WO2021086912A1 - Combined pikfyve and p38 map kinase inhibition for treating cancer - Google Patents

Abstract

Disclosed is a method for treating a cancer in a mammal in need thereof, comprising administering to the mammal a combination of effective amount of an autophagy inhibitor and a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor, wherein the effective amount is sufficient to reduce or inhibit proliferation of cancer cells or to cause the death of cancer cells.

Description

COMBINED PIKFYVE AND P38 MAP KINASE INHIBITION FOR TREATING

CANCER

CROSS-REFERENCE TO A RELATED APPLICATION

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 62/927,966, filed October 30, 2019, the disclosure of which is incorporated by reference herein in its entirety for all purposes.

STATEMENT REGARDING

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under Grant Numbers HD000506 and HD000507 awarded by the National Institutes of Health. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] Autophagy is a cellular process responsible for degradation of damaged or unnecessary cellular organelles and proteins resulting in the release of amino acids, sugars, fatty acids, and nucleosides that are recycled for macromolecular synthesis and energy production. This recycling system is particularly important during starvation. Cancer cells are thought to use autophagy as a source of energy in unfavorable metastatic or compact tumor environment and thus become autophagy-dependent for proliferation and survival.

The idea that inhibition of autophagy can increase the effectiveness of cancer therapies has been studied for decades; chloroquine and the closely related hydroxychloroquine, the only available autophagy inhibitors, were studied in numerous clinical trials (1). However, decades of research have failed to produce successful clinical application, perhaps because chloroquine and its derivatives are not specific autophagy inhibitors but also inhibit other cellular functions such as endocytosis with unexpected side effects on organs, such as the kidney (2).

[0004] PIKfy ve is a lipid kinase that phosphorylates the 5- site of the phosphatidylinositol molecule involved in regulation of a variety of endosomal and membrane trafficking pathways (6). Over the past 15 years, newly discovered chemical inhibitors revealed that PIKfy ve activity is essential for normal lysosomal function, including heterotypic fusion with autophagosomes. Thus, PIKfyve inhibition leads to an accumulation of enlarged lysosomes readily visible under light microscopy and prevents recycling of cellular components through autophagy. PIKfyve inhibitors YM201636, Apilimod, and compound 1 (described herein) have been shown to exhibit selective toxicity against certain cancer cell lines in vitro. Still, only a handful of cancers thus far have been investigated as potential targets for therapeutic intervention with PIKfyve inhibitors.

[0005] p38 mitogen-activated protein kinases (p38 MAPKs) are a class of evolutionarily conserved serine/threonine mitogen-activated protein kinases that transfer extracellular signals to the intracellular machinery to regulate cell growth, differentiation, migration, autophagy, apoptosis, etc. Along with c-Jun N-terminal kinase (JNK), they are described as stress-activated protein kinases (SAPKs) because they are activated by a wide range of stress-inducing stimuli (7). Several recent studies provide new insights into how SAPK pathways function in the control of the balance of autophagy and apoptosis in response to genotoxic stress suggesting that modulation of their activity be considered as a way to improve cancer therapy success (8). Recent clinical trials have utilized the p38 MAPK inhibitor Ralimetinib (LY2228820) in combination with traditional chemotherapies with the rationale that p38 MAPK pathway activity has a compensatory function when malignant cells respond to stressors which helps them survive cytotoxic cancer therapies. The foregoing shows that there exists an unmet need for treating cancer, particularly by increasing the selectivity of autophagy inhibitors in autophagy-dependent cancer cells versus normal cells.

BRIEF SUMMARY OF THE INVENTION

[0006] The invention provides a method for treating a cancer in a mammal in need thereof, comprising administering to the mammal a combination of an effective amount of an autophagy inhibitor and an effective amount of a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor, wherein the effective amount is sufficient to reduce or inhibit proliferation of cancer cells or to cause the death of cancer cells. The method of the present invention has one or more of the following characteristics or advantages.

[0007] Cancer cells have variant sensitivity to PIKfyve inhibition that is inversely correlated with their level of p38 MAPK activity. Moreover, the results suggest a consistently similar relationship between sensitivity to PIKfyve and p38 MAPK inhibition, suggesting a functional link between the two pathways. p38 MAPK protein and phosphorylation levels in tumors may also serve as biomarkers for sensitivity to PIKfyve inhibitors. [0008] p38 MAPK inhibition synergistically enhances the selective anti-cancer activity of

PIKfyve inhibitors. Combined p38 MAPK and PIKfyve inhibition has synergistic anti proliferative effect in a wide variety of cancers in vitro, including those of lung, colon, bone, brain, breast, and cervix origins.

[0009] The synergistic cytotoxic effect is not restricted to specific PIKtyve or p38 MAPK chemical inhibitors, but is based on the functional cooperation of the two pathways. The combined inhibition causes dramatic synergistic inhibition of autophagy that selectively kills cancer cells. Therefore, combinations of PIKtyve and p38 MAPK inhibitors elicit a synergistic anti-cancer killing effect.

[0010] Combined treatment with PIKfyve and p38 MAPK inhibitors more than doubles the length of time and magnitude of autophagy inhibition in cancer cells. Such a long-term effect reduces the frequency of patient treatment while maintaining full anti-cancer toxicity. [0011] Cytotoxicities of the general autophagy inhibitors chloroquine and hydroxychloroquine are also synergistically enhanced by p38 MAPK inhibition to selectively target cancer cells.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0012] FIG. 1 shows that human cell lines have different sensitivity to PIKfyve inhibition by compound 1. Twelve cell lines were treated with indicated concentrations of compound 1 or vehicle (DMSO). After 3 days, attached and floating cells were collected and total number of cells was counted. Results are plotted as fraction of vehicle-treated samples for each cell line. To plot the concentrations of compound 1 used in logarithmic scale, the vehicle was set to 0.001 mM instead of 0 pM.

[0013] FIG. 2A and 2B show that the specific inhibition of PIKfyve activity is solely responsible for the cytotoxic effects of compound 1, compound 4, Vacuolin-1 and Apilimod. Wildtype and mutant N1939K PIKfyve were stably expressed in A375 melanoma cells. The number of cells was counted after 3 days of incubation with IC75 concentrations of PIKfyve inhibitors (3.75 pM compound 1, 6 pM compound 4, 5 pM Vacuolin-1, 0.3 pM Apilimod, 12.5 pM YM201636). FIG. 2A depicts in vitro images which show vacuolization in wildtype PIKfyve expressing A375 cells treated with compound 1, compound 4, Vacuolin-1,

Apilimod, and YM201636. Compound 1, compound 4, Vacuolin-1, and Apilimod did not cause vacuoles in N1939K mutant PIKfyve A375 cells. The mutant did not affect YM201636-induced vacuolization. FIG. 2B depicts the effect of PIKfyve mutant on cytotoxicity of PIKfyve inhibitors. Mutant PIKfyve confers resistance to compound 1, compound 4, Vacuolin-1, and Apilimod. YM201636 activity was not affected by the mutant. [0014] FIG. 3 shows that human cells have different steady state p38 MAPK protein levels and p38 MAPK phosphorylation. Still, the p38 MAPK stress response pathway was activated in all tested cells in response to UV irradiation induced DNA damage. Twelve human cell lines were irradiated with 80 Joules of UV light and collected 30 minutes later. The phosphorylation levels of MKK3/6, p38 MAPK, and Hsp27 were determined by immunoblotting to determine the steady state (-UV) and activated levels (+UV) of p38 MAPK stress response pathway marker proteins.

[0015] FIG. 4 shows that human cells have different sensitivity to p38 MAPK inhibition by SB202190. Twelve cell lines were treated with indicated concentrations of SB202190 or vehicle (DMSO). After 3 days, attached and floating cells were collected and total number of cells was counted. The results are plotted as fraction of vehicle-treated samples for each cell line. To plot the concentrations of SB202190 used in logarithmic scale, the vehicle was set to 0.001 mM instead of 0 pM.

[0016] FIG. 5 A and 5B show that the sensitivity of human cells to PIKfyve and p38

MAPK inhibition correlates inversely with the cellular levels of p38 MAPK phosphorylation. Open circles are cancer cell lines and closed circles are normal fibroblasts. FIG. 5A: Quantification of immunoblot determination of steady state p38 MAPK phosphorylation levels (Fig. 3) and the compound 1 and SB202190 IC50 values (Figs. 1 and 4) of 12 human cell lines. FIG. 5B depicts the IC50 values of compound 1 versus basal p38 MAPK phosphorylation levels. A strong inverse correlation was observed between sensitivity to compound 1 and steady state p38 MAPK phosphorylation. FIG. 5C depicts the IC50 values of Compound 1 IC50 values versus SB202190 IC50 values. A strong correlation was observed between sensitivity to compound 1 and sensitivity to SB202190.

[0017] FIG. 6 A and 6B show that cancer cells are sensitive to PIKfyve and p38 MAPK inhibition while normal cells are resistant. Three cancer (HCT116, A549, and SF-295) and two normal (Hs27 and WI38) cell lines were treated with PIKfyve inhibitor (0.5 pM compound 1) and p38 MAPK inhibitor (lOpM SB202190) individually or in combination for 3 days and the total number of cells counted to determine the effect on cell survival. FIG. 6A depicts the effect of compound 1 and SB202190 individually and in combination on cell survival. Results are plotted as a fraction of the vehicle-treated cells counts for each line. FIG. 6B depicts the combined inhibition of PIKfyve and p38 MAPK caused severe morphological changes consistent with cell death in A549 cancer cells but not in Hs27 normal fibroblasts. [0018] FIGS. 7A-7E show that combinational treatment with compound 1 and SB202190 produces a selective synergistic effect on cell viability over a broad range of concentrations. FIG. 7A depicts a dosage matrix of A549 cells with compound 1 (0.03 - 4 mM) and SB202190 (1.25 - 20 pM) as determined using an ATP-based cell viability assay. FIG. 7B depicts the synergy of inhibitor combinations from (A). Synergy is calculated as the difference between the observed combination toxicity and expected toxicity if the combination were additive. FIG. 7C depicts the Chou-Talalay combinational index (Cl) which was calculated using the CompuSyn software (http://www.combosyn.com). Combinations with Cl values less than 1.0 are considered synergistic. FIG. 7D depicts a dosage matrix of Hs27 cells with compound 1 (0.03 - 4 pM) and SB202190 (1.25 - 20 pM) as determined using an ATP-based cell viability assay. FIG. 7E depicts the difference between Hs27 and A549 cell survival as reported in FIGS.

[0019] FIG. 8 shows synergistic effects from compound 1 and SB202190 in combination can be observed in multiple cell lines with diverse genetic and tissue origin profiles. Dosage matrices of six different cell lines (HCT116, SW480, SF-295, MDA-MB-231, HeLa, and SHP-77) with compound 1 and SB202190 as determined using an ATP-based cell viability assay. Synergy is calculated as the difference between the observed toxicity and expected toxicity if the combination were additive. The Chou-Talalay combinational index (Cl) was calculated using the CompuSyn software (http://www.combosyn.com). Combinations with Cl values less than 1.0 are considered synergistic.

