhbitor (e.g., CP-675,206, ipilimumab);
AdV-tk therapy; proteasome inhibitor (e.g., bortezomib (Velcade), NPI-0052);
thiazolidinedione (e.g., pioglitazone); NPC-1C; Aurora kinase inhibitor (e.g., R763/AS703569), CTGF
inhibitor (e.g., FG-3019); siGl2D LODER; and radiation therapy (e.g., tomotherapy, stereotactic radiation, proton therapy), surgery, and a combination thereof.
In certain embodiments, a combination of paclitaxel or a paclitaxel agent, and gemcitabine can be used with the hedgehog inhibitors. In some embodiments, the hedgehog inhibitor is used in combination with folfirinox to treat pancreatic cancer. Folfirinox comprises oxaliplatin 85 mg/m2 and irinotecan 180 mg/m2 plus leucovorin 400 mg/m2 followed by bolus fluorouracil (5-FU) 400 mg/m2 on day 1, then 5-FU 2,400 mg/m2 as a 46-hour continuous infusion.

Attorney Docket No. I2041-7000WO/3020PCT

An example of suitable therapeutics for use in combination with the hedgehog inhibitors for treatment of ovarian cancer includes, but is not limited to, a chemotherapeutic agent (e.g., paclitaxel or a paclitaxel agent; docetaxel;
carboplatin;
gemcitabine; doxorubicin; topotecan; cisplatin; irinotecan, TLK286, ifosfamide, olaparib, oxaliplatin, melphalan, pemetrexed disodium, SJG-136, cyclophosphamide, etoposide, decitabine); ghrelin antagonist (e.g., AEZS-130), immunotherapy (e.g., APC8024, oregovomab, OPT-821), tyrosine kinase inhibitor (e.g., EGFR inhibitor (e.g., erlotinib), dual inhibitor (e.g., E7080), multikinase inhibitor (e.g., AZD053O, JI-101, sorafenib, sunitinib, pazopanib), ON 01910.Na), VEGF inhibitor (e.g., bevacizumab, BIBF
1120, cediranib, AZD2171), PDGFR inhibitor (e.g., IMC-3G3), paclitaxel, topoisomerase inhibitor (e.g., karenitecin, Irinotecan), HDAC inhibitor (e.g., valproate, vorinostat), folate receptor inhibitor (e.g., farletuzumab), angiopoietin inhibitor (e.g., AMG 386), epothilone analog (e.g., ixabepilone), proteasome inhibitor (e.g., carfilzomib), IGF-1 receptor inhibitor (e.g., OSI 906, AMG 479), PARP inhibitor (e.g., veliparib, AGO 14699, iniparib, MK-4827), Aurora kinase inhibitor (e.g., MLN8237, ENMD-2076), angiogenesis inhibitor (e.g., lenalidomide), DHFR inhibitor (e.g., pralatrexate), radioimmunotherapeutic agnet (e.g., Hu3S 193), statin (e.g., lovastatin), topoisomerase 1 inhibitor (e.g., NKTR-102), cancer vaccine (e.g., p53 synthetic long peptides vaccine, autologous OC-DC vaccine), mTOR inhibitor (e.g., temsirolimus, everolimus), BCR/ABL inhibitor (e.g., imatinib), ET-A receptor antagonist (e.g., ZD4054), TRAIL
receptor 2 (TR-2) agonist (e.g., CS-1008), HGF/SF inhibitor (e.g., AMG 102), EGEN-001, Polo-like kinase 1 inhibitor (e.g., BI 6727), gamma-secretase inhibitor (e.g., R04929097), Wee-1 inhibitor (e.g., MK-1775), antitubulin agent (e.g., vinorelbine, E7389), immunotoxin (e.g., denileukin diftitox), SB-485232, vascular-disrupting agent (e.g., AVE8062), integrin inhibitor (e.g., EMD 525797), kinesin-spindle inhibitor (e.g., 4SC-205), revlimid, HER2 inhibitor (e.g., MGAH22), ErrB3 inhibitor (e.g., MM-121), radiation therapy; trabectadin, and combinations thereof.
An example of suitable therapeutics for use in combination with the hedgehog inhibitors for treatment of chronic myelogenous leukemia (CML) according to the invention includes, but is not limited to, a chemotherapeutic (e.g., cytarabine, hydroxyurea, clofarabine, melphalan, thiotepa, fludarabine, busulfan, etoposide, Attorney Docket No. I2041-7000WO/3020PCT
cordycepin, pentostatin, capecitabine, azacitidine, cyclophosphamide, cladribine, topotecan), tyrosine kinase inhibitor (e.g., BCR/ABL inhibitor (e.g., imatinib, nilotinib), ON 01910.Na, dual inhibitor (e.g., dasatinib, bosutinib), multikinase inhibitor (e.g., DCC-2036, ponatinib, sorafenib, sunitinib, RGB-286638)), interferon alfa, steroids, apoptotic agent (e.g., omacetaxine mepesuccinat), immunotherapy (e.g., allogeneic CD4+
memory Thl-like T cells/microparticle-bound anti-CD3/anti-CD28, autologous cytokine induced killer cells (CIK), AHN-12), CD52 targeting agent (e.g., alemtuzumab), hedgehog inhibitor (e.g., IPI-493, IPI-504, tanespimycin, STA-9090, AUY922, XL888), mTOR
inhibitor (e.g., everolimus), SMO antagonist (e.g., BMS 833923), ribonucleotide reductase inhibitor (e.g., 3-AP), JAK-2 inhibitor (e.g., INCB018424), Hydroxychloroquine, retinoid (e.g., fenretinide), cyclin-dependent kinase inhibitor (e.g., UCN-01), HDAC inhibitor (e.g., belinostat, vorinostat, JNJ-26481585), PARP
inhibitor (e.g., veliparib), MDM2 antagonist (e.g., R05045337), Aurora B kinase inhibitor (e.g., TAK-901), radioimmunotherapy (e.g., actinium-225 -labeled anti-CD33 antibody HuM195), Hedgehog inhibitor (e.g., PF-04449913), STAT3 inhibitor (e.g., OPB-31121), KB004, cancer vaccine (e.g., AG858), bone marrow transplantation, stem cell transplantation, radiation therapy, and combinations thereof.
An example of suitable therapeutics for use in combination with the hedgehog inhibitors for treatment of chronic lymphocytic leukemia (CLL) includes, but is not limited to, a chemotherapeutic agent (e.g., fludarabine, cyclophosphamide, doxorubicin, vincristine, chlorambucil, bendamustine, chlorambucil, busulfan, gemcitabine, melphalan, pentostatin, mitoxantrone, 5-azacytidine, pemetrexed disodium), tyrosine kinase inhibitor (e.g., EGFR inhibitor (e.g., erlotinib), BTK inhibitor (e.g., PCI-32765), multikinase inhibitor (e.g., MGCD265, RGB-286638), CD-20 targeting agent (e.g., rituximab, ofatumumab, R05072759, LFB-R603), CD52 targeting agent (e.g., alemtuzumab), prednisolone, darbepoetin alfa, lenalidomide, Bcl-2 inhibitor (e.g., ABT-263), immunotherapy (e.g., allogeneic CD4+ memory Thl -like T cells/microparticle-bound anti-CD3/anti-CD28, autologous cytokine induced killer cells (CIK)), HDAC
inhibitor (e.g., vorinostat, valproic acid, LBH589, JNJ-26481585, AR-42), XIAP inhibitor (e.g., AEG35156), CD-74 targeting agent (e.g., milatuzumab), mTOR inhibitor (e.g., everolimus), AT-101, immunotoxin (e.g., CAT-8015, anti-Tac(Fv)-PE38 (LMB-2)), Attorney Docket No. I2041-7000WO/3020PCT

CD37 targeting agent (e.g., TRU-0 16), radioimmunotherapy (e.g., 13 1 -tositumomab), hydroxychloroquine, perifosine, SRC inhibitor (e.g., dasatinib), thalidomide, P13K delta inhibitor (e.g., CAL-101), retinoid (e.g., fenretinide), MDM2 antagonist (e.g., R05045337), plerixafor, Aurora kinase inhibitor (e.g., MLN8237, TAK-901), proteasome inhibitor (e.g., bortezomib), CD-19 targeting agent (e.g., MEDI-551, MOR208), MEK inhibitor (e.g., ABT-348), JAK-2 inhibitor (e.g., INCB018424), hypoxia-activated prodrug (e.g., TH-302), paclitaxel or a paclitaxel agent, hedgehog inhibitor, AKT inhibitor (e.g., MK2206), HMG-CoA inhibitor (e.g., simvastatin), GNKG186, radiation therapy, bone marrow transplantation, stem cell transplantation, and a combination thereof.
An example of suitable therapeutics for use in combination with the hedgehog inhibitors for treatment of acute lymphocytic leukemia (ALL) includes, but is not limited to, a chemotherapeutic agent (e.g., prednisolone, dexamethasone, vincristine, asparaginase, daunorubicin, cyclophosphamide, cytarabine, etoposide, thioguanine, mercaptopurine, clofarabine, liposomal annamycin, busulfan, etoposide, capecitabine, decitabine, azacitidine, topotecan, temozolomide), tyrosine kinase inhibitor (e.g., BCR/ABL inhibitor (e.g., imatinib, nilotinib), ON 01910.Na, multikinase inhibitor (e.g., sorafenib)), CD-20 targeting agent (e.g., rituximab), CD52 targeting agent (e.g., alemtuzumab), hedgehog inhibitor (e.g., STA-9090), mTOR inhibitor (e.g., everolimus, rapamycin), JAK-2 inhibitor (e.g., INCB018424), HER2/neu receptor inhibitor (e.g., trastuzumab), proteasome inhibitor (e.g., bortezomib), methotrexate, asparaginase, CD-22 targeting agent (e.g., epratuzumab, inotuzumab), immunotherapy (e.g., autologous cytokine induced killer cells (CIK), AHN-12), blinatumomab, cyclin-dependent kinase inhibitor (e.g., UCN-01), CD45 targeting agent (e.g., BC8), MDM2 antagonist (e.g., R05045337), immunotoxin (e.g., CAT-8015, DT2219ARL), HDAC inhibitor (e.g., JNJ-26481585), JVRS-100, paclitaxel or apaclitaxel agent, STAT3 inhibitor (e.g., OPB-31121), PARP inhibitor (e.g., veliparib), EZN-2285, radiation therapy, steroid, bone marrow transplantation, stem cell transplantation, or a combination thereof.
An example of suitable therapeutics for use in combination with the hedgehog inhibitors for treatment of acute myeloid leukemia (AML) includes, but is not limited to, a chemotherapeutic agent (e.g., cytarabine, daunorubicin, idarubicin, clofarabine, Attorney Docket No. I2041-7000WO/3020PCT
decitabine, vosaroxin, azacitidine, clofarabine, ribavirin, CPX-35 1, treosulfan, elacytarabine, azacitidine), tyrosine kinase inhibitor (e.g., BCR/ABL
inhibitor (e.g., imatinib, nilotinib), ON 01910.Na, multikinase inhibitor (e.g., midostaurin, SU 11248, quizartinib, sorafinib)), immunotoxin (e.g., gemtuzumab ozogamicin), DT3881L3 fusion protein, HDAC inhibitor (e.g., vorinostat, LBH589), plerixafor, mTOR inhibitor (e.g., everolimus), SRC inhibitor (e.g., dasatinib), hedgehog inhbitor (e.g., STA-9090), retinoid (e.g., bexarotene, Aurora kinase inhibitor (e.g., BI 811283), JAK-2 inhibitor (e.g., INCB018424), Polo-like kinase inhibitor (e.g., BI 6727), cenersen, CD45 targeting agent (e.g., BC8), cyclin-dependent kinase inhibitor (e.g., UCN-01), MDM2 antagonist (e.g., R05045337), mTOR inhibitor (e.g., everolimus), LY573636-sodium, ZRx-101, MLN4924, lenalidomide, immunotherapy (e.g., AHN-12), histamine dihydrochloride, radiation therapy, bone marrow transplantation, stem cell transplantation, and a combination thereof.
An example of suitable therapeutics for use in combination with the hedgehog inhibitors for treatment of multiple myeloma (MM) includes, but is not limited to, a chemotherapeutic agent (e.g., melphalan, amifostine, cyclophosphamide, doxorubicin, clofarabine, bendamustine, fludarabine, adriamycin, SyB L-0501), thalidomide, lenalidomide, dexamethasone, prednisone, pomalidomide, proteasome inhibitor (e.g., bortezomib, carfilzomib, MLN9708), cancer vaccine (e.g., GVAX), CD-40 targeting agent (e.g., SGN-40, CHIR-12.12), perifosine, zoledronic acid, Immunotherapy (e.g., MAGE-A3, NY-ESO-1 , HuMax-CD38), HDAC inhibitor (e.g., vorinostat, LBH589, AR-42), aplidin, cycline-dependent kinase inhibitor (e.g., PD-0332991, dinaciclib), arsenic trioxide, CB3304, hedgehog inhibitor (e.g., KW-2478), tyrosine kinase inhibitor (e.g., EGFR inhibitor (e.g., cetuximab), multikinase inhibitor (e.g., AT9283)), VEGF
inhibitor (e.g., bevacizumab), plerixafor, MEK inhibitor (e.g., AZD6244), IPH2101, atorvastatin, immunotoxin (e.g., BB-10901), NPI-0052, radioimmunotherapeutic (e.g., yttrium Y 90 ibritumomab tiuxetan), STAT3 inhibitor (e.g., OPB-31121), MLN4924, Aurora kinase inhibitor (e.g., ENMD-2076), IMGN901, ACE-041, CK-2 inhibitor (e.g., CX-4945), radiation therapy, bone marrow transplantation, stem cell transplantation, and a combination thereof.

