Treatments and diagnostics for cancers

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 15-1330 Seq Listing_ST25.txt. The text file is about 7 KB, was created on Mar. 9, 2018, and is being submitted electronically via EFS-Web.

FIELD OF THE DISCLOSURE

The present disclosure provides treatments and diagnostics for cancers. The treatments utilize a combination therapy of Galeterone and a proteasome inhibitor. The diagnostics can measure androgen receptor (AR) cleavage products including AR-variant 7 (AR-V7) cleavage products, Poly (ADP-ribose) polymerase (PARP) cleavage products, and/or Spectrin .alpha.2 cleavage products or inhibition of DUB activities from a blood sample to monitor treatment efficacy for castration-resistant prostate cancer (CRPC) or multiple myeloma (MM).

BACKGROUND OF THE DISCLOSURE

Cancer (neoplasia) is characterized by deregulated cell growth and cell division. There are numerous types of cancers. As one example, prostate cancer, as its name indicates, is a cancer that develops in the prostate gland of the male reproductive system. Prostate cancer can be aggressive, in which cancer cells metastasize and move from the prostate gland to other parts of the body, such as the lymph nodes and the bones. It is the second leading cause of cancer-related death in men in the US, and its prevalence is increasing in developing countries.

More than 200,000 new cases of prostate cancer are diagnosed in the US each year. Of these, roughly 88% or 176,000 have localized disease that can be treated with surgery, radiation, cryotherapy or watchful waiting.

Prostate cancer growth is often driven by male sex hormones called androgens, which include testosterone. Because of this, a common treatment option for the 22% or 35,200 patients that cannot be treated with surgery, radiation, cryotherapy or watchful waiting is to lower the levels of androgens in the man's body. Androgen levels can be lowered by surgically removing the testicles or with drugs that stop the testicles, and to a lesser extent adrenal glands, from making androgens or block how they affect the body. This type of treatment is called hormone therapy or androgen-deprivation therapy.

Unfortunately, about 40,000 patients each year begin to fail hormone therapy or become hormone refractory. That is, they develop castration-resistant prostate cancer (CRPC) or hormone refractory prostate cancer (HRPC).

Treatment options for prostate cancer are very limited once the disease becomes resistant to hormonal therapy. In the past few years, docetaxel was the only treatment option for patients with CRPC.

Recently, the FDA approved a chemotherapeutic drug, cabazitaxel, for clinical management of CRPC. Cabazitaxel has shown survival benefits for patients with CRPC. The drug is used to treat men with advanced prostate cancer after treatment with other anticancer agents, including docetaxel, have failed to curtail cancer progression. Thus, cabazitaxel is mostly administered to patients when docetaxel is no longer effective.

Although cabazitaxel has been shown to increase the overall survival of prostate cancer patients, it has serious adverse side effects. These include low white blood cell count, low red blood cell count, low blood platelet count, nausea, vomiting, constipation and diarrhea, and decreased appetite, shortness of breath, tiredness and hair loss.

Galeterone also named TOK-001 or VN/124-1, is a small molecule, oral drug that is capable of disrupting androgen receptor signaling. In preclinical studies, Galeterone has been shown to selectively inhibit cytochrome C.sub.17.alpha.-hydroxylase/C.sub.17-20-lyase (CYP17) lyase to prevent biosynthesis of androgens, antagonize testosterone binding to the androgen receptor (AR), and degrade the AR protein. Galeterone is the first drug in development that has been shown to have all three properties. At the time of this application's filing, Galeterone is undergoing a Phase III clinical trial for the treatment of metastatic CRPC.

SUMMARY OF THE DISCLOSURE

The current disclosure provides compositions and methods to potentiate treatment with Galeterone for a variety of cancers. The compositions and methods utilize administration of proteasome inhibitors, which, as disclosed herein, can be used to potentiate the beneficial anti-cancer effects of Galeterone and/or to reduce toxicity (e.g., lower the required dose) of administered Galeterone. Without being bound by theory, administration of proteasome inhibitors sensitizes cancer cells to the beneficial anti-cancer effects of Galeterone.

The current disclosure also provides systems and methods to monitor anti-cancer effects of CRPC treatments (e.g., Galeterone administration) by measuring blood levels of cleavage products of androgen receptor (AR), Poly (ADP-ribose) polymerase (PARP), and/or Spectrin .alpha.2, and more particularly, in particular embodiments, AR-variant 7 (AR-V7) cleavage products including 53 kDa and 41 kDa AR-V7 fragments, PARP cleavage products including 89 kDa and 65 kDa PARP fragments, and Spectrin .alpha.2 cleavage products including 120 kDa and 150 kDa Spectrin .alpha.2 fragments.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A. Western blotting with anti-N20/AR antibody to examine levels of AR and AR-V7 proteins in various prostate cancer cell lines. FIG. 1B. Inhibition of calpain or caspase could rescue AR-V7 degradation induced by Gal. Lanes 1-4, 22Rv1 cells were treated with 20 .mu.M of Gal (DMSO as a control) without or with a calpain inhibitor, 40 .mu.M of calpeptin or PD 156060 (PD) for 66 h. Lanes 5-8, 22Rv1 cells treated with 20 .mu.M of Gal (DMSO as a control) without or with 50 .mu.M of the calpain inhibitor ALLM or a pan-caspase inhibitor for up to 72 h. This was followed by Western blotting with specific AR-V7 antibody, with Actin as a loading control.

FIGS. 2A and 2B: Gal induces activation of caspase-3 and calpain followed by AR-V7 degradation. 22Rv1 cells were treated with 20 .mu.M Gal for up to 72 h. FIG. 2A. Western blotting. (a, b) AR-V7 and AR protein levels determined by AR-V7 specific antibody and N20 antibody, respectively. (c) PARP/p89 is a caspase-3 cleavage fragment while PARP/p65 is a calpain cleavage product. (d) Caspase-3 cleavage fragment of Spectrin .alpha.2/p120, and a 150 fragment by either caspase-3 or calpain cleavage. (e) Autolysis of calpain small subunit (Cal Sm) to p28 fragment. (f) Calpain cleavage fragment of Calpastatin/p70. FIG. 2B. Caspase-3 activity assay.

FIGS. 3A-3D: Proteasome Inhibition could not block, instead increase Gal-Induced AR-V7 degradation and PARP cleavage. 22Rv1 cells were treated with 20 .mu.M Gal with or without BTZ at indicated concentrations (DMSO as a control) for 72 h (FIG. 3A, 3B) or 48 h (FIG. 3C, 3D), followed by Western blotting with specific AR-V7 mAb and PARP (FIG. 3A, 3C) and proteasome activity (FIG. 3B, 3D).

FIGS. 4A-4D: Gal inhibits the proteasomal activities in CRPC cells. 22Rv1 cells were treated with 5-20 .mu.M Gal for 24 h (FIG. 4A) or 48 h (FIG. 4B), or VCaP cells treated with 5-20 .mu.M Gal for 72 h (FIG. 4C), with 100 nM BTZ as a control, followed by assaying proteasomal CT-like, PGPH-like, and T-like activities. (FIG. 4D) 22Rv1 cells were treated with 20 .mu.M Gal for up to 72 h, followed by assaying CT-like activity.

FIGS. 5A-5D: FIG. 5A--Cellular localizations of AR and AR-V7 as well as calpain/caspase substrate proteins and their cleavage fragments in Gal-treated R22v1. 22Rv1 cells were treated with 20 .mu.M Gal for up to 72 h, followed by isolating nuclear (Nuc) and cytosolic (Cyto) fractions and analysis in Western blotting (35 .mu.g/lane) using below antibodies: (a, b) Anti-AR-V7 monoclonal antibody, shorter and longer exposures, respectively; (c) Anti-AR (N20); (d) Anti-PARP; (e) Anti-Spectrin .alpha.2; (f) Anti-calpain small subunit; (g) Anti-Calpastatin; (h) Anti-actin. (FIG. 5B-D) Nuclear AR-V7 cleavage fragment(s) and effects of calpain/caspase inhibitors in Gal-treated 22Rv1 cells. B, Cleavage of AR-V7 in nuclei. 22 Rv1 cells, with androgen starvation for 24 h, were treated with 20 .mu.M of Gal for indicated hours, followed by isolation of nuclear (Nu) and cytosolic (Cyto) fractions and Western blotting with specific antibodies indicated. The 53 kDa fragment was detected by the AR-V7-specific monoclonal antibody. (C, D) Inhibitors of calpain and caspases block the nuclear AR-V7 degradation in Gal-treated CRPC cells. 22 Rv1 cells, without (C) or with androgen starvation for 24 h (D), were treated with 20 .mu.M of Gal in the absence or presence of 40 .mu.M of calpain inhibit calpeptin (CAPT) (C, D), 40 .mu.M of pan-caspase inhibitor IV (Cas IV) (C), or 20 .mu.M of caspase inhibitor Z-VAD (D) for different time points. Nuclear fractions were isolated and separated by Western blotting to determine AR-V7 levels. Actin was used as a loading control.

FIG. 6 provides an exemplary human AR sequence (SEQ ID NO: 1).

FIG. 7 provides an exemplary human AR variant sequence (SEQ ID NO: 2) (ref. 66).

FIG. 8 provides an exemplary human Spectrin .alpha.2 sequence (SEQ ID NO: 3).

FIG. 9 provides an exemplary human PARP sequence (SEQ ID NO: 4).

FIGS. 10A-10E. Gal inhibits 19S proteasome-associated DUB, but not 20S proteasomal catalytic activities. Purified 20S proteasome (FIG. 10A), purified 26S proteasome (FIG. 10B), or protein extract of 22Rv1 cells (FIG. 10C) was incubated with Gal (.mu.M) or BTZ (nM) at indicated concentrations for 15 min, and the proteasome activities were determined. (FIG. 10D and FIG. 10E) Purified 26S proteasome was incubated with Gal (20 .mu.M or as indicated) for 15 min, and then Ub-AMC substrate was added to determine DUBs activity. Purified 20S proteasome was used a control; BG, background (10D).

FIGS. 11A-11D. Gal induces DUB inhibition and protein oxidization in castrate-resistant prostate cancer cells. 22Rv1 cells were treated with 20 .mu.M galeterone (Gal) for up to 72 h, followed by measuring levels of DUB activity (FIG. 11A), proteasomal CT-like activity (FIG. 11B), Western blotting (FIG. 11C) and caspase-3 activity (FIG. 11D). In C, (a) Cell lysates were derivatized with 2,4-dinitrophenylhydrazine (DNPH), followed by Western blotting to determine the levels of oxidized proteins (Oxi-P). Actin was used a load control (b). AR-V7 levels were determined by AR-V7 specific antibody (c). PARP/p89 is caspase-3 cleavage fragment while PARP/p65 is calpain cleavage product (d). (e) Autolysis of calpain small subunit (Cal Sm) to p28 fragment. (f) Actin, loading control.

FIGS. 12A-E. Gal and BTZ combination induced greater levels of growth inhibition and cell death in CRPC cells. (FIG. 12A) 22Rv1 cells were treated with Gal at indicated concentrations in the presence of different concentrations of bortezomib (BTZ) for 24 h, cell proliferation was determined by MTT. (FIG. 12B-C) Morphological changes after bortezomib and Gal combination treatment for 24 h. 22Rv1 cells were treated with Gal (20 .mu.M) in the presence of indicated concentrations (nM) of bortezomib (BTZ) for 24 h, morphological changes (12B) and crystal violet staining (12C) were determined. In 12C, cells were fixed with formalin, and then stained with violet overnight. (FIG. 12D-E) 22Rv1 cells were treated with Gal (20 .mu.M) in the presence of indicated concentrations (nM) of bortezomib (BTZ) for 48 h, morphological changes (12D) and crystal violet staining (12E) were determined.

FIGS. 13A-C. Gal overcomes BTZ resistance in MM. BTZ-resistant MM 8826 cells were treated with Gal (10-20 .mu.M) in combination with BTZ (2.5-40 nM) for 24 h (FIG. 13A, FIG. 13B from two independent experiments). Cell proliferation was determined by MTS. (FIG. 13C) Multiple myeloma 8826 cells were treated with 0.5-4 nM bortezomib (BTZ) in the presence of 10 .mu.M of Gal for 24 h. Cell death was determined by detection of PARP cleavage. Increased levels of PARP cleavage were seen in the combination treated cells. Actin was used as loading control.

DETAILED DESCRIPTION

Cancer (neoplasia) is characterized by deregulated cell growth and cell division. There are numerous types of cancers. Examples of cancers include acoustic neuroma, adenocarcinoma, astrocytoma, basal cell cancer, bile duct cancer, bladder cancer, brain cancer, breast cancer, bronchogenic cancer, central nervous system cancer, cervical cancer, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, colon cancer, craniopharyogioma, ependymoma, Ewing's tumor, fibrosarcoma, glandular cancer, glioma, hairy cell leukemia, hemangioblastoma, hepatocellular carcinoma, hepatoma, kidney cancer, leiomyosarcoma, liver cancer, liposarcoma, lung cancer, melanoma, medulloblastoma, medullary cancer, medullary thyroid cancer, menangioma, mesothelioma, multiple myeloma (MM), myxosarcoma, neuroblastoma, non-Hodgkin's lymphoma, oligodendroglioma, osteogenic sarcoma, ovarian cancer, papillary adenocarcinomas, papillary thyroid cancer, pancreatic cancer, pheochromocytomas papillary cancer, pineal cancer, prolymphocytic leukemia, prostate cancer (including castration-resistant prostate cancer), renal cell cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, sebaceous gland cancer, seminoma, skin cancer, squamous cell cancer, sweat gland cancer, synovioma, testicular cancer, and/or Wilms' tumor.

As one example, multiple myeloma (MM) is a cancer of plasma cells, a type of white blood cell normally responsible for producing antibodies. In multiple myeloma, collections of abnormal plasma cells accumulate in the bone marrow, where they interfere with the production of normal blood cells. Most cases of multiple myeloma also feature the production of a paraprotein--an abnormal antibody which can cause kidney problems. Bone lesions and hypercalcemia (high blood calcium levels) are also often encountered. Multiple myeloma is considered to be incurable but treatable. Remissions may be induced with steroids, chemotherapy, proteasome inhibitors, immunomodulatory drugs such as thalidomide or lenalidomide, and stem cell transplants. Radiation therapy is sometimes used to reduce pain from bone lesions.

Another example of a cancer is prostate cancer. Prostate cancer, as its name indicates, is a cancer that develops in the prostate gland of the male reproductive system. Prostate cancer can be aggressive, in which cancer cells metastasize and move from the prostate gland to other parts of the body, such as the lymph nodes and the bones. It is the second leading cause of cancer-related death in men in the US, and its prevalence is increasing in developing countries.

More than 200,000 new cases of prostate cancer are diagnosed in the US each year. Of these, roughly 88% or 176,000 have localized disease that can be treated with surgery, radiation, cryotherapy or watchful waiting.

Prostate cancer growth is often driven by male sex hormones called androgens, which include testosterone. Because of this, a common treatment option for the 22% or 35,200 patients that cannot be treated with surgery, radiation, cryotherapy or watchful waiting is to lower the levels of androgens in the man's body. Androgen levels can be lowered by surgically removing the testicles or with drugs that stop the testicles from making androgens or block how they affect the body. This type of treatment is called hormone therapy or androgen-deprivation therapy.

Unfortunately, about 40,000 patients each year begin to fail hormone therapy or become hormone refractory. That is, they develop castration-resistant prostate cancer (CRPC) or hormone refractory prostate cancer (HRPC). CRPC is particularly prostate cancer that continues to grow despite the suppression of male hormones that fuel the growth of prostate cancer cells.

Androgen receptors (AR), also known as NR3C4 (nuclear receptor subfamily 3, group C, member 4), are a type of nuclear receptor that are activated by binding either of the androgenic hormones, testosterone, or dihydrotestosterone in the cytoplasm. Upon binding, AR translocates into the cell nucleus. The AR is most closely related to the progesterone receptor, and progestins in higher dosages can block AR.