[0020] FIG. 9 A and 9B show that multiple structurally diverse PIKfyve and p38 MAPK inhibitors produce consistent synergistic anti-cancer effects that are not limited to a particular chemical structure but are due to functional cooperation between the PIKfyve and p38 MAPK pathways. Synergy is reported as the difference between the observed toxicity and expected toxicity if a combination has a simple additive effect. FIG. 9A depicts cell counts plotted as a fraction of the vehicle treated cells counts for each sample observed after treatment of A549 and Hs27 cells with five PIKfyve inhibitors (0.375 mM compound 1, 0.6 pM compound 4, 0.5 pM Vacuolin-1, 30 nM Apilimod, and 1.25 pM YM201636) alone or in combination with 7.5 pM SB202190 for 3 days. FIG. 9B depicts cell counts plotted as a fraction of the vehicle treated cells counts for each sample observed after treatment of A549 and Hs27 cells with five p38 MAPK inhibitors (7.5 pM SB202190, 7.5 pM LY2228820, 7.5 mM Skepinone-L, 5 mM TAK-715, and 10 mM BIRB-796) alone or in combination with 0.375 mM compound 1 for 3 days.

[0021] FIG. 10A-10C show Western blot of autophagy -related proteins that demonstrates that PIKfy ve and p38 MAPK inhibition has a synergistic inhibitory effect on autophagy.

Time dynamics of (A) p62, (B) LC3, and (C) LAMP2 protein changes upon treatment with compound 1, SB202190, and the two inhibitors together are shown.

[0022] FIG. 11A and 1 IB show that p38 MAPK inhibitors disrupt autophagy and have cooperative effects when combined with a PIKfy ve inhibitor. FIG. 11 A depicts Western blot (A) of A549 cells treated with 7.5 mM SB202190 and 7.5 mM LY2228820 individually or in combination with 0.375 mM compound 1 for 1 day. FIG. 1 IB depicts the result of protein quantification from (A).

[0023] FIG. 12 shows that Bafilomycin A1 prevents vacuole accumulation caused by PIKfy ve and p38 MAPK inhibition demonstrating that both pathways inhibit autophagy by related mechanisms. A549 cancer cells were treated with 50 nM Bafilomycin A or vehicle (DMSO) and 1 hour later 7.5 mM SB202190 or 0.375 mM compound 1 were added individually or in combination for 6 more hours.

[0024] FIG. 13A and 13B show that the combined inhibition of p38 MAPK and PIKfyve has a prolonged inhibitory effect on autophagy that persists even after the inhibitors were removed. A549 cancer cells were treated with 7.5 mM SB202190 or 0.375 mM compound 1 individually or in combination for 1 day, then were washed to remove the inhibitors and samples were collected 1 and 2 days later. The protein level of the p62 autophagy marker was determined by immunoblotting (FIG. 13 A) to quantify the relative degree of autophagy inhibition (FIG. 13B).

[0025] FIG. 14A and 14B show that autophagy inhibitors chloroquine and hydroxychloroquine have synergistic anti-cancer effect when combined with the p38 MAPK inhibitor SB202190. Dosage matrices were generated using ATP-based cell viability assay. Synergy is calculated as the difference between the observed toxicity and expected toxicity if the combination has simple additive effect. The Chou-Talalay combinational index (Cl) was calculated using the CompuSyn software (http://www.combosyn.com). Cl values less than 1.0 are considered synergistic. Dosage matrices of A549 and Hs27 cell lines are compared for each inhibitor combination. FIG. 14A depicts the dosage matrix for Chloroquine (1.25 - 20 mM) with SB202190 (2.5 - 7.5 mM). FIG. 14B depicts the dosage matrix for Hydroxychloroquine (1.25 - 20 mM) with SB202190 (2.5 - 7.5 mM). [0026] FIG. 15 shows the structures of exemplary PIKfyve inhibitors compound 1, Vacuolin-1, Apilimod, compound 4, and YM201636, p38 MAPK inhibitors SB202190, LY2228820, TAK-715, BIRB-796, and Skepinone-L, and autophagy inhibitors chloroquine and hydroxychloroquine.

[0027] FIG. 16 shows immunoblot analysis of the phosphorylation of MKK3/6, p38 MAPK, HSP27, and H2AX proteins and the amount of p38 MAPK, PIKfyve, and TFEB proteins in exponentially growing and UV-irradiated cells (30 minutes after 80 Joules) from 18 human lines.

[0028] FIG. 17A-D show that p38 MAPK phosphorylation inversely correlates with sensitivity to PIKfyve inhibition. All cells were collected, counted, and analyzed by FACS. The number of viable cells for each treatment was presented as a percentage from the control viable cells “Cell Survival (%). FIG. 17A shows the calculated IC50 concentrations (50% reduction in viability) of compound 1 and SB202190 and quantified p38 MAPK basal phosphorylation levels for 18 cell lines. FIG. 17B show a plot of the p38 MAPK basal phosphorylation levels versus compound 1 IC50 values for 18 cell lines. FIG. 17C shows a plot of the p38 MAPK basal phosphorylation levels versus SB202190 IC50 values for 18 cell lines. FIG. 17D shows a plot of compound 1 versus SB202190 IC50 values for 18 cell lines. [0029] FIG. 18A-F show that p38 MAPK and PIKfyve inhibitors synergistically and selectively reduce cancer cell viability. FIG. 18A show a compound 1 and SB202190 dosage matrix performed with SW480 cells cultured for three days with the indicated drug concentrations. All cells were collected, counted, and analyzed by FACS. The number of viable cells for each treatment was presented as a percentage from the control viable cells “Cell Survival (%)”. FIG. 18B shows the synergy above additive as calculated by subtracting the individual toxi cities of compound 1 and SB202190 from the toxicity of their combinations. Positive numbers indicate a synergistic effect above the expected additive effect. FIG. 18C shows the Chou-Talalay combinational index (Cl) as calculated for each compound 1 and SB202190 combination in FIG. 18A. The degree of synergism is measured by the deviation of Cl from 1: the smaller the Cl, the larger the synergistic effect. FIG. 18E shows images and FACS generated DNA size histograms with “dead cells (%)” for one inhibitor combination. FIG. 18F shows the cell survival (%) and dead cells (%) results from a compound 1 and SB202190 dosage matrix generated with Hs27 cells under the same conditions used in FIG. 18A. [0030] FIG. 19A-F show that p39 MAPK and PIKfyve inhibitors synergistically and selectively reduce the viability of multiple cancer cell types. The compound 1 and SB202190 dosage matrices were performed with the indicated cell lines by culturing for three days with the indicated drug concentrations. All cells were collected, counted, and analyzed by FACS. The number of viable cells for each treatment was presented as a percentage from the control viable cells (“cell survival (%)”). Synergy above additive was calculated by subtracting the individual toxicities of compound 1 and SB202190 from the toxicity of their combination. Positive numbers indicate a synergistic effect above the expected additive effect. The Chou-Talalay combinational index (Cl) was calculated for each compound 1 and SB202190 combination in the dosage matrix. The degree of synergism was measured by the deviation of Cl form 1: the smaller the Cl, the larger the synergistic effect. “Dead cells (%)” indicate the percentage of dead cells caused by each treatment.

[0031] Fig. 20A-J show that p38 MAPK and PIKfyve inhibitors synergistically block autophagy -mediated protein degradation and maturation of lysosomal enzymes. SW480 cells were cultured with either 0.125 mM compound 1 or 5 pM SB202190 or both together for the time indicated. The amounts of p62 (FIG. 20A and 20E), LC3 (FIG. 20B and 20F), LAMP2 (FIG. 20C and 20G) and precursor and mature cathepsin D (FIG. 20H, 201, and 20J) proteins were determined by immunoblot, normalized by histone content (FIG. 20D), and plotted as a percentage of the protein present at time zero.

[0032] FIG. 21A-B show that specific inhibition of PIKfyve and p38 MAPK activities are responsible for the effects on cellular viability and autophagy. SW480 and Hs27 cell lines were cultured for 3 days with the indicated inhibitors. All cells were collected, counted, and the number of cells cultured with inhibitor reported as a percentage of the number of cells cultured with vehicle. FIG. 21 A shows the results with PIKfyve inhibitors (0.125 pM compound 1, 0.3 pM compound 4, 0.25 pM Vacuolin, 30 nM Apilimod, 0.6 pM YM201636) present either alone (individual inhibitors) or together with 5 pM SB202190. FIG. 21B shows the results with p38 MAPK inhibitors (5 pM SB202190, 5 pM LY2228820, 5 pM Skepinone-L, 3.5 pM TAK-715, 7.5 pM BIRB-796) were present either alone (individual inhibitors) or together with 0.125 pM compound 1. The “above additive effect” was calculated as in FIG. 18. Values are average of three independent experiments and error bars represent SEM.

[0033] FIG. 22A-B shows that specific inhibition of PIKfyve and p38 MAPK activities are responsible for the effects on cellular viability and autophagy. FIG. 22A shows that p38 MAPK was depleted with the indicated concentrations of p38 siRNA or control siRNA and incubated with 0.375 mM compound 1. Cells were collected after 3 days, and FIG. 22B shows counts plotted as a percentage of the control siRNA treated cells. Error bars indicate SEM.

[0034] FIG. 23A-B show that specific inhibition of PIKfyve and p38 MAPK activities are responsible for the effects on cellular viability and autophagy. FIG. 23A shows immublot of cells cultured for 1 day or 3 days with individual p38 MAPK inhibitors or p38 MAPK inhibitors in combination with compound 1. FIG. 23B shows quantification of the amount of p62 protein on day 3 as a percentage of the amount in vehicle treated cells after normalization by histone content.

[0035] FIG. 24A-G show that combined PIKfyve and p38 MAPK inhibition synergistically inhibits xenograft tumor growth. Mice bearing xenografts of SW480 colon carcinoma cells were injected daily with either vehicle, 20 mg/kg compound 1, 12.5 mg/kg SB202190, or 20 mg/kg compound 1 + 12.5 mg/kg SB202190. FIG. 24A shows tumor volumes as a function of time in mice bearing SW480 cell tumors treated with the four treatments. FIG. 24B shows the relative average tumor volumes on day 22 for each treatment group. FIG. 24C shows the average weight of the mice in each group during the experiment. FIG. 24D shows images of representative tumors from each group compared with a centimeter scale ruler and Hematoxylin and Eosin and Ki-67 staining of tumor sections from each group. FIG. 24E shows Ki-67 positive tumor cells as a percentage of those in tumors from vehicle injected mice. A minimum of 1000 cells were counted per sample.

[0036] FIG. 24F shows analysis of total cell lysates from tumors depicted in FIG. 24D as analyzed by immunoblot for the indicated proteins. FIG. 24G shows the p62 protein amount plotted as a percentage of vehicle sample after normalization by histone content. Error bars indicates SEM.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The invention provides a method for treating a cancer in a mammal in need thereof, comprising administering to the mammal a combination of an effective amount of an autophagy inhibitor and an effective amount of a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor, wherein the effective amount is sufficient to reduce or inhibit proliferation of cancer cells or to cause the death of cancer cells. [0038] Advantageously, the autophagy inhibitor and the p38 MAPK inhibitor yield a synergistic effect in treating cancer.

