WO2015143072A1

WO2015143072A1 - Methods for inhibiting proliferation of cancer cells and uses thereof

Info

Publication number: WO2015143072A1
Application number: PCT/US2015/021314
Authority: WO
Inventors: Kivanc Birsoy; David M. Sabatini
Original assignee: Whitehead Institute For Biomedical Research
Priority date: 2014-03-18
Filing date: 2015-03-18
Publication date: 2015-09-24

Abstract

Disclosed herein are methods for inhibiting the growth or survival of cholesterol auxotrophic cancer cells, for example, by limiting the availability of extracellular cholesterol in the cholesterol auxotrophic cancer cells. In some aspects, the disclosure provides a method of inhibiting growth or survival of a cancer cell that is cholesterol auxotrophic due to inadequate squalene epoxidase (SQLE) expression, the method comprising limiting the availability of extracellular cholesterol in a cancer cell that is cholesterol auxotrophic due to inadequate SQLE expression, thereby inhibiting growth or survival of the cancer cell that is cholesterol auxotrophic due to inadequate SQLE expression.

Description

M BTHODS FOR INH IB ITING PROLIFERATION OF CANCER CELLS AND USES

THEREOF

GOVERNMENT SUPPORT

[ I ] This application claims the benefit of U.S. Provisional Application No.

61 /955,007, filed on March 18, 2014, the entire teachings of which are incorporated herein by reference.

GOVERNMENT SUPPORT

[2] This invention was made with government support under CA103866 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[3 | Cancer cell metabolism is altered in various ways, which could be exploited for therapeutic purposes. These alterations, unlike normal cells, include auxotrophies for nonessential nutrients, whereby depletion or transport inhibition of the corresponding nutrient could be used as a therapeutic approach to target cancer cell survival or growth.

SUMMARY OF THE INVENTION

[4] In some aspects, the disclosure provides a method of inhibiting growth or survival of a cancer cell that is cholesterol auxotrophic due to inadequate squalene epoxidase (SQLE) expression, the method comprising limiting the availability of extracellular cholesterol in a cancer cell that is cholesterol auxotrophic due to inadequate SQLE expression, thereby inhibiting growth or survival of the cancer cell that is cholesterol auxotrophic due to inadequate SQLE expression.

[5] In some embodiments, inadequate SQLE expression comprises a decreased level of SQLE expression in the cancer cell relative to the level of SQLE expression in a cholesterol protrophic cancer cell. In some embodiments, inadequate SQLE expression comprises the absence of SQLE expression.

[6] In some embodiments, limiting the availability of extracellular cholesterol in the cancer cell occurs in vitro. In some embodiments, limiting the availability of extracellular cholesterol in the cancer cell comprises contacting the cancer cell with an effective amount of an agent that inhibits cholesterol uptake into the cancer cell. In some embodiments, the agent inhibits the expression level or activity of low density lipoprotein receptor (LDLR) in the cancer cell . In some embodiments, the agent is selected from the group consisting of an antibody, an antisense oligonucleotide, a short hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (mi RNA).

| 7 | In some embod i ments, l im iti ng the avai labi lity of extracellular cholesterol in the cancer cell occurs in vivo. In some embodi ments, limiting the availability of extracellular cholesterol in the cancer cell comprises administering to a subject an effective amount of a cholesterol lowering agent. In some embodiments, the cholesterol lowering agent is selected from the group consisting of small organic or inorganic molecules; saccharides; oligosaccharides; polysaccharides; a biological macromolecule selected from the group consisting of peptides, proteins, peptide analogs and derivatives; peptidomimetics; nucleic acids selected from the group consisting of siRNAs, shRNAs, antisense RNAs, ribozymes, and aptamers; an extract made from biological materials selected from the group consisting of bacteria, plants, fungi, animal cells, and animal tissues; naturally occurring or synthetic compositions; and any combination thereof.

[8] In some embodiments, the cholesterol lowering agent is selected from the group consisting of an agent that i) inhibits intestinal absorption of cholesterol, ii) inhibits the level or activity of 3-hydroxy-3-methylglutaryl-coenzyrne A (HMG-CoA) reductase, iii) increases cholesterol metabol ism, iv) decreases formation and/or secretion of cholesterol esters, v) increases the ratio of high density lipoprotein (HDL) to LDL, and vi) upregulates expression of l iver LDLR receptors. In some embodiments, the agent that i) inhibits intestinal absorption of cholesterol is selected from the group consisting of a Niemann-Pick C l -Like (NPC 1 L1 ) protein inhibitor, a phytosterol, and a phytostanol. In some embodiments, the agent that ii) inhibits the level or activity of HMG-CoA reductase is selected from the group consisting of a tocotrienol and a statin. In some embodiments, the agent that iii) increases cholesterol metabolism is selected from the group consisting of a bile acid sequestrant and a 7-alpha-hydroxylase activator. In some embodiments, the agent that iv) decreases formation and/or secretion of cholesterol esters comprises an acyl-CoA acyl transferase inhibitor. In some embodiments, the agent that v) increases the ratio of HDL to LDL comprises a cholesterylester transfer protein (CETP) inhibitor. In some embodiments, the agent that vi) upregulates expression of liver LDLR receptors comprises a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor.

[9] In some aspects, the disclosure provides a method of rendering a cancer cell cholesterol auxotrophic, the method comprising contacting a cancer cell with an agent that inhibits the expression level or activity of SQLE in the cancer cell, wherein the agent inhibits the expression level or activity of SQLE in the cancer cell, thereby rendering the cancer cell cholesterol auxotrophic.

[10] In some embodiments, the agent is selected from the group consisting of small organic or inorganic molecules; saccharides; oligosaccharides; polysaccharides; a biological macromolecule selected from the group consisting of peptides, proteins, peptide analogs and derivatives; peptidomimetics; nucleic acids selected from the group consisting of siRNAs, shRNAs, antisense RNAs, ribozymes, and aptamers; an extract made from biological materials selected from the group consisting of bacteria, plants, fungi, animal cells, and animal tissues; naturally occurring or synthetic compositions; and any combination thereof.

[ 1 1 ] In some embod iments, the agent is selected from the group consisting of i) an antimycotic compound, i i) an al lylamine, iii) a squalene derivative, iv) a natural compound, and v) derivati ves of any of i)-iv). In some embodiments, the antimycotic compound comprises a

wherein R is selected from the group consisting of

[ 12] In some embodiments, the antimycotic compound comprises a compound of

wherein R is selected from the group consisting of

In some embodiments, the allylamine comprises a compound of formula (II)

wherein R is selected from the group consisting of

[ 14] In some embodiments, the allylamine comprises a compound of formula (III)

wherein R is selected from the group consisting of H, CH₃, CH₂CH₃, CH₂CH₂CH₃, CH₂CHCH_2; CH₂CCH, and C₃H₆.

[ 15] In some embodiments, the allylamine comprises a compound of formula (IV)

wherein R is selected from the group consisting of H, CH₃, CH₂CH , CF₃, OH, nitrile, formyl, hydroxymethyl, phenyl , 2-furyl, 2-oxazolyl, 2-thiazolyl, 5-oxazolyl, 5-thiazolyl, 1 -pyrrolyl, and 3-thienyl .

allylamine comprises a compound of formula (V)

wherein R is selected from the group consisting of

7 in some embod iments, the al lylamine comprises a compound of formula (VI)

wherein R is selected from the group consisting of

and

18] In some embodiments, the allylamine comprises a compound of formula (VII)

wherein R is selected from the group consisting of OCH₃, SCH₃, OH, SH, CONH₂, COCF3, CCH, F, CH3, CHO, CN, and CHCH₂.

[ 19] In some embodiments, the allylamine comprises a compound of formula (VIII)

wherein R is selected from the group consisting of CH₃, OCH₃, CF₃, and CN.

comprises

In some embodiments, the allylamine comprises a compound of formula (IX)

(IX)

wherein R is selected from the group consisting of CH₃ and OCH₃.

[22] In some embodiments, the squalene derivative is selected from the group consisting of

[23] In some embodiments, the squalene derivative comprises a compound of formula

wherein R is selected from the group consisting of OH, CH₂CH₂OH, CH₂OOH, CH₂SH, CH₂NH-

, wherein R is

CHCF,,

[25] In some embodiments, the squalene derivative comprises a compound of formula

(XI)

wherein R is selected from the group consisting of CH₂OH, CHO, and CH₂NH₂.

[26] In some embodiments, the natural compound comprises a plant extract selected from the group consisting of Agrimonia pilosa extract, Aleurites fordii extract, Euphorbia jolkini extract, Lagerstroemia indica extract, Camellia sinensis extract, Allium sativum extract, Rheum palmatum extract, Cynara scolymus extract, Fraxinus excelsior extract, and Peumus boldus extract. In some embodiments, a derivative of any of the above natural compounds or plant extracts is used.

[27] In some embodiments, the natural compound is selected from the group consisting of (~)-epigallocatechin-3-0-gallate (EGCG), (-)-epicatechin-3-0-gallate (ECG), (-)- epigallocatechin (EGC), and (-)-epicatechin (EC), gallic acid.

[28] In some embodiments, the natural compound is a compound of formula (XII)

wherei n R is selected from the group consisting of OH, -0(CH2)7CH3, -0(CH2) 1 1 CH3,

[29] In some embodiments, the natural compound comprises a garlic compound selected from the group consisting of a selenium compound, a tellurium compound, and an allyl compound. In some embodiments, the selenium compound is selected from the group consisting of selenocysti ne, selenite, selenium dioxide, and methylselenol. In some embodiments, the tel lurium compound is selected from the group consisting of tellurite, tellurium dioxide, and dimethyltelluride. In some embodiments, the al lyl compound is selected from the group consisting of S-allylcysteine, alliin, 1 ,3-diallyltrisulfane, and 1 ,2-diallyldisulfane. In some embodiments,the contacting occurs in vitro. In some embodiments, the contacting occurs in vivo. In some embodiments, the method further includes identifying the cancer cell as cholesterol protrophic. [30] In some aspects, the disclosure provides a method of inhibiting growth or survival of a cancer cel l, the method comprising: a) contacting the cancer cell with an effective amount of a first agent that inhibits the level or activity of SQLE in the cancer cell, thereby rendering the cancer cell cholesterol auxotrophic; and b) contacting the cholesterol auxotrophic cancer cell with an effective amount of a second agent that limits the availability of extracellular cholesterol in the cancer cell, thereby inhibiting growth or survival of the cancer cell.

[31 ] In some aspects, the disclosure provides a method of classifying a tumor as cholesterol auxotrophic, the method comprising: a) determining the level of an expression product of the SQLE gene in a sample obtained from the tumor; and b) comparing the level determined in a) with a control level, wherein if the level determined in a) is less than the control level, the level is correlated with cholesterol auxotrophy, thereby classifying the tumor as cholesterol auxotrophic.

[32] In some embodiments, the sample comprises tumor tissue. In some embodiments, the tumor is from a cancer selected from the group consisting of ovarian cancer, uterine cancer (e.g., endometrium adenocarcinoma), bladder cancer, lung cancer, cervical cancer, breast cancer, prostate cancer, gliomas, fibrosarcomas, retinoblastomas, melanomas, soft tissue sarcomas, osteosarcomas, leukemias, stomach cancer (e.g., adenocarcinoma), colon cancer, carcinoma of the kidney, gastrointestinal cancer, salivary gland cancer, pancreatic cancer, Hodgkin's disease, non-Hodgkin's lymphomas (e.g., anaplastic large cell lymphoma), acute and chronic lymphocytic leukemias, multiple myeloma, neuroblastoma, Wilms' tumor, testicular cancer, soft-tissue sarcomas, chronic lymphocytic leukemia, primary macroglobulinemia, chronic granulocytic leukemia, primary brain carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinomas, malignant melanomas, choriocarcinomas, mycosis fungoides, head and neck carcinomas, osteogenic sarcoma, pancreatic carcinomas, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, genitourinary carcinomas, thyroid carcinomas, esophageal carcinomas, malignant hypercalcemia, cervical hyperplasia, renal cell carcinomas, endometrial carcinomas, polycythemia vera, essential thrombocytosis, adrenal cortex carcinomas, skin cancer, and prostatic carcinomas.

[33] In some embodiments, the expression product comprises a mRNA or a polypeptide. In some embodiments, determining the level of the expression product in the sample comprises performing a hybridization based assay, polymerase chain reaction assay, sequencing, ELISA assay, Western blot, mass spectrometry, or immunohistochemistry. [34] In some aspects, the disclosure provides a method of diagnosing a tumor in a subject as a cholesterol auxotrophic tumor, the method comprising: a.) determining the level of an expression product of the SQLE gene in a sample obtained from the subject; and b) comparing the level determined in a) with a control level, wherein if the level determined in a) is less than the control level, the level is indicative of cholesterol auxotrophy, thereby diagnosing the tumor in the subject as a cholesterol auxotrophic tumor.

[35] In some embodiments, the sample comprises tumor tissue. In some embodiments, the tumor is from a cancer selected from the group consisting of ovarian cancer, uterine cancer (e.g., endometrium adenocarcinoma), bladder cancer, lung cancer, cervical cancer, breast cancer, prostate cancer, gliomas, fibrosarcomas, retinoblastomas, melanomas, soft tissue sarcomas, osteosarcomas, leukemias, stomach cancer (e.g., adenocarcinoma), colon cancer, carcinoma of the kidney, gastrointestinal cancer, salivary gland cancer, pancreatic cancer, Hodgkin's disease, non-Hodgkin's lymphomas (e.g., anaplastic large cell lymphoma), acute and chronic lymphocytic leukemias, multiple myeloma, neuroblastoma, Wilms' tumor, testicular cancer, soft-tissue sarcomas, chronic lymphocytic leukemia, primary macroglobulinemia, chronic granulocytic leukemia, primary brain carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinomas, malignant melanomas, choriocarcinomas, mycosis fungoides, head and neck carcinomas, osteogenic sarcoma, pancreatic carcinomas, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, genitourinary carcinomas, thyroid carcinomas, esophageal carcinomas, malignant hypercalcemia, cervical hyperplasia, renal cell carcinomas, endometrial carcinomas, polycythemia vera, essential thrombocytosis, adrenal cortex carci nomas, skin cancer, and prostatic carcinomas.

[36J In some embodiments, the expression product comprises a mRNA or a polypeptide. In some embodiments, determining the level of the expression product in the sample comprises performing a hybridization based assay, polymerase chain reaction assay, sequencing, ELISA assay, Western blot, mass spectrometry, or immunohistochemistry.

[37] In some aspects, the disclosure provides a method of determining whether a subject with a tumor is a suitable candidate for treatment with a cholesterol lowering agent, the method comprising: a) assessing the level of expression of a SQLE gene product in a tumor sample obtained from the subject; and b) comparing the level determined in a) to a control level, wherein if the level determined in a) is less than the control level, then the level in a) indicates that the tumor is a cholesterol auxotrophic tumor, thereby determining that the subject with the tumor is a suitable candidate for treatment with a cholesterol lowering agent. [38] In some embodiments, the sample comprises tumor tissue. In some embodiments, the tumor is a from a cancer selected from the group consisting of ovarian cancer, uterine cancer (e.g., endometrium adenocarcinoma), bladder cancer, lung cancer, cervical cancer, breast cancer, prostate cancer, gliomas, fibrosarcomas, retinoblastomas, melanomas, soft tissue sarcomas, osteosarcomas, leukemias, stomach cancer (e.g., adenocarcinoma), colon cancer, carcinoma of the kidney, gastrointestinal cancer, salivary gland cancer, pancreatic cancer, Hodgkin's disease, non-Hodgkin's lymphomas (e.g., anaplastic large cell lymphoma), acute and chronic lymphocytic leukemias, multiple myeloma, neuroblastoma, Wilms' tumor, testicular cancer, soft-tissue sarcomas, chronic lymphocytic leukemia, primary macroglobulinemia, chronic granulocytic leukemia, primary brain carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinomas, malignant melanomas, choriocarcinomas, mycosis fungoides, head and neck carcinomas, osteogenic sarcoma, pancreatic carcinomas, acute granulocytic leukemia, hairy cell leukem i a, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, genitourinary carcinomas, thyroid carcinomas, esophageal carcinomas, malignant hypercalcemia, cervical hyperplasia, renal cell carcinomas, endometrial carcinomas, polycythemia vera, essential thrombocytosis, adrenal cortex carcinomas, skin cancer, and prostatic carcinomas. In some embodiments, the sample comprises an anaplastic large cell lymphoma tumor sample.

[39] In some embodiments, the expression product comprises a mRNA or a polypeptide. In some embodiments, determining the level of the expression product in the sample comprises performing a hybridization based assay, polymerase chain reaction assay, sequencing, ELISA assay, Western blot, mass spectrometry, or immunohistochemistry.

[40] In some aspects, the disclosure provides a method of treating a cholesterol auxotrophic cancer in a subject in need thereof, the method comprising administering to a subject suffering from a cholesterol auxotrophic cancer an effective amount of a composition which limits the availability of extracellular cholesterol in the cholesterol auxotrophic cancer cells in the subject, wherein the composition inhibits the growth or survival of the cholesterol auxotrophic cancer cel ls, thereby treating the subject's cholesterol auxotrophic cancer.