The AR contains a N-terminal domain (NTD), a DNA binding domain (DBD), a hinge region (H) and a C-terminal domain (CTD). The CTD contains the Ligand Binding Domain (LBD) and the Transcriptional Activation Function 2 Domain (AF2). Androgen Receptor Variants (ARVs) lack the LBD and are constituently active in the absence of ligand (AR-V3, AR-V4, AR-V5, AR-V7 and AR-V12). A human AR sequence can be found at, for example, Accession: AAA51772.1 and this sequence is provided in FIG. 6. A human variant AR sequence can be found at Accession: ACN39559.1 and this sequence is provided in FIG. 7.

Among the contributors to CRPC are AR splice variants that lack the LBD. Instead, AR splice variants have small amounts of unique sequences derived from cryptic exons or from out of frame translation. Androgen Receptor Variant 7 (AR-V7) is constitutively active and is expressed under conditions consistent with CRPC. AR-V7 is reported to regulate a transcriptional program that is similar but not identical to that of AR. However, it is unknown whether these differences are due to the unique sequences in AR-V7, or simply to loss of the LBD.

Galeterone, also named TOK-001 and VN/124-1, is a small molecule, oral drug that is capable of disrupting androgen receptor signaling. In preclinical studies, Galeterone has been shown to selectively inhibit CYP17 lyase to prevent biosynthesis of androgens, antagonize testosterone binding to the androgen receptor (AR), and degrade the AR protein. Galeterone is the first drug in development that has been shown to have all three properties. At the time of this application's filing, Galeterone is undergoing a Phase III clinical trial for the treatment of metastatic CRPC.

Galeterone has been shown to be effective against the full length AR (fAR) and AR-V7, causing tumor regression in preclinical models. Activities are shown by reductions in Prostate-specific antigen (PSA), Transmembrane protease, serine 2 (TMPRSS2) and Homeobox protein Nkx-3.1 (NKX3.1).

Particular embodiments of Galeterone have a molecular formula of C.sub.26H.sub.32N.sub.2O and a molecular weight of about 388.55 g/mol. Particular embodiments of Galeterone include (3S,8R,9S,10R,13S,14S)-17-(benzimidazol-1-yl)-10,13-dimethyl-2,3,4,7,8,9,- 11,12,14,15-decahydro-1H-cyclopenta[a]phenanthren-3-ol. Particular embodiments of Galeterone include 3-.beta.-Hydroxy17-(1H-benzimidazol-1-yl)androsta-5,16-diene).

Particular embodiments of Galeterone include compounds of formula (I)

##STR00001##

Particular embodiments of Galeterone include analogs which include pharmaceutically acceptable salts, N-oxides, active metabolites, prodrugs, or solvates of Formula 1.

Particular embodiments of Galeterone include analogs which include compounds of formula (II) or a pharmaceutically acceptable salt, N-oxide, active metabolite, prodrug, or solvate thereof

##STR00002## wherein R.sub.1 is H or acetyl; and R.sub.2 is pyridyl or benzimidazolyl. Additional embodiments of Galeterone analogs are disclosed in US 2014/0288037.

The current disclosure provides compositions and methods to potentiate the anti-cancer effects of Galeterone. Potentiating the anti-cancer effects of Galeterone means that at least one anti-cancer effect of Galeterone is increased, and in particular embodiments is statistically-significantly increased. The potentiation can be used to increase the anti-cancer effect of Galeterone, or, in particular embodiments, to lower the required dose of Galeterone to reduce potential side effects or toxicities of drug administration or allow for more Galeterone to be tolerated for greater drug concentration and subsequently greater efficacy.

The compositions and methods that potentiate the anti-cancer effects of Galeterone utilize administration of proteasome inhibitors in combination with Galeterone. Without being bound by theory, administration of proteasome inhibitors sensitizes cancer cells to the beneficial anti-cancer effects of Galeterone because Galeterone itself is a proteasome inhibitor. Galeterone also inhibits deubiquitinating enzymes (DUBs).

The 19S proteasome subunit (PA700/19S) plays essential roles in processing ubiquitylated substrates; it can bind, deubiquitylate, and unfold ubiquitylated proteins, which then translocate into the proteolytic chamber of the 20S proteasome for degradation. The 20S proteasome subunit (20S) is a 700-kDa proteasome that has multiple peptidase activities that function through a new type of proteolytic mechanism involving a threonine active site. The 26S proteasome degrades ubiquitylated proteins. It includes the 20S proteasome and the PA700/19S complex.

Deubiquitinating enzymes (DUBs) are also known as deubiquitinating peptidases, deubiquitinating isopeptidases, deubiquitinases, ubiquitin proteases, ubiquitin hydrolases, and ubiquitin isopeptidases. DUBs are a large group of proteases that cleave ubiquitin from proteins and other molecules. Ubiquitin is attached to proteins in order to regulate the degradation of proteins via the proteasome and lysosome; coordinate the cellular localization of proteins; activate and inactivate proteins; and modulate protein-protein interactions. DUBs can reverse these effects by cleaving the peptide or isopeptide bond between ubiquitin and its substrate protein. In humans there are nearly 100 DUB genes, which can be classified into two main classes: cysteine proteases and metalloproteases. The cysteine proteases include ubiquitin-specific proteases (USPs), ubiquitin C-terminal hydrolases (UCHs), Machado-Josephin domain proteases (MJDs) and ovarian tumor proteases (OTU). The metalloprotease group includes the Jab1/Mov34/Mpr1 Pad1 N-terminal+ (MPN+) (JAMM) domain proteases.

Proteasome inhibitors are molecules that block the activity of proteasomes, which are proteolytic complexes that degrade cytosolic and nuclear proteins into small peptide units. Examples of proteasome inhibitors include lactacystin, bortezomib, disulfiram, epigallocatechin-3-gallate, salinosporamide A, carfilzomib, oprozomib (ONX 0912), CEP-18770, laxomib (MLN9708), epoxomicin, .beta.-lapachone, and MG132. Bortezomib is a first-generation proteasome inhibitor. Examples of second-generation proteasome inhibitors include MLN9708, CEP-18770, carfilzomib, salinosporamide A (NPI-0052, marizomib), oprozomib (ONX 0912) and immunoproteasome inhibitors (e.g., UK-101, IPSI-001, YU-102, ONX 0914, PR-924 (IPSO). Bortezomib has been approved in the US for the treatment of multiple myeloma. Moreover, proteasomal inhibitors such as Brotezamide, MG132, and .beta.-lapachone inhibit the 20S proteasome. Therefore, these drugs inhibit the entire proteasomal activity including the 26S (which includes the 20S and 19S) and the 20S proteasomal complexes.

In particular embodiments, cancer cells (e.g., CRPC cells, MM cells) are sensitized to the beneficial anti-cancer effects of Galeterone due to activation of calpains/caspases. For example, in CRPC cells, calpains/caspases cleave AR and the variant AR (e.g., AR-V7), resulting in cancer cell death.

Calpains belong to a family of calcium-dependent, non-lysosomal cysteine proteases (proteolytic enzymes) expressed ubiquitously in mammals and many other organisms. Amongst protein substrates, tertiary structure elements rather than primary amino acid sequences are likely responsible for directing cleavage to a specific substrate. Amongst peptide and small-molecule substrates, the most consistently reported specificity is for small, hydrophobic amino acids (e.g. leucine, valine and isoleucine) at the P2 position, and large hydrophobic amino acids (e.g. phenylalanine and tyrosine) at the P1 position. Calpains have been implicated in necrotic and apoptotic cell death.

The calpain family includes two major isoforms, calpain I and calpain II which require .mu.M and mM Ca.sup.2+ concentrations to initiate activity. An increase in intracellular Ca.sup.2+ level is thought to trigger a cascade of biochemical processes including calpain activation. Once activated, calpains degrade membrane, cytoplasmic and nuclear substrates, leading to the breakdown of cellular architecture and finally apoptosis.

Like calpains, caspases are cysteine proteases. Caspases are a family of cysteine aspartic proteases or cysteine-dependent aspartate-directed proteases that play an important role in apoptosis, necrosis, and inflammation. Caspases are essential in cellular processes including apoptosis, in development and in stages of adult life. Some caspases are also required in the immune system for the maturation of lymphocytes. Failure of apoptosis has been shown to contribute to tumor development, chemoresistance and autoimmune diseases. It has been shown that caspases and calpains interact with each other to increase or decrease apoptosis.

There are two types of apoptotic caspases, the initiator (apical) caspases and the effector (executioner) caspases. The initiator caspases cleave inactive pro-forms of effector caspases, thereby activating them. The effector caspases subsequently cleave other protein substrates within the cell to trigger the apoptotic process. The initiation of this caspase cascade is regulated by caspase inhibitors.

Examples of initiator caspases include Caspase-2 (CASP2), Caspase-8 (CASP8), Caspase-9 (CASP9), and Caspase-10 (CASP10). Examples of effector caspases include Caspase-3 (CASP3), Caspase-6 (CASP6), and Caspase-7 (CASP7). Other caspases that are not classified as an initiator or effector caspases include Caspase-4 (CASP4), Caspase-5 (CASP5), and Caspase-1 (CASP1). CASP4 and CASP5 are inflammatory enzymes and together with CASP1 are involved in T-cell maturation. Other names for these exemplary caspases include the following: "ICE" for CASP1; "ICH-1" for CASP2; "CPP32," "Yama," and "apopain" for CASP3; "ICE(rel)II," "TX," and "ICH-2" for CASP4; "ICE(rel)III," and "TY" for CASP5; "Mch2" for CASP6; "Mch3," "ICE-LAP3," and "CMH-1" for CASP7; "FLICE," "MACH," and "Mch5" for CASP8; "ICE-LAP6," and "Mch6" for CASP9; and "Mch4," and "FLICE-2" for CASP10.

As suggested, the current disclosure describes a combination therapy of Galeterone and a proteasome inhibitor. A combination therapy refers to those situations in which two or more different active ingredients are administered in overlapping regimens so that the subject is simultaneously exposed to both agents in therapeutically effective amounts.

Combination therapies can be used to treat subjects (humans, veterinary animals (dogs, cats, reptiles, birds, etc.) livestock (horses, cattle, goats, pigs, chickens, etc.) and research animals (monkeys, rats, mice, fish, etc.).

Therapeutically effective amounts of combination therapies disclosed herein have an anti-cancer effect. Cancer (medical term: malignant neoplasm) refers to a class of diseases in which a group of cells display uncontrolled growth (division beyond the normal limits), invasion (intrusion on and destruction of adjacent tissues), and sometimes metastasis. "Metastasis" refers to the spread of cancer cells from their original site of proliferation to another part of the body. For solid tumors, the formation of metastasis is a very complex process and depends on detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membranes to enter the body cavity and vessels, and then, after being transported by the blood or lymph, infiltration of target organs. Finally, the growth of a new tumor, i.e. a secondary tumor or metastatic tumor, at the target site depends on angiogenesis. Tumor metastasis often occurs even after the removal of the primary tumor because tumor cells or components may remain and develop metastatic potential.

A "tumor" is a swelling or lesion formed by an abnormal growth of cells (called neoplastic cells or tumor cells). A "tumor cell" is an abnormal cell that divides by a rapid, uncontrolled cellular proliferation and continues to divide after the stimuli that initiated the new division cease. Tumors show partial or complete lack of structural organization and functional coordination with the normal tissue, and usually form a distinct mass of tissue, which may be either benign, pre-malignant or malignant.

As used herein, an anti-cancer effect refers to a biological effect, which can be manifested by a decrease in tumor volume, a decrease in the number of cancer cells, a decrease in the number of metastases, an increase in life expectancy, or a decrease of various physiological symptoms associated with the cancerous condition. An anti-cancer effect can also be manifested by a decrease in recurrence or an increase in the time before recurrence. In particular embodiments, an anti-cancer effect also includes a statistically-significant up-regulation in AR cleavage products (e.g., 53 kDa and 41 kDa AR-V7 fragments) in a subject following administration of an active ingredient.

For administration, therapeutically effective amounts (also referred to herein as doses) can be initially estimated based on results from in vitro assays and/or animal model studies. Such information can be used to more accurately determine useful doses in subjects of interest. Particularly useful pre-clinical tests include measure of calpain activation, caspase activation, cell growth, and/or cell viability.

Calpain activation can be assessed by increased calpain activity using its substrates Ac-LLY-AFC and N-succinyl-LY-AMC (abcam; 28) in a cell-free activity assay and by the production of specific calpain substrate cleavage fragments, e.g., calpain small subunit/p28 (17), Calpastatin/p70 (18), 65-kDa fragment of PARP (19), and fragments of the microtubule-associated protein Tau in Western blotting (29).

Caspase activation can be measured by increased activities using specific substrates to caspase-9, -8 and 3 (cell-free activity assays), increased levels of the active caspases/decreased levels of pro-caspases (Western blotting and immunostaining), and the production of specific caspase substrate cleavage fragments, e.g., PARP/p89 (12-13, 21-27), and 120-kDa fragments of .alpha.-spectrin (Western blotting) (16).

Cell growth can be determined by MTT and colony formation assays. Cell counting as a golden standard can be performed routinely to determine cell doubling times and growth rates (12-13, 21-27). Cell viability can be determined by trypan blue exclusion and LDH release assays (30).

Spectrin .alpha.2 and PARP are caspase cleavage substrates. Spectrin .alpha.2 (all-spectrin) is a 285 kDa scaffolding protein expressed in most eukaryotic cells. The Spectrin heterodimer comprising all-Spectrin and any of five .beta.-Spectrins have been found to play various roles in cellular processes including formation and maintenance of specialized plasma membrane domains, structural support of the plasma membrane and the maintenance of cell shape, and as a tumor-suppressor protein involved in TGF-.beta.-SMAD regulation, a scaffold upon which calcium-mediated and tyrosine-phosphatase signal transduction pathways converge. An exemplary human Spectrin .alpha.2 amino acid sequence can be found at UniProtKB-Q13813 and this sequence is provided in FIG. 8.

Poly (ADP-ribose) polymerase (PARP) is a family of proteins involved in a number of cellular processes involving mainly DNA repair and programmed cell death. PARP is found in the cell's nucleus. PARPs detect and signal single-strand DNA breaks (SSB) to the enzymatic machinery involved in the SSB repair. PARP activation is an immediate cellular response to metabolic, chemical, or radiation-induced DNA SSB damage.

PARP is composed of four domains of interest: a DNA-binding domain, a caspase-cleaved domain (see below), an auto-modification domain, and a catalytic domain. The DNA-binding domain is composed of two zinc finger motifs. PARP binds sites with single-strand breaks through its N-terminal zinc fingers and recruits other proteins such as XRCC1, DNA ligase III, DNA polymerase beta, and a kinase to the site to repair the break.

Examples of proteins of the PARP family include: PARP1, PARP2, VPARP (PARP4), Tankyrase-1 and -2 (PARP-5a or TNKS, and PARP-5b or TNKS2, respectively) which are confirmed to have PARP activity; and PARP3, PARP6, TIPARP (or "PARP7"), PARP8, PARP9, PARP10, PARP11, PARP12, PARP14, PARP15, and PARP16. An exemplary human PARP1 amino acid sequence can be found at Accession: NP_001609.2, and this sequence is provided in FIG. 9.

The actual dose amount administered to a particular subject can be determined by a physician, veterinarian, or researcher taking into account parameters such as physical, physiological and psychological factors including target, body weight, stage of prostate cancer, type of prostate cancer, previous or concurrent therapeutic interventions, idiopathy of the subject, and route of administration.

