WO2015027171A1

WO2015027171A1 - Methods for predicting toxicity in response to treatment with a drug by assessing activation of the sterol regulatory binding protein (srebp) pathway

Info

Publication number: WO2015027171A1
Application number: PCT/US2014/052320
Authority: WO
Inventors: Sarah S. Bacus
Original assignee: Quintiles Transnational Corp
Current assignee: Iqvia Inc
Priority date: 2013-08-23
Filing date: 2014-08-22
Publication date: 2015-02-26
Anticipated expiration: 2016-02-23

Abstract

The present disclosure provides methods for predicting whether a drug (e.g., a tyrosine kinase inhibitor) is likely to be toxic to a cell or biological sample by assessing whether pathways (e.g., the SREBP pathway) that lead to the production of fatty acids and/or cholesterol are activated in the cell and/or biological sample upon treatment of the cell or biological sample with the drug. Such methods may be used to select cells or biological samples predicted to be responsive (i.e., sensitive) to treatment with a drug for treatment with the drug.

Description

METHODS FOR PREDICTING TOXICITY IN RESPONSE TO TREATMENT WITH A DRUG BY ASSESSING ACTIVATION OF THE STEROL REGULATORY BINDING

PROTEIN (SREBP) PATHWAY

BACKGROUND

[0001] Biomarkers have been employed to determine those patients that are most likely to respond to a particular therapy, including therapies directed to EGFR. For example, the presence of K-Ras mutations is associated with a lack of treatment response to gefitinib and erlotinib. Therefore, the presence of a K-Ras gene mutation has been used as a marker for selecting those patients who will not benefit from anti-EGFR therapy. Accordingly, therapeutic regimens that target EGFR may not be administered to patients predicted to be unresponsive to the regimen. The advent of targeted therapy has vastly improved the treatment and prognosis of multiple types of cancer. The goal of these agents is to improve specificity in the elimination of tumor cells while minimizing toxicity on healthy cells. Despite this goal, unexpected cardiotoxicity has arisen for many targeted compounds. For example, imatinib mesylate, a Bcr-AbI, PDGF, SCF, and c-kit inhibitor for the treatment of chronic myelogenous leukemia, is associated with left ventricular dysfunction and severe congestive heart failure in patients. These adverse cardiac events were not initially detailed on the prescribing label and only became apparent after drug approval. Earlier recognition of the potential for cardiotoxicity in the drug development lifecycle would be beneficial for both patient care as well as for the generation of safer therapies. SUMMARY

[0002] The present disclosure provides methods for predicting whether a drug (e.g., a tyrosine kinase inhibitor) is likely to be toxic to a cell or biological sample by assessing whether pathways (e.g., the SREBP pathway) that lead to the production of fatty acids and/or cholesterol are activated in the cell and/or biological sample upon treatment of the cell or biological sample with the drug. Such methods may be used to select cells or biological samples predicted to be responsive (i.e., sensitive) to treatment with a drug for treatment with the drug.

[0003] The present disclosure provides methods for predicting whether a drug is likely to be toxic to a cell comprising: contacting the cell with the drug; and determining whether a Sterol Regulatory Binding Protein (SREBP) pathway in the cell contacted with the drug is activated, wherein the drug is predicted to be toxic to the cell where the SREBP pathway is activated in the cell contacted with the drug or wherein the drug is predicted to not be toxic to the cell where the SREBP pathway is not activated in the cell contacted with the drug.

[0004] In some embodiments of each or any of the above or below mentioned embodiments, activation of the SREBP pathway is identified by increased expression of one or more genes in the SREBP pathway.

[0005] In some embodiments of each or any of the above or below mentioned embodiments, the one or more genes are selected from the group consisting of: SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and hydroxymethylglutaryl- CoA reductase (HMGCR).

[0006] In some embodiments of each or any of the above or below mentioned embodiments, the drug is a tyrosine kinase inhibitor.

[0007] In some embodiments of each or any of the above or below mentioned embodiments, the toxicity is cardiotoxicity or hepatotoxicity.

[0008] In some embodiments of each or any of the above or below mentioned embodiments, the cell is a myocardiocyte or a hepatocyte.

[0009] The present disclosure provides methods for contacting a cell with a drug such as a tyrosine kinase inhibitor, the method comprising: contacting the cell with the drug where the SREBP pathway is activated in the cell upon treatment of the cell with the drug.

[0010] The present disclosure provides methods for contacting a cell with a drug such as a tyrosine kinase inhibitor, the method comprising: determining if a SREBP pathway in the cell is activated upon contact with the drug, and contacting the cell with the drug where the SREBP pathway is activated in the cell upon treatment of the cell with the drug.

[0011] In some embodiments of each or any of the above or below mentioned embodiments, activation of the SREBP pathway is identified by increased expression of one or more genes in the SREBP pathway.

[0012] In some embodiments of each or any of the above or below mentioned embodiments, the one or more genes are selected from the group consisting of: SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase

(FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and hydroxymethylglutaryl-

CoA reductase (HMGCR).

[0013] In some embodiments of each or any of the above or below mentioned embodiments, the drug is a tyrosine kinase inhibitor.

[0014] In some embodiments of each or any of the above or below mentioned embodiments, the toxicity is cardiotoxicity or hepatotoxicity. [0015] In some embodiments of each or any of the above or below mentioned embodiments, the cell is a myocardiocyte or a hepatocyte.

[0016] The present disclosure also provides method for treating a cell with a drug, the method comprising: contacting the cell with the drug where the SREBP pathway is activated in the cell upon treatment with the drug.

[0017] In some embodiments of each or any of the above or below mentioned embodiments, the methods further comprise determining whether a Sterol Regulatory Binding Protein (SREBP) pathway in the cell contacted with the drug is activated.

[0018] In some embodiments of each or any of the above or below mentioned embodiments, activation of the SREBP pathway is identified by increased expression of one or more genes in the SREBP pathway.

[0019] In some embodiments of each or any of the above or below mentioned embodiments, the one or more genes are selected from the group consisting of: SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and hydroxymethylglutaryl- CoA reductase (HMGCR).

[0020] In some embodiments of each or any of the above or below mentioned embodiments, the drug is a receptor tyrosine kinase inhibitor.

[0021] In some embodiments of each or any of the above or below mentioned embodiments, the toxicity is cardiotoxicity or hepatotoxicity.

[0022] In some embodiments of each or any of the above or below mentioned embodiments, the cell is a myocardiocyte or a hepatocyte.

[0023] The present disclosure also provides methods for treating a cell with a drug, the method comprising: contacting the cell with the drug where the SREBP pathway is not activated in the cell upon treatment with the drug.

[0024] In some embodiments of each or any of the above or below mentioned embodiments, activation of the SREBP pathway is identified by increased expression of one or more genes in the SREBP pathway.

[0025] In some embodiments of each or any of the above or below mentioned embodiments, the one or more genes are selected from the group consisting of: SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and hydroxymethylglutaryl- CoA reductase (HMGCR).

[0026] In some embodiments of each or any of the above or below mentioned embodiments, the drug is a receptor tyrosine kinase inhibitor. [0027] In some embodiments of each or any of the above or below mentioned embodiments, the toxicity is cardiotoxicity or hepatotoxicity.

[0028] In some embodiments of each or any of the above or below mentioned embodiments, the cell is a myocardiocyte or a hepatocyte.

[0029] The present disclosure also provides methods for selecting a cell for treatment with a drug, the method comprising: determining whether a Sterol Regulatory Binding Protein (SREBP) pathway in the cell is activated upon treatment of the cell with the drug; and selecting the cell for treatment with the drug where the SREBP pathway is activated in the cell contacted with the drug or not selecting the cell for treatment with the drug where the SREBP pathway is not activated in the cell contacted with the drug.

[0030] In some embodiments of each or any of the above or below mentioned embodiments, activation of the SREBP pathway is identified by increased expression of one or more genes in the SREBP pathway.

[0031] In some embodiments of each or any of the above or below mentioned embodiments, the one or more genes are selected from the group consisting of: SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and hydroxymethylglutaryl- CoA reductase (HMGCR).

[0032] In some embodiments of each or any of the above or below mentioned embodiments, the drug is a receptor tyrosine kinase inhibitor.

[0033] In some embodiments of each or any of the above or below mentioned embodiments, the toxicity is cardiotoxicity or hepatotoxicity.

[0034] In some embodiments of each or any of the above or below mentioned embodiments, the cell is a myocardiocyte or a hepatocyte.

[0035] The present disclosure also provides methods for treating a patient with a drug, the method comprising: obtaining a biological sample from the patient; contacting one or more cells in the biological sample with a drug; determining whether a Sterol Regulatory Binding Protein (SREBP) pathway in one or more of the contacted cells are activated upon treatment with the drug; and selecting the drug that activates the Sterol Regulatory Binding Protein (SREBP) pathway; and treating the patient with the drug that activates the Sterol Regulatory Binding Protein (SREBP) pathway.

[0036] In some embodiments of each or any of the above or below mentioned embodiments, activation of the SREBP pathway is identified by increased expression of one or more genes in the SREBP pathway.

[0037] In some embodiments of each or any of the above or below mentioned embodiments, the one or more genes are selected from the group consisting of: SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and hydroxymethylglutaryl- CoA reductase (HMGCR).

[0038] In some embodiments of each or any of the above or below mentioned embodiments, the drug is a receptor tyrosine kinase inhibitor.

[0039] In some embodiments of each or any of the above or below mentioned embodiments, the toxicity is cardiotoxicity or hepatotoxicity.

[0040] In some embodiments of each or any of the above or below mentioned embodiments, the cell is a myocardiocyte or a hepatocyte.

[0041] In some embodiments of each or any of the above or below mentioned embodiments, the patient is a cancer patient.

[0042] The present disclosure also provides methods for conducting a clinical trial, the method comprising: obtaining biological samples from one or more subjects; contacting one or more cells in the biological samples from the subjects with a drug; determining whether a Sterol Regulatory Binding Protein (SREBP) pathway in one or more of the contacted cells in the biological samples from the subjects are activated upon treatment with the drug; and enrolling subjects in the clinical trial where the Sterol Regulatory Binding Protein (SREBP) pathway is activated in one or more of cells in the biological sample upon contact of the cells with the drug.

[0043] In some embodiments of each or any of the above or below mentioned embodiments, activation of the SREBP pathway is identified by increased expression of one or more genes in the SREBP pathway.

[0044] In some embodiments of each or any of the above or below mentioned embodiments, the one or more genes are selected from the group consisting of: SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and hydroxymethylglutaryl- CoA reductase (HMGCR).

[0045] In some embodiments of each or any of the above or below mentioned embodiments, the drug is a receptor tyrosine kinase inhibitor.

[0046] In some embodiments of each or any of the above or below mentioned embodiments, the toxicity is cardiotoxicity or hepatotoxicity.

[0047] In some embodiments of each or any of the above or below mentioned embodiments, the cell is a myocardiocyte or a hepatocyte.

[0048] In some embodiments of each or any of the above or below mentioned embodiments, the patient is a cancer patient. [0049] The present disclosure also provides methods for screening one or more drugs for likelihood of being cardiotoxic, the method comprising: contacting a cell with the drug; and determining whether a Sterol Regulatory Binding Protein (SREBP) pathway in the cell contacted with the drug is activated.