[0039] In certain embodiments, the autophagy inhibitor is a compound of formula (I):

wherein R¹ is (a) -NR³R⁴ wherein R³ and R⁴ are independently H, optionally substituted C1-C6 alkyl, bisphenylmethyl, or optionally substituted C6-Cioaryl, (b) morpholino, or (c) 2-pyridyl-(CH2)n-0-, wherein n is an integer of from 1 to about 10,

R² is optionally substituted C6-C10 aryl or a group of the formula: R⁵CH=N- wherein R⁵ is C6-C10 aryl or heteroaryl,

X is CH orN, or a tautomer thereof; a compound of formula (II):

or a compound of formul

wherein R⁶ is H or OH, or a pharmaceutically acceptable salt thereof.

[0040] In an aspect, the autophagy inhibitor is a compound of formula (I) and X is N. [0041] In certain aspects, R¹ is morpholinyl, and R² is optionally substituted C6-C10 aryl. [0042] In certain particular aspects, the autophagy inhibitor is:

[0043] In certain aspects, R² is R⁵CH=N-, R¹ is -NR³R⁴, R³ is H, and R⁴ is optionally substituted C6-C10 aryl.

[0044] In a particular aspect, the autophagy inhibitor is:

[0045] In certain aspects, R² is R⁵CH=N, R¹ is -NR³R⁴, R³ H, and R⁴ is bisphenylmethyl. [0046] In a particular aspect, the autophagy inhibitor is:

[0047] In an aspect of formula (I), X is CH.

[0048] In certain aspects, R² is R⁵CH=N- and wherein R¹ is morpholinyl.

[0049] In certain particular aspects, the autophagy inhibitor is:

[0050] In a particular aspect, the autophagy inhibitor is:

[0051] In a particular aspects, the autophagy inhibitor is:

[0052] In certain particular aspects, the autophagy inhibitor is:

[0053] In certain aspects, the p38 MAPK inhibitor is selected from: [0054] In an aspect, the cancer is an autophagy -dependent cancer.

[0055] In certain aspects, the autophagy inhibitor is a PIKfy ve inhibitor.

[0056] In an aspect, the cancer is a malignant, metastatic cancer.

[0057] In certain aspects, the cancer is selected from non-small cell lung carcinoma, lung small cell carcinoma, colorectal carcinoma, breast adenocarcinoma, cervix adenocarcinoma, brain glioblastomal carcinoma, malignant melanoma, thyroid carcinoma, ovarian carcinoma, and leukemia.

[0058] In an aspect, the method selectively kills cancer cells.

[0059] In certain aspects, the cancer cells are selected from breast cancer cells, malignant melanoma cells, colorectal carcinoma cells, thyroid papillary carcinoma cells, glioma cells, ovarian serous carcinoma cells, lung adenocarcinoma cells, hairy cell leukemia cells, or cervical carcinoma cells.

[0060] Referring now to terminology used generically herein, the term “alkyl” means a straight-chain or branched alkyl substituent containing from, for example, 1 to about 6 carbon atoms, preferably from 1 to about 4 carbon atoms, more preferably from 1 to 2 carbon atoms. Examples of such substituents include methyl, ethyl, propyl, isopropyl, «-butyl, .sec-butyl isobutyl, tert- butyl, pentyl, isoamyl, hexyl, and the like.

[0061] The term “aryl” refers to an unsubstituted or substituted aromatic carbocycbc substituent, as commonly understood in the art, and the term “C6-C10 aryl” includes phenyl and naphthyl. It is understood that the term aryl applies to cyclic substituents that are planar and comprise 4n+2 p electrons, according to HiickeTs Rule.

[0062] The term “heteroaryl” refers to a monocyclic or bicyclic (i.e.., fused heteroaryl)

5- or 6-membered ring system as described herein, wherein the heteroaryl group is unsaturated and satisfies HiickeTs rule. Non-limiting examples of suitable heteroaryl groups include furanyl, thiopheneyl, pyrrolyl, pyrazolyl, imidazolyl, 1,2,3-triazolyl, 1 ,2,4-triazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, l,3,4-oxadiazol-2-yl, l,2,4-oxadiazol-2-yl, 5- methyl-l,3,4-oxadiazole, 3-methyl-l,2,4-oxadiazole, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, benzofuranyl, benzothiopheneyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolinyl, benzothiazolinyl, and quinazolinyl. The heteroaryl group is optionally substituted with 1, 2, 3, 4, or 5 substituents as recited herein such as with alkyl groups such as methyl groups, ethyl groups, and the like, halo groups such as chloro, or hydroxyl groups, with aryl groups such as phenyl groups, naphthyl groups and the like, wherein the aryl groups can be further substituted with, for example halo, dihaloalkyl, trihaloalkyl, nitro, hydroxy, alkoxy, aryloxy, amino, substituted amino, alkylcarbonyl, alkoxy carbonyl, arylcarbonyl, aryloxy carbonyl, thio, alkylthio, arylthio, and the like, wherein the optional substituent can be present at any open position on the heterocyclyl or heteroaryl group, or with benzo groups, to form a group of, for example, benzofuran or indolyl.

[0063] The phrase “pharmaceutically acceptable salt” is intended to include non-toxic salts synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington ’s Pharmaceutical Sciences , 18th ed., Mack Publishing Company, Easton, PA, 1990, p. 1445, and Journal of Pharmaceutical Science, 66, 2-19 (1977).

[0064] Suitable bases include inorganic bases such as alkali and alkaline earth metal bases, such as those containing metallic cations such as sodium, potassium, magnesium, calcium and the like. Non-limiting examples of suitable bases include sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate. Suitable acids include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as /Mol uenesul Tonic. methanesulfonic acid, benzenesulfonic acid, oxalic acid, / bromophenylsul Tonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, maleic acid, tartaric acid, fatty acids, long chain fatty acids, and the like. Preferred pharmaceutically acceptable salts of inventive compounds having an acidic moiety include sodium and potassium salts. Preferred pharmaceutically acceptable salts of inventive compounds having a basic moiety (such as a dimethylaminoalkyl group) include hydrochloride and hydrobromide salts. The compounds of the present invention containing an acidic or basic moiety are useful in the form of the free base or acid or in the form of a pharmaceutically acceptable salt thereof.

[0065] It should be recognized that the particular counterion forming a part of any salt of this invention is usually not of a critical nature, so long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole.

[0066] It is further understood that the above compounds and salts may form solvates, or exist in a substantially uncomplexed form, such as the anhydrous form. As used herein, the term "solvate" refers to a molecular complex wherein the solvent molecule, such as the crystallizing solvent, is incorporated into the crystal lattice. When the solvent incorporated in the solvate is water, the molecular complex is called a hydrate. Pharmaceutically acceptable solvates include hydrates, alcoholates such as methanolates and ethanolates, acetonitrilates and the like. These compounds can also exist in polymorphic forms.

[0067] In any of the above embodiments, the compound or salt can exist in one or more tautomeric forms. The term "tautomer" as used herein includes two or more interconvertable compounds resulting from at least one formal migration of a hydrogen atom and at least one change in valency (e.g., a single bond to a double bond, a triple bond to a single bond, or vice versa). The exact ratio of the tautomers depends on several factors, including temperature, solvent, and pH. Tautomerizations (i.e., the reaction providing a tautomeric pair) may catalyzed by acid or base. Exemplary tautomerizations include keto-to-enol; amide-to-imide; lactam-to-lactim; enamine-to-imine; and enamine-to-(a different) enamine tautomerizations. In an example, when R³ is a group of the formula: R⁴CH=N- and R⁴ includes a CH group bonded to the CH of R⁴CH=N-, such as -CH-CH=N-, a tautomer can be represented by the formula: -C=CH-NH-. Thus, the following structural representations are tautomeric to each other:

[0068] The dose administered to a mammal, particularly, a human, in accordance with the present invention should be sufficient to effect the desired response. Such responses include reversal or prevention of the adverse effects of the disease for which treatment is desired or to elicit the desired benefit. One skilled in the art will recognize that dosage will depend upon a variety of factors, including the age, condition, and body weight of the human, as well as the source, particular type of the disease, and extent of the disease in the human. The size of the dose will also be determined by the route, timing and frequency of administration as well as the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular compound and the desired physiological effect. It will be appreciated by one of skill in the art that various conditions or disease states may require prolonged treatment involving multiple administrations.

[0069] Suitable doses and dosage regimens can be determined by conventional range- finding techniques known to those of ordinary skill in the art. Generally, treatment is initiated with smaller dosages that are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. The present inventive method typically will involve the administration of about 0.1 to about 300 mg of one or more of the compounds described above per kg body weight of the animal or mammal.

[0070] Any of the autophagy inhibitors can be administered in combination with any of the p38 MAPK inhibitors, e.g., simultaneously, sequentially, e.g., the autophagy inhibitor administered before the p38 MAPK inhibitor or vice-versa, or cyclically. In some embodiments, it is suitable to administer two or more separate and distinct formulations, one of which contains the autophagy inhibitor and the other contains the p38 MAPK inhibitor.

The separate and distinct formulations can be administered simultaneously, or the formulations can be administered separately at different time periods. In some embodiments, the autophagy inhibitor and the p38 MAPK inhibitor can be administered in combination in the same formulation, i.e., in a single dosage form. [0071] The therapeutically effective amount of the compound or compounds administered can vary depending upon the desired effects and the factors noted above. Typically, dosages of each of the compounds individually in the combination treatment (i.e., the autophagy inhibitor and the p38 MAPK inhibitor) can be between 0.01 mg/kg and 250 mg/kg of the subject’s body weight, and more typically between about 0.05 mg/kg and 100 mg/kg, such as from about 0.2 to about 80 mg/kg, from about 0.5 to about 40 mg/kg or from about 0.5 to about 30 mg/kg of the subject’s body weight. Thus, unit dosage forms can be formulated based upon the suitable ranges recited above and the subject’s body weight. The term “unit dosage form” as used herein refers to a physically discrete unit of therapeutic agent appropriate for the subject to be treated.

[0072] Alternatively, dosages are calculated based on body surface area and from about 1 mg/m² to about 200 mg/m², such as from about 5 mg/m² to about 100 mg/m² will be administered to the subject per day. In particular embodiments, administration of the therapeutically effective amount of the compound or compounds involves administering to the subject from about 5 mg/m² to about 50 mg/m², such as from about 10 mg/m² to about 40 mg/m² per day. It is currently believed that a single dosage of the compound or compounds (i.e., the autophagy inhibitor and the p38 MAPK inhibitor) either separately or in combination is suitable; however, a therapeutically effective dosage of the autophagy inhibitor and/or the p38 MAPK inhibitor can be supplied over an extended period of time or in multiple doses per day. Thus, unit dosage forms also can be calculated using a subject’s body surface area based on the suitable ranges recited above and the desired dosing schedule. [0073] The autophagy inhibitor and the p38 MAPK inhibitor desirably are administered in the form of a pharmaceutical composition or pharmaceutical compositions. In an embodiment, the autophagy inhibitor and the p38 MAPK inhibitor are each separately administered in the form of pharmaceutical compositions.