Exemplary doses can include 0.05 mg/kg to 5.0 mg/kg of Galeterone and/or a proteasome inhibitor. For certain indications, the total daily dose can be 0.05 mg/kg to 30.0 mg/kg Galeterone and/or a proteasome inhibitor administered to a subject one to three times a day, including administration of total daily doses of about 0.05-3.0, 0.1-3.0, 0.5-3.0, 1.0-3.0, 1.5-3.0, 2.0-3.0, 2.5-3.0, and 0.5-3.0 mg/kg/day of administration forms of Galeterone and/or a proteasome inhibitor using 60-minute oral, intravenous or other dosing. In one particular example, doses can be administered QD or BID to a subject with, e.g., total daily doses of 1.5 mg/kg, 3.0 mg/kg, or 4.0 mg/kg of a composition with up to 92-98% wt/v of Galeterone and/or a proteasome inhibitor.

Additional useful doses can often range from 0.1 to 5 .mu.g/kg or from 0.5 to 1 .mu.g/kg. In other examples, a dose can include 1 .mu.g/kg, 5 .mu.g/kg, 10 .mu.g/kg, 15 .mu.g/kg, 20 .mu.g/kg, 25 .mu.g/kg, 30 .mu.g/kg, 35 .mu.g/kg, 40 .mu.g/kg, 45 .mu.g/kg, 50 .mu.g/kg, 55 .mu.g/kg, 60 .mu.g/kg, 65 .mu.g/kg, 70 .mu.g/kg, 75 .mu.g/kg, 80 .mu.g/kg, 85 .mu.g/kg, 90 .mu.g/kg, 95 .mu.g/kg, 100 .mu.g/kg, 150 .mu.g/kg, 200 .mu.g/kg, 250 .mu.g/kg, 350 .mu.g/kg, 400 .mu.g/kg, 450 .mu.g/kg, 500 .mu.g/kg, 550 .mu.g/kg, 600 .mu.g/kg, 650 .mu.g/kg, 700 .mu.g/kg, 750 .mu.g/kg, 800 .mu.g/kg, 850 .mu.g/kg, 900 .mu.g/kg, 950 .mu.g/kg, 1000 .mu.g/kg, 0.1 to 5 mg/kg or from 0.5 to 1 mg/kg. In other examples, a dose can include 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, 150 mg/kg, 200 mg/kg, 250 mg/kg, 350 mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 550 mg/kg, 600 mg/kg, 650 mg/kg, 700 mg/kg, 750 mg/kg, 800 mg/kg, 850 mg/kg, 900 mg/kg, 950 mg/kg, 1000 mg/kg, or more.

Doses referred to herein can include Galeterone and a proteasome inhibitor collectively or individually.

Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., hourly, every 2 hours, every 3 hours, every 4 hours, every 6 hours, every 9 hours, every 12 hours, every 18 hours, daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, or monthly).

Galeterone and a proteasome inhibitor can be administered simultaneously or within a selected time window, such as within 10 minutes, 1 hour, 3 hour, 10 hour, 15 hour, 24 hour, or 48 hour time windows or when the complementary active ingredient is within a clinically-relevant therapeutic window.

In particular embodiments, Galeterone and a proteasome inhibitor can be used in conjunction with other cancer treatments. For example, Galeterone and a proteasome inhibitor can be administered in combination with, for example, a gonadotropin-releasing hormone agonist or antagonist (e.g., Lupron, Zoladex (Goserelin), Degarelix, Ozarelix, ABT-620 (Elagolix), TAK-385 (Relugolix), EP-100 or KLH-2109); a phosphoinositide 3-kinase (PI3K) inhibitor, a TORC inhibitor, or a dual PI3K/TORC inhibitor (e.g., BEZ-235, BKM120, BGT226, BYL-719, GDC0068, GDC-0980, GDC0941, GDC0032, MK-2206, OSI-027, CC-223, AZD8055, SAR245408, SAR245409, PF04691502, WYE125132, GSK2126458, GSK-2636771, BAY806946, PF-05212384, SF1126, PX866, AMG319, ZSTK474, Cal101, PWT33597, LY-317615 (enzastaurin hydrochloride), CU-906, or CUDC-907); a CYP17 inhibitor in addition to Galeterone (e.g., abiraterone acetate (Zytiga), TAK-700 (orteronel), or VT-464); prednisone; an osteoprotective agent; a radiation therapy; a kinase inhibitor (e.g. MET, VEGFR, EGFR, MEK, SRC, AKT, RAF, FGFR, CDK4/6); Provenge, Prostvac, Ipilimumab, a PD-1 inhibitor; a taxane or tubulin inhibitor; an anti-STEAP-1 antibody; a heat shock protein 90 (HSP90) or heat shock protein 27 (HSP27) pathway modulator; an anti-androgen (e.g. bicalutamide); and/or immunotherapy.

As suggested, the active ingredients of combination therapies disclosed herein can be formulated into compositions. Each active ingredient can be formulated into its own composition for administration or active ingredients can be formulated into the same composition.

For injection, compositions can be formulated as aqueous solutions, such as in buffers including Hanks' solution, Ringer's solution, or physiological saline. The aqueous solutions can contain formulatory agents such as suspending, stabilizing, and/or dispersing agents. Examples of suitable aqueous and non-aqueous carriers, which may be employed in the injectable formulations 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 ethyloleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of selected particle size in the case of dispersions, and by the use of surfactants.

Injectable formulations may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the compositions.

Alternatively, the composition can be in lyophilized form and/or provided in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. Lyophilized compositions can include less than 5% water content; less than 4.0% water content; or less than 3.5% water content.

In particular embodiments, the composition can be in a unit dosage form, such as in a suitable diluent in sterile, hermetically sealed ampoules or sterile syringes.

In particular embodiments, in order to prolong the effect of a composition, it is desirable to slow the absorption of the active ingredient(s) following injection. Compositions can be formulated as sustained-release systems utilizing semipermeable matrices of solid polymers containing at least one administration form. Various sustained-release materials have been established and are well known by those of ordinary skill in the art. Sustained-release systems may, depending on their chemical nature, release active ingredients following administration for a few weeks up to over 100 days.

In particular embodiments, delayed absorption can be accomplished by dissolving or suspending the active ingredient(s) in an oil vehicle. In particular embodiments, administration forms can be formulated as depot preparations. Depot preparations can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salts. In addition, prolonged absorption of the injectable composition may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

Injectable depot forms can be made by forming microencapsule matrices of administration forms in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of administration form to polymer, and the nature of the particular polymer employed, the rate of administration form release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable depot formulations are also prepared by entrapping the active ingredient(s) in liposomes or microemulsions which are compatible with body tissue.

Alternatively, delayed absorption of a composition can be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the active ingredient(s) then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form.

Compositions can also be administered with anesthetics including ethanol, bupivacaine, chloroprocaine, levobupivacaine, lidocaine, mepivacaine, procaine, ropivacaine, tetracaine, desflurane, isoflurane, ketamine, propofol, sevoflurane, codeine, fentanyl, hydromorphone, marcaine, meperidine, methadone, morphine, oxycodone, remifentanil, sufentanil, butorphanol, nalbuphine, tramadol, benzocaine, dibucaine, ethyl chloride, xylocaine, and/or phenazopyridine.

Compositions can also be formulated for oral administration. For ingestion, compositions can take the form of tablets, pills, lozenges, sprays, liquids, and capsules formulated in conventional manners. Ingestible compositions can be prepared using conventional methods and materials known in the pharmaceutical art. For example, U.S. Pat. Nos. 5,215,754 and 4,374,082 relate to methods for preparing swallowable compositions. U.S. Pat. No. 6,495,177 relates to methods to prepare chewable supplements with improved mouthfeel. U.S. Pat. No. 5,965,162, relates to compositions and methods for preparing comestible units which disintegrate quickly in the mouth.

Ingestible compositions may have a shape containing no sharp edges and a smooth, uniform and substantially bubble free outer coating. Coatings of ingestible compositions can be derived from a polymeric film. Such film coatings reduce the adhesion of the compositions to the inner surface of the mouth and can aid in masking potential unpleasant tastes. Coatings can also protect the compositions from atmospheric degradation. Exemplary polymeric films include vinyl polymers, cellulosics, acrylates and methacrylates, natural gums and resins such as zein, gelatin, shellac and acacia. Other common excipients used in ingestible compositions include sucrose, fructose, lactose, glucose, lycasin, xylitol, lactitol, erythritol, mannitol, isomaltose, dextrose, polydextrose, dextrin, compressible cellulose, compressible honey, compressible molasses, fondant or gums, vegetable oils, animal oils, alkyl polysiloxanes, corn starch, potato starch, pre-gelatinized starches, stearic acid, calcium stearate, magnesium stearate, zinc stearate, benzoic acid, and colorants

For administration by inhalation (e.g., nasal or pulmonary), the compositions can be formulated as aerosol sprays for pressurized packs or a nebulizer, with the use of suitable propellants, e.g. dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetra-fluoroethane.

As suggested, nanoparticle formulations for a variety of administration routes can also be used.

Any composition disclosed herein can advantageously include any other pharmaceutically acceptable carriers which include those that do not produce significantly adverse, allergic, or other untoward reactions that outweigh the benefit of administration, whether for research, prophylactic, and/or therapeutic treatments. Exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Moreover, formulations can be prepared to meet sterility, pyrogenicity, general safety, and purity standards as required by U.S. FDA Office of Biological Standards and/or other relevant foreign regulatory agencies.

Exemplary generally used pharmaceutically acceptable carriers include any and all bulking agents or fillers, solvents or co-solvents, dispersion media, coatings, surfactants, antioxidants (e.g., ascorbic acid, methionine, vitamin E), preservatives, isotonic agents, absorption delaying agents, salts, stabilizers, buffering agents, chelating agents (e.g., EDTA), gels, binders, disintegration agents, and/or lubricants. Fillers and excipients are commercially available from companies such as Aldrich Chemical Co., FMC Corp, Bayer, BASF, Alexi Fres, Witco, Mallinckrodt, Rhodia, ISP, and others.

In particular embodiments, the compositions can include, for example, 25 .mu.g/mL or mg-5 mg/mL or mg, 50 .mu.g/mL or mg-5 mg/mL or mg, 100 .mu.g/mL or mg-5 mg/mL or mg, 150 .mu.g/mL or mg-5 mg/mL or mg, 200 .mu.g/mL or mg-5 mg/mL or mg, 250 .mu.g/mL or mg-5 mg/mL or mg, 300 .mu.g/mL or mg-5 mg/mL or mg, 350 .mu.g/mL or mg-5 mg/mL or mg, 400 .mu.g/mL or mg-5 mg/mL or mg, 450 .mu.g/mL or mg-5 mg/mL or mg, 500 .mu.g/mL or mg-5 mg/mL or mg, 550 .mu.g/mL or mg-5 mg/mL or mg, 600 .mu.g/mL or mg-5 mg/mL or mg, 650 .mu.g/mL or mg-5 mg/mL or mg, 700 .mu.g/mL or mg-5 mg/mL or mg, 750 .mu.g/mL or mg-5 mg/mL or mg, 800 .mu.g/mL or mg-5 mg/mL or mg, 850 .mu.g/mL or mg-5 mg/mL or mg, 900 .mu.g/mL or mg-5 mg/mL or mg, 950 .mu.g/mL or mg-5 mg/mL or mg, 1 mg/mL or mg-5 mg/mL or mg, 1.5 mg/mL or mg-5 mg/mL or mg, 2 mg/mL or mg-5 mg/mL or mg, 2.5 mg/mL or mg-5 mg/mL or mg, 3 mg/mL or mg-5 mg/mL or mg, 3.5 mg/mL or mg-5 mg/mL or mg, 4 mg/mL or mg-5 mg/mL or mg, 4.5 mg/mL or mg-5 mg/mL or mg, 25 .mu.g/mL or mg-2.5 mg/mL or mg, 50 .mu.g/mL or mg-2.5 mg/mL or mg, 100 .mu.g/mL or mg-2.5 mg/mL or mg, 150 .mu.g/mL or mg-2.5 mg/mL or mg, 200 .mu.g/mL or mg-2.5 mg/mL or mg, 250 .mu.g/mL or mg-2.5 mg/mL or mg, 300 .mu.g/mL or mg-2.5 mg/mL or mg, 350 .mu.g/mL or mg-2.5 mg/mL or mg, 400 .mu.g/mL or mg-2.5 mg/mL or mg, 450 .mu.g/mL or mg-2.5 mg/mL or mg, 500 .mu.g/mL or mg-2.5 mg/mL or mg, 550 .mu.g/mL or mg-2.5 mg/mL or mg, 600 .mu.g/mL or mg-2.5 mg/mL or mg, 650 .mu.g/mL or mg-2.5 mg/mL or mg, 700 .mu.g/mL or mg-2.5 mg/mL or mg, 750 .mu.g/mL or mg-2.5 mg/mL or mg, 800 .mu.g/mL or mg-2.5 mg/mL or mg, 850 .mu.g/mL or mg-2.5 mg/mL or mg, 900 .mu.g/mL or mg-2.5 mg/mL or mg, 950 .mu.g/mL or mg-2.5 mg/mL or mg, 1 mg/mL or mg-2.5 mg/mL or mg, 1.5 mg/mL or mg-2.5 mg/mL or mg, 2 mg/mL or mg-2.5 mg/mL or mg, 25 .mu.g/mL or mg-1 mg/mL or mg, 50 .mu.g/mL or mg-1 mg/mL or mg, 100 .mu.g/mL or mg-1 mg/mL or mg, 150 .mu.g/mL or mg-1 mg/mL or mg, 200 .mu.g/mL or mg-1 mg/mL or mg, 250 .mu.g/mL or mg-1 mg/mL or mg, 300 .mu.g/mL or mg-1 mg/mL or mg, 350 .mu.g/mL or mg-1 mg/mL or mg, 400 .mu.g/mL or mg-1 mg/mL or mg, 450 .mu.g/mL or mg-1 mg/mL or mg, 500 .mu.g/mL or mg-1 mg/mL or mg, 550 .mu.g/mL or mg-1 mg/mL or mg, 600 .mu.g/mL or mg-1 mg/mL or mg, 650 .mu.g/mL or mg-1 mg/mL or mg, 700 .mu.g/mL or mg-1 mg/mL or mg, 750 .mu.g/mL or mg-1 mg/mL or mg, 800 .mu.g/mL or mg-1 mg/mL or mg, 850 .mu.g/mL or mg-1 mg/mL or mg, 900 .mu.g/mL or mg-1 mg/mL or mg, 950 .mu.g/mL or mg-1 mg/mL or mg, 25 .mu.g/mL or mg-750 .mu.g/mL or mg, 50 .mu.g/mL or mg-750 .mu.g/mL or mg, 100 .mu.g/mL or mg-750 .mu.g/mL or mg, 150 .mu.g/mL or mg-750 .mu.g/mL or mg, 200 .mu.g/mL or mg-750 .mu.g/mL or mg, 250 .mu.g/mL or mg-750 .mu.g/mL or mg, 300 .mu.g/mL or mg-750 .mu.g/mL or mg, 350 .mu.g/mL or mg-750 .mu.g/mL or mg, 400 .mu.g/mL or mg-750 .mu.g/mL or mg, 450 .mu.g/mL or mg-750 .mu.g/mL or mg, 500 .mu.g/mL or mg-750 .mu.g/mL or mg, 550 .mu.g/mL or mg-750 .mu.g/mL or mg, 600 .mu.g/mL or mg-750 .mu.g/mL or mg L, 650 .mu.g/mL or mg-750 .mu.g/mL or mg, 700 .mu.g/mL or mg-750 .mu.g/mL or mg, 25 .mu.g/mL or mg-500 .mu.g/mL or mg, 50 .mu.g/mL or mg-500 .mu.g/mL or mg, 100 .mu.g/mL or mg-500 .mu.g/mL or mg, 150 .mu.g/mL or mg-500 .mu.g/mL or mg, 200 .mu.g/mL or mg-500 .mu.g/mL or mg, 250 .mu.g/mL or mg-500 .mu.g/mL or mg, 300 .mu.g/mL or mg-500 .mu.g/mL or mg, 350 .mu.g/mL or mg-500 .mu.g/mL or mg, 400 .mu.g/mL or mg-500 .mu.g/mL or mg, 450 .mu.g/mL or mg-500 .mu.g/mL or mg, 25 .mu.g/mL or mg-250 .mu.g/mL or mg, 50 .mu.g/mL or mg-250 .mu.g/mL or mg, 100 .mu.g/mL or mg-250 .mu.g/mL or mg, 150 .mu.g/mL or mg-250 .mu.g/mL or mg, 200 .mu.g/mL or mg-250 .mu.g/mL or mg, 25 .mu.g/mL or mg-100 .mu.g/mL or mg, or 50 .mu.g/mL or mg-100 .mu.g/mL or mg of one or more of the active ingredients.