Attorney Docket No. I2041-7000WO/3020PCT

An example of suitable therapeutics for use in combination with the hedgehog inhibitors for treatment of head and neck cancer includes, but is not limited to, a chemotherapeutic (e.g., paclitaxel or a paclitaxel agent, carboplatin, docetaxel, amifostine, cisplantin, oxaliplatin, docetaxel), tyrosine kinase inhibitors (e.g., EGFR
inhibitor (e.g., erlotinib, gefitinib, icotinib, cetuximab, panitumumab, zalutumumab, nimotuzumab, necitumumab, matuzumab, cetuximab), dual inhibitor (e.g., lapatinib, neratinib, vandetanib, BIBW 2992, multikinase inhibitor (e.g., XL-647)), VEGF inhibitor (e.g., bevacizumab), reovirus, radiation therapy, surgery, and a combination thereof.
An example of suitable therapeutics for use in combination with the hedgehog inhibitors for treatment of prostate cancer includes, but is not limited to, a chemotherapeutic agent (e.g., docetaxel, carboplatin, fludarabine), hormonal therapy (e.g., flutamide, bicalutamide, nilutamide, cyproterone acetate, ketoconazole, aminoglutethimide, abarelix, degarelix, leuprolide, goserelin, triptorelin, buserelin), tyrosine kinase inhibitor (e.g., dual kinase inhibitor (e.g., lapatanib), multikinase inhibitor (e.g., sorafenib, sunitinib)), VEGF inhibitor (e.g., bevacizumab), TAK-700, cancer vaccine (e.g., BPX-101, PEP223), lenalidomide, TOK-001, IGF-1 receptor inhibitor (e.g., cixutumumab), TRC105, Aurora A kinase inhibitor (e.g., MLN8237), proteasome inhibitor (e.g., bortezomib), OGX-011, radioimmunotherapy (e.g., HuJ591-GS), HDAC
inhibitor (e.g., valproic acid, SB939, LBH589), hydroxychloroquine, mTOR
inhibitor (e.g., everolimus), dovitinib lactate, diindolylmethane, efavirenz, OGX-427, genistein, IMC-3G3, bafetinib, CP-675,206, abiraterone (CB-7598, cytochrome P450 17 alpha-hydroxylase- 17, 20-lyase inhibitor (CYP17), CB-7598 and prednisone, radiation therapy, surgery, androgen ablation, or a combination thereof. Androgen ablation can include goserelin acetate and leuprolide acetate.
In some embodiments, the hedgehog inhibitor is used in combination with an mTOR inhibitor. "mTOR inhibitor" as used herein refers to an agent that directly or indirectly target, decreases or inhibits the activity/function of an mTOR
kinase (mammalian Target Of Rapamycin). Some reports have described a role of mTOR in chondrocyte differentiation, see e.g., Brown, R. E. (2004) Annals of Clinical &
Laboratory Science 34:397-399; Chan, S. (2004) Br J Cancer 91(8):1420-4; and Geryk-Hall, M. et al. (2009) Curr Oncol Rep. 11(6):446-53. mTOR inhibitors suitable for use in Attorney Docket No. I2041-7000WO/3020PCT
the invention are described in numerous references, including but not limited to: WO
94/02136 (16-0-substituted derivatives); U.S. Pat. No. 5,258,389 (40-0-substituted derivatives); WO 94/9010 (O-aryl and O-alkyl derivatives); WO 92/05179 (carboxylic acid esters); U.S. Pat. Nos. 5,118,677 and 5,118,678 (amide esters); U.S. Pat.
No.
5,118,678 (carbamates); U.S. Pat. No. 5,100,883 (fluorinated esters); U.S.
Pat. No.
5,151,413 (acetals); U.S. Pat. No. 5,120,842 (silyl esters); WO 93,11130 (methylene derivatives); WO 94/02136 (methoxy derivatives); WO 94/02385 and WO 95/14023 (alkenyl derivatives); U.S. Pat. No. 5,256,790 (32-0-dihydro or substituted derivatives);
EP 96/02441; U.S. 2004/023562 (carbohydrate derivatives); U.S. Pat. No.
4,316,885 (mono and diacylated derivatives); U.S. Pat. No. 5,120,725 (bicylic derivatives); U.S.
Pat. No. 5,120,727 (rapamycin dimers); EP 467606 (27-oximes of rapamycin);
U.S. Pat.
No. 5,023,262 (42-oxo analogs); U.S. Pat. No. 5,177,203 (arylsulfonates and sulfamates);
U.S. Pat. No. 5,177,203. In addition, various rapamycin prodrugs have been described in U.S. Pat. Nos. 4,650,803; 5,672,605; 5,583,189; 5,527,906; 5,457,111;
5,995,100; and 6,146,658. Of particular interest for use in treatment methods are derivatives described in patents owned by Novartis (U.S. Pat. Nos. 5,665,772; 5,912,253; 5,985,890;
5,912,253;
6,200,985; 6,384,046; and 6,440,990), Ariad (WO 96/41865); and Wyeth (U.S.
Pat. Nos.
5,362,718; 6,399,625; 6,399,627; 6,432,973; 6,440,991; 6,677,357; and 6,680,718).
Exemplary mTOR inhibitors, include, but are not limited to, rapamycin, temsirolimus (TORISEL ), everolimus (RAD001, AFINITOR ), ridaforolimus, AP23573, AZD8055, BEZ235, BGT226, XL765, PF-4691502, GDC0980, SF1126, OSI-027, G5K1059615, KU-0063794, WYE-354, INK128, temsirolimus (CCI-779), Palomid 529 (P529), PF-04691502, or PKI-587. In one embodiment, the mTOR inhibitor inhibits TORCI and TORC2. Examples of TORCI and TORC2 dual inhibitors include, e.g., OSI-027, XL765, Palomid 529, and INK128.
In some embodiments, the hedgehog inhibitor is used in combination with an inhibitor of insulin-like growth factor receptor (IGF-1R). IGF-1R (also known as EC
2.7.112, CD 221 antigen) belongs to the family of transmembrane protein tyrosine kinases (Ullrich et al., Cell, 61: 203-212, (1990), LeRoith et al., Endocrin.
Rev., 16: 143-163 (1995); Traxler, Exp. Opin. Ther. Patents, 7: 571-588 (1997); Adams et al., Cell.
Mol. Life. Sci., 57: 1050-1063 (2000)), and is involved in childhood growth ((Liu et al., Attorney Docket No. I2041-7000WO/3020PCT

Cell, 75: 59-72 (1993); Abuzzahab et al.; NEngl JMed, 349: 2211-2222 (2003)).
The IGF system has been implicated in several cancers. See, for example, Pollak et al., Nat Rev Cancer, 4: 505-518 (2004); Yee, British J. Cancer, 94: 465-468 (2006);
Bohula et al., Anti-Cancer Drugs, 14: 669-682 (2003); Surmacz, Oncogene, 22: 6589-97 (2003);
Bahr and Groner, Growth Hormone and IGF Research 14: 287-295 (2004);
Guillemard and Saragovi, Current Cancer Drug Targets, 4: 313-326 (2004); Jerome et al., Seminars in Oncology 31/1 Suppl. 3 (54-63) (2004); Zhang and Yee, Breast Disease, 17:

(2003); Samani and Brodt, Surgical Oncology Clinics of North America, 10: 289-(2001); Nahta et al., Oncologist, 8: 5-17 (2003); Dancey and Chen, Nature Reviews, 5:
649-659 (2006); Jones et al., Endocr. Relat. Cancer, 11:793-814 (2004);
Schedin, Nature Reviews, 6: 281-290 (2006); Thorne and Lee, Breast Disease, 17: 105-114 (2003);
Minchinton and Tannock, Nature Reviews, 6: 583-592 (2006); and Kurmasheva and Houghton, Biochim. Biophys. Acta, 1766: 1-22 (2006). A role for IGF-1R
signaling in sarcomas (e.g., chondrosarcomas, chordoma Ewing sarcoma, or osteosarcoma), is described in, e.g., Matsumura, T. et al. (2000) J. Orthop Res. 18(3):351-5;
Ho, L. et al.
(2009) Cancer Cell 16:126-136; Sommer, J. et al. (2010) J. Pathol. 220(5):608-17;
Geryk-Hall, M. et al. (2009) Curr Oncol Rep. 11(6):446-53.
Inhibitory peptides targeting IGF-1R have been generated that possess anti-proliferative activity in vitro and in vivo (Pietrzkowski et al., Cancer Res., 52:6447-6451 (1992); Haylor et al., J. Am. Soc. Nephrol., 11:2027-2035 (2000)). Growth can also be inhibited using peptide analogues of IGF-I (Pietrzkowski et al., Cell Growth &Diff., 3:
199-205 (1992); Pietrzkowski et al., Mol. Cell. Biol., 12: 3883-3889 (1992)).
In addition, dominant-negative mutants of IGF-1R (Li et al., J. Biol. Chem., 269: 32558-32564 (1994);
Jiang et al., Oncogene, 18: 6071-6077 (1999); Scotlandi et al., Int. J.
Cancer, 101: 11-16 (2002); Seely et al., BMC Cancer, 2: 15 (2002)) can reverse the transformed phenotype, inhibit tumorigenesis, and induce loss of the metastatic phenotype. A C-terminal peptide of IGF-1R has been shown to induce apoptosis and significantly inhibit tumor growth (Reiss et al., J. Cell. Phys., 181:124-135 (1999)). Also, a soluble form of IGF-1R inhibits tumor growth in vivo (D'Ambrosio et al., Cancer Res., 56: 4013-4020 (1996)).
Thus, antagonists or inhibitors of IGF-1R that can be used in combination with the therapies disclosed herein include, but are not limited to, small molecule Attorney Docket No. I2041-7000WO/3020PCT

antagonists (e.g., GSK1904529A), antibody antagonists, IGF-1R peptide antagonists, or anti-sense or other nucleic acid antagonists. Exemplary IGF-1R inhibitors include, but are not limited to, BMS-536924, GSK1904529A, AMG 479, MK-0646, cixutumumab, OSI 906, figitumumab (CP-751,871), and BIIB022.
In one embodiment, the IGF-1R antagonist is GSK1904529A described in, e.g., Sabbatini, P. et al. (2009) Mol Cancer Ther 8(10):2811-20; Sabbatini, P. et al. (2009) Clin Cancer Res. 3058. Additional small-molecule inhibitors of IGF-1R are described, e.g., in Garcia-Echeverria et al., Cancer Cell, 5: 231-239 (2004); Mitsiades et al., Cancer Cell, 5: 221-230 (2004); and Carboni et al., Cancer Res, 65: 3781-3787 (2005).
Further, compounds have been developed that disrupt receptor activation, such as, for example, Vasilcanu et al., Oncogene, 23: 7854-7862 (2004), which describes a cyclolignan, picropodophyllin, which appears to be specific for IGF-1R (Gimita et al., Cancer Res, 64:
236-242 (2004); Stromberg et al., Blood, 107: 669-678 (2006)).
Nordihydroguaiaretic acid (NDGA) also disrupts IGF-1R function (Youngren et al., Breast Cancer Res Treat, 94: 37-46 (2005)). Further examples of small-molecule inhibitors include WO
2002/102804; WO 2002/102805; WO 2004/55022; U.S. Pat. No. 6,037,332; WO
2003/48133; US 2004/053931; US 2003/125370; U.S. Pat. No. 6,599,902; U.S. Pat.
No.
6,117,880; WO 2003/35619; WO 2003/35614; WO 2003/35616; WO 2003/35615; WO
1998/48831; U.S. Pat. No. 6,337,338; US 2003/0064482; U.S. Pat. No. 6,475,486;
U.S.
Pat. No. 6,610,299; U.S. Pat. No. 5,561,119; WO 2006/080450; WO 2006/094600;
and WO 2004/093781 See also WO 2007/099171 (bicyclo-pyrazole inhibitors) and WO
2007/099166 (pyrazolo-pyridine derivative inhibitors). See also (Hubbard et al., AACR-NCI-EORTC Int ConfMol Targets Cancer Ther (October 22-26, San Francisco) 2007, Abst A227) on Abbott Corporation's molecule A-928605.
Exemplary peptides that antagonize IGF-1R or treat cancer involving IGF-I
include those described by U.S. Pat. No. 6,084,085; U.S. Pat. No. 5,942,489;
WO
2001/72771; WO 2001/72119; US 2004/0086863; U.S. Pat. No. 5,633,263; and US
2003/0092631. See also U.S. Pat. No. 7,173,005; Bioworld Today published May 19, 2006 (Vol. 17, page 1).
Exemplary anti-sense and nucleic acids that antagonize IGF-1R are described, e.g., in Wraight et al., Nat. Biotech., 18: 521-526 (2000); U.S. Pat. No. 5,643,788;
U.S. Pat.

Attorney Docket No. I2041-7000WO/3020PCT

No. 6,340,674; US 2003/0031658; U.S. Pat. No. 6,340,674; U.S. Pat. No.
5,456,612; U.S.
Pat. No. 5,643,788; U.S. Pat. No. 6,071,891; WO 2002/101002; CN 1237582A; CN
111709713; WO 1999/23259; WO 2003/100059; US 2004/127446; US 2004/142895; US
2004/110296; US 2004/006035; US 2003/206887; US 2003/190635; US 2003/170891;
US 2003/096769; U.S. Pat. No. 5,929,040; U.S. Pat. No. 6,284,741; US
2006/0234239;
and U.S. Pat. No. 5,872,241. Further, US 2005/0255493 discloses reducing IGF-expression by RNA interference using short double-stranded RNA.
In some embodiments, the hedgehog inhibitor is used in combination with a tyrosine kinase inhibitor (e.g., a receptor tyrosine kinase (RTK) inhibitor).
Exemplary tyrosine kinase inhibitor include, but are not limited to, an epidermal growth factor (EGF) pathway inhibitor (e.g., an epidermal growth factor receptor (EGFR) inhibitor), a vascular endothelial growth factor (VEGF) pathway inhibitor (e.g., a vascular endothelial growth factor receptor (VEGFR) inhibitor (e.g., a VEGFR-1 inhibitor, a VEGFR-2 inhibitor, a VEGFR-3 inhibitor)), a platelet derived growth factor (PDGF) pathway inhibitor (e.g., a platelet derived growth factor receptor (PDGFR) inhibitor (e.g., a PDGFR-13 inhibitor)), a RAF-1 inhibitor, a KIT inhibitor and a RET inhibitor.
In some embodiments, the anti-cancer agent used in combination with the hedgehog inhibitor is selected from the group consisting of. axitinib (AGO13736), bosutinib (SKI-606), cediranib (RECENTIN TM, AZD2171), dasatinib (SPRYCEL , BMS-354825), erlotinib (TARCEVA ), gefitinib (IRESSA ), imatinib (Gleevec , CGP57148B, STI-571), lapatinib (TYKERB , TYVERB ), lestaurtinib (CEP-701), neratinib (HKI-272), nilotinib (TASIGNA ), semaxanib (semaxinib, SU5416), sunitinib (SUTENT , SU11248), toceranib (PALLADIA ), vandetanib (ZACTIMA , ZD6474), vatalanib (PTK787, PTK/ZK), trastuzumab (HERCEPTIN ), bevacizumab (AVASTIN ), rituximab (RITUXAN ), cetuximab (ERBITUX ), panitumumab (VECTIBIX ), ranibizumab (Lucentis ), nilotinib (TASIGNA ), sorafenib (NEXAVAR ), alemtuzumab (CAMPATH ), gemtuzumab ozogamicin (MYLOTARG ), ENMD-2076, PCI-32765, AC220, dovitinib lactate (TK1258, CHIR-258), BIBW 2992 (TOVOKTM) SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120 (VARGATEF ), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154, CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, XL228, Attorney Docket No. I2041-7000WO/3020PCT

AEE788, AG-490, AST-6, BMS-599626, CUDC-101, PD153035, pelitinib (EKB-569), vandetanib (zactima), WZ3146, WZ4002, WZ8040, ABT-869 (linifanib), AEE788, AP24534 (ponatinib), AV-951(tivozanib), axitinib, BAY 73-4506 (regorafenib), brivanib alaninate (BMS-582664), brivanib (BMS-540215), cediranib (AZD2171), CHIR-258 (dovitinib), CP 673451, CYC116, E7080, Ki8751, masitinib (AB1010), MGCD-265, motesanib diphosphate (AMG-706), MP-470, OSI-930, Pazopanib Hydrochloride, PD 173074,nSorafenib Tosylate(Bay 43-9006), SU 5402, TSU-68(SU6668), vatalanib, XL880 (GSK1363089, EXEL-2880). Selected tyrosine kinase inhibitors are chosen from sunitinib, erlotinib, gefitinib, or sorafenib. In one embodiment, the tyrosine kinase inhibitor is sunitinib.
In some embodiments, the hedgehog inhibitor is used in combination with folfirinox to treat the cancers and metastatic growths described herein, e.g., pancreatic cancer. Folfirinox comprises oxaliplatin 85 mg/m2 and irinotecan 180 mg/m2 plus leucovorin 400 mg/m2 followed by bolus fluorouracil (5-FU) 400 mg/m2 on day 1, then 5-FU 2,400 mg/m2 as a 46-hour continuous infusion.
In some embodiments, the hedgehog inhibitor is used in combination with a P13K
inhibitor. In one embodiment, the P13K inhibitor is an inhibitor of delta and gamma isoforms of P13K. In another embodiment, the hedgehog is used in combination with a dual PI3K/mTOR inhibitor. Exemplary P13K inhibitors that can be used in combination are described in, e.g., WO 2010/036380; WO 2010/006086, WO 09/114870, WO
05/113556. Additional P13K inhibitors that can be used in combination with the pharmaceutical compositions, include but are not limited to, GSK 2126458, GDC-0980, GDC-0941, Sanofi XL147, XL756, XL147, PF-46915032, BKM 120, CAL-101, CAL
263, SF1126, PX-886, and a dual P13K inhibitor (e.g., Novartis BEZ235). In one embodiment, the P13K inhibitor is an isoquinolinone. In one embodiment, the inhibitor is INK1197 or a derivative thereof. In other embodiments, the P13K
inhibitor is INK1117 or a derivative thereof.
In some embodiments, the hedgehog inhibitor is administered in combination with a BRAF inhibitor, e.g., GSK2118436, RG7204, PLX4032, GDC-0879, PLX4720, and sorafenib tosylate (Bay 43-9006), and/or other anti-cancer agents..

