WO2008036254A2

WO2008036254A2 - Autophagic compounds and tyrosine kinase inhibitors for treating cancer

Info

Publication number: WO2008036254A2
Application number: PCT/US2007/020179
Authority: WO
Inventors: Jeffrey Settleman; Sreenath V. Sharma; Ultan Mcdermott
Original assignee: The General Hospital Corporation
Priority date: 2006-09-18
Filing date: 2007-09-18
Publication date: 2008-03-27
Also published as: WO2008036254A3

Abstract

The invention provides combinations of autophagy modulators and inhibitors of the epidermal growth factor receptor and methods of using these combinations for the treatment subjects having cancer.

Description

AUTOPHAGIC COMPOUNDS AND TYROSINE KINASE INHIBITORS FOR

TREATING CANCER

RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No.

60/845,311, entitled QUINOLINE COMPOUNDS AND THEIR USE IN TREATING CANCER, filed September 18, 2006. The entire contents of the aforementioned application are hereby incorporated herein by reference.

GOVERNMENT SUPPORT

Research supporting this application was carried out by the United States of America as represented by the Secretary, Department of Health and Human Services. This research has been funded in part with Federal funds from the National Institutes of Health (NIH), under contract No. NIH ROl CAl 15830. Accordingly, the government has certain rights in the invention.

INCORPORATION BY REFERENCE

Any of the applications or patents cited in this text, as well as each document or reference cited in each of the applications and patents (including during the prosecution of each issued patent; "application cited documents"), and each of the PCT and foreign applications or patents corresponding to and/or claiming priority from any of these applications and patents, and each of the documents cited or referenced in each of the application cited documents, are hereby expressly incorporated herein by reference, and may be employed in the practice of the invention. More generally, documents or references are cited in this text, either in a Reference List before the claims, or in the text itself; and, each of these documents or references ("herein cited references"), as well as each document or reference cited in each of the herein cited references (including any manufacturer's specifications, instructions, etc.), is hereby expressly incorporated herein by reference.

BACKGROUND OF THE INVENTION

Tyrosine kinase inhibitors encompass a large and continually expanding class of pharmaceutical compounds. Because it has been shown that certain tyrosine kinases may be mutated or overexpressed in many human cancers such as brain, lung, epithelial, squamous cell, bladder, gastric, breast, colorectal, pancreatic, head and neck, oesophageal, gynecological and thyroid cancers, the rationale to continue to develop and refine inhibitors of the tyrosine kinase receptor has been supported by the successful development and use of such inhibitors. For example, tyrosine kinase inhibitors have been described in U.S. Pat. No. 5,593,997, incorporated herein by reference in its entirety, which teaches erlotinib, a quinazoline derivative, for use as an inhibitor of the epidermal growth factor receptor ("EGFR") for the treatment of solid tumors, such as non-small cell lung cancer (NSCLC), pancreatic cancer, breast cancer, and neck cancer. STI-571 is a tyrosine kinase inhibitor indicated for the treatment of chronic myelogenous leukemia (CML), prostate tumors, and gastrointestinal stromal tumors, among others. Gefitinib, described in U.S. Pat. No. 6,919,340, and incorporated herein by reference in its entirety, is an inhibitor of EGFR, and has been used for the treatment of cancers which over-express EGFR, including non-small cell lung cancer (NSCLC), and other solid tumors such as breast tumors.

Although the targeted small molecule EGFR inhibitors provide a new line of cancer treatment, they have been found clinically effective in only a relatively small fraction of cancer patients. Further, resistance to small molecule tyrosine kinase inhibitors has emerged as a significant clinical problem (Clark J et al. 2005). Selective kinase inhibitors (KIs) induce dramatic clinical responses in some cancers; however, responses are invariably limited by acquired drug resistance.

Accordingly, there is a need in the art to improve the efficacy of the EGFR inhibitors, and to extend the efficacy of the EGFR inhibitors to a greater patient population.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of inhibiting growth of tumor cells in a subject, comprising contacting said tumor cells with a cytotoxically effective amount of a compound that raises intralysosomal acidification in combination with an inhibitor of the epidermal growth factor receptor, thereby inhibiting growth of the tumor cells in the subject.

In one embodiment, the compound that raises intralysosomal acidification is an autophagy modulator. In another aspect, the invention provides, a method of inhibiting growth of tumor cells in a subject, comprising administering to the subject a cytotoxically effective amount of an autophagy modulator, in combination with an inhibitor of the epidermal growth factor receptor, thereby inhibiting growth of the tumor cells in the subject.

In another aspect, the invention features a method of inhibiting growth of tumor cells that are kinase inhibitor resistant in a subject, comprising administering to the subject a cytotoxically effective amount of a compound that raises intralysosomal acidification in combination with an inhibitor of the epidermal growth factor receptor, thereby inhibiting growth of the tumor cells that are resistant to kinase inhibitor compounds in the subject.

In one embodiment, the compound that raises intralysosomal acidification is an autophagy modulator.

In another aspect, the invention features a method of inhibiting growth of tumor cells that are resistant to kinase inhibitory compounds in a subject, comprising administering to the subject a cytotoxically effective amount of an autophagy modulator, in combination with an inhibitor of the epidermal growth factor receptor, thereby inhibiting growth of the of tumor cells that are resistant to kinase inhibitor compounds in the subject. In one embodiment, the kinase inhibitor compounds are receptor tyrosine kinase inhibitors or non-receptor tyrosine kinase inhibitors, or combinations thereof.

In another embodiment, the compound that raises intralysosomal acidification is selected from a quinoline compound or bafilomycin Al.

In another particular embodiment, the autophagy inhibitor is selected from a quinoline compound or rapamycin. In a further embodiment, the quinoline compound is selected from the group consisting of: chloroquine, hydroxychloroquine and primaquine.

In another embodiment, the inhibitor of the epidermal growth factor receptor inhibits the activity of the receptor. In another particular embodiment, the inhibitor of the epidermal growth factor receptor inhibits the expression of the receptor.

In a further embodiment, the inhibitor of the epidermal growth factor receptor is an antagonist of the receptor. In another embodiment, the inhibitor of the epidermal growth factor receptor is selected from the group consisting of: gefitinib, erlotinib, cetuximab, and panitumumab.

In one embodiment of any of the above-mentioned aspects, the tumor cells contain an amplified epidermal growth factor receptor gene.

In another embodiment of any of the above-mentioned aspects, the tumor cells are associated with a cancer selected from the group consisting of: lung, brain, epithelial, colorectal, breast, pancreatic, head and neck cancer, esophageal, gastric and prostate. In another embodiment of any of the above-mentioned aspects, the tumor is a solid tumor.

In another embodiment of any of the above-mentioned aspects, the tumor cells are resistant to one or more inhibitors of the epidermal growth factor receptor. In a further embodiment, the one or more inhibitors of the epidermal growth factor receptor is selected from the group consisting of gefitinib, erlotinib, cetuximab and panitumumab.

In another embodiment of any of the above-mentioned aspects, the combination of said compound that raises intralysosomal acidification with said inhibitor of the epidermal growth factor receptor results in a decrease in growth of tumor cells that is greater than the decrease in growth of tumor cells that results from treatment with either said compound that raises intralysosomal acidification or inhibitor or the epidermal growth factor receptor alone.

In another aspect, the invention features a method of treating or preventing cancer in a subject comprising administering to the subject a cytotoxically effective amount of a compound that raises intralysosomal acidification in combination with an inhibitor of the epidermal growth factor receptor, thereby treating the cancer in the subject.

In another aspect, the invention features a method of treating or preventing a drug resistant cancer in a subject comprising administering to the subject a cytotoxically effective amount of a compound that raises intralysosomal acidification in combination with an inhibitor of the epidermal growth factor receptor, thereby treating the drug resistant cancer in the subject.

In one embodiment, the compound that raises intralysosomal acidification is an autophagy modulator. In another embodiment, the drug resistant cancer is resistant to kinase inhibitory compounds.

In a further embodiment, the kinase inhibitory compounds are receptor tyrosine kinase inhibitors or non-receptor tyrosine kinase inhibitors, or combinations thereof.

In another embodiment, the compound that raises intralysosomal acidification is selected from a quinoline compound or bafilomycin Al .

In another embodiment, the autophagy modulator is selected from a quinoline compound and rapamycin. In a related embodiment, the quinoline compound is selected from the group consisting of: chloroquine, hydroxychloroquine and primaquine.

In another further embodiment, the inhibitor of the epidermal growth factor receptor inhibits the activity of the receptor.

In a further embodiment, the inhibitor of the epidermal growth factor receptor inhibits the expression of the receptor. In a related embodiment, the inhibitor of the epidermal growth factor receptor is an antagonist of the receptor.

In a further embodiment, the inhibitor of the epidermal growth factor receptor is selected from the group consisting of gefitinib, erlotinib, cetuximab and panitumumab. In another further embodiment, the cancer selected from the group consisting of: lung, brain, epithelial, colorectal, breast, pancreatic, head and neck, esophageal, gastric and prostate cancer.

In another embodiment, the subject has a cancerous tumor.

In another embodiment, the cells of said tumor contain an amplified epidermal growth factor receptor gene. Ina related embodiment, the tumor is a solid tumor. In another embodiment, the tumor is resistant to one or more inhibitors of the epidermal growth factor receptor.

In another embodiment, the one or more inhibitors of the epidermal growth factor receptor is selected from the group consisting of gefitinib, erlotinib, cetuximab and panitumumab.

In another embodiment, the combination of said compound that raises intralysosomal acidification and inhibitor of the epidermal growth factor receptor decreases the size of said tumor. In a further embodiment, the combination of said compound that raises intralysosomal acidification and inhibitor of the epidermal growth factor receptor results in treatment of cancer that is greater than the treatment with either said compound that raises intralysosomal acidification or inhibitor of the epidermal growth factor receptor alone.

In another aspect, the invention features a method of treating a cancerous tumor in a subject having a tumor comprising administering to the subject a cytotoxically effective amount of a compound that raises intralysosomal acidification in combination with an inhibitor of the epidermal growth factor receptor, wherein the combination results in a decrease in tumor size that is greater than the decrease in tumor size that results from treatment with either said compound that raises intralysosomal acidification or inhibitor of the epidermal growth factor receptor alone.

In one embodiment, the compound that raises intralysosomal acidification is an autophagy modulator. In another aspect, the invention features a method of treating a cancerous tumor in a subject having a tumor comprising administering to the subject a cytotoxically effective amount of an autophagy modulator in combination with an inhibitor of the epidermal growth factor receptor, wherein the combination results in a decrease in tumor size that is greater than the decrease in tumor size that results from treatment with either said autophagy modulator or inhibitor of the epidermal growth factor receptor alone.

In one embodiment, the cancerous tumor is drug resistant.

In another embodiment, the cancerous tumor is drug resistant to kinase inhibitory compounds. In another embodiment, the kinase inhibitory compounds are receptor tyrosine kinase inhibitors or non-receptor tyrosine kinase inhibitors, or combinations thereof.

In another further embodiment, the compound that raises intralysosomal acidification is selected from a quinoline compound or bafilomycin Al. In a related embodiment, the quinoline compound is selected from the group consisting of chloroquine, hydroxychloroquine and primaquine.

In another embodiment, the inhibitor of the epidermal growth factor receptor inhibits the expression of the receptor. In another embodiment, the inhibitor of the epidermal growth factor receptor is an antagonist of the receptor.

In another further embodiment, the inhibitor of the epidermal growth factor is selected from the group consisting of gefitinib, erlotinib, cetuximab and panitumumab.

In another embodiment, the tumor contains cells having an amplified epidermal growth factor receptor gene.

In a further embodiment, the tumor is associated with a cancer selected from the group consisting of: lung, brain, epithelial, colorectal, breast, pancreatic, head and neck, esophageal, gastric and prostate cancer.

In another embodiment, the tumor is resistant to one or more inhibitors of the epidermal growth factor receptor.

In another embodiment, the inhibitor of the epidermal growth factor is selected from the group consisting of: gefitinib, erlotinib, cetuximab, and panitumumab. In various embodiments, the methods of the invention further comprise obtaining the compound(s) recited in the methods, e.g., compounds that raise intralysosomal acidification and/or inhibitors of epidermal growth factor receptor

In another aspect, the invention features a pharmaceutical composition comprising a therapeutically effective amount of a cytotoxic compound that raises intralysosomal acidification, an inhibitor of the epidermal growth factor receptor, and a pharmaceutically acceptable carrier.

In a further aspect the invention features a pharmaceutical composition comprising a therapeutically effective amount of a cytotoxic compound that modulates autophagy, an inhibitor of the epidermal growth factor receptor, and a pharmaceutically acceptable carrier.

In one embodiment, the cytotoxic quinoline compound is selected from the group consisting of chloroquine, hydroxychloroquine and primaquine.

In another embodiment, the inhibitor of the epidermal growth factor receptor is selected from the group consisting of: gefitinib, erlotinib, cetuximab, panitumumab. In another further embodiment, the cytotoxic compound that raises intralysosomal acidification is between about 60 to about 90% weight of the composition and the inhibitor of the epidermal growth factor receptor is between about 10 to about 40% weight of the composition. In another embodiment, the cytotoxic compound that modulates autophagy is between about 60 to about 90% weight of the composition and the inhibitor of the epidermal growth factor receptor is between about 10 to about 40% weight of the composition. In another embodiment, the ratio of the cytotoxic compound that raises intralysosomal acidification to the inhibitor of the epidermal growth factor receptor is between about 60: 40 to about 90: 10.