In particular embodiments, ratios of active ingredients can include: (Galeterone:proteasome inhibitor (and whether in the same or different compositions)): 1:0.0001; 1:0.001; 1:0.005; 1:0.0075; 1:0.01; 1:0.05; 1:0.075; 1:0.1; 1:0.5; 1:0.75; 1:1; 1:1.25; 1:1.5; 1:1.75; 1:8; 1:1.2; 1:1.25; 1:1.3; 1:1.35; 1:1.4; 1:1.5; 1:1.75; 1:2; 1:3; 1:4; 1:5; 1:6:1:7; 1:8; 1:9; 1:10; 1:15; 1:20; 1:30; 1:40; 1:50; 1:60; 1:70; 1:80; 1:90; 1:100; 1:200; 1:300; 1:400; 1:500; 1:600; 1:700; 1:800; 1:900; 1:1000.

The current disclosure also provides diagnostics for CRPC treatment efficacy including systems and methods to monitor the anti-cancer effects of a CRPC therapeutic. As described herein, therapeutically effective doses of active ingredients, such as Galeterone and/or Galeterone in combination with a proteasome inhibitor results in the anti-cancer effect of cleavage of androgen receptors (ARs), including the AR-V7 variant. Detection of AR cleavage products, therefore, is indicative of an anti-cancer effect following active ingredient(s) administration. In particular embodiments, the diagnostics disclosed herein detect (e.g., measure and/or determine) levels of AR-V7 fragments, including 53 kDa and 41 kDa AR-V7 fragments. An up-regulation of AR fragments demonstrates (e.g., is indicative of) an anti-cancer effect.

In particular embodiments, the diagnostics disclosed herein detect PARP fragments and/or Spectrin .alpha.2 fragments in addition to or instead of AR fragments. The PARP fragments can be 65 kDa (p65) and 89 kDa (p89), and the Spectrin .alpha.2 fragments can be 120 kDa (p120) and 150 kDa (p150i or p150). Without being bound by theory, Galeterone treatment activates caspase-3 which produces PARP fragments of 89 kDa (PARP/p89) and Spectrin .alpha.2 fragments of 120 kDa (Spectrin .alpha.2/p120). Galeterone treatment activates caspase-3 and calpain which produces Spectrin .alpha.2 fragments of 150 kDa (Spectrin .alpha.2/p150i or p150). Galeterone treatment also activates calpain which produces PARP fragments of 65 kDa (p65). Thus, an up-regulation of these fragments is indicative of and can be used to monitor treatment efficacy.

The diagnostics disclosed herein include detecting AR fragments, PARP fragments, and/or spectrin .alpha.2 from subject samples. The detection can be qualitative or quantitative. Detected AR cleavage products can be compared to a reference level.

Methods of detecting AR, PARP and Spectrin .alpha.2 cleavage products (e.g. fragments) are well known in the art. In particular embodiments, the fragments levels can be detected using techniques for measuring and determining the serum levels of proteins. Such methods are well-known in the art and include, e.g., immunoassays based on antibodies to proteins encoded by the genes, aptamers, or molecular imprints (e.g., Western blots and ELISA). Alternatively, a suitable method can be selected to determine the activity of proteins. Such assays include protease assays, kinase assays, phosphatase assays, and reductase assays, among many others.

In embodiments, the detection of the cleavage products of AR, PARP, and/or Spectrin .alpha.2 in circulating tumor cells (in blood) can be used as to predict resistance to enzalutamide, abiraterone, and other AR-targeted agents. A Western blot assay can be used to detect the blood levels of AR or AR-V7 cleavage fragments, PARP, and/or Spectrin .alpha.2 that could then be used in selection criteria to monitor and select patients in a clinical trial as well as to monitor the activity of Gal and proteasome inhibitor.

In particular embodiments, a "reference level" can refer to a standardized value for AR, AR-V (e.g., AR-V7), PARP, and/or Spectrin .alpha.2 cleavage product biomarkers which represents a level not associated with any disease; a level associated with a particular stage of the disease (i.e., hormone dependent or hormone refractory (CRPC)); or a level associated with a particular subject at the time of diagnosis, at the beginning of treatment, or at a time point during a treatment, such as at a time point following Galeterone and proteasome inhibitor administration. The reference level can be a universal reference level which is useful across a variety of testing locations or can be a reference level specific for the testing location and specific assay used to measure the cleavage product biomarkers. In particular embodiments, the reference level is derived from (i) an individual who does not have prostate cancer; (ii) a group of individuals who do not have prostate cancer; (iii) a subject before diagnosis of prostate cancer; or (iv) a subject at the time of diagnosis, at the beginning of a treatment regimen for prostate cancer or at particular time points during a treatment. Reference levels for a subject can also be related to time points of a subject not undergoing treatments to monitor the natural progression or regression of the disease.

In particular embodiments, the levels of AR, AR-V (e.g., AR-V7), PARP, and/or Spectrin .alpha.2 cleavage product biomarkers, can be determined sequentially over time. In particular embodiments, the cleavage product biomarker levels can be determined 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times and every remaining integer up to 100 times or more. In a subject at risk of having CRPC, the cleavage product biomarkers, levels can be determined weekly, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, or every 11 months, or yearly to determine if the subject has prostate cancer or to determine if a treatment has been effective or ineffective, or a prostate cancer is progressing or regressing (i.e., each measure can provide an intra-subject reference level).

In a subject undergoing treatment for prostate cancer, the AR, AR-V (e.g., AR-V7), PARP, and/or Spectrin .alpha.2 cleavage product biomarker levels can be determined weekly, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, or every 11 months, or yearly to determine if the prostate cancer in the subject has progressed, has regressed, or has been successfully or unsuccessfully treated. In particular embodiments, a single determination of AR and/or AR-V7 cleavage product, PARP, and/or Spectrin .alpha.2 biomarkers is used in the disclosed methods.

Diagnostics disclosed herein may be particularly useful to predict when a subject may become refractory to hormone treatments.

In particular embodiments, the biomarkers are measured and determined from a biological sample such as blood, plasma, or serum. The blood sample could contain peripheral blood.

Reductions in the described measures can also indicate spontaneous remission of the disease and/or the effectiveness of a treatment regimen.

As used herein, "unchanged" measures are evaluated in relation to a previous comparison in the same subject and denote a failure to achieve a statistically significant change in a score towards or away from a reference level in the particular subject.

The present disclosure further provides for kits including one or more treatment options (e.g., combination therapies) and/or diagnostic assays for practicing any of the methods disclosed herein. The kits may include a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, biological products, lab developed tests, etc., which notice reflects approval by the agency of the manufacture, use or sale for human administration and/or testing. Treatment portions of the kits may include active ingredient(s) in a ready-to-use form and/or a form that requires preparation before administration (e.g., lyophilized). Diagnostic portions of the kits may utilize any necessary or appropriate polypeptides, conjugates, antibodies, polynucleotides, expression vectors, cells, methods, compositions, systems, and/or apparatuses useful for the detection of AR or AR-V fragments such as the 53 kDa and/or the 41 kDa fragment of AR-V7, PARP, and/or Spectrin .alpha.2. The kits may also include syringes, pipettes, antiseptics, tubing, gloves, diluents, etc. as well as instructions for practicing any method described herein which may include relevant reference levels. Optionally reference levels are determined using the detection kits.

In particular embodiments of the kits, the kit includes one of more diagnostic assays for detecting intact AR-V7 and AR-V7 fragments such as the 53 kDa and/or the 41 kDa fragments. In particular embodiments of the kits, the kit includes one of more diagnostic assays for detecting PARP cleavage products including 89 kDa and 65 kDa PARP fragments, and Spectrin .alpha.2 cleavage products including 120 kDa and 150 kDa Spectrin .alpha.2 fragments.

In particular embodiments, the kit includes three or more diagnostic assays, four or more diagnostic assays, five or more diagnostic assays, six or more diagnostic assays, seven or more diagnostic assays, or eight or more diagnostic assays.

The Exemplary Embodiments and Example below are included to demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

Exemplary Embodiments

1. A method of providing an anti-cancer effect in a subject having cancer (e.g., castration-resistant prostate cancer (CRPC) or multiple myeloma (MM)) comprising administering therapeutically effective amounts of Galeterone and a proteasome inhibitor to the subject, thereby providing an anti-cancer effect in the subject. 2. A method of embodiment 1 wherein Galeterone has the structure

##STR00003## 3. A method of embodiment 1 or 2 wherein the proteasome inhibitor is selected from .beta.-lapachone, bortezomib, brotezamide, carfilzomib, CEP-18770, disulfiram, epigallocatechin-3-gallate, epoxomicin, lactacystin, laxomib (MLN9708), MG132, MLN9708, oprozomib (ONX 0912), salinosporamide A (NPI-0052, marizomib), or an immunoproteasome inhibitor. 4. A method of embodiment 3 wherein the proteasome inhibitor is bortezomib. 5. A method of potentiating the anti-cancer effect of Galeterone comprising administering a therapeutically effective amount of a proteasome inhibitor in combination with a therapeutically effective amount of Galeterone to a subject in need thereof. 6. A method of embodiment 5 wherein the subject in need thereof has castration-resistant prostate cancer (CRPC) or multiple myeloma (MM). 7. A method of embodiment 5 or 6 wherein Galeterone has the structure

##STR00004## 8. A method of any of embodiments 5-7 wherein the proteasome inhibitor is selected from .beta.-lapachone, bortezomib, brotezamide, carfilzomib, CEP-18770, disulfiram, epigallocatechin-3-gallate, epoxomicin, lactacystin, laxomib (MLN9708), MG132, MLN9708, oprozomib (ONX 0912), salinosporamide A (NPI-0052, marizomib), or an immunoproteasome inhibitor. 9. A method of embodiment 8 wherein the proteasome inhibitor is bortezomib. 10. A composition comprising Galeterone and a proteasome inhibitor. 11. A composition of embodiment 10 wherein Galeterone has the structure

##STR00005## 12. A composition of embodiment 10 or 11 wherein the proteasome inhibitor is selected from .beta.-lapachone, bortezomib, brotezamide, carfilzomib, CEP-18770, disulfiram, epigallocatechin-3-gallate, epoxomicin, lactacystin, laxomib (MLN9708), MG132, MLN9708, oprozomib (ONX 0912), salinosporamide A (NPI-0052, marizomib), or an immunoproteasome inhibitor. 13. A composition of embodiment 12 wherein the proteasome inhibitor is bortezomib. 14. A method of monitoring an anti-cancer effect of a castration-resistant prostate cancer (CRPC) therapeutic composition following its administration to a subject comprising detecting an androgen receptor (AR, e.g. SEQ ID NOs: 1-2) cleavage product, a Poly (ADP-ribose) polymerase (PARP, e.g. SEQ ID NO: 4) cleavage product, and/or a Spectrin all (e.g. SEQ ID NO: 3) cleavage product in a sample obtained from the subject following the administration wherein presence of the AR cleavage product, PARP cleavage product, and/or Spectrin .alpha.II cleavage product is indicative of the anti-cancer effect. 15. A method of embodiment 14 wherein the AR cleavage product is an AR variant (e.g. SEQ ID NO: 2) cleavage product. 16. A method of embodiment 15 wherein the AR variant cleavage product is an AR-variant 7 (AR-V7) cleavage product. 17. A method of embodiment 16 wherein the AR-V7 cleavage product is a 53 kDa or a 41 kDa AR-V7 fragment. 18. A method of any of embodiments 14-17 wherein the PARP cleavage product is PARP/p65 and/or PARP/p89. 19. A method of any of embodiments 14-18 wherein the Spectrin .alpha.II cleavage product is Spectrin .alpha.II/p120 and/or Spectrin .alpha.II/p150 or 150i. 20. A method of monitoring an anti-cancer effect of a castration-resistant prostate cancer (CRPC) therapeutic composition following its administration to a subject comprising detecting a PARP cleavage product (e.g. cleavage product of SEQ ID NO: 4) and/or a Spectrin .alpha.2 cleavage product (e.g. cleavage product of SEQ ID NO: 3) in a sample obtained from the subject following the administration wherein presence of the PARP cleavage product and/or Spectrin .alpha.2 cleavage product is indicative of the anti-cancer effect. 21. The method of embodiment 20, wherein the PARP cleavage product is PARP/p65 and/or PARP/p89. 22. The method of embodiment 20 or 21, wherein the Spectrin .alpha.2 cleavage product is Spectrin .alpha.2/p120 and/or Spectrin .alpha.2/p150 or p150i. 23. A kit for practicing a method of any of embodiments 1-9 or 14-22, alone or in combination. 24. A kit comprising a composition of any of embodiments 10-13, alone or in combination. 25. A method to target 19S proteasome-associated DUBs and 20s proteasome comprising administering therapeutically effective amounts of Galeterone and a proteasome inhibitor in vivo or in vitro thereby targeting 19S proteasome-associated DUBs with Galeterone and 20s proteasome with the proteasome inhibitor. 26. A method of embodiment 25 wherein Galeterone has the structure

##STR00006## 27. A method of embodiment 25 or 26 wherein the proteasome inhibitor is selected from .beta.-lapachone, bortezomib, brotezamide, carfilzomib, CEP-18770, disulfiram, epigallocatechin-3-gallate, epoxomicin, lactacystin, laxomib (MLN9708), MG132, MLN9708, oprozomib (ONX 0912), salinosporamide A (NPI-0052, marizomib), or an immunoproteasome inhibitor. 28. A method of embodiment 27 wherein the proteasome inhibitor is bortezomib. 29. A method to target DUBs in solid and/or liquid tumors comprising administering a therapeutically effective amount of Galeterone to a subject in need thereof thereby targeting DUBs in solid and/or liquid tumors. 30. A method of embodiment 29 wherein the targeting provides an anti-cancer effect. 31. A method of embodiment 29 or 30 wherein Galeterone has the structure

##STR00007## 32. A method of any of embodiments 29-31 further comprising administering a therapeutically effective amount of a proteasome inhibitor. 33. A method of embodiment 32 wherein the proteasome inhibitor is selected from .beta.-lapachone, bortezomib, brotezamide, carfilzomib, CEP-18770, disulfiram, epigallocatechin-3-gallate, epoxomicin, lactacystin, laxomib (MLN9708), MG132, MLN9708, oprozomib (ONX 0912), salinosporamide A (NPI-0052, marizomib), or an immunoproteasome inhibitor. 34. A method of embodiment 33 wherein the proteasome inhibitor is bortezomib.

Example 1

Background: Abiraterone (Abi) acetate and Enzalutamide (Enz) are two newly FDA-approved antiandrogen receptor (anti-AR) agents for treating castrate-resistant prostate cancer (CRPC) (1-3).

Abiraterone (Abi) is a steroidal antiandrogen, specifically an androgen synthesis inhibitor, used in combination with prednisone in metastatic CRPC. After an expedited six-month review, abiraterone acetate was approved by the US Food and Drug Administration (FDA) in April 2011. In phase III clinical trials, it extended median survival to 14.8 months versus 10.9 months placebo, and the trial was stopped early because of the successful outcome.