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In some embodiments, the hedgehog inhibitor is administered in combination with a MEK inhibitor, e.g., ARRY-142886, GSKI 120212, RDEA436, RDEA119/BAY
869766, AS703026, AZD6244 (selumetinib), BIX 02188, BIX 02189, CI-1040 (PD184352), PD0325901, PD98059, and U0126, and/or other anti-cancer agents.
In some embodiments, the hedgehog inhibitor is administered in combination with a JAK2 inhibitor, e.g., CEP-701, INCB18424, CP-690550 (tasocitinib).
In some embodiments, one or more of the following agents are used in combination with the hedgehog inhibitors described herein: inhibitors of B-Raf (e.g., Sorafenib, PLX4032), Mek (e.g., PD 032901), Erk (e.g., PD98059), Cdk4/6 (e.g., PD
0332991), and EGFR (e.g., Tarceva )).
In some embodiments, the hedgehog inhibitor is administered in combination with paclitaxel or a paclitaxel agent, e.g., TAXOL , protein-bound paclitaxel (e.g., ABRAXANE ), and/or other anti-cancer agents.
"Paclitaxel" as used herein refers to a compound having the following structure:

Ame O OH
Me Me Me H

O N H O
Me O
OH O O yme OH

or a pharmaceutically acceptable salt thereof.
Paclitaxel marketed as TAXOL (Bristol-Myers Squibb, Princeton, NJ) is formulated in the nonionic surfactant Cremophor EL (polyoxyethylated castor oil) and ethanol to enhance drug solubility (Dorr et al., Ann. Pharmacother., (1994) 28: S11-S 14). Cremophor EL can add to paclitaxel's toxic effects by producing or contributing to the well-described hypersensitivity reactions that commonly occur during infusion, affecting 25-30% of treated patients (Weiss et al., J. Clin. Oncol. (1990) 8:

and Rowinsky et al., N. Eng. J. Med. (1995) 332:1004-1014). To minimize the incidence Attorney Docket No. I2041-7000WO/3020PCT

and severity of these reactions, premedication with histamine 1 and 2 blockers, as well as glucocorticoids (e.g., dexamethasone), has become standard practice (Finley et al., Ann.
Pharmacother. (1994) 28: S27-S30). The cumulative side effects of dexamethasone used as a premedication can add to treatment-related morbidity and, in some instances, result in early discontinuation of therapy. Cremaphor EL can also contribute to chronic paclitaxel toxic effects, such as peripheral neuropathy (Windebank et al., J.
Pharmacol.
Exp. Ther. (1994) 268: 1051-1056). An additional problem arising from the Cremophor and ethanol solvent is the leaching of plasticizers from PVC bags and infusion sets in routine clinical use (Waugh et al., Am. J. Hosp. Pharm. (1991) 48: 1520-1524).
Consequently, paclitaxel marketed as TAXOL must be prepared and administered in either glass bottles or non-PVC infusion systems and with in-line filtration.
These problematic issues have spurred interest in the development of new formulations of paclitaxel with improved solubility in aqueous solutions.
A "paclitaxel agent" as used herein refers to a formulation of paclitaxel (e.g., for example, TAXOL ) or a paclitaxel equivalent (e.g., for example, a prodrug of paclitaxel). Exemplary paclitaxel equivalents include, but are not limited to, nanoparticle albumin-bound paclitaxel (ABRAXANE , marketed by Abraxis Bioscience), docosahexaenoic acid bound-paclitaxel (DHA-paclitaxel, Taxoprexin, marketed by Protarga), polyglutamate bound-paclitaxel (PG-paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX, marketed by Cell Therapeutic), the tumor-activated prodrug (TAP), ANG 105 (Angiopep-2 bound to three molecules of paclitaxel, marketed by ImmunoGen), paclitaxel-EC-1 (paclitaxel bound to the erbB2-recognizing peptide EC- 1; see Li et al., Biopolymers (2007) 87:225-230), and glucose-conjugated paclitaxel (e.g., 2'-paclitaxel methyl 2-glucopyranosyl succinate, see Liu et al., Bioorganic & Medicinal Chemistry Letters (2007) 17:617-620). In certain embodiments, the paclitaxel agent is a paclitaxel equivalent. In certain embodiments, the paclitaxel equivalent is ABRAXANE .
In certain embodiments, the hedgehog inhibitor and the additional anti-cancer agent are administered concurrently (i.e., administration of the two agents at the same time or day, or within the same treatment regimen) or sequentially (i.e., administration of one agent over a period of time followed by administration of the other agent for a second period of time, or within different treatment regimens).

Attorney Docket No. I2041-7000WO/3020PCT

In certain embodiments, the hedgehog inhibitor and the additional anti-cancer agent are administered concurrently. For example, in certain embodiments, the hedgehog inhibitor and the additional anti-cancer agent are administered at the same time. In certain embodiments, the hedgehog inhibitor and the additional anti-cancer agent are administered on the same day. In certain embodiments, the hedgehog inhibitor is administered after the additional anti-cancer agent on the same day or within the same treatment regimen. In certain embodiments, the hedgehog inhibitor is administered before the additional anti-cancer agent on the same day or within the same treatment regimen.
In certain embodiments, a hedgehog inhibitor is concurrently administered with additional anti-cancer agent for a period of time, after which point treatment with the additional anti-cancer agent is stopped and treatment with the hedgehog inhibitor continues.
In other embodiments, a hedgehog inhibitor is concurrently with the additional anti-cancer agent for a period of time, after which point treatment with the hedgehog inhibitor is stopped and treatment with the additional anti-cancer agent continues.
In certain embodiments, the hedgehog inhibitor and the additional anti-cancer agent are administered sequentially. For example, in certain embodiments, the hedgehog inhibitor is administered after the treatment regimen of the additional anti-cancer agent has ceased. In certain embodiments, the additional anti-cancer agent is administered after the treatment regimen of the hedgehog inhibitor has ceased.
Certain exemplary embodiments of two- or three-way combination therapies are provided below based on the combination of a second agent (e.g., the paclitaxel agent) and a hedgehog inhibitor. These are intended to be illustrative of combination treatments that can be modified and/or applied to other combination therapies disclosed herein.
In one aspect, the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor are administered concurrently. In certain embodiments, the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor are concurrently administered on the same day to the patient. In certain embodiments, the second agent (e.g., the paclitaxel agent) is administered first, provided that the hedgehog inhibitor is also administered on the same day to the patient. In other embodiments, the hedgehog inhibitor is Attorney Docket No. I2041-7000WO/3020PCT
administered first, provided that the second agent (e.g., the paclitaxel agent) is also administered on the same day to the patient. In yet other embodiments, the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor are administered simultaneously (i.e., at the same time) on the same day to the patient. Alternatively, in certain embodiments, the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor are administered on different days and/or on different schedules (e.g., one administered daily while the other is administered weekly), provided that this treatment regimen for both begin and end on the same day.
In another aspect, the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor are administered sequentially. For example, in certain embodiments, the hedgehog inhibitor is administered after administration of the second agent (e.g., the paclitaxel agent) has ceased. In certain embodiments, the hedgehog inhibitor is administered immediately after administration of the second agent (e.g., the paclitaxel agent) has ceased (i.e., on the same day as treatment with the second agent (e.g., the paclitaxel agent) has ceased), or, in certain embodiments, there is a period of time (e.g., one day, two days, one week, two weeks, one month, two months, six months, one year, etc.) between the end of the second agent (e.g., the paclitaxel agent) administration and the beginning of the hedgehog inhibitor administration. Alternatively, in certain embodiments, the second agent (e.g., the paclitaxel agent) is administered after administration of the hedgehog inhibitor has ceased. In certain embodiments, the second agent (e.g., the paclitaxel agent) is administered immediately after administration of the hedgehog inhibitor has ceased (i.e., on the same day as treatment with the hedgehog inhibitor has ceased), or, in certain embodiments, there is a period of time (e.g., one day, two days, one week, two weeks, one month, two months, six months, one year, etc.) between the end of the hedgehog inhibitor administration and the beginning of the second agent (e.g., the paclitaxel agent) administration.
In certain embodiments, the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor are administered concurrently for a first period of time, followed by administration of the hedgehog inhibitor for a second period of time (i.e., with administration of second agent (e.g., the paclitaxel agent) ceased). In certain embodiments, administration of the hedgehog inhibitor continues immediately after (i.e., Attorney Docket No. I2041-7000WO/3020PCT

on the same day as) concurrent administration of the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor ceases. Alternatively, in certain embodiments, treatment with the hedgehog inhibitor begins after a period of time (e.g., one day, two days, one week, two weeks, one month, two months, six months, one year, etc.) after concurrent administration of the second agent and the hedgehog inhibitor ceases. In each of these embodiments, the hedgehog treatment regimen during the second period of time can be the same as the treatment regimen when the hedgehog inhibitor is concurrently administered with the second agent (e.g., the paclitaxel agent) during the first period of time, or the hedgehog treatment regimen during the second period of time can be different than the treatment regimen when the hedgehog inhibitor is concurrently administered with the second agent (e.g., the paclitaxel agent) during the first period of time.
In other embodiments, the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor are administered concurrently for a first period of time, followed by administration of the second agent (e.g., the paclitaxel agent) for a second period of time (i.e., with administration of the hedgehog inhibitor ceased). In certain embodiments, administration of the second agent (e.g., the paclitaxel agent) continues immediately after (i.e., on the same day as) concurrent administration of the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor ceases. Alternatively, in certain embodiments, administration of the second agent (e.g., the paclitaxel agent) begins after a period of time (e.g., one day, two days, one week, two weeks, one month, two months, six months, one year, etc.) after concurrent administration of the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor ceases. In each of these embodiments, the second agent (e.g., the paclitaxel agent) treatment regimen during the second period of time can be the same as the treatment regimen when the second agent (e.g., the paclitaxel agent) is concurrently administered with the hedgehog inhibitor during the first period of time, or the second agent (e.g., the paclitaxel agent) treatment regimen during the second period of time can different than the treatment regimen when the second agent (e.g., the paclitaxel agent) is concurrently administered with the hedgehog inhibitor during the first period of time.

Attorney Docket No. I2041-7000WO/3020PCT
In certain embodiments, the second agent (e.g., the paclitaxel agent) is administered to the patient for a first period of time, followed by concurrent administration of the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor for a second period of time. In certain embodiments, concurrent administration of the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor begins immediately following (i.e., on the same day as) administration of the second agent (e.g., the paclitaxel agent) ceases. Alternatively, in certain embodiments, concurrent administration of the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor begins after a period of time (e.g., one day, two days, one week, two weeks, one month, two months, six months, one year, etc.) after administration of the second agent (e.g., the paclitaxel agent) ceases. In each of these embodiments, the second agent (e.g., the paclitaxel agent) treatment regimen during the second period of time can be the same as the treatment regimen during the first period of time, or the second agent (e.g., the paclitaxel agent) treatment regimen during the second period of time can be different than the treatment regimen during the first period of time. In some embodiments, administration of the hedgehog inhibitor continues after the concurrent administration has ceased (i.e., administration of second agent (e.g., the paclitaxel agent) for a first period of time, followed by concurrent administration of the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor for a second period of time, followed by administration of the hedgehog inhibitor for a third period of time). In other embodiments, administration of the second agent (e.g., the paclitaxel agent) continues after the concurrent administration has ceased (i.e., administration of second agent (e.g., the paclitaxel agent) for a first period of time, followed by concurrent administration of the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor for a second period of time, followed by administration of the second agent (e.g., the paclitaxel agent) for a third period of time).
In certain embodiments, the hedgehog inhibitor is administered to the patient for a first period of time, followed by concurrent administration of the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor for a second period of time. In certain embodiments, concurrent administration of the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor begins immediately following (i.e., on the same day as) administration of the hedgehog inhibitor ceases. Alternatively, in certain embodiments, Attorney Docket No. I2041-7000WO/3020PCT
concurrent administration of the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor begins after a period of time (e.g., one day, two days, one week, two weeks, one month, two months, six months, one year, etc.) after administration of the hedgehog inhibitor ceases. In each of these embodiments, the hedgehog inhibitor treatment regimen during the second period of time can be the same as the treatment regimen during the first period of time, or the hedgehog inhibitor treatment regimen during the second period of time can be different than the treatment regimen during the first period of time. In some embodiments, administration of the hedgehog inhibitor continues after the concurrent administration has ceased (i.e., administration of hedgehog inhibitor for a first period of time, followed by concurrent administration of the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor for a second period of time, followed by administration of the hedgehog inhibitor for a third period of time). In other embodiments, administration of the second agent (e.g., the paclitaxel agent) continues after the concurrent administration has ceased (i.e., administration of hedgehog inhibitor for a first period of time, followed by concurrent administration of the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor for a second period of time, followed by administration of the second agent (e.g., the paclitaxel agent) for a third period of time).
Also provided are methods of treating a cancer in a patient by administering to the patient a therapeutically effective amount of second agent (e.g., the paclitaxel agent), a therapeutically effective amount of a hedgehog inhibitor, and a therapeutically effective amount of an additional therapeutic agent. The second agent (e.g., the paclitaxel agent), hedgehog inhibitor and additional therapeutic agent can be concurrently administered, sequentially administered, or can be administered using a combination of concurrent and sequential administration.
In some embodiments, the second agent (e.g., the paclitaxel agent), hedgehog inhibitor and an additional therapeutic agent (e.g., a third agent) are administered concurrently. For example, in certain embodiments, the second agent (e.g., the paclitaxel agent), the hedgehog inhibitor and the third agent are administered on the same day to the patient, or, in certain embodiments, are administered simultaneously on the same day to the patient. Alternatively, in certain embodiments, the second agent (e.g., the paclitaxel agent), the hedgehog inhibitor and the third agent are administered on different days Attorney Docket No. I2041-7000WO/3020PCT

and/or on different schedules (e.g., one administered daily or every other day while the others are administered weekly), provided that the treatment regimen for all begin and end on the same day.
In certain embodiments, the second agent (e.g., the paclitaxel agent), the hedgehog inhibitor and the third agent are administered sequentially. For example, in certain embodiments, the additional therapeutic agent is administered for a first period of time, followed by concurrent administration of the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor for a second period of time. In certain embodiments, concurrent administration of the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor begins immediately following (i.e., on the same day as) administration of the third agent ceases. Alternatively, in certain embodiments, concurrent administration of the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor begins after a period of time (e.g., one day, two days, one week, two weeks, one month, two months, six months, one year, etc.) after administration of the third agent ceases. The current methods also contemplate administration of the hedgehog inhibitor for a first period of time followed by administration of the second agent (e.g., the paclitaxel agent) and the third agent for a second period of time, as well as administration of the second agent (e.g., the paclitaxel agent) for a first period of time followed by administration of the hedgehog inhibitor and the third agent for a second period of time. In any of these embodiments, any one of the second agent (e.g., the paclitaxel agent), hedgehog inhibitor, and/or third agent can be administered for a third period of time following the concurrent administration in the second period of time.
Additionally, the second agent (e.g., the paclitaxel agent), the hedgehog inhibitor or the third agent can be administered for a first period of time, followed by concurrent administration of the second agent (e.g., the paclitaxel agent), hedgehog inhibitor and the third agent for a second period of time. For example, the second agent (e.g., the paclitaxel agent) can be administered for a first period of time, followed by concurrent administration of the second agent (e.g., the paclitaxel agent), hedgehog inhibitor and the third agent for a second period of time. Following concurrent administration, any one of the second agent (e.g., the paclitaxel agent), hedgehog inhibitor, and/or the third agent can be administered for a third period of time.

Attorney Docket No. I2041-7000WO/3020PCT

In some instances, the second agent (e.g., the paclitaxel agent), hedgehog inhibitor and the third agent can be administered for a first period of time, followed by administration of one or two of the second agent (e.g., the paclitaxel agent), hedgehog inhibitor and the third agent for a second period of time (i.e., administration of one or two of the second agent (e.g., the paclitaxel agent), hedgehog inhibitor and additional therapeutic agent can cease while administration of the other agent(s) continues). For example, the second agent (e.g., the paclitaxel agent), hedgehog inhibitor and the third agent can be administered for a first period of time, followed by administration of the hedgehog inhibitor for a second period of time. Following the second period of time, any one of the second agent (e.g., the paclitaxel agent), hedgehog inhibitor, and/or the third agent can be administered for a third period of time.
Also provided are methods of extending relapse free survival in a pancreatic cancer patient who is undergoing or has undergone cancer therapy by administering a therapeutically effective amount of a second agent (e.g., the paclitaxel agent) and a therapeutically effective amount of a hedgehog inhibitor, and optionally, a therapeutically effective amount of a third agent.
As described herein, the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor can be concurrently administered, sequentially administered or can be administered using a combination of concurrent and sequential administration.
In some embodiments, the second agent (e.g., the paclitaxel agent) and/or the hedgehog inhibitor are administered concurrently with the cancer therapy. In instances of concurrent administration, the second agent (e.g., the paclitaxel agent) and/or the hedgehog inhibitor can continue to be administered after the cancer therapy has ceased. In other embodiments, the second agent (e.g., the paclitaxel agent) and/or the hedgehog inhibitor are administered after cancer therapy has ceased (i.e., with no period of overlap with the cancer therapy). The second agent (e.g., the paclitaxel agent) and/or the hedgehog inhibitor can be administered immediately after cancer therapy has ceased, or there can be a gap in time (e.g., up to about a day, a week, a month, six months, or a year) between the end of cancer therapy and the administration of the second agent (e.g., the paclitaxel agent) and/or the hedgehog inhibitor. Treatment with the second agent (e.g., the paclitaxel agent) and/or the hedgehog inhibitor can continue for as long as relapse-free Attorney Docket No. I2041-7000WO/3020PCT

survival is maintained (e.g., up to about a day, a week, a month, six months, a year, two years, three years, four years, five years, or longer).
Also provided are methods of extending relapse free survival in a pancreatic cancer patient who had previously undergone cancer therapy by administering a therapeutically effective amount of a second agent (e.g., the paclitaxel agent) and a therapeutically effective amount of a hedgehog inhibitor to the patient after the cancer therapy has ceased. As described herein, the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor can be concurrently administered, sequentially administered, or can be administered using a combination of concurrent and sequential administration.
The second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor can be administered immediately after cancer therapy has ceased, or there can be a gap in time (e.g., up to about a day, a week, a month, six months, or a year) between the end of cancer therapy and the administration of the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor.
Also provided are methods for treating (e.g., reducing the amount or occurrence of) or preventing pancreatic tumor and/or metastasis in a patient by administering a therapeutically effective amount of a second agent (e.g., the paclitaxel agent) and a therapeutically effective amount of a hedgehog inhibitor. As described herein, the second agent (e.g., the paclitaxel agent) and the hedgehog inhibitor can be concurrently administered, sequentially administered, or can be administered using a combination of concurrent and sequential administration.
In other embodiments, the hedgehog inhibitor and the combination therapies described herein can be used further in combination with one or more of: other chemotherapeutic agents, radiation, or surgical procedures.