In another embodiment, the ratio of the cytotoxic compound that modulates autophagy to the inhibitor of the epidermal growth factor receptor is between about 60: 40 to about 90: 10.

In another aspect, the invention features a packaged pharmaceutical comprising a cytotoxic compound that raises intralysosomal acidification and an inhibitor of the epidermal growth factor receptor and associated instructions for using said cytotoxic inhibitor of lysosomal acidification and inhibitor of the epidermal growth factor receptor to treat cancer.

In one embodiment, the cytotoxic compound that raises intralysosomal acidification is selected from the group consisting of: chloroquine, hydroxychloroquine and primaquine.

In another embodiment, the inhibitor of the epidermal growth factor receptor is selected from the group consisting of: gefitinib, erlotinib, cetuximab, panitumumab.

In a further embodiment, the cytotoxic compound that raises intralysosomal acidification is between about 60 to about 90% weight of the composition and the inhibitor of the epidermal growth factor receptor is between about 10 to about 40% weight of the composition. In another embodiment the ratio of the cytotoxic compound that raises intralysosomal acidification to the inhibitor of the epidermal growth factor receptor is between about 60:40 to about 90:10.

In another embodiment, the instructions are for use in accordance with the method of any one of the above-mentioned aspects. In another aspect, the invention features a kit comprising a cytotoxic compound that raises intralysosomal acidification and an inhibitor of the epidermal growth factor receptor and associated instructions for using said cytotoxic compound that raises intralysosomal acidification and inhibitor of the epidermal growth factor receptor to treat cancer. In one embodiment, the cytotoxic compound that raises intralysosomal acidification is selected from the group consisting of: chloroquine, hydroxychloroquine and primaquine.

In another further embodiment, the cytotoxic compound that raises intralysosomal acidification is between about 60 to about 90% weight of the composition and the inhibitor of the epidermal growth factor receptor is between about 10 to about 40% weight of the composition. In another embodiment, the ratio of the compound that raises intralysosomal acidification to the inhibitor of the epidermal growth factor receptor is between about 60:40 to about 90:10.

In another further embodiment, the compound that raises intralysosomal acidification is an autophagy modulator. In another aspect, the invention features a kit comprising an autophagy modulator and an inhibitor of the epidermal growth factor receptor and associated instructions for using said an autophagy modulator and inhibitor of the epidermal growth factor receptor to treat cancer.

In one embodiment, the autophagy modulator is selected from the group consisting of: chloroquine, hydroxychloroquine and primaquine.

In another embodiment, the autophagy modulator is between about 60 to about 90% weight of the composition and the inhibitor of the epidermal growth factor receptor is between about 10 to about 40% weight of the composition.

In another further embodiment, the ratio of the autophagy modulator to the inhibitor of the epidermal growth factor receptor is between about 60:40 to about 90:10.

In another further embodiment, the kit of any of the above-mentioned aspects further comprises a packaging means thereof.

These and other objects of the invention will be described in further detail in connection with the detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

The following Detailed Description, given by way of example, but not intended to limit the invention to specific embodiments described, may be understood in conjunction with the accompanying drawings, which are briefly described as follows.

Figure 1 is a graph showing the response of human cancer cell lines expressing different levels of EGFR to chloroquine (CQ) treatment. Cell lines and their corresponding levels of EGFR expression are indicated in the graph legend. Relative cell survival was measured with a MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide] assay and is indicated as a percent on the Y-axis.

Figures 2 and 2A are graphs showing PC9 cells treated with gefitinib alone, erlotinib alone, chloroquine alone, or any combination thereof. Relative cell survival was measured with MTT assay and is indicated as a percent on the Y-axis.

Figure 3 is a graph showing the ability of chloroquine to sensitize HT- 1666 cells to killing by gefitinib. Gefitinib concentration (μM) is indicated on the X-axis. The inset shows relative cell survival in the absence of EGFR inhibitor and in the absence of chloroquine as compared to cell survival in the absence of EGFR inhibitor and the presence of chloroquine. The data presented in the inset shows that chloroquine alone does not affect cell survival. Figure 4 is a graph showing the ability of chloroquine to sensitize G7 cells to killing by gefitinib. Gefitinib concentration (μM) is indicated on the X-axis. The inset shows relative cell survival in the absence of EGFR inhibitor and the absence of chloroquine as compared to cell survival in the absence of EGFR inhibitor and the presence of chloroquine. The data presented in the inset shows that chloroquine alone does not affect cell survival.

Figure 5 is a graph showing the ability of chloroquine to sensitize NCI-H1975 cells to killing by gefitinib. Gefitinib concentration (μM) is indicated on the X-axis. The inset shows relative cell survival in the absence of EGFR inhibitor and the absence of chloroquine as compared to cell survival in the absence of EGFR inhibitor and the presence of chloroquine. The data presented in the inset shows that chloroquine alone does not affect cell survival.

Figure 6 is a graph showing the ability of chloroquine to sensitize HT- 1666 cells to killing by erlotinib. Erlotinib concentration (μM) is indicated on the X-axis. The inset shows relative cell survival in the absence of EGFR inhibitor and the absence of chloroquine as compared to cell survival in the absence of EGFR inhibitor and the presence of chloroquine. The data presented in the inset shows that chloroquine alone does not affect cell survival.

Figure 7 (A - E) is five graphs showing the effects of combination chloroquine and erlotinib (TV) treatment in a panel of non-small cell lung cancer cells. Cell lines used are: (A) PC-9, (B) 1755, (C) CaIu 1, (D) 1568, (E) A431. Concentration of erlotinib treatment (TV, nM)) is indicated on X-axis. Concentration of chloroquine (CQ) treatment (μM) is indicated in the graph legends. Cell survival is represented as fraction of control. Figure 8 (A - C) are three graphs showing of the results of a dose response study done on a panel of non-small cell lung cancer (NSCLC) cells. Cell lines are indicated in the graph legends. Panel (C) highlights the cell lines that are sensitive or resistant to chloroquine treatment. Concentration of chloroquine treatment (μM) is indicated on the X-axis. Figure 9 are two graphs showing the results of a dose response study done on a panel of gastric (A) and pancreatic (B) cancer cell lines. Cell lines are indicated in the graph legends. Concentration of chloroquine treatment (μM) is indicated on the X- axis.

Figure 10 (A - C) shows the effect of the autophagy inhibitor hydroxychloroquine on the development of acquired resistance to erlotinib in PC9 cells. Shown in Panel (A) 10⁶ PC9 cells were either untreated, treated with 5μM of hydroxychloroquine (5μM HCQ), treated with 2μM erlotinib (2μM ERL) or treated with 2μM erlotinib plus 5μM hydroxychloroquine (2μM ERL + 5μM HCQ). After 6 days, cells were fixed and stained with SytoόO (top set of plates) or Giemsa (bottom set of plates). The 2μM ERL and 2μM ERL + 5μM HCQ treated plates were similarly fixed and stained after 33 days of treatment (treatments were repeated every three days). The experiment was performed in triplicate and representative stained plates are shown. In panels (B) and (C) the SytoόO stained plates were scanned, quantitated and the results are represented in histograms. The data is expressed as a percentage of surviving cells relative to cells in untreated controls (B) or percentage of surviving cells in the 2μM ERL + 5μM HCQ relative to cells in the 2μM ERL plates (C). Error bars represent standard deviation from the mean value.

Figure 11 (A - E) shows resistance to EGFR KIs is reversible, involves persistent Akt activation, and engagement of autophagy. Panel (A) is a graph that shows the survival curves of PC9 and several gefitinib-resistant (GR) PC9 cell lines treated for 4 days with the indicated concentrations of gefitinib. Each data point represents the average value determined from four identically treated samples. The data are expressed as a percentage of surviving cells relative to untreated controls. Panel (B) is a graph that shows the survival curves of PC9 and one gefitinib-resistant PC9 cell line, GR7, after withdrawal from gefitinib and after varying number of passages (p) in drug-free medium. Cell sensitivity was determined by treating cells with the indicated concentrations of gefitinib for 4 days. Each data point represents the average value determined from four identically treated samples. The data are expressed as a percentage of surviving cells relative to untreated controls. Error bars represent standard deviation from the mean value. Similar findings were obtained from multiple independently-generated clones. Panel (C) shows the results of immunoblotting experiments. Cell lysates from PC9 and the gefitinib-resistant clone GR7 taken out of drug for 7 passages and treated for 2h with the indicated concentrations of gefitinib were analyzed by SDS-PAGE followed by immunoblotting using antibodies directed against the indicated signal transduction proteins. The identity of the cell lines and other relevant experimental details is indicated at the top of each lane, the relative migrations of the relevant proteins are indicated on the right- hand side of each autoradiogram, and to the left of each autoradiogram are indicated the relative migration of molecular weight standards. Similar findings were obtained from multiple independently-generated clones. Panel (D) shows the results of immunoblotting experiments. Cell lysates from PC9 cells and the gefitinib-resistant clone GR7 after 3 (3°) or 33 passages (33°) in drug-free medium, were treated for 2h with the indicated concentrations of gefitinib, and then were analyzed by SDS-PAGE followed by immunoblotting using antibodies directed against the indicated signal transduction proteins. In Panel (E), PC9 cells, one erlotinib-resistant clone and two gefitinib-resistant PC9 clones were transfected with a plasmid expressing a GFP-C3 fusion protein, and plated in chamber-well slides. When cells reached 50% confluence, they were fixed with 4% para-formaldehyde. Localization of GFP-LC3 was carried out by fluorescence microscopy. Varying levels of GFP signal within the cell population most likely reflects the fact that pools of transfected cells were examined in each case.

Figure 12 is a graph showing the tyrosine kinase inhibitor (TKI) sensitivity of erlotinib-resistant PC9 cell lines. Survival curves of PC9 and several erlotinib- resistant (ER) PC9 cell lines treated for 4 days with the indicated concentrations of erlotinib. Each data point represents the average value determined from four identically treated samples. The data are expressed as a percentage of surviving cells relative to untreated controls. Figure 13 shows DNA sequence analysis of the relevant region of ex on 19 of the EGFR gene in PC9 and several gefitinib-resistant PC9 clones showing the presence of DE746-A750 deletion mutation. Electropherograms showing the sequence of a relevant portion of exon 19 of the EGFR gene from PC9 cells and several gefitinib-resistant PC9 clones. PC9 cells harbor an in- frame deletion of 15 bp that results in a deletion of 5 amino acids spanning amino acids 746 to 750 within the kinase domain of the EGFR protein. The nucleotide sequence of the deleted region is also shown, hi all the gefitinib-resistant PC9 clones a similar deletion was observed, confirming that the KI-resistant clones are derived from PC9 cells.

Figure 14 shows HPLC analysis showing absence of the T790M mutation in the gefitinib-resistant PC9 clones. HPLC analysis was performed of DNA fragments generated by NIaIII digestion of the PCR-amplified exon 20 region of EGFR from PC9 and one gefitinib-resistant PC9 clone. As a positive control, DNA from NCI- HI 975 with a known T790M in cis was similarly analyzed and shows a third diagnostic NIa III fragment indicating the presence of T790M in these samples. This diagnostic third NIa III fragment is missing from the PC9 or gefitinib-resistant PC9 DNA samples. Similar analysis was performed on DNA from several other gefitinib- resistant PC9 clones with identical results (data not shown).

Figure 15 (A - D) is four graphs showing further characterization of gefitinib- and erlotinib-resistant PC9 clones. In Panel (A) PC9 cells or their counterparts selected for their ability to grow in the absence of serum (SFPC9) were plated at a density of 100,000 cells per well in a 12-well dish in complete medium containing serum (to allow the cells to attach). The following day, cells were treated with the indicated concentrations of erlotinib in quadruplicate for 72h in serum-containing medium. At the end of this treatment period, cells were fixed, stained with SytoόO, and quantitated. Each data point represents the average value determined from four identically treated samples. The data are expressed as a percentage of surviving cells relative to untreated controls. Error bars represent the standard deviation from the mean value. Panel (B) is a graph showing the results of experiments where PC9 cells and several gefitinib- or erlotinib-resistant PC9 cells were either untreated (open boxes), treated with 10 μM of chloroquine (black boxes), or treated with 10 μM of hydroxychloroquine (grey boxes), in quadruplicate. The data are expressed as a percentage of surviving cells relative to untreated controls. Error bars represent the standard deviation from the mean value. Panel (C) is a graph showing the results of experiments where PC9 cells (white boxes) and the gefitinib-resistant PC9 clone GR7 taken out of drug for 10 passages (grey boxes) were treated with 2μM of erlotinib (black boxes) in the presence of varying concentrations of hydroxychloroquine, in quadruplicate. The data are expressed as a percentage of surviving cells relative to untreated controls. Error bars represent the standard deviation from the mean value. Panel (D) is a graph showing the results of experiments where PC9 cells and the gefitinib-resistant PC9 clone GR7 taken out of drug for 10 passages were treated as follows in quadruplicate for 96h: Untreated PC9 cells (black box); PC9 cells treated with 0.02nM Bafilomycin Al (grey box); untreated GR7 cells (white box); GR7 cells treated with 2 μM erlotinib (blue box); GR7 cells treated with 0.02 nM Bafilomycin Al (green box); GR7 cells treated with 2 μM erlotinib plus 0.02 nM Bafilomycin Al (red box). The data are expressed as a percentage of surviving cells relative to untreated controls. Error bars represent the standard deviation from the mean value.