[0074] Overall, the requirements for effective pharmaceutical carriers for parenteral compositions are well known to those of ordinary skill in the art. See, e.g., Banker and Chalmers, eds., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company, Philadelphia, pp. 238-250 (1982), and Toissel, ASHP Handbook on Injectable Drugs, 4^th ed., pp. 622-630 (1986). Such solutions can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The sensitizer and/or apoptosis-inducing ligand may be administered in physiologically acceptable ampoules in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane-4-methanol, ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

[0075] Oils useful in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils useful in such formulations include peanut, soybean, sesame, cottonseed, com, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0076] Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-beta-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.

[0077] The parenteral formulations can contain preservatives and buffers. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5 to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.

The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[0078] Topical formulations, including those that are useful for transdermal drug release, are well-known to those of skill in the art and are suitable in the context of the invention for application to skin. Topically applied compositions are generally in the form of liquids, creams, pastes, lotions and gels. Topical administration includes application to the oral mucosa, which includes the oral cavity, oral epithelium, palate, gingival, and the nasal mucosa. In some embodiments, the composition contains at least one sensitizer and a suitable vehicle or carrier. It may also contain other components, such as an anti-irritant.

The carrier can be a liquid, solid or semi-solid. In embodiments, the composition is an aqueous solution. Alternatively, the composition can be a dispersion, emulsion, gel, lotion or cream vehicle for the various components. In one embodiment, the primary vehicle is water or a biocompatible solvent that is substantially neutral or that has been rendered substantially neutral. The liquid vehicle can include other materials, such as buffers, alcohols, glycerin, and mineral oils with various emulsifiers or dispersing agents as known in the art to obtain the desired pH, consistency and viscosity. It is possible that the compositions can be produced as solids, such as powders or granules. The solids can be applied directly or dissolved in water or a biocompatible solvent prior to use to form a solution that is substantially neutral or that has been rendered substantially neutral and that can then be applied to the target site. In embodiments of the invention, the vehicle for topical application to the skin can include water, buffered solutions, various alcohols, glycols such as glycerin, lipid materials such as fatty acids, mineral oils, phosphoglycerides, collagen, gelatin and silicone based materials.

[0079] Formulations suitable for oral administration can consist of (a) liquid solutions, such as a therapeutically effective amount of the sensitizer dissolved in diluents, such as water, saline, or orange juice, (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules, (c) powders, (d) suspensions in an appropriate liquid, and (e) suitable emulsions. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and com starch. Tablet forms can include one or more of lactose, sucrose, mannitol, com starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.

[0080] In an embodiment, the invention provides a pharmaceutical composition comprising an autophagy inhibitor, a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor, and a pharmaceutically acceptable carrier, wherein the composition provides a synergistic amount of the autophagy inhibitor and the p38 MAPK inhibitor, that is effective to reduce or inhibit proliferation of cancer cells or to cause the death of cancer cells, in a mammal if the composition is administered to the mammal,, wherein the autophagy inhibitor is a compound of formula (I):

wherein R¹ is (a) -NR³R⁴ wherein R³ and R⁴ are independently H, optionally substituted C1-C6 alkyl, bisphenylmethyl, or optionally substituted C6-C10 phenyl, (b) morpholino, or (c) 2-pyridyl-(CH2)n-0-, wherein n is an integer of from 1 to about 10, R² is optionally substituted C6-C10 phenyl or a group of the formula:

R⁵CH=N- wherein R⁵ is C6-C10 aryl, heteroaryl, or fused bi cyclic heteroaryl,

X is CH orN, or a tautomer thereof; a compound of formula (II): or a compound of formula (III):

wherein R is H or OH, or a pharmaceutically acceptable salt thereof, and wherein the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor is selected from:

[0081] In certain embodiments, the autophagy inhibitor is selected from: [0082] In these embodiments, the pharmaceutically acceptable carrier can be as described herein in connection with pharmaceutical compositions. The dosages of the autophagy inhibitor and the p38 MAPK inhibitor can be as described herein in connection with the dosages of the autophagy inhibitor and the p38 MAPK inhibitor as individually administered. [0083] In another embodiment, the invention provides a kit comprising an autophagy inhibitor and a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor in a package, wherein the kit comprises a synergistic amount of the autophagy inhibitor and p38 MAPK inhibitor that is effective to reduce or inhibit proliferation of cancer cells or to cause the death of cancer cells in a mammal, wherein the autophagy inhibitor is a compound of formula (I):

wherein R¹ is (a) -NR³R⁴ wherein R³ and R⁴ are independently H, optionally substituted C1-C6 alkyl, bisphenylmethyl, or optionally substituted C6-C10 phenyl, (b) morpholino, or (c) 2-pyridyl-(CH2)n-0-, wherein n is an integer of from 1 to about 10, R² is optionally substituted C6-C10 phenyl or a group of the formula:

R⁵CH=N- wherein R⁵ is C6-C10 aryl, heteroaryl, or fused bi cyclic heteroaryl,

X is CH orN, or a tautomer thereof; a compound of formula (II):

or a compound of formula (III):

wherein R is H or OH, or a pharmaceutically acceptable salt thereof,

[0084] In certain embodiments, the autophagy inhibitor is selected from:

[0085] The kit may comprise a container comprising the combination (i.e., of the autophagy inhibitor and the p38 MAPK inhibitor). Suitable containers include, for example, bottles, vials, syringes, blister pack, and the like. The container may be formed from a variety of materials such as glass or plastic. The container may hold the combination which is effective for treating the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).

[0086] The kit may further comprise a label or package insert on or associated with the container. The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. In an embodiment, the label or package inserts indicates that the composition comprising the autophagy inhibitor and/or the p38 MAPK inhibitor can be used to treat a disorder such as cancer. The label or package insert may also indicate that the composition can be used to treat other disorders. [0087] In certain embodiments, the kits are suitable for the delivery of solid oral forms of the autophagy inhibitor and the p38 MAPK inhibitor, such as tablets or capsules. In these embodiments, the kit preferably includes a number of unit dosages. Such kits can include a card having the dosages oriented in the order of their intended use. One example of such a kit is a "blister pack". Blister packs are well known in the packaging industry and are widely used for packaging pharmaceutical unit dosage forms. If desired, a memory aid can be provided, for example in the form of numbers, letters, or other markings or with a calendar insert, designating the days in the treatment schedule in which the dosages can be administered.

[0088] In another embodiment, a kit may comprise (a) a first container with an autophagy inhibitor contained therein; and (b) a second container with a p38 MAPK inhibitor contained therein. Alternatively, or in addition, the kit may further comprise a third container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection, phosphate-buffered saline, Ringer's solution and dextrose solution. The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0089] The kit may further comprise directions for the administration of the autophagy inhibitor and the p38 MAPK inhibitor. For example, the kit may further comprise directions for the simultaneous, sequential or separate administration of the autophagy inhibitor and the p38 MAPK inhibitor to a patient in need thereof.

[0090] In certain other embodiments, the kit may comprise a container for containing the separate compositions such as a divided bottle or a divided foil packet, however, the separate compositions may also be contained within a single, undivided container. In certain embodiments, the kit comprises directions for the administration of the separate components. The kit form is particularly advantageous when the separate components are preferably administered in different dosage forms (e.g., oral and parenteral), are administered at different dosage intervals, or when titration of the individual components of the combination is desired by the prescribing physician.

[0091] In another embodiment, the invention provides a kit comprising separate containers of an autophagy inhibitor and a p38 MAPK inhibitor for use in combination to treat or prevent cancer. In another embodiment, the invention provides a kit comprising separate containers in a single package pharmaceutical composition for use in combination to treat or prevent cancer, which comprises in one container a pharmaceutical composition comprising an effective amount of an autophagy inhibitor and in a second container a pharmaceutical composition comprising an effective amount of a p38 MAPK inhibitor.

[0092] Autophagy Inhibitors

[0093] The autophagy inhibitor for use in the present method is not particularly limited and can be any agent capable of inhibiting autophagy in a cell. Non-limiting examples of suitable autophagy inhibitors include a compound of formula (I), a compound of formula (II), a compound of formula (III), chloroquine, hydroxychloroquine, flubendazole, hexachlorophene, propidium iodide, bepridil, clomiphene citrate (Z,E), GBR 12909, propafenone, metixene, dipivefrin, fluvoxamine, dicyclomine, dimethisoquin, ticlopidine, memantine, bromhexine, norcyclobenzaprine, diperodon, nortriptyline, tetrachlorisophthalonitrile, phenylmercuric acetate, benzethonium, niclosamide, monensin, bromperidol, levobunolol, dehydroisoandosterone 3-acetate, sertraline, tamoxifen, reserpine, hexachlorophene, dipyridamole, harmaline, prazosin, lidoflazine, thiethylperazine, dextromethorphan, desipramine, mebendazole, canrenone, chlorprothixene, maprotiline, homochlorcyclizine, loperamide, nicardipine, dexfenfluramine, nilvadipine, dosulepin, biperiden, denatonium, etomidate, toremifene, tomoxetine, clorgyline, zotepine, beta-escin, tridihexethyl, ceftazidime, methoxy-6-harmalan, melengestrol, albendazole, rimantadine, chlorpromazine, pergolide, cloperastine, prednicarbate, haloperidol, clotrimazole, nitrofural, iopanoic acid, naftopidil, methimazole, trimeprazine, ethoxy quin, clocortolone, doxycycline, pirlindole mesylate, doxazosin, deptropine, nocodazole, scopolamine, oxybenzone, halcinonide, oxybutynin, miconazole, clomipramine, cyproheptadine, doxepin, dyclonine, salbutamol, flavoxate, amoxapine, fenofibrate, pimethixene, and the like, a pharmaceutically acceptable salt thereof, and mixtures thereof.

[0094] p38 MAPK Inhibitor

[0095] The p38 MAPK inhibitor (p38 MAP kinase inhibitor) for use in the present method is not particularly limited and can be any suitable p38 MAPK inhibitor. A p38 MAPK inhibitor is considered to specifically bind to one member of p38 MAPK (e.g., p38a, r38b, r38g, or r38d) if the inhibitor binds to the specific member of p38 MAPK with a greater affinity than for an irrelevant polypeptide. In some embodiments, the inhibitor binds to one member of p38 MAPK (e.g., r38a, r38b, r38g, or r38d) with at least 5, e.g., at least 10, or at least 50 times greater affinity than for the irrelevant polypeptide. In some embodiments, the inhibitor binds to one member of p38 MAPK (e.g., r38a, r38b, r38g, or r38d) with at least 100, e.g., at least 1,000, or at least 10,000 times greater affinity than for the irrelevant polypeptide. Such binding may be determined by methods well known in the art. In some embodiments, the inhibitor has an affinity (as measured by a dissociation constant, KD) for a specific member of p38 MAPK (e.g., r38a, r38b, r38g, or r38d) of at least Kb⁷ M, e.g., 10⁸ M, 10⁹ M, 10 ¹⁰ M, or 10 ¹¹ M.