[ 4 1 1 In some embodiments, the composition comprises at least one cholesterol lowering agent. In some embodi ments, the cholesterol lowering agent is selected from the group consisting of an agent that i) inhibits intestinal absorption of cholesterol, ii) inhibits the level or activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, iii) increases cholesterol metabolism, iv) decreases formation and/or secretion of cholesterol esters, v) increases the ratio of high density lipoprotein (HDL) to LDL, and vi) upregulates expression of liver LDLR receptors. In some embodiments, the agent that i) inhibits intestinal absorption of cholesterol is selected from the group consisting of a N iemann-Pick C I -Like (NPC 1 L 1 ) protein i nhibitor, a phytosterol, and a phytostanol . In some embodiments, the agent that ii) inhibits the level or acti vity of H MG-CoA reductase is selected from the group consisting of a tocotrienol and a statin. In some embodiments, the agent that iii) increases cholesterol metabolism is selected from the group consisting of a bile acid sequestrant and a 7-alpha-hydroxylase activator. In some embodiments, the agent that iv) decreases formation and/or secretion of cholesterol esters comprises an acyl-CoA acyl transferase inhibitor. In some embodiments, the agent that v) increases the ratio of HDL to LDL comprises a cholesterylester transfer protein (CETP) inhibitor. In some embodiments, the agent that vi) upregulates expression of liver LDLR receptors comprises a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor. In some embodiments, the method further includes administering to the subject an effective amount of at least one chemotherapeutic agent. In some embodiments, the method includes diagnosing the subject as having a cholesterol auxotrophic cancer.

[42] In some aspects, the disclosure provides a method of identifying a candidate agent that renders a cancer cell cholesterol auxotrophic, comprising: a) contacting a cancer cell that expresses SQLE with a test agent; b) measuring the expression level of SQLE in the presence and absence of the test agent; and c) determining whether the test agent renders the cancer cell cholesterol auxotrophic, wherein if the level measured in b) in the presence of the test agent is decreased compared to the level measured in b) in the absence of the test agent, the test agent is identified as a candidate agent that renders the cancer cell cholesterol auxotrophic.

[43] In some embodiments, the method includes performing an assay to confirm that the candidate agent that renders the cancer cell cholesterol auxotrophic. In some embodiments, the assay comprises a cell competition assay in lipoprotein depleted serum media. In some embodiments, the assay comprises: a) incubating cholesterol protrophic cancer cells in a lipoprotein depleted serum media; b) contacting the cholesterol protrophic cancer cells with a candidate agent that renders a cancer cell cholesterol auxotrophic; and c) assessing the growth of the cholesterol protrophic cancer cells in the lipoprotein depleted serum media after being contacted with the candidate agent, wherein if the growth of the cholesterol protrophic cancer cells is inhibited in the lipoprotein depleted serum media after being contacted with the candidate agent, then the candidage agent renders the cancer cells cholesterol auxotrophic.

[44 ] In some aspects, the disclosure provides a composition comprising: a) at least one cholesterol loweri ng agent; and b) at least one chemotherapeutic agent. [45] In some aspects, the disclosure provides a composition comprising: a) at least one agent that inhibits the level or activity of SQLE; b) at least one cholesterol lowering agent; and c) at least one chemotherapeutic agent.

[46] In some aspects, the disclosure provides a kit comprising: a) at least one cholesterol lowering agent; and b) at least one chemotherapeutic agent.

[47] In some aspects, the disclosure provides a kit comprising: a) at least one agent that inhibits the level or activity of SQLE; b) at least one cholesterol lowering agent; and c) at least one chemotherapeutic agent.

[48] In some aspects, the disclosure provides a kit comprising: a) at least one cancer cell that auxotrophic due to inadequate expression of SQLE in the cancer cell; and b) a test agent.

[49 ] In some embodiments, the at least one cancer cell is selected from the group consisting of Snu- 1 , Raj i, Daudi, IT716, and U937. In some embodiments, the at least one cancer cell is engineered to be auxotrophic due to inadequate expression of SQLE in the cancer cell. In some embodiments, the kit includes a lipoprotein conditioned media, in some embodiments, the kit includes instructions for assessing whether the test agent is capable of limiting the availability of extracellular cholesterol in the at least one cancer cell.

[50] The practice of the present invention will typically employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant nucleic acid (e.g., DNA) technology, immunology, and RNA interference (RNAi) which are within the skill of the art. Non-limiting descriptions of certain of these techniques are found in the following publications: Ausubel, F., et al, (eds.), Current Protocols in Molecular Biology, Current Protocols in Immunology, Current Protocols in Protein Science, and Current Protocols in Cell Biology, al l John Wi ley & Sons, N.Y., edition as of December 2008; Sambrook, Russel l, and Sambrook, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001 ; Harlow, E. and Lane, D., Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988; Freshney, R.I., "Culture of Animal Cells, A Manual of Basic Technique", 5th ed., John Wiley & Sons, Hoboken, NJ, 2005. Non-limiting information regarding therapeutic agents and human diseases is found in Goodman and Gilman's The Pharmacological Basis of

Therapeutics, 1 1th Ed., McGraw Hill, 2005, Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw-Hiil/Appleton & Lange; 10th ed. (2006) or 1 1th edition (July 2009). Non-limiting information regarding genes and genetic disorders is found in McKusick, V.A. : Mendelian Inheritance in Man. A Catalog of Human Genes and Genetic Disorders. Baltimore: Johns Hopkins University Press, 1 998 ( 12th edition) or the more recent online database: Online Mendelian Inheritance in Man, OMIM™. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD), as of May 1 , 2010, World Wide Web URL: http://www.ncbi .nlm.nih.gov/omim/ and in Online Mendelian Inheritance in Animals (OMIA), a database of genes, inherited disorders and traits in animal species (other than human and mouse), at http://omia.angis.org.au/contact.shtml. All patents, patent applications, and other publications (e.g., scientific articles, books, websites, and databases) mentioned herein are incorporated by reference in their entirety. In case of a conflict between the specification and any of the incorporated references, the specification (including any amendments thereof, which may be based on an incorporated reference), shall control. Standard art-accepted meanings of terms are used herei n unless indicated otherwise. Standard abbreviations for various terms are used herein.

BR I FF DESCRIPTION OF TH E DRAWINGS

[5 1 j FIGS. 1 A, 1 B, 1 C, and 1 D demonstrate the cell competition assay used to identify cancer cells that are cholesterol auxotrophic due to inadequate SQLE expression. FIG. 1 A is a schematic representation of an exemplary cell competition assay scheme of the disclosure. FIG. I B is a graph showing the results of a cell competition assay conducted under lipoprotein depleted conditions. FIG . 1 C is a graph confirming that cholesterol auxotrophic cancer cells depend on extracellular cholesterol for growth. FIG. I D is a graph showing that cholesterol supplementation rescues the growth defect of cholesterol auxotrophic cancer cells observed under lipoprotein depleted conditions.

[52] FIGS. 2A, 2B, 2C, 2D and 2E demonstrate the identification of cholesterol auxotrophic SNU-1 cancer cells. FIG. 2A depicts SQLE, EBP, and CYP51 Al expression analysis of cell lines from the CCLE database. FIG. 2B is a graph showing that cancer cells with low expression of SQLE, EBP and CYPS l A 1 are sensitive to cholesterol depletion. FIG. 2C is a graph showing squalene accumulation in the SNU- 1 cancer cell line. FIG. 2D is a graph showing that overexprssion of SQLE in the SNU- 1 cancer cell l ine rescues the growth defect observed under l ipoprotein depleted conditions. FIG. 2E demonstrates that treatment with a polyclonal LDLR antibody (R&D) inhibits growth of cholesterol auxotrophic cancer cells. [53] FIG. 3 is a graph showing the relative cell number of cancer cells treated with 20 ^ig/ml of anti-LDLR monoclonal antibody for 6 days in the presence and absence of extracellular cholesterol.

[54] FIGS. 4A and 4B demonstrate that squalene accumulates in cancer cell lines with low levels of SQLE (FIG. 4A) and that SQLE low cancer cell lines are enriched with the anaplastic large cell lymphoma cancer subtype (FIG. 4B).

[55] The patent or appl ication file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

DETAILED DESCRIPTION OF THE INVENTION

[56] Aspects of the disclosure relate to methods, compositions, kits, and agents for inhibiting the growth or survival of cancer cells (e.g., cholesterol auxotrophic cancer cells). Work described herein surprisingly and unexpectedly demonstrates that certain cancer cells exhibit cholesterol auxotrophy due to inadequate expression, or improper functioning, of a cholesterol biosynthesis gene known as squalene epoxidase (SQLE), i.e., the cholesterol auxotrophic cancer cells are unable to synthesize intracellular cholesterol and therefore are dependent on extracellular cholesterol for growth and/or survival .

[57] Accordingly, in some aspects, the disclosure provides a method of inhibiting growth or survival of a cancer cell that is cholesterol auxotrophic due to inadequate squalene epoxidase (SQLE) expression comprising limiting the availability of extracellular cholesterol in a cancer cel l that is cholesterol auxotrophic due to inadequate SQLE expression, thereby inhibiting growth or survi val of the cancer cell that is cholesterol auxotrophic due to inadequate SQLE expression.

[58] The expressions "inhibit", "modulate", "increase", "decrease" or the like, e.g., which denote quantitative differences between two states, refer to at least statistically significant differences between the two states. For example, "inhibiting growth or survival of a cancer cell" means that the rate of growth of the cancer cells or the fraction of cancer cells surviving after treatment will be at least statistically significantly different from the cancers cells in which growth or survival is not inhibited by use of a method, composition, or agent of the disclosure. Such terms are applied herein to, for example, rates of cell proliferation, percentages of surviving cells, levels of expression, levels of transcriptional or translational activity, and levels of enzymatic or protein activity. [59] As used herei n, "cholesterol" refers to any form of cholesterol that maintains the growth or survival of a cholesterol auxotrophic cancer cell when it is transported to and taken up into the cholesterol auxotrophic cancer cell, including, for example, cholesterol, esterified forms of cholesterol, cholesterol present in lipoproteins, e.g., chylomicrons, very low density lipoprotein (VLDL), low density lipoprotein (LDL), and intermediate density lipoprotein (IDL).

[60] The expressions "cholesterol auxotrophic", "cholesterol auxotrophy", and the like, in the context of "cancer cells" refer to the inability of the cells to synthesize enough cholesterol for their growth or survival. The expressions "cholesterol protrophic", "cholesterol protrophy", and the like, in the context of "cancer cells" refer to the ability of the cancer cells to synthesize enough cholesterol for their growth or survival. The expression "inadequate SQLE expression" in the context of "cholesterol auxotrophic" cancer cells means that the expression levels of SQLE in the cancer cells are insufficient for the cancer cells to synthesize enough intracel lular cholesterol for the cancer cells to proliferate and/or survive. In some embodiments, i nadequate SQLE expression encompasses expression of a defective SQLE enzyme or protein, i.e., the level of SQLE mRNA or protein produced is equivalent to levels found in cholesterol protrophic cancer cells but the SQLE enzyme is catalytically inactive, e.g., due to a mutation in the catalytic core.

[61 ] In some embodiments, inadequate SQLE expression comprises a decrease in the level of expression of SQLE in the cholesterol auxtrophic cancer cell relative to the level of expression of SQLE in a cholesterol protrophic cancer cell. The disclosure contemplates any decrease in the level of expression of SQLE in a cancer cell that results in the cancer cell becoming cholesterol auxotrophic. In some embodiments, inadequate SQLE expression comprises a decrease in the level of expression of SQLE in the cholesterol auxotrophic cancer cell of at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more relative to level of expression of SQLE in a cholesterol protrophic cancer cell.

[62] In some embod iments, inadequate SQLE expression comprises a decrease in the level of expression of SQLE in the cholesterol auxotrophic cancer cell of at least 1 .1 fold, at least 1 .2 fold, 1 .3 fold, at least 1 .4 fold, at least 1 .5 fold, at least 1 .6 fold, at least 1.7 fold, at least 1 .8 fold, at least 1 .9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 100 fold, or more relative to level of expression of SQLE in a cholesterol protrophic cancer cell . [63] In some embodiments, i nadequate SQLE expression comprises a decrease in the level of expression of SQLE mRNA. In some embodiments, inadequate SQLE expression comprises a decrease in the level of expression of SQLE mRNA in the cholesterol auxotrophic cancer cell of at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 1 5%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more relative to level of expression of SQLE mRNA in a cholesterol protrophic cancer cell. In some embodiments, inadequate SQLE expression comprises a decrease in the level of expression of SQLE mRNA in the cholesterol auxotrophic cancer cell of at least 1 . 1 fold, at least 1 .2 fold, 1 .3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1 .8 fold, at least 1.9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 100 fold, or more relative to level of expression of SQLE mRNA in a cholesterol protrophic cancer cell.

[64] In some embodiments, inadequate SQLE expression comprises a decrease in the level of expression of SQLE protein. In some embodiments, inadequate SQLE expression comprises a decrease in the level of expression of SQLE protein in the cholesterol auxotrophic cancer cell of at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 1 %, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more relative to level of expression of SQLE protein in a cholesterol protrophic cancer cell. In some embodiments, inadequate SQLE expression comprises a decrease in the level of expression of SQLE protein in the cholesterol auxotrophic cancer cell of at least 1 .1 fold, at least 1 .2 fold, 1 .3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1 .8 fold, at least 1.9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 100 fold, or more relative to level of expression of SQLE protein in a cholesterol protrophic cancer cell.

[65] In some embodiments, inadequate SQLE expression comprises a decrease in the level of epoxidase activity of SQLE enzyme. In some embodiments, inadequate SQLE expression comprises a decrease in the level of epoxidase activity of SQLE enzyme in the cholesterol auxotrophic cancer cell of at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 1 5%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more relative to level of epoxidase activity of SOLE enzyme in a cholesterol protrophic cancer ceil. In some embodiments, inadequate SQLE expression comprises a decrease in the level of epoxidase activity of the SQLE enzyme in the cholesterol auxotrophic cancer cell of at least 1 .1 fold, at least 1.2 fold, 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1 .6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 100 fold, or more relative to level of epoxidase activity of SQLE enzyme in a cholesterol protrophic cancer cell.

[66] In some embodiments, inadequate SQLE expression comprises the absence SQLE expression in the cholesterol auxotrophic cancer cell. In some embodiments, the absence of SQLE expression comprises the absence of expression of SQLE mRNA. In some embodiments, the absence of SQLE expression comprises the absence of expression of SQLE protein.

[67] The expression "intracellular cholesterol" refers to cholesterol that is synthesized inside a cell (e.g., cancer cell). The expression "extracellular cholesterol" in the context of a "cholesterol auxotrophic cancer cell" refers to cholesterol that is not synthesized inside the cholesterol auxotrophic cancer cell, e.g., circulating cholesterol.

[68] Cytochrome P450, family 51 , subfamily A, polypeptide 1 (CYP51 A 1 ; NCBI Reference Sequence: NG_007968.1 ) encodes a member of the cytochrome P450 superfamily of enzymes, which are monooxygenases which catalyze reactions involved in metabolism and synthesis of cholesterol, steroids, and other lipids. CYP51 Al is an endoplasmic reticulum protein that aids cholesterol synthesis by catalyzing the removal of a 14alpha-methyl group from lanosterol , Genes homologous to CYP51 A l can be found in fungi, plants, and animals. Two known transcript variants of CYP5 1 A l encoding isoforms have been identified in humans, incl uding a 3208 bp li near mRNA referred to as CYP5 1 A l , transcript variant 1 (NCBI Reference Sequence: NM_000786.3), and a 2934 bp linear mRNA referred to as CYP51 A 1 , transcript variant 2 (NCBI Reference Sequence: NM_001 146152.1 ). Without wishing to be bound by theory, it is believed that low expression of CYP51 A1 in a cell may induce an auxotrophy in the cell (e.g., cholesterol auxotrophy).

[69] In some aspects, a cancer cell exhibits cholesterol auxotrophy due to inadequate expression, or improper functioning, of the cholesterol biosynthesis gene known as CYP5 1 Al .

[70] In some aspects, the disclosure provides a method of inhibiting growth or survival of a cancer cell that is cholesterol auxotrophic due to inadequate CYP51 A l expression comprising limiting the availability of extracellular cholesterol in a cancer cell that is cholesterol auxotrophic due to inadequate CYP51 A l expression, thereby inhibiting growth or survival of the cancer cell that is cholesterol auxotrophic due to inadequate CYP5 1 A l expression. [71 ] The expression "inadequate CYP51A I expression" in the context of "cholesterol auxotrophic" cancer cells means that the expression levels of CYP51A1 in the cancer cells are insufficient for the cancer cells to synthesize enough intracellular cholesterol for the cancer cells to proliferate and/or survive. In some embodiments, inadequate CYP51A1 expression encompasses expression of a defective CYP51A1 enzyme or protein, i.e., the level of CYP51 Al mRNA or protein produced is equivalent to levels found in cholesterol protrophic cancer cells but the CYP5 1 A l enzyme is catalytically inactive, e.g., due to a mutation in the catalytic core.

[72] In some embodiments, inadequate CYP51 Al expression comprises a decrease in the level of expression of CYP5 1 A l in the cholesterol auxtrophic cancer cell relative to the level of expression of CYP5 1 A l in a cholesterol protrophic cancer cell. The disclosure contemplates any decrease in the level of expression of CYP5 1 A 1 in a cancer cell that results in the cancer cell becom i ng cholesterol auxotrophic. In some embodiments, inadequate CYP51 A l expression comprises a decrease in the level of expression of CYP51 A1 in the cholesterol auxotrophic ^' cancer cell of at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 1 5%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more relative to level of expression of CYP51A1 in a cholesterol protrophic cancer cell.

[73] In some embodiments, inadequate CYP51 A1 expression comprises a decrease in the level of expression of CYP51 Al in the cholesterol auxotrophic cancer cell of at least 1.1 fold, at least 1 .2 fold, 1.3 fold, at least 1 .4 fold, at least 1 .5 fold, at least 1.6 fold, at least 1.7 fold, at least 1 .8 fold, at least 1 .9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 100 fold, or more relative to level of expression of CYP51 A l in a cholesterol protrophic cancer cell.