EXEMPLIFICATION

The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

Attorney Docket No. I2041-7000WO/3020PCT
Example 1: Activity in the Hedgehog Pathway Hedgehog pathway specific cancer cell killing effects can be ascertained using the following assay. C3H1OT1/2 cells differentiate into osteoblasts when contacted with the sonic hedgehog peptide (Shh-N). Upon differentiation, these osteoblasts produce high levels of alkaline phosphatase (AP) which can be measured in an enzymatic assay (Nakamura et at., 1997 BBRC 237: 465). Compounds that block the differentiation of C3H1OT1/2 into osteoblasts (a Shh dependent event) can therefore be identified by a reduction in AP production (van der Horst et at., 2003 Bone 33: 899). The assay details are described below.
Cell Culture Mouse embryonic mesoderm fibroblasts C3H1OT1/2 cells (obtained from ATCC) were cultured in Basal MEM Media (Gibco/Invitrogen) supplemented with 10% heat inactivated FBS (Hyclone), 50 units/ml penicillin and 50ug/ml streptomycin (Gibco/Invitrogen) at 37 C with 5% CO2 in air atmosphere.

Alkaline Phosphatase Assay C3H1OT1/2 cells were plated in 96 wells with a density of 8x103 cells/well.
Cells were grown to confluence (72 hrs.). After sonic hedgehog (250ng/ml) and/or compound treatment, the cells were lysed in 110 tL of lysis buffer (50 mM Tris pH 7.4, 0.1%
TritonX100), plates were sonicated and lysates spun through 0.2 gm PVDF plates (Corning). 40 gL of lysates was assayed for AP activity in alkaline buffer solution (Sigma) containing lmg/ml p-Nitrophenyl Phosphate. After incubating for 30 min at 37 C, the plates were read on an Envision plate reader at 405 nm. Total protein was quantified with a BCA protein assay kit from Pierce according to manufacturer's instructions. AP activity was normalized against total protein. Using the above-described assay, IPI-926 was shown to be an antagonist of the hedgehog pathway with an IC50 less than 20 nM.

Attorney Docket No. I2041-7000WO/3020PCT
H
O

H O
H

~ N%,.
H H
IPI-926 (also referred to herein as Compound 42) Example 2: Pancreatic Cancer Monotherapy Model The activity of IPI-926 was tested in a human pancreatic model. BxPC-3 cells were implanted subcutaneously into the flanks of the right legs of mice. On day 42 post-tumor implant, the mice were randomized into two groups to receive either Vehicle (30%
HPBCD) or IPI-926. IPI-926 was dosed at 40mg/kg/day. After receiving 25 daily doses, IPI-926 statistically reduced tumor volume growth by about 40% when compared to the vehicle control (p=0.0309) (see Figure 1).
At the end of the study, the tumors were harvested 4 hours post the last dose to evaluate an on target response by q-RT-PCR analysis of the Hedgehog pathway genes.
As shown in Figure 2A, Human Gli-1 was not modulated in either the vehicle or the treated group. However, murine Gli-1 mRNA levels were significantly down-regulated in the IPI-926 treated group when compared to the vehicle treated group (see Figure 2B).
Example 3: Pancreatic Cancer Concurrent Combination Therapy Model Animals bearing BxPC-3 pancreatic cancer xenografts were treated with the chemotherapeutic drug gemcitabine in concurrent combination with IPI-926.
Gemcitabine was administered at a dose of 100 mg/kg twice weekly by intraperitoneal injection while IPI-926 was administered at a dose of 40 mg/kg daily by oral gavage. As shown in Figure 3, under these conditions the tumors showed a 33% response to gemcitabine alone, a 55% response to IPI-926 alone, and a 67% response to the combination of IPI-926 and gemcitabine.
In another model, Animals bearing MiaPaCa pancreatic cancer xenografts were treated with the chemotherapeutic drug gemcitabine in concurrent combination with IPI-Attorney Docket No. I2041-7000WO/3020PCT
926. Gemcitabine was administered at a dose of 100 mg/kg once weekly by intraperitoneal injection while IPI-926 was administered at a dose of 40 mg/kg daily by oral gavage. As shown in Figure 4, under these conditions the tumors showed a 52%
response to gemcitabine alone, a 50% response to IPI-926 alone, and a 70%
response to the combination of IPI-926 and gemcitabine.

Example 4: Lung Cancer Concurrent Combination Therapy Model To test the activity of IPI-926 in a human small cell lung cancer tumor model, LX22 cells were implanted subcutaneously into the flank of the right leg of male Ncr nude mice. LX22 is primary xenograft model of SCLC derived from chemo-naive patients, which has been maintained by mouse to mouse passaging. This tumor responds to etoposide/carboplatin chemotherapy in way that closely resembles a clinical setting.
LX22 regresses during chemotherapy treatment, goes through a period of remission, and then begins to recur.
Animals bearing LX-22 small cell lung cancer xenografts were treated with the chemotherapeutic drugs etoposide and carboplatin in concurrent combination with IPI-926. In this experiment, etoposide was administered at a dose of 12 mg/kg by intravenous route on three consecutive days followed by a single administration two weeks after the initial dose. Carboplatin was administered at a dose of 60 mg/kg weekly for three weeks by intravenous injection. IPI-926 was administered at a dose of 40 mg/kg daily by oral gavage either at the same time as etoposide/carboplatin or immediately following etoposide/carboplatin treatment. As shown in Figure 5, under these conditions the tumors showed an overall 40% response to all treatments when compared to those animals receiving etoposide/carboplatin alone.
Example 5: Chemo-Resistant Recurrence Model In the LX22 model, IPI-926 single agent activity and its ability to modulate the chemo-resistant recurrence were tested. On day 32 post tumor implant, mice were randomized into three dosing groups to receive vehicle (30% HBPCD), IPI-926, or the chemotherapy combination of etoposide and carboplatin (E/P). IPI-926 was administered at a dose of 40mg/kg/day, etoposide was administered i.v. at 12mg/kg on days 34, 35, 36, Attorney Docket No. I2041-7000WO/3020PCT

and 48, and carboplatin was administered i.v. at 60mg/kg on days 34, 41, and 48, post tumor implant. After 16 consecutive doses there was no measurable difference between the group treated with IPI-926 and the vehicle treated group (see Figure 6).
On day 50, the E/P treated mice were further randomized to receive either vehicle (30%
HPBCD) or IPI-926 follow-up treatment. IPI-926 was administered at 40mg/kg/day. As shown in Figure 6, after 35 consecutive doses of IPI-926, there was a substantial delay in tumor recurrence in the treated group (82%), compared to the vehicle group (p=0.0101).
Example 6: Colon Cancer Combination Therapy Model Animals bearing Colo205 colon cancer xenografts were treated with the chemotherapeutic drug 5-fluorouracil in combination with IPI-926. 5-fluorouracil was administered at a dose of either 50 mg/kg or 100 mg/kg as a once weekly intraperitoneal injection for two weeks. IPI-926 was administered at 40 mg/kg as a daily oral gavage for 21 days. Under these conditions the tumors showed a 68% to 5-fluorouracil alone or in combination with IPI-926.

Example 7: Colon Cancer Chemo-resistant Recurrence Models Animals are implanted with SW620 colon cancer cells. Tumor bearing animals are administered paclitaxel for such a time that their tumors respond to chemotherapy treatment. These animals are randomized into two groups, one receiving vehicle and one receiving IPI-926. Tumor response to the different therapies is determined as discussed herein.
Alternatively, Colo205 colon cancer cells are implanted into experimental animals.
Tumor bearing animals will be administered 5-fluorouracil for such a time that their tumors respond to chemotherapy treatment. These animals are then randomized into two groups, one receiving vehicle and one receiving IPI-926. Tumor response to the different therapies is determined as discussed herein.

Example 8: Ovarian Cancer Models Mice bearing IGROV-1 ovarian cancer xenografts were treated with daily doses of IPI-926 at 40 mg/kg for 21 consecutive days. No substantive effect on tumor growth Attorney Docket No. I2041-7000WO/3020PCT
was observed at this dosage with this particular ovarian cancer cell xenograft. In a further study, mice bearing IGROV-1 ovarian cancer xenografts were treated with 5 consecutive daily doses of paclitaxel at 15 mg/kg followed by IPI-926 at 40 mg/kg for 21 consecutive days. Again, no substantive effect on tumor growth was observed at these dosages with this particular ovarian cancer cell xenograft.
To determine if other ovarian cancer cell types respond to treatment with IPI-926, SKOV-3, OVCAR-4 or OVCAR-5 ovarian cancer cells are implanted into experimental animals. To determine the effect of monotherapy and concurrent combination therapy, tumor bearing animals are administered paclitaxel or carboplatin alone, IPI-926 alone, or IPI-926 and paclitaxel or carboplatin in combination. To determine the effect of sequential combination therapy, tumor bearing animals are administered paclitaxel or carboplatin for such a time that their tumors respond to chemotherapy treatment. These animals are then randomized into two groups, one receiving vehicle and one receiving IPI-926. Tumor response to the different therapies is determined as discussed herein.

Example 9: Bladder Cancer Models To determine the effect of monotherapy and concurrent combination therapy, animals are implanted with UMUC-3 bladder cancer cells. Tumor bearing animals are then administered gemcitabine/cisplatin alone, IPI-926 alone, or the three agents in combination. Tumor response to the different therapies is determined as discussed herein.
To determine the effect of sequential combination therapy, animals are implanted with UMUC-3 bladder cancer cells, and tumor bearing animals are then administered a combination of gemcitabine and cisplatin for such a time that their tumors respond to chemotherapy treatment. These animals are then randomized into two groups, one receiving vehicle and one receiving IPI-926. Tumor response to the different therapies is determined as discussed herein.
Alternatively, SW780 bladder cancer cells are implanted into experimental animals. To determine the effect of monotherapy and concurrent combination therapy, tumor bearing animals are administered gemcitabine/cisplatin alone, IPI-926 alone, or the three agents in combination. To determine the effect of sequential combination therapy, tumor bearing animals are administered a combination of gemcitabine and cisplatin for Attorney Docket No. I2041-7000WO/3020PCT

such a time that their tumors respond to chemotherapy treatment. These animals are then randomized into two groups, one receiving vehicle and one receiving IPI-926.
Tumor response to the different therapies is determined as discussed herein.

Example 10: Non-Small Cell Cancer Models To determine the effect of monotherapy and concurrent combination therapy, animals are implanted with NCI-H1650 non-small cell lung cancer cells. Tumor bearing animals are then administered gefitinib alone, IPI-926 alone, or the two agents in combination. Tumor response to the different therapies is determined as discussed herein.
To determine the effect of sequential combination therapy, animals are implanted with NCI-H1650 non-small cell lung cancer cells, and tumor bearing animals are then administered gefitinib for such a time that their tumors respond to gefitinib treatment.
These animals are then randomized into two groups, one receiving vehicle and one receiving IPI-926. Tumor response to the different therapies is determined as discussed herein.

Example 11: Hedgehog Lit and Induction Studies Follow up studies in the LX22 model were designed to examine Hh pathway modulation by IPI-926 post etoposide and carboplatin (E/P) treatment. As described in Example 4 above, animals bearing LX22 small cell lung cancer xenografts were treated with etoposide and carboplatin. A single dose of IPI-926 (40mg/kg) was administered 24 hours prior to each time point collected. Naive tumors were collected from five animals for baseline levels prior to chemotherapy treatment. Tumors from four animals were collected on days 1, 4, 7, and 10, and tumors from three animals were collected on day 14. Samples were collected for q-RT-PCR analysis and histology/
immunohistochemistry evaluation. RNA was extracted and q-RT-PCR analysis was completed by first converting to cDNA then using the one-step master mix (FAST
method on 7900).
The results of this study showed that Hh ligand, specifically Indian Hh (IHH), was up-regulated in the human tumor cells and the surrounding murine stroma cells following chemotherapy, as measured both by RT-PCR and immunohistochemistry (see Attorney Docket No. I2041-7000WO/3020PCT

Figures 7A and 7B). In addition, stromal-derived murine Gli-1 and tumor-derived human Gli-1 were induced in response to tumor-derived ligand. Murine Gli-1 expression remained elevated compared to the expression level in naive tumors for at least 14 days post the cessation of E/P treatment and was inhibited by administration of IPI-926 (see Figure 8A), while human Gli-1 expression was not affected by administration of (see Figure 8B). Without wishing to be bound to any theory, it is believed that up-regulation of tumor-derived Hh ligand post-chemotherapy can confer upon the surviving cell population a dependency upon the Hh pathway that is important for tumor recurrence. These findings are consistent with the observed paracrine cross-talk between the tumor and the surrounding stroma previously shown to be important for Hh signaling (Yauch et al., 2008, Nature 455:406-410).

Example 12: Hedgehog Lit and Induction Studies Induction of Hh ligand post chemotherapy was also studied in other cancer tumor models. In vivo, mice bearing UMUC-3 bladder cancer xenografts were treated with 100mg/kg gemcitabine once-weekly for 4 weeks. Tumors showed increased IHH
expression similar to that observed in the LX22 model 24 hours post administration of the final dose (see Figures 9A and 9B). In vitro studies showed that in UMUC-3 cells exposed to either doxorubicin or gemcitabine for 12-24 hours, all 3 Hh ligands (Sonic, Indian and Desert) were up-regulated (see doxorubicin data in Figure 10).
Additional in vitro studies showed that IHH expression was increased in A2780 ovarian cancer cells after treatment with carboplatin, while Sonic Hh (SHH) expression was not affected (see Figure 11), and expression of both IHH and SHH were increased in IGROV-1 cells treated with docetaxel, with SHH being up-regulated to a greater degree (See Figure 12).
Further in vitro studies showed that in small cell lung cancer H82 cells, SHH
is up-regulated by docetaxel but not carboplatin, while IHH is not up-regulated by either agent (see Figure 13).
To determine if cellular stresses other than chemotherapy up-regulate Hh ligand expression, UMUC-3 cells were exposed in vitro to various stressors including hypoxia.
Compared to normoxic controls, SHH ligand expression was increased at both the RNA
and protein level (see Figure 14).

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In summary, multiple tumor types exhibit up-regulation of Hh ligands post chemotherapy. The type of Hh ligand that is up-regulated (i.e., Sonic, Indian and/or Desert) and the degree of up-regulation vary depending upon the tumor type and the chemotherapeutic agent. Without wishing to be bound to any theory, these results suggest that stress (including chemotherapy) induces Hedgehog ligand production in tumor cells as a protective or survival mechanism. The results further suggest that a surviving sub-population can be dependent upon the Hh pathway and thus can be susceptible to Hh pathway inhibition. Taken together, these results indicate that Hedgehog inhibition can increase relapse free survival in clinical indications (such as small cell lung cancer, non-small cell lung cancer, bladder cancer, colon cancer, or ovarian cancer) that are initially chemo-responsive but eventually relapse.