[0096] In some embodiments, the p38 MAPK inhibitor is a small molecule, such as a small organic molecule, which typically has a molecular weight less than 5,000 kDa. Suitable small molecules include those that bind to one or more family members of p38 MAPK (e.g., r38a, r38b, r38g, or r38d) or a fragment thereof. Non-limiting examples of suitable p38 MAPK inhibitors include SB203580 (4-[5-(4-Fluorophenyl)-2-[4-(methylsulfonyl)phenyl]- 1H- pyridinyl imidazol-4-yl]pyridine); SKF-86002 (6-(4-Fluorophenyl)-2,3-dihydro-5-(4- pyridinyl)imidazo[2,l- b]thiazole dihydrochloride); SB-242235 (l-(4-piperidinyl)-4-(4- fluorophenyl)-5-(2-methoxy-4- heterocycles pyrimidinyl)Imidazole); RWJ-67657 (4-[4-(4- Fluorophenyl)-l-(3-phenylpropyl)-5-(4-pyridinyl)- lH-imidazol-2-yl]-3-butyn-l-ol); SB 239065; RO3201195 (S-[5-amino-l-(4-fluorophenyl)-lH-pyrazol-4-yl]-[3-(2,3- pyridyl dihydroxypropoxy)phenyl]methanone); BIRB-796 (l-[5-tert-butyl-2-(4- methylphenyl)pyrazol-3-yl]-3-[4-(2- morpholin-4-ylethoxy)naphthalen-l-yl]urea)); VX-745 (5-(2,6-dichlorophenyl)-2-(2,4-difluorophenylthio)-6H- pyrimido[l,6-b]pyridazin-6-one); SB202190 (4-[4-(4-fluorophenyl)-5-pyridin-4-yl-l,3-dihydroimidazol-2- ylidene]cyclohexa- 2,5-dien-l-one); VX-702 (6-(N-carbamoyl- 2,6-difluoroanilino)-2-(2,4- difluorophenyl)pyridine-3- carboxamide); LY2228820 (5-[2-tert-butyl-4-(4-fluorophenyl)- lH-imidazol-5-yl]-3-(2,2- dimethylpropyl)imidazo[4,5-b]pyridin-2-amine; methanesulfonic acid); PH-797804 (3-[3-bromo-4-[(2,4-difluorophenyl)methoxy]-6-methyl-2- oxopyridin-1- yl]-N,4-dimethylbenzamide); VX-702 (6-(N-carbamoyl-2,6-difluoroanilino)-2-(2,4- difluorophenyl)pyridine-3-carboxamide); L-167307 (4-[2-(4-Fluorophenyl)-5-[4- (methylsulfinyl)phenyl]- (lH-pyrrol-3-yl]pyridine imidazole)-pyridinyloxazole); RPR- 200765A (((2r,5r)-2-(4-(4-fluorophenyl)-5-(pyridin-4-yl)-lH-imidazol- 2-yl)-5-methyl-l,3- dioxan-5-yl)(morpholino)methanone methanesulfonate), RPR-238677; FR167653 (l-(7-(4- fluorophenyl)-l,2,3,4-tetrahydro-8-(4- pyridyl)pyrazolo(5,l-c)(l,2,4)triazin-2-yl)-2- phenylethanedione sulphate monohydrate); TAK-715; Skepinone-L; and SB-239063 (trans-1- (4-hydroxycyclohexyl)-4-(4-fluorophenyl)-5-(2- methoxypyridimidin-4-yl)imidazole),and combinations thereof.

[0097] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. [0098] MATERIALS & METHODS

[0099] Cell Culture

[0100] Cells were obtained from the American Type Culture Collection [A375 (CRL- 1619), Hs27 (CRL-1634), WI38 (CCL-75), BJ (CRL-2522), A549 (CCL-185), SW480 (CCL-228), MDA-MB-231 (HTB-26), SHP-77 (CRL-2195), HeLa (CCL-2), G361 (CRL- 1424), VMM39 (CRL-3230), Daudi (CCL-213), U20S (HTB-96), HCT116 (CCL-247)], OVCAR3 (HTB-161), IMR90 (CCL-186), MRC5 (CCL-171), from Coriell (AG02262), and from the National Cancer Institute [SF-295 (NCI-60 tumor line panel)] . To compare inhibitors' effect on cell growth and survival, all cells in experiment were seeded at same density [7 x 10⁴ cells/well per 6-well plates; 2.3 x 10³ cells/well in 96-well plates] and cultured in high glucose Dulbecco’s Modified Eagle’s medium supplemented with L- glutamine, sodium pyruvate and 10% fetal calf serum at 37°C with 5% C02. 1 day later, the indicated inhibitor was added at 1:1000 dilution. Cells were collected or ATP production determined after 3 days incubation. To generate stable cell lines expressing wild type and mutant PIKfyve, A375 cells were transduced with viral particles obtained in Phoenix packaging line of retroviral vector pOP containing full length wild type or mutant human PIKfyve fused with Flag and HA tag peptides at the N terminus, and were selected using 2 pg/ml puromycin (Sigma).

[0101] Chemical Inhibitors

[0102] Vacuolin-1, YM201636, Apilimod, Chloroquine, Hydroxychloroquine,

SB202190, BIRB-796, Skepinone-L, LY2228820, and Tak-715 (Cayman Chemical), and compounds 1 and 4 (SPECS) were dissolved in dimethyl sulfoxide (DMSO) at 20 mM stock concentration and diluted further as necessary to add to culture media at 1 : 1000 dilution. [0103] Western Immuno-blotting

[0104] Equal numbers of cells were collected, lysed in Laemmli sample buffer, and subjected to electrophoresis and protein immuno-blotting using the NuPAGE polyacrylamide gel system (ThermoScientific). The filters were probed with specific primary antibodies and peroxidase-linked species-specific secondary antibodies (Amersham Biosciences), and the signals visualized using West-DURA enhanced chemiluminescence kit (ThermoScientific). The following primary antibodies were used: anti-LC3 (#12741), anti-p62 (#8025), anti- MKK3/6-p (#12280), anti-Hsp27-p (#2401), anti-p38 MAPK-p (#9211), anti-p38 MAPK (#9212) from Cell Signaling Technology; from Santa Cruz Biotechnology; anti-LAMP2 (#25631) from AbCam. [0105] Cell count-based quantification of cell viability

[0106] Cells were seeded at a density of 7x10⁴ cells/well in 6-well plates. One day after plating, old media was aspirated and fresh media containing the appropriate drug(s) was added to each well. 72 hours after treatment, floating and attached cells were collected and counted in a hemocytometer. lxl 0⁵ cells were processed for FACS analysis to determine the percentage of those with less than 2N DNA content. Cells with less than 2N DNA were considered dead and cell counts were adjusted accordingly to reflect the percentage of live cells.

[0107] Fluorescence Activated Cell Sorting (FACS)

[0108] Cells were washed with phosphate buffered saline, and 2x10⁵ cells were stained with propidium iodide and subjected to FACS analysis [13] using a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA) in accordance with the manufacturer instructions. Special attention was given to ensuring that cell clusters were well separated by suspension and that the 2N DNA content peaks were coincident in the resulting cellular DNA histograms in all analysis. Data were analyzed using FlowJo software (FlowJo, LLC).

[0109] Dosage matrix by ATP -based quantification of cell viability [0110] The extent of functional cooperation between PIKfyve and p38 MAPK inhibitors in reducing cells survival was evaluated by generation of a dose response matrix of a wide range of concentrations of the two drugs. 2.3 xlO³ cells per well were plated in 96 well plates (a density that maintains exponential growth during the length of the experiment) and one day later the two inhibitors were added without removing any media to limit cellular stress and prevent uneven treatment of wells. For each dose combination, the survival of the tested cells after three days with inhibitors was determined by measuring the levels of produced cellular ATP using CellTiter-Glo Luminescent Cell Viability Assay kit (Promega) per manufacturer's instructions. The results were analyzed using the CompuSyn software (http://www.combosyn.com) to generate Chou-Talalay Combination Index [14, 15] for each inhibitor combination to determine the extent of observed additive effect and the range of concentrations that have synergistic effect.

Human cells have variant sensitivity to PIKfyve inhibition which correlates with basal p38 MAPK phosphorylation

[0111] Previous studies have shown that PIKfyve inhibition causes varying degree of inhibition of human cancer cell proliferation. Reported IC50 concentrations of the PIKfyve inhibitor compound 1 varied 200-fold between tested cell lines in a recent study [4] Six-point concentration titrations were performed to determine the sensitivity of 12 human cell lines to compound 1. Results similarly demonstrated a range of sensitivities, with IC50 values spanning 0.15 - 2.0 mM (Fig. 1). Compound 1 and other inhibitors have been shown to have high binding affinity to PIKfyve in cell-free assays [4] In order to confirm PIKfyve is the relevant target of compound 1 in live cells and responsible for the observed toxicity, a mutated PIKfyve protein was expressed in A375 melanoma cells. The PIKfyve N1939K mutation was recently shown to confer resistance against the PIKfyve inhibitor Apilimod, proving PIKfyve inhibition alone is responsible for the observed cytotoxic effects [3] When treated with high dosages of compound 1, compound 4, and Vacuolin-1, mutant PIKfyve- expressing A375 cells were unaffected, while the growth of wildtype PIKfyve-expressing cells was inhibited up to 70% and dramatic vacuolization consistent with PIKfyve inhibition was observed (FIG. 2). The results demonstrate that the compound 1, compound 4, and Vacuolin-1 inhibitors are practically single-target specific, even in high concentrations, indicating that inhibition of PIKfyve is responsible for the observed effects on autophagy and cell viability.

[0112] Levels of p38 MAPK pathway proteins were quantified for all 12 human cell lines via immunoblot (FIG. 3). While all 12 cell lines possessed a functional p38 MAPK pathway which responded appropriately to UV radiation, the basal levels differed between cell lines. A strong inverse correlation between cell sensitivity to compound 1 and the baseline level of p38 MAPK phosphorylation was observed (FIG. 5A-B). The most resistant lines had the highest levels of p38 MAPK phosphorylation, while the most sensitive lines had the lowest levels, demonstrating a 10-fold range in the levels of activating T180/Y182 site phosphorylation. A similar correlation was also observed between the sensitivity of human cell lines to compound 1 and their sensitivity to SB202190, a p38 MAPK inhibitor, which suggests a functional link between the PIKfyve and p38 MAPK pathways (FIG. 4, FIG. 5C). All 9 cancer cell lines were found to have relatively low levels of p38 MAPK protein and phosphorylation and be sensitive to PIKfyve and p38 MAPK inhibition. However, the tested 3 normal lines demonstrated the opposite, high levels of p38 MAPK protein and phosphorylation and resistance to PIKfyve and p38 MAPK inhibition, supporting the hypothesis that combined inhibition of p38 MAPK and PIKfyve will have selective anti cancer effect. Taken together, the data reveal a strong correlation between p38 MAPK phosphorylation and sensitivity to inhibition of PIKfyve. Thus, p38 MAPK protein and phosphorylation levels in tumors may also serve as biomarkers for sensitivity to PIKfy ve inhibitors.