[74] In some embodiments, inadequate CYP51 A1 expression comprises a decrease in the level of expression of CYP51 Al mRNA. As used herein, "CYP51 A l mRNA" is intended to encompass variants or isoforms, e.g., CYP5 1 A 1 , transcript variant 1 , mRNA, CYP51 A 1 , transcri pt variant 2, mRNA, etc. In some embodiments, inadequate CYP51A 1 expression comprises a decrease in the level of expression of CYP51 A I in the cholesterol auxotrophic cancer cell of at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 1 5%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more relative to level of expression of CYP51 A l mRNA in a cholesterol protrophic cancer cell. In some embodiments, inadequate CYP51A 1 expression comprises a decrease in the level of expression of CYP51 Al mRNA in the cholesterol auxotrophic cancer cell of at least 1 . 1 fold, at least 1.2 fold, 1 .3 fold, at least 1 .4 fold, at least 1 .5 fold, at least 1 .6 fold, at least 1 .7 fold, at least 1 .8 fold, at least 1 .9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 1 00 fold, or more relative to level of expression of CYP51 A 1 mRNA in a cholesterol protrophic cancer cell.

[75] In some embodiments, inadequate CYP51 A1 expression comprises a decrease in the level of expression of CYP51A1 protein. In some embodiments, inadequate SQLE expression comprises a decrease in the level of expression of CYP51 Al protein in the cholesterol auxotrophic cancer cell of at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more relative to level of expression of CYP51 Al protein in a cholesterol protrophic cancer cell. In some embodiments, inadequate CYP51 A 1 expression comprises a decrease in the level of expression of CYP51A1 protein in the cholesterol auxotrophic cancer cell of at least 1 .1 fold, at least 1 .2 fold, 1 .3 fold, at least 1.4 fold, at least 1.5 fold, at least 1 .6 fold, at least 1 .7 fold, at least 1 .8 fold, at least 1.9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 100 fold, or more relative to level of expression of CYP51 A 1 protein in a cholesterol protrophic cancer cell .

[76] In some embod iments, inadequate CYP51 A l expression comprises a decrease in the level of demethylase activity of CYP5 1 A l enzyme. In some embodiments, inadequate CYP51 A 1 expression comprises a decrease in the level of demethylase activity of CYP51 Al enzyme in the cholesterol auxotrophic cancer cell of at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more relative to level of demethylase activity of CYP51 Al enzyme in a cholesterol protrophic cancer cell. In some embodiments, inadequate CYP51 A l expression comprises a decrease in the level of demethylase activity of the CYP5 1 Al enzyme in the cholesterol auxotrophic cancer cell of at least 1 .1 fold, at least 1 .2 fold, 1 .3 fold, at least 1 .4 fold, at least 1 .5 fold, at least 1.6 fold, at least 1.7 fold, at least 1 .8 fold, at least 1 .9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 1 0 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 100 fold, or more relative to level of demethylase activity of CYP5 1 A l enzyme in a cholesterol protrophic cancer [77] In some embodiments, inadequate CYP5 1 A l expression comprises a decrease in the rate or turnover of the removal of the 14alpha-methyl group from lanosterol. In some embodiments, inadequate CYP51 Al expression comprises a decrease rate or turnover of the removal of the 14alpha-methyl group from lanosterol in the cholesterol auxotrophic cancer cell of at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more relative to the rate or turnover of the removal of the 14alpha-methyl group from lanosterol of CYP5 1 A l enzyme in a cholesterol protrophic cancer cell. In some embodi ments, inadequate CYP51 A l expression comprises a decrease in the decrease rate or turnover of the removal of the 14alpha-methyl group from lanosterol in the cholesterol auxotrophic cancer cell of at least 1 . 1 fold, at least 1 .2 fold, 1 .3 fold, at least 1.4 fold, at least 1.5 fold, at least 1 .6 fold, at least 1 .7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 100 fold, or more relative to decrease rate or turnover of the removal of the 1 4alpha-methyl group from lanosterol of the CYP51 A l enzyme in a cholesterol protrophic cancer cell.

[78] In some embodiments, inadequate CYP51 A l expression comprises the absence CYP51 A 1 expression in the cholesterol auxotrophic cancer cell. In some embodiments, the absence of CYP51A 1 expression comprises the absence of expression of CYP51 Al mRNA. In some embodiments, the absence of CYP51 A l expression comprises the absence of expression of CYP5 1 A 1 protein.

[79] In some aspects, the disclosure provides a method of inhibiting growth or survival of a cancer cel l that is cholesterol auxotrophic due to inadequate SQLE and/or CYP51 A1 expression comprising limiting the availabil ity of extracellular cholesterol in a cancer cell that is cholesterol auxotrophic due to inadequate SQLE and/or CYP51 A1 expression, thereby inhibiting growth or survival of the cancer cell that is cholesterol auxotrophic due to inadequate SQLE and/or CYP5 1 A 1 expression.

[80] In some aspects, the disclosure provides a method of inhibiting growth or survival of a cancer cell that is cholesterol auxotrophic due to inadequate SQLE and CYP51 A l expression comprising limiting the availability of extracellular cholesterol in a cancer cell that is cholesterol auxotrophic due to inadequate SQLE and CYP51A1 expression, thereby inhibiting growth or survival of the cancer cell that is cholesterol auxotrophic due to inadequate SQLE and CYP51A1 expression. [81 ] In some aspects, the disclosure provides a method of inhibiting growth or survival of a cancer cell that is cholesterol auxotrophic due to inadequate SQLE or CYP5 1 A 1 expression comprising limiting the availability of extracellular cholesterol in a cancer cell that is cholesterol auxotrophic due to inadequate SQLE or CYP51A1 expression, thereby inhibiting growth or survival of the cancer cell that is cholesterol auxotrophic due to inadequate SQLE or CYP51 A l expression.

[82] Emopamil binding protein (sterol isomerase) (EBP) (NCBI Reference Sequence: NM_006579.2) is an integral membrane protein of the endoplasmic reticulum possessing high affinity binding for the antiischemic phenylalkylamine Ca2+ antagonist [3H]emopamil and the photoaffinity label [3H]azidopamil. EBP exhibits similarity to sigma receptors and could be a member of the superfamily of high affinity drug-binding proteins in the endoplasmic reticulum of various tissues. Interestingly, EBP shares structural features with drug transporting proteins (e.g., bacterial and eukaryotic). EBP contains four putative transmembrane segments and two conserved gl utamate residues which are thought to be involved in the transport of cationic amphi l ics. EB P also contains a high content of aromatic amino acid residues (at least 23%) in its transmembrane segments, which may be involved in drug transport by the P-glycoprotein.

Mutations in the EBP gene cause Chondrodysplasia punctate 2 (CDPX2, also known as Conradi- Hunermann syndrome).

[83] Aspects of the disclosure involve limiting the availability of extracellular cholesterol in cholesterol auxotrophic cancer cells, for example, to inhibit the growth or survival of the cholesterol auxotrophic cancer cells. The disclosure contemplates limiting the availability of extracellular cholesterol in cholesterol auxotrophic cancer cells by any amount that results in the inhibition of growth or survival of the cholesterol auxotrophic cells.

[84] In some embodiments, the availability of extracellular cholesterol in cholesterol auxotrophic cancer cells is limited by at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 1 0%, at least 1 5%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more relative to the availability of extracel lular cholesterol in the cholesterol auxotrophic cancer cells in the absence of l imiting the availabi lity of extracellular cholesterol in the cells utilizing a method, composition, kit or agent described herein. In some embodiments, the availability of extracellular cholesterol in cholesterol auxotrophic cancer cells is limited by at least 1. 1 fold, at least 1.2 fold, 1.3 fold, at least 1.4 fold, at least 1 .5 fold, at least 1 .6 fold, at least 1 .7 fold, at least 1.8 fold, at least 1 .9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 100 fold, or more relative to the availability of extracellular cholesterol in the cholesterol auxotrophic cancer cells in the absence of l imiting the availabil ity of extracellular cholesterol in the cells utilizing a method, composition, kit or agent described herein. In some embodiments, the availability of extracellular cholesterol in cholesterol auxotrophic cancer cells is completely limited, i .e., the cholesterol auxotrophic cancer cel ls are deprived of extracellular cholesterol.

[85] Without wishing to be bound by theory, the inhibition of growth or survival of cholesterol auxotrophic cancer cells is expected to be proportionate to the extent the availability of extracellular cholesterol is limited in the cholesterol auxotrophic cancer cells, ΐη some embodiments, limiting the availability of extracellular cholesterol in cholesterol auxotrophic cancer cells inhibits the growth of the cancer cells by at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 1 5%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more. In some

embodiments, limiting the availability of extracellular cholesterol in cholesterol auxotrophic cancer cells inhibits the growth of the cancer cells by at least 1. 1 fold, at least 1.2 fold, 1.3 fold, at least 1.4 fold, at least 1 .5 fold, at least 1 .6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 100 fold, or more. In some embodiments, l i miting the avai labi lity of extracellular cholesterol in cholesterol auxotrophic cancer cells compl etely inhi bits the growth of the cholesterol auxotrophic cancer cel ls.

[86] In some embodiments, limiting the availability of extracellular cholesterol in cholesterol auxotrophic cancer cells inhibits the survival of the cancer cells by at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more. In some embodiments, limiting the availability of extracellular cholesterol in cholesterol auxotrophic cancer cells inhibits the survival of the cancer cells by at least 1.1 fold, at least 1 .2 fold, 1.3 fold, at least 1 .4 fold, at least 1.5 fold, at least 1.6 fold, at least 1 .7 fold, at least 1.8 fold, at least 1 .9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 100 fold, or more. In some embodiments, limiting the availability of extracellular cholesterol in cholesterol auxotrophic cancer cells completely inhibits the survival of the cholesterol auxotrophic cancer cel ls, i.e., all the affected cholesterol auxotrophic cancer cells are destroyed. [87] The methods, compositions, kits, and agents can be employed both in vitro or in vivo to limit the availability of extracellular cholesterol in cholesterol auxotrophic cancer cells. The expression "limiting the availability of extracellular cholesterol" in a cancer cell refers to decreasing extracellular cholesterol transport to and uptake into the cancer cell. The mechanisms by which cholesterol is absorbed, synthesized, metabolized, and transported to and into cells are well understood by the skilled artisan (see, e.g., Dietschy and Wilson, "Regulation of Cholesterol Metabol ism", N Engl J Med 1 970; 282: 1 179-1 183; Goldstein and Brown, "Lipoprotein receptors, cholesterol metabol ism, and atherosclerosis", Archives of Pathology 1975; 99(4): 181- 184;

Chobanian and Hollander, "Body cholesterol metabolism in man. I. The equilibrium of serum and tissue cholesterol", J Clin Invest 1962; 41 (9) 1732- 1737; Goldstein and Borwn, "Familial hypercholesterolemia: A genetic regulatory defect in cholesterol metabolism", The American J of Medicine 1 975; 58(2): 147-50; Bengoechea-Alonso and Ericsson, "SREBP in signal transduction: cholesterol metabol ism and beyond", Current Opinion in Cell Biology 2007; 19(2):21 5-222; Nestel et al., "Cholesterol metabolism in human obesity", Journal of Clinical Investigations 1973 ; 52( 10):2389; Chen, "Role of cholesterol metabolism in cell growth", Federation proceedings 1 984; 43( 1 ): 126; Russell, "Cholesterol biosynthesis and metabolism", Cardiovascular drugs and therapy 1992;6(2): 103-1 10; Cook, RP, "Cholesterol chemistry, biochemistery, and pathology", Cholesterol chemistry, biochemistry, and pathology. 1 958; Grundy, SM. "Cholesterol metabolism in man", Western Journal of Medicine, 128( 1): 13; Dietschy, JM. "Regulation of cholesterol metabolism in man and other species", Klinische Wochenschrift 1984; 62(8):338-345; Wood et al., "Dietary regulation of cholesterol metabolism", The Lancet 1966; 288(7464):604-607; Hu et al., "Cellular cholesterol delivery, intracellular processing and utilization for biosynthesis of steroid hormones", Nutrition & Metabolism 2010; 7:47; and Santos and Schulze, "Lipid metabolism in cancer", FEBS Journal 201 2; 279:2610-2623). The disclosure contemplates any suitable method o f l i miting the avai lability of extracel lular cholesterol in a cholesterol auxotrophic cancer cell. Suitable such mechanisms involve exploiti ng or targeting the mechanisms by which cholesterol is absorbed, synthesized, metaboli zed, and transported to and into cells.

[88] In some embodiments, limiting the availability of extracellular cholesterol in a cholesterol auxotrophic cancer cell comprises contacting the cancer cell with an effective amount of agent that inhibits cholesterol uptake into the cancer cell. The disclosure contemplates the use of any agent that is capable of inhibiting cholesterol uptake into the cancer cell. Exemplary agents include, but are not limited to, small organic or inorganic molecules; saccharides;

oligosaccharides; polysaccharides; a biological macromolecule selected from the group consisting of peptides, proteins, peptide analogs and derivati ves; peptidomimetics; nucleic acids selected from the group consisting of si RNAs, shRNAs, antisense RNAs, ribozymes, and aptamers; an extract made from biological materials selected from the group consisting of bacteria, plants, fungi, animal cells, and animal tissues; naturally occurring or synthetic compositions; and any combination thereof.

[89] In some embodiments, the agent that inhibits cholesterol uptake into a cell comprises an agent that inhibits the level or activity of low density lipoprotein receptor (LDLR). It is understood that when a cell requires extracellular cholesterol (e.g., a cholesterol auxotrophic cancer cell), it synthesizes LDL receptors which are translated by ribosomes on the endoplasmic reticulum, modified by the Golgi apparatus, and are transported in vesicles to the cell surface where they bind to ligand (e.g., low density lipoprotein, apoprotein B 100, apoE protein from chylomicron remnants, and VLDL remnants), and internalize the ligand (e.g., LDL particles) from the bloodstream into the cell via a process known as receptor-mediated endocytosis.

[90] It is contemplated that the agents can inhibit the level or activity of LDLR by any suitable mechanism, e.g., decreasing LDLR expression (e.g., transcription of LDLR mRNA or translation of LDLR mRNA into LDLR protein), decreasing transport of synthesized LDLR from the ER to the cell surface, decreasing LDLR recycling, interfering with receptor-mediated endocytosis of the LDLR, etc.

[91 ] In some embodiments, the agent that inhibits the level or activity of LDLR comprises a short hairpin RNA (shRNA). Examples of LDLR shRNA include, without limitation, LDLR shRNA plasmid (commercially available from Santa Cruz Biotechnology, Dallas, TX), LDLR shRNA lenti viral particles (commercially available from Santa Cruz Biotechnology, Dallas, TX), lentiviral or non-viral shRNA constructs (commercially available from GeneCopoeia, Rockville, MD), and LDLR shRNA constructs in retroviral vectors (commercially available from OriGene Rockville, MD).

[92] In some embodiments, the agent that inhibits the level or activity of LDLR comprises a small interfering RNA (siRNA). Exemplary si RNA include, without limitation, LDLR siRNA (commercially available from Santa Cruz Biotechnology, Dallas, TX) and LDLR si R NA duplexes (commercially available from OriGene, Rockville, MD).

[93 J In some embod iments, the agent that inhibits the level or activity of LDLR compri ses an interfering RN A agent (RNAi). Exemplary LDLR RNAi include, without l imitation, LDLR pre-design chimera RNAi commercially available from Abnova and

M ISSION® csiR A (commercially available from Sigma-Aldrich, St. Louis, MO). [94] In some embodiments, the agent that inhibits the level or activity of LDLR comprises an antibody. In some embodiments, the antibody comprises a monoclonal antibody to the human LDL receptor, such as the monoclonal antibodies described in U.S. Pat. No. 6,849,720. Additional exemplary LDLR antibodies are commercially available (e.g., from Epitomics (Burl ingame, CA ), Abnova (Walnut, CA), Abeam (Cambridge, MA), EMD Mil lipore (Billerica, MA), R and D Systems (United Kingdom), Abbiotec (San Diego, CA), Proteintech Group (Ch icago, IL), Thermo Scienli llc Pierce Products (Rockford, I L), GeneTex (Irvine, CA), Creative Biomart (Shirley, NY), Sino Biological (Daxing, China), Novus Biologicals (Littleton, CO), OriGene (Rockville, MD), US Biological, LifeSpan, ATCC, and Santa Cruz Biotechnology (Dallas, TX)).

[95] In some embodiments, limiting the availability of extracellular cholesterol in the cancer cell comprises administering to a subject an effective amount of a cholesterol lowering agent. As used herein, a "cholesterol lowering agent" refers to an agent that decreases the amount of circulating cholesterol in a subject, e.g., plasma cholesterol levels. Numerous cholesterol lowering agents are known in the art. Exemplary cholesterol lowering agents which are of use include agents that i) inhibit intestinal absorption of cholesterol, ii) inhibit the level or activity of 3-hydroxy-3~methylglutaryl-coenzyme A (HMG-CoA) reductase, iii) increase cholesterol metabolism, iv) decrease formation and/or secretion of cholesterol esters, v) increase the ratio of high density lipoprotein (HDL) to LDL, and vi) upregulate expression of liver LDLR receptors.

[96] It is understood that dietary cholesterol is absorbed in the intestines before entering the systemic circulation. Without wishing to be bound by theory, it is believed that inhibiting intestinal absorption of cholesterol can limit the availability of extracellular cholesterol in cholesterol auxotrophic cancer cells, for example, by reducing circulating LDL-cholesterol levels, thereby decreasing the transport of cholesterol to cholesterol auxotrophic cancer cells.

[97] The disclosure contemplates the use of any agent that is capable of inhibiting intestinal absorption of cholesterol. Exemplary agents that inhibit intestinal absorption of cholesterol include, for example, Niemann-Pick C l -Like (NPCI LI ) protein inhibitors, phytosterol, and phytostanols.