Figure 16 (A - D) shows the effect of various inhibitors of lysosomal acidification autophagy modulators on acquired resistance to KIs. Panel (A) is a graph showing the percent survival when PC9 cells and gefitinib- or erlotinib-resistant PC9 cells were either untreated (grey boxes) or treated with 10 μM of chloroquine (black boxes). Results are shown from experiments done in quadruplicate. The data is expressed as a percentage of surviving cells relative to untreated controls. Error bars represent the standard deviation from the mean value. In Panel (B) 10⁶ PC9 cells were either, untreated, treated with 5 μM of hydroxychloroquine (5μM HCQ), 2μM erlotinib (2μM ERL), or 2 μM erlotinib plus 5 μM hydroxychloroquine (2μM ERL + 5μM HCQ). After 6 days, the untreated and 5 μM HCQ treated plates had reached confluence and were fixed and stained with Giemsa. The 2 μM ERL and 2 μM ERL + 5 μM HCQ treated plates were similarly fixed and stained after 33 days of treatment (drug treatments were repeated every three days in fresh medium). Experiments were performed in triplicate and representative stained plates are shown. Parallel sets of SytoόO stained plates were scanned, quantified, and the results are represented in the histograms at the bottom of each figure in Panels (B) and (C). Data are expressed as a percentage of surviving cells in the 2 μM ERL + 5 μM HCQ relative to cells in the 2 μM ERL plates. Error bars represent the standard deviation from the mean value. In parallel, 10⁶ PC9 cells were treated with 5 μM hydroxychloroquine for 33 days, with 6 passages. At the end of 33 days the 5 μM hydroxychloroquine treated plates were fixed and stained. They appeared confluent and were indistinguishable from the untreated (6d) or the 5 μM HCQ (6d) plates (data not shown). In Panel (C), 10⁶ PC9 cells were either untreated, treated with 0.2 μM of rapamycin (0.2μM RAPA), 2 μM erlotinib (2μM ERL), or 2 μM erlotinib plus 0.2 μM rapamycin (2μM ERL + 0.2μM RAPA). After 6 days, the untreated and 0.2 μM rapamycin treated plates had reached confluence and were fixed and stained with Giemsa (top plates). The 2 μM erlotinib and 2 μM erlotinib + 0.2 μM rapamycin treated plates were similarly fixed and stained after 29 days of treatment (bottom plates). Drug treatments were repeated every three days. The experiment was performed in triplicate and representative stained plates are shown. The colonies on the bottom two plates (2μM erlotinib and 2μM erlotinib + 0.2μM rapamycin) were counted and the results are represented in the histograms. Error bars represent the standard deviation from the mean value. In Panel (D), 10⁶ M- 14 melanoma cells were either, untreated, treated with 5 μM of hydroxychloroquine (5μM HCQ), 2 μM AZ628 (2μM AZ628), 2 μM AZ628 plus 5 μM hydroxychloroquine (2μM AZ628 + 5μM HCQ). After 6 days, the untreated and 5 μM HCQ treated plates had reached confluence and were fixed and stained with Giemsa (top plates). The 2 μM AZ628 and 2 μM AZ628 + 5 μM HCQ treated plates were similarly fixed and stained after 39 days of treatment (bottom plates). Drug treatments were repeated every three days in complete medium. The experiment was performed in triplicate and representative stained plates are shown. The colonies on the bottom two plates (2μM AZ628 and 2μM AZ628+5μM HCQ) were counted and the results are represented in the histograms. Error bars represent the standard deviation from the mean value.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods of treating cancer by administering compounds that raises intralysosomal acidification, for example quinoline compounds, in combination with one or more inhibitors of the epidermal growth factor receptor. The invention further provides methods of treating cancer by administering inhibitors of autophagy in combination with one or more inhibitors of the epidermal growth factor receptor. The methods of the invention are particularly useful for treating cancers that are resistant to kinase inhibitors.

The combinations of the invention are used in methods to treat cancer in a subject. The new combinations of compounds provide optimized therapy to treat cancer more effectively than treatment with either of the compounds alone. For example, the therapy is optimized to provide a decrease in tumor size that is greater than the decrease in tumor size that results from treatment with either of the compounds alone. In other words, the combinations featured by the invention provide synergistic effects.

I. Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application, including definitions will control.

The term "autophagy" is meant to refer to a cellular process that allows cells to sequester cytoplasmic contents through the formation of double-membraned vesicles, termed autophagasomes, and target them for degradation through the fusion of autophagosomes with lysosomes, creating single membrane autolysosomes. In certain examples, antineoplastic therapy induces autophagy in cancer cells. High levels of autophagic vesicles have been noted in some forms of degenerative muscle disease, and in degenerative nervous system diseases like Huntington's, Parkinson's, Alzheimer's and ALS, (Lou Gehrig's disease).

The term "autophagy modulator" is intended to mean a stimulus, for example a compound or composition that controls, regulates, activates or inactivates the process of autophagy as defined herein. For example, the process of autophagy can change from inactive to active in the presence of a compound that activates the autophagic process. Manifestations of activation of autophagy include high levels of autophagic vesicles. In exemplary embodiments, autophagy modulators of the invention are quinoline compounds as described herein.

The term "quinoline compound" is intended to mean quinoline, also known as 1-azanaphthalene, 1-benzazine or benzo[b]pyridine. The term "quinoline compound" also includes quinoline compounds, analogs and family members including but not limited to chloroquine, hydroxychloroquine, primaquine, isoquinoline, pyridine, naphthalene, and indole. The term "quinoline compound" also includes a compound that includes and/or is related to the structure:

"Quinoline compound" also means compounds that increase the pH of intracellular organelles such as the lysosome, endosome and trans Golgi network. An "increase in pH" means any increase in pH as compared to the pH in the absence of a quinoline compound.

The term "obtaining" as in "obtaining the quinoline compound" or "obtaining the epidermal growth factor receptor inhibitor" is intended to include purchasing, synthesizing or otherwise acquiring the quinoline compound or epidermal growth factor receptor inhibitor (or indicated substance or material).

The term "cytotoxic" is intended to refer to compounds that cause cell death.

A "cytotoxic quinoline compound" is a quinoline compound as defined herein that has a harmful or lethal effect on a cell of a tumor, for example, causes cell death. A "cytotoxically effective amount" is a dosage of a quinoline compound that has a harmful or lethal effect on a cell of a tumor when administered to a subject having cancer and/or a tumor.

A "modulator" is meant to refer to a compound that has the ability to control, regulate, or activate a physiological response within a cell that ultimately changes the state of the cell. For example, an autophagic state can change from active to inactive in the presence of stimuli that inactivates autophagy. In certain preferred embodiments, the modulator is an inhibitor.

As used herein, "inhibits" means decreases by at least 5%, for example, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%, for example, as compared to the level in the absence of a cytotoxic quinoline compound in combination with an inhibitor of the epidermal growth factor receptor.

As used herein, "inhibits" also means decreases by at least 1-fold, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000-fold or more, for example, as compared to the level in the absence of a cytotoxic quinoline compound in combination with an inhibitor of the epidermal growth factor receptor.

The terms "carcinoma" or "cancer" refer to lesions that are cancerous. Examples include, but are not limited to, malignant melanomas, lung, brain, epithelial, squamous cell, bladder, gastric, breast, prostate, colorectal, pancreatic, head and neck, oesophageal, gynecological and thyroid cancers. "Cancer" is a term for diseases in which abnormal cells divide without control. Cancer cells can invade nearby tissues and can spread through the bloodstream and lymphatic system to other parts of the body. There are several main types of cancer. Carcinoma is cancer that begins in the skin or in tissues that line or cover internal organs. Sarcoma is cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is cancer that starts in blood-forming tissue such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the bloodstream. Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system.

As used herein, a "cancer cell" or a "tumor cell" refers to an abnormal cell that divides without control.

The term "tumor" is intended to include an abnormal mass or growth of cells or tissue that results when cells divide abnormally and/or exhibit an extended period of growth or an extended period of viability as compared to a normal cell.

As used herein, a "solid tumor" refers to an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors may be benign (not cancerous), or malignant (cancerous). Different types of solid tumors are named for the cellular composition. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (cancers of the blood) generally do not form solid tumors.

The term "treating" cancer refers to decreasing the size, number or growth rate of malignant or cancerous cells. "Treating" cancer also refers to the killing of malignant, or cancerous, cells. "Treating cancer" also means decreasing the size or growth rate of a tumor. By treating is meant causing tumor cell death, or causing tumor cell death in the subject. Alternatively, "treating" cancer means arresting or otherwise ameliorating symptoms of cancer in the subject.

As used herein, "decreases" means decreases by at least 5%, for example, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%, for example, as compared to the level in the absence of a cytotoxic quinoline compound in combination with an inhibitor of the epidermal growth factor receptor.

As used herein, "decreases" also means decreases by at least 1-fold, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000-fold or more, for example, as compared to the level in the absence of a cytotoxic quinoline compound in combination with an inhibitor of the epidermal growth factor receptor.

The term "resistant" in the context of cancer treatment refers to the renewed and unregulated proliferation cancer cells in the presence of a treatment that was once effective to reduce the unregulated proliferation.

The phrase "epidermal growth factor receptor inhibitor" or "inhibitor of the epidermal growth factor receptor" or "antagonist of the epidermal growth factor receptor" is intended to refer to compounds that decrease, as defined herein, or otherwise interfere with, for example modify or change, the activity of the epidermal growth factor receptor under normal or disease conditions. That is, an epidermal growth factor receptor inhibitor or antagonist decreases, as defined herein, or modifies any one of EGFR activity or expression as compared to activity or expression in the absence of inhibitor or antagonist. EGFR inhibitors useful according to the invention include but are not limited to gefitinib, erlotinib, cetuximab, and panitumumab. Inhibitors of the epidermal growth factor receptor may function by providing either direct inhibition of the EGFR, or indirect inhibition of kinases activated in the EGFR signaling pathway (e.g., kinases activated either upstream or downstream of the EGFR signaling cascade).

The term "amplified" as used herein refers to either overexpression of nucleotide sequences corresponding to a gene, for example the epidermal growth factor receptor gene or multiple copies of the epidermal growth factor receptor gene in a single cell. For example, a tumor cell of the invention may contain an amplified gene, for example the epidermal growth factor receptor gene.

As used herein, "amplified" means increased by at least 5%, for example, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%, as compared to the unamplified level.

As used herein, "amplified" also means increased by at least 1-fold, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000- fold or more, as compared to the unamplified level. As used herein, "tumor cell growth" refers to an increase in the size of a tumor or an increase in the number of tumors, an increase in the rate of tumor growth or an increase in the duration of the period of tumor growth.

The term "anti-tumor effect" refers to any measurable decrease in tumor size, tumor number or tumor growth, for example growth rate or duration of the growth period.

An "anti-tumor effect" also means having the effect of killing tumor or cancer cells such that they are no longer viable as detected by methods known in the art, including but not limited to the MTT assay described hereinbelow. The term "in combination with" is intended to refer to all forms of administration that provide the cytotoxic quinoline compound and the inhibitor of the epidermal growth factor receptor together, and can include sequential administration, in any order, or administration of a pre-mixed combination of a quinoline compound and an inhibitor of the epidermal growth factor receptor. In one embodiment, the invention is performed with a combination consisting essentially of a cytotoxic quinoline compound and an inhibitor of the epidermal growth factor receptor.

The term "subject" is intended to include vertebrates, preferably a mammal. "Mammal" includes, but is not limited to, human, mouse, rat, sheep, monkey, goat, rabbit, hamster, horse, cow, or pig. The term "synergistic" is intended to refer to the effect of two compounds, wherein the effect is greater combined that the effect of each compound individually. As used herein, the cytotoxic quinoline compound and the inhibitor of the epidermal growth factor receptor are synergistic when the combination results in a decrease in tumor size that is greater than the decrease in tumor size that results from treatment with the cytotoxic quinoline compound or inhibitor of the epidermal growth factor receptor alone.

A method of "administration" useful according to the invention includes but is not limited to topical application, intravenous drip or injection, subcutaneous, intramuscular, intraperitoneal, intracranial and spinal injection, ingestion via the oral route, inhalation, trans-epithelial diffusion (such as via a drug-impregnated, adhesive patch) or by the use of an implantable, time-release drug delivery device, which may comprise a reservoir of exogenously-produced agent or may, instead, comprise cells that produce and secrete the therapeutic agent. Additional methods of administration are provided hereinbelow in the section entitled "Dosage and Administration." In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

Other definitions appear in context throughout this disclosure.

II. Quinoline Compounds

The invention provides methods of treating cancer by administering quinoline compounds in combination with an inhibitor of the epidermal growth factor receptor. The combinations of the invention are referred to interchangeably throughout this disclosure as therapeutic or pharmaceutical compositions. A quinoline compound useful according to the invention includes quinoline, quinoline analogs and quinoline family members. A quinoline compound useful according to the invention includes but is not limited to quinoline, chloroquine, hydroxychloroquine, primaquine, isoquinoline, pyridine, naphthalene, and indole. A "quinoline compound" also includes compounds that increase the pH of intracellular organelles, such as the lysosome, endosome and trans golgi network. Chloroquine and hydroxychloroquine, for example, are well known anti-malarial drugs. It is known that anti-malarial drugs, including chloroquine and hydroxychloroquine, exert their effects by increasing the pH of intracellular organelles, such as the lysosome, endosome, and trans golgi network (Fox, RI 1993; Fox RI 1996). Thus, a quinoline compound according to the invention may also have anti-malarial activity.