[0050] In some embodiments of each or any of the above or below mentioned embodiments, the methods further comprise selecting the drug that activates the Sterol Regulatory Binding Protein (SREBP) pathway.

[0051] In some embodiments of each or any of the above or below mentioned embodiments, activation of the SREBP pathway is identified by increased expression of one or more genes in the SREBP pathway.

[0052] In some embodiments of each or any of the above or below mentioned embodiments, the one or more genes are selected from the group consisting of: SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and hydroxymethylglutaryl- CoA reductase (HMGCR).

[0053] In some embodiments of each or any of the above or below mentioned embodiments, the drug is a receptor tyrosine kinase inhibitor.

[0054] In some embodiments of each or any of the above or below mentioned embodiments, the toxicity is cardiotoxicity or hepatotoxicity.

[0055] In some embodiments of each or any of the above or below mentioned embodiments, the cell is a myocardiocyte or a hepatocyte.

[0056] The present disclosure also provides an in-vitro method for determining drug-induced cell toxicity, the method comprising: identifying whether a Sterol Regulatory Binding Protein (SREBP) pathway in a cell contacted with the drug is activated in response to treatment with a drug, wherein the drug is predicted to be toxic to the cell where the SREBP pathway is activated in the cell contacted with the drug or wherein the drug is predicted to not be toxic to the cell where the SREBP pathway is not activated in the cell contacted with the drug.

[0057] In some embodiments of each or any of the above or below mentioned embodiments, activation of the SREBP pathway is identified by increased expression of one or more genes in the SREBP pathway.

[0058] In some embodiments of each or any of the above or below mentioned embodiments, the one or more genes are selected from the group consisting of: SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and hydroxymethylglutaryl- CoA reductase (HMGCR). [0059] In some embodiments of each or any of the above or below mentioned embodiments, the drug is a receptor tyrosine kinase inhibitor.

[0060] In some embodiments of each or any of the above or below mentioned embodiments, the toxicity is cardiotoxicity or hepatotoxicity.

[0061] In some embodiments of each or any of the above or below mentioned embodiments, the cell is a myocardiocyte or a hepatocyte.

[0062] The present disclosure also provides a test panel for determining whether a drug is toxic to a cell, the test panel comprising: a test for determining whether a Sterol Regulatory Binding Protein (SREBP) pathway in a cell is activated upon contact of the cell with the drug, wherein the drug is predicted to be toxic to the cell where the SREBP pathway is activated in the cell contacted with the drug or wherein the drug is predicted to not be toxic to the cell where the SREBP pathway is not activated in the cell contacted with the drug.

[0063] In some embodiments of each or any of the above or below mentioned embodiments, a test comprises a microarray comprising oligonucleotides that specifically bind one or more genes are selected from the group consisting of: SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and hydroxymethylglutaryl-CoA reductase (HMGCR).

[0064] The present disclosure also provides a kit for assessing toxicity of a drug, the kit comprising: a set of instructions for using the test panel as disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0065] The foregoing summary, as well as the following detailed description of the disclosure, will be better understood when read in conjunction with the appended figures. For the purpose of illustrating the disclosure, shown in the figures are embodiments which are presently preferred. It should be understood, however, that the disclosure is not limited to the precise arrangements, examples and instrumentalities shown.

[0066] Figure 1 : Crizotinib, sunitinib, and nilotinib activate cell death mechanisms in human cardiomyocytes (HCM). A. HCMs were treated with a wide dose range of crizotinib, sunitinib, erlotinib, and nilotinib (0.01 μΜ - 10μΜ) for 72 hours. Cell viability was determined using Hoechst staining, and total cell counts were done using the Celllnsight Spot Detector bioapplication as detailed in Materials and Methods. B. HCMs were treated for 24 hours with 0.3μΜ - 10μΜ of each tyrosine kinase inhibitor and assayed for caspase activation using the Promega Caspase-Glo 3/7™ assay. C. HCMs were treated for 24 h with 0.3μΜ - 10μΜ of each tyrosine kinase inhibitor. Reactive oxygen species generation was examined using the probe dihydroethidium to detect superoxide production. All experiments were done in triplicate. Fold-change calculations for each study reflect values from drug-treated cells relative to DMSO controls. Data represent mean ± SEM. * indicates p<0.05 as compared to control.

[0067] Figure 2: Tyrosine kinase inhibitors (TKi) have differential effects on lipid accumulation and the activation of acetyl CoA carboxylase (ACC). A. Human cardiomyocytes (HCMs) were treated with either DMSO or 3μΜ of crizotinib, sunitinib, eriotinib, or nilotinib for 48 hours. Lipid accumulation was determined using Oil Red-0 staining. Images are 40x with 100x insets; panel representative of n=3 experiments. B. Phosphorylation of ACC was determined following 24 hours treatment with increasing doses of each TKi by Western blot analysis. Protein lysates were collected, and "^g total protein was resolved on SDS-PAGE. Blots were probed for phospho-ACC (Ser79) and actin. Blot is representative of three independent experiments.

[0068] Figure 3: Crizotinib activates the cholesterol pathway and increases cholesterol production in human cardiomyocytes (HCMs). A. RNA was collected from HCM treated for 24 hours with DMSO or 3μΜ crizotinib, sunitinib, eriotinib, or nilotinib. Transcriptome analysis was done using the Affymetrix GeneChip® Human Genome U 133A 2.0 Array. Graphical representation depicts the upregulation of genes involved in the sterol regulatory binding (SREBP) pathway. The table reports the fold-changes in the major genes associated with cholesterol synthesis following treatment. B. qRT-PCR was performed to confirm SREBP pathway gene upregulation as shown by crizotinib and sunitinib transcriptome analysis. C. Total intracellular cholesterol levels were determined in HCMs treated for 48 hours with DMSO or 3μΜ crizotinib, sunitinib, eriotinib, or nilotinib using the Abeam Cholesterol/Cholesteryl Ester Quantification Kit. Data represents mean ± SEM from n=3 experiments. * indicates p<0.05 as compared to control.

DETAILED DESCRIPTION

[0069] Tyrosine kinase inhibitors including, for example, EGFR inhibitors (e.g. trastuzumab, Eriotinib, or Gefitinib, etc.) are often used in the treatment of diseases and/or disorders such as cancer. However, such drugs may be toxic including, for example, cardiotoxic or hepatotoxic. As such, certain targeted therapy options may be associated with undesirable side effects. Thus, biomarkers and methods are desired which can be used to predict and/or determine whether a drug is likely to be toxic to a cell or biological sample (e.g., a biological sample obtained from a patient).

[0070] The inventors of the instant disclosure have unexpectedly discovered that cells in which the SREBP pathway is activated upon contact (e.g., administration or treatment) with a drug are likely to exhibit drug induced toxicity (i.e., the drug is likely to be toxic to the cells) as compared to cells in which the SREBP pathway is not activated upon contact with the drug. Without wishing to be bound by theory, it is believed that cells in which the SREBP pathway is activated produce fatty acids and cholesterol upon treatment with the drug. The present methods may be used to select a drug for administration to a subject (e.g., a cancer patient) that is predicted to not be toxic to the subject. Such methods may comprise contacting a biological sample obtained from the subject with a drug, determining is the SREBP pathway is activated in cells in the biological sample, and selecting a drug for administration to the subject that does not activate the SREBP pathway.

[0071] The present disclosure provides methods for predicting whether a drug (e.g., a tyrosine kinase inhibitor) is likely to be toxic to a cell comprising: contacting the cell with the drug; and determining whether a Sterol Regulatory Binding Protein (SREBP) pathway in the cell contacted with the drug is activated, wherein the drug is predicted to be toxic to the cell where the SREBP pathway is activated in the cell contacted with the drug or wherein the drug is predicted to not be toxic to the cell where the SREBP pathway is not activated in the cell contacted with the drug. In an embodiment, activation of the SREBP pathway may be identified by increased expression of one or more genes in the SREBP pathway including, for example, SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and/or hydroxymethylglutaryl-CoA reductase (HMGCR). The drug may be predicted or determined to be toxic to the cell where activation of the SREBP pathway in the cell is elevated as compared to a control sample or is greater than a threshold. The threshold may be set at a level of activation of the SREBP pathway (e.g., the expression level of genes in the SREBP pathway) in a control sample above which a drug is known to be toxic to a cell and below which a drug is known to not be toxic to a cell. In some embodiments, the threshold is set at a level of activation of the SREBP pathway in a control cell above which the drug is toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cells(s) treated with the drug. In some embodiments, the threshold is set at a level of activation of the SREBP pathway in a control cell below which the drug is toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cell(s) treated with the drug.

[0072] The present disclosure also provides method for treating a cell with a drug (e.g., a tyrosine kinase inhibitor), the method comprising: contacting the cell with the drug where the SREBP pathway is activated in the cell upon treatment with the drug. In an embodiment, activation of the SREBP pathway may be identified by increased expression of one or more genes in the SREBP pathway including, for example, SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and/or hydroxymethylglutaryl-CoA reductase (HMGCR). The drug may be predicted or determined to be toxic to the cell where activation of the SREBP pathway in the cell is elevated as compared to a control sample or is greater than a threshold. The threshold may be set at a level of activation of the SREBP pathway {e.g., the expression level of genes in the SREBP pathway) in a control sample above which a drug is known to be toxic to a cell and below which a drug is known to not be toxic to a cell. In some embodiments, the threshold is set at a level of activation of the SREBP pathway in a control cell above which the drug is toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cells(s) treated with the drug. In some embodiments, the threshold is set at a level of activation of the SREBP pathway in a control cell below which the drug is toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cell(s) treated with the drug.

[0073] The present disclosure also provides methods for selecting a cell for treatment with a drug (e.g., a tyrosine kinase inhibitor), the method comprising: determining whether a Sterol Regulatory Binding Protein (SREBP) pathway in the cell is activated upon treatment of the cell with the drug; and selecting the cell for treatment with the drug where the SREBP pathway is activated in the cell contacted with the drug or not selecting the cell for treatment with the drug where the SREBP pathway is not activated in the cell contacted with the drug. In an embodiment, activation of the SREBP pathway may be identified by increased expression of one or more genes in the SREBP pathway including, for example, SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and/or hydroxymethylglutaryl-CoA reductase (HMGCR). The drug may be predicted or determined to be toxic to the cell where activation of the SREBP pathway in the cell is elevated as compared to a control sample or is greater than a threshold. The threshold may be set at a level of activation of the SREBP pathway (e.g., the expression level of genes in the SREBP pathway) in a control sample above which a drug is known to be toxic to a cell and below which a drug is known to not be toxic to a cell. In some embodiments, the threshold is set at a level of activation of the SREBP pathway in a control cell above which the drug is toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cells(s) treated with the drug. In some embodiments, the threshold is set at a level of activation of the SREBP pathway in a control cell below which the drug is toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cell(s) treated with the drug.