Combinational treatment with PIKfyve and p38 MAPK inhibitors demonstrates selective anti-cancer cytotoxicity

[0113] Since lower p38 MAPK activity is associated with higher sensitivity to PIKfyve inhibition, chemical inhibition of p38 MAPK may enhance the toxicity of PIKfyve inhibitors. A p38 MAPK inhibitor, SB202190, was added in combination with compound 1 to five cell lines and cellular viability was measured after 3 days (FIG. 6). In each cancer cell line, addition of SB202190 increased the toxicity of the PIKfyve inhibitor compound 1, indicating the interaction of the two inhibitors has a cooperative cytotoxic effect (FIG. 6A). Moreover, both the PIKfyve and p38 MAPK inhibitors caused accumulation of vacuoles, suggesting a possible similar mechanism of action (FIG. 6B). The normal fibroblast cell lines, Hs27 and WI38, demonstrated remarkable resistance to the PIKfyve and p38 MAPK inhibitors in combination. On average, viability of the tested cancer cell lines was below 10%, while the average viability of the fibroblasts was only minimally affected, approximately 90% of the untreated cells. Such a selective anti-cancer cytotoxicity suggests the combination of PIKfyve and p38 MAPK inhibition could be a novel efficient anti-cancer therapy.

Combinational treatment with PIKfyve and p38 MAPK inhibitors increases cytotoxicity in a synergistic and selective manner over a broad range of concentrations [0114] To determine the extent of functional cooperation between p38 MAPK and PIKfyve inhibitors, a dosage matrix of compound 1 and SB202190 was generated utilizing an ATP -based cell viability assay. Dosage matrices were created for two cell lines, A549 non small cell lung carcinoma and Hs27 normal foreskin fibroblasts, to determine the extent of the selective anti-cancer toxicity when PIKfyve and p38 MAPK inhibitors are combined. Results were analyzed by calculation of the difference between the observed toxicity and the expected toxicity of the two drugs together at the given concentrations (FIG. 7A-B) and by calculation of the Chou-Talalay Combination Index (Cl), a measure extensively used to analyze the pharmacological interaction between two drugs (FIG. 7C) [9] The dosage matrix of A549 with compound 1 and SB202190 revealed a broad range of concentrations which reduce cell viability far below the expected additive toxicity, and with Cl values of less than 1, which together prove synergy. As a whole, toxicity greater than 50% above the expected was observed, with a range of combinations of mild and intermediate concentrations of compound 1 and SB202190 that produced similarly impressive effects. At the same concentrations, Hs27 cells were largely unaffected at all tested combinations of compound 1 and SB202190 concentrations (FIG. 7D). The synergistic interaction between compound 1 and SB202190 was also observed in multiple cancer cell lines of different tissue origin. A549 (non-small cell lung carcinoma), SHP-77 (lung small cell carcinoma), HCT116, SW480 (colorectal carcinomas), MDA-MB-231 (breast adenocarcinoma), HeLa (cervix adenocarcinoma), and SF-295 (brain glioblastoma) cell lines all demonstrated ranges of synergistic effect despite the vast differences in tissue of origin, cancer type and genetic profile (FIG. 7, 8). While the concentrations producing synergistic effects differed between each cell line, the ability of the two inhibitors to synergistically reduce cell viability in each case supports the usefulness of this treatment against a broad spectrum of malignancies. Structurally diverse PIKfyve and p38 MAPK inhibitors produce consistent synergistic anti-cancer effects

[0115] Multiple PIKfyve and p38 MAPK inhibitors were tested in combination for their ability to synergistically reduce cell viability in order to determine whether the phenotype observed with compound 1 and SB202190 treatment is limited to any one unique inhibitor combination. Five PIKfyve inhibitors were tested in combination with SB202190, and five p38 MAPK inhibitors were tested in combination with compound 1. A549 and Hs27 cell lines were treated with inhibitor concentrations which reduced A549 cell survival by an average of 30% individually (FIG. 9). Results demonstrated that each inhibitor combination was capable of synergistically reducing cell viability; the observed toxicity was on average 25% higher than the expected additive toxicity of the two drugs together. Moreover, YM201636 is not structurally similar to the other four PIKfyve inhibitors tested but produced the same synergistic effect, supporting the conclusion that the synergism is not due to a unique characteristic of compound 1 or other structurally similar drugs but is a direct result of PIKfyve kinase inhibition. Likewise, all tested p38 MAPK inhibitors demonstrated similar synergistic effects in combination with compound 1. As the five p38 MAPK inhibitors are all quite structurally distinct, the relevant common target is p38 MAPK. In summary, these results prove that inhibition of PIKfyve and p38 MAPK activity produces a synergistic effect that selectively reduces cancer cell viability and proliferation and is not dependent upon a unique drug interaction or side effect.

PIKfyve and p38 MAPK inhibitor combinational treatment has synergistic and prolonged inhibitory effect on autophagy [0116] In time courses of A549 cells treated with compound 1, SB202190, and the combination of the two drugs, markers of autophagic flux (LC3, LAMP2, and p62) accumulated with all treatments (FIG. 10). In order to confirm p38 MAPK inhibition can cause disruption of autophagy, A549 cells were treated with a different p38 MAPK inhibitor, LY2228820, and tested via Western blot for accumulation of p62 and LC3. Despite having different chemical structures, both SB202190 and LY2228820 caused accumulation of LC3 and p62, which increased upon addition of compound 1, indicating a common disruption of autophagy as a result of PIKfyve and p38 MAPK inhibition (FIG. 11). Likewise, V-ATPase inhibitor Bafilomycin A1 was shown to prevent vacuole formation resulting from Apilimod, a PIKfyve inhibitor [10] Pretreatment of A549 cells with Bafilomycin A1 similarly prevented vacuolization caused by treatment with compound 1 and SB202190, further supporting a common autophagy related mechanism (FIG. 12). Because individual inhibition of both PIKfyve and p38 MAPK pathways leads to autophagy disruption, the dramatic reduction in cell viability of the combinational treatment is likely due to dramatic synergistic disruption of autophagy.

[0117] Accumulation of LC3, p62, and LAMP2 proteins occurred faster and persisted at high levels longer with the combinational treatment compared to either inhibitor alone (FIG. 10). With compound 1 and SB202190 alone, LC3 protein levels accumulated up to 8- and 6- fold above the untreated cells, respectively. When treated in combination, total cellular LC3 accumulated to an enormous 87-fold above the control with high levels of protein remaining for over 5 days. p62 protein, a marker of autophagic disruption, conveyed a similar story. p62 levels increased 5- and 3-fold over two days with individual treatment of compound 1 and SB202190, respectively. Using the combinational treatment, p62 levels increased more rapidly and by 16-fold above the control non-treated cells. This accumulation persisted for up to six days, three times longer than either inhibitor alone. Synergistic inhibition of PIKfyve and p38 MAPK activity both exacerbate and prolongs the effect on biochemical autophagy markers, and therefore the effect on autophagic disruption.

[0118] Further, the synergistic effect of PIKfyve and p38 MAPK inhibitor combinational treatment persisted for days even after the chemical inhibitors are physically removed from the environment. Results demonstrate that p62 levels practically disappeared 2 days after the individual inhibitors are removed from the environment, indicating the cells recovered quickly and fully (FIG. 13). When cells were treated for only one day with the combination of compound 1 and SB202190, p62 protein levels persisted for 2 days at almost 70% the level of cells which were treated consistently. Proliferation inhibition and dramatic vacuolization also continued, indicating the cells do not recover even when the inhibitors are no longer present in the culture environment. These data suggest that once initiated, the effects of the combinational treatment persist independently and do not require maintenance of high dosages of the inhibitors. This inhibitor combination is practical for clinical usage as treatment could be administered less frequently while maintaining potent long-lasting cytotoxicity, even after the inhibitors have been eliminated from the blood stream.

General inhibitors of autophagy, chloroquine and hydroxychloroquine, also have synergistic inhibitory effect on cancer cell survival when combined with p38 MAPK inhibitors

[0119] Since the combined p38 MAPK and PIKfyve inhibition had a synergistic inhibitory effect on autophagy, the effect of other non-PIKfy ve-mediated mechanisms of autophagy inhibition was also investigated. The most studied and well-characterized inhibitors of autophagy are chloroquine and its close derivative hydroxychloroquine, which, according to current literature, disrupt autophagy independent of PIKtyve activity [11] ATP- based dosage matrices of chloroquine or hydroxychloroquine in combination with SB202190 revealed a strong synergistic, as well as selective, anti-cancer cytotoxic effect over a wide range of concentrations of both compounds (FIG. 14). These results confirmed that the main functional reason for the observed synergistic effect of PIKfyve and p38 MAPK inhibition is through cooperative inhibition of autophagy. Consequently, p38 MAPK inhibition will likely synergistically enhance the cytotoxic effect of any method of autophagy inhibition. Given the relatively low toxicity and well-understood pharmacological properties of chloroquine and its derivatives, as well as the large amount of clinical study data collected over the years, this combinational treatment could be more readily implemented in clinical settings.

Low p38 MAPK phosphorylation levels correlate with high sensitivity to PIKfyve and p38 MAPK inhibition

[0120] Biochemical analysis of 12 cancer and 6 normal human cell lines confirmed that cells resistant to PIKfyve inhibition have consistently high basal levels of p38 MAPK pathway protein phosphorylation, while sensitive lines have lower levels, as shown in FIG.

16.

[0121] Quantification of the p38 MAPK phosphorylation as shown in FIG. 17B revealed that higher steady-state levels are invariably associated with higher resistance to PIKfyve and p38MAPK inhibition. The consistent correlation between the sensitivities to PIKfyve and p38 MAPK inhibitors of each line as shown in FIG. 17D suggested the existence of an important functional link between the two cellular pathways.

PIKfyve and p38 MAPK inhibitors synergistically reduced proliferation and viability of cancer cells but not of normal cells

[0122] To determine if combined inhibition is beneficial, a dosage matrix using multiple concentrations of compound 1 in combination with multiple concentrations of SB202190 was created using SW480 colon carcinoma cells and is shown in FIG. 18A-G. Cell viability was reported as a normalized percentage of each treatment compared to the control. After three days of treatment, cells were counted manually to determine the rate of proliferation and then a sample was analyzed by FACS. Cells containing less than 2N DNA content were assumed have fragmented DNA, and therefore either dead or undergoing cell death. Thus, cells with less than 2N DNA content were removed from the total cell number in the well to obtain the number of viable cells. This method provided precise measurements of both cell proliferation and survival. Compound 1 and SB202190 in combination dramatically reduced SW480 cell viability as shown in FIG. 18 A.