In one embodiment, a quinoline compound is cytotoxic and has a harmful or lethal effect on a cell or a tumor, for example, the quinoline compound causes cell death.

A cytotoxic quinoline compound according to the invention can be any quinoline compound that has cellular cytotoxicity, as determined, for example, by the MTT assay described hereinbelow. Exemplary cytotoxic quinoline compounds according to the invention include chloroquine, hydroxychloroquine, and primaquine. Quinoline compounds according to the invention can be used at 0, 0.1 μM, 0.2 μM, 0.3 μM. 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45μM, 50 μM, or any concentration within the range of 0 - lmm, preferably 0-500 μM, more preferably 0- 250 μM and even more preferably 0-50 μM. The cytotoxic quinoline compound can also be administered in combination with an inhibitor of the epidermal growth factor receptor, for example in a ratio in the range of 1:1-1 :5, 1 :1-1:10, 1 :1-1:25, 1:1-1 :50. 1:1-1 :100, 1 :1-1:500, 1:1-1 :1000, 1 :1-1 :10,000, 5:1-1 :1, 10:1-1 :1, 25:1-1-1, 50:1-1 :1, 100:1-1 :1, 500:1-1:1, 1000:1-1:1 or 10,000:1-1:1. In another embodiment, the ratio of the cytotoxic quinoline compound to the inhibitor of the epidermal growth factor receptor is between about 60:40 to about 90:10. In one embodiment, the combination of the cytotoxic quinoline compound and the inhibitor of the epidermal growth factor receptor are administered such that the cytotoxic quinoline compound is between about 60 to about 90% (for example, 60, 61, 62, 63, 64, 65, 70, 75, 80, 85, 86, 87, 88, 89, 90%) weight of the composition and the inhibitor of the epidermal growth factor receptor is between about 10 to about 40% (for example, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 36, 37, 38, 39, 40%) weight of the composition. The ratio of the cytotoxic quinoline compound to the inhibitor of the epidermal growth factor receptor is such that the combination of compounds treats cancer, for example by reducing the size of a tumor or by killing tumor cells more effectively, than either compound alone.

In one embodiment, the compounds are administered to a subject. In another embodiment, the compounds are administered directly to tumor cells of the subject.

III. Autophagy

Autophagy is a protein degradation system in which cellular proteins and organelles are sequestered, delivered to lysosomes, and digested by lysosomal hydrolases. In normal cells, autophagy functions to maintain homeostasis by eliminating excessive or unnecessary proteins and injured or aged organelles, autophagy is observed under physiological conditions such as nutrient starvation and in some pathological conditions, including myopathy, neuronal degeneration, infectious disease, and cancer (Cuervo AM. Autophagy: In sickness and in health. Trends Cell Biol 2004; 14:70-7; Shintani T, Klionsky DJ. Autophagy in health and disease: A double-edged sword. Science 2004; 306:990-5; Kondo Y, Kanzawa T, Sawaya R, Kondo S. The role of autophagy in cancer development and response to therapy. Nat Rev Cancer 2005; 5:726-34). Various anticancer agents have been shown to induce autophagy in certain types of cancer cells, including tamoxifen and other anti-estrogen agents, and rapamycin (Takeuchi H, Kondo Y, Fujiwara K, Kanzawa T, Aoki H, Mills GB, Kondo S. Synergistic augmentation of rapamycin- induced autophagy in malignant glioma cells by phosphatidylinositol 3-kinase/protein kinase B inhibitors. Cancer Res 2005; 65:3336-46). Selective kinase inhibitors induce dramatic clinical responses in some cancers; however, responses are invariably limited by acquired drug resistance. As reported herein autophagy modulators, such as hydroxychloroquine and rapamycin, at clinical concentrations, virtually eliminate acquired resistance to kinase inhibitors, suggesting a combination drug strategy to prevent resistance. The observation that autophagy is one of the antitumor effects of anti-cancer therapies supports to the instant invention and the use of autophagy modulators, for example autophagy inhibitors, as part of a combination treatment strategy for cancers, and in particular drug resistant cancers.

IV. Inhibitor of the Epidermal Growth Factor Receptor

Uncontrolled proliferation of tumor cells is a hallmark of cancer. In many types of cancer, mutations in genes that activate cellular signal transduction pathways contribute to enhanced proliferation and survival of cancer cells. One well- characterized example is the protein tyrosine kinase (PTK) signaling pathway. PTK activity is normally tightly controlled and regulated; however mutation in the PTK enzyme leads to perturbation in signaling, and deregulation of the highly precise mechanisms that normally regulate cell growth and survival. Because tyrosine kinase activity is tightly regulated in normal cells, but dysregulated due to mutation in some cancers, the tyrosine kinases are attractive candidates for molecularly targeted therapies. Tyrosine kinases require ATP for their enzymatic activity, and thus small molecules that mimic ATP can bind to mutant kinases and inactivate them.

EGFR is a tyrosine kinase of the ErbB family that has been shown to be upregulated in a number of cancers, including NSCLC, brain, breast, head and neck, gastric, colorectal, esophageal, prostate, bladder, renal, pancreatic, and ovarian cancers (Salomon D et al. 1995 ). Gefitinib and erlotinib are small molecules that selectively inhibit epidermal growth factor receptor (EGFR) tyrosine kinase activity.

"Gefitinib" refers to the compound imidazo[l,2-a]pyrazin-8-ylamines and related compounds, which when appropriately substituted, are modulators of kinase activity, described in U.S. Pat. No. 6,919,340. "Erlotinib" refers to the compound 4-aminopyrazolo-[3,4-d]pyrimidine and 4- aminopyrazolo[3,4-d]pyridine and related compounds, which are tyrosine kinase inhibitors, as described in U.S. Pat. No. 5,593,997.

Gefitinib and Erlotinib are molecular targeted therapies that have both been used in the treatment of locally advanced or metastatic NSCLC (Comis, RL. 2005). Often, these therapies are used either alone or in combination with other agents as second or third line treatments after first round chemotherapy. For example, the IDEAL-2 study indicated that gefitinib monotherapy may be given as a single-agent, third-line therapy to patients with advanced NSCLC who have received and progressed on prior chemotherapy (Kris MG et al. 2003). Gefitinib has been used in combination with other chemotherapeutic agents, as reported in the ENTACT-I (Giaccone G et al. 2004) and INTACT-2 (Herbst RS et al. 2004) studies, which describe the use of gefitinib in combination with gemcitabine and cisplatin in advanced non-small-cell lung cancer, and gefitinib in combination with paclitaxel and carboplatin in advanced non-small-cell lung cancer, respectively.

The inhibitor of the EGFR can be any known in the art. It can function by inhibition of either the receptor expression or activity. Additional EGFR inhibitors useful herein include cetuximab and panitumumab. Inhibitors of the epidermal growth factor receptor may function by providing either direct inhibition of the EGFR, or indirect inhibition of kinases activated in the EGFR signaling pathway (e.g., kinases activated either upstream or downstream of the EGFR signaling cascade). EGFR inhibitors useful according to the invention include but are not limited to the agents presented in Tables 1 and 2 below. Tables 1 and 2 also provide dosages useful according to the invention.

TABLE 1: EGFR INHIBITORS

MONOCLONAL ANTIBODIES

TABLE 2: EGFR INHIBITORS SMALL MOLECULE KINASE INHIBITORS

Other small molecule kinase inhibitors include, but are not limited to, Lapatinib (Tykerb) for breast, renal, gastric, and head and neck cancers; ZD6474 (Zactima) for non-small cell lung cancer and thyroid cancers; STI571; AMG706 for gastrointestinal stromal tumors; AG-013736; lestaurtinib for acute myelogenous leukemia; XL647; XL999; MLN518 for acute myelogenous leukemia; PKC412; AMN 107 for chronic myelogenous leukemia and Philadelphia chromosome positive acute lymphoblastic leukemia; hypereosinophilic syndrome; systemic mastocytosis; AEE788; OSI-930; MP-412 for non-small cell lung cancer, metastatic breast cancer, pancreatic cancer, head and neck cancer; and hormone refractory prostate cancer and OSI-817. V. Cancers

The invention is useful for treating all types of cancers including but not limited to adrenal cancer, AIDS-related lymphoma, anal cancer, ataxia-telangiectasia, bladder cancer, brain tumors, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, endometrial and uterine cancer, esophageal cancer, Ewing's sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, gestational trophoblastic disease, choriocarcinoma, Hairy cell leukemia, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leukemia including acute lymphocytic leukemia and acute myelogenous leukemia, Li-Fraumeni syndrome, liver cancer, lung cancer, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, medulloblastoma, melanoma, mesothelioma, metastases, myelomas, myeloproliferative disorders, neuroblastoma, Non-Hodgkin's disease, non-small cell lung cancer, oropharyngeal cancers, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma and other soft-tissue sarcomas, osteosarcoma, small intestine cancer, small-cell lung cancer, testicular cancer, thymoma, thyroid cancer, urethal cancer, vaginal cancer, vulvar cancer and Wilms'tumor.

The term "treating" cancer refers to decreasing the size, number, growth rate or duration of the growth period of malignant or cancerous cells. "Treating" cancer also refers to the killing of malignant, or cancerous, cells. "Treating cancer" also means decreasing the size or growth rate of a tumor. By treating is meant causing tumor cell death, or causing tumor cell death in the subject. Alternatively, "treating" cancer means arresting or otherwise ameliorating symptoms of cancer in the subject. The efficacy of treatment can be assessed by the MTT assay using 3-(4,5- dimethylthiazol-2-yl)-2,50diphenyl-l-butene (SIGMA, USA). The MTT assay is well known to one skilled in the art, and is performed as described by Carmichael et al., and incorporated herein by reference in its entirety. The efficacy of treatment according to a method of the invention can also be assessed by monitoring tumor size or weight by methods well known in the art including but not limited to CT-scan. In one embodiment, the decrease in tumor size following administration of a quinoline compound and an anti-tumor agent is greater than the decrease in tumor size that results from treatment with the cytotoxic quinoline compound or inhibitor of the epidermal growth factor receptor, alone. VI. Pharmaceutical Compositions and Formulations

The invention provides for compositions and formulations comprising a quinoline compound in combination with an inhibitor of the epidermal growth factor receptor admixed with a physiologically compatible carrier. As used herein,

"physiologically compatible carrier" refers to a physiologically acceptable diluent such as water, phosphate buffered saline, or saline, and further may include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are materials well known in the art. The invention also provides for pharmaceutical compositions comprising a quinoline compound in combination with an inhibitor of the epidermal growth factor receptor. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carrier preparations that can be used pharmaceutically. The pharmaceutical compositions of the invention may be formulated as sustained release or extended release formulations.

Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for ingestion by the subject.

Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethyl cellulose; and gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.

Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers. Pharmaceutical formulations for parenteral administration include aqueous solutions of active compounds. For injection, the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer' solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

For nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention may be manufactured in a manner known in the art, e.g. by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc... Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder in lmM-50 mM histidine, 0.1%- 2% sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with buffer prior to use. After pharmaceutical compositions comprising a compound of the invention formulated in an acceptable carrier have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition with information including amount, frequency and method of administration. The dosages administered will vary from patient to patient; a "therapeutically effective dose" can be determined, for example, by monitoring the size or growth rate, or the duration of the growth period of a tumor, tumor number, cancer cell number, viability, growth rate and the duration of the growth period of a cancer cell. A therapeutically effective dose refers to a dose wherein the combination of compounds has a synergistic effect on the treatment of cancer.

In the treatment of cancer, a therapeutically effective dosage regimen should be used. By "therapeutically effective", one refers to a treatment regimen sufficient to decrease tumor size or tumor number, decrease the rate of tumor growth or kill the tumor. Alternatively, a "therapeutically effective regimen" may be sufficient to arrest or otherwise ameliorate symptoms of the cancer. Generally, in the treatment of cancer, an effective dosage regimen advantageously provides the medication over a period of time to achieve noticeable therapeutic effects.

The pharmaceutical composition may be formulated from a range of preferred doses, as necessitated by the condition of the patient being treated. For example, the cytotoxic quinoline compound can preferably be about 60%, 61%, 62%, 63%, 64%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, and any percentage between 60% and 90%, of the weight of the composition. The inhibitor of the EGFR can be between about 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 36%, 37%, 38%, 39%, 40%, and any percentage between 10% and 40%, of the weight of the composition. In another formulation, the ratio of the cytotoxic quinoline compound to the inhibitor of the EGFR is between about 60:40 to about 90:10, or any ratio therein.