Example 13: Immunohistochemistry (IHC) Studies to Detect Sonic Heft ehot (SHH) To determine if the Sonic Hedgehog (SHH) ligand can be detected on human neuroendocrine samples by immunohistochemistry analysis, a neuroendocrine tissue microarray (TMA) HTMA17 (obtained from Dana-Farber Cancer Institute (DFCI)) was tested for SHH immunostaining. Sections for staining were first de-paraffinized and hydrated in a series of graded alcohols, and then processed through the Heat-Induced Epitope Retrieval (HIER) in citrate buffer (pH=6) for 20 minutes at 120 C and under pressure. Next, samples were allowed to reach room temperature and were prepared for SHH immunohistochemistry (IHC). Rabbit anti-human SHH antibody (EP1190Y) (Abeam catalog # ab5328 1) was used as the primary antibody at 1:2000 dilution. Rabbit-on-Rodent Polymer (Biocare catalog # RMR622L) was used as the secondary polymer system. DAB Liquid Substrate Buffer+ Chromogen System (DAKO catalog # K3468) was used for developing and detection of the staining.
The data were scored based on the percentage of staining that was calculated by gross observation for each TMA spot. The data were organized by grouping the samples by the organ of origin (benign versus tumor tissue). As shown in Figures 15A-15G, the expression of SHH ligand was detected in multiple primary neuroendocrine cancers of different organs of origin, including appendix, duodenum, ileum, pancreas, rectum, small intestine and lung.

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Example 14: Efficacy of IPI-926 in Neuroendocrine Cancer Model To determine the efficacy of IPI-926 in a neuroendocrine cancer (NET) model, Bon-1 cells were implanted into male Ncr nude mice (5x106 cells/mouse). Bon-1 (obtained from Purdue University) is a human pancreatic neuroendocrine cancer cell line and was maintained in RPMI1460 medium supplemented with 10% FBS and 1%
penicillin/streptomycin. Treatment with IPI-926 was initiated once the tumor volume reached approximately 200 mm3. Tumor bearing mice were treated with a single dose of IPI-926 in 5% HPBCD at 40mg/kg by oral gavage (8mIkg).
RT-PCR analysis Tumors were collected at 24, 48 and 72 hours after the single dose treatment of IPI-926 and were snap frozen for qRT-PCR analysis. Non-treated tumors were collected and served as the control for comparison to the IPI-926-treated tumors. Total RNA was extracted from all tumors using a standard TRIZOL (Invitrogen) method and cleaned up using RNEASY Mini Kit (Qiagen). Next, the RNA was converted to cDNA and 50 ng cDNA was used in each reaction/sample for qRT-PCR analysis of the expression of murine Gli l, human Glil, and human Hedgehog ligands (Sonic Hh (SHH) and Indian Hh (IHH)). All samples were tested in duplicate.
As shown in Figures 16A-16B, the expression of stromal-derived murine Gli-1 mRNA was down-regulated up to 72 hours after treatment with IPI-926, while the expression of tumor-derived human Gli-1 was not significantly modulated in response to IPI-926. As shown in Figures 16C-16D, the expression of both human Sonic Hedgehog (hSHH) and Indian Hedgehog (hIHH) was not significantly affected in tumors treated with IPI-926. These results suggest that Hh ligand secreted by the tumor cell is activating the surrounding stromal cell micro environment in a paracrine signaling cascade occurring between the tumor cell and the stroma. Activation of Hh pathway plays an important role in maintaining the tumor microenvironment in pancreatic NETs.

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Sonic Hedgehog (SHH) immunostaining Tumors were excised and fixed for 24 hours in 10% Neutral Buffered Formalin.
The next day samples were switched to storage in 70% ethanol. Tumors were processed and embedded according to standard protocols. The embedded tumor tissues were sectioned into 5um sections for SHH immunohistochemistry (IHC). Sections for staining were first de-paraffinized and hydrated in a series of graded alcohols, and then put through the Heat-Induced Epitope Retrieval (HIER) in citrate buffer (pH=6) for minutes at 120 C and under pressure. Next, samples were allowed to reach room temperature and were prepared for SHH immunohistochemistry (IHC). Rabbit anti-human SHH antibody (EP1190Y) (Abeam catalog # ab53281) was used as the primary antibody at 1:2000 dilution. Rabbit-on-Rodent Polymer (Biocare catalog #
RMR622L) was used as the secondary polymer system. DAB Liquid Substrate Buffer+
Chromogen System (DAKO catalog # K3468) was used for developing and detection of the staining.
As shown in Figure 17, expression of human SHH was detected in the human tumor cells and the surrounding murine stroma cells.

Example 15: Efficacy of IPI-926, Alone or in Combination with Sunitinib in a Neuroendocrine Cancer Model To determine the efficacy of IPI-926, alone or in combination with sunitinib, in a neuroendocrine cancer (NET) model, Bon-1 cells were implanted into male Ncr nude mice (5x106 cells/mouse). Before implantation Bon-1 cells were cultured in medium supplemented with 10% FBS and 1 % penicillin/streptomycin. Treatment was initiated once tumor volume reached approximately 200 mm3.
On day 13 post tumor cell implant, mice were randomized into three dosing groups to receive vehicle control (5% HPBCD), IPI-926 in 5% HPBCD, sunitinib (in water), or sunitinib in combination with IPI-926. From day 13 through day 33, was administered at a dose of 40mg/kg every other day by oral gavage (8m1/kg).
Sunitinib was administered at a dose of 40mg/kg every day by oral gavage (8m1/kg).
Thus, in this experiment, mice bearing Bon-1 pancreatic neuroendocrine cancer cells received a total of ten doses of IPI-926 and/or twenty doses of sunitinib.
Bodyweight and tumor measurements were taken three times a week. Tumor measurements were made in Attorney Docket No. I2041-7000WO/3020PCT

two dimensions (width x length) using calipers and the tumor volume equals to length x width2/2. Body weight loss greater than 20% from the initial day of treatment or tumor volumes greater than 3000 mm3 resulted in euthanasia. Samples for analysis were collected 24 hours post the final dose. Tumors collected were snap frozen for analytical evaluation and qRT-PCR analysis. For histopathology tumors were fixed in 10%
formalin for 24 hours prior to transferring the samples into 70% ethanol.
As shown in Figure 18, on the final day of tumor measurement, IPI-926 alone group showed 35% tumor growth inhibition (TGI) when compared to the control group, while sunitinib (shown as "Sutent") alone group showed 60% TGI when compared to the control group. Mice treated with the combination of sunitinib and IPI-926 showed a 72%
TGI. All %TGIs compared to the vehicle control were statistically significant (p =
0.0096 for IPI-926 alone group; p = 0.0028 for sunitinib alone group; p =
0.0002 for the combination group of IPI-926 and sunitinib). The combination group of IPI-926 and sunitinib did not show a statistically significant %TGI when compared to IPI-926 (p =
0.1631) or sunitinib alone (p = 0.3593) group. These results indicate a statistically significant increase in efficacy in reducing tumor volume growth when IPI-926 and sunitinib were administered in combination, compared to the control group.

Example 16A: Efficacy of IPI-926 in Neoadjuvant and Adjuvant Therapy in a Rat Syngeneic Chondrosarcoma Model Chondrosarcoma a therapeutic challenge:
Chondrosarcomas constitute a heterogeneous group of neoplasms that have in common the production of cartilage-like matrix by the tumor cells. Clinical management of these second most common types of skeletal malignancies has remained largely unchanged over the last 3 decades. It is generally believed that, because of their extracellular matrix, low percentage of dividing cells, and poor vascularity, chondrogenic tumors are relatively chemo- and radiotherapy resistant. Thus, surgery still prevails as the primary treatment modality of this tumor. Improving chondrosarcoma clinical management is a challenging problem and developing innovative therapeutic approaches is an important goal in the treatment of patients with chondrosarcoma.

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Inhibition of Hh pathway for sarcoma treatment.
The Hedgehog (Hh) gene is important in the signaling pathways of proliferation and differentiation during embryonic development. There is evidence that uncontrolled activation of this pathway results in specific types of cancer and that inhibition of Hh signaling is able to suppress tumour growth. Preclinical studies using inhibitors of hedgehog signaling in chondrosarcoma and osteosarcoma cell lines provided evidence for the potency of Hh-inhibitors as future agents for musculoskeletal sarcoma treatment.
Inhibiting Hh pathway is believed to have antitumor activity, and can be used to limit or prevent sarcoma invasion (local and metastatic). Previous studies established that inhibiting mTOR pathway had a strong antitumor activity towards chondrosarcoma (not shown). Based on these data, a combination of Hh inhibitor and an inhibitor of mTOR
constitutes an attractive combination for an additive antitumor effect.
Described below are in vivo experiments (in clinical relevant settings) aim to evaluate whether Hh-specific inhibitor IPI-926 (also referred to herein as Compound 42) used alone or in combination with a specific inhibitor of mTOR could inhibit chondrosarcoma progression. To assess this, a rat syngeneic chondrosarcoma model can be characterized in two main settings:
^ Setting 1. Evaluate IPI-926 as curative treatment for chondrosarcoma. The efficacy of IPI-926 on tumor growth can be evaluated in a syngeneic chondrosarcoma model. In this setting, the treatment will start 10 days after tumor implantation (cf Experimental protocol). The therapeutic efficacy of IPI-926 (used alone or in combination with mTOR inhibitor) can be compared to the one of conventional chemotherapy (adryamicin) and to an inhibitor of mTOR.
^ Setting 2. Evaluate IPI-926 therapeutic efficiency on "relapsed"
chondrosarcoma.
In a second set of experiments, the effects of IPI-926 and IPI-926-based drugs combination on tumor growth that occurred after intralesional curettage can be evaluated.
In these series, an intralesionnal curretage will be performed on the rats with progressive tumors (i.e. with tumor volume of 1000 mm). In this setting the treatment will start one day after curettage. As in the first setting, therapeutic efficacy of IPI-926 will be Attorney Docket No. I2041-7000WO/3020PCT

compared to the one of conventional chemotherapy (adriamycin) and to an inhibitor of mTOR.
For each setting, the comparison of therapeutic efficiency of IPI-926 and conventional cytotoxic agent therapy will use tumor volume evolution (MRI and tumor measure using a caliper), tumor necrosis percentage and mitotic index, tumor MVD
quantification and overall survival analysis between the IPI-926 treated and control groups.
IPI-926 is expected to have a beneficial effect in vivo reducing chondrosarcoma tumor progression. A slower tumor progression in the IPI-926-treated groups in comparison to the other groups is expected. In a second step, the same protocol could be conducted on an osteosarcoma model to evaluate the antitumor and antimetastatic effect of IPI-926 used alone or in combination with an mTOR inhibitor.
The data generated herein provide a strong experimental rationale for designing further studies to evaluate the benefit of an addition of IPI-926 for adjuvant treatment of patients with chondrosarcoma or relapsed/refractory osteosarcoma.

EXPERIMENTAL DESIGN
Animal models Two orthotopic sarcoma models are briefly presented below.
Chondrosarcoma model Transplantable orthotopic Schwarm chondrosarcoma model can be used. Tumors are grafted on 21- to 28-day-old Sprague-Dawley rats according to a method previously described. Shorty, using a lateral approach, a 6-8 mm3 tumor fragment is placed contiguous to tibial diaphysis after periostal abrasion; then the cutaneous and muscular wounds are sutured. Tumors can be detected at the graft site 8-11 days after transplantation by palpation and MRI imaging (A) of Figure 20.
Histological analysis classified this model as a grade II chondrosarcoma characterized by synthesis of cartilage, a. lobular pattern (B), and the presence of mitotic cells (C), of Figure 20.
Osteosarcoma model: an intramedular and metastatic osteosarcoma model in rat has been developed. Briefly, small tumor fragments (100 mm3) taken from a hyperproliferative osteogenic tumor area., are grafted on 3-weeks old immunocofnpetent Attorney Docket No. I2041-7000WO/3020PCT

rats. Using a lateral approach, a tumor fragment is placed within the femoral diaphysis of the animals; then the cutaneous and muscular wounds are sutured. 'Tumors can be detected 9 days post transplantation in 95% of the animals, at the graft site by palpation and metabolic and morphologic imaging with '3FDG PET Scan. This model mimics its human counterpart in term of aggresiveness, vascularisation of the primary tumour, and haematogenous spread of the primary tumour to the lungs; 21-24. days post transplantation, lung metastasis are detected in 80`;-0 of the animals. In parallel to conventional histological analysis, multimodal imaging techniques ('8F-FDG
PET, 18FNa, MRI 18FMISO scintigraphies) have been used sucessfully to evaluate non-invasively tumor volume, metabolism, hypoxia evolution and tumor bone synthesis.
^ Treatment schedules.
^ Two treatment schedules can be conducted:

I IPI-926 as curative treatment for sarcoma. Ten days after tumor transplantation animals can be divided into the following treatment groups (8 animals/group; 5 treatment groups) (i) IPI-926 (ii) Adryamicine (iii) mTOR inhibitor, (iv) IPI-926 + mTOR
inhibitor (v) control (saline). Each rat in the treated or control groups can be given the corresponding treatment (at the same frequency and using the same administration route) started at day 10 after implantation until euthanasia (day 40). Animals can be sacrificed when tumor become too bulky and when life. of the animal would be threatened.

The dose and frequency of administration of IPI-926 and IPI-926-based drug combination can be chosen based on IPI-926 rat PK/PD studies.
II) IPI-926 as adiuvant treatment for sarcoma. To assess the efficiency of IPI-926 to prevent tumor relapse, in a second setting, treatment can start 1 day after intralesional tumor curretage. In this setting, intralesional curretage can be performed on animals with progressive tumors (i.e. when tumors will reach a volume of 1000 mm3). One day after surgery, the animals will be divided into the following treatment groups (8 animals/group;
5 treatment groups): (i) IPI-926 (ii) Adryamicine (iii) mTOR inhibitor, (iv) IPI-926+mTOR inhibitor (v) control (saline). Rats in IPI-926 treated-groups will receive IPI-926-based combination (IPI-926 dose and frequency currently under determination), started at day 1 after intralesional curetage until euthanasia (day 40).
Animals from the Attorney Docket No. I2041-7000WO/3020PCT
adrrarnicin, fnTOR inhibitor and the control groups will be given the corresponding solution at the same frequency. Animals will be sacrificed when tumor become too bulky and when life of the animal would be threatened.

Assessment ofIPI-926 therapeutic efficacy.
Tumors can be measured twice a week with a calliper and tumor volume can he calculated according to the Carlsonn's formula. Chondrosarcoma evolution throughout treatment will also be followed using MRI. Imaging sessions will be performed on animals at the beginning of treatment (TO), then even, ten days till euthanasia. At the end of the experiments, tumor, muscle, bone, lungs from all the animals will be collected.
Samples of the collected tissues will be snap-frozen and stored at -80 for immunohistological and molecular analyses.
The imaging observations can be correlated to immunohistochemistry analyses performed on tumor and tissue sections of animals from treated and control groups. For this purpose, anti-MMPs, CD3 1, Glut-1, Ki67 antibodies will be used. Tumor necrosis induced by IPI-926 will be assessed by microscopic examination of H&E-stained tumor specimens collected at the time of euthanasia. For each tumor, necrosis and mitotic index will be estimated on whole transverse sections from the '/4 distal, middle and'/4 proximal of the tumor and expressed as percentage of whole tumor volume according to the system.
Analysis of calcification/bone differentiation markers as well as markers of invasiveness will be performed by RT-PCRq using appropriate sets of primers i.e (Runx2, type I, II
collagene, sox9, Indian Hh integrins).
Given the number of animals involved in the study, the experiments can be performed in two steps. In the first step, the efficiency of IPI-926 as curative treatment in chondrosarcoma will be evaluated. Then in a second step, the effect of IPI-926 on relapsed chondrosarcoma will be assessed. The two settings of administration represent two clinically different situations and are complementary.