[0074] The present disclosure also provides methods for treating a patient with a drug (e.g., a tyrosine kinase inhibitor), the method comprising: obtaining a biological sample from the patient; contacting one or more cells in the biological sample with a drug; determining whether a Sterol Regulatory Binding Protein (SREBP) pathway in one or more of the contacted cells are activated upon treatment with the drug; and selecting the drug that activates the Sterol Regulatory Binding Protein (SREBP) pathway; and treating the patient with the drug that activates the Sterol Regulatory Binding Protein (SREBP) pathway. In an embodiment, activation of the SREBP pathway may be identified by increased expression of one or more genes in the SREBP pathway including, for example, SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and/or hydroxymethylglutaryl-CoA reductase (HMGCR). The drug may be predicted or determined to be toxic to the cell where activation of the SREBP pathway in the cell is elevated as compared to a control sample or is greater than a threshold. The threshold may be set at a level of activation of the SREBP pathway (e.g., the expression level of genes in the SREBP pathway) in a control sample above which a drug is known to be toxic to a cell and below which a drug is known to not be toxic to a cell. In some embodiments, the threshold is set at a level of activation of the SREBP pathway in a control cell above which the drug is toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cells(s) treated with the drug. In some embodiments, the threshold is set at a level of activation of the SREBP pathway in a control cell below which the drug is toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cell(s) treated with the drug.

[0075] The present disclosure also provides methods for conducting a clinical trial, the method comprising: obtaining biological samples from one or more subjects; contacting one or more cells in the biological samples from the subjects with a drug (e.g., a tyrosine kinase inhibitor); determining whether a Sterol Regulatory Binding Protein (SREBP) pathway in one or more of the contacted cells in the biological samples from the subjects are activated upon treatment with the drug; and enrolling subjects in the clinical trial where the Sterol Regulatory Binding Protein (SREBP) pathway is activated in one or more of cells in the biological sample upon contact of the cells with the drug. In an embodiment, activation of the SREBP pathway may be identified by increased expression of one or more genes in the SREBP pathway including, for example, SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and/or hydroxymethylglutaryl-CoA reductase (HMGCR). The drug may be predicted or determined to be toxic to the cell where activation of the SREBP pathway in the cell is elevated as compared to a control sample or is greater than a threshold. The threshold may be set at a level of activation of the SREBP pathway (e.g., the expression level of genes in the SREBP pathway) in a control sample above which a drug is known to be toxic to a cell and below which a drug is known to not be toxic to a cell. In some embodiments, the threshold is set at a level of activation of the SREBP pathway in a control cell above which the drug is toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cells(s) treated with the drug. In some embodiments, the threshold is set at a level of activation of the SREBP pathway in a control cell below which the drug is toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cell(s) treated with the drug.

[0076] The present disclosure also provides methods for screening one or more drugs (e.g., a tyrosine kinase inhibitor) for likelihood of being cardiotoxic, the method comprising: contacting a cell with the drug; and determining whether a Sterol Regulatory Binding Protein (SREBP) pathway in the cell contacted with the drug is activated. In an embodiment, activation of the SREBP pathway may be identified by increased expression of one or more genes in the SREBP pathway including, for example, SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and/or hydroxymethylglutaryl-CoA reductase (HMGCR). The drug may be predicted or determined to be toxic to the cell where activation of the SREBP pathway in the cell is elevated as compared to a control sample or is greater than a threshold. The threshold may be set at a level of activation of the SREBP pathway (e.g., the expression level of genes in the SREBP pathway) in a control sample above which a drug is known to be toxic to a cell and below which a drug is known to not be toxic to a cell. In some embodiments, the threshold is set at a level of activation of the SREBP pathway in a control cell above which the drug is toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cells(s) treated with the drug. In some embodiments, the threshold is set at a level of activation of the SREBP pathway in a control cell below which the drug is toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cell(s) treated with the drug.

[0077] The present disclosure also provides an in-vitro method for determining drug-induced cell toxicity, the method comprising: identifying whether a Sterol Regulatory Binding Protein (SREBP) pathway in a cell contacted with the drug (e.g., a tyrosine kinase inhibitor) is activated in response to treatment with a drug, wherein the drug is predicted to be toxic to the cell where the SREBP pathway is activated in the cell contacted with the drug or wherein the drug is predicted to not be toxic to the cell where the SREBP pathway is not activated in the cell contacted with the drug. In an embodiment, activation of the SREBP pathway may be identified by increased expression of one or more genes in the SREBP pathway including, for example, SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and/or hydroxymethylglutaryl-CoA reductase (HMGCR). The drug may be predicted or determined to be toxic to the cell where activation of the SREBP pathway in the cell is elevated as compared to a control sample or is greater than a threshold. The threshold may be set at a level of activation of the SREBP pathway (e.g., the expression level of genes in the SREBP pathway) in a control sample above which a drug is known to be toxic to a cell and below which a drug is known to not be toxic to a cell. In some embodiments, the threshold is set at a level of activation of the SREBP pathway in a control cell above which the drug is toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cells(s) treated with the drug. In some embodiments, the threshold is set at a level of activation of the SREBP pathway in a control cell below which the drug is toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cell(s) treated with the drug.

[0078] The present disclosure also provides a test panel for determining whether a drug (e.g., a tyrosine kinase inhibitor) is toxic to a cell, the test panel comprising: a test for determining whether a Sterol Regulatory Binding Protein (SREBP) pathway in a cell is activated upon contact of the cell with the drug, wherein the drug is predicted to be toxic to the cell where the SREBP pathway is activated in the cell contacted with the drug or wherein the drug is predicted to not be toxic to the cell where the SREBP pathway is not activated in the cell contacted with the drug. In an embodiment, activation of the SREBP pathway may be identified by increased expression of one or more genes in the SREBP pathway including, for example, SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and/or hydroxymethylglutaryl-CoA reductase (HMGCR). The drug may be predicted or determined to be toxic to the cell where activation of the SREBP pathway in the cell is elevated as compared to a control sample or is greater than a threshold. The threshold may be set at a level of activation of the SREBP pathway (e.g., the expression level of genes in the SREBP pathway) in a control sample above which a drug is known to be toxic to a cell and below which a drug is known to not be toxic to a cell. In some embodiments, the threshold is set at a level of activation of the SREBP pathway in a control cell above which the drug is toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cells(s) treated with the drug. In some embodiments, the threshold is set at a level of activation of the SREBP pathway in a control cell below which the drug is toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cell(s) treated with the drug.

[0079] The present disclosure provides methods for predicting whether a drug

(e.g., a tyrosine kinase inhibitor) is likely to be toxic to a cell comprising: contacting the cell with the drug; and determining whether levels of cholesterol and/or fatty acids in the cell contacted with the drug increase, wherein the drug is predicted to be toxic to the cell where the levels of cholesterol and/or fatty acids increase in the cell contacted with the drug or wherein the drug is predicted to not be toxic to the cell where the levels of cholesterol and/or fatty acids decrease or remain unchanged in the cell contacted with the drug. [0080] The present disclosure also provides methods for predicting whether a drug {e.g., a tyrosine kinase inhibitor) is likely to be toxic to a test cell, comprising: generating a genetic signature of one or more expressed genes in the test cell (e.g., transcriptosome analysis); comparing the genetic signature of the test cell to a library of genetic signatures for one or more control cells treated with the drug, wherein the drug is determined to be toxic to the test cell where the genetic signature of the test cell is similar to the genetic profile of a control cell in the library that was treated with the drug and where the drug was toxic to the control cell.

[0081] A drug may be considered toxic to a cell or biological sample if the dug induces apoptosis, and/or decreases cell proliferation of the cell and/or biological sample. Toxicity of a drug may also be measured as a reduction in size of the cell or biological sample. In some embodiments, a drug may be considered toxic to a cell and/or biological sample where there is a greater than 50%, 60%, 70%, 80%, 90% or 95% likelihood that the drug will be toxic to the cell and/or biological sample. In some embodiments, a drug may be considered toxic to a cell or biological sample if the dug induces apoptosis, decreases cell proliferation of the cell and/or biological sample as compared to a control cell/control biological sample. Toxicity of a drug may also be measured as a reduction in size of the cell or biological sample as compared to the control cell or control biological sample. In some embodiments, the drug may be considered toxic to a cell and/or biological sample where there is a greater than 50%, 60%, 70%, 80%, 90% or 95% likelihood that the drug will be toxic to the cell and/or biological sample.

[0082] A drug may be predicted to be toxic to a subject including, for example, a human patient, if the dug induces apoptosis, decreases cell proliferation, or induces an immune response against a cell and/or biological sample obtained from the subject or patient. Toxicity of the drug may also be measured as a reduction in size of the cell or biological sample.

[0083] In some embodiments, "treating" or "treatment" of a disease, disorder, or condition includes at least partially: (1 ) preventing the disease, disorder, or condition, i.e. causing the clinical symptoms of the disease, disorder, or condition not to develop in a mammal that is exposed to or predisposed to the disease, disorder, or condition but does not yet experience or display symptoms of the disease, disorder, or condition; (2) inhibiting the disease, disorder, or condition, i.e., arresting or reducing the development of the disease, disorder, or condition or its clinical symptoms; or (3) relieving the disease, disorder, or condition, i.e., causing regression of the disease, disorder, or condition or its clinical symptoms. The treating or treatment of a disease or disorder may include treating or the treatment of cancer. [0084] The term "treatment of cancer" refers to administration to a mammal afflicted with a cancerous condition and refers to an effect that alleviates the cancerous condition by killing the cancerous cells, but also to an effect that results in the inhibition of growth and/or metastasis of the cancer.

[0085] An "effective amount," as used herein, refers to the amount of an active composition that is required to confer a therapeutic effect on the subject. A "therapeutically effective amount," as used herein, refers to a sufficient amount of an agent or a compound being administered which will relieve to some extent one or more of the symptoms of the disease, disorder, or condition being treated. In some embodiments, the result is a reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, in some embodiments, an "effective amount" for therapeutic uses is the amount of the composition including a compound as disclosed herein required to provide a clinically significant decrease in disease symptoms without undue adverse side effects. In some embodiments, an appropriate "effective amount" in any individual case is determined using techniques, such as a dose escalation study. The term "therapeutically effective amount" includes, for example, a prophylactically effective amount. In other embodiments, an "effective amount" of a compound disclosed herein, such as a compound of Formula (A) or Formula (I), is an amount effective to achieve a desired pharmacologic effect or therapeutic improvement without undue adverse side effects. In other embodiments, it is understood that "an effect amount" or "a therapeutically effective amount" varies from subject to subject, due to variation in metabolism, age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician.

[0086] The SREBP pathway may be identified/determined to be activated in a cell/biological sample where one or more genes (e.g., as detected by mRNA expression) or one or more proteins in the SREBP pathway (e.g., SREBP2 (SEQ ID NO: 1 , SEQ ID NO: 2), insulin induced gene 1 (INSIG 1 ; SEQ ID NO: 3, SEQ ID NO: 4), fatty acid synthase (FASN; SEQ ID NO: 5, SEQ ID NO: 6), fatty acid desaturase (FADS2; SEQ ID NO: 7, SEQ ID NO: 8), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2; SEQ ID NO: 9, SEQ ID NO: 10), and hydroxymethylglutaryl-CoA reductase (HMGCR; SEQ ID NO: 1 1 , SEQ ID NO: 12) are expressed at an increased level as compared to the expression of the one or more genes and/or proteins in a control cell/biological sample. In an embodiment, the SREBP pathway may be identified/determined to be activated in a cell/biological sample where the expression level of one or more genes and/or proteins in the SREBP pathway is 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 1 1 x, 12x, 13x, 14x, 15x or more than the expression level of the one or more genes and/or proteins in a control cell/biological sample. Alternatively, the SREBP pathway may be identified/determined to be activated in a cell/biological sample where the expression level of one or more genes and/or proteins in the SREBP pathway is greater than a threshold. In a further embodiment, the threshold may be set at the level of expression of one or more genes and/or proteins in the SREBP pathway in a control cell/biological sample.