The cytotoxic quinoline compound can also be administered in combination with an inhibitor of the epidermal growth factor receptor for example in a ratio in the range of about 1 :1-1:5, 1 :1-1:10, 1:1-1 :25, 1 :1-1 :50. 1:1-1 :100, 1:1-1:500, 1:1-1 :1000, 1 :1-1 :10,000, 5:1-1:1, 10:1-1 :1, 25:1-1-1, 50:1-1:1, 100:1-1 :1, 500:1-1:1, 1000:1-1 :1 or 10,000: 1-1:1. In another embodiment, the ratio of the cytotoxic quinoline compound to the inhibitor of the epidermal growth factor receptor is between about 60:40 to about 90:10. In one embodiment, the combination of the cytotoxic quinoline compound and the inhibitor of the epidermal growth factor receptor are administered such that the cytotoxic quinoline compound is between about 60 to about 90% (for example, about 60, 61, 62, 63, 64, 65, 70, 75, 80, 85, 86, 87, 88, 89, 90%) weight of the composition and the inhibitor of the epidermal growth factor receptor is between about 10 to about 40% (for example, about 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 36, 37, 38, 39, 40%) weight of the composition. The ratio of the cytotoxic quinoline compound to the inhibitor of the epidermal growth factor receptor is such that the combination of compounds treats cancer, for example by reducing the size of a tumor or by killing tumor cells more effectively, than either compound alone. The actual dosage employed may be varied depending upon the requirements of the patient and the severity of the condition being treated. Determination of the proper dosage for a given condition is within the skill of the art. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small amounts until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired. Intermittent therapy (e.g., one week out of three weeks or three out of four weeks) may also be used.

VII. Mode of Administration

A combination of a quinoline compound and an EGFR inhibitor, can be administered to the patient, preferably in a biologically compatible solution or a pharmaceutically acceptable delivery vehicle, by ingestion, injection, inhalation, or any number of other methods. The cytotoxic quinoline compound/inhibitor of the epidermal growth factor receptor combinations may be administered systemically or locally in a ratio wherein administration results in treatment of cancer, wherein the treatment is more effective than administration of either compound alone. That is, the combination of compounds has a synergistic effect, as defined herein. Systemic administration of the composition according to the invention may be performed by methods of whole-body drug delivery that are well known in the art. These include, but are not limited to, administration orally as well as by intravenous, intramuscular, or subcutaneous routes. In some cases it may be desirable to administer the composition directly to the airways in the form of an aerosol. Localized administration of a therapeutic compound according to the invention is preferably by a drip device, drug pump, or drug-saturated solid matrix from which the composition can diffuse once implanted at the target site.

The cytotoxic quinoline compound/inhibitor of the epidermal growth factor receptor combinations of the invention may also be administered parenterally or

Solutions or suspensions of these active compounds as a free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxy-propylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The form is sterile and fluid to the extent that easy syringability exists. It is preferably stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. It is contemplated that global administration of a therapeutic composition to an animal is not needed in order to achieve a highly localized effect. Localized administration of a therapeutic composition according to the invention is preferably by injection, catheter or by means of a drip device, drug pump or drug-saturated solid matrix from which the composition can diffuse implanted at the target site. When a tissue that is the target of treatment according to the invention is on a surface of an organism, topical administration of a pharmaceutical composition is possible. For example, antibiotics are commonly applied directly to surface wounds as an alternative to oral or intravenous administration, which methods necessitate a much higher absolute dosage in order to counter the effect of systemic dilution, resulting both in possible side-effects in otherwise unaffected tissues and in increased cost. Compositions comprising a therapeutic composition which are suitable for topical administration can take one of several physical forms, as summarized below:

(i) A liquid, such as a tincture or lotion, which may be applied by pouring, dropping or "painting" (i.e. spreading manually or with a brush or other applicator such as a spatula) or injection. (ii) An ointment or cream, which may be spread either manually or with a brush or other applicator (e.g. a spatula), or may be extruded through a nozzle or other small opening from a container such as a collapsible tube.

(iii) A dry powder, which may be shaken or sifted onto the target tissue or, alternatively, applied as a nebulized spray.

(iv) A liquid-based aerosol, which may be dispensed from a container selected from the group that comprises pressure-driven spray bottles (such as are activated by squeezing), natural atomizers (or "pump-spray" bottles that work without a compressed propellant) or pressurized canisters. (v) A carbowax or glycerin preparation, such as a suppository, which may be used for rectal or vaginal administration of a therapeutic composition.

Note that in some cases, the surface in question is internal; in such a case, topical application would comprise taking the therapeutic composition via an oral route, whether in liquid, gel or solid form. Systemic administration of a therapeutic composition according to the invention may be performed by methods of whole-body drug delivery well known in the art. These include, but are not limited to, intravenous drip or injection, subcutaneous, intramuscular, intraperitoneal, intracranial and spinal injection, ingestion via the oral route, inhalation, trans-epithelial diffusion (such as via a drug- impregnated, adhesive patch) or by the use of an implantable, time-release drug delivery device. Note that injection may be performed either by conventional means (i.e. using a hypodermic needle) or by hypospray (see Clarke and Woodland, 1975, Rheumatol. Rehabil, 14: 47-49).

Systemic administration is advantageous when a pharmaceutical composition is delivered to a target tissue that is widely-dispersed, inaccessible to direct contact or, while accessible to topical or other localized application, is resident in an environment (such as the digestive tract) wherein the native activity of the nucleic acid or other agent might be compromised, e.g. by digestive enzymes or extremes of pH.

A therapeutic composition of use in the invention can be given in a single- or multiple dose. A multiple dose schedule is one in which a primary course of administration can include 1-10 separate doses, followed by other doses given at subsequent time intervals to maintain and or reinforce the level of the therapeutic agent. Such intervals are dependent on the continued need of the recipient for the therapeutic agent, and/or the half-life of a therapeutic agent. The efficacy of administration may be assayed by monitoring the reduction in the levels of a symptom indicative or associated with cancer which it is designed to inhibit. Administration of the cytotoxic quinoline compound/inhibitor of the epidermal growth factor receptor combinations can be sequential, in either order, or concomitant, for example, in a single dosage form.

A therapeutically effective regimen may be sufficient to arrest or otherwise ameliorate symptoms of a disease. An effective dosage regimen advantageously provides the regulatory drug over a period of time to achieve noticeable therapeutic effects wherein symptoms are reduced to a clinically acceptable standard or ameliorated. The symptoms are specific for the disease in question. For example, when the disease is associated with tumor formation, the claimed invention is successful when tumor growth is arrested, or tumor mass is decreased by at least 50% and preferably 75%.

VIII. Kits or Pharmaceutical Systems

The present compositions may be assembled into kits or pharmaceutical systems for use in the treatment of cancer. Kits or pharmaceutical systems according to this aspect of the invention comprise a carrier means, such as a box, carton, tube or the like, having in close confinement therein one or more container means, such as vials, tubes, ampules, bottles and the like. The kits or pharmaceutical systems of the invention may also comprise associated instructions for using the agents of the invention, for example an agent that treats cancer and/or inhibits tumor cell growth. Having now generally described the invention, the same will be more readily understood through reference to the following Examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

EXAMPLES The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Example 1: Human Cancer Cell Lines Expressing Different Levels of EGFR are Sensitive to Killing by Chloroquine

In this example, the sensitivity of cell lines to chloroquine was determined. The effects of treatment with chloroquine were examined in various human cancer cell lines. Chloroquine, an inhibitor of lysosomal acidification, is well-known for its use in the treatment of malaria and rheumatologic disorders. The effects of chloroquine on human tumor cell lines were examined. Figure 1 shows cells derived from different human cancers expressing variable levels of the epidermal growth factor receptor (EGFR) that were treated with a range of concentrations of chloroquine for a period of 5 days. Cell lines used in the experiments, and the expression levels of EGFR per cell, as previously reported in the literature, included: A431 (1,000,000 EGFR/ cell), PC-9 (100,000 EGFR/ cell), H-358 (10,000 EGFR/ cell), H- 1666 (barely detectable), H- 1650 (detectable), and H- 1975 (detectable). Here, all tumor cell lines were tested for sensitivity to a range of chloroquine (CQ) concentrations, from 0 - 25 uM, in the presence or absence of an EGFR inhibitor, in a cell survival assay. Chloroquine was added to 15,000 cells at the beginning of the experiment, and every 2 days thereafter in fresh tissue culture media. Cell survival at the end of the experimental period was determined by MTT [3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide] assay. In cell lines that harbor amplified levels of EGFR, such as A431, PC-9 and H-358, treatment with chloroquine alone exerted a killing effect. Such effect is especially visible in the A431 and PC-9 non- small cell lung cancer (NSCLC) cell lines, which express the highest levels of EGFR. These data indicate that chloroquine has a potential therapeutic value in human tumors that harbor amplified EGFR.

Example 2: Cbloroquine Sensitizes Cells to Killing by Gefitinib

In this example, the effects of chloroquine in combination with gefitnib were examined. Gefitnib is a targeted small molecule EGFR inhibitor of the tyrosine kinase activity which prevents EGFR autophosphorylation. Although gefitinib has been used in the treatment of certain cancers, including NSCLC, some cancer types remain resistant to gefitinib treatment. Additionally, the toxic effects of gefitinib at high doses remain problematic. Figures 2 and 2A shows that when PC-9 NSCLC cells, which harbor overactive EGFR activities, are treated with chloroquine alone, they do not die. Likewise, when PC-9 NSCLC cells are treated with gefitinib alone, they are resistant to killing. However, when chloroquine was combined with a targeted small molecule inhibitor of EGFR, such as gefitinib, the previously gefitinib -resistant cells became sensitive to killing by the combination of agents. The ability of chloroquine to sensitize cells to killing by gefitinib was tested in various cell lines that are normally insensitive to gefitinib. Cell lines used included HT- 1666 expressing the wild-type EGFR (Figure 3), cell-line G7 (derived from NCI-H1650) with acquired resistance to gefitinib (Figure 4), NCI-H1975, a cell line that contains an activating mutation in

EGFR (L858R), but is resistant to gefitinib by virtue of a secondary mutation (L858R) in the EGFR (Figure 5), and PC-9 cells, which are resistant to killing by gefitinib and express high levels of EGFR (Figure 2 and 2A). 15,000 cells were treated with different amounts of gefitinib, with or without various concentrations of chloroquine over a period of 5 days. Gefitinib and chloroquine were added every 2 days in fresh tissue culture media at the indicated concentrations. Cell survival at the end of the experimental period was determined by MTT assays. Figure 3 shows that HT- 1666 cells which harbor wild type EGFR are sensitive to killing by gefitinib when treated alone with the agent; however when gefitinib therapy is combined with chloroquine, cell death increases dramatically. The amount of gefitinib needed to kill 50% of the cells is decreased by more than 4-fold. Figure 4 depicts a gefitinib resistant cell line, NCI-Hl 650. These data show that concentrations of gefitinib (i.e. 10 um) that will not cause cell death alone, when combined with chloroquine, result in cell death. The same is true in NCI-Hl 975 cells, a line that is resistant to gefitinib treatment. When gefitinib is combined with chloroquine, the previously death resistant NCI-Hl 975 cells are killed. Likewise, PC-9 cells overexpress high levels of EGFR and do not respond to gefitinib treatment (Figure 2 and 2A); however when treated with chloroquine in combination with gefitinib, these cells die.

Taken together, these data indicate that combination therapy of chloroquine and a targeted small molecule EGFR inhibitor may be clinically effective for treatment of certain tumor types resistant to treatment with a targeted small molecule EGFR inhibitor alone. Example 3: Chloroquine Sensitizes Cells to Killing by Erlotinib

In this example, the effects of chloroquine in combination with erlotinib were examined.

Erlotinib is another targeted small molecule EGFR inhibitor. The ability of chloroquine to sensitize cells to killing by erlotinib was tested in HT- 1666 cells expressing the wild-type EGFR. These cells are normally insensitive to erlotinib. Figure 6 shows the results of 15,000 cells treated with different amounts of erlotinib, with or without various concentrations of chloroquine for 5 days. There was no decrease in cell survival in the cells that did not receive the combination of chloroquine and erlotinib; however, when the therapies were combined, there was a decrease in cell survival. As above, erlotinib and chloroquine were added every 2 days in fresh tissue culture media. Cell survival at the end of the experimental period was determined by MTT assays. Figure 2, right panel, shows that PC-9 cells, which are resistant to killing by erlotinib and express high levels of EGFR, become responsive to erlotinib and die when treated with a combination of chloroquine and erlotinib. The effect of the combination of erlotinib and chloroquine treatment was examined in a panel of non-small cell lung cancer cells (NSCLC) after 72 hours of treatment with the combination therapy. The cell lines tested all express EGFR and include PC-9, 1755, CaIu 1, 1568 and A431 cell lines (see Figure 7). Chloroquine was given at 0, 1 OμM and 20 μM concentrations, with and without concomitant erlotinib treatment. Cell survival at the end of the experimental period was determined by MTT assays, and results are represented as the fraction of control cells that survive.

Chloroquine is highly related to the drug hydroxychloroquine. Thus, the effects of hydroxychloroquine on the killing of gefitinib and erlotinib-resistant PC-9 cells were compared to the effects of chloroquine. Figure 2 and 2 A shows that overall, hydroxychloroquine has the same effects on PC-9 cells as treatment with chloroquine.