Abi acetate, an orally active agent, is converted in vivo to Abi, a specific and irreversible inhibitor of CYP17, resulting in significant decrease in testosterone levels (1-3). This is because CYP17 catalyzes two sequential reactions: (a) the conversion of pregnenolone and progesterone to their 17.alpha.-hydroxy derivatives by its 17.alpha.-hydroxylase activity, and (b) the subsequent formation of dehydroepiandrosterone (DHEA) and androstenedione, respectively, by its 17,20-lyase activity. DHEA and androstenedione are androgens and precursors of testosterone. Inhibition of CYP17 activity by abiraterone thus decreases circulating levels of androgens such as DHEA, testosterone, and dihydrotestosterone (DHT).

Enzalutamide (Enz) is a synthetic, non-steroidal pure antiandrogen which was developed by the pharmaceutical company Medivation for the treatment of metastatic CRPC. It was FDA-approved in August, 2012. PSA level decreased more than 50% in 40/65 chemo-naive patients and 38/75 chemotherapy-treated patients. In November 2011, this trial was stopped early after an interim analysis revealed that patients given the drug lived for approximately 5 months longer than those taking placebo.

Although both Abi and Enz have shown impressive results, resistance in CRPC patients has already been observed (1-3). One of the major resistance mechanisms is related to constitutively active forms of ARs with C-terminal loss or lacking of a functional LBD (4). AR-V7 is the most common form of C-terminal loss (4).

Galeterone (TOK-001 or VN/124-1) is a novel steroidal antiandrogen under development by Tokai Pharmaceuticals for the treatment of prostate cancer. Galeterone acts by disrupting the androgen receptor signaling pathway. This pathway is activated by the binding of male hormones (also known as androgens), such as testosterone and dihydrotestosterone (DHT) to androgen receptors in prostate cancer cells. Galeterone disrupts the activation of the androgen receptor pathway in three ways: Androgen receptor degradation, CYP17 enzyme inhibition and inhibition of androgen binding to the androgen receptor.

Galeterone has been tested in a Phase 2 clinical trial (ARMOR2) as a treatment for patients with CRPC (6-7). It has been found that Abi- and Enz refractory CRPC patients with C-terminal AR loss remain responsive to Gal (6-7). A Gal Phase 3 trial, the first precision medicine-based trial in AR-V7-positive metastatic CRPC patients started in the summer of 2015.

Gal has been shown to induce degradation of AR and AR-V7 proteins under preclinical conditions (8). Further, it was suggested that Gal-induced AR-V7 (and AR) protein depletion was through degradation by proteasomes (9), for which one of the supporting evidence is that Gal-induced AR-V7 degradation could be inhibited by 5 .mu.M of MG132 (9). However, MG132 at 5 .mu.M can inhibit both proteasome (IC.sub.50=100 nM) and calpain (IC.sub.50=1.2 .mu.M) activities (10-11). Therefore Gal's mechanism of action requires careful investigation.

The medicinal compound Celastrol, which has a chemical structure related to that of Gal, is a potent proteasome inhibitor which suppresses human PC growth in nude mice, associated with decreased AR protein (12; the Cancer Res Cover Story). Celastrol and other proteasome inhibitors can decrease levels of AR protein in prostate cancer cells via activation of a calpain-like activity (13). In the study described below, the effect of Gal on the proteasome, calpain and AR-V7 protein in CRPC cells was examined.

It was first confirmed that VCaP and 22Rv1 CRPC cells express high AR-V7 protein (14-15), compared to LNCaP and C4-2B cells (FIG. 1A) and that treatment of 20 .mu.M Gal for 66-72 h decreased AR-V7 protein expression (FIG. 1B, lanes 2 vs. 1, and 6 vs. 5) (8-9).

Gal treatment causes activation of calpains and caspases prior to the decrease in AR-V7 protein expression. Detailed kinetics of Gal-induced AR-V7 protein depletion (FIG. 2A) was investigated next. 22Rv1 cells were treated with 20 .mu.M of Gal for up to 72 h, followed by Western blotting with specific anti-AR-V7 monoclonal antibody (Precision, AG10008, that can selectively detect AR-V7 protein) and anti-AR N20 antibody (that can recognize both full length AR, AR-V7 and other AR-V proteins). Levels of full length AR (AR-FL) protein were decreased after 48 h; however, AR-V7 protein expression remained high after up to 56 h and decreased significantly between 64-68 h, and became undetectable at 72 h (FIG. 2A, a).

Possible activation of caspases and calpains in these CRPC cells during Gal treatment was investigated. As shown in FIG. 2B, caspase-3 activity was increased after 24 h and reached to the highest after 48 h. Consistently, the production of caspase-3-specific cleavage substrates, PARP fragment of 89 kDa (PARP/p89) and Spectrin .alpha.2/p120 (16) were detected along with caspase-3 activation (FIG. 2B, c-d). Both caspase-3 and calpain can also cleave Spectrin .alpha.2 into fragments with a similar size, 150 kDa (p150i or p150) (FIG. 2A, d; 16).

Calpain activation is associated with the production of a 28 kDa cleavage fragment (p28) of calpain small subunit (17). It was found that in Gal-treated cells, a p28 fragment appeared between 48 and 72 h, as detected by a specific antibody to the 30 kDa calpain small subunit (FIG. 2A, e). Consistently, another calpain cleavage product, Calpastatin/p70 (18) was detected at the same time points (FIG. 2A, f). It has been shown that calpain cleaves PARP into a 65 kDa fragment (19); two peaks of PARP/p65, at approximately 4 and 68 hours, respectively (FIG. 2A, c) were found, which suggests early and late calpain activations. These results show that Gal induces activation of both caspase-3 and (early) calpain prior to AR-V7 depletion.

Gal-induced AR-V7 loss can be blocked by a specific inhibitor to calpain or caspases. Whether calpains and/or caspases are involved in Gal-induced AR-V7 degradation was further determined. As shown in FIG. 1B, addition of any one of the three specific calpain inhibitors, calpeptin (Z-Leu-Nle-CHO), PD150606 or N-acetyl-L-leucyl-L-leucyl-L-methioninal (ALLM), was able to rescue AR-V7 loss partially (FIG. 1B, lanes 1-7). Furthermore, a pan-caspase inhibitor was able to suppress Gal-induced AR-V7 decrease (FIG. 1B; lanes 8 vs. 6). These results support the contribution of calpains & caspases to AR-V7 protein depletion by Gal treatment.

The FDA-approved, specific 20S proteasome inhibitor Bortezomib is unable to block, rather increasing Gal-induced AR-V7 degradation. To confirm the involvement of proteasomes in Gal-induced AR-V7 protein degradation (8-9), Bortezomib (BTZ), the first FDA approved specific 20S proteasome inhibitor (20) was used. Reproducibly, 72 h treatment with 20 .mu.M Gal caused loss of AR-V7 protein in 22Rv1 cells (FIG. 3A, lanes 2 vs. 1; also see FIGS. 1B, 2A). However, addition of BTZ at up to 40 nM did not rescue the loss of AR-V7 protein by Gal (FIG. 3A, lanes 3-6 vs. 2 vs. 1); as a control, BTZ alone caused a dose-dependent inhibition of proteasomal chymotrypsin (CT)-like activity (FIG. 3B, DMSO bars). In addition to the inability to inhibit Gal-mediated AR-V7 protein degradation, BTZ alone (without Gal) caused a dose-dependent decrease in AR-V7 protein expression (FIG. 3A, lanes 7-10 vs. 1), demonstrating that specific proteasome inhibition leads to AR-V7 protein depletion.

FIG. 2A shows that Gal treatment induced AR-V7 protein depletion after 64 h, but not 48 h or earlier. It was then investigated, under such a condition, whether BTZ could sensitize the CRPC cells to Gal by increasing AR-V7 protein degradation. While again Gal treatment for 48 h was not sufficient to decrease expression of AR-V7 protein (FIG. 3C, lanes 2 vs. 1), addition of BTZ, especially at 20 nM to Gal-treated cells resulted in a dose-dependent AR-V7 protein depletion (FIG. 3C, lanes 3-6 vs. 2 vs. 1). As a control, BTZ treatment for 48 h caused proteasomal CT-like inhibition (FIG. 3D, DMSO bars). Therefore, the proteasome inhibitor BTZ not only caused AR-V7 depletion but also increased Gal-induced AR-V7 degradation, suggesting the beneficial use of combination therapies of Gal and second generation proteasome inhibitors for CRPC patients. Consistently, in the same experiment, increased levels of PARP cleavage were detected in the combination treated groups,

Gal treatment of CRPC cells causes a reduction in levels of all three proteasome activities. Since both Gal and BTZ decrease levels of AR-V7 protein expression (FIGS. 1-3; refs. 8-9), whether Gal treatment alone affects levels of cellular proteasome activities in CRPC cells was examined. Exposure of 22Rv1 cells to 5-20 .mu.M Gal for 24 h resulted in a dose-dependent decrease in levels of proteasomal CT-like and peptidyl-glutamyl peptidehydrolyzing (PGPH)-like, but not trypsin (T)-like activities (FIG. 4A; 100 nM BTZ as a control); after 48 h treatment, all three proteasomal activities were decreased in a Gal dose-dependent fashion (FIG. 4B). When VCaP cells were treated with 5-20 .mu.M Gal for 72 h, a dose-dependent inhibition of the three proteasomal activities was again observed (FIG. 4C). To study how early Gal causes proteasome inhibition, a detailed kinetic experiment in 22Rv1 cells was performed, and it was found that addition of Gal caused a time-dependent proteasome CT inhibition: 10% between 2-24 h, 30% at 48 h and >50% after 64 h (FIG. 4D). Therefore, Gal-induced proteasome inhibition occurs prior to activation of calpain/caspases and loss of AR-V7 protein (FIGS. 4 vs. 2).

Because both Gal and BTZ cause proteasome inhibition (FIG. 4; 20), it was then determined the effect of their combination on CRPC cellular proteasome activities. It was found that the combination of Gal and BTZ resulted in a further inhibition of the proteasomal CT-like activity, compared to each drug alone (FIG. 3B, D), supporting the idea that both Gal and BTZ inhibit the proteasome activity independently.

Cleavage of nuclear AR-V7 protein into fragments of 53 kDa and 41 kDa after Gal treatment: Cellular localizations of AR-V7/AR proteins, calpain/caspase substrate proteins and their cleavage fragments was also examined using cytosolic and nuclear fractions isolated from Gal-treated R22v1 cells (FIG. 5A). By using AR-V7 specific monoclonal antibody, it was observed that, prior to Gal addition, most of AR-V7 protein was in the nuclear fraction (FIG. 5A, a, b, lanes 1 vs. 6). After 8-24 h treatment with Gal, AR-V7 band decreased, associated with an appearance of at least two lower bands of 53 kDa and 41 kDa, both of which were increased at 48 h (FIG. 5A, a, b, lanes 2-4). At 72 h, the nuclear ARV7 was decreased significantly, while mainly the 53 kDa fragment was detected (FIG. 5A, a, b, lane 5). The levels of cytosolic AR-V7 protein were decreased only at 72 h (FIG. 5A, a, b, lanes 6-10). When anti-AR amino terminal (N20) antibody was used (FIG. 5A, c), a decrease in both cytosolic and nuclear AR-FL protein expression was detected after Gal treatment. Anti-N20 antibody also detected several potential AR-Vs in both fractions, all of which were decreased at 72 h (FIG. 5A, c). The caspase 3-specific product PARP/p89 was found in nuclear fraction (FIG. 5A, d), while the calpain cleavage product Calpastatin/p70 was found mainly in the cytosolic fraction (FIG. 5A, g). Spectrin .alpha.2 fragments of 150 kDa (caspase-3 or calpain cleavage fragment) and 120 kDa (caspase-3 cleavage product) as well as calpain small/p28 (calpain cleavage product) were detected in both cytosolic and nuclear fractions (between 24-72 h) (FIG. 5A, e-f). (FIG. 5B-D).

22Rv1 cells were first androgen-starved for 24 h, followed by Gal treatment for up to 56 h and preparation of cellular fractions (FIG. 5B). Consistent with previous report (20), androgen starvation did not change AR-V7 primary nuclear localization (FIG. 5B, a). Nuclear AR-V7 was again decreased in the time-dependent manner. A cleaved band of .about.53 kDa was again detected in the nuclear fraction after 24-48 h treatment (FIG. 5B, a). A similar fragment was also detected by using the anti-AR (N20) antibody between 8 and 48 h (FIG. 5B, b). Along with nuclear AR-V7 decrease was activation of nuclear calpain, evidenced by increased levels of its specific cleavage substrates, Cal Sm/p28 and Calpastatin/p70 (FIG. 5B, d-e). These calpain cleavage fragments were also found in the cytosolic fraction (FIG. 2A, d-e). The caspase-3-specific cleavage fragment PARP/p89 was found only in the nuclear fraction (FIG. 5B, c). These results indicate that calpain and caspase activation are correlated with cleavage of AR-V7 protein in CRPC nuclei.

Inhibitors of calpain and caspases block the nuclear AR-V7 degradation in Gal-treated CRPC cells. Then we tested whether an inhibitor of calpain or caspase could rescue AR-V7 degradation induced by Gal treatment. 22Rv1 cells were treated with Gal in the absence or presence of the specific calpain inhibitor calpeptin or a pan-caspase inhibitor, followed by isolation of cytosolic and nuclear fractions. As shown in FIG. 5C, Gal induced the nuclear AR-V7 depletion after 56 h or 72 h, which could be blocked by addition of calpeptin, a calpain inhibitor at both time points (lanes 4-5 vs. 1-3; a pan-caspase inhibitor IV was able to rescue Gal-induced nuclear AR-V7 loss significantly at 56 h, but only slightly at 72 h (FIG. 5C, lanes 6-7). Similarly, when androgen-starved 22Rv1 cells were used for another inhibitor experiment, Gal treatment decreased levels of nuclear AR-V7 protein after 40, 48 and 64 h (FIG. 5D, lanes 1-4). Addition of Calpeptin partially rescued AR-V7 loss by Gal (FIG. 5D, lanes 5-7), while another pan-caspase inhibitor Z-VAD was able to block the Gal-induced AR-V7 depletion (FIG. 5D, lanes 8-10). The inhibitor experiments further support the involvement of calpain and caspases in the depletion of AR-V7 protein during Gal treatment.

The described results show that Gal treatment of CRPC cells: (i) decreased cellular proteasome activity (FIGS. 3-4); (ii) activated calpains and caspases (FIGS. 1-2); (iii) caused AR-V7 degradation (FIGS. 1-3; consistent with Refs. 8-9); (iv) Gal-induced AR-V7 depletion is likely due to degradation by calpains and caspases but not 20S proteasome; (v) BTZ sensitizes CRPC cells to Gal-induced AR-V7 degradation, associated with increased proteasome inhibition; (vi) For the first time, specific AR-V7 fragments of 53 kDa and 41 kDa were detected, associated with AR-V7 decrease in the nuclei of R22v1 cells after Gal treatment (FIG. 5); (vii) Nuclear AR-V7 cleavage was accompanied by activation of caspase-3 and calpain (FIG. 5).

Example 2

Galeterone inhibits activities of 19S proteasome-associated Deubiquitinating enzymes (DUBs) in castrate resistant prostate cancer (CRPC) cells and sensitizes bortezomib-resistant multiple myeloma (MM) cells.

Previously it was found that Gal inhibits the chymotrypsin-like (CT-like) activity of the proteasome in prostate cancer cells. Whether Gal is a direct inhibitor of the proteasome was of interest. All of the three proteasomal activities of purified 20S or 26S proteasomes in the presence of Gal were determined. As shown in FIGS. 10A and 10B, Gal was unable to block all the three proteasomal activities (CT, PGPH and T-like activities) when purified 20S or 26S proteasomes were used. When a protein extract of 22Rv1 cells was used, Gal could inhibit the CT-like activity of the proteasome at high concentrations (50 or 100 .mu.M) (FIG. 10C). These results suggest that Gal might target the 19S proteasome rather than the 20S proteasome. Then deubiquitinase (DUBs) activity associated with the 19S proteasome was determined by using the purified 26S proteasome. As a control, purified 20S proteasome showed no DUBs activity (FIG. 10D). In comparison, the purified 26S proteasome showed 3-fold DUBs activity, which was suppressed when incubated with Gal (FIG. 10D). When this experiment was repeated, it was shown that Gal inhibited DUBs activity of the purified 26S proteasome in a dose-dependent fashion; Gal at a low dose (10 .mu.M) could efficiently inhibit DUBs activity (FIG. 10E).