[0087] Variants and/or isoforms of the genes and/or proteins in the SREBP pathway (e.g., SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and hydroxymethylglutaryl-CoA reductase (HMGCR)) may include a polynucleotide or protein possessing a nucleotide or amino acid sequence that possess at least 90% sequence identity, more preferably at least 91 % sequence identity, even more preferably at least 92% sequence identity, still more preferably at least 93% sequence identity, still more preferably at least 94% sequence identity, even more preferably at least 95% sequence identity, still more preferably at least 96% sequence identity, even more preferably at least 97% sequence identity, still more preferably at least 98% sequence identity, and most preferably at least 99% sequence identity, to the wild type form of the gene or protein.

[0088] Sequence identity or percent identity is intended to mean the percentage of the same nucleotides or residues shared between two sequences, when the two sequences are aligned using the Clustal method [Higgins et al, Cabios 8:189-191 (1992)] of multiple sequence alignment in the Lasergene biocomputing software (DNASTAR, INC, Madison, Wis.). In this method, multiple alignments are carried out in a progressive manner, in which larger and larger alignment groups are assembled using similarity scores calculated from a series of pairwise alignments. Optimal sequence alignments are obtained by finding the maximum alignment score, which is the average of all scores between the separate residues in the alignment, determined from a residue weight table representing the probability of a given amino acid change occurring in two related proteins over a given evolutionary interval. Penalties for opening and lengthening gaps in the alignment contribute to the score. The default parameters used with this program are as follows: gap penalty for multiple alignments 0; gap length penalty for multiple alignments 0; k-tuple value in pairwise alignments ; gap penalty in pairwise alignment=3; window value in pairwise alignment=5; diagonals saved in pairwise alignment=5. The residue weight table used for the alignment program is PAM250 [Dayhoff, et al., in Atlas of Protein Sequence and Structure, Dayhoff, Ed., NDRF, Washington, Vol. 5, suppl. 3, p. 345, (1978)].

[0089] In one embodiment the cancer may be selected from the group consisting of: oral cancer, prostate cancer, rectal cancer, non-small cell lung cancer, lip and oral cavity cancer, liver cancer, lung cancer, anal cancer, kidney cancer, vulvar cancer, breast cancer, oropharyngeal cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, urethra cancer, small intestine cancer, bile duct cancer, bladder cancer, ovarian cancer, laryngeal cancer, hypopharyngeal cancer, gallbladder cancer, colon cancer, colorectal cancer, head and neck cancer, glioma; parathyroid cancer, penile cancer, vaginal cancer, thyroid cancer, pancreatic cancer, esophageal cancer, Hodgkin's lymphoma, leukemia-related disorders, mycosis fungoides, and myelodysplastic syndrome.

[0090] In another embodiment the cancer may be non-small cell lung cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, or head and neck cancer. In yet another embodiment the cancer may be a carcinoma, a tumor, a neoplasm, a lymphoma, a melanoma, a glioma, a sarcoma, or a blastoma.

[0091] In one embodiment the carcinoma may be selected from the group consisting of: carcinoma, adenocarcinoma, adenoid cystic carcinoma, adenosquamous carcinoma, adrenocortical carcinoma, well differentiated carcinoma, squamous cell carcinoma, serous carcinoma, small cell carcinoma, invasive squamous cell carcinoma, large cell carcinoma, islet cell carcinoma, oat cell carcinoma, squamous carcinoma, undifferentiatied carcinoma, verrucous carcinoma, renal cell carcinoma, papillary serous adenocarcinoma, merkel cell carcinoma, hepatocellular carcinoma, soft tissue carcinomas, bronchial gland carcinomas, capillary carcinoma, bartholin gland carcinoma, basal cell carcinoma, carcinosarcoma, papilloma/carcinoma, clear cell carcinoma, endometrioid adenocarcinoma, mesothelial, metastatic carcinoma, mucoepidermoid carcinoma, cholangiocarcinoma, actinic keratoses, cystadenoma, and hepatic adenomatosis.

[0092] In another embodiment the tumor may be selected from the group consisting of: astrocytic tumors, malignant mesothelial tumors, ovarian germ cell tumors, supratentorial primitive neuroectodermal tumors, Wilms tumors, pituitary tumors, extragonadal germ cell tumors, gastrinoma, germ cell tumors, gestational trophoblastic tumors, brain tumors, pineal and supratentorial primitive neuroectodermal tumors, pituitary tumors, somatostatin-secreting tumors, endodermal sinus tumors, carcinoids, central cerebral astrocytoma, glucagonoma, hepatic adenoma, insulinoma, medulloepithelioma, plasmacytoma, vipoma, and pheochromocytoma.

[0093] In yet another embodiment the neoplasm may be selected from the group consisting of: intraepithelial neoplasia, multiple myeloma/plasma cell neoplasm, plasma cell neoplasm, interepithelial squamous cell neoplasia, endometrial hyperplasia, focal nodular hyperplasia, hemangioendothelioma, and malignant thymoma. In a further embodiment the lymphoma may be selected from the group consisting of: nervous system lymphoma, AIDS-related lymphoma, cutaneous T-cell lymphoma, non-Hodgkin's lymphoma, lymphoma, and Waldenstrom's macroglobulinemia. In another embodiment the melanoma may be selected from the group consisting of: acral lentiginous melanoma, superficial spreading melanoma, uveal melanoma, lentigo maligna melanomas, melanoma, intraocular melanoma, adenocarcinoma nodular melanoma, and hemangioma. In yet another embodiment the sarcoma may be selected from the group consisting of: adenomas, adenosarcoma, chondosarcoma, endometrial stromal sarcoma, Ewing's sarcoma, Kaposi's sarcoma, leiomyosarcoma, rhabdomyosarcoma, sarcoma, uterine sarcoma, osteosarcoma, and pseudosarcoma. In one embodiment the glioma may be selected from the group consisting of: glioma, brain stem glioma, and hypothalamic and visual pathway glioma. In another embodiment the blastoma may be selected from the group consisting of: pulmonary blastoma, pleuropulmonary blastoma, retinoblastoma, neuroblastoma, medulloblastoma, glioblastoma, and hemangiblastomas.

[0094] Biological samples or test cells that may be used in the methods of the present disclosure may include tissues, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject {e.g., a patient). Preferably, biological samples comprise cells, most preferably tumor cells, that are isolated from body samples, such as, but not limited to, smears, sputum, biopsies, secretions, cerebrospinal fluid, bile, blood, serum, lymph fluid, urine and faeces, or tissue which has been removed from organs, such as breast, lung, intestine, skin, cervix, prostate, and stomach. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes.

Determination of Sterol Regulatory Binding Protein (SREBP) Pathway Activation

[0095] A number of methodologies may be employed to determine or assess (i.e., detect) whether the sterol regulatory binding protein (SREBP) pathway has been activated in a cell or biological sample upon treatment of the cell or biological sample with a drug. Such activation of the SREBP pathway may be detected at the protein level and/or nucleic acid level. Those skilled in the art will appreciate that the methods indicated below represent some of the preferred ways in which activation of the SREBP pathway may be determined and/or quantitated and in no manner limit the scope of methodologies that may be employed. Those skilled in the art will also be able to determine operative and optimal assay conditions for each determination by employing routine experimentation. Such methods may include but are not limited to in situ hybridization (ISH), Western blots, ELISA, immunoprecipitation, immunofluorescence, flow cytometry, northern blots, PCR, immunocytochemistry (I HC) and microarray analysis (e.g., transcriptosome analysis). In an embodiment, the expression level of one or more genes in the SREBP pathway (e.g., SREBP2 (SEQ ID NO: 1 ), insulin induced gene 1 (INSIG 1 ; SEQ ID NO: 2), fatty acid synthase (FASN; SEQ ID NO: 3), fatty acid desaturase (FADS2; SEQ ID NO: 4), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2; SEQ ID NO: 5), and hydroxymethylglutaryl-CoA reductase (HMGCR; SEQ ID NO: 6) may be determined in order to assess whether the SREBP pathway is activated.

[0096] In another embodiment, the methods may further involve obtaining a control sample and detecting activation of the SREBP pathway in this control sample, such that the activation of the SREBP pathway in the control sample is determined. A negative control sample is useful if there is an absence of activation of the SREBP pathway, whereas a positive control sample is useful if there is a presence of activation of the SREBP pathway. For the negative control, the sample may be from the same individual as the test sample (i.e. different location such as tumor versus non-tumor) or may be from a different individual known to have an absence of activation of the SREBP pathway.

[0097] Nucleic acid-based techniques for assessing expression of polynucleotides are well known in the art and include, for example, determining the expression level of one or more genes in the SREBP pathway including, but not limited to, SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and hydroxymethylglutaryl-CoA reductase (HMGCR).

[0098] Many expression detection methods use isolated RNA encoding one or more proteins in the SREBP pathway. Any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from cells (see, e.g., Ausubel et al., ed., (1987-1999) Current Protocols in Molecular Biology (John Wiley & Sons, New York). Additionally, large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single- step RNA isolation process of Chomczynski (1989, U.S. Pat. No. 4,843, 155).

[0099] In one embodiment, the mRNA from a biological sample is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip array.

[00100] An alternative method for determining the level of a mRNA (e.g., a mRNA expressed from a gene in the SREBP pathway) in a cell involves the process of nucleic acid amplification, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991 ) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1 173-1 177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6:1 197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects of the disclosure, biomarker expression may be assessed by quantitative fluorogenic RT-PCR (i.e., the TaqMan® System). Such methods typically may utilize pairs of oligonucleotide primers that are specific for the mRNA. Methods for designing oligonucleotide primers specific for a known sequence are well known in the art.

[00101] Expression levels of RNA (e.g., a RNA expressed from a gene in the SREBP pathway) may be monitored using a membrane blot (such as used in hybridization analysis such as Northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids) (see, e.g., U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677, 195 and 5,445,934). The detection of expression of a gene in the SREBP pathway may also comprise using nucleic acid probes in solution.

[00102] In one embodiment of the disclosure, microarrays are used to detect expression of one or more genes in the SREBP pathway. Microarrays are particularly well suited for this purpose because of the reproducibility between different experiments. DNA microarrays provide one method for the simultaneous measurement of the expression levels of large numbers of genes. Each array consists of a reproducible pattern of capture probes attached to a solid support. Labeled RNA or DNA may be hybridized to complementary probes on the array and then detected by laser scanning. Hybridization intensities for each probe on the array are determined and converted to a quantitative value representing relative gene expression levels (see, e.g., U.S. Pat. Nos. 6,040, 138, 5,800,992, 6,020,135, 6,033,860, and 6,344,316). High-density oligonucleotide arrays are particularly useful for determining the gene expression profile for a large number of RNAs in a sample.