[98] Any suitable NPCI L I inhibitor can be used as long as it limits the availability of extracellular cholesterol. NPC I LI inhibitors are described in the literature (see, e.g., Betters and Yu, "Transporters as Drug Targets: Discovery and Development of NPC I LI Inhibitors",

Translation^ Medicine, 2009; 87( 1 ): 1 1 7-124). In some embodiments, the NPCI LI inhibitor comprises ezetimibe. In some embodiments, the NPC I LI inhibitor comprises spiroimidazolidinone NPC I LI inhibitor (see, e.g., Howell et al, "Spiroimidazolidinone NPCI LI inhibitors. Part 2: Structure-activity studies and in vivo efficacy", Bioorganic & Medicinal Chemistry Letters 2010; 20(23):6929-6932). Additional NPC I LI inhibitors include, without limitation, NPC I LI inhibitors described in WIPO Pub. No. WO/2006/01 5365, and the NPCI LI antagonists described in US Pub. No. 201 0/01 19525.

[99] Exemplary phytosterols include, without limitation, sitosterol, sitostanol, campesterol, campestanol, taraxasterol, stigmasterol, clionastanol, brassicastanol and brassicasterol . Exemplary phytostanols include, without limitation, sitostanol, campestanol, clionastanol and brassicastanol . Phytosterols and phytostanol compositions of use are described i n U. S. Pat. No. 6,677,327.

1 1 001 It is bel ieved that inh i biting the level or activity of HMG-Co A reductase can limit the availabi l ity of extracellular cholesterol in cholesterol auxotrophic cancer cells, HMG-CoA reductase inhi bition blocks cholesterol synthesis, inducing hepatic LDL receptor upregulation to compensate for decreased cholesterol availability. The LDL receptors draw LDL and VLDL out of circulation and into the liver where cholesterol is reprocessed into bile salts. The disclosure contemplates the use of any agent that inhibits the level or activity of HMG-CoA reductase. Exemplary agents that inhibits the level or activity of HMG-CoA reductase include tocotrienol and statins, for example. Tocotrienols include, without limitation, delta-tocotrienol and gamma- tocotrienol. Exemplary statins include, without limitation, atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin.

[ 101 ] Jt is believed that modulating cholesterol metabolism can limit the availability of extracellular cholesterol in cholesterol auxotrophic cancer cells. As used herein, "cholesterol metabolism" refers to the metabolic conversion of cholesterol into another molecule. As used herein, "modulating cholesterol metabolism" refers to inducing the metabolic conversion of cholesterol into a molecule that lowers cholesterol levels or preventing the metabolic conversion or modification of cholesterol into a form that facilitates its transport to cholesterol auxotrophic cancer cells. The disclosure contemplates the use of any agent that is capable of modulating cholesterol metabolism in a way that limits the availability of extracellular cholesterol in cholesterol auxotrophic cancer cells.

[ 102] In some embodiments, an agent that modulates cholesterol metabolism is a bile acid sequestrant. Bile acid sequestrants have been described (see, e.g., Einarsson et al., "Bile acid sequestrants: mechanisms of action o bile acid and cholesterol metabolism", Eur J Clin Pharmacol 1991 ;40( 1 ): S53-S58). Briefly, bi le acid sequestrants bind to bile acids in the gut and prevent reabsorption of cholesterol . This shunts endogenous cholesterol into the production of more bi le acids, thereby lowering cholesterol levels. Any suitable bile acid sequestrant can be used to limit the avai lability of extracellular cholesterol in cholesterol auxotrophic cancer cells. Exemplary such bile acid sequestrants include, without limitation, cholestyramine, colestipol, and colesevelam.

[ 103] In some embodiments, an agent that modulates cholesterol metabolism is a 7- alpha-hydroxylase activator. The regulation of cholesterol 7-alpha-hydroxylase has been described (see, e.g., Wang and Chiang, "Regulation of cholesterol 7 alpha-hydroxylase in the liver. Cloning, sequencing, and regulation of cholesterol 7 alpha-hydroxylase mRNA", J. Biol. Chem. 1990;264: 1 2012-12019). 7-alpha-hydroxylase is an enzyme involved in cholesterol metabolism. Hepatic cholesterol 7-alpha-hydroxylase catalyzes the conversion of cholesterol into 7alpha cholesterol, which is considered the rate limiting step in the conversion of cholesterol into bile acids. It is believed that the increase of cholesterol 7-alpha-hydroxylase activity results in the decrease of blood serum cholesterol, thereby limiting the availability of extracellular cholesterol .

[ 1 04] A cholesterol 7a-hydroxylase activator can enhance the activity of cholesterol 7a- hydroxylase, thus enhance the conversion of cholesterol into 7a-cholesterol. Cholesterol 7a- hydroxylase activators can act on the cholesterol 7a-hydroxylase directly or indirectly by increasing the activity of enzymes and cofactors involved in the activation of cholesterol 7a- hydroxylase or decrease the activity of enzymes or cofactors involved in the down-regulation of cholesterol 7a-hydroxylase (e.g. by effecting enzymes involved in the phosphorylation and dephosphorylation of cholesterol 7a-hydroxylase) or increasing the cholesterol 7a-hydroxy!ase gene transcription or cholesterol 7a hydroxylase RNA translation.

[ 105] Exemplary 7-alpha-hydroxylase activators include, without limitation, 25- hydroxycholesterol and 26-hydroxycholesterol (see, e.g., WIPO Publication WO 91 /15213). In some embodiments, the 7-alpha-hydroxylase activator comprises a composition that includes exogenous 7-alpha-hydroxylase enzyme, for example, a nanoparticle comprising a core having a metal and/or a polymer, and a LDL binding agent, 7-alpha-hydroxylase or an enzymatically active fragment thereof, and nicotinamide adenine dinucleotide (NADH) attached to the core, as described in US Pub. No. 2008/007573 1 .

[ 1 06] In some embodiments, an agent that modulates cholesterol metabolism comprises an agent that decreases formation and/or secretion of cholesteryl esters. [ 107] Acyl-CoA cholesterol acy [transferase (ACAT) is an enzyme that catalyzes the intracellular esterification of cholesterol and formation of cholesteryl esters. In the intestines, ACAT mediates esterification of cholesterol that is absorbed in the intestines before it is incorporated into chylomicrons. In the liver, ACAT-mediated esterification plays a role in the production and release of apoB-containing lipoproteins. It is believed that ACAT inhibitors limit the availabil ity of extracellular cholesterol by decreasing the secretion of cholesteryl esters and apolipoprotein B, thereby decreasing cholesterol transported to the systemic circulation in plasma l ipoproteins (LDL).

[ 108] ACAT inhibitors can act on ACAT directly, or indirectly by decreasing the activity of one or more enzymes or cofactors involved in the activation of ACAT or increasing the activity of one or more enzymes or cofactors involved in the down regulation of ACAT or decreasing the ACAT gene transcription or ACAT RNA translation.

[ 109] The disclosure contemplates the use of any ACAT inhibitor for limiting the availability of extracellular cholesterol. Exemplary ACAT inhibitors include, without limitation, aminosulfonyl urea ACAT inhibitors, such as those described in U.S. Pat. No. 5,214,206, tetrazole-substituted urea ACAT inhibitors, such as those described in U.S. Pat. No. 5,362,744, sulfonate ACAT inhibitors, such as those described in U.S. Pat. No. 5,5 10,379, amide tetrazole ACAT inhibitors, such as those described in U.S. Pat. No. 5,461 ,049, carbamate ACAT inhibitors, such as those described in U.S. Pat. No. 5,087,726, sulfonamide tetrazole ACAT inhibitors, such as those described in U.S. Pat. No. 5,239,082, beta-carboxy sulfonamide ACAT inhibitors, such as those described in U.S. Pat. No. 5,491 , 1 70, anilide ACAT inhibitors, such as those described in HP Pal. App. EP0807627, oxysul onyl urea ACAT inhibitors, such as those described in U.S. Pat. No. 5,364,882, and phosphonamide ACAT inhibitors, such as those described in U.S. Pat. No. 6,0 1 7,905.

[ 1 1 0] Exemplary acyl-CoA acyl transferase inhibitors include, without limitation, CI- 976 (Parke-Davis), CP- 1 1 3818 (Pfizer), PD- 138142- 1 5 (Parke-Davis). In some embodiments, the ACAT inhibitor comprises 3 '-acetoxy-4'-tigloyloxy-3 '-4'-dihydroseselin or a derivative thereof, as described in WHO Pub. No. WO/2008/01 5950. In some embodiments, the ACAT inhibitor comprises a Form A poymorph of N-benzyl-N'-(2,6-diisopropyl-phenyl)-N-isopropyl- malonamide, as described in U.S. Pat. No. 7,981 ,936.

[ I l l ] In some embodiments, the agent that modulates cholesterol metabolism comprises a microRNA (miRNA). In some embodiments, the miRNA comprises miR-122, miR-33a, miR- 33 b, miR-758, and miR- 1 06b, as described in Table 1 of Rotllan and Fernandez-Hernando, " icroRNA Regulation of Cholesterol Metabolism", Cholesterol 2012; Article ID 847849.

[1 12] It is believed that increasing the ratio of HDL to LDL can limit the availability of extracellular cholesterol in cholesterol auxotrophic cancer cells. It should be appreciated that the ratio of HDL to LDL can be increased in many ways, for example, increasing HDL levels or decreasing LDL levels. The disclosure contemplates the use of any agent that is capable of increasing the ratio of HDL to LDL in a way that limits the availability of extracellular cholesterol in cholesterol auxotrophic cancer cells.

[ 1 1 3] In some embodiments, the agent that increases the ratio of HDl to LDL comprises a cholesterylester transfer protein (CETP) inhibitor. CTEP transfers cholesterol from HDL cholesterol to VLDL or LDL cholesterol in a process known as reverse cholesterol transport, resulting in higher HDL levels. Exemplary CETP inhibitors include, without limitation, anacetrapib, evacetrapib, torcetrapib, and dalcetrapib.

[ 1 1 4] It is bel ieved that upregulating expression of liver LDLR receptors can l imit the avai lability of extracell ular cholesterol in cholesterol auxotrophic cancer cells. The disclosure contemplates the use of any agent that is capable of upregulating expression of liver LDLR receptors in a way that limits the availability of extracellular cholesterol in cholesterol auxotrophic cancer cells.

[1 1 5] In some embodiments, the agent that upregulates expression of liver LDLR comprises a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor. In some embodiments, the PCSK9 inhibitor comprises an antibody. In some embodiments, the PCSK9 inhibitor comprises a monoclonal antibody that binds to PCSK9 in the vicinity of the catalytic domain that interacts with the LDLPv. In some embodiments, the PCSK9 inhibitor comprises an anti-PCS 9 antibody or fragment disclosed in U.S. Pub. No. 2013/0302399.

[ 1 1 6] In some embodiments, the PCSK9 inhibitor comprises AMG 145. In some embodiments, the PCSK9 inhibitor comprise l D05-IgG2. In some embodiments, the PCSK9 inhibitor comprises SAR236553/R EGN727. In some embodiments, the PCSK9 inhibitor comprises a peptide mi metic of the EGFA domain of the LDLR which binds to PCS 9. In some embodiments, the peptide mimetic of the EGFA domain of the LDLR comprises an EGF-A peptide (see, e.g., Shan el a/.,, "PCS 9 binds to multiple receptors and can be functionally inhibited by an EGF-A peptide", Biochemical and Biophysical Research Communications 2008; 375( 1 ):69-73). In some embodiments, the PCSK9 inhibitor comprises an antisense

oligonucleotide that increases LDLR expression and decreases circulating cholesterol levels. In some embodiments, the PCSK9 inhibitor comprises ISIS 394814 (see, e.g., Graham, et al., "Antisense inhibition of proprotein convertase subtilisin/kexin type 9 reduces serum LDL in hyperl i pidemic mice", ,/. of Lipid Research 2007; 48:763-767). In some embodiments, the antisense ol igonucl eotide comprises an antisense oligo targeting PCS 9 described in U.S. Pat. No. 8, 563,528. In some embodiments, the PCS 9 inhibitor comprises a locked nucleic acid that reduces PCS 9 mRNA levels. In some embodiments, the PCSK9 inhibitor comprises a locked nucleic acid antisense oligonucleotide complementary to PCS 9 mRNA (accession # NMl 74936 and NM l 53565) comprising a 13-nucleotide long gapmer having a sequence GTctgtggaaGCG (uppercase LNA, lowercase DNA) and phosphorotioate internucleoside linkages, (see, e.g., Gupta el al. "A locked nucleic acid antisense oligonucleotide (LNA) silences PCSK9 and enhances LDLR expression in vitro and in vivo", PLoS ONE 2010; 5(5):el 0682). In some embodiments, the PCSK9 inhibitor comprises ALN-PCS, an RNAi agent that inhibits PCSK9 (see, e.g., Frank- amenetsky et al, "Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman primates". Proc. Natl. Acad. Sci. U.S.A. 2008; 105(33): 1 19 15-20). Examples of such RNAi agents are shown in Table 1 below (SEQ ID NOS: 1 -8).

[ 1 1 7] Table 1 - siRN A agents targeting PCSK9

P m^r^ m _^ — — ' ~~—™- ^■— · ^■— — " ~ ' '

5C-..V 10») TVS TPi *! aiSGASUUU*UyC XAAiir*tfr ULfC£G.MUAAACliC:AC¾CdT¾1T

PCS*; SSiS gf'UTRI J ^'OMesli Py 3 PS iXits ei V CA Tfi tiuc.-'AWcojSiiwaCsui/dT'riT AAG.^¾ S.<Ai>iSUC:jAGAft.J¥* T

PCS-CJ MJ 2 0Me aB ¾' 3 l¾ a*£»fe««*,C*m ASC- CuAucsjsaiASAiActiF*!!! G«UCufK;S a ijA ACa)dT*

[ 1 1 8] In some embodiments, the siRNA is formulated in a LNP lipidoid nanoparticle as is described in Frank-Kamenetsky et al. 2008.

[ 1 1 9] It should be appreciated that any of the agents that inhibit cholesterol transport to or uptake into cholesterol auxotrophic cancer cells (e.g., agents that inhibit the LDLR expression or activity), cholesterol lowering agents described herein, and/or SQLE inhibitors can be employed individually or in combination to limit the availability of extracellular cholesterol in cholesterol auxotrophic cancer cells. In some embodiments, the method comprises contacting a cholesterol auxotrophic cancer cell with an agent that inhibits LDLR expression or activity and a cholesterol lowering agent. In some embodiments, the method comprises administering to a subj ect suffering from a cholesterol auxotrophic cancer an agent that inhibits LDLR expression or acti vity and a cholesterol lowering agent. In some embodiments, the method comprises contacting cholesterol auxotrophic cancer cells with two or more agents that inhibit LDLR expression or activity. In some embodiments, the method comprises contacting cholesterol auxotrophic cancer cells with three or more agents that inhibit LDLR expression or activity. In some embodiments, the method comprises contacting cholesterol auxotrophic cancer cells with four or more agents that inhibit LDLR expression or activity. In some embodiments, the method comprises contacting cholesterol auxotrophic cancer cells with five or more agents that inhibit LDLR expression or activity.

1 120] In some embodiments, the method comprises administering to a subject suffering from a chol esterol auxotrophi c cancer two or more cholesterol lowering agents described herein. I n some embodiments, the method comprises administering to a subject suffering from a cholesterol auxotrophic cancer three or more cholesterol lowering agents described herein. In some embodiments, the method comprises administering to a subject suffering from a cholesterol auxotrophic cancer four or more cholesterol lowering agents described herein. In some embodiments, the method comprises administering to a subject suffering from a cholesterol auxotrophic cancer five or more cholesterol lowering agents described herein. In some embodiments, the method comprise administering to a subject suffering from a cholesterol auxotrophic cancer at least five, at least six, at least seven, at least eight, at least nine, or at least ten or more cholesterol lowering agents described herein.

[ 121 ] Aspects of the disclosure relate to rendering cancer cells cholesterol auxotrophic. Those skilled in the art will appreciate that rendering a cancer cell cholesterol auxtrophic may be useful, e.g., to make cancer cel ls susceptible to treatment with cholesterol lowering agents alone, e.g., to inhibit the growth or survival of the resulting cholesterol auxotrophic cells, or in combination with another anticancer therapy, e.g., chemotherapy.

[ 1 22] In some aspects, the disclosure provides a method of rendering a cancer cell cholesterol auxotrophic, the method comprising contacting a cancer cell with an agent that inhibits the expression level or activity of SQLE in the cancer cell, wherein the agent inhibits the expression level or activity of SQLE in the cancer cell, thereby rendering the cancer cell cholesterol auxotrophic.

[ 123] In some aspects, the disclosure provides a method of rending a cancer cell cholesterol auxotrophic, the method comprising contacting a cancer cell with an agent that inhibits the expression level or activity of CYP51 Al in the cancer cell, wherein the agent inhibits the expression level or activity of CYP51 Al in the cancer cell, thereby rendering the cancer cell cholesterol auxotrophic. f 1 24] In some aspects, the disclosure provides a method of rending a cancer cell cholesterol auxotrophic, the method comprising contacting a cancer cell with an agent that inhibits the expression level or activity of SQLE and/or CYP51 A l in the cancer cell, wherein the agent inhibits the expression level or activity of SQLE and/or CYP51 A l in the cancer cell, thereby rendering the cancer cell cholesterol auxotrophic.

[125] In some aspects, the disclosure provides a method of rending a cancer cell cholesterol auxotrophic, the method comprising contacting a cancer cell with an agent that inhibits the expression level or activity of SQLE and CYP51 Al in the cancer cell, wherein the agent inhibits the expression level or activity of SQLE and CYP51 A 1 in the cancer cell, thereby rendering the cancer cell cholesterol auxotrophic.

[ 126] In some aspects, the disclosure provides a method of rending a cancer cell cholesterol auxotrophic, the method comprising contacting a cancer cell with an agent that inhibits the expression level or activity of SQLE or CYP5 1A 1 in the cancer cell, wherein the agent inhibits the expression level or activity of SQLE or CYP51 A 1 in the cancer cell, thereby renderi ng the cancer cel l cholesterol auxotrophic.