It was next determined whether DUBs activity could be inhibited in cultured CRPC cells after Gal treatment. As shown in FIG. 11A, DUBs activity was inhibited by Gal at as early as 2 h after Gal treatment. Sustained DUBs inhibition was detected for up to 48 h (FIG. 11A). The levels of proteasomal CT-like activities were inhibited in a time-dependent fashion, but later than DUBs inhibition (FIG. 11B). Oxidized proteins were accumulated by Gal treatment before 2 h, and sustained accumulation was detected up to 72 h (FIG. 11C, a). AR-V7 protein level remained subtle changes up to 56 h and decreased after 64-68 h, and completely depleted after 72 h (FIG. 11C, c). Caspase-3 activity was increased after 24 h treatment with Gal and reached to 5-fold activation after 48 h (FIG. 11D). Consistently, caspase-3-specific cleavage fragments, 89 kD fragment of PARP (PARP/p89) were also detected between 48 and 72 h (FIG. 11C, d). Calpain activation is associated with self-autolysis of its small subunit. A cleaved fragment (28 kD) of calpain small subunit (Cal Sm/p28) was detected during 48-72 h treatment with Gal (FIG. 11C, e). The above results suggest that Gal is an inhibitor of DUBs associated with the 19S proteasomes in CRPC cells. This finding has important clinical significance.

It was next determined if the combinational treatment of Gal and BTZ would induce greater growth inhibition and cell death in CRPC cells. Gal treatment alone for 24 h did not inhibit cell proliferation in 22Rv1 cells. In the presence of bortezomib, cell growth was greatly inhibited by Gal at 20 .mu.M (MTT assay; FIG. 12A). Gal at 20 .mu.M in combination with bortezomib 5 or 10 nM generated greater inhibitory effects on cell proliferation compared to each drug alone (FIG. 12A). Consistently, Bortezomib in combination with Gal induced greater morphological changes (indicator of cell death) as shown by more detached cells or apoptotic cells in co-treatment compared to each drug alone (FIGS. 12B and 12D for 24 and 48 h, respectively). Synergistic effects of Gal in combination with bortezomib were shown. After 24 h (FIG. 12B) and 48 h (FIG. 12D) treatment, bortezomib (2.5 or 5 nM) or Gal alone did not cause apparent morphological changes, but their combination caused apparent or complete cell death. These synergistic effects were reflected in violet staining, showing less staining cells in the combination compared to each drug alone (FIGS. 12C and 12E for 24 and 48 h, respectively).

The combination of Gal and BTZ causes greater proteasome inhibition, more AR-V7 depletion and more CRPC cell death, compared to each drug alone. It was then determined whether Gal could sensitize BTZ-resistant multiple myeloma cells. BTZ-resistant MM cells were sensitive to Gal treatment alone at 10 or 20 .mu.M (FIG. 13A-B); Combination of Gal and BTZ showed greater inhibition on BTZ-resistant MM cells than each drug alone in both Gal dose- and BTZ dose-dependent manners, as evident in two independent experiments (FIG. 13A-B). Next we determined whether Gal and BTZ combination could increase higher levels of cell death in the resistant MM cells. 8826-7BR is a multiple myeloma cell line developed resistance to bortezomib. Gal in combination with bortezomib induced more cell death as shown by stronger cleaved PARP bands generated by cotreatment (FIG. 13C, lanes 7-10) compared with each alone (lane 2 or lanes 3-6). Therefore, Gal is able to overcome bortezomib resistance in MM. This finding has high translational potential to clinics.

In brief, the disclosed studies indicate that Gal acts as an inhibitor of the 19S proteasome-associated DUBs, which can be used to overcome the resistance of 20S inhibitor BTZ for the treatment of MM and other liquid tumors.

As will be understood by one of ordinary skill in the art, while exemplary sequences of proteins described herein are provided, additional homologous and/or variant sequences can be obtained from publicly available databases. Such sequences are included within the teachings of the current disclosure. Homologous sequences are those with shared ancestry. Shared ancestry can occur based on a speciation event (orthologs) or a duplication event (paralogs). Variant sequences include sequences having one or more amino acid additions, deletions, stop positions, or substitutions, as compared to a protein sequence disclosed elsewhere herein. Variants can include allelic forms, representing minor changes in amino acid composition. Homologous sequences and variant sequences will typically exhibit the same qualitative biological activity and elicit a substantially similar response as reference proteins provided herein. Screening of homologues and variants can be performed using assays described elsewhere herein.

Homologous sequences and variant sequences included within the scope of the disclosure can have at least 80% sequence identity; at least 85% sequence identity; at least 90% sequence identity; at least 95% sequence identity or at least 99% sequence identity with an exemplary reference sequence disclosed herein. "Percent (%) amino acid sequence identity" with respect to the sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or ALIGN-2. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.

As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms "include" or "including" should be interpreted to recite: "comprise, consist of, or consist essentially of." The transition term "comprise" or "comprises" means includes, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase "consisting of" excludes any element, step, ingredient or component not specified. The transition phrase "consisting essentially of" limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. A material effect would cause a statistically-significant reduction in a combination therapy's ability to cause an anti-CRPC or MM effect or to detect 53 kDa and 41 kDa AR-V7 fragments in a blood sample from a subject.

Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term "about" has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of .+-.20% of the stated value; .+-.19% of the stated value; .+-.18% of the stated value; .+-.17% of the stated value; .+-.16% of the stated value; .+-.15% of the stated value; .+-.14% of the stated value; .+-.13% of the stated value; .+-.12% of the stated value; .+-.11% of the stated value; .+-.10% of the stated value; .+-.9% of the stated value; .+-.8% of the stated value; .+-.7% of the stated value; .+-.6% of the stated value; .+-.5% of the stated value; .+-.4% of the stated value; .+-.3% of the stated value; .+-.2% of the stated value; or .+-.1% of the stated value.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

The terms "a," "an," "the" and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents, printed publications, journal articles and other written text throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching.

In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and/or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3.sup.rd Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