[00103] Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Pat. No. 5,384,261 . Although a planar array surface is preferred, the array may be fabricated on a surface of virtually any shape or even a multiplicity of surfaces. Arrays may be peptides or nucleic acids on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate, see U.S. Pat. Nos. 5,770,358, 5,789, 162, 5,708, 153, 6,040,193 and 5,800,992. Arrays may be packaged in such a manner as to allow for diagnostics or other manipulation of an all-inclusive device (see, e.g., U.S. Pat. Nos. 5,856,174 and 5,922,591 ).

[00104] In one approach, total mRNA isolated from the biological sample may be converted to labeled cRNA and then hybridized to an oligonucleotide array. Each sample may be hybridized to a separate array. Relative transcript levels may be calculated by reference to appropriate controls present on the array and in the sample.

[00105] In a particular embodiment, the level of mRNA expressed by a gene in the SREBP pathway can be determined both by in situ and by in vitro formats in a biological sample using methods known in the art. Many expression detection methods use isolated RNA. For in vitro methods, any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from tumor cells (see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York 1987-1999). Additionally, large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski (see, e.g., U.S. Pat. No. 4,843, 155).

[00106] The isolated mRNA expressed by a gene in the SREBP pathway can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymeRASe chain reaction analyses and probe arrays. One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the disclosure are described herein. Hybridization of an mRNA with the probe indicates that the gene is being expressed.

[00107] In one format, the mRNA may be immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probe(s) are immobilized on a solid surface and the mRNA may be contacted with the probe(s), for example, in an Affymetrix gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of mRNA encoded by a gene in the SREBP pathway.

[00108] An alternative method for determining the level of mRNA (expressed by a gene in the SREBP pathway) in a cell/biological sample involves the process of nucleic acid amplification, e.g., by RT-PCR (see, e.g., U.S. Pat. No. 4,683,202), ligase chain reaction (Barany, 1991 , Proc. Natl. Acad. Sci. USA, 88:189-193), self sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86:1 173-1 177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology 6:1 197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

[00109] For in situ methods, mRNA does not need to be isolated from the cells prior to detection. In such methods, a cell or tissue sample may be prepared/processed using known histological methods. The sample may be then immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA expressed by a gene in the SREBP pathway.

[00110] In another embodiment of the present disclosure, a protein expressed by a gene in the SREBP pathway may be detected. A preferred agent for detecting protein expressed by a gene in the SREBP pathway is an antibody capable of binding to such a protein or a fragment thereof, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment or derivative thereof can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that may be directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.

[00111] Antibody fragments may comprise a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al. (1995) Protein Eng. 8(10):1057-1062); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize 35 readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and may be still capable of cross-linking antigen.

[00112] Detection of antibody binding can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesteRASe; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include lucifeRASe, luciferin, and aequorin; and examples of suitable radioactive material include ¹²⁵l, ¹³¹1, ³⁵S, or ³H.

[00113] In regard to detection of antibody staining in the immunocytochemistry methods of the disclosure, there also exist in the art, video-microscopy and software methods for the quantitative determination of an amount of multiple molecular species (e.g., biomarker proteins) in a biological sample wherein each molecular species present may be indicated by a representative dye marker having a specific color. Such methods are also known in the art as a colorimetric analysis methods. In these methods, video- microscopy may be used to provide an image of the biological sample after it has been stained to visually indicate the presence of a particular biomarker of interest. Some of these methods, such as those disclosed in U.S. Patent Application Ser. Nos. 09/957,446 and 10/057,729, disclose the use of an imaging system and associated software to determine the relative amounts of each molecular species present based on the presence of representative color dye markers as indicated by those color dye markers' optical density or transmittance value, respectively, as determined by an imaging system and associated software. These techniques provide quantitative determinations of the relative amounts of each molecular species in a stained biological sample using a single video image that may be deconstructed into its component color parts.

[00114] The antibodies used to practice the disclosure are selected to have high specificity for a protein in the SREBP pathway. Methods for making antibodies and for selecting appropriate antibodies are known in the art (see, e.g., Celis, ed. (in press) Cell Biology & Laboratory Handbook, 3rd edition (Academic Press, New York)). In some embodiments, commercial antibodies directed to specific RAS proteins may be used to practice the disclosure. The antibodies of the disclosure may be selected on the basis of desirable staining of cytological, rather than histological, samples. That is, in particular embodiments the antibodies are selected with the end sample type (i.e., cytology preparations) in mind and for binding specificity.

[00115] One of skill in the art will recognize that optimization of antibody titer and detection chemistry may be needed to maximize the signal to noise ratio for a particular antibody. Antibody concentrations that maximize specific binding to a protein in the SREBP pathway and minimize non-specific binding (or background) can be determined. In particular embodiments, appropriate antibody titers for use in cytology preparations are determined by initially testing various antibody dilutions on formalin-fixed paraffin- embedded normal and high-grade cervical disease tissue samples. Optimal antibody concentrations and detection chemistry conditions are first determined for formalin-fixed paraffin-embedded tissue samples. The design of assays to optimize antibody titer and detection conditions is standard and well within the routine capabilities of those of ordinary skill in the art. After the optimal conditions for fixed tissue samples are determined, each antibody may be then used in cytology preparations under the same conditions. Some antibodies require additional optimization to reduce background staining and/or to increase specificity and sensitivity of staining in the cytology samples.

[00116] Furthermore, one of skill in the art will recognize that the concentration of a particular antibody used to practice the methods of the disclosure will vary depending on such factors as time for binding, level of specificity of the antibody for the protein in the SREBP pathway, and method of body sample preparation. Moreover, when multiple antibodies are used, the required concentration may be affected by the order in which the antibodies are applied to the sample, i.e., simultaneously as a cocktail or sequentially as individual antibody reagents. Furthermore, the detection chemistry used to visualize antibody binding to a biomarker of interest must also be optimized to produce the desired signal to noise ratio.

[00117] Proteins from tumor cells can be isolated using techniques that are well known to those of skill in the art. The protein isolation methods employed can, for example, be such as those described in Harlow and Lane (Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

[00118] A variety of formats can be employed to determine whether a sample contains a protein that binds to a given antibody. Examples of such formats include, but are not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis and enzyme linked immunoabsorbant assay (ELISA). A skilled artisan can readily adapt known protein/antibody detection methods for use in determining whether tumor cells express a biomarker of the present disclosure.

[00119] One skilled in the art will know many other suitable carriers for binding antibody or antigen, and will be able to adapt such support for use with the present disclosure. For example, protein isolated from tumor cells can be run on a polyacrylamide gel electrophoresis and immobilized onto a solid phase support such as nitrocellulose. The support can then be washed with suitable buffers followed by treatment with the detectably labeled antibody. The solid phase support can then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on the solid support can then be detected by conventional means.

[00120] For ELISA assays, specific binding pairs can be of the immune or nonimmune type. Immune specific binding pairs are exemplified by antigen-antibody systems or hapten/anti-hapten systems. There can be mentioned fluorescein/anti-fluorescein, dinitrophenyl/anti-dinitrophenyl, biotin/anti-biotin, peptide/anti-peptide and the like. The antibody member of the specific binding pair can be produced by customary methods familiar to those skilled in the art. Such methods involve immunizing an animal with the antigen member of the specific binding pair. If the antigen member of the specific binding pair is not immunogenic, e.g., a hapten, it can be covalently coupled to a carrier protein to render it immunogenic. Non-immune binding pairs include systems wherein the two components share a natural affinity for each other but are not antibodies.

[00121] The present disclosure also includes methods for fixing cells and tissue samples for analysis. Generally, neutral buffered formalin may be used. Any concentration of neutral buffered formalin that can fix tissue or cell samples without disrupting the epitope can be used. In one embodiment a solution of about 10 percent may be used. Preferably, the method includes suitable amounts of phosphatase inhibitors to inhibit the action of phosphatases and preserve phosphorylation. Any suitable concentration of phosphatase inhibitor can be used so long as the biopsy sample is stable and phosphatases are inhibited, for example 1 mM NaF and/or Na3V04 can be used. In one method a tissue sample or tumor biopsy may be removed from a patient and immediately immersed in a fixative solution which can and preferably does contain one or more phosphatase inhibitors, such as NaF and/or Na3V04. Preferably, when sodium orthovanadate is used it is used in an activated or depolymerized form to optimize its activity.

[00122] Depolymerization can be accomplished by raising the pH of its solution to about 10 and boiling for about 10 minutes. The phosphatase inhibitors can be dissolved in the fixative just prior to use in order to preserve their activity. [00123] Fixed samples can then be stored for several days or processed immediately. To process the samples into paraffin after fixing, the fixative can be thoroughly rinsed away from the cells by flushing the tissue with water. The sample can be processed to paraffin according to normal histology protocols which can include the use of reagent grade ethanol. Samples can be stored in 70% ethanol until processed into paraffin blocks. Once samples are processed into paraffin blocks they can be analyzed histochemically for virtually any antigen that is stable to the fixing process.

[00124] In preferred embodiments, protein (e.g., expressed by a gene in the SREBP pathway) staining may be detected, measured and quantitated automatically using automated image analysis equipment. Such equipment can include a light or fluorescence microscope, and image-transmitting camera and a view screen, most preferably also comprising a computer that can be used to direct the operation of the device and store and manipulate the information collected, most preferably in the form of optical density of certain regions of a stained tissue preparation. Image analysis devices useful in the practice of this disclosure include but are not limited to the CAS 200 (Becton Dickenson, Mountain View, Calif), Chromavision or Tripath systems. Using such equipment the quantity of the target epitope in unknown cell samples can be determined using any of a variety of methods that are known in the art. The cell pellets can be analyzed by eye such that the optical density reading of the control cells can be correlated to a manual score such as 0, 1 +, 2+ or 3+, as in Table 1 below which shows the correlation between quantitative image analysis data measured in optical density (OD) and manual score.

[00125] Automated (computer-aided) image analysis systems known in the art can augment visual examination of biological samples. In a representative system, the cell or tissue sample may be exposed to detectably labeled reagents specific for a protein in the SREBP pathway, and the magnified image of the cell may be then processed by a computer that receives the image from a charge-coupled device (CCD) or camera such as a television camera. Such a system can be used, for example, to detect and measure expression and activation levels of Her1 , pHER1 HER2, HER3, and pERK in a sample. Additional biomarkers are also contemplated by this disclosure. This methodology provides more accurate diagnosis of cancer and a better characterization of gene expression in histologically identified cancer cells, most particularly with regard to expression of tumor marker genes or genes known to be expressed in particular cancer types and subtypes (i.e., different degrees of malignancy). This information permits a more informed and effective regimen of therapy to be administered, because drugs with clinical efficacy for certain tumor types or subtypes can be administered to patients whose cells are so identified. [00126] In practicing the method of the present disclosure, detection procedures can be carried out by a technician in the laboratory. Alternatively, the detection procedures can be carried out using automated systems. In either case, staining procedures for use according to the methods of this disclosure are performed according to standard techniques and protocols well-established in the art.

[00127] In one embodiment of the present invention, amplification-based assays can be used to measure copy number of the mRNA expressed from a gene in the SREBP pathway. In such amplification-based assays, the corresponding nucleic acid sequence from the gene in the SREBP pathway acts as a template in an amplification reaction (for example, PolymeRASe Chain Reaction or PCR). In a quantitative amplification, the amount of amplification product will be proportional to the amount of template in the original sample. Comparison to appropriate controls provides a measure of the copy-number of the mRNA expressed from the gene in the SREBP pathway, corresponding to the specific probe used. The presence of a higher level of amplification product, as compared to a control, is indicative of increased levels of the mRNA expressed from a gene in the SREBP pathway.