[ 1 271 As used herein, "contacting" refers to any means of introducing an agent or composition into sufficient proximity with a target cell for the agent to exert its intended effect on the cell, including chemical and physical means, whether the agent or composition physically contacts the cell directly or is introduced into an environment in which the cell is present. For example, contacting includes binding of an agent to an extracellular domain of a receptor (e.g., LDLR) and exerting its effects in that way. "Contacting" encompasses methods of exposing a cell, delivering to a cell, or "loading" a cell with an agent or composition by viral or non-viral vectors, and wherein such agent is bioactive upon delivery. The method of delivery will be chosen for the particular agent and use. Parameters that affect delivery, as is known in the medical art, can include, inter alia, the cell type affected (e.g. tumor), and cellular location. In some embodiments, contacting includes administering the agent to a subject. In some embodiments, contacting refers to exposing a cancer cell line or an environment in which the cell line is located (e.g., cell culture) to one or more agents described herein, e.g., agents that limit the availabi lity of extracellular cholesterol in cholesterol auxotrophic cancer cells, agents that inhibit cholesterol transport to and into cholesterol auxotrophic cancer cells, chemotherapeutic agents, cholesterol lowering agents, or SQLE inhibitors.

[ 128] Aspects of the disclosure involve inhibiting the expression level or activity of squalene epoxidase (SQLE). The squalene epoxidase gene (SQLE; Gene ID: 671 3 ; mRNA accession NM_003129; protein accession NP_003120.2) encodes an enzyme that utilizes NADPH and oxygen to catalyze the oxidation of squalene to 2,3-oxidosqualene (squalene epoxide), in the first oxygenation step of sterol biosynthesis. The chemical reaction catalyzed by squalene epoxidase is shown below.

[ 130] Agents that inhibit the expression level or activity of SQLE are also referred to herein as "SQLE inhibitors". The disclosure contemplates the use of any SQLE inhibitor that is capable of rendering a cancer cell cholesterol auxotrophic by inhibiting the expression level or activity of SQLE in the cancer cell . An SQLE inhibitor can be evaluated for its ability to render a cancer cel l cholesterol auxotrophic by incubating a cholesterol protrophic cancer cell in a l ipoprotein depleted media with the SQLE inhibitor, and assessing the growth or survival of the protrotrophic cancer cell in the presence of the SQLE inhibitor, wherein if the growth or survival of the protrophic cancer cel l in the l ipoprotein depleted media decreases in the presence of the SQLE inhibitor then the SQLE inhibitor has rendered the cancer cell cholesterol auxotrophic. Suitable SQLE inhibitors may decrease the growth or survival of the protrophic cancer cell in lipoprotein depleted media by at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1.1 fold, at least 1.2 fold, 1.3 fold, at least 1 .4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, or more relative to the growth or survival of the protrophic cancer cell in the lipoprotein depleted media in the absence of the SOLE inhibitor.

[ 13 1 ] In some embodiments, the SOLE inhibitor inhibits the level or activity of SQLE in the cancer cell by at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 1 5%, at least 20%o, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more relative to the expression level or activity of SQLE in the cancer cell prior to being contacted with the SQLE inhibitor. In some embodiments, the SQLE inhibitor inhibits the level or activity of SQLE in the cancer cell by at least 1 .1 fold, at least 1.2 fold, 1.3 fold, at least 1 .4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1 .8 fold, at least 1.9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 100 fold, or more relative to the expression level or activity of SQLE in the cancer cell prior to being contacted with the SQLE inhibitor. In some embodiments, the SQLE inhibitor completely abolishes the expression or activity of SQLE in the cancer cell.

[ 1 32] Exemplary SQLE inhibitors include, without limitation, small organic or inorganic molecules; saccharides; oligosaccharides; polysaccharides; a biological macromolecule selected from the group consisting of peptides, proteins, peptide analogs and derivatives;

peptidomimetics; nucleic acids selected from the group consisting of siRNAs, shRNAs, antisense RNAs, ribozymes, and aptamers; an extract made from biological materials selected from the group consisting of bacteria, plants, fungi, animal cells, and animal tissues; naturally occurring or synthetic compositions; and any combination thereof.

[ 133] Small organic molecules capable of inhibiting the expression level or activity of SQLE are described in the literature (see, e.g., Belter et al, "Squalene monooxygenase - a target for hypercholesterolemic therapy", Biological Chemistry 201 1 ; 392(12): 1053-1075). Exemplary such agents include, without limitation, antimycotic compounds, allylamines, squalene derivatives, natural compounds, and analogs or derivatives of any of these.

[ 1 34] In some embodiments, the antimycotic compound comprises a compound of ί J

wherein R is selected from the group consisting of

[ 135] In some embodiments, the antimycotic compound comprises a compound of

(Π)

wherein R is selected from the group consistin

the allylamine comprises a compound of formula (II)

wherein R is selected from the rou consistin of

[137] In some embodiments, the allylamine comprises a compound of formula (III)

wherein R is selected from the group consisting of H, CH₃, CH₂CH₃, CH₂CH2CH₃, CH2CHCH2, CH₂CCH, and C₃H₆.

[ 1 38] In some embodiments, the allylamine comprises a compound of formula (IV)

wherein R is selected from the group consisting of H, CH₃, CH₂CH₃, CF₃, OH, nitrile, formyl, hydroxymethyl, phenyl, 2-furyl, 2-oxazolyl, 2-thiazolyl, 5-oxazolyl, 5-thiazolyl, 1 - pyrrolyl, and 3-thienyl.

[ 139] In some embodiments, the allylamine comprises a compound of formula (V)

ted from the group consisting of

In some embodiments, the all lamine comprises a compound of formula (VI)

wherein R is selected from the group consisting of and

[ 1 4 1 ] In some embodiments, the allylamine comprises a compound of formula (VII)

wherein R is selected from the group consisting of OCH₃, SCH₃, OH, SH, CONH₂,

3, ν_^ν-,π, r, 13, tuw, v.,IN, nu iL-ii2-

[ 142] In some embodiments, the allylamine comprises a compound of formula (VIII)

comprises

[ 144] In some embodiments, the allylamine comprises a compound of formula (IX)

wherei n R is selected from the group consisting of CH₃ and OCH₃.

[ 145 ] Any suitable squalene derivative can be used to inhibit the level or activity of SQLE. In some embodiments, the squalene derivative is selected from the group consisting of

HO^'" V ^"X,

[146] In some embodiments, the squalene derivative comprises a compound of formula

wherein R is selected from the group consisting of OH, CH₂CH₂OH, CH₂OOH, CH2SH, CH2NH-C-C3H5, and CHCF₂.

wherein R is CHCF₂.

[ 148] In some embodiments, the squalene derivative comprises a compound of formula

(XI)

[ 149] Any natural compound that is capable of inhibiting the level or activity of SQLE can be used as an SQLE inhibitor of the disclosure. In some embodiments, the natural compound comprises a plant extract selected from the group consisting of Agrimonia pilosa extract, Aleiirites fordii extract, Euphorbia jolkini extract, Lagerstroemia indica extract, Camellia sinensis extract, Allium sativum extract, Rheum palmalum extract, Cynara scolymus extract, Fraxinus excelsior extract, and Peumus boldus extract. [1 50] In some embodiments, the natural compound includes, without limitation, (-)- epigallocatechin-3-O-gallate (EGCG), (-)-epicatechin-3-0-gallate (ECG), (-)-epigallocatechin (EGC), and (-)-epicatechin (EC), gallic acid.

[ 1 5 1 ] In some embodiments, the natural compound is a compound of formula (XII)

wherein R is selected from the group consisting of OH, -0(CH2)7CH3, -0(CH2) 1 1 CH3,

and

[ 1 52] In some embodiments, the natural compound comprises a garlic compound sel ected from the group consisti ng of a selenium compound, a tellurium compound, and an allyl compound. Exemplary selenium compounds include, without limitation, selenocystine, selenite, selenium dioxide, and methylselenol . Exemplary tel lurium compounds include, without l imitation, tellurite, tellurium dioxide, and dimethyltelluride. Exemplary allyl compounds include, without limitation, S-allylcysteine, alliin, 1 ,3-diallyltrisulfane, and 1 ,2-diallyldisulfane.

[ 153] In some embodiments, the SOLE inhibitor comprises an anti-SQLE antibody. A variety of anti-SQLE antibodies are commercially available. Examples of anti-SQLE antibodies include, without limitation, polyclonal antibody (Proteintech Group), anti-squalene epoxidase poiycionai antibody (Bioss), squalene epoxidase (S- 1 7) polyclonal antibody (commercially available from Santa Cruz Biotechnology, Dallas, TX), squalene epoxidase (H-300) poiycionai (commercially available from Santa Cruz Biotechnology, Dallas, TX), squalene epoxidase (L-23) polyclonal (commerci ally avai lable from Santa Cruz Biotechnology, Dallas, TX), anti- ERG 1 /SQLE polyclonal (LifeSpan Biosciences), anti-SQLE antibody (commercially available from Sigma-Aldrich, St. Louis, MO).

[ 1 54] In some embodiments, the SQLE inhibitor comprises shRNA. Exemplary SQLE shRNA include, without limitation, lentiviral or non-viral shRNA constructs (commercially available from GeneCopoeia, Rockville, MD), 29mer shRNA constructs in retroviral vectors (commercially available from OriGene, Rockville, MD) SQLE shRNA plasmid (commercially available from Santa Cruz Biotechnology, Dallas, TX), and SQLE shRNA lentiviral particles (commercially available from Santa Cruz Biotechnology, Dallas, TX).

[1 55] In some embodiments, the SQLE inhibitor comprises siRNA. Exemplary SQLE siRNA include, without limitation, 27mer siRNA duplexes (commercially available from

OriGene, Rockville, MD), SQLE siRNA (commercially available from Santa Cruz Biotechnology, Dal las, TX), and MISSION© esiRNA (commercial ly available from Sigma-Aldrich, St. Louis, MO).

1 56] Aspects of the d isclosure involve inhibiting the expression level or activity of CYP5 1 A l (also known as lanosterol 14alpha-demethylase). CYP5 1 A 1 is an enzyme that catalyzes the conversion of lanosterol to 4,4-dimethylcholesta-8(9), 14,24-trien-3 -ol, via the removal of the C- 14 al ha-methyl group from lanosterol (shown below).

[ 1 8] This demethylation step is considered the initial checkpoint in the conversion of lanosterol to other sterols used in cel ls, i .e., it is an essential step in sterol biosynthesis.

[ 1 59] Agents that inhibit the expression level or activity (e.g., demethylase activity) of CYP5 1 A I are also re ferred to herein as "CYP51 A 1 inhibitors". The disclosure contemplates the use of any CYP51 A1 inhibitor that is capable of rendering a cancer ceil cholesterol auxotrophic by inhibiting the expression level or activity of CYP51A 1 in the cancer cell. An CYP51 Al inhibitor can be evaluated for its ability to render a cancer cell cholesterol auxotrophic by incubating a cholesterol protrophic cancer cell in a lipoprotein depleted media with the CYP51 Al inhibitor, and assessing the growth or survival of the protrotrophic cancer cell in the presence of the CYP5 1 A l inhibitor, wherein if the growth or survival of the protrophic cancer cell in the l i poprotei n depleted med ia decreases in the presence of the CYP5 1 A 1 inhibitor then the CYP5 1 A l inhi bi tor has rendered the cancer cell cholesterol auxotrophic. Suitable CYP51 A 1 inhibitors may decrease the growth or survival of the protrophic cancer cell in lipoprotein depleted media by at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 1 5%, at least 20%, at least 25%o, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1. 1 fold, at least 1.2 fold, 1.3 fold, at least 1.4 fold, at least 1 .5 fold, at least 1.6 fold, at least 1 .7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, or more relative to the growth or survival of the protrophic cancer cell in the lipoprotein depleted media in the absence of the CYP5 1 A 1 inhibitor.

[ 160] In some embodiments, the CYP51 Al inhibitor inhibits the level or activity of CYP51 A l in the cancer cell by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 1 0%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%), at least 90%, or more relative to the expression level or activity of CYP5 1 A 1 in the cancer cell prior to being contacted with the CYP5 1 A 1 inhibitor. In some embodiments, the SQLE inhibitor inhibits the level or activity of CYP51 A l in the cancer cell by at least 1 . 1 fold, at least 1 .2 fold, 1 .3 fold, at least 1.4 fold, at least 1.5 fold, at least 1 .6 fold, al least 1 .7 fold, at least 1 .8 fold, at least 1 .9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 1 00 fold, or more relative to the expression level or activity of CYP51 Al in the cancer cell prior to being contacted with the SQLE inhibitor. In some embodiments, the CYP51 A l inhibitor completely abolishes the expression or activity of CYP51 A l in the cancer cell .

[ 161 ] Exemplary CYP51 A1 inhibitors include, without limitation, small organic or inorganic molecules; saccharides; oligosaccharides; polysaccharides; a biological macromolecule selected from the group consisting of peptides, proteins, peptide analogs and derivatives;

[ 162] The activity of the CYP51 A l gene and/or protein products has been reported to be regulated by azole anti-fungal agents such as ketoconazole and fluconazole (Matsuura et al., 2005, J. Biol . Chem. 280 :9088-96) and oxysterols (Stromstedt et al., Arch. Biochem. Biophys. 1 996, 329:73-8 1 ). In some embodi ments, the CYP5 1 A l inhibitor comprises fenticonazole nitrate (CA S # 73 1 5 1 -29-28). In some embodiments, the CYP51 A 1 inhibitor comprises cyproconazole (CA S # 9436 1 -06-5). In some embodiments, the CYP5 1 A l inhi bitor comprises clotrimazole (CAS # 23593-75- 1 ). In some embodiments, the CYP51 Al inhibitor comprises voriconazole-d3 (CAS # 12 1 7661 - 14-7).

[ 163] In some embodiments, the CYP51 Al inhibitor comprises 2-((3,4- dich!orophenethyl)(propyl)amino)- l -(pyridin-3-yl)ethanol (LEK-935).

[ 164] In some instances, the cancer cell is identified as cholesterol protrophic before contacting the cancer cell with at least one agent that inhibits the expression level or activity of SQLE to render the cancer cell cholesterol auxotrophic.

[ 165] In some instances, the cancer cell is identified as cholesterol protrophic before contacting the cancer cell with at least one agent that inhibits the expression level or activity of CYP51 Al to render the cancer cell cholesterol auxotrophic.

[166] In some aspects, the disclosure provides a method of inhibiting growth or survival of a cancer cell, the method comprising: a) contacting the cancer cell with an effective amount of a first agent that inhibits the level or activity of SQLE in the cancer cell, thereby rendering the cancer cell cholesterol auxotrophic; and b) contacting the cholesterol auxotrophic cancer cell with an effective amount of a second agent that limits the availability of extracellular cholesterol in the cancer cel l, thereby inhi biting growth or survival of the cancer cel l.

[ 167] In some aspects, the disclosure provides a method of inhibiting growth or survival of a cancer cell, the method comprising: a) contacting the cancer cell with an effective amount of a first agent that inhibits the level or activity of CYP51 A1 in the cancer cell, thereby rendering the cancer cell cholesterol auxotrophic; and b) contacting the cholesterol auxotrophic cancer cell with an effective amount of a second agent that limits the availability of extracellular cholesterol in the cancer cell, thereby inhibiting growth or survival of the cancer cell.

[ 168] In some aspects, the disclosure provides a method of inhibiting growth or survival of a cancer cell, the method comprising: a) contacting the cancer cell with an effective amount of a first agent that inhibits the level or activity of SQLE and/or CYP51 A l in the cancer cell, thereby rendering the cancer cell cholesterol auxotrophic; and b) contacting the cholesterol auxotrophic cancer cell with an effective amount of a second agent that limits the availability of extracellular cholesterol in the cancer cell, thereby inhibiting growth or survival of the cancer cell.

[ 169] In some aspects, the disclosure provides a method of inhibiting growth or survival of a cancer cell, the method comprising: a) contacting the cancer cell with an effective amount of a first agent that inhibits the level or activity of SQLE and CYP51 Al in the cancer cell, thereby rendering the cancer cell cholesterol auxotrophic; and b) contacting the cholesterol auxotrophic cancer cell with an effective amount of a second agent that limits the availability of extracellular cholesterol in the cancer cell, thereby inhibiting growth or survival of the cancer cell.

[ 170] In some aspects, the disclosure provides a method of inhibiting growth or survival of a cancer cell, the method comprising: a) contacting the cancer cell with an effective amount of a first agent that inhibits the level or activity of SQLE or CYP51 A 1 in the cancer cell, thereby renderi ng the cancer cel l cholesterol auxotrophic; and b) contacti ng the cholesterol auxotrophic cancer cel l with an e ffective amount of a second agent that limits the availability of extracellular cholesterol in the cancer cel l, thereby inhibiting growth or survival of the cancer cell.

[ 1 71 ] Aspects of the disclosure involve contacting cells with or administering to subjects an effective amount an agent. As used herein, "an effective amount" refers to an amount of the agents or the compounds mentioned, which result in successful treatment, e.g., limit the availabil ity of extracellular cholesterol in cholesterol auxotrophic cancer cells, decrease circulating cholesterol levels, render cancer cells cholesterol auxotrophic, inhibit the growth or survival of cholesterol auxotrophic cells, etc., i.e., to effectively inhibit, treat the syndromes of a cancer or retard or reverse the rate of tumor proliferation or prevent the formation of a cancer or a tumor. In some embodiments, an "effective amount" is a "cholesterol lowering amount". As used herein, "cholesterol lowering amount" means a sufficient amount of an agent to provide the desired cholesterol lowering effect. For example, in some embodiments, "a cholesterol lowering amount" means that dose of agent effective to inhibit the growth or survival of cancerous cells or tumor to render the cancerous cel ls or tumor more susceptible to an anticancer therapy, e.g., at least one chemothcrapeutic agent.