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Example 16B: IPI-926 Affects the Growth and Survival of Osteosarcomas and Chondrosarcomas In Vivo This Example provides experimental evidence of the effects of IPI-926 on the growth and survival of osteosarcomas and chondrosarcomas in vivo.
Osteosarcoma Xenograft Models The mRNA expression of Hh ligand and Hh receptor in tumor cells and stromal cells from osteosarcoma xenograft models (Xenograft models A-C) was quantified using human and mouse specific primers. Elevated expression of IHH mRNA and Gli l mRNA
was detected in tumor cells compared to tumor-stromal associated cells (data not shown).
The expression of PTCH1 mRNA was elevated in both tumor cells and tumor-associated stromal cells (data not shown).
IPI-926 decreased tumor growth and possibly vascularization is osteosarcoma xenograft models relative to control animals. For example, for Xenograft A, the mean tumor weight was decreased to 1.83 grams in IPI-926-treated animals, compared to 2.49 grams in controls, with a P-value of 0.23. For Xenograft C, the mean tumor volume was decreased to 2.95 cm3 in IPI-926-treated animals, compared to 5.19 cm3 in controls, with a P-value of 0.04; the mean tumor weight was decreased to 2.05 grams in IPI-926-treated animals, compared to 3.34 grams in controls, with a P-value of 0.05. No difference in volume or weight was observed for treated Xenograft B.
Figures 21A-21D show the effects of IPI-926 in decreasing Hh signaling in tumor and stromal cells of osteosarcoma xenograft models. Figures 21A-21B show a decrease in PTCH1 and Gli l mRNA expression in tumor cells from Xenograft A and B
aninals treated with IPI-926 compared to controls. Similar decreases in PTCH1 and Glil mRNA
expression is detected in stromal cells treated with IPI-926 compared to controls (Figures 21C-21D). No change in tumor cell Hh signaling was detected in Xenograft B.
Figures 22A-22D show the effects of IPI-926 in proliferation and apoptosis in osteosarcoma xenograft models. Figures 22A and 22C show a decrease in proliferation of tumor cells detected by the percentage of cells showing Ki-67 staining in two different animals in response to IPI-926 compared to controls. Figures 22B and 22D show an increase in apoptosis detected by Tunel Staving in response to IPI-926 compared to controls.

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The results shown herein support both autocrine and a paracrine mechanisms of Hh signaling in osteosarcomas, both of which are mediated, at least in part, by Smoothened (Smo) receptor. Smo activity can be inhibited by IPI-926.

Chondrosarcoma Xenograft Models Chondrosarcomas express high levels of Hh pathway genes such as IHH, PTCH1 and Glil (Tiet et al. (2006) American J. Pathology 168(1):321-330). Hh ligand increases the proliferation of primary chondrosarcoma cells in vitro.
To evaluate the relevance of these findings to in vivo models, primary chondrosarcoma xenografts as described herein were treated with IPI-926 at 40 mg/kg;
PO QD x 5. This study evaluated the effects of IPI-926 on tumor volume, pharmacodynamics of Hh pathway inhibition in tumor and stroma, and tumor morphology. IPI-926 administered at 40 mg QD (oral daily treatment for 6-10 weeks) was found to decrease tumor volume by 37-55% in three independent tumor models.
IPI-926 was shown to have a direct effect in inhibiting the growth of tumor cells, and not through the stroma. Figure 23 is a bar graph showing the inhibition of expression of human Gli I and PTCH1 in human cells. Thus, chondrosarcoma provides an example of a solid tumor having a Hh ligand-dependent signaling directly to tumor cells. Hh inhibition has also been shown to have an effect on the surrounding tumor stroma. Upon treatment with IPI-926, calcification is detected in treated samples, which show little to no detectable chondrocytes (data not shown). In contrast, many chondrocytes are detected in untreated primary tumors (data not shown).
IPI-926-treated tumors show a tumor growth inhibition of 44%, p+0.0123, compared to other chemotherapies of primary chondrosarcoma xenografts (Figure 24).
Treatment of the primary chondrosarcoma xenografts studies summarized in Figure 24 were initiated one month after tumor implant into NSG mice. Oral IPI-926 was administered at 40 mg/kg for 5 days/week for a total of three weeks. Other treatment groups in this study included: Triparanol at 400 mg/kg administered orally for days/week; doxorubicin administered at 5 mg/kg administered by i.v. every other day for 3 days/week; cisplatin administered at 8 mg/kg once a week, and DAPT
administered at 10 mg/kg administered by i.p. every other day for 3 days/week. Animals treated with Attorney Docket No. I2041-7000WO/3020PCT
IPI-926 were the only group showing a statistically significant change in human Hh pathway (Gli l) gene expression compared to the control group (Figure 25).
In summary, the Hh pathway plays a significant role in the biology of chondrocytes and in chondrosarcoma. IPI-926 leads to tumor growth inhibition in I' chondrosarcoma tumor xenografts. Inhibition of Hh pathway in chondrosarcoma xenografts leads to morphology changes Example 17: Combination Study of a Paclitaxel Agent and a Hed2eho2 Inhibitor in L3.6p1 Tumor Bearing Mice This Example describes the effect of Abraxane alone or in combination with IPI-926 (HC1 salt) in L3.6p1 tumor bearing animals.

Experimental Design Mouse model Five week old male Ncr nude mice (weight 20-25 g) were purchased from Taconic Farms, Inc. (Hudson, NY).
Cell lines L3.6p1 is a pancreatic tumor model purchased from ATCC. The cells were cultured in advanced DMEM supplemented with 10% FBS and 1%P/S. Cells were harvested with trypsin and a viable cell count was performed using trypan blue exclusion of dead cells. Cells were re-suspended in DMEM (no serum) and subcutaneously implanted at 2x106 cells /100uL/ mouse into the right flank.

Mouse model experiments On Day 9 post tumor cell implant, mice were randomized into three dosing groups to receive vehicle (in 5% hydroxypropyl beta cyclodextrin (HPBCD)) (N = 7), ABRAXANE alone (in saline) (N = 8), or a combination of ABRAXANE (in saline) +
IPI-926 (in 5% HPBCD) (N = 7). From Day 9 through Day 27, IPI-926 was dosed at 40mg/kg orally every other day (QOD), for a total of ten doses. On Day 9 ABRAXANE was dosed at 2 mg/kg i.v., and on Days 13, 20, and 27 ABRAXANE
was dosed at 20mg/kg i.v (on Day 9, there was a miscalculation during the preparation of Attorney Docket No. I2041-7000WO/3020PCT

the ABRAXANE dose. On Day 13, 20 and 27, the calculation was corrected, and the full dose of 20mg/kg was administered on those days). On Day 27, the ABRAXANE
alone and ABRAXANE + IPI-926 groups received their final dose of treatment.
Samples for analysis were collected 24 hours post the final dose.
The control animals were taken down on Day 20 due to multiple animals having ulcertions in their tumors, which is a criteria for the animal to be sacrificed under the IACUC guidelines. On Day 20, the ABRAXANE alone group showed 27% tumor growth inhibition (TGI) when compared to the control group receiving vehicle, while the ABRAXANE + IPI-926 combination group showed 68% TGI when compared to the control group receiving vehicle. On the final day of dosing (Day 27) the ABRAXANE
+ IPI-926 combination group showed a 71% TGI when compared to the ABRAXANE
group. Using the JMP stats program, a means comparison Student's T Test was run on all groups for both Day 20 and Day 27, and all % TGIs reported were found to be statistically significant. The results of this experiment are summarized in Figure 26 and Table 1 below. The data shows a statistically significant increase in efficacy in when ABRAXANE and IPI-926 were dosed in combination, compared to control or ABRAXANE alone.

Table 1 Comparison Day %TGI P Value Control v. ABRAXANE 20 27% 0.08 Control v. ABRAXANE + IPI-926 20 68% 0.003 ABRAXANE v.ABRAXANE + IPI-926 20 56% 0.0138 27 71% 0.001 Immunohistochemical Analysis Phospho histone 3 (PH3) is a nuclear marker of cells in the late G2/M phase.
Paclitaxel inhibits the depolymerization of tubules which can arrest cells in the late G2/M
phase, and can prevent cells from undergoing mitosis. If IPI-926 enhances tumor dug levels, then an increase in PH3 staining will be detected in the IPI-926 +
Abraxane treated tumor sections.
On day 27, 24 hours after the last dose of IPI-926 and ABRAXANE , tumors were collected and fixed overnight in 10% neutral buffered formalin. The next day Attorney Docket No. I2041-7000WO/3020PCT

samples were switched to storage in 70% ETOH. Tumors were processed and embedded according to standard protocols. The embedded tumor tissues were cut into 5um sections for immunostaining of Phospho Histone 3 (PH3). Sections for staining were deparaffinized and hydrated in a series of xylene and 100%, 95% and 70% ETOH
washes. Sections were immersed in citrate buffer and heated under pressure for minutes. Samples were allowed to reach room temperature and were prepared for immunostaining on the DAKO autostainer. The rabbit anti-human PH3 (ser10) (cell signaling#9701L) was used at 1:100. The Rabbit on Rodent polymer (Biocare#RMR622L) was used as the secondary polymer system. DAB liquid substrate buffer + Chromogen system (DAKO#K3468) was used for developing and detection of the stain.
PH3 stained sections were scanned using the Aperio scanner system, and images were subjected to morphometric analysis (see Figure 27A; 200x images of PH3 staining on the L3.6p1 tumor model). The Genie pattern recognition tool and nuclear algorithm were used to quantitate the % PH3 positive neoplastic nuclei per stained tumor section (Figure 27B). A count of the positive neoplasm nuclei showed an increase of 30% PH3 positive in the combination IPI-926 + ABRAXANE group, versus 20% in the ABRAXANE group alone. These data demonstrate an increase of PH3 staining in L3.6p1 tumors treated with the combination of IPI-926 + ABRAXANE and suggests enhancement of ABRAXANE delivery when used in combination with IPI-926 compared to control or ABRAXANE alone.

PK data of endpoint tumors Experiments were conducted in order to measure paclitaxel levels in L3.pl tumors treated with the combination of IPI-926 and ABRAXANE or ABRAXANE alone, and in order to determine if treatment with IPI-926 leads to increased levels of paclitaxel in the tumors. On day 27, twenty-four hours after the last dose of IPI-926 and ABRAXANE , 50-300mg of the L3.6p1 tumor was snap frozen from each mouse for pharmacokinetic (PK) evaluation of tumor paclitaxel levels. PK analysis of these tumors indicated a 28% increase of tumor paclitaxel levels (ng/g of tissue) in the ABRAXANE
+ IPI-926 combination treated tumors versus the ABRAXANE alone treated animals Attorney Docket No. I2041-7000WO/3020PCT
(the vehicle control group was below the level of detection for paclitaxel).
The experiments, similar to the above-described PH3 experiments, show that IPI-926 was able enhance paclitaxel levels to the L3.6p1 tumors.

Example 18: Combination Study of An Exemplary Paclitaxel Agent And Exemplary Hedgehog Inhibitor In ASPC-1 Tumor-Bearing Ncr Nude Mice This Example describes the combination effects of treating ASPC-1 tumor bearing mice with IPI-926 (HCl salt) and ABRAXANE or paclitaxel.

Experimental Design Mouse Model ASPC-1 is a pancreatic tumor model.
Cell line Cells were cultured in advanced RPMI 8226 supplemented with 10% FBS and 1%P/S. Cells were purchased from ATCC. Cells were harvested using trypsin, and viability was assessed by trypan blue exclusion. Cells were implanted 5x106 cells/mouse/100uL into the right flank, subcutaneously.

Experiment and Results Treatment was initiated once tumor volumes reached between -200mm3. IPI-926 was administered in 5% HPBCD @ 40mg/kg (8 mL/kg) by oral gavage every other day, QOD. ABRAXANE or Paclitaxel in saline were administered at 20mgA/kg i.v. with a 27 gauge needle QIW. Bodyweight and tumor measurements were taken twice weekly.
Body weight loss greater than 20% from the initial day of treatment or tumor volumes greater than 3000mm3 resulted in euthanasia. The study design is summarized in Table 2.
On day 20 post tumor cell implant, the mice were randomized into six dosing groups to receive vehicle control, IPI-926 alone, Abraxane +/- IPI-926 or paclitaxel +/-IPI-926 (Figures 28A-28B). IPI-926 was administered at 40mg/kg orally QOD, from day 21 to 41 for a total of 11 doses total. Both Abraxane and paclitaxel were dosed on days 21, 28 and 35 at 20mg/kg i.v. oncer per week, QIW. On day 41, the last dose of was administered and re-growth was monitored. Table 3 summarizes the % tumor growth inhibition (TGI) obtained on day 41 of all the test groups versus the control Attorney Docket No. I2041-7000WO/3020PCT
group, and p values calculated using the JMP stats program (a means comparison Student's T test).
Similar to Example 17 in the L3.6p1 xeongraft model, Abraxane + IPI-926 showed a synergistic effect when dosed in combination, compared to the Vehicle control, IPI-926 alone, or Abraxane alone group. Tumor re-growth was monitored and the Abraxane + IPI-926 group showed at least a 15 day delay in reaching the same tumor volume as the IPI-926 or Abraxane alone treated groups.
Although not as significant, there was a combination effect when IPI-926 and paclitaxel were combined. On day 41 the combination of IPI-926 and paclitaxel was not significantly different from the paclitaxel group alone, however, on day 48, during the regrowth phase of the study, there was a 47% TGI that was statistically significant (p=0.006).
Table 2 Group Compound Dose Route Dose volume mg/ml N
mpk/day (ml/kg) 1 Control (5% HPBCD) 0 PO 8 0 8 2 IPI-926 (5% HPBCD) 40 PO 8 5 8 3 Abraxane (saline) 20 IV 8 2.5 8 4 IPI-926 (5% HPBCD) 40 PO 8 5 8 4 Abraxane (saline) 20 IV 8 2.5 8 5 Paclitaxel (saline) 20 IV 8 2.5 8 6 IPI-926 (5% HPBCD) 40 PO 8 5 8 6 Paclitaxel (saline) 20 IV 8 2.5 8 Table 3 Group % TGI p value Control v. IPI-926 38.5% 0.0008 Control v. Abraxane 34.4% 0.0024 Control v. Abraxane + IPI-926 76.7% <0.001 Control v. paclitaxel 56.4% <0.001 Control v. paclitaxel + IPI-926 73.3% <0.001 paclitaxel vs paclitaxel + IPI-926 38.7% 0.1207 IPI-926 v. paclitaxel + IPI-926 56.6% 0.0022 Attorney Docket No. I2041-7000WO/3020PCT

IPI-926 v. Abraxane + IPI-926 62.3% 0.0009 Abraxane v. Abraxane + IPI-926 64.4% 0.0003 Example 19: Thee-way Combination Study of a Paclitaxel Agent, a Hedgehog Inhibitor and an Additional Therapeutic Agent in L3.6pl Tumor-bearing Mice This Example describes the combination effect of Abraxane , IPI-926 (HC1 salt) and gemcitabine (GEMZAR) in L3.6p1 tumor bearing animals.

Experimental Design Mouse model Five week old male Ncr nude mice (weight 20-25 g) were purchased from Taconic Farms, Inc. (Hudson, NY).

Cell lines L3.6p1 is a pancreatic tumor model purchased from ATCC. The cells were cultured in advanced DMEM supplemented with 10% FBS and 1%P/S. Cells were harvested with trypsin and a viable cell count was performed using trypan blue exclusion of dead cells. Cells were re-suspended in DMEM (no serum) and subcutaneously implanted at 2x106 cells /100uL/ mouse into the right flank.

Experiment and Results On day 10 post tumor cell implant, the mice were randomized into eight dosing groups to receive vehicle, IPI-926 alone, Abraxane alone, Gemzar alone, Abraxane +
IPI-926, Gemzar + IPI-926, Abraxane + Gemzar, and Abraxane + Gemzar + IPI-(see Figure 29A for cummulative results. Figure 29B depicts a subset of the results of Figure 29A). IPI-926 was dosed at 40mg/kg orally QOD, from day 10 to 31 for a total of 11 doses administered. Starting on Day 10, Abraxane was dosed on Days 10, 17 and 24 at 20mg/kg i.v. oncer per week, QI W. Starting on Day 10, Gemzar was dosed on Days 10, 13, 17, 20, 24 and 31 at 100mg/kg i.p. twice per week (Tuesday/Friday schedule).
After Day 31, the study was continued in order to monitor survival while on treatment Attorney Docket No. I2041-7000WO/3020PCT

(see Figure 29C for cummulative results. Figure 29D depicts a subset of the results of Figure 29C).
All animals in the vehicle control group were sacrificed by Day 26. When compared to the vehicle control, the group that resulted in the most significant percent tumor growth inhibtion (%TGI) was the Abraxane + IPI-926 group. On Day 26 the Abraxane + IPI-926 group showed an 83.3% TGI when compared to vehicle control.
Abraxane alone, Abraxane + Gemzar, and Abraxane + Gemzar + IPI-926 all showed a %TGI of 61.3%, 57.2% and 66.5% respectively, when compared to the vehicle control. In this particular model there was no statistical significant single agent activity of IPI-926 or Gemzar and there was no added benefit in combining the two agents (see Figures 29A and 29C and Tables 4-5). Using the JMP stats program, a means comparison Student's T Test was run on all groups for Day 26. Table 4 summarizes the % tumor growth inhibition (TGI) obtained on day 26 of all the test groups, and p values calculated using the JMP stats program (a means comparison Student's T test).
Time to tumor progression was also recorded (Figures 29C-29D and Table 5).
Progression while on treatment was measured as the time it took for each tumor to reach 1000mm3. Once a tumor measured 1000mm3, the animal was sacrificed and the day was recorded to plot the data as a Kaplan-Meier curve. Mean time to progression was found significantly increased in the Abraxane + IPI-926 combination group when compared to all other groups.