REFERENCES

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SEQUENCE LISTINGS

1

41917PRTHomo sapiens 1Met Glu Val Gln Leu Gly Leu Gly Arg Val Tyr Pro Arg Pro Pro Ser1 5 10 15Lys Thr Tyr Arg Gly Ala Phe Gln Asn Leu Phe Gln Ser Val Arg Glu 20 25 30Val Ile Gln Asn Pro Gly Pro Arg His Pro Glu Ala Ala Ser Ala Ala 35 40 45Pro Pro Gly Ala Ser Leu Leu Leu Leu Gln Gln Gln Gln Gln Gln Gln 50 55 60Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Glu Thr Ser65 70 75 80Pro Arg Gln Gln Gln Gln Gln Gln Gly Glu Asp Gly Ser Pro Gln Ala 85 90 95His Arg Arg Gly Pro Thr Gly Tyr Leu Val Leu Asp Glu Glu Gln Gln 100 105 110Pro Ser Gln Pro Gln Ser Ala Leu Glu Cys His Pro Glu Arg Gly Cys 115 120 125Val Pro Glu Pro Gly Ala Ala Val Ala Ala Ser Lys Gly Leu Pro Gln 130 135 140Gln Leu Pro Ala Pro Pro Asp Glu Asp Asp Ser Ala Ala Pro Ser Thr145 150 155 160Leu Ser Leu Leu Gly Pro Thr Phe Pro Gly Leu Ser Ser Cys Ser Ala 165 170 175Asp Leu Lys Asp Ile Leu Ser Glu Ala Ser Thr Met Gln Leu Leu Gln 180 185 190Gln Gln Gln Gln Glu Ala Val Ser Glu Gly Ser Ser Ser Gly Arg Ala 195 200 205Arg Glu Arg Ser Gly Ala Pro Thr Ser Ser Lys Asp Asn Tyr Leu Gly 210 215 220Gly Thr Ser Thr Ile Ser Asp Asn Ala Lys Glu Leu Cys Lys Ala Val225 230 235 240Ser Val Ser Met Gly Leu Gly Val Glu Ala Leu Glu His Leu Ser Pro 245 250 255Gly Glu Gln Leu Arg Gly Asp Cys Met Tyr Ala Pro Leu Leu Gly Val 260 265 270Pro Pro Ala Val Arg Pro Thr Pro Cys Ala Pro Leu Ala Glu Cys Lys 275 280 285Gly Ser Leu Leu Asp Asp Ser Ala Gly Lys Ser Thr Glu Asp Thr Ala 290 295 300Glu Tyr Ser Pro Phe Lys Gly Gly Tyr Thr Lys Gly Leu Glu Gly Glu305 310 315 320Ser Leu Gly Cys Ser Gly Ser Ala Ala Ala Gly Ser Ser Gly Thr Leu 325 330 335Glu Leu Pro Ser Thr Leu Ser Leu Tyr Lys Ser Gly Ala Leu Asp Glu 340 345 350Ala Ala Ala Tyr Gln Ser Arg Asp Tyr Tyr Asn Phe Pro Leu Ala Leu 355 360 365Ala Gly Pro Pro Pro Pro Pro Pro Pro Pro His Pro His Ala Arg Ile 370 375 380Lys Leu Glu Asn Pro Leu Asp Tyr Gly Ser Ala Trp Ala Ala Ala Ala385 390 395 400Ala Gln Cys Arg Tyr Gly Asp Leu Ala Ser Leu His Gly Ala Gly Ala 405 410 415Ala Gly Pro Gly Ser Gly Ser Pro Ser Ala Ala Ala Ser Ser Ser Trp 420 425 430His Thr Leu Phe Thr Ala Glu Glu Gly Gln Leu Tyr Gly Pro Cys Gly 435 440 445Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 450 455 460Gly Gly Gly Gly Gly Gly Glu Ala Gly Ala Val Ala Pro Tyr Gly Tyr465 470 475 480Thr Arg Pro Pro Gln Gly Leu Ala Gly Gln Glu Ser Asp Phe Thr Ala 485 490 495Pro Asp Val Trp Tyr Pro Gly Gly Met Val Ser Arg Val Pro Tyr Pro 500 505 510Ser Pro Thr Cys Val Lys Ser Glu Met Gly Pro Trp Met Asp Ser Tyr 515 520 525Ser Gly Pro Tyr Gly Asp Met Arg Leu Glu Thr Ala Arg Asp His Val 530 535 540Leu Pro Ile Asp Tyr Tyr Phe Pro Pro Gln Lys Thr Cys Leu Ile Cys545 550 555 560Gly Asp Glu Ala Ser Gly Cys His Tyr Gly Ala Leu Thr Cys Gly Ser 565 570 575Cys Lys Val Phe Phe Lys Arg Ala Ala Glu Gly Lys Gln Lys Tyr Leu 580 585 590Cys Ala Ser Arg Asn Asp Cys Thr Ile Asp Lys Phe Arg Arg Lys Asn 595 600 605Cys Pro Ser Cys Arg Leu Arg Lys Cys Tyr Glu Ala Gly Met Thr Leu 610 615 620Gly Ala Arg Lys Leu Lys Lys Leu Gly Asn Leu Lys Leu Gln Glu Glu625 630 635 640Gly Glu Ala Ser Ser Thr Thr Ser Pro Thr Glu Glu Thr Thr Gln Lys 645 650 655Leu Thr Val Ser His Ile Glu Gly Tyr Glu Cys Gln Pro Ile Phe Leu 660 665 670Asn Val Leu Glu Ala Ile Glu Pro Gly Val Val Cys Ala Gly His Asp 675 680 685Asn Asn Gln Pro Asp Ser Phe Ala Ala Leu Leu Ser Ser Leu Asn Glu 690 695 700Leu Gly Glu Arg Gln Leu Val His Val Val Lys Trp Ala Lys Ala Leu705 710 715 720Pro Gly Phe Arg Asn Leu His Val Asp Asp Gln Met Ala Val Ile Gln 725 730 735Tyr Ser Trp Met Gly Leu Met Val Phe Ala Met Gly Trp Arg Ser Phe 740 745 750Thr Asn Val Asn Ser Arg Met Leu Tyr Phe Ala Pro Asp Leu Val Phe 755 760 765Asn Glu Tyr Arg Met His Lys Ser Arg Met Tyr Ser Gln Cys Val Arg 770 775 780Met Arg His Leu Ser Gln Glu Phe Gly Trp Leu Gln Ile Thr Pro Gln785 790 795 800Glu Phe Leu Cys Met Lys Ala Leu Leu Leu Phe Ser Ile Ile Pro Val 805 810 815Asp Gly Leu Lys Asn Gln Lys Phe Phe Asp Glu Leu Arg Met Asn Tyr 820 825 830Ile Lys Glu Leu Asp Arg Ile Ile Ala Cys Lys Arg Lys Asn Pro Thr 835 840 845Ser Cys Ser Arg Arg Phe Tyr Gln Leu Thr Lys Leu Leu Asp Ser Val 850 855 860Gln Pro Ile Ala Arg Glu Leu His Gln Phe Thr Phe Asp Leu Leu Ile865 870 875 880Lys Ser His Met Val Ser Val Asp Phe Pro Glu Met Met Ala Glu Ile 885 890 895Ile Ser Val Gln Val Pro Lys Ile Leu Ser Gly Lys Val Lys Pro Ile 900 905 910Tyr Phe His Thr Gln 9152645PRTHomo sapiens 2Met Glu Val Gln Leu Gly Leu Gly Arg Val Tyr Pro Arg Pro Pro Ser1 5 10 15Lys Thr Tyr Arg Gly Ala Phe Gln Asn Leu Phe Gln Ser Val Arg Glu 20 25 30Val Ile Gln Asn Pro Gly Pro Arg His Pro Glu Ala Ala Ser Ala Ala 35 40 45Pro Pro Gly Ala Ser Leu Leu Leu Gln Gln Gln Gln Gln Gln Gln Gln 50 55 60Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln65 70 75 80Gln Gln Gln Gln Gln Glu Thr Ser Pro Arg Gln Gln Gln Gln Gln Gln 85 90 95Gly Glu Asp Gly Ser Pro Gln Ala His Arg Arg Gly Pro Thr Gly Tyr 100 105 110Leu Val Leu Asp Glu Glu Gln Gln Pro Ser Gln Pro Gln Ser Ala Leu 115 120 125Glu Cys His Pro Glu Arg Gly Cys Val Pro Glu Pro Gly Ala Ala Val 130 135 140Ala Ala Ser Lys Gly Leu Pro Gln Gln Leu Pro Ala Pro Pro Asp Glu145 150 155 160Asp Asp Ser Ala Ala Pro Ser Thr Leu Ser Leu Leu Gly Pro Thr Phe 165 170 175Pro Gly Leu Ser Ser Cys Ser Ala Asp Leu Lys Asp Ile Leu Ser Glu 180 185 190Ala Ser Thr Met Gln Leu Leu Gln Gln Gln Gln Gln Glu Ala Val Ser 195 200 205Glu Gly Ser Ser Ser Gly Arg Ala Arg Glu Ala Ser Gly Ala Pro Thr 210 215 220Ser Ser Lys Asp Asn Tyr Leu Gly Gly Thr Ser Thr Ile Ser Asp Asn225 230 235 240Ala Lys Glu Leu Cys Lys Ala Val Ser Val Ser Met Gly Leu Gly Val 245 250 255Glu Ala Leu Glu His Leu Ser Pro Gly Glu Gln Leu Arg Gly Asp Cys 260 265 270Met Tyr Ala Pro Leu Leu Gly Val Pro Pro Ala Val Arg Pro Thr Pro 275 280 285Cys Ala Pro Leu Ala Glu Cys Lys Gly Ser Leu Leu Asp Asp Ser Ala 290 295 300Gly Lys Ser Thr Glu Asp Thr Ala Glu Tyr Ser Pro Phe Lys Gly Gly305 310 315 320Tyr Thr Lys Gly Leu Glu Gly Glu Ser Leu Gly Cys Ser Gly Ser Ala 325 330 335Ala Ala Gly Ser Ser Gly Thr Leu Glu Leu Pro Ser Thr Leu Ser Leu 340 345 350Tyr Lys Ser Gly Ala Leu Asp Glu Ala Ala Ala Tyr Gln Ser Arg Asp 355 360 365Tyr Tyr Asn Phe Pro Leu Ala Leu Ala Gly Pro Pro Pro Pro Pro Pro 370 375 380Pro Pro His Pro His Ala Arg Ile Lys Leu Glu Asn Pro Leu Asp Tyr385 390 395 400Gly Ser Ala Trp Ala Ala Ala Ala Ala Gln Cys Arg Tyr Gly Asp Leu 405 410 415Ala Ser Leu His Gly Ala Gly Ala Ala Gly Pro Gly Ser Gly Ser Pro 420 425 430Ser Ala Ala Ala Ser Ser Ser Trp His Thr Leu Phe Thr Ala Glu Glu 435 440 445Gly Gln Leu Tyr Gly Pro Cys Gly Gly Gly Gly Gly Gly Gly Gly Gly 450 455 460Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Glu Ala Gly Ala Val Ala465 470 475 480Pro Tyr Gly Tyr Thr Arg Pro Pro Gln Gly Leu Ala Gly Gln Glu Ser 485 490 495Asp Phe Thr Ala Pro Asp Val Trp Tyr Pro Gly Gly Met Val Ser Arg 500 505 510Val Pro Tyr Pro Ser Pro Thr Cys Val Lys Ser Glu Met Gly Pro Trp 515 520 525Met Asp Ser Tyr Ser Gly Pro Tyr Gly Asp Met Arg Leu Glu Thr Ala 530 535 540Arg Asp His Val Leu Pro Ile Asp Tyr Tyr Phe Pro Pro Gln Lys Thr545 550 555 560Cys Leu Ile Cys Gly Asp Glu Ala Ser Gly Cys His Tyr Gly Ala Leu 565 570 575Thr Cys Gly Ser Cys Lys Val Phe Phe Lys Arg Ala Ala Glu Gly Lys 580 585 590Gln Lys Tyr Leu Cys Ala Ser Arg Asn Asp Cys Thr Ile Asp Lys Phe 595 600 605Arg Arg Lys Asn Cys Pro Ser Cys Arg Leu Arg Lys Cys Tyr Glu Ala 610 615 620Gly Met Thr Leu Gly Glu Lys Phe Arg Val Gly Asn Cys Lys His Leu625 630 635 640Lys Met Thr Arg Pro 64532472PRTHomo sapiens 3Met Asp Pro Ser Gly Val Lys Val Leu Glu Thr Ala Glu Asp Ile Gln1 5 10 15Glu Arg Arg Gln Gln Val Leu Asp Arg Tyr His Arg Phe Lys Glu Leu 20 25 30Ser Thr Leu Arg Arg Gln Lys Leu Glu Asp Ser Tyr Arg Phe Gln Phe 35 40 45Phe Gln Arg Asp Ala Glu Glu Leu Glu Lys Trp Ile Gln Glu Lys Leu 50 55 60Gln Ile Ala Ser Asp Glu Asn Tyr Lys Asp Pro Thr Asn Leu Gln Gly65 70 75 80Lys Leu Gln Lys His Gln Ala Phe Glu Ala Glu Val Gln Ala Asn Ser 85 90 95Gly Ala Ile Val Lys Leu Asp Glu Thr Gly Asn Leu Met Ile Ser Glu 100 105 110Gly His Phe Ala Ser Glu Thr Ile Arg Thr Arg Leu Met Glu Leu His 115 120 125Arg Gln Trp Glu Leu Leu Leu Glu Lys Met Arg Glu Lys Gly Ile Lys 130 135 140Leu Leu Gln Ala Gln Lys Leu Val Gln Tyr Leu Arg Glu Cys Glu Asp145 150 155 160Val Met Asp Trp Ile Asn Asp Lys Glu Ala Ile Val Thr Ser Glu Glu 165 170 175Leu Gly Gln Asp Leu Glu His Val Glu Val Leu Gln Lys Lys Phe Glu 180 185 190Glu Phe Gln Thr Asp Met Ala Ala His Glu Glu Arg Val Asn Glu Val 195 200 205Asn Gln Phe Ala Ala Lys Leu Ile Gln Glu Gln His Pro Glu Glu Glu 210 215 220Leu Ile Lys Thr Lys Gln Asp Glu Val Asn Ala Ala Trp Gln Arg Leu225 230 235 240Lys Gly Leu Ala Leu Gln Arg Gln Gly Lys Leu Phe Gly Ala Ala Glu 245 250 255Val Gln Arg Phe Asn Arg Asp Val Asp Glu Thr Ile Ser Trp Ile Lys 260 265 270Glu Lys Glu Gln Leu Met Ala Ser Asp Asp Phe Gly Arg Asp Leu Ala 275 280 285Ser Val Gln Ala Leu Leu Arg Lys His Glu Gly Leu Glu Arg Asp Leu 290 295 300Ala Ala Leu Glu Asp Lys Val Lys Ala Leu Cys Ala Glu Ala Asp Arg305 310 315 320Leu Gln Gln Ser His Pro Leu Ser Ala Thr Gln Ile Gln Val Lys Arg 325 330 335Glu Glu Leu Ile Thr Asn Trp Glu Gln Ile Arg Thr Leu Ala Ala Glu 340 345 350Arg His Ala Arg Leu Asn Asp Ser Tyr Arg Leu Gln Arg Phe Leu Ala 355 360 365Asp Phe Arg Asp Leu Thr Ser Trp Val Thr Glu Met Lys Ala Leu Ile 370 375 380Asn Ala Asp Glu Leu Ala Ser Asp Val Ala Gly Ala Glu Ala Leu Leu385 390 395 400Asp Arg His Gln Glu His Lys Gly Glu Ile Asp Ala His Glu Asp Ser 405 410 415Phe Lys Ser Ala Asp Glu Ser Gly Gln Ala Leu Leu Ala Ala Gly His 420 425 430Tyr Ala Ser Asp Glu Val Arg Glu Lys Leu Thr Val Leu Ser Glu Glu 435 440 445Arg Ala Ala Leu Leu Glu Leu Trp Glu Leu Arg Arg Gln Gln Tyr Glu 450 455 460Gln Cys Met Asp Leu Gln Leu Phe Tyr Arg Asp Thr Glu Gln Val Asp465 470 475 480Asn Trp Met Ser Lys Gln Glu Ala Phe Leu Leu Asn Glu Asp Leu Gly 485 490 495Asp Ser Leu Asp Ser Val Glu Ala Leu Leu Lys Lys His Glu Asp Phe 500 505 510Glu Lys Ser Leu Ser Ala Gln Glu Glu Lys Ile Thr Ala Leu Asp Glu 515 520 525Phe Ala Thr Lys Leu Ile Gln Asn Asn His Tyr Ala Met Glu Asp Val 530 535 540Ala Thr Arg Arg Asp Ala Leu Leu Ser Arg Arg Asn Ala Leu His Glu545 550 555 560Arg Ala Met Arg Arg Arg Ala Gln Leu Ala Asp Ser Phe His Leu Gln 565 570 575Gln Phe Phe Arg Asp Ser Asp Glu Leu Lys Ser Trp Val Asn Glu Lys 580 585 590Met Lys Thr Ala Thr Asp Glu Ala Tyr Lys Asp Pro Ser Asn Leu Gln 595 600 605Gly Lys Val Gln Lys His Gln Ala Phe Glu Ala Glu Leu Ser Ala Asn 610 615 620Gln Ser Arg Ile Asp Ala Leu Glu Lys Ala Gly Gln Lys Leu Ile Asp625 630 635 640Val Asn His Tyr Ala Lys Asp Glu Val Ala Ala Arg Met Asn Glu Val 645 650 655Ile Ser Leu Trp Lys Lys Leu Leu Glu Ala Thr Glu Leu Lys Gly Ile 660 665 670Lys Leu Arg Glu Ala Asn Gln Gln Gln Gln Phe Asn Arg Asn Val Glu 675 680 685Asp Ile Glu Leu Trp Leu Tyr Glu Val Glu Gly His Leu Ala Ser Asp 690 695 700Asp Tyr Gly Lys Asp Leu Thr Asn Val Gln Asn Leu Gln Lys Lys His705 710 715 720Ala Leu Leu Glu Ala Asp Val Ala Ala His Gln Asp Arg Ile Asp Gly 725 730 735Ile Thr Ile Gln Ala Arg Gln Phe Gln Asp Ala Gly His Phe Asp Ala 740 745 750Glu Asn Ile Lys Lys Lys Gln Glu Ala Leu Val Ala Arg Tyr Glu Ala 755 760 765Leu Lys Glu Pro Met Val Ala Arg Lys Gln Lys Leu Ala Asp Ser Leu 770 775 780Arg Leu Gln Gln Leu Phe Arg Asp Val Glu Asp Glu Glu Thr Trp Ile785 790 795 800Arg Glu Lys Glu Pro Ile Ala Ala Ser Thr Asn Arg Gly Lys Asp Leu 805 810 815Ile Gly Val Gln Asn Leu Leu Lys Lys His Gln Ala Leu Gln Ala Glu 820 825 830Ile Ala Gly His Glu Pro Arg Ile Lys Ala Val Thr Gln Lys Gly Asn 835 840 845Ala Met Val Glu Glu Gly His Phe Ala Ala Glu Asp Val Lys Ala Lys 850 855 860Leu His Glu Leu Asn Gln Lys Trp Glu Ala Leu Lys Ala Lys Ala Ser865 870 875 880Gln Arg Arg Gln Asp Leu Glu Asp Ser Leu Gln Ala Gln Gln Tyr Phe 885 890 895Ala Asp Ala Asn Glu Ala Glu Ser Trp Met Arg Glu Lys Glu Pro Ile 900