[ 1 72] CLASSIFICA TION OF DISEASES AND SUBJECTS

[ 1 73] Aspects of the disclosure relate to classification of diseases and/or subjects, for example, to inform clinical decision making.

[ 174] In some aspects, the disclosure provides a method of classifying a tumor as cholesterol auxotrophic, the method comprising: a) determining the level of an expression product of the SQLE gene in a sample obtained from the tumor; and b) comparing the level determined in a) with a control level, wherein if the level determined in a) is less than the control level, the level is correlated with cholesterol auxotrophy, thereby classifying the tumor as cholesterol auxotrophic.

[ 1 75 ] In some aspects, the disclosure provides a method of classifying a tumor as cholesterol auxotrophic, the method comprising: a) determining the level of an expression product of the CYP51 A l gene in a sample obtained from the tumor; and b) comparing the level determined in a) with a control level, wherein if the level determined in a) is less than the control level, the level is correlated with cholesterol auxotrophy, thereby classifying the tumor as cholesterol auxotrophic.

[ 176] In some aspects, the disclosure provides a method of classifying a tumor as cholesterol auxotrophic, the method comprising: a) determining the level of an expression product of the SQLE and/or CYP5 1 A l genes in a sample obtained from the tumor; and b) comparing the level determined in a) with a control level, wherein if the level determined in a) is less than the control level, the level is correlated with cholesterol auxotrophy, thereby classifying the tumor as cholesterol auxotrophic.

[ 1 77 J In some aspects, the disclosure provides a method of classifying a tumor as cholesterol auxotrophic, the method comprising: a) determining the level of an expression product o f the SQLE and CY P5 I A 1 genes in a sample obtai ned from the tumor; and b) comparing the level determined in a) with a control level, wherein if the level determined in a) is less than the control level, the level is correlated with cholesterol auxotrophy, thereby classifying the tumor as cholesterol auxotrophic.

[ 1 78] in some aspects, the disclosure provides a method of classifying a tumor as cholesterol auxotrophic, the method comprising: a) determining the level of an expression product of the SQLE or CYP5 1 Al genes in a sample obtained from the tumor; and b) comparing the level determined in a) with a control level, wherein if the level determined in a) is less than the control level, the level is correlated with cholesterol auxotrophy, thereby classifying the tumor as cholesterol auxotrophic.

[ 179] A biological sample used in the methods described herein will typically comprise or be derived from cells isolated from a subject. The cells will typically comprise cells isolated from tumors. Exemplary tumors are derived from ovarian cancer, uterine cancer (e.g., endometrium adenocarcinoma), bladder cancer, lung cancer, cervical cancer, breast cancer, prostate cancer, gl iomas, fi brosarcomas, retinoblastomas, melanomas, soft tissue sarcomas, osteosarcomas, leukemias, stomach cancer (e.g., adenocarcinoma), colon cancer, carcinoma of the kidney, gastrointestinal cancer, salivary gland cancer, pancreatic cancer, Hodgkin's disease, non-Hodgkin's lymphomas (e.g., anaplastic large cell lymphoma), acute and chronic lymphocytic leukemias, multiple myeloma, neuroblastoma, Wilms' tumor, testicular cancer, soft-tissue sarcomas, chronic lymphocytic leukemia, primary macroglobulinemia, chronic granulocytic leukemia, primary brain carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinomas, malignant melanomas, choriocarcinomas, mycosis fungoides, head and neck carcinomas, osteogenic sarcoma, pancreatic carcinomas, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, genitourinary carcinomas, thyroid carcinomas, esophageal carcinomas, malignant hypercalcemia, cervical hyperplasia, renal cell carcinomas, endometrial carcinomas, polycythemia vera, essential thrombocytosis, adrenal cortex carcinomas, skin cancer, and prostatic carcinomas.

[1 80] Samples can be, e.g., surgical samples, tissue biopsy samples, fine needle aspiration biopsy samples, core needle samples. The sample may be obtained using methods known in the art. A sample can be subjected to one or more processing steps. In some embodiments the sample is frozen and/or fixed. In some embodiments the sample is sectioned and/or embedded, e.g., in paraffin. In some embodiments, tumor cells, e.g., epithelial tumor cells, are separated from at least some surrounding stromal tissue (e.g., stromal cells and/or extracellular matrix). Cells of interest can be isolated using, e.g., tissue microdissection, e.g., laser capture microdissection. It should be appreciated that a sample can be a sample isolated from any of the subjects described herein.

[ 1 8 1 ] In some embodiments, cells of the sample are lysed. Nucleic acids or polypeptides may be isolated. In some embodiments RNA, optionally isolated from a sample, is reverse transcri bed and/or amplified. A wide variety of solution phase or solid phase methods are avai lable for detection of RNA, e.g., SQLE mRNA. Suitable methods include e.g., hybridization- based approaches (e.g., uclease protection assays, Northern blots, microarrays, in situ

hybridization), amplification-based approaches (e.g., reverse transcription polymerase chain reaction (which can be a real-time PCR reaction), or sequencing (e.g., RNA-Seq, which uses high throughput sequencing techniques to quantify RNA transcripts (see, e.g., Wang, Z., et al. Nature Reviews Genetics 10, 57-63, 2009)). In some embodiments of interest a quantitative PCR (qPCR) assay is used. Other methods include electrochemical detection, bioluminescence-based methods, fluorescence-correlation spectroscopy, etc. 1 1 82 ] Aspects of the methods described herein involve detecting the levels of expression products, e.g., using the level of an expression product of the SQLE gene and/or CYP51 A l gene as an indicator of cholesterol auxotrophy. Levels of expression products of the SQLE and/or CYP51 A l gene may be assessed using any suitable method. Either mRNA or protein level may be measured. A "polypeptide", "peptide" or "protein" refers to a molecule comprising at least two covalently attached amino acids. A polypeptide can be made up of naturally occurring amino acids and peptide bonds and/or synthetic peptidomimetic residues and/or bonds. Polypeptides described herein include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells.

[ 1 83] Exemplary methods for measuring mRNA include hybridization based assays, polymerase chain reaction assay, sequencing, in situ hybridization, etc. Exemplary methods for measuring protein levels include ELISA assays, Western blot, mass spectrometry, or i mmunohislochemistry. It wil l be understood that suitable controls and normalization procedures can be used to accurately quantify SQLE and/or CYP5 1 A l expression. Values can also be normalized to account for the fact that different samples may contain different proportions of a cell type of interest, e.g., cancer cells, versus non-cancer cells.

[ 184] in some aspects, the disclosure provides a method of diagnosing a tumor in a subject as a cholesterol auxotrophic tumor comprising: a) determining the level of an expression product of the SQLE gene in a sample obtained from the subject; and b) comparing the level determined in a) with a control level, wherein if the level determined in a) is less than the control level, the level is indicative of cholesterol auxotrophy, thereby diagnosing the tumor in the subject as a cholesterol auxotrophic tumor.

[ 185] In some aspects, the disclosure provides a method of diagnosing a tumor in a subject as a cholesterol auxotrophic tumor comprising: a) determining the level of an expression product of the CYP51 A l gene in a sample obtained from the subject; and b) comparing the level determined in a) with a control level, wherein if the level determined in a) is less than the control level, the level is indicative of cholesterol auxotrophy, thereby diagnosing the tumor in the subject as a cholesterol auxotrophic tumor.

[ 1 86] In some aspects, the disclosure provides a method of diagnosing a tumor in a subject as a cholesterol auxotrophic tumor comprising: a) determining the level of an expression product of the SQLE gene and/or CYP51 A1 gene in a sample obtained from the subject; and b) comparing the level determined in a) with a control level, wherein if the level determined in a) is less than the control level, the level is indicative of cholesterol auxotrophy, thereby diagnosing the tumor in the subject as a cholesterol auxotrophic tumor.

[ 1 87] In some aspects, the disclosure provides a method of diagnosing a tumor in a subject as a cholesterol auxotrophic tumor comprising: a) determining the level of an expression product of the SQLE gene and CYP5 1 A l gene in a sample obtained from the subject; and b) comparing the level determined in a) with a control level, wherein if the level determined in a) is less than the control level, the level is indicative of cholesterol auxotrophy, thereby diagnosing the tumor in the subject as a cholesterol auxotrophic tumor.

[ 188] In some aspects, the disclosure provides a method of diagnosing a tumor in a subject as a cholesterol auxotrophic tumor comprising: a) determining the level of an expression product of the SQLE gene or CYP5 1 A l gene in a sample obtained from the subject; and b) comparing the level determined in a) with a control level, wherein if the level determined in a) is less than the control level, the level is indicative of cholesterol auxotrophy, thereby diagnosing the tumor in the subject as a cholesterol auxotrophic tumor.

[ 189] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents. In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human. Preferably, the subject is a mammal . The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used, for example, as subjects that represent animal models of disease. A subject can be male or female.

[ 190] In some embodiments, the subject is a subject suffering from cancer, e.g., ovarian cancer, uterine cancer (e.g., endometrium adenocarcinoma), bladder cancer, lung cancer, cervical cancer, breast cancer, prostate cancer, gliomas, fibrosarcomas, retinoblastomas, melanomas, soft tissue sarcomas, osteosarcomas, leukemias, stomach cancer (e.g., adenocarcinoma), colon cancer, carcinoma of the kidney, gastrointestinal cancer, salivary gland cancer, pancreatic cancer, Hodgkin's disease, non-Hodgkin's lymphomas (e.g., anaplastic large cell lymphoma), acute and chronic lymphocytic leukemias, multiple myeloma, neuroblastoma, Wilms' tumor, testicular cancer, soft-tissue sarcomas, chronic lymphocytic leukemia, primary macroglobulinemia, chronic granulocytic leukemia, primary brain carcinoma, malignant pancreatic insulinoma, malignant carci noid carcinomas, malignant melanomas, choriocarcinomas, mycosis fungoides, head and neck carcinomas, osteogenic sarcoma, pancreatic carcinomas, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, genitourinary carcinomas, thyroid carcinomas, esophageal carcinomas, malignant hypercalcemia, cervical hyperplasia, renal cell carcinomas, endometrial carcinomas, polycythemia vera, essential thrombocytosis, adrenal cortex carcinomas, skin cancer, and prostatic carcinomas.

[ 191 ] In some embodiments, the subject is a subject at risk of developing cancer, e.g., ovarian cancer, uterine cancer (e.g., endometrium adenocarcinoma), bladder cancer, lung cancer, cervical cancer, breast cancer, prostate cancer, gliomas, fibrosarcomas, retinoblastomas, melanomas, soft tissue sarcomas, osteosarcomas, leukemias, stomach cancer (e.g.,

adenocarcinoma), colon cancer, carcinoma of the kidney, gastrointestinal cancer, salivary gland cancer, pancreatic cancer, Hodgkin's disease, non-Hodgkin's lymphomas (e.g., anaplastic large cell lymphoma), acute and chronic lymphocytic leukemias, multiple myeloma, neuroblastoma, Wilms' tumor, testicular cancer, soft-tissue sarcomas, chronic lymphocytic leukemia, primary macroglobulinemia, chronic granulocytic leukemia, primary brain carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinomas, malignant melanomas, choriocarcinomas, mycosis fungoides, head and neck carcinomas, osteogenic sarcoma, pancreatic carcinomas, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, genitourinary carcinomas, thyroid carcinomas, esophageal carcinomas, malignant hypercalcemia, cervical hyperplasia, renal cell carcinomas, endometrial carcinomas,

polycythemia vera, essential thrombocytosis, adrenal cortex carcinomas, skin cancer, and prostatic carcinomas. In some embodiments, the cancer is anaplastic large cell lymphoma.

[ 192] In some embodiments, the subject is suffering from or at risk of developing a cholesterol auxotrophic cancer selected from the group consisting of ovarian cancer, uterine cancer (e.g., endometrium adenocarcinoma), bladder cancer, lung cancer, cervical cancer, breast cancer, prostate cancer, gliomas, fibrosarcomas, retinoblastomas, melanomas, soft tissue sarcomas, osteosarcomas, leukemias, stomach cancer (e.g., adenocarcinoma), colon cancer, carci noma of the kidney, gastrointestinal cancer, sal ivary gland cancer, pancreatic cancer, Hodgkin's disease, non-Hodgkin's lymphomas (e.g., anaplastic large cell lymphoma), acute and chronic lymphocytic leukemias, multiple myeloma, neuroblastoma, Wilms' tumor, testicular cancer, soft-tissue sarcomas, chronic lymphocytic leukemia, primary macroglobulinemia, chronic granulocytic leukemia, primary brain carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinomas, malignant melanomas, choriocarcinomas, mycosis fungoides, head and neck carcinomas, osteogenic sarcoma, pancreatic carcinomas, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, genitourinary carcinomas, thyroid carci nomas, esophageal carcinomas, malignant hypercalcemia, cervical hyperplasia, renal cell carcinomas, endometrial carcinomas, polycythemia vera, essential thrombocytosis, adrenal cortex carcinomas, skin cancer, and prostatic carcinomas. In some embodiments, the cholesterol auxotrophic cancer is anaplastic large cell lymphoma.

[ 193] In some embodiments, the subject is a cancer patient presenting with

hypercholesterolemia or symptoms of hypercholesterolemia.

[ 194] In some embodiments, the subject is a cancer patient presenting with a lipid disorder or symptoms of a lipid disorder.

[ 195] In some embodiments, the subject is a cancer patient presenting with elevated triglyceride levels.

[ 1 96] In some embodiments, the subject has a genetic predisposition for a lipid disorder.

[197] In some aspects, the disclosure provides a method of determining whether a subject with a tumor is a suitable candidate for treatment with a cholesterol lowering agent, the method comprising: a) assessing the level of expression of a SQLE gene product in a tumor sample obtained from the subject; and b) comparing the level determined in a) to a control level, wherei n i f (he level determined in a) is less than the control level, then the level in a) indicates that the tumor is a cholesterol auxotrophic tumor, thereby determining that the subject with the tumor is a suitable candidate for treatment with a cholesterol lowering agent. In some embodiments, the method includes administering an agent that limits the availability of extracellular cholesterol to the subject, e.g., a cholesterol lowering agent. In some embodiments, the method includes administering a cholesterol lowering agent to the subject in combination with an SQLE inhibitor.

[ 198] In some aspects, the disclosure provides a method of determining whether a subject with a tumor is a suitable candidate for treatment with a cholesterol lowering agent, the method comprising: a) assessing the level of expression of a CYP51A1 gene product in a tumor sample obtained from the subject; and b) comparing the level determined in a) to a control level, wherein if the level determined in a) is less than the control level, then the level in a) indicates that the tumor is a cholesterol auxotrophic tumor, thereby determining that the subject with the tumor is a suitable candidate for treatment with a cholesterol lowering agent. In some embodi ments, the method incl udes administering an agent that limits the availability of extracel lular cholesterol to the subject, e.g., a cholesterol lowering agent. In some embodiments, the method includes administering a cholesterol lowering agent to the subject in combination with a CYP51 A l inhibitor.

[ 199] In some embodiments, the CYP5 1A1 gene product comprises CYP51 A1 transcript variant 1 mRNA. Tn some embodiments, the CYP51A1 gene product comprises CYP5 1 A 1 transcript variant 2 mRNA.

[200] METHODS OF TREA TMENT

[201 ] Aspects of the d isclosure relate to methods for treating diseases, e.g., cholesterol auxotroph ic cancer, and compositions, kits and agents for use in the methods. In some aspects, the disclosure provides a method of treating a cholesterol auxotrophic cancer in a subject in need thereof, the method comprising administering to a subject suffering from a cholesterol auxotrophic cancer an effective amount of a composition which limits the availability of extracellular cholesterol in the cholesterol auxotrophic cancer cells in the subject, wherein the composition inhibits the growth or survival of the cholesterol auxotrophic cancer cells, thereby treating the subject's cholesterol auxotrophic cancer.

[202] In some aspects, the disclosure provides a method of treating a cancer in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising an SQLE inhibitor and a cholesterol lowering agent.

[203] In some aspects, the disclosure provides a method of treating a cancer in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising an CYP51 A l inhibitor and a cholesterol lowering agent.

[204] As used herein, the expressions "treating" or "treatment" or "to treat" or

"alleviati ng" or "to al leviate" refer to therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder (e.g., cancer). Treatment also encompasses prophylactic or preventative measures that prevent or slow the development of a targeted pathologic condition or disorder. Subjects in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented.

[205] A subject is successfully "treated" according to the methods of the present invention if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in the tumor size; inhibition of or an absence of cancer cell infiltration into peripheral organs including the spread of cancer cells into soft tissue and bone; inhibition of or an absence of tumor or cancer cell metastasis; inhibition or an absence of cancer growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumorigenicity; reduction in the number or frequency of cancer stem cells; increased sensitivity of cancer cells when treated with cholesterol lowering agents, reduced circulating cholesterol levels; or some combination of effects.

[206] As used herein, the term "administering," refers to the placement of an agent or composition as disclosed herein into a subject by a method or route which results in delivery to a site of action. Compositions comprising agents that i) inhibit cholesterol transport to or uptake into cholesterol auxotrophic cancer cells, ii) limit the availability of extracellular cholesterol in cholesterol auxotrophic cancer cells, iii) SQLE inhibitors, and iv) cholesterol lowering agents disclosed herein, can be administered by any appropriate route which results in an effective treatment in the subject. Compositions comprising agents that i) inhibit cholesterol transport to or uptake i nto cholesterol auxotrophic cancer cells, ii) limit the availabil ity of extracellular cholesterol in cholesterol auxotrophic cancer cells, ii i) CYP5 I A l inhibitors, and iv) cholesterol lowering agents disclosed herei n, can be administered by any appropriate route which results in an effective treatment in the subject.