Table 4 TGI P Value control v. IPI-926 - 0.8444 control v. Gemzar 13.6 % 0.0946 control v. Gemzar + IPI-926 4.8 % 0.5341 control v. Abraxane 61.3% <.001 control v. Abraxane + IPI-926 83.3 % <.001 control v. Abraxane + Gemzar 57.2 % <.001 control v. Abraxane + Gemzar + IPI-926 66.2 % <.001 Abraxane v. Abraxane + IPI-926 56.8 % 0.0062 Abraxane + Gemzar v. Abraxane + IPI-926 60.9 % 0.0014 Attorney Docket No. I2041-7000WO/3020PCT
Abraxane + Gemzar + IPI-926 v. Abraxane + IPI-926 50.4 % 0.0314 Table 5 Treatment Group Number failed Censored Mean survival (days) Std Error IPI-926 8 0 24 1.059607 Abraxane + Gemzar 8 0 39 2.281525 Abraxane + Gemzar + IPI-926 8 0 42 2.982089 Abraxane 6 1 40 3.666251 Abraxane + IPI-926 7 0 53 3.123316 Gemzar 7 0 26 1.459196 Gemzar + IPI-926 7 0 25 1.375193 Control 8 0 23 0.811469 Test Chi2 DF Prob>ChiSq Log-Rank 73.7486573 7 <.0001 Wilcoxon 59.1713141 7 <.0001 Example 20: Tumor Perfusion Experiments This Example evaluated whether the synergistic effect of the combination of IPI-926 (HC1 salt) and ABRAXANE is by enhanced drug delivery of ABRAXANE to the tumor through the effect of IPI-926 on the mouse stroma via increased tumor perfusion.
Mouse model Five week old male Ncr nude mice (weight 20-25 g) were purchased from Taconic Farms, Inc. (Hudson, NY).

Cell lines L3.6p1 is a pancreatic tumor model purchased from ATCC. The cells were cultured in advanced DMEM supplemented with 10% FBS and 1%P/S. Cells were harvested with trypsin and a viable cell count was performed using trypan blue exclusion of dead cells. Cells were re-suspended in DMEM (no serum) and subcutaneously implanted at 2x106 cells /100uL/ mouse into the right flank.

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Experiment and Results Tumor perfusion was directly measured in IPI-926 (HC1 salt) treated and untreated animals using contrast enhanced ultrasound. The L3.6pl tumor cell line was injected subcutaneously and treatment with IPI-926 was initiated. IPI-926 or vehicle was administered orally at 40 mg/kg for seven consecutive days. On the eighth day, animals were subjected to ultrasound image analysis using perfusion contrast enhancement (microbubbles) during the imaging procedure.
In tumor bearing animals treated with IPI-926, the ultrasound data showed greater tumor perfusion with IPI-926 (compare Figures 30A/30C with 30B/30D). Vehicle treated animals imaged via ultrasound show less contrast agent than the IPI-926 treated animals. The time to reach peak contrast was measured and showed a decrease in the IPI-926 treated animals compared to vehicle. On average, the peak time for contrast agent levels decreased from 11.0 seconds to 4.75 seconds in the vehicle versus treated animals, respectively, (p=0.0321) (Table 6). These data suggest that the synergistic effect of the combination of IPI-926 and ABRAXANE is likely enhanced drug delivery of ABRAXANE to the tumor through the effect of IPI-926 on the mouse stroma via increased tumor perfusion.

Table 6 Mouse # Treatment Time to Peak (s) C1 M 1 Control 16 C1 M2 Control 13 C1M3 Control 8 C1M4 Control 7 C2M1 IPI-926 *3 C2M2 IPI-926 *4 C2M3 IPI-926 *6 C2M4 IPI-926 *6 * Statistically significant compared to control values, p<0.05 Student T test Attorney Docket No. I2041-7000WO/3020PCT
Example 21: Measurement of Gli-1 Levels Figure 31 depicts the results of Q-RT-PCR analysis of excised IPI-926 treated tumors of Example 17 (L3.6p1 pancreatic cell lines) and Example 18 (ASPC-1 pancreatic cell lines). Q-RT-PCR analysis revealed inhibition of murine Gli-1 with IPI-treament. Human Hh ligand was detected and human Gli-1 levels were not modulated with treatment. These data indicate that Hh paracrine signaling can occur in a paracrine manner in pancreatic xenograft tumor models, where the tumor cells are providing Hh ligand and activate murine Gli l, which is inhibited by IPI-926 treatment.

Example 22: Head and Neck Cancer Model The aim of Example 22 was to elucidate the relevance of the Hedgehog pathway in head and neck squamous cell carcinoma (HNSCC) and the effect of the hedgehog inhibitor, IPI-926, in combination with ERBITUX (cetuximab) in a direct patient tumor model (DPTM) of HNSCC, and the role of cancer stem cells (CSC) in relapses after therapy.
An unbiased global pathway analysis on a HNSCC gene expression data set with 42 HNSCC and 14 head and neck normal tissues (GSE6791) compared pathways enriched in the cancer vs. normal classes with Gene Set Enrichment Analysis (GSEA) and showed that the Hedgehog pathway was enriched in the cancer phenotype compared to normal. To test the activation of the Hedgehog pathway we stained for the ligand sonic hedgehog (SHH) protein expression a tissue microarray (TMA) of 30 HNSCC and 10 normal tissue samples. 35% of the cancers were negative or faint, and 65% were positive or strongly positive; 78% of normal tissues were negative or faint and 22%
were positive with no strong positive samples. Six tumor samples from three DPTM (CUH002, CUHN004, and CUHNO 13) and three cell-line derived xenografts (HN11, HN12, and UMMC22) were positive or strongly positive. Treatment in vivo of the three DTPM cases with ERBITUX (cetuximab), IPI-926, and the combination showed in all three cases that ERBITUX -treated tumors re-grew 4-8 weeks post-therapy, whereas combination-treated mice were homogenously relapse-free three months post-therapy (Figure 32).
To address whether ERBITUX treatment leads to an increase in putative cancer stem cells (CSC), we measured putative CSC subpopulations CD24/44 and Attorney Docket No. I2041-7000WO/3020PCT

in ERBITUX -treated tumors in all three DTPM cases by flow cytometry. We found accumulation of these cell types in ERBITUX treated tumors but absolute decrease of CSC in combination-treated tumors. Isolated CD24/44 and CD24/ALDH positive cells each generated tumors at a higher rate than negative cells despite a 100-fold cell number dilution, and the key Hh signaling component SMO and the GLI1 transcription factor genes were 200-fold and 700-fold more highly expressed in CSC than in negative cells.
Conclusion: The Hedghog pathway is active in HNSCC, with over-expression seen in the CSC subpopulation where data suggest autologous signaling. When combined with ERBITUX , Hh pathway inhibition with IPI-926 diminishes CSC
(which can be, in part, responsible for repopulation of the original tumor) prevents relapse of tumor growth of HNSCC.

Example 23: Non-Small Cell Lung Cancer NCI-H1650 Xenograft Model Post Gefitinib Therapy This Example evaluates the activity of IPI-926 in the NCI-H1650 tumor xenograft model post targeted therapy with gefitinib.
Model NCI-H1650 lung carcinoma cell line (ATCC #CRL-5883) is an adenocarcinoma that was isolated from a 27 year old Caucasian male smoker in 1987. These cells have an acquired mutation in the EGFR tyrosine kinase domain (E746 - A750 deletion).
This mutation makes them sensitive to EGFR-tyrosine kinase inhibitors such as gefitinib.
H1650 cells were obtained from ATCC and cultured in RPMI 1640 supplemented with 1% pen/strep and 10% fetal bovine serum. Cells were harvested with trypsin and a viable cell count was performed using trypan blue exclusion of dead cells. Cells were resuspended in RPMI 1640 (no serum) and subcutaneously implanted at 2 x 106 cell/ l00uL/mouse into the right flank of a 5-6 week old male athymic mice (Taconic NcrNu-M).

Study overview Once tumor volumes reached between 150-200 mm3 mice were randomized and treatment was initiated. Randomized mice were treated with vehicle (5% HPBCD), mg/kg gefitinib p.o QD for 7 days then followed by either 40 mg/kg IPI-926 or vehicle.

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Dosing Groups (1) vehicle (5% HPBCD); (2) gefitinib (1% carboxymethylcellulose) @ 40mg/kg p.o QD, followed by vehicle (5% HPBCD); (3) gefitinib (1%
carboxymethylcellulose) @
40mg/kg p.o QD, followed by IPI-926 (5% HPBCD) @ 40mg/kg QOD.
Dosing Regimen IPI-926 p.o Q.O.D for 3 weeks at dose volume of 8 ml/kg; gefitinib p.o Q.D for days at dose volume of 8 ml/kg.

Experiment and Results On day 34 post tumor cells implant, mice were randomized in two dosing groups receiving either vehicle p.o Q.D, or gefitinib (40 mg/kg p.o, Q.D). On day 41 the gefitinib treated mice were then randomized and received either vehicle p.o Q.D, or IPI-926 (40 mg/kg, p.o Q.O.D) for 25 days. Samples for analysis were collected 24 hours post the final dose. On day 67 the gefitinib followed-by IPI-926 (gefitinib 4 IPI-926) group showed 65% tumor growth inhibition (TGI) when compared to gefitinib followed-by vehicle (gefitinib 4 vehicle) group (Figure 33).
Using the JMP stats program, a means comparison Student's T Test was run on all groups and all % TGI reported were statistically significant. The TGIs and p values are summarized in Table 7 below. The data from this study show a statistically significant increase in tumor growth inhibition when IPI-926 is dosed post regression with gefitinib.
Table 7 Comparison %TGI p Value vehicle v. gefitinib 4 vehicle 11 % 0.4152 vehicle v. gefitinib 4 IPI-926 69 % 0.0018 gefitinib 4 vehicle v. 65 % 0.0104 gefitinib 4 IPI-926 Example 24: Non-Small Cell Lung Cancer HCC827 Xenot raft Model Post Gefitinib Therapy Attorney Docket No. I2041-7000WO/3020PCT

This Example evaluates the activity of IPI-926 in the HCC827 tumor xenograft model post targeted therapy with gefitinib.
Model HCC827 tumor cells were isolated from patients with non-small lung cancer (NSCLC). These cells have an acquired mutation in the EGFR tyrosine kinase domain (E746 - A750 deletion). This mutation makes them sensitive to targeted therapy with gefitinib, a tyrosine kinase inhibitor. HCC827 cells were obtained from ATCC
and cultured in RPMI 1640 supplemented with 1 % pen/strep and 5% fetal bovine serum.
Cells were harvested with trypsin and a viable cell count was performed using trypan blue exclusion of dead cells. Cells were resuspended in RPMI 1640 (no serum) and subcutaneously implanted at 5 x 106 cell/100uL/mouse into the right flank of 5-6 week old male athymic mice (Taconic NcrNu-M).
Study overview Once tumor volumes reached between 150-200mm3 mice were randomized and treatment were initiated. Randomized mice were treated with vehicle (5% HPBCD) or 10 mg/kg gefitinib p.o QD for 3 days then followed by either 40 mg/kg IPI-926 or vehicle.
Dosing Groups (1) vehicle (5% HPBCD); (2) gefitinib (1% carboxymethylcellulose) @ 10mg/kg p.o QD, followed by vehicle; (3) gefitinib @ 10mg/kg p.o QD, followed by IPI-926 (5%
HPBCD) (@ 40mg/kg QOD; (4) IPI-926 (5% HPBCD) @ 40 mg/kg p.o. Q.O.D.
Dosing Regimen Gefitinib p.o QD for 3 days at dose volume of 8 ml/kg; IPI-926 p.o QOD for 3 weeks at dose volume of 8 m1/kg.
Experiment and Results On day 18 post tumor cells implant, mice were randomized in three dosing groups receiving either vehicle (p.o. Q.D), gefitinib (40 mg/kg p.o. Q.D) or IPI-926 (40mg/kg p.o. Q.O.D). On day 20 the gefitinib treated mice were then randomized and received either vehicle (p.o. Q.D) or IPI-926 (40 mg/kg, p.o Q.O.D) for 36 days.
Samples for analysis were collected 24 hours post the final dose. On day 56 the gefitinib followed-by IPI-926 (gefitinib 4 IPI-926) group showed 70% tumor growth inhibition (TGI) when compared to gefitinib followed-by vehicle (gefitinib 4 vehicle) group (Figure 29).

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Using the JMP stats program, a means comparison Student's T Test was run on all groups and all % TGI reported were statistically significant. The TGIs and p values are summarized in Table 8 below. The data from this study show a statistically significant increase in tumor growth inhibition when IPI-926 is dosed post regression with gefitinib.

Table 8 Comparison %TGI p Value Vehicle v. gefitinib 4 vehicle 44 % 0.3 Vehicle v. gefitinib 4 IPI- 83 % < 0.02 gefitinib 4 vehicle v. 70 % < 0.03 gefitinib 4 IPI-926 IPI-926 v. 79 % < 0.02 gefitinib 4 IPI-926 Example 25: Hh Pathway Profile Expression In Non-Small Cell Lung Cancer NCI-H1650 Xenot raft Model Post Gefitinib Regression The goal of this Example was to understand the in vivo Hh pathway expression profile immediately post-gefitinib treatment.

Model NCI-H1650 lung carcinoma cell line (ATCC #CRL-5883) is an adenocarcinoma that was isolated from a 27 year old Caucasian male smoker in 1987. These cells have an acquired mutation in the EGFR tyrosine kinase domain (E746 - A750 deletion).
This mutation makes them sensitive to EGFR-tyrosine kinase inhibitors such as gefitinib.
H1650 cells were obtained from ATCC and cultured in RPMI 1640 supplemented with 1% pen/strep and 10% fetal bovine serum. Cells were harvested with trypsin and a viable cell count was performed using trypan blue exclusion of dead cells. Cells were resuspended in RPMI 1640 (no serum) and subcutaneously implanted at 2 x 106 cell/ l00uL/mouse into the right flank of a 5-6 week old male athymic mice (Taconic NcrNu-M).
Study overview Attorney Docket No. I2041-7000WO/3020PCT

Once tumor volumes reached between 150-250mm3 mice were randomized and treatment was initiated. Randomized mice were treated with vehicle (5% HPBCD), mg/kg gefitinib p.o QD x 5 days or when tumor regress 50%, then followed by 40 mg/kg IPI-926 or vehicle.
Dosing Groups (1) vehicle (5% HPBCD); (2) gefitinib (1% carboxymethylcellulose)@ 40mg/kg p.o QD, followed by vehicle; (3) gefitinib @ 40mg/kg p.o QD, followed by IPI-926 (5%
HPBCD) @ 40mg/kg QOD.
Dosing Regimen IPI-926 p.o. QD for 1, 4, 7 or 10 days at dose volume of 8 mlkg; gefitinib p.o.
QD for 5 days at dose volume of 8 mlkg.
Experiment and Results On days 1, 4, 7 and 10 post-gefitinib treatment tumor samples were analyzed for hedgehog ligand modulation. The data from this study indicates that human hedgehog ligands IHh and DHh are up-regulated post gefitinib treatment (Figure 35 and Table 9) and that IPI-926 inhibits the up-regulation of stromal cell Glil and G1i2 (Figure 36). For example, murine Gli l is up-regulated post therapy compared to vehicle treated tumor and down modulated upon IPI-926 treatment. Murine G1i2 is up-regulated post target therapy when compared to vehicle and down modulated upon IPI-926 treatment.
In NCSLC xenograft models NCI-H1650 of Example 23, IPI-926 significantly inhibits tumor re-growth post-gefitinib therapy. Example 25 data indicates that Hh ligands are upregulated post-gefitinib therapy in this xenograft model, and that the hedgehog inhibitor IPI-926 down regulates stromal Gli I and Gli2. The Example 23 and Example 25 data combined suggest that therapeutic inhibition of the Hh signaling pathway is an important strategy to extend progression free survival in patients who initially respond to therapy but later relapse and provide a rationale for evaluating IPI-926 in patients with NSCLC.