[00128] Methods of "quantitative" amplification are well known to those of skill in the art. For example, quantitative PCR involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction. Detailed protocols for quantitative PCR are provided, for example, in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.

[00129] Real time PCR is another amplification technique that can be used to determine gene copy levels or levels of mRNA expression of any gene in the SREBP pathway. (See, e.g., Gibson et al., Genome Research 6:995-1001 , 1996; Heid et al., Genome Research 6:986-994, 1996). Real-time PCR evaluates the level of PCR product accumulation during amplification. This technique permits quantitative evaluation of mRNA levels in multiple samples. For gene copy levels, total genomic DNA is isolated from a sample. For mRNA levels, mRNA is extracted from tumor and normal tissue and cDNA is prepared using standard techniques. Real-time PCR can be performed, for example, using a Perkin Elmer/Applied Biosystems (Foster City, Calif.) 7700 Prism instrument. Matching primers and fluorescent probes can be designed for genes of interest using, for example, the primer express program provided by Perkin Elmer/Applied Biosystems (Foster City, Calif.). Optimal concentrations of primers and probes can be initially determined by those of ordinary skill in the art, and control (for example, beta-actin) primers and probes may be obtained commercially from, for example, Perkin Elmer/Applied Biosystems (Foster City, Calif.)- To quantitate the amount of the specific nucleic acid of interest in a sample, a standard curve is generated using a control. Standard curves may be generated using the Ct values determined in the real-time PCR, which are related to the initial concentration of the nucleic acid of interest used in the assay. Standard dilutions ranging from 10-10⁶ copies of the gene of interest are generally sufficient. In addition, a standard curve is generated for the control sequence. This permits standardization of initial content of the nucleic acid of interest in a tissue sample to the amount of control for comparison purposes.

[00130] Methods of real-time quantitative PCR using TaqMan probes are well known in the art. Detailed protocols for real-time quantitative PCR are provided, for example, for RNA in: Gibson et al., 1996, A novel method for real time quantitative RT- PCR. Genome Res., 10:995-1001 ; and for DNA in: Heid et al., 1996, Real time quantitative PCR. Genome Res., 10:986-994.

[00131] A TaqMan-based assay also can be used to quantify MET polynucleotides. TaqMan based assays use a fluorogenic oligonucleotide probe that contains a 5' fluorescent dye and a 3' quenching agent. The probe hybridizes to a PCR product, but cannot itself be extended due to a blocking agent at the 3' end. When the PCR product is amplified in subsequent cycles, the 5' nuclease activity of the polymeRASe, for example, AmpliTaq, results in the cleavage of the TaqMan probe. This cleavage separates the 5' fluorescent dye and the 3' quenching agent, thereby resulting in an increase in fluorescence as a function of amplification.

[00132] Differential expression of mRNA expressed from a gene in the SREBP pathway may also be measured using a nucleic acid microarray. In this method, single- stranded nucleic acids (e.g., cDNAs, oligonucleotides, etc.) are plated, or arrayed, on a solid support. The solid support may be a material such as glass, silica-based, silicon- based, a synthetic polymer, a biological polymer, a copolymer, a metal, or a membrane. The form or shape of the solid support may vary, depending on the application. Suitable examples include, but are not limited to, slides, strips, plates, wells, microparticles, fibers (such as optical fibers), gels, and combinations thereof. The arrayed immobilized sequences are generally hybridized with specific DNA probes from the cells of interest. Fluorescently labeled cDNA probes may be generated through incorporation of fluorescently labeled deoxynucleotides by reverse transcription of RNA extracted from the cells of interest. The probes are hybridized to the immobilized nucleic acids on the microchip under highly stringent conditions. After stringent washing to remove non- specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera. Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance. With dual color fluorescence, separately labeled cDNA probes generated from two sources of RNA are hybridized pairwise to the array. The relative abundance of the transcripts from the two sources corresponding to each specified molecular marker is thus determined simultaneously. Microarray analysis may be performed by commercially available equipment, following manufacturer's protocols, such as by using the Affymetrix GenChip technology, or Incyte's microarray technology.

[00133] Differential expression of a mRNA expressed from a gene in the SREBP pathway may also be measured using Northern blotting. For this, RNA samples are first separated by size via electrophoresis in an agarose gel under denaturing conditions. The RNA is then transferred to a membrane, crosslinked, and hybridized, under highly stringent conditions, to a labeled DNA probe. After washing to remove the non-specifically bound probe, the hybridized labeled species are detected using techniques well known in the art. The probe may be labeled with a radioactive element, a chemical that fluoresce when exposed to ultraviolet light, a tag that is detected with an antibody, or an enzyme that catalyses the formation of a colored or a fluorescent product. A comparison of the relative amounts of RNA detected in the different cells will reveal whether the expression of the molecular marker is changed in the cancer cell.

[00134] Nuclease protection assays may also be used to monitor the differential expression of a mRNA expressed from a gene in the SREBP pathway in cancer and control cells. In nuclease protection assays, an antisense probe hybridizes in solution to an RNA sample. The antisense probe may be labeled with an isotope, a fluorophore, an enzyme, or another tag. Following hybridization, nucleases are added to degrade the single-stranded, unhybridized probe and RNA. An acrylamide gel is used to separate the remaining protected double-stranded fragments, which are then detected using techniques well known in the art. Again, qualitative differences in expression may be detected.

[00135] Differential expression of a mRNA expressed from a gene in the SREBP pathway may also be measured using in situ hybridization. This type of hybridization uses a labeled antisense probe to localize a particular mRNA in cells of a tissue section. The hybridization and washing steps are generally performed under highly stringent conditions. The probe may be labeled with a fluorophore or a small tag (such as biotin or digoxigenin) that may be detected by another protein or antibody, such that the labeled hybrid may be visualized under a microscope. The transcripts of a molecular marker may be localized to the nucleus, the cytoplasm, or the plasma membrane of a cell.

[00136] Expression of the mRNA expressed from a gene in the SREBP pathway will generally be measured in a cancer cell relative to a control cell. The cell may be isolated from a subject so that expression of the marker may be examined in vitro. The type of biopsy used to isolated cells can and will vary, depending upon the location and nature of the cancer.

[00137] A sample of cells, tissue, or fluid may be removed by needle aspiration biopsy. For this, a fine needle attached to a syringe is inserted through the skin and into the organ or tissue of interest. The needle is typically guided to the region of interest using ultRASound or computed tomography (CT) imaging. Once the needle is inserted into the tissue, a vacuum is created with the syringe such that cells or fluid may be sucked through the needle and collected in the syringe. A sample of cells or tissue may also be removed by incisional or core biopsy. For this, a cone, a cylinder, or a tiny bit of tissue is removed from the region of interest. This type of biopsy is generally guided by CT imaging, ultRASound, or an endoscope. Lastly, the entire cancerous tumor may be removed by excisional biopsy or surgical resection.

[00138] RNA, protein, or DNA may be extracted from the biopsied cells or tissue to permit analysis of the expression of a molecular marker using methods described above in section (l)(d). The biopsied cells or tissue may also be embedded in plastic or paraffin, from which nucleic acids may be isolated. The expression of a molecular marker may also be performed in the biopsied cells or tissue in situ {e.g., in situ hybridization, immunohistochemistry).

[00139] Expression of a molecular marker may also be examined in vivo in a subject. A particular mRNA or protein may be labeled with fluorescent dye, a bioluminescent marker, a fluorescent semiconductor nanocrystal, or a short-lived radioisotope, and then the subject may be imaged or scanned using a variety of techniques, depending upon the type of label.

Methods for Predicting Drug Toxicity

[00140] The present disclosure provides methods for predicting whether a drug (e.g., a tyrosine kinase inhibitor) is likely to be toxic to a cell comprising: contacting the cell with the drug; and determining whether a Sterol Regulatory Binding Protein (SREBP) pathway in the cell contacted with the drug is activated, wherein the drug is predicted to be toxic to the cell where the SREBP pathway is activated in the cell contacted with the drug or wherein the drug is predicted to not be toxic to the cell where the SREBP pathway is not activated in the cell contacted with the drug. In an embodiment, activation of the SREBP pathway may be identified by increased expression of one or more genes in the SREBP pathway including, for example, SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and/or hydroxymethylglutaryl-CoA reductase (HMGCR). The drug may be predicted or determined to be toxic to the cell where activation of the SREBP pathway in the cell is elevated as compared to a control sample or is greater than a threshold. The threshold may be set at a level of activation of the SREBP pathway {e.g., the expression level of genes in the SREBP pathway) in a control sample above which a drug is known to be toxic to a cell and below which a drug is known to not be toxic to a cell. In some embodiments, the threshold is set at a level of activation of the SREBP pathway in a control cell above which the drug is toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cells(s) treated with the drug. In some embodiments, the threshold is set at a level of activation of the SREBP pathway in a control cell below which the drug is toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cell(s) treated with the drug. In an embodiment, one or more cells may be isolated from a population of cells and treated with the drug. SREBP activation may be measured in the isolated and treated cells and used to preduict how the other cells (i.e., cells that were not treated with the drug) in the population will respond to treatment with the drug.

[00141] In some embodiments, a drug is toxic to a cell or biological sample where the mean GI50 of the drug is 0.10 μΜ or less (e.g., 0.10 μΜ, 0.50 μΜ, or 0.10 μΜ).

[00142] In some embodiments, a drug (e.g., a tyrosine kinase inhibitor) is likely to be toxic to a cell where the SREBP pathway is activated (e.g., expression levels of one or more genes in the SREBP pathway are increased) as compared to a control cell or is above a threshold. In a further embodiment, the threshold may be set at an expression level of one or more genes in the SREBP pathway above which a drug is known to be toxic to a cell and below which a drug is known to not be toxic to a cell. In yet a further embodiment, the threshold is set at an expression level of one or more genes in the SREBP pathway above which the drug is toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cells (i.e., control cells) treated with the drug. In an alternative embodiment, the threshold is set at an expression level of one or more genes in the SREBP pathway below which the drug is not toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cells (i.e., control cells) treated with the drug.

Methods for Screening and/or Identifying a Drug

[00143] The present disclosure provides methods for screening and identifying a drug that is likely to not be toxic to a cell or biological sample including, for example, a tyrosine kinase inhibitor.

[00144] The present disclosure provides methods for screening one or more drugs to determine or predict whether the drug will be toxic to a cell or biological sample. Such drugs that are determined or predicted to not be toxic to a cell may be identified and selected for use in contacting or treating the cell while drugs assessed or predicted to be toxic to the cell may be identified not to be used in contacting or treating the cell. The methods may comprise contacting the cell with the drug; and determining whether a Sterol Regulatory Binding Protein (SREBP) pathway in the cell contacted with the drug is activated, wherein the drug is predicted to be toxic to the cell where the SREBP pathway is activated in the cell contacted with the drug or wherein the drug is predicted to not be toxic to the cell where the SREBP pathway is not activated in the cell contacted with the drug; and optionally selecting the drug determined to not be toxic to the cell for contacting or treating the cell. The drug may be predicted or determined to be toxic to the cell where activation of the SREBP pathway in the cell is elevated as compared to a control sample or is greater than or less than a threshold. The threshold may be set at a level of activation of the SREBP pathway (e.g., the expression level of genes in the SREBP pathway) in a control sample above which a drug is known to be toxic to a cell and below which a drug is known to not be toxic to a cell. In some embodiments, the threshold is set at a level of activation of the SREBP pathway in a control cell above which the drug is toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cells(s) treated with the drug. In some embodiments, the threshold is set at a level of activation of the SREBP pathway in a control cell below which the drug is toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cell(s) treated with the drug. In some embodiments, the threshold may be set at a level of SREBP activation in a control cell above which a drug is known to be toxic and below which a drug is known to not be toxic. In some embodiments, the threshold is set at a level SREBP activation in a control cell above which the drug is toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cells. In some embodiments, the threshold is set at a level of SREBP activation in a control cell below which the drug is not toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cells. For example, a threshold may be set at the minimal level of SREBP activation in a control cell where the drug is toxic to the cell. Such a threshold may be an average or median obtained from two or more control cells treated with the drug. In some embodiments, the test cell and control cell are from the same specimen. In some embodiments, the test cell and control cell are from the different specimens.

[00145] In an embodiment, two or more drugs may be screened against a single cell type on a substrate (e.g., screened on an array). In alternative embodiments, two or more cells may be screened against a single drug on a substrate (e.g., screened on an array). In other embodiments, two or more drugs may be screened against two or more cell types on a substrate (e.g., screened on an array). Methods for Predicting and/or Determining if a Drug is Toxic to a Subject

[00146] The present disclosure provides methods for predicting and/or determining the likelihood that a drug is toxic to a subject. Such methods may be used to select drugs predicted or determined to not be toxic to the subject for treatment of the subject.

[00147] The methods may comprise contacting the cell or biological sample obtained form a subject with the drug; and determining whether a Sterol Regulatory Binding Protein (SREBP) pathway in the cell or biological sample contacted with the drug is activated, wherein the drug is predicted to be toxic to the subject where the SREBP pathway is activated in the cell or biological sample contacted with the drug or wherein the drug is predicted to not be toxic to the cell or biological sample where the SREBP pathway is not activated in the cell or biological sample contacted with the drug. The drug may be predicted or determined to be toxic to the cell or biological sample where activation of the SREBP pathway in the cell or biological sample is elevated as compared to a control sample or is greater than or less than a threshold. The threshold may be set at a level of activation of the SREBP pathway (e.g., the expression level of genes in the SREBP pathway) in a control sample above which a drug is known to be toxic to a cell or biological sample and below which a drug is known to not be toxic to a cell or biological sample. In some embodiments, the threshold is set at a level of activation of the SREBP pathway in a control cell or biological sample above which the drug is toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cells(s) or biological samples treated with the drug. In some embodiments, the threshold is set at a level of activation of the SREBP pathway in a control cell or biological sample below which the drug is toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cell(s) or biological samples treated with the drug. In some embodiments, the threshold may be set at a level of SREBP activation in a control cell or biological sample above which a drug is known to be toxic and below which a drug is known to not be toxic. In some embodiments, the threshold is set at a level SREBP activation in a control cell or biological sample above which the drug is toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cells or biological samples. In some embodiments, the threshold is set at a level of SREBP activation in a control cell or biological sample below which the drug is not toxic to 50%, 60%, 70%, 80%, 90%, or 95% of cells or biological samples. For example, a threshold may be set at the minimal level of SREBP activation in a control cell or biological sample where the drug is toxic to the cell or biological sample. Such a threshold may be an average or median obtained from two or more control cells or biological samples treated with the drug. Optionally, a drug predicted to not be toxic to a subject may be administered to the subject. Assays and Kits

[00148] The present disclosure provides assays and kits for assessing {e.g., determining) or predicting whether a drug will be toxic including, for example, the likelihood that a drug will be toxic to a cell or biological sample. Such assays and kits may determine if the SREBP pathway is activated in a cell or biological sample.

[00149] In an embodiment, the assays or kits determine if the SREBP pathway is activated in a cell by determining if expression of one or more genes in the SREBP pathway including, for example, SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and/or hydroxymethylglutaryl-CoA reductase (HMGCR) is elevated including, as compared to a control drug and/or cell.

[00150] In addition, the assays or kits may include instructional materials containing directions (i.e., protocols) for the practice of the methods of the methods disclosed herein. While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.

EXAMPLES

Example 1 : Certain Drugs Induce Cardiomyocyte Death

[00151] Four FDA-approved tyrosine kinase inhibitors with known clinical cardiotoxicity outcomes (Table 1 ) were screened for cardiotoxicity potential.

Table 1 : Summary of FDA-approved tyrosine kinase inhibitors

CARDIOVASCULAR QT interval LVEF Reports of Black box TOXICITY prolongation declines, myocardial warning for prolonged QT infarction/ischemia QT intervals, (2.3% incidence) prolongation Torsade de and sudden Pointes death

Cmax (μΜ) 0.73 - 1.06 0.5 - 1 .4 3.0 - 10.0 3.0 - 4.27

[00152] The effect of treatment with increasing concentrations of crizotinib, sunitinib, erlotinib, and nilotinib on the viability of human cardiomyocytes (HCMs) at 72 hours after initial treatment of the cells was determined in order to evaluate cardiotoxicity. Briefly, cell viability was determined for HCMs treated for 72 hours with DMSO or with a dose course that encompassed the Cmax range for crizotinib, sunitinib, nilotinib, and erlotinib (10nM - 10μΜ). After treatment, cells were stained with the nuclear dye Hoechst (ThermoFisher, Pittsburgh, PA) and fixed in 4% formaldehyde (JT Baker, Phillipsburg, NJ). Total cell count was determined using the spot detector bioapplication (v4) on the Thermo Scientific Celllnsight High Content platform (ThermoFisher). The spot detector bioapplication allows for cell enumeration by applying user-defined parameters for cell shape and fluorescence intensity of the Hoechst stain to detect cells. All objects within these limits are then counted for the entire well, thus providing a total cell count. Cell viability graphs represent the mean fold-change, calculated as the cell count of treatment condition relative to the cell count for DMSO control, for 3 independent studies.

[00153] All four tested tyrosine kinase inhibitors induced cell death at the highest drug concentration tested (10μΜ) (Fig. 1A). However, crizotinib, sunitinib, and nilotinib also significantly decreased cell viability at lower, more physiologically relevant, concentrations. Compared to the DMSO control, 3μΜ of drug resulted in a cell viability of 31 .6±0.84%, 77.3±2.4%, and 85.0±1.7% for crizotinib, nilotinib, and sunitinib respectively. Additionally, 1 μΜ sunitinib significantly reduced cell viability (83.4±2.8%). In comparison, erlotinib did not induce cardiomyocyte death at concentrations less than 10μΜ. These data show that crizotinib most potently induced cardiomyocyte death while sunitinib and nilotinib also led to significant cardiac cell loss at concentrations less than 10μΜ.

Example 2: Certain Drugs Lead to Increased Lipid Accumulation in Cardiomyocytes

[00154] Lipid accumulation in cardiac cells is associated with weakened cardiac function, and notably cardiac contractile dysfunction. Thus, assessing lipid droplet formation within cardiomyocytes reflects another possible mechanism of injury induced by drug treatment. To this end, lipid accumulation was examined in HCM using Oil Red-0 staining following a 48 hour treatment with 3μΜ of each drug. While nilotinib and erlotinib slightly decreased lipid accumulation, both sunitinib and crizotinib induced marked elevation of lipid formation (Fig. 2A).

Example 3: Differential Effects of Drugs on AMPK Activation

[00155] The increased lipids induced by sunitinib and crizotinib could be due to drug-induced changes in lipid metabolism. As such, the regulation of lipids in treated HCMs (i.e., HCMs treated with crizotinib, sunitinib, eriotinib, and nilotinib) was assessed by determining if the drug had an effect on the phosphorylation of acetyl CoA carboxylase (ACC), the critical, rate-limiting enzyme in fatty acid biosynthesis. The phosphorylation status of ACC typically reflects changes in fatty acid metabolism. Without wishing to be bound by a theory of the invention, it is hypothesized that phosphorylation of ACC inhibits its activity, resulting in reduced fatty acid synthesis while dephosphorylation of ACC increases its activity, resulting in increased fatty acid synthesis and lipid accumulation. Thus, to assess levels of phosphorylated ACC, HCMs were treated with increasing doses of each drug for 24 hours and Western blots were performed on cell lysates. Crizotinib, nilotinib and, to a lesser extent, eriotinib, increased the phosphorylation of ACC in a dose- dependent manner while sunitinib led to a dose-dependent decrease in the phosphorylation of ACC (Fig. 3B) Example 4: Toxic Drugs Activate the SREBP Pathway in Cardiac Cells

[00156] The above ACC data corresponds with the lipid levels following treatment with sunitinib (decreased p-ACC; increase in lipids) as well as nilotinib and eriotinib (increased p-ACC; no increase in lipids). However, crizotinib led to an increased p-ACC status, which would suggest decreased lipid accumulation rather than the dramatic increase in lipid accumulation seen in HCM. Therefore, other mechanisms may be responsible for the marked elevation of lipids in crizotinib-treated cells.

[00157] To determine other possible mechanisms of lipid accumulation, transcriptome analysis was performed on RNA isolated from HCM treated for 24 hours with DMSO or 3μΜ of each drug. Briefly, HCM were treated for 24 hours with DMSO or 3.0 μΜ crizotinib, sunitinib, eriotinib, or nilotinib. After treatment, RNA was harvested using the miRcury RNA isolation kit (Exiqon, Woburn, MA) according to the instructions provided by the manufacturer. Product purity and quality were monitored with a NanoDrop spectrophotometer (Wilmington, DE). RNA samples were sent to University of California Los Angeles core facility for microarray analysis using the Affymetrix GeneChip® Human Genome U133A 2.0 Array (Santa Clara, CA). Genego's Metacore software (Thomson Reuters, Carlsbad, CA) was used to assess significantly altered signaling pathways and/or networks. Using GeneGo's MetaCore pathway analysis software, it was discovered that the Sterol Regulatory Binding Protein (SREBP) pathway, which plays a key role in lipid and cholesterol synthesis, was one of the most highly up-regulated pathways in crizotinib, and to a lesser extent, sunitinib-treated cells (Fig. 3A). Neither eriotinib nor nilotinib significantly affected the SREBP pathway.

[00158] The up-regulation of several important genes in the SREBP pathway by crizotinib, including SREBP2, insulin induced gene 1 (INSIG1 ), fatty acid synthase (FASN), fatty acid desaturase 2(FADS2), hydroxymethylglutaryl CoA synthase 2 (HMGCS2), and hydroxymethylglutaryl CoA reductase (HMGCR), was confirmed by qRT-PCR analysis (Fig. 3B). Briefly, RNA used for transcriptome analysis was also used for quantitative RT- PCR. cDNA reverse transcription was performed using ABI's MultiScribe Reverse Transcriptase assays (Life Technologies, Grand Island, NY). RT-PCR was performed on ABI's 7900HT fast real-time PCR system using ABI Taqman arrays for INSIG1 , HMGCR, FADS2, FASN, SREBF2, and ACTIN (reference gene). The AACT method was used to calculate fold change relative to DMSO control. Although transcriptome analysis showed that sunitinib also up-regulated two members of this pathway (FADS2 and HMGCS1 ), the induction was far less potent than that shown with crizotinib, and only FADS2 could be confirmed by qRT-PCR.