[207] In some embodiments, the composition comprises at least one cholesterol lowering agent described herein. In some embodiments, the method includes administering to the subject an effective amount of at least one chemotherapeutic agent. A "chemotherapeutic agent" is used to connote a compound or composition that is administered in the treatment of cancer. Chemotherapeutic agents useful in methods, compositions, and kits disclosed herein include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, tri ethyl enemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamime; nitrogen mustards such as chlorambuci l, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethami ne, meehlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, predni mustine, trofos lamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotem ustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo~L- norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenol ic acid, nogalamycin, ol ivomycins, peplomycin, potfiromycin, puromycin,

quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fiudarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6- azauridine, carmofur, cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU ; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenishers such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone;

elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;

podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol ; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); taxoids, e.g. paclitaxel and docetaxel ; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine;

platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide; ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide;

daunomyci n; aminopterin; xeloda; ibandronate; CPT1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylornithi ne; retinoi c acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or deri vatives of any o f the above. Chemotherapeutic agents also include anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti- estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4- hydroxytamoxifen, trioxifene, keoxifene, LY 1 1701 8, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, ni!utamide, bica!utamide, !eupro!ide, and gosere!in; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[208] In some embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. Topoisomerase inhibitors are chemotherapy agents that interfere with the action of a

topoisomerase enzyme (e.g., topoisomerase I or II). Topoisomerase inhibitors include, but are not limited to, doxorubicin HCl, daunorubicin citrate, mitoxantrone HCl, actinomycin D, etoposide, topotecan HCl, teniposide, and irinotecan, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these.

[209] In some embodiments, the chemotherapeutic agent is an anti-metabolite. An antimetabolite is a chemical with a structure that is similar to a metabolite required for normal biochemical reactions, yet different enough to interfere with one or more normal functions of cells, such as cell division. Anti-metabolites include, but are not limited to, gemcitabine, fluorouracil, capecitabine, methotrexate sodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these.

[210] In certain embodiments, the chemotherapeutic agent is an antimitotic agent, including, but not limited to, agents that bind tubulin. In some embodiments, the agent is a taxane. In certain embodiments, the agent is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel. In certain alternative embodiments, the antimitotic agent comprises a vinca alkaloid, such as vincristine, binblastine, vinorelbine, or vindesine, or pharmaceutically acceptable salts, acids, or derivatives thereof.

[21 1 ] In some embodiments, the method includes administering radiotherapy to the subject. The term "radiation therapy" is used interchangeably with the term "radiotherapy". In some embodiments, the radiation is one of x-ray and gamma ray. For example, but not by way of limitation, x-ray radiation can be administered; for example, high-energy megavoltage (radiation of greater than 1 MeV energy) can be used for deep tumors, and electron beam and orthovoltage x-ray radiation can be used for skin cancers. Gamma ray emitting radioisotopes, such as radioactive isotopes of radium, cobalt and other elements may also be administered to expose tissues to radiation. Any radiation therapy protocol can be used depending upon the type of cancer to be treated. Radiation therapy as used herein includes both ionizing and non-ionizing radiation. Non-ionizing radiation may be used, for example, in connection with photodynamic therapy ("PDT") and PDT-photosensitizing agents.

[ 2 1 2] In some embodi ments, the method includes administering a targeted inhibitor therapy. The expression "targeted inhibitor therapy" incl udes the use of therapeutic agents which can alter the expression and/or activation state of proteins or molecules deregulated in a disease state, e.g., cancer. The skilled artisan will be able to readily determine suitable targeted inhibitor therapies based on the type of cancer to be treated.

[213] In some embodiments, the method includes diagnosing the subject as having a cholesterol auxotrophic cancer, for example, according to the methods described herein.

[214] In some embodiments, the method includes selecting a subject that does not have a cholesterol auxotrophic cancer, administering an SQLE inhibitor to the subject, e.g., to render the subject's cancer cells cholesterol auxotrophic, and then administering a cholesterol lowering agent to the subject. [215/ SCREENING METHODS

[21 6] Aspects of the disclosure relate to identifying candidate agents that render cancer cells cholesterol auxotrophic, for example, to make cancer cells susceptible to growth or survival inhibition via treatment with cholesterol lowering agents. In some aspects, a method of identifying a candidate agent that renders a cancer cell cholesterol auxotrophic comprises a) contacting a cancer cell that expresses SQLE with a test agent; b) measuring the expression level of SQLE in the presence and absence of the test agent; and c) determining whether the test agent renders the cancer cell cholesterol auxotrophic, wherein if the level measured in b) in the presence of the test agent is decreased compared to the level measured in b) in the absence of the test agent, the test agent is identified as a candidate agent that renders the cancer cell cholesterol auxotrophic.

[217] In some aspects, a method of identifying a candidate agent that renders a cancer cell cholesterol auxotrophic comprises a) contacting a cancer cell that expresses CYP51 A l with a test agent; b) measuring the expression level of CYP51 A l in the presence and absence of the test agent; and c) determining whether the test agent renders the cancer cell cholesterol auxotrophic, wherein if the level measured in b) i n the presence of the test agent is decreased compared to the level measured in b) in the absence of the test agent, the test agent is identified as a candidate agent that renders the cancer cell cholesterol auxotrophic.

[218] In some embodiments, the method includes performing an assay to confirm that the candidate agent that renders the cancer cell cholesterol auxotrophic. In some embodiments, the confirmatory assay comprises a cell competition assay in lipoprotein depleted serum media. In some embodiments, the assay comprises: a) incubating cholesterol protrophic cancer cells in a lipoprotein depleted serum media; b) contacting the cholesterol protrophic cancer cells with a candidate agent that renders a cancer cell cholesterol auxotrophic; and c) assessing the growth or survival of the cholesterol protrophic cancer cells in the lipoprotein depleted serum media after being contacted with the candidate agent, wherein if the growth or survival of the cholesterol protrophic cancer cells is inhibited in the lipoprotein depleted serum media after being contacted with the candidate agent, then the candidage agent renders the cancer cel ls cholesterol

auxotroph ic.

[2191 COMPOSITIONS

[220] Any agent or combinations of agents described herein can be formulated into one or more compositions. In some aspects, the disclosure provides a composition comprising a) at least one cholesterol lowering agent; and b) at least one chemotherapeutic agent. In some embodiments, the composition further comprises an agent that inhibits the level or activity of the LDLR. In some embodiments, the composition comprises a pharmaceutically acceptable carrier, diluents or excipient.

1221 ] In some aspects, the disclosure provides a composition comprising a) at least one agent that i nhi bits the level or activity of SQLE; b) at least one cholesterol lowering agent; and c) at least one chemotherapeutic agent.

[ 222 ] In some aspects, the disclosure provides a composition comprising a) at least one agent that inhi bits the level or activity of CYP5 1 A 1 ; b) at least one cholesterol lowering agent; and c) at least one chemotherapeutic agent.

[223] In some aspects, the disclosure provides a composition comprising a) at least one agent that inhibits the level or activity of SQLE, b) at least one agent that inhibits the level or activity of CYP51 Al ; c) at least one cholesterol lowering agent; and d) at least one

chemotherapeutic agent.

[224] KITS

[225] The disclosure further provides packaged products and kits, including an agent or composition described herein, packaged into suitable packaging material. In some embodiments, a packaged product or kit includes an agent or composition described herein in unit dosage form.

[226] In some aspects, a packaged product or kit includes a label, such as a list of the contents of the package, or instructions for using the kit e.g., instructions for identifying a candidate agent that renders a cancer cell cholesterol auxotrophic, administering an agent or composition, e.g., to inhibit the growth or survival of a cholesterol auxotrophic cancer cell, or screening for a compound or agent, e.g., that limits the availability of extracellular cholesterol in cholesterol auxotrophic cancer cells.

[227] In certain embodiments, a packaged product or kit includes a container, such as a sealed pouch or shipping container, or an article of manufacture, for example, to carry out an assay described herein, such as a tissue culture dish, tube, flask, roller bottle or plate (e.g., a single multi-well plate or dish such as an 8, 16, 32, 64, 96, 384 and 1536 multi-well plate or dish).

[228] The term "packaging material" refers to a physical structure housing the product or components of the kit. The packaging material can maintain the components sterilely, and can be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules, etc.). A label or packaging insert can be included, listing contents or appropriate written instructions, for example, practicing a method of the disclosure. [229] A packaged product or kit can therefore include instructions for practicing any of the methods of the disclosure described herein. For example, a cancer cell can be included in a tissue culture dish, tube, flask, roller bottle or plate (e.g., a single multi-well plate or dish such as an 8, 16, 32, 64, 96, 384 and 1536 multi-well plate or dish) together with instructions, e.g., for rendering the cancer cell cholesterol auxotrophic or identifying agents that inhibit the growth or survival of cholesterol auxotrophic cancer cells by limiting the availability of extracellular cholesterol in the cholesterol auxotrophic cancer cells.

[230] Instructions may be on "printed matter," e.g., on paper or cardboard within the kit, on a label affixed to the package, kit or packaging material, or attached to a tissue culture dish, tube, flask, roller bottle, plate (e.g., a single multi-well plate or dish such as an 8, 16, 32, 64, 96, 384 and 1 536 multi-well plate or dish) or vial containing a component of the kit. Instructions may comprise voice or video tape and additionally be included on a computer readable medium, such as a disk (floppy diskette or hard disk), optical CD such as CD- or DVD-ROM/RAM, magnetic tape, electrical storage media such as RAM and ROM and hybrids of these such as

magnetic/optical storage media.

[231] Disclosed kits can optionally include additional components, such as buffering agent, a preservative, or a reagent. Each component of the kit can be enclosed within an individual container or in a mixture and all of the various containers can be within single or multiple packages,

[232] In some aspects, a kit of the disclosure comprises a) at least one cholesterol lowering agent; and b) at least one chemotherapeutic agent.

[ 233] In some aspects, a kit of the disclosure comprises a) at least one agent that inhibits the level or acti vity of SQLE; b) at least one cholesterol lowering agent; and c) at least one chemotherapeutic agent.

[234] In some aspects, a kit of the disclosure comprises a) at least one agent that inhibits the level or activity of CYP51 A 1 ; b) at least one cholesterol lowering agent; and c) at least one chemotherapeutic agent.

[235] In some aspects, a kit of the disclosure comprises a) at least one cancer cell that is auxotrophic due to inadequate expression of SQLE in the cancer cell; and b) a test agent. In some aspects, a kit of the disclosure comprises a) at least one cancer cell that is auxotrophic due to inadequate expression of CYP51A1 in the cancer cell; and b) a test agent.

[236] Exemplary cholesterol auxotrophic cancer cells include, without limitation, Snu-1 , Raji, Daudi, H716, and U937. In some embodiments, the at least one cancer cell is engineered to be auxotrophic due to inadequate expression of SQLE in the cancer cell. In some embodiments, the at least one cancer cell is engineered to be auxotrophic due to inadequate expression of

CY P5 1 A 1 in the cancer cell . In some embodiments, the kit comprises a lipoprotein conditioned media. In some embodiments, the kit includes instructions for assessing whether the test agent is capable of limiting the availability of extracellular cholesterol in the at least one cancer cell. In some embodiments, the instructions comprise directions for instructing a user to incubate the at least one cancer cell in the lipoprotein conditioned media with the test agent, and to assess the viability (e.g., growth or survival) of the cancer cell in the lipoprotein conditioned media in the presence of the test agent compared to the viability of the cancer cell in the lipoprotein conditioned media in the absence of the test agent.

* * *

[237] One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein, The detai ls of the description and the examples herein are representative of certain embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modi fications therein and other uses wi ll occur to those skil led in the art. These modifications are encompassed within the spi rit of the invention. It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[238] The articles "a" and "an" as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to include the plural referents. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention provides all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed cl aims is i ntroduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. It is contemplated that all embodiments described herein are applicable to all different aspects of the invention where appropriate. It is also contemplated that any of the embodiments or aspects can be freely combined with one or more other such embodiments or aspects whenever appropriate. Where elements are presented as lists, e.g., in Markush group or similar format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the i nvention consist, or consist essentially of, such elements, features, etc. For purposes of simpl icity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. For example, any one or more nucleic acids, polypeptides, cells, species or types of organism, disorders, subjects, or combinations thereof, can be excluded.

[239] Where the claims or description relate to a composition of matter, e.g., a nucleic acid, polypeptide, cell, or non-human transgenic animal, it is to be understood that methods of making or using the composition of matter according to any of the methods disclosed herein, and methods of using the composition of matter for any of the purposes disclosed herein are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where the claims or description relate to a method, e.g., it is to be understood that methods of making compositions useful for performing the method, and products produced according to the method, are aspects of the i nvention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[240] Where ranges are given herein, the invention includes embodiments in which the endpoints are included, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other is excluded. It should be assumed that both endpoints are included unless indicated otherwise. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also understood that where a series of numerical values is stated herein, the invention includes embodiments that relate analogously to any intervening value or range defined by any two values in the series, and that the lowest value may be taken as a minimum and the greatest value may be taken as a maximum. Numerical values, as used herein, include values expressed as percentages. For any embodiment of the invention in which a numerical value is prefaced by "about" or "approximately", the invention includes an embodiment in which the exact value is recited. For any embodiment of the invention in which a numerical value is not prefaced by "about" or "approximately", the invention includes an embodiment in which the value is prefaced by "about" or "approximately". "Approximately" or "about" generally includes numbers that fall within a range of 1 % or in some embodiments within a range of 5% of a number or in some embodiments within a range of 10% of a number in either direction (greater than or less than the number) unless otherwise stated or otherwise evident from the context (except where such number would impermissibly exceed 1 00% of a possible value). It should be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one act, the order of the acts of the method is not necessari ly limited to the order in which the acts of the method are recited, but the invention includes embodiments in which the order is so limited. It should also be understood that unless otherwise indicated or evident from the context, any product or composition described herein may be considered "isolated".

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[241 ] EXAMPLES

[242] Example 1 : Cell Competition Assay Used to Identify Cancer Cells That Are Cholesterol Auxotrophic Due to Inadequate SQLE Expression

[243] A cell competition assay was conducted in lipoprotein depleted serum media, which lacks lipids as well as sterols. These experiments identified a group of cancer cells ( 10- 15% out of 28 cell lines) dependent on extracel lular lipoprotein supplementation (Snu-1 , Raji, Daudi, H71 6, U937). Addition of free LDL or cholesterol to this subset of cancer cells indeed can rescue their growth defect under l ipoprotein depleted conditions. Furthermore, we identified that extremely low expression ( or no expression) of a cholesterol biosynthesis gene, SQLE, is responsi ble for cholesterol auxotrophy in SNU- 1 cancer cells. Consistent with this defect, squalene levels are significantly higher and re-expression of SQLE gene in SNU- 1 cells rescues growth defect under lipoprotein depleted conditions. We were able to obtain similar "SQLE low" cancer cells and confirmed their dependence to external lipoproteins. Finally, as a therapy approach, we blocked cholesterol uptake of sensitive cancer cells using anti-LDLR antibodies. Incubation of cancer cells with an anti-LDLR antibody inhibits growth of lipoprotein depletion sensitive cancer cells and not others. [244] Example 2: Characterization of Squaiene Levels in Cancer Cell Lines

[245 ] First, polar metabolites were extracted from different cancer cell lines using 80% methanol . ^'The polar metabol ites were analyzed using LC-MS to determine squaiene levels. As is shown i n F IG. 4A, squaiene levels are significantly upregulated in cancer cell lines exhibiting low levels of squaiene monooxygenase. Secondly, SQLE expression levels were analyzed in - 1000 cancer cel l lines using the Cancer Cel l Line Encyclopedia. Immunoblotting was also performed in various cancer cell lines to determine SQLE protein levels in those cancer cell lines As shown in FIG. 4B, cancer cell lines exhibiting low SQLE expression levels are enriched with the anaplastic large cell lymphoma cancer subtype.

Claims

What is claimed is:

1 . A method of inhibiting growth or survival of a cancer cell that is cholesterol auxotrophic due to inadequate squalene epoxidase (SQLE) expression, the method comprising limiting the availability of extracellular cholesterol in a cancer cell that is cholesterol auxotrophic due to inadequate SQLE expression, thereby inhibiting growth or survival of the cancer cell that is cholesterol auxotrophic due to inadequate SQLE expression.

2. The method of claim 1 , wherein inadequate SQLE expression comprises a decreased level of SQLE expression in the cancer cell relative to the level of SQLE expression in a cholesterol protrophic cancer cell.

3. The method of claim 1 , wherein inadequate SQLE expression comprises the absence of SQLE expression.

4. The method of any one of claims 1 to 3, wherein limiting the availability of extracellular cholesterol in the cancer cell occurs in vitro.

5. The method of claim 4, wherein limiting the availability of extracellular cholesterol in the cancer cell comprises contacting the cancer cell with an effective amount of an agent that inhibits cholesterol uptake into the cancer cell.

6. The method of claim 5, wherein the agent inhibits the expression level or activity of low density lipoprotein receptor (LDLR) in the cancer cell.

7. The method of claim 6, wherein the agent is selected from the group consisting of an antibody, an antisense oligonucleotide, a short hairpin RNA (shRNA), a small interfering RNA (si RNA), a micro RNA (miRNA).

8. The method of any one of claims 1 to 3, wherein limiting the availability of extracellular cholesterol in the cancer cell occurs in vivo.

9. The method of claim 8, wherein limiting the availabil ity of extracellular cholesterol in the cancer cel l comprises adm i nistering to a subject an effective amount of a cholesterol lowering agent.

10. The method of claim 9, wherein the cholesterol lowering agent is selected from the group consisting of small organic or inorganic molecules; saccharides; oligosaccharides;

polysaccharides; a biological macromolecule selected from the group consisting of peptides, proteins, peptide analogs and derivatives; peptidomimetics; nucleic acids selected from the group consisting of siRNAs, shRNAs, antisense RNAs, ribozymes, and aptamers; an extract made from biological materials selected from the group consisting of bacteria, plants, fungi, animal cells, and animal tissues; naturally occurring or synthetic compositions; and any combination thereof.