These data indicate that combination therapy of chloroquine and targeted small molecule EGFR inhibitors may be clinically effective for treatment of certain tumor types that are resistant to monotherapy. Further, these data suggest that chloroquine alone may be an effective anti-cancer therapy. Example 4: Treatment with Chloroquine Alone is Able to Inhibit Cell Growth in EGFR Overexpressing Cancer Cell Lines

To further investigate a role for chloroquine monotherapy to treat tumors over- expressing EGFR, dose response studies were done on a number of cancer cell lines, including a panel of NSCLC cells (Figure 8), and gastric and pancreatic cancer cell lines (Figure 9). Cells were treated over a period of 72 hours. Figure 8, panels A and B show the results of a large number of NSCLC cell lines treated with chloroquine at concentrations up to 25 μM. While some cell lines did not respond to chloroquine treatment, some lines, such as PC-9, get, 1568 were responsive, as represented by cell survival as a fraction of control. Panel C shows a further dose study using chloroquine at concentrations up to 50 μM. While the 1975 cell line was resistant to chloroquine treatment, the graph shows that the other cell lines tested were sensitive to chloroquine at varying doses. Figure 9 expands these studies to gastric (Panel A) and pancreatic (Panel B) cell lines. Cell lines were treated with chloroquine at concentrations up to 25 μM over a period of 8 days with fresh agent and medium added every two days. The gastric cell lines Nugc 3, AGS, and the pancreatic cell line HPAC were identified as sensitive to chloroquine treatment alone. Again, some cells were found to be resistant (see Panel B). Taken together, these results provide rationale to use chloroquine both in combination with EGFR inhibitors and alone as a chemotherapeutic agent.

Example 5: Chloroquine compounds are effective against acquired resistance to kinase inhibitors.

The following methods were used in the experiments described in Examples 5 and 6.

Methods Cell Lines. PC9 cells expressing the EGFR exon 19 deletion mutation (DE746-A750) were provided by Dr. Kazuto Nishio (National Cancer Center Hospital, Tokyo) and were maintained in RPMI 1640 containing 10% fetal-bovine serum, penicillin and streptomycin (100 U/ml and 100 g/ml, respectively). Unless otherwise stated, gefitinib- and erlotinib-resistant PC9 clones (designated GR and ER) were maintained in RPMI 1640 containing 10% fetal-bovine serum, penicillin and streptomycin (100 U/ml and 100 g/ml, respectively) and lμM gefitinib or 2.5μM erlotinib, respectively. The serum- free PC9 clones were propagated in the above-mentioned medium minus serum.

Antibodies and other reagents. Rabbit polyclonal antibodies against phospho-Akt (Ser 473) and EGFR were purchased from Biosource and Santa Cruz Biotechnology, respectively. Mouse monoclonal antibodies against Phospho-EGFR (Tyr 1068), as well as rabbit polyclonal antibody against phospho-p44/42 MAP kinase (Thr 202/Tyr 204), phospho-Akt (Ser 473), and antibodies directed against their non-phosphorylated counterparts, were purchased from Cell Signaling Technology. Secondary antibodies included HRP conjugated anti-mouse and anti-rabbit antibodies, and were purchased from Cell Signaling Technology. GFP-LC3 plasmid was provided by Dr. Sheila Thomas (BIDMC/Harvard Medical School).

Inhibitors. Gefitinib (Astra-Zeneca Pharmaceuticals, Macclesfield, UK) and erlotinib (from the Massachusetts General Hospital pharmacy), was resuspended in DMSO at a stock concentration of 10 mM. The experimental BRAF inhibitor AZ628 was synthesized by Astra-Zeneca Pharmaceuticals (Waltham, MA), and was resuspended in DMSO at a stock concentration of 10 mM. Chloroquine and hydroxychloroquine (Sigma- Aldrich) were resuspended in sterile water at a stock concentration of 10 mM. Bafilomycin (Calbiochem) was resuspended in DMSO at a concentration of 1 mM. Rapamycin (Biomol) was resuspended in DMSO at a concentration of 10 mM. All inhibitors were stored in small aliquots at -200C.

Cell Survival Assays. Approximately 30,000 cells were plated in a well of a 12-well cluster dish. 24 h after plating, media was removed from the wells and replaced with media containing drugs. Fresh media was replaced every 2 days and this process was repeated every two days until the untreated control well reached confluence. At this point the media was removed, the cells were washed once with Phosphate Buffered Saline (PBS) and then fixed for 15 min at room temperature with 4% formaldehyde in PBS. Cells were then washed twice with PBS and stained with the fluorescent nucleic acid stain, Syto60 (1 nM in PBS; excitation and emission wavelengths of 652 and 678nm; Molecular Probes) for 15 min at room temperature. The dye was then removed and the cell monolayers were washed three times with PBS. Quantitation of fluorescent signal intensity was carried out at 700nm, using an Odyssey Infrared Imager (Li-Cor Biosciences). Each experiment was performed in quadruplicate. The results shown represent the average of the four values in comparison with untreated controls. Error bars represent the standard deviation of the 4 values from the mean. In some cases (e.g., Figure IA), quantitation of cell viability was performed using the MTS assay (CellTiter 96 Aqueous One Solution Cell Proliferation Assay; Pierce).

Generation ofgefitinib- and erlotinib-resistant PC9 clones. Approximately 10⁵ PC9 cells were plated in five 10cm plates and allowed to adhere for 24h. The attached cells were then treated with either 1 μM gefitinib or 2.5 μM erlotinib, which was added to the cells along with fresh media. Fresh media containing drug was added to the cells every 3 days until clones of drug-resistant cells appeared. Typically, approximately 10 clones per dish appeared after about 30 days of drug selection. Two to three clones were picked from each dish using cloning cylinders and propagated progressively in 3, 6, and 10 cm dishes, as individual cell lines, and were maintained in the presence of EGFR TKIs (1 μM gefitinib or 2.5 μM erlotinib). Once the clones were deemed to be stably drug-resistant they were further characterized for morphology, sensitivity to gefitinib or erlotinib, or biochemical characterization. In some circumstances, as indicated in figure legend, cells were withdrawn from the selecting drug, but "stock cultures" were always maintained in either 1 μM gefitinib or 2.5 μM erlotinib.

Generation of serum-free variants ofPC9. Approximately 10⁵ PC9 cells were plated in five separate 10 cm plates and allowed to adhere for 24h. The attached cells were then transferred to fresh medium without serum. Fresh serum-free media was added to the cells every 3 days until most of the cells died out and isolated clones of cells appeared. Typically, 3-5 clones per dish began to appear after about 30 days of incubation in serum- free medium. Two of these clones were picked using cloning cylinders and propagated progressively in 3, 6, and 10 cm dishes as individual cell lines in the absence of serum. For passaging of the clones, adherent cells were dissociated with trypsin that was subsequently inactivated with trypsin inhibitor from soybean. Serum- free PC9 clones were propagated for 4 to 5 months to ensure their stability. Once the clones were deemed to be stably growing in serum-free media they were further characterized with respect to their sensitivity to gefitinib or erlotinib. Cell Harvesting and Protein Analysis. PC9 and gefitinib- or erlotinib-resistant PC9 derived cells were cultured in a 10 cm dish and treated variously as indicated in the figures. Following each treatment, cells were placed on ice, scraped in their own media and collected by low-speed refrigerated centrifugation. Cell pellets were then washed once in ice-cold PBS and stored at -80 C until ready for analysis by SDS-PAGE and immunoblotting. Briefly, cells were lysed in NP40 lysis buffer (1% NP40; 20 mM TRIS-HCl pH 7.5; 2 mM EDTA; 137 mM NaCl, 10% glycerol) containing the protease inhibitors Aprotinin, Leupeptin, NaF, Na3 VO4 and PMSF. The lysate was cleared by centrifugation at 15,000 rpm for 5 minutes and the protein samples were quantitated by BCA protein assay (Pierce). 20 μg of total protein were resuspended in Laemmli sample buffer and the proteins were separated by electrophoresis on 10% SDS- polyacrylamide protein gels. Proteins were transferred to nitrocellulose membranes (0.45 μm Protran- Schleicher & Scheull) and the non-specific protein binding sites were blocked by incubating filters in 5% non-fat dry milk resuspended in Tris-Buffered- Saline (TBS) containing 0.1% Tween (TBS-T). Filters were then incubated overnight with the appropriate primary antibody in TBS-T containing 5% bovine serum albumin (BSA). Next day, the filters were washed three times in TBS-T and incubated with the respective HRP-coηjugated secondary antibodies for 1 hour at room temperature. The filters were then washed three times in TBS-T and the specific protein bands were visualized by Supersignal West Pico chemiluminescence (Pierce).

DNA Sequence analysis. Genomic DNA was isolated from cell lines using the Gentra purification system according to the manufacturer's protocol. The EGFR kinase domain (Exons 18-24) was amplified from genomic DNA by PCR-amplification with primers. PCR products were digested with exonuclease I and shrimp alkaline phosphatase (United States Biochemical, Cleveland, OH) followed by bidirectional sequencing using BigDye vl.l (Applied Biosystems, Foster City, CA) in combination with an ABI3100 sequencer (Applied Biosystems). Electropherograms were analyzed using Sequence Navigator software (Applied Biosystems).

Allele Specific mutation analysis. Exon 20 was amplified by PCR from genomic DNA of PC9 and several of the gefitinib- and erlotinib-resistant PC9 clones. A positive control consisted of genomic DNA from the NSCLC cell line NCI-H1975 that had previously been shown to harbor a T790M mutation 1. The amplified DNA was digested with NIa in and the digestion products were analyzed by HPLC. The presence of a T790M mutation creates a novel NIa III restriction site and results in the appearance of a novel peak that was readily detected in DNA from NCI-H 1975 cells.

PhosphoScan Analyses. Comparative tyrosine phosphoproteome analyses were performed with the PC9 cells and two gefitinib- and two erlotinib-resistant PC9 clones using the PhosphoScan™ technology (Cell Signaling Technology, Danvers MA). Briefly, cells from ten sub-confluent 10 cm plates of each cell line were collected, lysed and digested with proteases and the tyrosine phosphorylated peptides in the cell lysates were immunoaffinity purified using anti-phosphotyrosine antibody conjugated to protein G agarose beads. Tyrosine phosphopeptides were subsequently eluted from the beads and identified by liquid chromatography/tandem mass spectrometry and analyzed using proprietary software developed by Cell Signaling Technology.

Clonogenicity assays to assess the emergence of resistance. To assess the effect of agents such as hydroxychloroquine on the emergence of acquired resistance to erlotinib (e.g. Figure 16B), approximately 10⁶ PC9 cells were plated in triplicate under various conditions (untreated, 2 μM erlotinib, 5 μM hydroxychloroquine, 2 μM erlotinib + 5 μM hydroxychloroquine). After 24h the various drugs were added to the cells in fresh medium and the process was repeated every three days. By the end of the 6th day the untreated cells and cells treated with 5 μM hydroxychloroquine alone had reached confluence and were therefore fixed with methanol and stained with Giemsa. The 2 μM erlotinib and plates treated with the combination of 2 μM erlotinib and 5 μM hydroxychloroquine were cultured for 33 days with change of media every 3 days. At the end of this period, these plates were fixed and stained as described above. The procedure used to assess the effect of hydroxychloroquine on the emergence of acquired resistance to the experimental BRAF inhibitor, AZ628 in the melanoma-derived cell line, M- 14 (Figure 16C) was identical to those described for PC9 cells, above. The 2 μM AZ628 and plates treated with the combination of 2 μM AZ628 and 5 μM hydroxychloroquine were cultured for 39 days with change of media every 3 days, before they were fixed in methanol and stained with Giemsa. To assess the effect of Rapamycin on the emergence of resistance to erlotinib, PC9 cells were treated exactly as described for hydroxychloroquine except that the concentration of Rapamycin used was 200 nM. Transfection and GFP-LCi localization analyses. Transfections of PC9 and its gefitinib- or erlotinib-resistant derivatives were performed with an Amaxa electroporator using reagents and protocols provided by the manufacturer (Amaxa). Cells were transfected with 1 μg of the GFP-LC3 plasmid and 6-8 hours following transfection, the cells were washed and transferred to fresh culture medium containing 1 μM puromycin and 1 μM or 2.5 μM gefitinib or erlotinib (for the resistant lines). Following puromycin selection for 6 days, stable populations of PC9 and gefitinib- and erlotinib-resistant PC9 cells stably expressing GFP-LC3 were obtained. Cells were subsequently removed from puromycin for at least two passages before fluorescence microscopy was performed to determine the localization of the GFP-LC3. Briefly, cells expressing the GFP-LC3 fusion protein were plated in 2 well chamber slides and allowed to grow to 50% confluence. Next, cells were fixed with 4% para-formaldehyde and a coverslip was placed on the slide containing the cells and observed under an inverted fluorescence microscope (Nikon ECLIPSE 9Oi) at 6OX magnification and photographed using a Canon CCD camera equipped with epifluorescence capability. Images were processed using Nikon NIS Elements software.

Selective kinase inhibitors (KIs) induce dramatic clinical responses in some cancers; however, responses are invariably limited by acquired drug resistance. The results reported herein demonstrate a cell culture model of acquired KI resistance that reveals a reversible "drug-tolerant" state. This reversible "drug tolerant" state may precede stable drug resistance, and may involve autophagy, a primitive cell survival mechanism. Autophagy modulators, such as hydroxychloroquine and rapamycin, at clinical concentrations, virtually eliminate acquired resistance to kinase inhibitors, suggesting a combination drug strategy to prevent resistance.

Epidermal growth factor receptor (EGFR) kinase inhibitors (erlotinib and gefitinib) induce dramatic clinical responses in a subset of non-small cell lung cancer (NSCLC) patients, but responses are invariably limited by acquired drug resistance. Two established genetic resistance mechanisms involve a secondary T790M EGFR mutation and MET gene amplification, and both have been successfully modeled in cell culture (Pao et al. 2005; Engelman et al. 2007). However, several clinical reports describe KI-responsive patients that eventually experience disease progression, and after a "drug holiday", demonstrate a second response to KI re-treatment (Kurata. et al. 2004; Yano et al. 2005; Yokouchi et al. 2007), implicating a reversible, and possibly, non-genetic "drug-tolerant" state.

In these experiments, Kl-resistance in NSCLC was determined using the KI- sensitive EGFR-mutant PC9 human NSCLC cell line. PC9 cells cultured continuously in KIs yielded 500-fold drug-resistant clones at a frequency of 1 in 5,000 cells (Figure 1 Ia and Figure 12). The clones retain the activating EGFR mutation (Figure 13), and do not acquire the T790M secondary mutation, (Figure 14) or MET gene amplification (not shown), implicating a distinct resistance mechanism. The high frequency of Kl-resistant clones suggested a non-mutational, and therefore, possibly reversible mechanism. Indeed, the resistant cells re-acquire KI sensitivity during propagation in drug-free media, and after 29 passages, drug sensitivity is completely restored (Figure 1 Ib).

In Kl-resistant clones, EGFR expression is unaltered (data not shown), and while gefitinib efficiently suppresses EGFR and ERK phosphorylation, phosphorylation of the survival effector Akt is virtually unaffected by drug (Figure 1 Ic), suggesting engagement of an EGFR-independent survival mechanism. Moreover, in cells that re-acquire drug sensitivity, Akt phosphorylation is attenuated by drug, indicating that in these "re-sensitized" cells, survival signaling becomes re- coupled to EGFR (Figure 1 Id). To identify alternative survival mechanisms in Kl- resistant lines, a whole-phospho-tyrosine proteomic analysis was performed, which revealed substantially reduced phosphorylation of many signaling adapters in Kl- resistant cells. This is shown in Table 3, below. The Table shows the results of PhosphoScan analysis of activated adapters that are attenuated in EGFR Kl-resistant PC9 clones. Here, two gefitinib- and two erlotinib-resistant PC9 clones were subjected to PhosphoScan analysis. The various signaling adapters that were tyrosine phosphorylated in the parental PC9 cells but not in the resistant clones were tabulated. The list includes several signaling adaptors that were under-phosphorylated in all of the Kl-resistant clones relative to parental PC9 cells. Table 3: ADAPTERS ACTIVE IN PC9 CELLS BUT NOT IN THE RESISTANT CELLS

CBL <E3 Ugase EGFR endocytosis)

DLG 3

EPS 8 (EGFR signaling through Rac and trafficking through Rab5)

IRS 2 (connects EGFR to PI3K activation)

GAB 1 (connects RTKs such as EGFR to PI3K activation)

ITSN 2 (connects endocytosis to signaling)

PAG

PARD 3 (endocyiosis)

SAP 97

SH2D3A

SLAP 130 (Integπn signaling)

SHC 1 (connects EGFR to ERK activation)

SPRY 4 (inhibitor of EGFR) .

WIRE

ErbB3 (partners with EGFR to activate PI3K)

The lack of activated adapters demonstrates that severe adapter deprivation simulates starvation conditions and trigger a consequent response.

Example 6: Autophagy modulators as effective agents for preventing the development of acquired resistance to EGFR-targeted inhibitors.

A widely utilized starvation response in mammalian cells involves autophagy, a primitive survival mechanism in which cells consume their own macromolecules when nutrients are limiting (Kurata et al. 2007). Several recent reports implicate autophagy inhibitors as potential cancer therapeutic agents, either alone or in combination with other drugs (Kondo et al. 2005; Rubensztein et al. 2007). A defining characteristic of cells undergoing autophagy is translocation of the autophagy component Atg8/LC3 to autophagosomes. Numerous LC3-positive puncta, indicative of autophagasomes, were detected throughout the cytoplasm of KI-resistant (but not parental) PC9 cells, indicating the engagement of autophagy (Figure 1 Ie). Further evidence of a "starvation response'Vautophagy mechanism came from the establishment of serum-independent PC9 cells. When transferred to serum- free media, the vast majority of PC9 cells die rapidly, however, serum-independent clones were established, which exhibit substantially reduced KI sensitivity (Figure 15a).

To directly test the hypothesis that KI-resistant cells survive drug exposure by engaging autophagy, their sensitivity to autophagy inhibitors was examined. Chloroquine is a commonly used autophagy inhibitor that blocks the fusion of autophagosomes to lysosomes (Kroemer, G. & Jaattela, M 2005). Chloroquine does not detectably affect the growth or viability of PC9 cells but is uniformly toxic to KI- resistant clones (Figure 16a). Similar results were obtained with the related drug hydroxychloroquine (Plaquenil) (Figure 16B and 16C), and with the alternative autophagy inhibitor, Bafilomycin Al (Figure 16D). A potential role for autophagy inhibitors in preventing acquired resistance to these drugs was examined.

Remarkably, co-treatment of PC9 cells with hydroxychloroquine and erlotinib virtually eliminates the emergence of KI-resistant colonies (Figure 16B).

Previous studies demonstrated that insufficient or excessive autophagy is equally detrimental to cancer cell survival (Levine 2007). Therefore, the effect of autophagy "super-induction" on KI resistance was examined using an established autophagy inducer, rapamycin (Noda, T. & Ohsumi, Y 1998) . Rapamycin does not detectably affect PC9 cells, whereas co-treatment with rapamycin and erlotinib virtually eliminates the emergence of erlotinib-resistant colonies (Figure 16C). Together, these results demonstrate that autophagy modulators can be highly effective clinical agents for preventing the development of acquired resistance to EGFR- targeted inhibitors.

To determine whether modulating autophagy could impact drug resistance beyond EGFR inhibitors in lung cancer, a second model of acquired drug resistance was used. Melanoma cell lines harboring an activating allele of the BRAF kinase (V600E), which is seen in -70% of melanomas (Davies. 2002), are exquisitely sensitive to the selective BRAF inhibitor AZ628 (unpublished observation). However, as with EGFR Kl-treated PC9 cells, AZ628-treated melanoma cells eventually yield drug-resistant colonies, and co-treatment of drug-naϊve cells with AZ628 plus hydroxychloroquine severely attenuates the emergence of AZ628- resistant clones (Figure 16D). These findings demonstrate that autophagy underlies acquisition of resistance to other kinase inhibitors, and that clinical use of autophagy modulators combined with kinase inhibitors can constitute a broadly applicable strategy to prevent the emergence of drug-resistant tumor cells.

In a related experiment, shown in Figure 10, 10⁶ PC9 cells were either untreated, treated with 5μM of hydroxychloroquine (5μM HCQ), treated with 2μM erlotinib (2μM ERL) or treated with 2μM erlotinib plus 5μM hydroxychloroquine (2μM ERL + 5μM HCQ). After 6 days, the untreated and 5μM HCQ treated plates had reached confluence and were fixed and stained with Syto60 (top set of plates) or Giemsa (bottom set of plates). The 2μM ERL and 2μM ERL + 5μM HCQ treated plates were similarly fixed and stained after 33 days of treatment (treatments were repeated every three days). The experiment was performed in triplicate and representative stained plates are shown. The SytoόO stained plates were scanned, quantitated and the results are represented in the histograms at the bottom of the figure. The data is expressed as a percentage of surviving cells relative to cells in untreated controls (left histogram) or percentage of surviving cells in the 2μM ERL + 5μM HCQ relative to cells in the 2μM ERL plates. Error bars represent standard deviation from the mean value.

Taken together, the results presented herein demonstrate a reversible resistance mechanism, and further demonstrate that a small subset of cells within a tumor population exists in a distinct functional state characterized by their ability to tolerate external stresses such as growth factor deprivation or drug exposure. Such a state may be called a "drug-tolerant" state. This "drug-tolerant" state may be restricted to a subset of cells prone to rapidly engage the autophagy process, and could allow such cells to withstand an initial onslaught of drug to promote survival for a sufficient period of time before more permanent resistance mechanisms can be established, consequently yielding a therapeutic opportunity. This may be analogous to antibiotic-tolerant bacterial subpopulations, known as "persisters", which can endure potentially lethal stresses(Balaban et al. 2004). Persisters tend to be slower growing cells, whose survival within a more rapidly growing cell population is ensured by their ability to revert to a non-persister state. Thus, the "burden" of protecting the bulk population from eradication is shared among the whole population(Lewis K. 2007). An analogous sub-population of cells within tumors may similarly contribute to drug tolerance as a means of ensuring the long-term survival of the tumor.

The results presented herein suggest that such a drug-tolerant state may precede stable drug resistance, possibly explaining the successful re-treatment of some EGFR KI-treated NSCLCs after a drug holiday. These observations demonstrate that autophagy modulators such as rapamycin and hydroxychloroquine can be highly effective when used in combination with EGFR KIs, and other selective kinase inhibitors, both to overcome a drug-tolerant state, once established, and to prevent drug tolerance, thereby inhibiting the emergence of drug resistance. Equivalents

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

REFERENCES

Blume- Jensen, P and Hunter, T. Oncogenic Kinase Signalling. Nature 411, 355-365 (2001).

Clark J, Cools J, and Gilliland, JG. EGFR Inhibition in Non-Small Cell Lung Cancer: Resistance, Once Again, Rears its Ugly Head. PLoS Med. 2005 March; 2(3): e75.

Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 47(4), 936-42.1987. Comis RL. The Oncologist, Vol. 10, No. 7, 467-470, August 2005

Fox RI. Mechanism of Action of Hydroxychloroquine as an Antirheumatic Drug. Seminars in Arthritis and Rheumatism. 23, 82-91. 1993.

Fox, RI. Mechanism of action in Autoimmune Disease and Prospects for Drug Development. Lupus. 5 Suppl. 1, S4-10. 1996. Giaccone G, Herbst RS, Manegold C, Scagliotti G, Rosell R, Miller V, Natale

RB, Schiller JH, Von Pawel J, Pluzanska A, Gatzmeier U, Grous J, Ochs JS, Averbuch SD, WoIfMK, Rennie P, Fandi A, Johnson DH. Gefitinib in combination with gemcitabine and cisplatin in advanced non-small-cell lung cancer: a phase III trial— INTACT 1. JCHn Oncol 22(5), 777-84.2004. Herbst RS, Giaccone G, Schiller JH, Natale RB, Miller V, Manegold C,

Scagliotti G, Rosell R, OHfFI, Reeves JA, WoIfMK, Krebs AD, Averbuch SD, Ochs JS, Grous J, Fandi A, Johnson DH. Gefitinib in combination with paclitaxel and carboplatin in advanced non-small-cell lung cancer: a phase III trial — INTACT 2. J Clin Oncol 22(5), 785-94.2004. Kris MG, Natale RB, Herbst RS, Lynch TJ Jr, Prager D, Belani CP, Schiller

JH, Kelly K, Spiridonidis H, Sandler A, Albain KS, Cella D, WoIfMK, Averbuch SD, Ochs JJ, Kay AC. Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer: a randomized trial. JAMA. 16, 2149-58.2003. Salomon DS, Brandt R, Ciardiello F, et al. Epidermal growth factor-related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol. 1995;19:183-232. 1995.

Pao, W. et al. PLoS Med 2, e73 (2005).

Engelman, J. A. et al. Science 316, 1039-43 (2007). Kurata, T. et al. Ann Oncol 15, 173-4 (2004).

Yano, S. et al. Oncol Res 15, 107-11 (2005).

Yokouchi, H. et al. BMC Cancer 7, 51 (2007).

Yoshimoto, A., Inuzuka, K., Kita, T., Kawashima, A. & Kasahara, K. Am J Med Sci 333, 221-5 (2007).

Klionsky, DJ. Nature 431, 31-2 (2004).

Kondo, Y., Kanzawa, T., Sawaya, R. & Kondo, S. Nat Rev Cancer 5, 726-34 (2005).

Rubinsztein, D.C., Gestwicki, J.E., Murphy, L.O. & Klionsky, DJ. Nat Rev Drug Discov 6, 304-12 (2007).

Kroemer, G. & Jaattela, M. Nat Rev Cancer 5, 886-97 (2005).

Levine, B. Nature 446, 745-7 (2007).

Noda, T. & Ohsumi, Y. J Biol Chem 273, 3963-6 (1998).

Davies, H. et al. Nature 417, 949-954 (2002). Balaban, N.Q., Merrin, J., Chait, R., Kowalik, L. & Leibler, S. Science 305,

1622-5 (2004).

Lewis, K. Nat Rev Microbiol 5, 48-56 (2007).

Claims

WE CLAIM:

1. A method of inhibiting growth of tumor cells in a subject, comprising contacting said tumor cells with a cytotoxically effective amount of a compound that raises intralysosomal acidification in combination with an inhibitor of the epidermal growth factor receptor, thereby inhibiting growth of the tumor cells in the subject.

2. The method of claim 1 , wherein the compound that raises intralysosomal acidification is an autophagy modulator.

3. A method of inhibiting growth of tumor cells in a subject, comprising administering to the subject a cytotoxically effective amount of an autophagy modulator, in combination with an inhibitor of the epidermal growth factor receptor, thereby inhibiting growth of the tumor cells in the subject.

4 A method of inhibiting growth of tumor cells that are kinase inhibitor resistant in a subject, comprising administering to the subject a cytotoxically effective amount of a compound that raises intralysosomal acidification in combination with an inhibitor of the epidermal growth factor receptor, thereby inhibiting growth of the tumor cells that are resistant to kinase inhibitor compounds in the subject.

5. The method of claim 5, wherein the compound that raises intralysosomal acidification is an autophagy modulator.

6. A method of inhibiting growth of tumor cells that are resistant to kinase inhibitory compounds in a subject, comprising administering to the subject a cytotoxically effective amount of an autophagy modulator, in combination with an inhibitor of the epidermal growth factor receptor, thereby inhibiting growth of the of tumor cells that are resistant to kinase inhibitor compounds in the subject.

7. The method of claim 4 or 6, wherein the kinase inhibitor compounds are receptor tyrosine kinase inhibitors or non-receptor tyrosine kinase inhibitors, or combinations thereof.

8. The method of claim 1 or 4 wherein the compound that raises intralysosomal acidification is selected from a quinoline compound or bafilomycin Al.

9. The method of any one of claims 2, 3, 5 or 6, wherein the autophagy inhibitor is selected from a quinoline compound or rapamycin.

10. The method of claim 9, wherein said quinoline compound is selected from the group consisting of: chloroquine, hydroxychloroquine and primaquine.

11. The method of claim 3 or 6, wherein said inhibitor of the epidermal growth factor receptor inhibits the activity of the receptor.

12. The method of claim 3 or 6, wherein said inhibitor of the epidermal growth factor receptor inhibits the expression of the receptor.

13. The method of claim 3 or 6, wherein said inhibitor of the epidermal growth factor receptor is an antagonist of the receptor.

14. The method of claim 3 or 6 wherein said inhibitor of the epidermal growth factor receptor is selected from the group consisting of: gefitinib, erlotinib, cetuximab, and panitumumab.

15. The method of any one of claims 1-14, wherein said tumor cells contain an amplified epidermal growth factor receptor gene.

16. The method of any one of claims 1-15, wherein said tumor cells are associated with a cancer selected from the group consisting of: lung, brain, epithelial, colorectal, breast, pancreatic, head and neck cancer, esophageal, gastric and prostate.

17. The method of any one of claims 1-16, wherein said tumor is a solid tumor.

18. The method of any one of claims 1-17, wherein said tumor cells are resistant to one or more inhibitors of the epidermal growth factor receptor.

19. The method of claim 18, wherein said one or more inhibitors of the epidermal growth factor receptor is selected from the group consisting of: gefitinib, erlotinib, cetuximab, and panitumumab.

20. The method of any one of claims 1-19, wherein the combination of said compound that raises intralysosomal acidification with said inhibitor of the epidermal growth factor receptor results in a decrease in growth of tumor cells that is greater than the decrease in growth of tumor cells that results from treatment with either said compound that raises intralysosomal acidification or inhibitor or the epidermal growth factor receptor alone.

21. A method of treating or preventing cancer in a subject comprising administering to the subject a cytotoxically effective amount of a compound that raises intralysosomal acidification in combination with an inhibitor of the epidermal growth factor receptor, thereby treating the cancer in the subject.

22. A method of treating or preventing a drug resistant cancer in a subject comprising administering to the subject a cytotoxically effective amount of a compound that raises intralysosomal acidification in combination with an inhibitor of the epidermal growth factor receptor, thereby treating the drug resistant cancer in the subject.

23. The method of claim 21 or 22, wherein the compound that raises intralysosomal acidification is an autophagy modulator.

24. The method of claim 21 or 22, wherein the drug resistant cancer is resistant to kinase inhibitory compounds.

25. The method of claim 24, wherein the kinase inhibitory compounds are receptor tyrosine kinase inhibitors or non-receptor tyrosine kinase inhibitors, or combinations thereof.

26. The method of claim 21 or 22, wherein the compound that raises intralysosomal acidification is selected from a quinoline compound or bafilomycin Al.

27. The method of claim 23, wherein the autophagy modulator is selected from a quinoline compound and rapamycin.

28. The method of claim 26 or 27, wherein said quinoline compound is selected from the group consisting of: chloroquine, hydroxychloroquine and primaquine.

29. The method of claim 21 or 22, wherein said inhibitor of the epidermal growth factor receptor inhibits the activity of the receptor.

30. The method of claim 21 or 22, wherein said inhibitor of the epidermal growth factor receptor inhibits the expression of the receptor.

31. The method of claim 21 or 22, wherein said inhibitor of the epidermal growth factor receptor is an antagonist of the receptor.

32. The method of claim 21 or 22, wherein said inhibitor of the epidermal growth factor receptor is selected from the group consisting of: gefitinib, erlotinib, cetuximab, and panitumumab.

33. The method of claim 19 or 20, wherein the cancer selected from the group consisting of: lung, brain, epithelial, colorectal, breast, pancreatic, head and neck, esophageal, gastric and prostate cancer.

34. The method of claim 19 or 20, wherein said subject has a cancerous tumor.

35. The method of claim 34, wherein cells of said tumor contain an amplified epidermal growth factor receptor gene.

36. The method of claim 34, wherein said tumor is a solid tumor.

37. The method of claim 34, wherein said tumor is resistant to one or more inhibitors of the epidermal growth factor receptor.

38. The method of claim 37, wherein said one or more inhibitors of the epidermal growth factor receptor is selected from the group consisting of: gefitinib, erlotinib, cetuximab, and panitumumab.

39. The method of claim 21 or 22, wherein the combination of said compound that raises intralysosomal acidification and inhibitor of the epidermal growth factor receptor decreases the size of said tumor.

40. The method of claim 21 or 22, wherein the combination of said compound that raises intralysosomal acidification and inhibitor of the epidermal growth factor receptor results in treatment of cancer that is greater than the treatment with either said compound that raises intralysosomal acidification or inhibitor of the epidermal growth factor receptor alone.

41. A method of treating a cancerous tumor in a subject having a tumor comprising administering to the subject a cytotoxically effective amount of a compound that raises intralysosomal acidification in combination with an inhibitor of the epidermal growth factor receptor, wherein the combination results in a decrease in tumor size that is greater than the decrease in tumor size that results from treatment with either said compound that raises intralysosomal acidification or inhibitor of the epidermal growth factor receptor alone.

42. The method of claim 41, wherein the compound that raises intralysosomal acidification is an autophagy modulator.

43. A method of treating a cancerous tumor in a subject having a tumor comprising administering to the subject a cytotoxically effective amount of an autophagy modulator in combination with an inhibitor of the epidermal growth factor receptor, wherein the combination results in a decrease in tumor size that is greater than the decrease in tumor size that results from treatment with either said autophagy modulator or inhibitor of the epidermal growth factor receptor alone.

44. The method of claim 41 or 43, wherein the cancerous tumor is drug resistant.

45. The method of claim 44, wherein the cancerous tumor is drug resistant to kinase inhibitory compounds.

46. The method of claim 45, wherein the kinase inhibitory compounds are receptor tyrosine kinase inhibitors or non-receptor tyrosine kinase inhibitors, or combinations thereof.

47. The method of claim 41 , wherein the compound that raises intralysosomal acidification is selected from a quinoline compound or bafilomycin Al.

48. The method of claim 47, wherein said quinoline compound is selected from the group consisiting of: chloroquine, hydroxychloroquine and primaquine.

49. The method of claim 41 or 43, wherein said inhibitor of the epidermal growth factor receptor inhibits the activity of the receptor.

50. The method of claim 41 or 43, wherein said inhibitor of the epidermal growth factor receptor inhibits the expression of the receptor.

51. The method of claim 41 or 43 , wherein said inhibitor of the epidermal growth factor receptor is an antagonist of the receptor.

52. The method of claim 41 or 43, wherein said inhibitor of the epidermal growth factor is selected from the group consisting of: gefitinib, erlotinib, cetuximab, and panitumumab.

53. The method of claim 41 or 43, wherein said tumor contains cells having an amplified epidermal growth factor receptor gene.

54. The method of claim 41 or 43, wherein said tumor is associated with a cancer selected from the group consisting of: lung, brain, epithelial, colorectal, breast, pancreatic, head and neck, esophageal, gastric and prostate cancer.

55. The method of claim 41 or 43, wherein said tumor is resistant to one or more inhibitors of the epidermal growth factor receptor.

56. The method of claim 55, wherein said inhibitor of the epidermal growth factor is selected from the group consisting of: gefitinib, erlotinib, cetuximab, and panitumumab.

57. The method of claim any preceding claim, further comprising obtaining the cytotoxic inhibitor of lysosomal acidification and/or the inhibitor of the epidermal growth factor receptor.

58. A pharmaceutical composition comprising a therapeutically effective amount of a cytotoxic compound that raises intralysosomal acidification , an inhibitor of the epidermal growth factor receptor, and a pharmaceutically acceptable carrier.

59. A pharmaceutical composition comprising a therapeutically effective amount of a cytotoxic compound that modulates autophagy, an inhibitor of the epidermal growth factor receptor, and a pharmaceutically acceptable carrier.

60. The composition of claim 58 and 59, wherein said cytotoxic quinoline compound is selected from the group consisiting of: chloroquine, hydroxychloroquine and primaquine.

61. The composition of claim 58 or 59, wherein said inhibitor of the epidermal growth factor receptor is selected from the group consisting of gefitinib, erlotinib, cetuximab and panitumumab.

62. The composition of claim 58 or 59, wherein said cytotoxic compound that raises intralysosomal acidification is between about 60 to about 90% weight of the composition and the inhibitor of the epidermal growth factor receptor is between about 10 to about 40% weight of the composition.

63. The composition of claim 58 or 59, wherein said cytotoxic compound that modulates autophagy is between about 60 to about 90% weight of the composition and the inhibitor of the epidermal growth factor receptor is between about 10 to about 40% weight of the composition.

64. The composition of claim 58 or 59, wherein said ratio of the cytotoxic compound that raises intralysosomal acidification to the inhibitor of the epidermal growth factor receptor is between about 60: 40 to about 90: 10.

65. The composition of claim 58 or 59, wherein said ratio of the cytotoxic compound that modulates autophagy to the inhibitor of the epidermal growth factor receptor is between about 60: 40 to about 90: 10.

66. A packaged pharmaceutical comprising a cytotoxic compound that raises intralysosomal acidification and an inhibitor of the epidermal growth factor receptor and associated instructions for using said cytotoxic inhibitor of lysosomal acidification and inhibitor of the epidermal growth factor receptor to treat cancer.

67. The packaged pharmaceutical of claim 66, wherein said cytotoxic compound that raises intralysosomal acidification is selected from the group consisting of: chloroquine, hydroxychloroquine and primaquine.

68. The packaged pharmaceutical of claim 66, wherein said inhibitor of the epidermal growth factor receptor is selected from the group consisting of gefitinib, erlotinib, cetuximab and panitumumab.

69. The packaged pharmaceutical of claim 66, wherein said cytotoxic compound that raises intralysosomal acidification is between about 60 to about 90% weight of the composition and the inhibitor of the epidermal growth factor receptor is between about 10 to about 40% weight of the composition.

70. The packaged pharmaceutical of claim 66, wherein the ratio of the cytotoxic compound that raises intralysosomal acidification to the inhibitor of the epidermal growth factor receptor is between about 60:40 to about 90:10.

71. The packaged pharmaceutical of claim 66, wherein the instructions are for use in accordance with the method of any one of claims 1-57.

72. A kit comprising a cytotoxic compound that raises intralysosomal acidification and an inhibitor of the epidermal growth factor receptor and associated instructions for using said cytotoxic compound that raises intralysosomal acidification and inhibitor of the epidermal growth factor receptor to treat cancer.

73. The kit of claim 72, wherein said cytotoxic compound that raises intralysosomal acidification is selected from the group consisting of: chloroquine, hydroxychloroquine and primaquine.

74. The kit of claim 72, wherein said inhibitor of the epidermal growth factor receptor is selected from the group consisting of gefitinib, erlotinib, cetuximab and panitumumab.

75. The kit of claim 72, wherein said cytotoxic compound that raises intralysosomal acidification is between about 60 to about 90% weight of the composition and the inhibitor of the epidermal growth factor receptor is between about 10 to about 40% weight of the composition.

76. The kit of claim 75, wherein the ratio of the compound that raises intralysosomal acidification to the inhibitor of the epidermal growth factor receptor is between about 60:40 to about 90:10.

77. The kit of any one of claims 72-76 - 69, wherein the compound that raises intralysosomal acidification is an autophagy modulator.

78. A kit comprising an autophagy modulator and an inhibitor of the epidermal growth factor receptor and associated instructions for using said an autophagy modulator and inhibitor of the epidermal growth factor receptor to treat cancer.

79. The kit of claim 78, wherein said an autophagy modulator is selected from the group consisting of chloroquine, hydroxychloroquine and primaquine.

80. The kit of claim 78, wherein said inhibitor of the epidermal growth factor receptor is selected from the group consisting of gefitinib, erlotinib, cetuximab and panitumumab.

81. The kit of claim 78, wherein said an autophagy modulator is between about 60 to about 90% weight of the composition and the inhibitor of the epidermal growth factor receptor is between about 10 to about 40% weight of the composition.

82. The kit of claim 78, wherein the ratio of the autophagy modulator to the inhibitor of the epidermal growth factor receptor is between about 60:40 to about 90:10.

83. The kit of any one of claims 72-82, further comprising a packaging means thereof.