905 910Val Gly Ser Thr Asp Tyr Gly Lys Asp Glu Asp Ser Ala Glu Ala Leu 915 920 925Leu Lys Lys His Glu Ala Leu Met Ser Asp Leu Ser Ala Tyr Gly Ser 930 935 940Ser Ile Gln Ala Leu Arg Glu Gln Ala Gln Ser Cys Arg Gln Gln Val945 950 955 960Ala Pro Thr Asp Asp Glu Thr Gly Lys Glu Leu Val Leu Ala Leu Tyr 965 970 975Asp Tyr Gln Glu Lys Ser Pro Arg Glu Val Thr Met Lys Lys Gly Asp 980 985 990Ile Leu Thr Leu Leu Asn Ser Thr Asn Lys Asp Trp Trp Lys Val Glu 995 1000 1005Val Asn Asp Arg Gln Gly Phe Val Pro Ala Ala Tyr Val Lys Lys 1010 1015 1020Leu Asp Pro Ala Gln Ser Ala Ser Arg Glu Asn Leu Leu Glu Glu 1025 1030 1035Gln Gly Ser Ile Ala Leu Arg Gln Glu Gln Ile Asp Asn Gln Thr 1040 1045 1050Arg Ile Thr Lys Glu Ala Gly Ser Val Ser Leu Arg Met Lys Gln 1055 1060 1065Val Glu Glu Leu Tyr His Ser Leu Leu Glu Leu Gly Glu Lys Arg 1070 1075 1080Lys Gly Met Leu Glu Lys Ser Cys Lys Lys Phe Met Leu Phe Arg 1085 1090 1095Glu Ala Asn Glu Leu Gln Gln Trp Ile Asn Glu Lys Glu Ala Ala 1100 1105 1110Leu Thr Ser Glu Glu Val Gly Ala Asp Leu Glu Gln Val Glu Val 1115 1120 1125Leu Gln Lys Lys Phe Asp Asp Phe Gln Lys Asp Leu Lys Ala Asn 1130 1135 1140Glu Ser Arg Leu Lys Asp Ile Asn Lys Val Ala Glu Asp Leu Glu 1145 1150 1155Ser Glu Gly Leu Met Ala Glu Glu Val Gln Ala Val Gln Gln Gln 1160 1165 1170Glu Val Tyr Gly Met Met Pro Arg Asp Glu Thr Asp Ser Lys Thr 1175 1180 1185Ala Ser Pro Trp Lys Ser Ala Arg Leu Met Val His Thr Val Ala 1190 1195 1200Thr Phe Asn Ser Ile Lys Glu Leu Asn Glu Arg Trp Arg Ser Leu 1205 1210 1215Gln Gln Leu Ala Glu Glu Arg Ser Gln Leu Leu Gly Ser Ala His 1220 1225 1230Glu Val Gln Arg Phe His Arg Asp Ala Asp Glu Thr Lys Glu Trp 1235 1240 1245Ile Glu Glu Lys Asn Gln Ala Leu Asn Thr Asp Asn Tyr Gly His 1250 1255 1260Asp Leu Ala Ser Val Gln Ala Leu Gln Arg Lys His Glu Gly Phe 1265 1270 1275Glu Arg Asp Leu Ala Ala Leu Gly Asp Lys Val Asn Ser Leu Gly 1280 1285 1290Glu Thr Ala Glu Arg Leu Ile Gln Ser His Pro Glu Ser Ala Glu 1295 1300 1305Asp Leu Gln Glu Lys Cys Thr Glu Leu Asn Gln Ala Trp Ser Ser 1310 1315 1320Leu Gly Lys Arg Ala Asp Gln Arg Lys Ala Lys Leu Gly Asp Ser 1325 1330 1335His Asp Leu Gln Arg Phe Leu Ser Asp Phe Arg Asp Leu Met Ser 1340 1345 1350Trp Ile Asn Gly Ile Arg Gly Leu Val Ser Ser Asp Glu Leu Ala 1355 1360 1365Lys Asp Val Thr Gly Ala Glu Ala Leu Leu Glu Arg His Gln Glu 1370 1375 1380His Arg Thr Glu Ile Asp Ala Arg Ala Gly Thr Phe Gln Ala Phe 1385 1390 1395Glu Gln Phe Gly Gln Gln Leu Leu Ala His Gly His Tyr Ala Ser 1400 1405 1410Pro Glu Ile Lys Gln Lys Leu Asp Ile Leu Asp Gln Glu Arg Ala 1415 1420 1425Asp Leu Glu Lys Ala Trp Val Gln Arg Arg Met Met Leu Asp Gln 1430 1435 1440Cys Leu Glu Leu Gln Leu Phe His Arg Asp Cys Glu Gln Ala Glu 1445 1450 1455Asn Trp Met Ala Ala Arg Glu Ala Phe Leu Asn Thr Glu Asp Lys 1460 1465 1470Gly Asp Ser Leu Asp Ser Val Glu Ala Leu Ile Lys Lys His Glu 1475 1480 1485Asp Phe Asp Lys Ala Ile Asn Val Gln Glu Glu Lys Ile Ala Ala 1490 1495 1500Leu Gln Ala Phe Ala Asp Gln Leu Ile Ala Ala Gly His Tyr Ala 1505 1510 1515Lys Gly Asp Ile Ser Ser Arg Arg Asn Glu Val Leu Asp Arg Trp 1520 1525 1530Arg Arg Leu Lys Ala Gln Met Ile Glu Lys Arg Ser Lys Leu Gly 1535 1540 1545Glu Ser Gln Thr Leu Gln Gln Phe Ser Arg Asp Val Asp Glu Ile 1550 1555 1560Glu Ala Trp Ile Ser Glu Lys Leu Gln Thr Ala Ser Asp Glu Ser 1565 1570 1575Tyr Lys Asp Pro Thr Asn Ile Gln Ser Lys His Gln Lys His Gln 1580 1585 1590Ala Phe Glu Ala Glu Leu His Ala Asn Ala Asp Arg Ile Arg Gly 1595 1600 1605Val Ile Asp Met Gly Asn Ser Leu Ile Glu Arg Gly Ala Cys Ala 1610 1615 1620Gly Ser Glu Asp Ala Val Lys Ala Arg Leu Ala Ala Leu Ala Asp 1625 1630 1635Gln Trp Gln Phe Leu Val Gln Lys Ser Ala Glu Lys Ser Gln Lys 1640 1645 1650Leu Lys Glu Ala Asn Lys Gln Gln Asn Phe Asn Thr Gly Ile Lys 1655 1660 1665Asp Phe Asp Phe Trp Leu Ser Glu Val Glu Ala Leu Leu Ala Ser 1670 1675 1680Glu Asp Tyr Gly Lys Asp Leu Ala Ser Val Asn Asn Leu Leu Lys 1685 1690 1695Lys His Gln Leu Leu Glu Ala Asp Ile Ser Ala His Glu Asp Arg 1700 1705 1710Leu Lys Asp Leu Asn Ser Gln Ala Asp Ser Leu Met Thr Ser Ser 1715 1720 1725Ala Phe Asp Thr Ser Gln Val Lys Asp Lys Arg Asp Thr Ile Asn 1730 1735 1740Gly Arg Phe Gln Lys Ile Lys Ser Met Ala Ala Ser Arg Arg Ala 1745 1750 1755Lys Leu Asn Glu Ser His Arg Leu His Gln Phe Phe Arg Asp Met 1760 1765 1770Asp Asp Glu Glu Ser Trp Ile Lys Glu Lys Lys Leu Leu Val Gly 1775 1780 1785Ser Glu Asp Tyr Gly Arg Asp Leu Thr Gly Val Gln Asn Leu Arg 1790 1795 1800Lys Lys His Lys Arg Leu Glu Ala Glu Leu Ala Ala His Glu Pro 1805 1810 1815Ala Ile Gln Gly Val Leu Asp Thr Gly Lys Lys Leu Ser Asp Asp 1820 1825 1830Asn Thr Ile Gly Lys Glu Glu Ile Gln Gln Arg Leu Ala Gln Phe 1835 1840 1845Val Glu His Trp Lys Glu Leu Lys Gln Leu Ala Ala Ala Arg Gly 1850 1855 1860Gln Arg Leu Glu Glu Ser Leu Glu Tyr Gln Gln Phe Val Ala Asn 1865 1870 1875Val Glu Glu Glu Glu Ala Trp Ile Asn Glu Lys Met Thr Leu Val 1880 1885 1890Ala Ser Glu Asp Tyr Gly Asp Thr Leu Ala Ala Ile Gln Gly Leu 1895 1900 1905Leu Lys Lys His Glu Ala Phe Glu Thr Asp Phe Thr Val His Lys 1910 1915 1920Asp Arg Val Asn Asp Val Cys Thr Asn Gly Gln Asp Leu Ile Lys 1925 1930 1935Lys Asn Asn His His Glu Glu Asn Ile Ser Ser Lys Met Lys Gly 1940 1945 1950Leu Asn Gly Lys Val Ser Asp Leu Glu Lys Ala Ala Ala Gln Arg 1955 1960 1965Lys Ala Lys Leu Asp Glu Asn Ser Ala Phe Leu Gln Phe Asn Trp 1970 1975 1980Lys Ala Asp Val Val Glu Ser Trp Ile Gly Glu Lys Glu Asn Ser 1985 1990 1995Leu Lys Thr Asp Asp Tyr Gly Arg Asp Leu Ser Ser Val Gln Thr 2000 2005 2010Leu Leu Thr Lys Gln Glu Thr Phe Asp Ala Gly Leu Gln Ala Phe 2015 2020 2025Gln Gln Glu Gly Ile Ala Asn Ile Thr Ala Leu Lys Asp Gln Leu 2030 2035 2040Leu Ala Ala Lys His Val Gln Ser Lys Ala Ile Glu Ala Arg His 2045 2050 2055Ala Ser Leu Met Lys Arg Trp Ser Gln Leu Leu Ala Asn Ser Ala 2060 2065 2070Ala Arg Lys Lys Lys Leu Leu Glu Ala Gln Ser His Phe Arg Lys 2075 2080 2085Val Glu Asp Leu Phe Leu Thr Phe Ala Lys Lys Ala Ser Ala Phe 2090 2095 2100Asn Ser Trp Phe Glu Asn Ala Glu Glu Asp Leu Thr Asp Pro Val 2105 2110 2115Arg Cys Asn Ser Leu Glu Glu Ile Lys Ala Leu Arg Glu Ala His 2120 2125 2130Asp Ala Phe Arg Ser Ser Leu Ser Ser Ala Gln Ala Asp Phe Asn 2135 2140 2145Gln Leu Ala Glu Leu Asp Arg Gln Ile Lys Ser Phe Arg Val Ala 2150 2155 2160Ser Asn Pro Tyr Thr Trp Phe Thr Met Glu Ala Leu Glu Glu Thr 2165 2170 2175Trp Arg Asn Leu Gln Lys Ile Ile Lys Glu Arg Glu Leu Glu Leu 2180 2185 2190Gln Lys Glu Gln Arg Arg Gln Glu Glu Asn Asp Lys Leu Arg Gln 2195 2200 2205Glu Phe Ala Gln His Ala Asn Ala Phe His Gln Trp Ile Gln Glu 2210 2215 2220Thr Arg Thr Tyr Leu Leu Asp Gly Ser Cys Met Val Glu Glu Ser 2225 2230 2235Gly Thr Leu Glu Ser Gln Leu Glu Ala Thr Lys Arg Lys His Gln 2240 2245 2250Glu Ile Arg Ala Met Arg Ser Gln Leu Lys Lys Ile Glu Asp Leu 2255 2260 2265Gly Ala Ala Met Glu Glu Ala Leu Ile Leu Asp Asn Lys Tyr Thr 2270 2275 2280Glu His Ser Thr Val Gly Leu Ala Gln Gln Trp Asp Gln Leu Asp 2285 2290 2295Gln Leu Gly Met Arg Met Gln His Asn Leu Glu Gln Gln Ile Gln 2300 2305 2310Ala Arg Asn Thr Thr Gly Val Thr Glu Glu Ala Leu Lys Glu Phe 2315 2320 2325Ser Met Met Phe Lys His Phe Asp Lys Asp Lys Ser Gly Arg Leu 2330 2335 2340Asn His Gln Glu Phe Lys Ser Cys Leu Arg Ser Leu Gly Tyr Asp 2345 2350 2355Leu Pro Met Val Glu Glu Gly Glu Pro Asp Pro Glu Phe Glu Ala 2360 2365 2370Ile Leu Asp Thr Val Asp Pro Asn Arg Asp Gly His Val Ser Leu 2375 2380 2385Gln Glu Tyr Met Ala Phe Met Ile Ser Arg Glu Thr Glu Asn Val 2390 2395 2400Lys Ser Ser Glu Glu Ile Glu Ser Ala Phe Arg Ala Leu Ser Ser 2405 2410 2415Glu Gly Lys Pro Tyr Val Thr Lys Glu Glu Leu Tyr Gln Asn Leu 2420 2425 2430Thr Arg Glu Gln Ala Asp Tyr Cys Val Ser His Met Lys Pro Tyr 2435 2440 2445Val Asp Gly Lys Gly Arg Glu Leu Pro Thr Ala Phe Asp Tyr Val 2450 2455 2460Glu Phe Thr Arg Ser Leu Phe Val Asn 2465 247041014PRTHomo sapiens 4Met Ala Glu Ser Ser Asp Lys Leu Tyr Arg Val Glu Tyr Ala Lys Ser1 5 10 15Gly Arg Ala Ser Cys Lys Lys Cys Ser Glu Ser Ile Pro Lys Asp Ser 20 25 30Leu Arg Met Ala Ile Met Val Gln Ser Pro Met Phe Asp Gly Lys Val 35 40 45Pro His Trp Tyr His Phe Ser Cys Phe Trp Lys Val Gly His Ser Ile 50 55 60Arg His Pro Asp Val Glu Val Asp Gly Phe Ser Glu Leu Arg Trp Asp65 70 75 80Asp Gln Gln Lys Val Lys Lys Thr Ala Glu Ala Gly Gly Val Thr Gly 85 90 95Lys Gly Gln Asp Gly Ile Gly Ser Lys Ala Glu Lys Thr Leu Gly Asp 100 105 110Phe Ala Ala Glu Tyr Ala Lys Ser Asn Arg Ser Thr Cys Lys Gly Cys 115 120 125Met Glu Lys Ile Glu Lys Gly Gln Val Arg Leu Ser Lys Lys Met Val 130 135 140Asp Pro Glu Lys Pro Gln Leu Gly Met Ile Asp Arg Trp Tyr His Pro145 150 155 160Gly Cys Phe Val Lys Asn Arg Glu Glu Leu Gly Phe Arg Pro Glu Tyr 165 170 175Ser Ala Ser Gln Leu Lys Gly Phe Ser Leu Leu Ala Thr Glu Asp Lys 180 185 190Glu Ala Leu Lys Lys Gln Leu Pro Gly Val Lys Ser Glu Gly Lys Arg 195 200 205Lys Gly Asp Glu Val Asp Gly Val Asp Glu Val Ala Lys Lys Lys Ser 210 215 220Lys Lys Glu Lys Asp Lys Asp Ser Lys Leu Glu Lys Ala Leu Lys Ala225 230 235 240Gln Asn Asp Leu Ile Trp Asn Ile Lys Asp Glu Leu Lys Lys Val Cys 245 250 255Ser Thr Asn Asp Leu Lys Glu Leu Leu Ile Phe Asn Lys Gln Gln Val 260 265 270Pro Ser Gly Glu Ser Ala Ile Leu Asp Arg Val Ala Asp Gly Met Val 275 280 285Phe Gly Ala Leu Leu Pro Cys Glu Glu Cys Ser Gly Gln Leu Val Phe 290 295 300Lys Ser Asp Ala Tyr Tyr Cys Thr Gly Asp Val Thr Ala Trp Thr Lys305 310 315 320Cys Met Val Lys Thr Gln Thr Pro Asn Arg Lys Glu Trp Val Thr Pro 325 330 335Lys Glu Phe Arg Glu Ile Ser Tyr Leu Lys Lys Leu Lys Val Lys Lys 340 345 350Gln Asp Arg Ile Phe Pro Pro Glu Thr Ser Ala Ser Val Ala Ala Thr 355 360 365Pro Pro Pro Ser Thr Ala Ser Ala Pro Ala Ala Val Asn Ser Ser Ala 370 375 380Ser Ala Asp Lys Pro Leu Ser Asn Met Lys Ile Leu Thr Leu Gly Lys385 390 395 400Leu Ser Arg Asn Lys Asp Glu Val Lys Ala Met Ile Glu Lys Leu Gly 405 410 415Gly Lys Leu Thr Gly Thr Ala Asn Lys Ala Ser Leu Cys Ile Ser Thr 420 425 430Lys Lys Glu Val Glu Lys Met Asn Lys Lys Met Glu Glu Val Lys Glu 435 440 445Ala Asn Ile Arg Val Val Ser Glu Asp Phe Leu Gln Asp Val Ser Ala 450 455 460Ser Thr Lys Ser Leu Gln Glu Leu Phe Leu Ala His Ile Leu Ser Pro465 470 475 480Trp Gly Ala Glu Val Lys Ala Glu Pro Val Glu Val Val Ala Pro Arg 485 490 495Gly Lys Ser Gly Ala Ala Leu Ser Lys Lys Ser Lys Gly Gln Val Lys 500 505 510Glu Glu Gly Ile Asn Lys Ser Glu Lys Arg Met Lys Leu Thr Leu Lys 515 520 525Gly Gly Ala Ala Val Asp Pro Asp Ser Gly Leu Glu His Ser Ala His 530 535 540Val Leu Glu Lys Gly Gly Lys Val Phe Ser Ala Thr Leu Gly Leu Val545 550 555 560Asp Ile Val Lys Gly Thr Asn Ser Tyr Tyr Lys Leu Gln Leu Leu Glu 565 570 575Asp Asp Lys Glu Asn Arg Tyr Trp Ile Phe Arg Ser Trp Gly Arg Val 580 585 590Gly Thr Val Ile Gly Ser Asn Lys Leu Glu Gln Met Pro Ser Lys Glu 595 600 605Asp Ala Ile Glu His Phe Met Lys Leu Tyr Glu Glu Lys Thr Gly Asn 610 615 620Ala Trp His Ser Lys Asn Phe Thr Lys Tyr Pro Lys Lys Phe Tyr Pro625 630 635 640Leu Glu Ile Asp Tyr Gly Gln Asp Glu Glu Ala Val Lys Lys Leu Thr 645 650 655Val Asn Pro Gly Thr Lys Ser Lys Leu Pro Lys Pro Val Gln Asp Leu 660 665 670Ile Lys Met Ile Phe Asp Val Glu Ser Met Lys Lys Ala Met Val Glu 675 680 685Tyr Glu Ile Asp Leu Gln Lys Met Pro Leu Gly Lys Leu Ser Lys Arg 690 695 700Gln Ile Gln Ala Ala Tyr Ser Ile Leu Ser Glu Val Gln Gln Ala Val705 710 715 720Ser Gln Gly Ser Ser Asp Ser Gln Ile Leu Asp Leu Ser Asn Arg Phe 725 730 735Tyr Thr Leu Ile Pro His Asp Phe Gly Met Lys Lys Pro Pro Leu Leu 740 745 750Asn Asn Ala Asp Ser Val Gln Ala Lys Val Glu Met Leu Asp Asn Leu 755 760 765Leu Asp Ile Glu Val Ala Tyr Ser Leu Leu Arg Gly Gly Ser Asp Asp 770 775 780Ser Ser Lys Asp Pro Ile Asp Val Asn Tyr Glu Lys Leu Lys Thr Asp785 790 795 800Ile Lys Val Val Asp Arg Asp Ser Glu Glu Ala Glu Ile Ile Arg Lys 805 810 815Tyr Val Lys Asn Thr His Ala Thr Thr His Asn Ala Tyr Asp Leu Glu 820 825 830Val Ile Asp Ile Phe Lys Ile Glu Arg Glu Gly Glu Cys Gln Arg Tyr 835 840 845Lys Pro Phe Lys Gln Leu His Asn Arg Arg Leu Leu Trp His Gly Ser 850 855 860Arg Thr Thr Asn Phe Ala Gly Ile Leu Ser Gln Gly Leu Arg Ile Ala865 870 875 880Pro Pro Glu Ala Pro Val Thr

Gly Tyr Met Phe Gly Lys Gly Ile Tyr 885 890 895Phe Ala Asp Met Val Ser Lys Ser Ala Asn Tyr Cys His Thr Ser Gln 900 905 910Gly Asp Pro Ile Gly Leu Ile Leu Leu Gly Glu Val Ala Leu Gly Asn 915 920 925Met Tyr Glu Leu Lys His Ala Ser His Ile Ser Lys Leu Pro Lys Gly 930 935 940Lys His Ser Val Lys Gly Leu Gly Lys Thr Thr Pro Asp Pro Ser Ala945 950 955 960Asn Ile Ser Leu Asp Gly Val Asp Val Pro Leu Gly Thr Gly Ile Ser 965 970 975Ser Gly Val Asn Asp Thr Ser Leu Leu Tyr Asn Glu Tyr Ile Val Tyr 980 985 990Asp Ile Ala Gln Val Asn Leu Lys Tyr Leu Leu Lys Leu Lys Phe Asn 995 1000 1005Phe Lys Thr Ser Leu Trp 1010