1 1 . The method of claim 9, wherein the cholesterol lowering agent is selected from the group consisting of an agent that i) i nhibits intestinal absorption of cholesterol, ii) inhibits the level or activity of 3-hydroxy-3-methylglutaryl -coenzyme A (HMG-CoA) reductase, iii) increases cholesterol metabolism, iv) decreases formation and/or secretion of cholesterol esters, v) increases the ratio of high density lipoprotein (HDL) to LDL, and vi) upregulates expression of liver LDLR receptors.

12. The method of claim 1 1 , wherein the agent that i) inhibits intestinal absorption of

cholesterol is selected from the group consisting of a Niemann-Pick C l -Like (NPC1L1 ) protein inhibitor, a phytosterol, and a phytostanol.

13. The method of claim 1 1 , wherein the agent that ii) inhibits the level or activity of HMG- CoA reductase is selected from the group consisting of a tocotrienol and a statin.

14. The method of claim 1 1 , wherein the agent that iii) increases cholesterol metabolism is selected from the group consisting of a bile acid sequestrant and a 7-alpha-hydroxylase activator.

1 5. The method of claim 1 1 , wherein the agent that iv) decreases formation and/or secretion of cholesterol esters comprises an acyl-CoA acyl transferase inhibitor.

16. The method of claim 1 1 , wherein the agent that v) increases the ratio of HDL to LDL comprises a cholesterylester transfer protein (CETP) inhibitor.

1 7. The method of claim 1 1 , wherein the agent that vi) upregulates expression of liver LDLR receptors comprises a proprotein convertase subtil isin/kexin type 9 (PCS 9) inhibitor.

1 8. A method o f rendering a cancer cell cholesterol auxotrophic, the method comprising contacting a cancer cell with an agent that inhibits the expression level or activity of SQLE in the cancer cell, wherein the agent inhibits the expression level or activity of SQLE in the cancer cell, thereby rendering the cancer cell cholesterol auxotrophic. 9. The method of claim 1 8, wherein the agent is selected from the group consisting of small organic or inorganic molecules; saccharides; oligosaccharides; polysaccharides; a biological macromolecule selected from the group consisting of peptides, proteins, peptide analogs and derivatives; peptidomimetics; nucleic acids selected from the group consisting of siRNAs, shRNAs, antisense RNAs, ribozymes, and aptamers; an extract made from biological materials selected from the group consisting of bacteria, plants, fungi, animal cells, and animal tissues; naturally occurring or synthetic compositions; and any combination thereof.

The method of claims 1 7 or 1 8, wherein the agent is selected from the group consisting of i) an antimycotic compound, ii) an allylamine, iii) a squalene derivative, iv) a natural compound, and v) derivatives of any of i)-iv).

The method of claim 20, wherein the antimycotic compound comprises a compound of formula (I)

wherein R is selected from the group consisting of

22. The method of claim 20, wherein the antimycotic compound comprises a compound of formula (II)

wherein R is selected from the group consisting of

the allylamine comprises a compound of formula (Π)

(Π)

herein R is selected from the group consisting of

24. The method of claim 20, wherein the allylamine comprises a compound of formula (III)

wherein R is selected from the group consisting of H, CH₃, CH₂CH₃, CH2CH2CH3, CH₂CHCH2, CH₂CCH, and C₃H₆.

25. The method of claim 20, wherein the allylamine comprises a compound of formula (IV)

wherein R is selected from the group consisting of H, CH₃, CH₂CH₃, CF₃, OH, nitrile, formyl, hydroxy methyl, phenyl, 2-furyl, 2-oxazolyl, 2-thiazolyl, 5-oxazolyl, 5-thiazolyl, 1 -pyrrolyl, and 3-thienyl.

The method of claim 20, wherein the allylamine comprises a compound of formula (V)

the group consisting of

27. The method of claim 20, wherein the allylamine comprises a compound of formula (VI)

whe

R is selected from the group consisting of and

The method of claim 20, wherein the allylamine comprises a compound of formula (VI I)

wherein R is selected from the group consisting of OCH₃, SCH₃, OH, SH, CONH₂, COCF₃, CCH, F, CH₃, CHO, CN, and CHCH₂.

The method of claim 20, wherein the allylamine comprises a compound of formula (VIII)

(VI I I) wherein R is selected from the group consisting of CH₃, OCH₃, CF₃, and CN.

The method of claim 20, wherein the allylamine comprises

The method of claim 20, wherein the allylamine comprises a compound of formula (IX)

(IX)

wherein R is selected from the group consisting of CH₃ and OCH₃.

The method of claim 20, wherein the squalene derivative is selected from the group consisting of

, and

33. The method of claim 20, wherein the squalene derivative comprises a compound of

(X) wherein R is selected from the group consisting of OH, CH2CH₂OH, CH₂OOH, CH₂SH, CH₂NH-C-C3H5, and CHCFj,

34.

, wherein

R is CHCF₂.

35. The method of claim 20, wherein the squalene derivative comprises a compound of

(X I)

wherein R is selected from the group consisting of CH₂OH, CHO, and ΟΗ₂ΝΗ₂·

36. The method of claim 20, wherein the natural compound comprises a plant extract selected from the group consisting of Agrimonia pilosa extract, Aleurites fordii extract, Euphorbia jolkini extract, Lager sir oemia indica extract, Camellia sinensis extract, Allium sativum extract, Rheum palmatum extract, Cynara scolymus extract, Fraxinus excelsior extract, and Peumus boldus extract.

37. The method of claim 20, wherein the natural compound is selected from the group consisting of (-)-epigallocatechin-3-0-gal late (EGCG), (-)-epicatechin-3-0-gallate (ECG), (-)-epigal locatechin (EGG), and (-)-epicatechin (EC), gallic acid.

38. The method of claim 20, wherein the natural compound is a compound of formula (XII)

39. The method of claim 20, wherein the natural compound comprises a garlic compound selected from the group consisting of a selenium compound, a tellurium compound, and an allyl compound.

40. The method of claim 39, wherein the selenium compound is selected from the group

consisting of selenocystine, selenite, selenium dioxide, and methylselenol.

41 . The method of claim 39, wherein the tel lurium compound is selected from the group

consisting of tellurite, tellurium dioxide, and dimethyltel luride.

42. The method of claim 39, wherein the allyl compound is selected from the group consisting of S-allylcysteine, alliin, 1 ,3-diallyltrisulfane, and 1 ,2-diallyldisulfane

43. The method of any one of claims 18 to 42, wherein the contacting occurs in vitro.

44. The method of any one of claims 1 8 to 42, wherein the contacting occurs in vivo,

45. The method of any one of claims 1 8 to 44, further comprising identifying the cancer cell as cholesterol protrophic.

46. A method of inhibiting growth or survival of a cancer cell, the method comprising:

a) contacting the cancer cell with an effective amount of a first agent that inhibits the level or activity of SQLE in the cancer cell, thereby rendering the cancer cell cholesterol auxotrophic; and

b) contacting the cholesterol auxotrophic cancer cell with an effective amount of a second agent that limits the availability of extracellular cholesterol in the cancer cell, thereby inhibiting growth or survival of the cancer cell.

47. A method of classifying a tumor as cholesterol auxotrophic, the method comprising:

a) determining the level of an expression product of the SQLE gene in a sample obtained from the tumor; and

b) comparing the level determined in a) with a control level,

wherein if the level determined in a) is less than the control level, the level is correlated with cholesterol auxotrophy, thereby classifying the tumor as cholesterol auxotrophic.

48. The method of claim 47, wherein the sample comprises tumor tissue.

49. The method of claims 47 or 48, wherein the tumor is from a cancer selected from the group consisting of ovarian cancer, bladder cancer, lung cancer, cervical cancer, breast cancer, prostate cancer, gliomas, fibrosarcomas, retinoblastomas, melanomas, soft tissue sarcomas, osteosarcomas, leukemias, stomach cancer, colon cancer, carcinoma of the kidney, gastrointestinal cancer, salivary gland cancer, pancreatic cancer, Hodgkin's disease, non-Hodgkin's lymphomas, acute and chronic lymphocytic leukemias, multiple myeloma, neuroblastoma, Wilms' tumor, testicular cancer, soft-tissue sarcomas, chronic lymphocytic leukemia, primary macroglobulinemia, chronic granulocytic leukemia, primary brain carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinomas, malignant melanomas, choriocarcinomas, mycosis fungoides, head and neck carcinomas, osteogenic sarcoma, pancreatic carcinomas, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, genitourinary carcinomas, thyroid carcinomas, esophageal carcinomas, malignant hypercalcemia, cervical hyperplasia, renal cell carcinomas, endometrial carcinomas, polycythemia vera, essential thrombocytosis, adrenal cortex carcinomas, skin cancer, and prostatic carcinomas.

50. The method of any one of claims 47 to 49, wherein the expression product comprises a mRNA or a polypeptide.

5 1 . The method of any one of claims 47 to 50, wherein determining the level of the

expression product in the sample comprises perform ing a hybridization based assay, polymerase chain reaction assay, sequencing, ELISA assay, Western blot, mass spectrometry, or immunohistochemistry.

52. A method of diagnosing a tumor in a subject as a cholesterol auxotrophic tumor, the method comprising:

a) determining the level of an expression product of the SQLE gene in a sample obtained from the subject; and

b) comparing the level determined in a) with a control level, wherein if the level determined in a) is less than the control level, the level is indicative of cholesterol auxotrophy, thereby di agnosing the tumor in the subject as a cholesterol auxotrophic tumor.

53. The method of claim 52, wherein the sample comprises tumor tissue.

54. The method of claims 52 or 53, wherein the tumor is from a cancer selected from the group consisting of ovarian cancer, bladder cancer, lung cancer, cervical cancer, breast cancer, prostate cancer, gliomas, fibrosarcomas, retinoblastomas, melanomas, soft tissue sarcomas, osteosarcomas, leukemias, stomach cancer, colon cancer, carcinoma of the kidney, gastrointestinal cancer, salivary gland cancer, pancreatic cancer, Hodgkin's disease, non-Hodgkin's lymphomas, acute and chronic lymphocytic leukemias, multiple myeloma, neuroblastoma, Wilms' tumor, testicular cancer, soft-tissue sarcomas, chronic lymphocytic leukemia, primary macroglobulinemia, chronic granulocytic leukemia, pri mary brain carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinomas, malignant melanomas, choriocarcinomas, mycosis fungoides, head and neck carcinomas, osteogenic sarcoma, pancreatic carcinomas, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, genitourinary carcinomas, thyroid carcinomas, esophageal carcinomas, malignant hypercalcemia, cervical hyperplasia, renal cell carcinomas, endometrial carcinomas, polycythemia vera, essential thrombocytosis, adrenal cortex carcinomas, skin cancer, and prostatic carcinomas.

55. The method of any one of claims 52 to 54, wherein the expression product comprises a mRNA or a polypeptide.

56. The method of any one of claims 52 to 55, wherein determining the level of the

expression product in the sample comprises performing a hybridization based assay, polymerase chain reaction assay, sequencing, ELISA assay, Western blot, mass spectrometry, or immunohistochemistry.

57. A method of determining whether a subject with a tumor is a suitable candidate for treatment with a cholesterol lowering agent, the method comprising:

a) assessing the level of expression of a SQLE gene product in a tumor sample obtained from the subject; and

b) comparing the level determined in a) to a control level,

wherein if the level determined in a) is less than the control level, then the level in a) indicates that the tumor is a cholesterol auxotrophic tumor, thereby determining that the subject with the tumor is a suitable candidate for treatment with a cholesterol lowering agent.

58. The method of claim 57, wherein the sample comprises tumor tissue.

59. The method of claims 57 or 58, wherein the tumor is a from a cancer selected from the group consisting of ovarian cancer, bladder cancer, lung cancer, cervical cancer, breast cancer, prostate cancer, gliomas, fibrosarcomas, retinoblastomas, melanomas, soft tissue sarcomas, osteosarcomas, leukemias, stomach cancer, colon cancer, carcinoma of the kidney, gastrointestinal cancer, salivary gland cancer, pancreatic cancer, Hodgkin's disease, non-Hodgkin's lymphomas, acute and chronic lymphocytic leukemias, multiple myeloma, neuroblastoma, Wilms' tumor, testicular cancer, soft-tissue sarcomas, chronic lymphocytic leukemia, primary macroglobulinemia, chronic granulocytic leukemia, primary brain carcinoma, mal ignant pancreatic insul inoma, malignant carcinoid carci nomas, malignant melanomas, choriocarcinomas, mycosis fungoides, head and neck carcinomas, osteogenic sarcoma, pancreatic carcinomas, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, genitourinary carcinomas, thyroid carcinomas, esophageal carcinomas, malignant hypercalcemia, cervical hyperplasia, renal cell carcinomas, endometrial carcinomas, polycythemia vera, essential thrombocytosis, adrenal cortex carcinomas, skin cancer, and prostatic carcinomas.

60. The method of any one of claims 57 to 59, wherein the expression product comprises a mRNA or a polypeptide.

61 . The method of any one of claims 57 to 60, wherein determining the level of the

62. A method of treating a cholesterol auxotrophic cancer in a subject in need thereof, the method comprising administering to a subject suffering from a cholesterol auxotrophic cancer an effective amount of a composition which limits the availability of extracellular cholesterol in the cholesterol auxotrophic cancer cells in the subject, wherein the composition inhibits the growth or survival of the cholesterol auxotrophic cancer cells, thereby treating the subject's cholesterol auxotrophic cancer.

63. The method of claim 62, wherein the composition comprises at least one cholesterol lowering agent.

64. The method of claims 62 or 63, wherein the cholesterol lowering agent is selected from the group consisting of an agent that i) inhibits intestinal absorption of cholesterol, ii) inhibits the level or activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, iii) increases cholesterol metabolism, iv) decreases formation and/or secretion of cholesterol esters, v) increases the ratio of high density lipoprotein (HDL) to LDL, and vi) upregulates expression of liver LDLR receptors.

65. The method of any one of claims 62 to 64, wherein the agent that i) inhibits intestinal absorption of cholesterol is selected from the group consisting of a Niemann-Pick C l - Like (NPC 1 LI ) protein inhibitor, a phytosterol, and a phytostanol.

66. The method of any one of claims 62 to 65, wherein the agent that ii) inhibits the level or activity of HMG-CoA reductase is selected from the group consisting of a tocotrienol and a statin.

67. The method of any one of claims 62 to 66, wherein the agent that iii) increases cholesterol metabolism is selected from the group consisting of a bile acid sequestrant and a 7-alpha- hydroxylase activator.

68. The method of any one of claims 62 to 67, wherein the agent that iv) decreases formation and/or secretion of cholesterol esters comprises an acyl-CoA acyl transferase inhibitor.

69. The method of any one of claims 62 to 68, wherein the agent that v) increases the ratio of HDL to LDL comprises a cholesterylester transfer protein (CETP) inhibitor.

70. The method of any one of claims 62 to 69, wherein the agent that vi) upregulates

expression of liver LDLR receptors comprises a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhi bitor.

7 1 . The method of any one of claims 62 to 70, further comprising administering to the subject an effective amount of at least one chemotherapeutic agent.

72. The method of any one of claims 62 to 71 , further comprising diagnosing the subject as having a cholesterol auxotrophic cancer.

73. A method of identifying a candidate agent that renders a cancer cell cholesterol

auxotrophic, comprising:

a) contacting a cancer cell that expresses SQLE with a test agent; b) measuring the expression level of SQLE in the presence and absence of the test agent ; and

c) determining whether the test agent renders the cancer cell cholesterol auxotrophic, wherein if the level measured in b) in the presence of the test agent is decreased compared to the level measured in b) in the absence of the test agent, the test agent is identified as a candidate iigent that renders the cancer cell cholesterol auxotrophic.

74. The method of claim 73, further comprising performing an assay to confirm that the

candidate agent that renders the cancer cell cholesterol auxotrophic.

75. The method of claims 73 or 74, wherein the assay comprises a cell competition assay in lipoprotein depleted serum media.

76. The method of any one of claims 73 to 75, wherein the assay comprises:

a) incubating cholesterol protrophic cancer cells in a lipoprotein depleted serum media;

b) contacting the cholesterol protrophic cancer cells with a candidate agent that renders a cancer cel l cholesterol auxotrophic; and

c) assessing the growth of the cholesterol protrophic cancer cells in the lipoprotein depleted serum media after being contacted with the candidate agent, wherein if the growth of the cholesterol protrophic cancer cells is inhibited in the lipoprotein depleted serum media after being contacted with the candidate agent, then the candidage agent renders the cancer cells cholesterol auxotrophic.

77. A composition comprising:

a) at least one cholesterol lowering agent; and

b) at least one chemotherapeutic agent.

78. A composition comprising:

a) at least one agent that inhibits the level or activity of SQLE;

b) at least one cholesterol lowering agent; and

c) at least one chemotherapeutic agent.

79. A kit comprising:

a) at least one cholesterol lowering agent; and

b) at least one chemotherapeutic agent.

80. A kit comprising:

a) at least one agent that inhibits the level or activity of SQLE;

b) at least one cholesterol lowering agent; and

c) at least one chemotherapeutic agent.

8 1 . A kit comprisi ng:

a) at least one cancer cel l that auxotrophic due to inadequate expression of SQLE i n the cancer cell ; and

b) a test agent.

82. The kit of claim 81 , wherein the at least one cancer cell is selected from the group consisting of Snu- 1 , Raji, Daui, H716, and U937.

83. The kit of claim 81 , wherein the at least one cancer cell is engineered to be auxotrophic due to inadequate expression of SQLE in the cancer cell.

84. The kit of any one of claims 81 to 83, further comprising a lipoprotein conditioned media.

85. The kit of any one of claims 81 to 84, further comprising instructions for assessing whether the test agent is capable of limiting the availability of extracellular cholesterol in the at least one cancer cel l .