[00159] Activation of the SREBP fatty acid/cholesterol synthesis pathway as well as the increased lipid accumulation seen with Oil Red-O, which stains both hydrophobic lipids and esterified cholesterol, suggests that cholesterol synthesis may be induced by crizotinib and, to a lesser extent, by sunitinib. To assess this hypothesis, total cholesterol levels were evaluated in HCMs treated for 48 hours with DMSO or 3μΜ of each drug. Neither eriotinib nor nilotinib increased cholesterol levels in HCMs, which is concordant with the Oil Red-0 staining and transcriptome profiles and sunitinib only led to a 1 .8-fold increase. However, crizotinib induced a dramatic 5.7-fold increase in cholesterol levels as compared to DMSO control (Fig. 3C). These results suggest that crizotinib increases cholesterol production in HCMs. As such, without wishing to be bound by any theory of the invention, the toxicity observed upon treatment of HCMs with crizotinib is likely a result of increased cholesterol production.

Example 5: Predicting Toxicity of a Drug to a Mammalian Subject

[00160] The success of therapeutics in medicine and especially in a complex disease such as cancer depends on the correct diagnosis choice of drugs to treat a patient. This process requires knowledge of the specific patient markers that can be used to predict how the patient will respond to a given drug or class of drugs that share a common mechanism of action. The inventors of the instant application have shown that drugs that activate the SREBP pathway are toxic to a patent. Drugs that may be toxic to a mammalian subject may be identified as follows.

[00161] In an exemplary method, a biological sample is removed from a patient prior to treatment of the patient with a drug, such as a tyrosine kinase inhibitor, and analyzed for activation of the SREBP pathway upon treatment of the biological sample with the drug. If the SREBP pathway is activated in the biological sample (including one or more cells in the biological sample), the drug is predicted to be toxic to the patient and is not administered to the patient. In contrast, if the SREBP is not activated in the biological sample (including one or more cells in the biological sample), the drug is predicted to not be toxic to the patient and is selected for administration to the patient.

[00162] While the present disclosure has been described and illustrated herein by references to various specific materials, procedures and examples, it is understood that the disclosure is not restricted to the particular combinations of materials and procedures selected for that purpose. Numerous variations of such details can be implied as will be appreciated by those skilled in the art. It is intended that the specification and examples be considered as exemplary, only, with the true scope and spirit of the disclosure being indicated by the following claims. All references, patents, and patent applications referred to in this application are herein incorporated by reference in their entirety.

Claims

1. ) A method for predicting whether a drug is likely to be toxic to a cell comprising:

- contacting the cell with the drug; and

- determining whether a Sterol Regulatory Binding Protein (SREBP) pathway in the cell contacted with the drug is activated,

wherein the drug is predicted to be toxic to the cell where the SREBP pathway is activated in the cell contacted with the drug or wherein the drug is predicted to not be toxic to the cell where the SREBP pathway is not activated in the cell contacted with the drug.

2. ) The method of claim 1 , wherein activation of the SREBP pathway is identified by increased expression of one or more genes in the SREBP pathway.

3. ) The method of claim 2, wherein the one or more genes are selected from the group consisting of: SREBP2, insulin induced gene 1 (I NSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and hydroxymethylglutaryl-CoA reductase (HMGCR).

4. ) The method of claim 1 , wherein the drug is a receptor tyrosine kinase inhibitor.

5. ) The method of claim 1 , wherein the toxicity is cardiotoxicity or hepatotoxicity.

6. ) The method of claim 1 , wherein the cell is a myocardiocyte or a hepatocyte.

7. ) A method for treating a cell with a drug, the method comprising:

- contacting the cell with the drug where the SREBP pathway is activated in the cell upon treatment with the drug.

8. ) The method of claim 7 further comprising determining whether a Sterol Regulatory

Binding Protein (SREBP) pathway in the cell contacted with the drug is activated.

9. ) The method of claim 8, wherein activation of the SREBP pathway is identified by increased expression of one or more genes in the SREBP pathway.

10. ) The method of claim 9, wherein the one or more genes are selected from the group consisting of: SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and hydroxymethylglutaryl-CoA reductase (HMGCR).

1 1 . ) The method of claim 7, wherein the drug is a receptor tyrosine kinase inhibitor.

12. ) The method of claim 7, wherein the toxicity is cardiotoxicity or hepatotoxicity.

13.) The method of claim 7, wherein the cell is a myocardiocyte or a hepatocyte.

14.) A method for treating a cell with a drug, the method comprising:

- contacting the cell with the drug where the SREBP pathway is not activated in the cell upon treatment with the drug.

15.) The method of claim 14, wherein activation of the SREBP pathway is identified by increased expression of one or more genes in the SREBP pathway.

16. ) The method of claim 15, wherein the one or more genes are selected from the group consisting of: SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and hydroxymethylglutaryl-CoA reductase (HMGCR).

17. ) The method of claim 14, wherein the drug is a receptor tyrosine kinase inhibitor.

18. ) The method of claim 14, wherein the toxicity is cardiotoxicity or hepatotoxicity.

19. ) The method of claim 14, wherein the cell is a myocardiocyte or a hepatocyte.

20. ) A method for selecting a cell for treatment with a drug, the method comprising:

- determining whether a Sterol Regulatory Binding Protein (SREBP) pathway in the cell is activated upon treatment of the cell with the drug; and

- selecting the cell for treatment with the drug where the SREBP pathway is activated in the cell contacted with the drug or not selecting the cell for treatment with the drug where the SREBP pathway is not activated in the cell contacted with the drug

21 . ) The method of claim 20, wherein activation of the SREBP pathway is identified by increased expression of one or more genes in the SREBP pathway.

22. ) The method of claim 21 , wherein the one or more genes are selected from the group consisting of: SREBP2, insulin induced gene 1 (I NSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and hydroxymethylglutaryl-CoA reductase (HMGCR).

23. ) The method of claim 20, wherein the drug is a receptor tyrosine kinase inhibitor.

24. ) The method of claim 20, wherein the toxicity is cardiotoxicity or hepatotoxicity.

25.) The method of claim 20, wherein the cell is a myocardiocyte or a hepatocyte.

26. ) A method for treating a patient with a drug, the method comprising:

- obtaining a biological sample from the patient;

- contacting one or more cells in the biological sample with a drug;

- determining whether a Sterol Regulatory Binding Protein (SREBP) pathway in one or more of the contacted cells are activated upon treatment with the drug; and

- selecting the drug that activates the Sterol Regulatory Binding Protein (SREBP) pathway; and

- treating the patient with the drug that activates the Sterol Regulatory Binding Protein (SREBP) pathway.

27. ) The method of claim 26, wherein activation of the SREBP pathway is identified by increased expression of one or more genes in the SREBP pathway.

28. ) The method of claim 27, wherein the one or more genes are selected from the group consisting of: SREBP2, insulin induced gene 1 (I NSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and hydroxymethylglutaryl-CoA reductase (HMGCR).

29. ) The method of claim 26, wherein the drug is a receptor tyrosine kinase inhibitor.

30. ) The method of claim 26, wherein the toxicity is cardiotoxicity or hepatotoxicity.

31 . ) The method of claim 26, wherein the cell is a myocardiocyte or a hepatocyte.

32.) The method of claim 26, wherein the patient is a cancer patient.

33. ) A method for conducting a clinical trial, the method comprising:

- obtaining biological samples from one or more subjects;

- contacting one or more cells in the biological samples from the subjects with a drug;

- determining whether a Sterol Regulatory Binding Protein (SREBP) pathway in one or more of the contacted cells in the biological samples from the subjects are activated upon treatment with the drug; and

- enrolling subjects in the clinical trial where the Sterol Regulatory Binding Protein (SREBP) pathway is activated in one or more of cells in the biological sample upon contact of the cells with the drug.

34. ) The method of claim 33, wherein activation of the SREBP pathway is identified by increased expression of one or more genes in the SREBP pathway.

35. ) The method of claim 34, wherein the one or more genes are selected from the group consisting of: SREBP2, insulin induced gene 1 (I NSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and hydroxymethylglutaryl-CoA reductase (HMGCR).

36. ) The method of claim 33, wherein the drug is a receptor tyrosine kinase inhibitor.

37. ) The method of claim 33, wherein the toxicity is cardiotoxicity or hepatotoxicity.

38.) The method of claim 33, wherein the cell is a myocardiocyte or a hepatocyte.

39. ) The method of claim 33, wherein the patient is a cancer patient.

40. ) A method for screening one or more drugs for likelihood of being cardiotoxic, the method comprising:

- contacting a cell with the drug; and

- determining whether a Sterol Regulatory Binding Protein (SREBP) pathway in the cell contacted with the drug is activated.

41 . ) The method of claim 40 further comprising selecting the drug that activates the Sterol

Regulatory Binding Protein (SREBP) pathway.

42.) The method of claim 40, wherein activation of the SREBP pathway is identified by increased expression of one or more genes in the SREBP pathway.

43.) The method of claim 42, wherein the one or more genes are selected from the group consisting of: SREBP2, insulin induced gene 1 (I NSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and hydroxymethylglutaryl-CoA reductase (HMGCR).

44.) The method of claim 40, wherein the drug is a receptor tyrosine kinase inhibitor.

45. ) The method of claim 40, wherein the toxicity is cardiotoxicity or hepatotoxicity.

46. ) The method of claim 40, wherein the cell is a myocardiocyte or a hepatocyte.

47. ) An in-vitro method for determining drug-induced cell toxicity, the method comprising:

- identifying whether a Sterol Regulatory Binding Protein (SREBP) pathway in a cell contacted with the drug is activated in response to treatment with a drug, wherein the drug is predicted to be toxic to the cell where the SREBP pathway is activated in the cell contacted with the drug or wherein the drug is predicted to not be toxic to the cell where the SREBP pathway is not activated in the cell contacted with the drug.

48.) The method of claim 47, wherein activation of the SREBP pathway is identified by increased expression of one or more genes in the SREBP pathway.

49. ) The method of claim 48, wherein the one or more genes are selected from the group consisting of: SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and hydroxymethylglutaryl-CoA reductase (HMGCR).

50. ) The method of claim 47, wherein the drug is a receptor tyrosine kinase inhibitor.

51 . ) The method of claim 47, wherein the toxicity is cardiotoxicity or hepatotoxicity.

52. ) The method of claim 47, wherein the cell is a myocardiocyte or a hepatocyte. ) A test panel for determining whether a drug is toxic to a cell, the test panel comprising a test for determining whether a Sterol Regulatory Binding Protein (SREBP) pathway in a cell is activated upon contact of the cell with the drug,

) The test panel of claim 53, wherein a test comprises a microarray comprising oligonucleotides that specifically bind one or more genes are selected from the group consisting of: SREBP2, insulin induced gene 1 (INSIG 1 ), fatty acid synthase (FASN), fatty acid desaturase (FADS2), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and hydroxymethylglutaryl-CoA reductase (HMGCR). ) A kit for assessing toxicity of a drug, the kit comprising: a set of instructions for using the test panel of claim 53.