Table 9 Treatment Group IHh DHh p value p value Attorney Docket No. I2041-7000WO/3020PCT

gefitinib 4 vehicle (xlD) -- 0.0350 gefitinib 4 IPI-926 (x4D) 0.05 --gefitinib 4 vehicle (x7D) 0.0245 --gefitinib 4 IPI-926 (x7D) 0.0072 0.0306 gefitinib 4 vehicle (x10D) 0.05 --gefitinib 4 IPI-926 (x10D) 0.0073 <0.0001 The contents of all references, pending patent applications and published patent applications, cited throughout this application are hereby incorporated by reference in their entirety as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Example 26: Administration of IPI-926 and Avastin in BXPC-3 This example describes the effects of IPI-926 and Avastin on the vasculature and stroma of the BXPC3 tumor, a pancreatic tumor model.
Treatment can be initiated once tumor volume reaches 100-200mm3. Oral administration of IPI-926 will administered at 40mg/kg orally, QOD. Avastin will be administered at 5mg/kg i.p. two times per week. Post - two weeks of treatment, isolectin and maleimide will be injected i.v., 30 minutes prior to euthanizing.
Dosing Groups:
1. Vehicle 2. IPI-926 @ 40mg/kg/day, p.o, QOD
3. Avastin , i.p. @ 5mg/kg twice weekly 4. IPI-926 @ 40mg/kg/day, p.o. + Avastin, i.p. @ 5mg/kg twice weekly Table 10: Phase I of Treatment Grp Compound Dose Route Dose volume Drug conc N Total mgs x 1.2 # mpk/day (mikg) mg/ml extra per 7 days Attorney Docket No. I2041-7000WO/3020PCT
1 Vehicle (5% - PO 8 - 6 HPBCD) 2 IPI-926HC1 40 PO 8 5 6 34.56 3 Avastin 5 IP 8 0.625 6 2.16 4 IPI-926HC1 40 PO 8 5 6 34.56 4 Avastin 5 IP 8 0.625 6 2.16 Materials and Methods Cell Culture Cells were cultured in advanced RPMI 8226 supplemented with 1% FBS and 1%P/S. Cells were purchased from ATCC. Cells were harvested and implanted 1x107 cells/mouse.

B. Animals Strain: Ncr nudes Age: 5 weeks Sex: Males Weight: 20 - 25g N per group: dependent on dosing group Date ordered and vendor: 02/16/09 (Taconic) Date of receipt: 02/18/09 Date of implant: 02/23/09 Treatment start date: 03/31/09 Husbandry: Male mice are housed in groups of 4 per cage in suspended, stainless-steel cages and offered food and water ad libitum. Environmental controls for the animal room were set to maintain 18 to 26 C, a relative humidity of 30 to 70%, a minimum of 10 room air changes/hour, and a 12-hour light/12-hour dark cycle.

C. Test article IPI-926HCUIPA in 5% HPBCD
Avastin in saline D. Drug administration IPI-926 can be administered orally via a gavage at 8mIkg dose volume daily.
Avastin will be administered i.p. via a 27G needle at 8m1/kg volume, two times per week.

Attorney Docket No. I2041-7000WO/3020PCT
E. Endpoints Tumor collected will be snap frozen for analytical evaluation and RT-PCR (Gli, Ptch, Smo, HH and potential stem cell markers currently under development).
Histopathology: Tumors will be fixed in 10% formalin for 24hrs prior to transferring the samples into 70% ethanol.
Tumor measurements Twice weekly measurements, made in two dimensions (width x length) using calipers.
Tumor volume = length x width2/2 Mortality Clinical observations Body weights Experiment and Results Treatment was initiated once tumor volumes were on average 100mm3. IPI-926 was administered in 5% HPBCD @ 40mg/kg (8 mL/kg) by oral gavage every other day, QOD. AVASTIN or bevacizumab in saline were administered at 5mgA/kg i.p. with a 27 gauge needle 2x per week. Bodyweight and tumor measurements were taken twice weekly. Body weight loss greater than 20% from the initial day of treatment or tumor volumes greater than 3000mm3 resulted in euthanasia.
On day 31 post tumor cell implant, the mice were randomized into four dosing groups to receive vehicle control, IPI-926 alone, Avastin +/- IPI-926. IPI-926 was administered at 40mg/kg orally QOD for a total of 13 doses. Avastin was administered at 5mg/kg i.p. 2x/week for a total of 8 doses. Table 11 summarizes the % tumor growth inhibition (TGI) obtained on day 56 of all the test groups versus the control group, and p values calculated using the JMP stats program (a means comparison Student's T
test).
These results are summarized in Figure 37.

Table 11 Group % TGI p value Control v. IPI-926 36 0.0604 Control v. Avastin 42 0.0320 Attorney Docket No. I2041-7000WO/3020PCT

Control v. Avastin + IPI-926 72 0.0011 IPI-926 v. Avastin 9.5 0.7562 IPI-926 v. Avastin + IPI-926 55 0.0828 EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims (53)

1. A method of treating a hedgehog-associated cancer or tumor, in a subject in need of hedgehog inhibition, said method comprising administering to the subject a first anti-cancer agent and a second anti-cancer agent, in an amount sufficient to treat the cancer or tumor, wherein the first anti-cancer agent is a hedgehog inhibitor, and the hedgehog-associated cancer or tumor and the second anti-cancer agent are each selected from the group consisting of:
a) the hedgehog-associated cancer or tumor is a sarcoma and the second anti-cancer agent is chosen from one or more of: mTOR inhibitor, doxorubicin, cisplatin, ifosfamide, or methotrexate;
b) the hedgehog-associated cancer or tumor is a neuroendocrine cancer and the second anti-cancer agent is a tyrosine kinase inhibitor;
c) the hedgehog-associated cancer or tumor is a head and neck squamous cell cancer and the second anti-cancer agent is a tyrosine kinase inhibitor;
d) the hedgehog-associated cancer or tumor is a pancreatic cancer and the second anti-cancer agent is a paclitaxel agent;
e) the hedgehog-associated cancer or tumor is a pancreatic cancer and the second anti-cancer agent is a VEGF inhibitor; and e) the hedgehog-associated cancer or tumor is a lung cancer and the second anti-cancer agent is a tyrosine kinase inhibitor is chosen from sunitinib, erlotinib, gefitinib, or sorafenib.

2. A method of reducing or preventing a relapse of a hedgehog-associated cancer or tumor after therapy with a tyrosine kinase inhibitor in a subject, said method comprising administering to the subject a hedgehog inhibitor, in an amount sufficient to reduce or inhibit the cancer or tumor re-growth or relapse, wherein the subject is undergoing or has undergone therapy with the tyrosine kinase inhibitor, and wherein the hedgehog-associated cancer or tumor is a lung cancer or a head and neck squamous cell cancer.

3. The method of claim 1 or 2, wherein the hedgehog inhibitor targets one or more of the tumor cell, the tumor microenvironment, or other residual diseases that is responsive to a hedgehog ligand.

4. The method of claim 1 or 2, wherein the hedgehog inhibitor is a Smoothened antagonist.

5. The method of claim 1, wherein the hedgehog inhibitor is a compound of formula I:

or a pharmaceutically acceptable salt thereof.

6. The method of claim 2, wherein the hedgehog inhibitor is a compound of formula I:

or a pharmaceutically acceptable salt thereof.

7. The method of claim 1 or 5, wherein the sarcoma is chosen from one or more of a bone sarcoma, a soft tissue sarcoma, synovial sarcoma, liposarcoma, a musculoskeletal sarcoma, a cartilage sarcoma, a chondrosarcoma or an osteosarcoma.

8. The method of claim 7, wherein the hedgehog inhibitor is administered concurrently or sequentially with the mTOR inhibitor.

9. The method of claim 8, wherein the hedgehog inhibitor and the mTOR
inhibitor reduce or inhibit local or metastatic sarcoma invasion.

10. The method of claim 8, wherein the hedgehog inhibitor and the mTOR
inhibitor reduce or inhibit a relapsed or refractory osteosarcoma.

11. The method of claim 8, wherein the mTOR inhibitor is chosen from one or more of rapamycin, temsirolimus (TORISEL®), everolimus (RAD001, AFINITOR®), ridaforolimus, AP23573, AZD8055, BEZ235, BGT226, XL765, PF-4691502, GDC0980, SF1126 or OSI-027.

12. The method of claim 1 or 5, wherein the neuroendocrine cancer is chosen from one or more of a neuroendocrine cancer of the pancreas, lung, appendix, duodenum, ileum, rectum, or small intestine; a neuroendocrine cancer from the adrenal medulla, the pituitary, the parathyroids, thyroid endocrine islets, pancreatic endocrine islets, dispersed endocrine cells in the respiratory or gastrointestinal tract; or a functional or a non-functional carcinoid neuroendocrine cancer.

13. The method of claim 12, wherein the hedgehog inhibitor is administed concurrently or sequentially with the tyrosine kinase inhibitor.

14. The method of claim 13, wherein the hedgehog inhibitor is administered before initiating treatment with, or after ceasing treatment with, the tyrosine kinase inhibitor; or overlaps in part with each other, and either of which is continued as a single agent after cessation of treatment with the other.

15. The method of any of claims 1, 2, 5 or 6, wherein the hedgehog-associated cancer or tumor is a head and neck squamous cell cancer, and the tyrosine kinase inhibitor is an EGFR-tyrosine kinase inhibitor.

16. The method of claim 15, wherein the hedgehog inhibitor is administered concurrently or sequentially with the EGFR-tyrosine kinase inhibitor.

17. The method of claim 16, wherein the hedgehog inhibitor is administered after ceasing treatment with the EGFR-tyrosine kinase inhibitor; or overlaps in part with each other, and is continued as a single agent after cessation of treatment with the EGFR-tyrosine kinase inhibitor.

18. The method of any of claims 1, 2, 5 or 6, wherein the lung cancer is chosen from a small or a non-small lung cancer.

19. The method of claim 18, wherein the hedgehog inhibitor is administered concurrently or sequentially with the tyrosine kinase inhibitor, and wherein the tyrosine kinase inhibitor is gefitinib.

20. The method of claim 19, wherein the hedgehog inhibitor is administered after ceasing treatment with gefitinib; or overlaps in part with each other, and is continued as a single agent after cessation of treatment with gefitinib.

21. The method of claim 12, wherein the tyrosine kinase inhibitor is chosen from sunitinib, erlotinib, gefitinib, or sorafenib.

22. The method of claim 15, wherein the EGFR-tyrosine kinase inhibitor is a small molecule EGFR-tyrosine kinase inhibitor chosen from one or more of erlotinib, gefitinib, icotinib, lapatinib, neratinib, vandetanib, BIBW 2992 or XL-647.

23. The method of claim 15, wherein the EGFR-tyrosine kinase inhibitor is a monoclonal antibody chosen from cetuximab, panitumumab, zalutumumab, nimotuzumab necitumumab or matuzumab.

24. The method of claim 1 or 5, wherein the hedgehog-associated cancer or tumor is a pancreatic cancer and the second anti-cancer agent is a paclitaxel agent.

25. The method of claim 24, wherein the paclitaxel agent is chosen from an albumin-stabilized nanoparticle paclitaxel formulation or a liposomal paclitaxel formulation.

26. The method of claim 24, wherein the hedgehog inhibitor and the paclitaxel agent are administered sequentially or consecutively.

27. The method of claim 26, wherein the administration of the hedgehog inhibitor overlaps with the treatment with the paclitaxel agent, and continues after treatment with the paclitaxel or the paclitaxel agent has ceased.

28. The method of claim 26, wherein the hedgehog inhibitor and the paclitaxel agent are administered in combination with a third anti-cancer agent chosen from gemcitabine, cisplatin, epirubicin, 5-fluorouracil, a VEGF inhibitor (e.g., anti-VEGF
antibody), leucovorin, oxaplatin, or a combination thereof.

29. The method of claim 1, wherein the subject is a patient who is undergoing, or has undergone, treatment with a third anti-cancer agent, surgery and/or radiation.

30. The method of claim 2, wherein the subject is a patient who is undergoing, or has undergone, treatment with one or more additional anti-cancer agents, surgery and/or radiation.

31. The method of claim 29, wherein the third anti-cancer agent is chosen from one or more of an insulin-like growth factor receptor (IGF-1R) inhibitor, a PI3Kinhibitor, folfirinox, a BRAF inhibitor, a MEK inhibitor, a JAK2 inhibitor, a cytotoxic agent or cytostatic agent.

32. The method of claim 30, wherein the one or more anti-cancer agents are chosen from one or more of an insulin-like growth factor receptor (IGF-1R) inhibitor, a PI3Kinhibitor, folfirinox, a BRAF inhibitor, a MEK inhibitor, a JAK2 inhibitor, a cytotoxic agent or cytostatic agent.

33. The method of claim 31, wherein the hedgehog inhibitor and the second or third anti-cancer agent are administered concurrently in separate or the same pharmaceutical composition.

34. The method of claim 31, wherein the hedgehog inhibitor, and the second or the third anti-cancer agent are administered sequentially.

35. The method of claim 20, the tyrosine kinase inhibitor is administered to the subject at an oral dose of at least about 10 mg, about 25 mg, about 37.5 mg, about 50 mg, about 70 mg, about 87.5 mg, about 100 mg, about 125 mg, or about 150 mg per day.

36. The method of claim 20, wherein the tyrosine kinase inhibitor is administered to a subject daily for about one, two, three, four or more weeks.

37. The method of claim 5, further comprising a cancer treatment chosen from one or more of: a third anti-cancer agent, surgical or radiation procedures.

38. The method of claim 6, further comprising a cancer treatment chosen from one or more of: a third anti-cancer agent, surgical or radiation procedures.

39. The method of claim 37 or 38, wherein the hedgehog inhibitor is administered to the subject before the cancer treatment, concurrently with the cancer treatment, post-treatment, or during remission of the cancer.

40. The method of claim 1 or 5, wherein the hedgehog inhibitor is administered as one or more of:
a first line treatment for the hedgehog-associated cancer or tumor;
a second line treatment for the hedgehog-associated cancer or tumor;
a third or fourth line treatment for the hedgehog-associated cancer or tumor;
neoadjuvant therapy; or adjuvant therapy.

41. The method of any of claims 1, 2, 5 or 6, wherein the hedgehog inhibitor is administered to the subject prior to or following surgical excision/removal of the hedgehog-associated cancer or tumor.

42. The method of any of claims 1, 2, 5 or 6, wherein the hedgehog inhibitor is administered to the subject before, during, or after radiation treatment of the hedgehog-associated cancer or tumor.

43. The method of any of claims 1, 2, 5 or 6, further comprising the step of monitoring the subject for a change in one or more of. tumor size; hedgehog levels or signaling; stromal activation; levels of one or more cancer markers; the rate of appearance of new lesions; the appearance of new disease-related symptoms; the size of soft tissue mass; quality of life; or any other parameter related to clinical outcome.

44. The method of any of claims 1, 2, 5 or 6, further comprising the step of analyzing a nucleic acid or protein from the subject.

45. The method of claim 44, wherein one or more of. a hedgehog ligand, a nucleic acid encoding a hedgehog ligand, or an upstream or downstream component(s) of the hedgehog signaling are analyzed.

46. The method of claim 45, wherein the hedgehog ligand is detected in blood, urine, circulating tumor cells, a tumor biopsy or a bone marrow biopsy.

47. The method of any of claims 1, 2, 5 or 6, further comprising the step of evaluating a sample from the tumor, the cancer cell or the subject to detect the presence or absence of an alteration in an EGFR gene or gene product.

48. The method of claim 47, further comprising the step of identifying or select a tumor, a cancer cell, or a subject as having a likelihood to respond to a treatment comprising an EGFR inhibitor in combination with a hedgehog inhibitor, wherein the presence of the alteration in the EGFR gene or gene product is indicative of an increased responsiveness to the treatment.

49. The method of claim 48, wherein the alterations in the EGFR gene or gene product is an EGFR exon deletion, and/or exon mutation.

50. A composition for use in treating the hedgehog-associated tumor or cancer according to any of claims 1-49.

51. A pharmaceutical composition comprising a hedgehog inhibitor and a paclitaxel or a paclitaxel agent, a tyrosine kinase inhibitor, or an mTOR
inhibitor, in a pharmaceutically-acceptable carrier or excipient.

52. A method of making the pharmaceutical composition of claim 51.

53. A kit that comprising a hedgehog inhibitor and a paclitaxel or a paclitaxel agent, a tyrosine kinase inhibitor, or an mTOR inhibitor, and instructions for use for the treatment of a hedgehog-associated cancer or tumor.