[0123] To prove that compound 1 and SB202190 have pharmacological synergistic effect the Chou-Talalay Combination Index (Cl) method was utilized, which applies a median- effect equation based on the mass-action law (Chou, T. C., Cancer Res 70, 440-446 (2010)). The technique has been extensively tested and derived from mechanistic and mathematical considerations. The effect of each combination of a range of doses is compared with the individual drugs effects to calculate a Cl. Cl values of <1, =1, and >1 indicate synergistic, additive, and antagonistic effects, respectively. The degree of synergism is measured by the deviation of Cl from 1, the smaller the Cl, the stronger the synergistic effect of the combination is. The dosage matrix of SW480 with compound 1 and SB202190 revealed a broad range of concentrations which reduce cell viability far below the expected additive effect as shown in FIG. 18C. 75% of the tested combinations have Cl values of less than 0.5, and 22% have Cl values less than 0.2, which prove dramatic synergistic effect between the two inhibitors. The combination also caused dramatic increase in the fraction of dead cells reducing the survival of cancer cells to 0% with the highest tested concentrations.

Combined PIKfyve and p38 MAPK inhibitors in concentrations that caused dramatic reductions in SW480 cell survival, did not notably affect the viability of normal human fibroblasts Hs27 as shown in FIG. 18F. Similarly, the percentage of dead Hs27 cells in each treatment did not significantly increase under any treatment condition as shown in FIG. 18G. When contrasted with the effects on SW480 cells, these results suggested that compound 1 and SB202190 together selectively and dramatically reduce cancer cell survival while not affecting normal cells.

PIKfyve and p38 MAPK inhibitors synergistically reduced proliferation and viability of multiple cancer cell types

[0124] Combined PIKfyve and p38 MAPK inhibitors caused dramatic synergistic reductions in viability of multiple cancer cell lines with diverse origin and mutations, including HCT116 (colorectal carcinoma), U20S (osteosarcoma), A549 (non-small cell lung carcinoma), MDA-MB-231 (breast adenocarcinoma), and SF-295 (brain glioblastoma) while not affecting normal cells as WI38 (normal lung fibroblasts) as shown in FIG. 19A-F. Synergistic disruption of autophagy-mediated protein degradation and lysosomal function in cancer cells

[0125] Analysis of autophagy related protein reveals that combined inhibition of PIKfyve and p38MAPK synergistically inhibits autophagy-mediated protein degradation as demonstrated by the exacerbated and prolonged accumulation of autophagic, p62 and LC3, and lysosomal (Gonzalez-Polo, R. A., et al. , J Cell Sci 118, 3091-3102 (2005)). LAMP2 proteins, as shown in FIG. 20A-J. Moreover, it synergistically prevented the maturation of the lysosome cathepsin proteases necessary for protein degradation. Compound 1 alone caused abnormal accumulation of Cathepsin D precursor (52 kDa) (Zaidi, N., et al., Biochem Biophys Res Commun 376, 5-9 (2008)) that was only converted to mature form (34 kDa) after day 2. Combined compound 1 and SB202190 treatment prolonged the accumulation of the Cathepsin D precursor until day 4, when the precursor form was finally processed. Remarkably, the recovery of cathepsin maturation, and consequently their proteolytic activity, coincided with the precipitous degradation of p62, LC3 and LAMP2 proteins, suggesting restoration of autophagic protein degradation.

Specific inhibition of PIKfyve and p38 MAPK activities are responsible for the synergistic effects on cellular viability and autophagy

[0126] Multiple chemical inhibitors of PIKfyve and p38 MAPK are studied and used in the literature. To determine whether the synergism observed between compound 1 and SB202190 was unique to these two molecules, additional inhibitors were tested for their effect on cell survival using SW480 cancer cells and Hs27 normal fibroblasts as shown in FIG. 21 A-B. Compounds that have been characterized as specific PIKfyve inhibitors include compound 1, compound 4, Apilimod, Vacuolin-1 and YM201636 (Lu, Y., et al, Autophagy 10, 1895-1905 (2014); Sbrissa, D., et al Am J Physiol Cell Physiol 303, C436-446 (2012); Sharma, G., et al ,Autophagy, 1-25 (2019)). They were tested in combination with compounds characterized as p38 MAPK specific inhibitors. These include SB202190, BIRB- 796, Skepinone-L, LY2228820 and Tak-(Bain, J., et al, Biochem J 408, 297-315 (2007); Koeberle, S. C., et al, Nat Chem Biol 8, 141-143 (2011); Miwatashi, S., et al, JMed Chem 48, 5966-5979 (2005); Patnaik, A., et al, Clin Cancer Res 22, 1095-1102 (2016)) The results confirmed that synergistic and selective effects of compound 1 and SB202190 on cancer cell viability were not dependent on the chemical structure of a particular inhibitor, but on inhibition of PIKfyve and p38 MAPK activities. Furthermore, siRNA targeted against p38 MAPK (a isoform) also acted synergistically in the presence of compound 1 to inhibit cell proliferation as shown in FIG. 22A-B.

[0127] Some p38 MAPK inhibitors affect autophagy independently of p38 MAPK inhibition (Menon, M. B., et al, PLoS One 6, e23054 (2011)). To ascertain whether direct autophagy inhibition is necessary for synergistic enhancement of the effects of PIKfyve inhibition, SW480 cells were treated with five different p38 MAPK inhibitors alone and in combination with compound 1 as shown in FIG. 23A-B. Only SB202190 and LY2228820 alone induced accumulation of p62 within 24 hours, but all five p38 MAPK inhibitors acted synergistically with compound 1 to induce substantial accumulation of p62 within 1 day (FIG. 23B) and to maintain that accumulation for at least 3 days.

[0128] Taken together, the data show that any method of inhibiting PIKfyve and p38 MAPK activities induces synergistic inhibition of cancer cells survival. Moreover, it implies that newly developed inhibitors with improved potency or pharmacokinetic properties will very likely be able to induce the same effects.

Combined PIKfyve and p38 MAPK inhibition synergistically reduces tumor growth in mouse xenografts

[0129] To test the therapeutic potential of the synergism observed in vitro, mice bearing xenografts of SW480 colon carcinoma cells were treated with compound 1 and SB202190. SW480 cells were injected in the flanks of nude mice and the tumors were allowed to establish for 10 days, after which the mice were treated daily via intraperitoneal injection. Dosages of 20 mg/kg compound 1 and 12.5 mg/kg SB202190 were each found to have mild effects on reducing tumor growth. On average, compound 1 reduced tumor growth by 22%, and SB202190 reduced tumor growth by 28% (Fig. 24B). However, compound 1 and SB202190 in combination reduced tumor growth by 81%, which was 31% greater than the expected additive effect. No adverse effects were apparent from either mouse behavior or body weight, which were tracked daily (FIG. 24C).

[0130] Tumors were excised and subjected to histological and protein analyses to determine whether the synergistic effects of compound 1 and SB202190 on autophagy in vitro were reproducible in vivo. Hematoxylin and eosin staining of tumor slices revealed extensive cytoplasmic vacuolization when both compound 1 and SB202190 were administered together (FIG. 24D), consistent with vacuolization observed in vitro. Histological staining for Ki-67, a marker of actively proliferating cells, showed that tumors from mice treated with DMSO vehicle, compound 1 alone, or SB202190 alone were about 75% positive. Tumors from mice treated with both inhibitors together were only about 50% positive for Ki-67, consistent with synergistic inhibition of cell proliferation (FIG. 24D, 24E). The combined treated tumors also were enriched for LC3, p62, and LAMP2 proteins (FIG. 24F, 24G), consistent with the blockage in autophagy-dependent protein degradation that was observed in vitro. Phosphorylation of MKK3/6, the upstream kinase of p38 MAPK, increased strongly in SB202190 treated tumors, consistent with inhibition of p38 MAPK (Fig. 24F). Taken together, these results confirmed that the key effects of compound 1 and SB202190 observed in cultured cells were reproducible in tumors treated in vivo.

[0131] The synergistic disruption of autophagy and the dramatic reduction of tumor viability in vivo confirmed the therapeutic potential of combined inhibition of PIKfy ve and p38 MAPK.

[0132] The invention can be characterized by at least the following embodiments.

ASPECTS OR FEATURES OF THE INVENTION

[0133] 1. A method for treating a cancer in a mammal in need thereof, comprising administering to the mammal a combination of an effective amount of an autophagy inhibitor and an effective amount of a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor, wherein the effective amount is sufficient to reduce or inhibit proliferation of cancer cells or to cause the death of cancer cells.

[0134] 2. The method of aspect 1, wherein the autophagy inhibitor and the p38 MAPK inhibitor yield a synergistic effect in treating the cancer.

[0135] 3. The method of aspect 1 or 2, wherein the autophagy inhibitor is a compound of formula (I): wherein R¹ is (a) -NR³R⁴ wherein R³ and R⁴ are independently H, optionally substituted C1-C6 alkyl, bisphenylmethyl, or optionally substituted C6-C10 aryl, (b) morphobno, or (c) 2-pyridyl-(CH2)n-0-, wherein n is an integer of from 1 to about 10, R² is optionally substituted C6-C10 phenyl or a group of the formula:

R⁵CH=N- wherein R⁵ is C6-C10 aryl, heteroaryl, or fused bi cyclic heteroaryl,

X is CH orN, or a tautomer thereof; a compound of formula (II):

or a compound of formula (III):

wherein R is H or OH, or a pharmaceutically acceptable salt thereof.

[0136] 4. The method of any one of aspects 1-3, wherein the autophagy inhibitor is a compound of formula (I) and X is N.

[0137] 5. The method of aspect 3 or 4, wherein R¹ is morpholinyl, and R² is optionally substituted C6-C10 aryl.

[0138] 6. The method of aspect 4, wherein the autophagy inhibitor is: [0139] 7. The method of aspect 3 or 4, wherein R² is R⁵CH=N-, R¹ is -NR³R⁴, R³ is H, and R⁴ is optionally substituted C6-C10 aryl.

[0140] 8. The method of aspect 7, wherein the autophagy inhibitor is:

[0141] 9. The method of aspect 3 or 4, wherein R² is R⁵CH=N, R¹ is -NR³R⁴, R³ H, and

R⁴ is bisphenylmethyl.

[0142] 10. The method of aspect 9, wherein the autophagy inhibitor is:

[0143] 11. The method of any one of aspects 1-3, wherein X is CH.

[0144] 12. The method of aspect 11, wherein R² is R⁵CH=N- and wherein R¹ is morpholino.

[0145] 13. The method of aspect 12, wherein the autophagy inhibitor is:

[0146] 14. The method of aspect 11, wherein the autophagy inhibitor is:

[0148] 16. The method of any one of aspects 1-3, wherein the autophagy inhibitor is:

[0149] 17. The method of aspect 1 or 2, wherein the p38 MAPK inhibitor is selected

[0150] 18. The method of any one of aspects 1-17, wherein the cancer is an autophagy-dependent cancer.

[0151] 19. The method of any one of aspects 1-18, wherein the autophagy inhibitor is a

PIKlyve inhibitor.

[0152] 20. The method of any one of aspects 1-17, wherein the cancer is a malignant, metastatic cancer.

[0153] 21. The method of any one of aspects 1-20, wherein the cancer is selected from non-small cell lung carcinoma, lung small cell carcinoma, colorectal carcinoma, breast adenocarcinoma, cervix adenocarcinoma, brain glioblastomal carcinoma, malignant melanoma, thyroid carcinoma, ovarian carcinoma, and leukemia.

[0154] 22. The method of any one of aspects 1-20, wherein the method selectively kills cancer cells.

[0155] 23. The method of aspect 22, wherein the cancer cells are selected from breast cancer cells, malignant melanoma cells, colorectal carcinoma cells, thyroid papillary carcinoma cells, glioma cells, ovarian serous carcinoma cells, lung adenocarcinoma cells, hairy cell leukemia cells, or cervical carcinoma cells.

[0156] 24. A pharmaceutical composition comprising an autophagy inhibitor, a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor, and a pharmaceutically acceptable carrier, wherein the composition provides a synergistic amount of the autophagy inhibitor and the p38 MAPK inhibitor, that is effective to reduce or inhibit proliferation of cancer cells or to cause the death of cancer cells, in a mammal if the composition is administered to the mammal, wherein the autophagy inhibitor is a compound of formula (I):

wherein R¹ is (a) -NR³R⁴ wherein R³ and R⁴ are independently H, optionally substituted C1-C6 alkyl, bisphenylmethyl, or optionally substituted C6-C10 aryl, (b) morpholino, or (c) 2-pyridyl-(CH2)n-0-, wherein n is an integer of from 1 to about 10, R² is optionally substituted C6-C10 phenyl or a group of the formula:

R⁵CH=N- wherein R⁵ is C6-C10 aryl, heteroaryl, or fused bi cyclic heteroaryl,

X is CH orN, or a tautomer thereof; a compound of formula (II):

or a compound of formula (III): wherein R is H or OH, or a pharmaceutically acceptable salt thereof, and wherein the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor is

[0157] 25. The pharmaceutical composition of aspect 24, wherein the autophagy inhibitor is selected from: [0158] 26. A kit comprising an autophagy inhibitor and a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor in a package, wherein the kit comprises a synergistic amount of the autophagy inhibitor and p38 MAPK inhibitor that is effective to reduce or inhibit proliferation of cancer cells or to cause the death of cancer cells in a mammal, wherein the autophagy inhibitor is a compound of formula (I):

wherein R¹ is (a) -NR³R⁴ wherein R³ and R⁴ are independently H, optionally substituted C1-C6 alkyl, bisphenylmethyl, or optionally substituted C6-C10 aryl, (b) morpholino, or (c) 2-pyridyl-(CH2)n-0-, wherein n is an integer of from 1 to about 10, R² is optionally substituted C6-C10 phenyl or a group of the formula:

R⁵CH=N- wherein R⁵ is C6-C10 aryl, heteroaryl, or fused bi cyclic heteroaryl,

X is CH orN, or a tautomer thereof; a compound of formula (II):

or a compound of formula (III):

wherein R is H or OH, or a pharmaceutically acceptable salt thereof, and wherein the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor is

[0159] 27. The kit of aspect 26, wherein the autophagy inhibitor is selected from:

[0160] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0161] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0162] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. REFERENCES

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3. Gayle, S., et al, Identification of apilimod as a first-in-class PIKfyve kinase inhibitor for treatment of B-cell non-Hodgkin lymphoma. Blood, 2017. 129(13): p. 1768-1778.

4. Sharma, G., et al, A family of PIKFYVE inhibitors with therapeutic potential against autophagy-dependent cancer cells disrupt multiple events in lysosome homeostasis. Autophagy, 2019: p. 1-25.

5. Hou, J.Z., et al, Inhibition of PIKfyve using YM201636 suppresses the growth of liver cancer via the induction of autophagy. Oncol Rep, 2019. 41(3): p. 1971-1979.

6. Balia, T., Phosphoinositides: tiny lipids with giant impact on cell regulation. Physiol Rev, 2013. 93(3): p. 1019-137.

7. Wagner, E.F. and A.R. Nebreda, Signal integration by JNK and p38 MAPK pathways in cancer development. Nat Rev Cancer, 2009. 9(8): p. 537-49.

8. Sui, X., et al, p38 and JNK MAPK pathways control the balance of apoptosis and autophagy in response to chemotherapeutic agents. Cancer Lett, 2014. 344(2): p. 174-9.

9. Chou, T.C., Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev, 2006. 58(3): p. 621- 81.

10. Sbrissa, D., et al., Apilimod, a candidate anticancer therapeutic, arrests not only PtdIns(3,5)P2 but also PtdIns5P synthesis by PIKfyve and induces bafilomycin Al -reversible aberrant endomembrane dilation. PLoS One, 2018. 13(9): p. e0204532.

11. Solomon, V.R. and H. Lee, Chloroquine and its analogs: a new promise of an old drug for effective and safe cancer therapies. Eur J Pharmacol, 2009. 625(1-3): p. 220-33.

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Claims

CLAIM(S):

1. A combination of an effective amount of an autophagy inhibitor and an effective amount of a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor, for use in treating a cancer in a mammal in need thereof, wherein the effective amount is sufficient to reduce or inhibit proliferation of cancer cells or to cause the death of cancer cells.

2. The combination for use according to claim 1, wherein the autophagy inhibitor and the p38 MAPK inhibitor yield a synergistic effect in treating the cancer.

3. The combination for use according to claim 1 or 2, wherein the autophagy inhibitor is a compound of formula (I):

wherein R¹ is (a) -NR³R⁴ wherein R³ and R⁴ are independently H, optionally substituted C1-C6 alkyl, bisphenylmethyl, or optionally substituted C6-C10 aryl, (b) morpholino, or (c) 2-pyridyl-(CH2)n-0-, wherein n is an integer of from 1 to about 10, R² is optionally substituted C6-C10 phenyl or a group of the formula:

R⁵CH=N- wherein R⁵ is C6-C10 aryl, heteroaryl, or fused bi cyclic heteroaryl,

X is CH orN, or a tautomer thereof; a compound of formula (II):

or a compound of formula (III): or a pharmaceutically acceptable salt thereof.

4. The combination for use according to any one of claims 1-3, wherein the compound is of formula (I) and X is N.

5. The combination for use according to claim 3 or 4, wherein R¹ is morpholino, and R² is optionally substituted C6-C10 aryl.

6. The combination for use according to claim 4, wherein the compound is:

7. The combination for use according to claim 3 or 4, wherein R² is R⁵CH=N-, R¹ is -NR³R⁴, R³ is H, and R⁴ is optionally substituted C6-C10 aryl.

8. The combination for use according to claim 7, wherein the compound is:

9. The combination for use according to claim 3 or 4, wherein R² is R⁵CH=N, R¹ is -NR³R⁴, R³ H, and R⁴ is bisphenylmethyl.

10. The combination for use according to claim 9, wherein the compound is:

11. The combination for use according to any one of claims 1-3, wherein X is CH.

12. The combination for use according to claim 11, wherein R² is R⁵CH=N- and wherein R¹ is morpholino.

13. The combination for use according to claim 12, wherein the compound is:

14. The combination for use according to claim 11, wherein the compound is:

15. The combination for use according to any one of claims 1-3, wherein the compound is:

16. The combination for use according to any one of claims 1-3, wherein the compound is:

17. The combination for use according to claim 1 or 2, wherein the p38 MAPK inhibitor is selected from the group consisting of:

18. The combination for use according to any one of claims 1-17, wherein the cancer is an autophagy-dependent cancer.

19. The combination for use according to any one of claims 1-18, wherein the autophagy inhibitor is a PIKly ve inhibitor.

20. The combination for use according to any one of claims 1-17, wherein the cancer is a malignant, metastatic cancer.

21. The combination for use according to any one of claims 1-20, wherein the cancer is selected from the group consisting of non-small cell lung carcinoma, lung small cell carcinoma, colorectal carcinoma, breast adenocarcinoma, cervix adenocarcinoma, brain glioblastomal carcinoma, malignant melanoma, thyroid carcinoma, ovarian carcinoma, and leukemia.

22. The combination for use according to any one of claims 1-20, wherein the method selectively kills cancer cells.

23. The combination for use according to claim 22, wherein the cancer cells are selected from the group consisting of breast cancer cells, malignant melanoma cells, colorectal carcinoma cells, thyroid papillary carcinoma cells, glioma cells, ovarian serous carcinoma cells, lung adenocarcinoma cells, hairy cell leukemia cells, or cervical carcinoma cells.

24. A pharmaceutical composition comprising an autophagy inhibitor, a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor, and a pharmaceutically acceptable carrier, wherein the composition provides a synergistic amount of the autophagy inhibitor and the p38 MAPK inhibitor, that is effective to reduce or inhibit proliferation of cancer cells or to cause the death of cancer cells, in a mammal if the composition is administered to the mammal, wherein the autophagy inhibitor is a compound of formula (I):

wherein R¹ is (a) -NR³R⁴ wherein R³ and R⁴ are independently H, optionally substituted C1-C6 alkyl, bisphenylmethyl, or optionally substituted C6-C10 aryl, (b) morpholino, or (c) 2-pyridyl-(CH2)n-0-, wherein n is an integer of from 1 to about 10, R² is optionally substituted C6-C10 phenyl or a group of the formula:

R⁵CH=N- wherein R⁵ is C6-C10 aryl, heteroaryl, or fused bi cyclic heteroaryl,

X is CH orN, or a tautomer thereof; a compound of formula (II):

or a compound of formula (III):

wherein R is H or OH, or a pharmaceutically acceptable salt thereof, and wherein the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor is selected from the group consisting of:

25. The pharmaceutical composition of claim 24, wherein the autophagy inhibitor is selected from the group consisting of:

26. A kit comprising an autophagy inhibitor and a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor in a package, wherein the kit comprises a synergistic amount of the autophagy inhibitor and p38 MAPK inhibitor that is effective to reduce or inhibit proliferation of cancer cells or to cause the death of cancer cells in a mammal, wherein the autophagy inhibitor is a compound of formula (I):

wherein R¹ is (a) -NR³R⁴ wherein R³ and R⁴ are independently H, optionally substituted C1-C6 alkyl, bisphenylmethyl, or optionally substituted C6-C10 aryl, (b) morpholino, or (c) 2-pyridyl-(CH2)n-0-, wherein n is an integer of from 1 to about 10, R² is optionally substituted C6-C10 phenyl or a group of the formula:

R⁵CH=N- wherein R⁵ is C6-C10 aryl, heteroaryl, or fused bi cyclic heteroaryl, X is CH orN, or a tautomer thereof; a compound of formula (II):

wherein R is H or OH, or a pharmaceutically acceptable salt thereof, and wherein the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor is selected from the group consisting of:

27. The kit of claim 26, wherein the autophagy inhibitor is selected from the group consisting of: