JP2013542965A - Tumor treatment methods - Google Patents

Abstract

  The present invention relates to a method of treating a tumor in which PAK1 is overexpressed or amplified by co-administering a PAK1 inhibitor and a second anti-hyperproliferative agent.

Description

  The present invention discloses a method of treating a cancerous tumor that amplifies or overexpresses PAK1 by contacting the tumor with a PAK1 inhibitor in combination with a second anti-hyperproliferative agent.

  Protein kinases are a family of enzymes that catalyze the phosphorylation of the hydroxyl group of certain tyrosine, serine, or threonine residues in proteins. Typically, such phosphorylation dramatically changes the function of proteins, and thus protein kinases are central in the control of various cellular processes including metabolism, cell proliferation, cell differentiation, and cell survival. sell. These cellular process mechanisms target protein kinases and provide the basis for treating disease states arising from or involving these cellular process disorders. Examples of such diseases include but are not limited to cancer and diabetes.

  Protein kinases are divided into two types: protein tyrosine kinases (PTK) and serine-threonine kinases (STK). Both PTKs and STKs can be receptor protein kinases or non-receptor protein kinases. PAK is a family of non-receptor STKs. The serine / threonine protein kinase p21-activated protein kinase (PAK) plays an important role in cytoskeleton formation, cell morphogenesis, cellular processes and cell survival (Daniels et al., Trends Biochem. Sci. 1999 24: 350-355 ; Sells et al., Trends Cell. Biol. 1997 7: 162-167). The PAK family consists of six members that are subdivided into two groups: PAK4-6 (Group II) distinguished on the basis of the presence and sequence homology of autoinhibitory regions in PAK1-3 (Group I) and Group I PAKs . p21-activated kinases (PAKs) act as key mediators of Rac and Cdc42 GTPase function and pathways required for Ras-driven tumorigenesis. (Manser et al., Nature 1994 367: 40-46; B Dummler et al., Cancer Metathesis Rev. 2009 28: 51-63; R. Kumar et al., Nature Rev. Cancer 2006 6: 459-473).

  The invention comprises contacting a tumor with a PAK1 inhibitor and a second anti-hyperproliferative or anti-tumor agent selected from inhibitors of EGFR, Raf / MEK / ERK pathway, Src, Akt or an inhibitor of apoptosis protein or It relates to a method of treating a tumor or hyperproliferative condition wherein the tumor cell or hyperproliferating cell overexpresses or amplifies PAK1 by treating the patient thereby.

Analysis of PAK1 genome amplification and functional role in human breast tumors. (A) Genomic Identification of Significant Targets in Cancer (GISTIC) analysis of 11q13 copy number gain. Points were spaced proportionally and arranged in genome order. The vertical line represents the chromosome position of the PAK1 gene. The GISTIC Q-value of DNA gain is defined by the multiple test correction probability of gain frequency and the accidental average copy gain displayed as a negative log ₁₀ of Q-value for each probe set. (B) PAK1 DNA copy and mRNA expression dot plot shows the relationship of DNA copy number to 226507 at Affymetrix MAS 5.0 signal of 51 tumor samples. Pearson correlation statistics (0.75) are shown in the plot. The solid line represents the best fit line through these points. (C) The percentage increase in Annexin V positive cells after knockdown of PAK1 expression is shown for three breast cancer cell lines with localized PAK1 genomic amplification, MDA-MB-175, HCC1500 and MDA-MB-134 IV. Cells were harvested 3-5 days after transient transfection of pooled siRNA oligonucleotides. (D) Annexin V / PI. Fluorescence activated cell sorting analysis of MDA-MB-175 cells was cultured in the presence or absence of IPA-3 for 48 hours. Annexin V / PI staining was then performed to assess apoptosis / necrosis. Annexin V label (lower right quadrant) represents the population that underwent early apoptosis. Annexin V and PI double labels (upper right quadrant) represent cells that have already died due to apoptosis. Viable cells are represented in the lower left quadrant. The percentage of cells is shown for each quadrant. PAK1 is highly expressed in human lung tumors and plays a critical role in the amplification of squamous NSCLC cell lines. (A) Analysis of PAK1 mRNA expression in laser microdissection lung tissue. Data for Affymetrix probe 226507_at are plotted as mean (horizontal line), center 50% of data (box), and 95% confidence interval (line). Paired comparisons were performed by Student's t test. Compared with normal tissues (n = 9), PAK1 expression is significant in squamous NSCLC (n = 16; **, p = 0.0005) and adenocarcinoma NSCLC (n = 29; *, p = 0.008) It was big. The difference in PAK1 mRNA expression between squamous and adenocarcinoma NSCLC was not significant in this panel of lung tumors (p = 0.1). (B) Proliferation of a panel of squamous NSCLC cell lines was measured by [ ³ H] thymidine incorporation assay. EBC-1, NCI-H520, KNS-62, SK-MES-1 and NCI-H441 cells are either non-targeting control siRNA oligonucleotides (black column) or a pool of siRNA oligonucleotides against PAK1 and PAK2 (white column) Transfected with. The degree of proliferation under each condition was plotted as a percentage of the normalized non-target control value for each cell and presented as mean ± SD. (A) Accumulation of cells in G ₁ phase of the cell cycle, it is clear after PAK1 knockdown. NCI-H520. X1 cells were treated with 200 ng / mL Dox for 4 days and analyzed by propidium iodide staining and flow cytometry. (B) NCI-H520. X1 cells were serum starved for 24 hours and after growth in medium containing 10% serum, cell cycle re-entry was monitored by collecting cell lysates at the indicated times. Cell lysates were analyzed by immunoblotting using antibodies against PAK1, p27 ^Kip1 , E2F1 and actin. (C) ^Shows the percentage of cells with p27 ^Kip1 nuclear accumulation (2000 total cells per condition). Columns represent mean ± SD. *, P <0.05. **, p <0.0001. PAK1 is established NCI-H520. Required for growth of X1 and EBC-1 squamous NSCLC tumors. (A) NCI-H520 expressing inducible shRNA against LacZ, PAK1, PAK2 or PAK1 + PAK2. X1 cells were transplanted into the flank of athymic mice as described in Materials and Methods. Treatment in each experiment was initiated when the tumor size ranged from 200-250 mm ³ . Administration of 1 mg / mL doxycycline with drinking water is shPAK1 and shPAK1 + 2 NCI-H520. It resulted in inhibition of tumor growth in mice with X1 cells. Introduction of PAK2- or LacZ-specific shRNA did not affect tumor growth kinetics. No animal weight loss was observed. Data consists of 10 mice per treatment group and error bars represent standard error. When tumors reached a volume endpoint of 2000 mm ³ , individual mice were removed from the data plot: shLacZ control n = 5; shLacZ + Dox n = 3; shPAK1 control n = 2; shPAK2 control n = 5; shPAK2 + Dox n = 2 ShPAK1 + 2 control n = 5. Before administering doxycycline to inhibit PAK1 a group of mice with tumors (B) of equal size, was grown EBC-1 tumors expressing shLacZ or shPAK1 to 200-250mm ^3. Data consists of 10 mice per treatment group and error bars represent standard error. PAK1 inhibition reduces NF-κB pathway activation and, in combination with IAP antagonists, promotes apoptosis of NSCLC cells. (A) EBC-1-shPAK1 and -shLacZ cells were treated with BV6 IAP antagonist and 300 ng / mL doxycycline (Dox). The highest concentration of BV6 was 20 μM and 2-fold serial dilutions were evaluated in a 10 point dilution curve. Cells were preincubated in the presence of Dox for 3 days before adding BV6 for an additional 3 days. Cell cultures were then analyzed by the CellTiterGlo life / death discrimination assay. Data points were performed in quadruplicate. (B) Fluorescence activated cell sorting analysis for Annexin V and propidium iodide (PI) staining. EBC-1-shPAK1 cells were cultured for 72 hours with or without 300 ng / mL doxycycline (Dox), and further cultured for 24 hours with 5 μM BV6. Annexin V / PI staining and fluorescence activated cell sorting (FACS) analysis were then performed to assess apoptosis / necrosis. (A) Down-regulation of XIAP expression enhances the pro-apoptotic activity of PF-3758309 PAK small molecule inhibitors (PAK SMI; p <0.0001, Dunnett's t-test). Cells were transfected with non-targeting control (NTC) or XIAP-specific siRNA oligonucleotides for 48 hours and then treated with DMSO or PAK SMI for an additional 72 hours as described. Cell survival was determined by CellTiterGlo assay, and results represent the mean ± standard deviation from 3 experiments. (B) The combined antagonism of XIAP and PAK1 promotes effective cleavage of PARP and caspase-3. XIAP siRNA was transfected with DMSO or 5 mM PAK SMI prior to 72 hours of treatment. (A) Combined accumulation of cleaved PARP and caspase-3 by PAK1 and IAP antagonism. Cells were incubated at the indicated time points Dox and 5 μM, lysed and then used for Western blot analysis. (B) Double PAK1 and IAP inhibition results in a synergistic decrease in the viability of SK-MES-1 (squamous subtype) and NCI-H441 (adenocarcinoma subtype) NSCLC cells. Cellular ATP consumption was determined by CellTiterGlo after transient siRNA-mediated inhibition of the described PAK1 and 5 μM BV6 treatment. The inhibition of cell viability was significantly greater in the combination of PAK1 siRNA and BV6 than in the single agent (p <0.0001, Student's t test). In breast cancer cells with local genomic growth of PAK1, PAK1 inhibition induces caspase and polyADP ribose polymerase cleavage. (A) HCC-1500 cells were transiently transfected with individual or pooled siRNA oligonucleotides (100 nM) to introduce PAK1 knockdown. Apoptosis induction was monitored by collecting cell lysates after 48 hours and immunoblotting using antibodies against cleaved caspase-3, cleaved caspase-7 and cleaved PARP. (B) Ablation of PAK1 protein expression in MDA-MB-134 IV cells was also associated with a decrease in MEK1 (Ser298) and ERK1 / 2 (Thr202 / Tyr204) phosphorylation. Total protein and β-actin were used as controls. Combined PAK1 and IAP inhibition results in apoptosis of squamous NSCLC cells. (A) The percentage of annexin V-positive cells is shown for each treatment condition. (B) Cell apoptosis markers increased after gene disruption of PAK1 and IAP antagonist treatment for the indicated times. Cell lysates were analyzed by immunoblotting. PARP cleavage and caspase-3 / 6/7/9 activity were dramatically increased by combined Dox and BV6 treatment. The combination of ATP-competing pan-PAK inhibitor PF-3758309 (BW Murray et al. Proc. Nat. Acad. Sci. USA 2010 107 (20): 9446-9471 and IAP small molecule antagonists results in apoptosis of squamous NSCLC cells. (A) Catalytic inhibition of PAK1 by PF-3758309 treatment was tested with BV6 for in vitro combinatorial effects in EBC-1 cells using a 4-day CellTiterGlo viability assay Chou and Talalay (Chou calculating synergy TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors.Adv.Enzyme Regul. 1984; 22: 27-55) The level of synergy was determined: A strong synergy was observed as indicated by a combination index (CI) value ≦ 0.3 (CI 0.113). (B), wherein the 5μM of PAK inhibitor (Paki) and 5μM of BV6 combination of time, resulted in a dramatic induction of cell apoptosis marker. Combined effect of PAK1, PAK2, MEK and PI3K inhibitors on tumor cell viability. A CellTiter-Glo® (CTG) assay of cell viability was performed after PAK1 and PAK2 siRNA transfection and 3 days of compound treatment as described. GDC-0623 is a potent and highly selective inhibitor of MEK1 and MEK2. GDC-0941 is a potent inhibitor of class I PI3K isoforms and has a biochemical IC ₅₀ value of 3-75 nM for the four class I isoforms of PI3K. (A) Viability of SKMES-1 (KRAS ^N85K mutation) lung cancer cells treated with PAK1 and PAK2 siRNA oligonucleotides, 0.2 μM GDC-0623 and 0.5 μM GDC-0941. (B) Viability of Calu-6 (KRAS ^Q61K mutation) lung cancer cells treated with PAK1 and PAK2 siRNA oligonucleotides, 0.2 μM GDC-0623 and 0.4 μM GDC-0941. (C) Viability of Cal-120 (basal subtype) breast cancer cells treated with PAK1 and PAK2 siRNA oligonucleotides, 2 μM GDC-0623 and 2.5 μM GDC-0941. Combined control of apoptosis and proliferation biomarkers after combined PAK1, PAK2, MEK and PI3K inhibition in NSCLC cells. SKMES-1 (KRAS ^N85K mutation) NSCLC cells were treated with PAK1 and PAK2 siRNA oligonucleotides, 0.4 μM GDC-0623 and 1 μM GDC-0941 for 24 hours. Accumulation of cleaved caspase-3 and poly ADP ribose polymerase (PARP), and reduction of cyclin D1 protein was enhanced by a combination of PAK knockdown with inhibitors of MEK and PI3K pathways.

  The expression “a” or “an” entity as used herein refers to one or more entities; for example, a compound refers to one or more compounds or at least one compound. Thus, the terms “a” (or “an”), “one or more”, and “at least one” may be used interchangeably herein.

  As used herein and in the claims, the terms "include", "include", "include", "include" and "includes" are stated. Is intended to identify the presence of an additional trait, integer, component or process, but does not preclude the presence or addition of one or more other traits, integers, components, processes, or groups thereof.

  The terms "treat" and "treatment" refer to both therapeutic treatments, the purpose of which is to prevent or slow (reduce) undesirable physiological changes or disorders, such as cancer growth, development or spread. ) That is. For the purposes of the present invention, advantageous or desirable clinical outcomes include, whether detectable or undetectable, relief of symptoms, reduced degree of disease, stabilized (i.e., no worsening) condition of the disease, Includes delay or slowing of progression, reduction or alleviation of disease state, and remission (partial or total). “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already having the condition or disorder, as well as those prone to have the condition or disorder, or those whose condition or disorder is to be prevented.

  The expression “therapeutically effective amount” refers to (i) treating a particular disease, condition or disorder described herein, or (ii) one or more symptoms of a particular disease, condition or disorder. Means (iii) the amount of a compound of the invention that prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder. In the case of cancer, a therapeutically effective amount of the drug can reduce the number of cancer cells; it can reduce the size of the tumor; it can inhibit the invasion of cancer cells into surrounding organs (ie can be slowed to some extent). And preferably can be stopped); metastasis of the tumor can be inhibited (ie, can be slowed to some extent and preferably stopped); tumor growth can be inhibited to some extent; and / or associated with cancer One or more of the symptoms may be alleviated to some extent. If the drug can prevent and / or kill existing cancer cells from growing, the drug can be cytostatic and / or cytotoxic. For cancer treatment, efficacy can be measured, for example, by assessing time to disease progression (TTP) and / or determining response rate (RR).

  The term “synergistic” as used herein refers to a therapeutic combination that is more effective than the additive effect of two or more single agents. The determination of the synergistic interaction between the PAK1 inhibitor and the second anti-hyperproliferative agent can be based on the results obtained from the assays described herein. This combination provided by the present invention is evaluated in several assay systems and the data utilize standard programs to quantify synergy, additive and antagonism between anticancer drugs. Can be analyzed. A preferred program is that described by Chou and Talalay in "New Avenues in Developmental Cancer Chemotherapy," Academic Press, 1987, Chapter 2. A combined index value less than 0.8 indicates a synergistic effect, a value greater than 1.2 indicates antagonism, and values between 0.8 and 1.2 indicate additive effects. Combination therapy may exhibit a “synergistic effect” and is “synergistic”, that is, an effect obtained when the active ingredients used together are greater than the sum of the effects obtained with the separate compounds. The synergistic effect is that the active ingredients are: (1) simultaneously formulated and administered or delivered simultaneously as a combined unit dosage formulation, (2) delivered alternately or in parallel as separate formulations, or ( 3) It can be achieved by some other plan. When delivered in alternation therapy, a synergistic effect can be achieved when the compounds are administered or delivered sequentially by different injections, for example in separate syringes. In general, during alternation therapy, an effective dose of each active ingredient is administered sequentially, ie, serially, whereas in combination therapy, an effective amount of two abnormal active ingredients are administered together.

  The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A “tumor” includes one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoma. More specific examples of such cancers include squamous cell carcinoma (eg, epithelial squamous cell carcinoma), small cell lung cancer, non-small cell lung cancer (“NSCLC”), lung adenocarcinoma and lung squamous carcinoma Lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon Cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, renal or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma and head and neck cancer Is included.

  The term “carcinoma” refers to an invasive malignant tumor composed of transformed epithelial cells. The term “squamous cell carcinoma” (SCC) refers to squamous cells that can occur in many different organs including the skin, lips, mouth, esophagus, bladder, prostate, lung, vagina, and cervix. Refers to a subset of acting carcinomas. It is a malignant tumor of the squamous epithelium.

  Regardless of the mechanism of action, a “chemotherapeutic agent” is a biological (large molecule) or chemical (small molecule) compound useful in the treatment of cancer. Types of chemotherapeutic agents include but are not limited to alkylating agents, antimetabolites, spindle inhibitors plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, proteins, antibodies , Photosensitizers and kinase inhibitors. Chemotherapeutic agents include compounds used in “targeting therapy” and non-targeting conventional chemotherapy.

  Examples of chemotherapeutic agents include erlotinib (TARCEVA®, Genentech / OSI Pharm.), Bortezomib (VELCADE®, Millennium Pharm.), Fulvestrant (FASLODEX®, AstraZeneca), sunitib ( sunitib) (SUTENT (R), Pfizer / Sugen), letrozole (FEMARA (R), Novartis), imatinib mesylate (GLEEVEC (R), Novartis), finasnate (VATALANIB (R), Novartis) , Oxaliplatin (ELOXATIN®, Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (Sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®), GSK572016, Glaxo Smith Kline ), Lonafamib (SCH 66336), sorafenib (NEXAVAR®, Bayer Labs), gefitinib (IRE) SA®, AstraZeneca), AG1478, alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piperosulfan; benzodopa ), Carbocones, meturedopa and uredopa; aziridines; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaoramide and trimethylololomelamine ( Ethyleneimines and methylellamelamines including trimethylolomelamine; acetogenins (especially blatatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycin (especially cryptophycin 1 and cryptophysin 8); dolastatin Duocarmycin (including synthetic analogs, KW-2189 and CBI-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; chlorambucil; Chlormaphazine, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechlorethamine oxide hydrochloride, melphalan, nobenbitine (novembichin), phenesterine, prednimustine, trofosfamide (trofosfamide) , Nitrogen mustard such as uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; enediyne antibiotics etc. See, for example, calicheamicin, especially calicheamicin γ1I and calicheamicin ωI1, eg Agnew Chem Intl. Ed. Engl., 33: 183-186 (1994); dynemicin, dynemicin A (dynemicin A); bisphosphonates such as clodronate; esperamicin; as well as neocalcinostatin and related chromoproteins enediyne antibiotic luminophore), aclacinomycin (aclacinomysins), actinomycin, ausramycin (authramy cin), azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6 -Diazo-5-oxo-L-norleucine, ADRIAMYCIN® (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and dexoxorubicin), epirubicin, esorubicin, idarubicin , Mitomycins such as marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin Puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorobicin; such as methotrexate and 5-fluorouracil (5-FU) Anti-metabolites; folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiapurine, thioguanine; Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocitabine, floxuridine; erone), androgens such as drmostanolone propionate, epithiostanol, mepithiostan, testolactone; anti-adrenal drugs such as aminoglutethimide, mitotane, trilostane; folate represions such as frolinic acid Replenisher; acegraton; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine Demecolcine; diaziquone; elfomithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansine And maytansinoids such as ansamitocin; mitoguazone; mitoxantrone; mopidamnol; nitracrine; pentostatin; phennamet; pirarubicin; podophyllinic 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid acid); triaziquone; 2,2 ', 2 "-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine) Urethane; vindesine; dacarbazine; mannomustine; mitobro Mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids such as TAXOL (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, NJ) , ABRAXANE® (Cremophor-free), paclitaxel albumin engineered nanoparticle formulation (American Pharmaceutical Partners, Schaumberg, Ill.), And TAXOTERE® (doxetaxel, doxetaxel; Sanofi-Aventis); chlorambucil GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); mitoxanthone; vincristine; vinorelbine (NAVELBINE) Navelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®); ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylol Nitin (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of those mentioned above.

  The definition of “chemotherapeutic agent” includes (i) antihormonal agents that act to regulate or inhibit hormonal effects on tumors, antiestrogens and selective estrogen receptor modulators (SERM), such as tamoxifen (NOLVADEX®) (Including tamoxifen citrate), raloxifene, droloxifene, 4-hydroxy tamoxifen, trioxifene, keoxifene, LY11018, onapristone, and FARESTON® ) (Toremifene citrate); (ii) aromatase inhibitors that inhibit aromatase enzymes, which regulate estrogen production in the adrenal glands, such as 4 (5) -imidazoles, aminoglutethimide, MEGASE (registered) Trademark) (Megestrol AS ), AROMASIN® (Exemestane, Pfizer), formestanie, fadrozole, RIVISOR® (vorozole), FEMARA® (Letrozole, Novartis) and ARIMIDEX® (Iii) antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (1,3-dioxolane nucleoside cytosine analogues); (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those that suppress the expression of genes in signal transduction pathways associated with non-adherent cell growth, such as PKC-α, Raf and H-Ras; (vii) ribozyme, eg VEG Expression inhibitors (eg ANGIOZYME®) and HER2 expression inhibitors; (viii) gene therapy vaccines, eg vaccines such as ALLOVECTIN®, LEUVECTIN® and VAXID®; PROLEUKIN® rIL -2; topoisomerase 1 inhibitors such as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such as bevacizumab (AVASTIN®, Genentech); and (x) pharmaceuticals of the above Include acceptable salts, acids or derivatives.

  The definition of “chemotherapeutic agent” also includes therapeutic antibodies such as alemtuzumab (Campath), bevacizumab (Avastin®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX ( (Registered trademark), Amgen), rituximab (RITUXAN®, Genentech / Biogen Idec), pertuzumab (OMNITARG®, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), and Tositumomab (Bexxar, Corixia).

  Humanized monoclonal antibodies with therapeutic potential as chemotherapeutic agents in combination with the PI3K inhibitors of the invention include alemtuzumab, apolizumab, acelizumab, atolizumab, bapineuzumab, bevacizumab, bibatuzumab mertansin, cantuzumab mertansin, cedelizumab celtrizumab , Shidofushitsuzumabu, Shidotsuzumabu, daclizumab, eculizumab, efalizumab, epratuzumab, Erurizumabu, Ferubizumabu, fontolizumab, gemtuzumab ozogamicin, Inotsuzumabu ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, Motobizumabu, natalizumab, nimotuzumab, Norobizumabu, Numabizumabu , Ocrelizumab, omalizuma , Palivizumab, Pasukorizumabu, Pekufushitsuzumabu, Pekutsuzumabu, pertuzumab, pexelizumab, Raribizumabu, ranibizumab, Resuribizumabu, Resurizumabu, Reshibizumabu, Roberizumabu, Rupurizumabu, sibrotuzumab, siplizumab, Sontsuzumabu, Takatsuzumabu-Tetorakisetan, Tadoshizumabu, Tarizumabu, Tefibazumabu, tocilizumab, Torarizumabu, trastuzumab, Tsukotsuzumabu • Includes Sermoleukin, Tuxituzumab, Umabizumab, Urtoxazumab and Bicilizumab.

  The following abbreviations are used in this application: DCIS (ductal carcinoma in situ), SCLC (small cell lung cancer), NSCLC (non-small cell lung cancer); SCC (squamous cell carcinoma).

  PAK is involved in many pathways that are generally deregulated in human cancer cells. PAK1 is an element of the mitogen-activated protein kinase (MAPK), JUN N-terminal kinase (JNK), steroid hormone receptor, and nuclear factor κβ (NFκβ) signaling pathway, all involved in carcinogenesis. PAK activates MEK and RAF1 by phosphorylating them at serine 298 and serine 338, respectively. The increase in Ras-induced transformation by PAK1 correlated with its effect on signaling through the extracellular signal-regulated kinase (ERK) -MAPK pathway and was separable from the effect on the JNK or p38-MAPK pathway. (R. Kumar et al. Nature Rev. Cancer 2006 6: 459) Constitutive activation of the ERK / MEK pathway is involved in tumor formation, progression and survival and is characterized by unregulated growth, loss of control of apoptosis and poor prognosis Related to the aggressive phenotype. (J.A.Spicer, Expert Opin.Drug Discov. 2008 3: 7)

  Tumor formation and progression require inactivation of proapoptotic signals in cancer cells. PAK activity has been shown to down-regulate several important pro-apoptotic pathways. PAK1 phosphorylation of RAF1 induces RAF1 translocation to mitochondria, where it phosphorylates a BCL2-antagonist (BAD) of proapoptotic protein cell death. PAK1, PAK2, PAK4 and PAK5 also directly direct BAD in selected cell types such as CV-1 (Sigma) -derived SV40-bearing (COS) kidney, Chinese hamster (CHO) and human fetal kidney (HEK) 293T cells. It has been reported to be phosphorylated and inactivated. (R. Kumar et al., Supra) However, the relevant pathways downstream of PAK1 in human tumor cells remain only partially understood.

  PAK1 is widely expressed in various normal tissues; however, expression is markedly increased in ovarian, breast and bladder cancers. (S. Balasenthil et al., J. Biol. Chem. 2004 279: 4743; M. Ito et al., J. Urol. 2007 178: 1073; P. Schraml et al., Am. J. Pathol. 2003 163: 985) In luminal breast cancer , PAK1 genomic amplification is associated with resistance to tamoxifen treatment, possibly as a result of direct phosphorylation and ligand-independent transactivation of the estrogen receptor by PAK1. (S. K. Rayala et al., Cancer Res. 2006. 66: 1694-1701) PAK1 is an attractive target for the development of effective therapeutic agents for use in the treatment of hyperproliferative disorders. (R Kumar et supra)

Proliferation experiments-PAK1 genomic copy number and gene expression were determined for a large panel of breast, lung and head and neck tumors. PAK1 genome amplification is prevalent in luminal breast cancer, and PAK1 protein expression is associated with lymph node invasion and metastasis.

  Several genomic regions with copy number gains have been identified in breast cancer by a comparative genomic hybridization approach (J. Climent et al., Biochem. Cell Biol. 2007 85: 497-508; EH van Beers and PM Nederlof, Breast Cancer Res. 2006 8: 210). The 51 breast tumor assay for DNA copy number was varied using a high resolution single nucleotide polymorphism (SNP) assay, and the Genomic Identification of Significant Targets in Cancer (GISTIC) method (PM Haverty et al., Genes Chromosomes Cancer 2008 47: Data was analyzed using R. Beroukhim et al., Proc. Natl. Acad. Sci. USA 2007 104: 20007-20012 .. Amplification of the chromosome 11 region is shown in Figure 1A. GISTIC peaks are observed at 69 and 76 Mb, suggesting that the 11q13.5 region has two independent amplicons, which correspond to CCND1 amplification and are very well described genomic changes in breast cancer. (C. Dickson, et al., Cancer Lett. 1995 90: 43-50) The 76-Mb plateau carries the PAK1 gene (shown as a dotted line) The frequency of PAK1 amplification is 17% in this tumor panel (copy number? 2.5) and the copy number gain is good with mRNA expression. (Pearson correlation = 0.75; FIG. 1B) Similar results were also obtained in large panel (n = 165) breast tumors and were also analyzed for genomic amplification by high resolution SNP analysis. Spreading, average DNA copy number was highest in luminal hormone receptor positive tumors (7.7 average copy number) and lowest in basal breast tumors (2.8 average copy number) (Z. Kan et al., Nature). 2010 466: 869-873) These experiments suggest that PAK1 may be a tumor-promoting “drive” gene in the 76-Mb amplicon of chromosome 11. Although PAK1 expression is absent in normal breast epithelial cells , Detected in 39% of malignant cells of primary breast cancer.

  PAK1 protein expression levels and subcellular localization were confirmed by immunohistochemistry (IHC) staining of tissue microarrays. In parallel, the robust and selective IHC reactivity of PAK1 antibody was confirmed in cancer cell lines by immunoblot analysis of protein extracts from these cells. PAK1 protein expression for 226 primary breast cancer, 15 DCIS, 32 breast cancer lymph node metastases, 97 NSCLC, 27 SCLC and 130 head and neck squamous cell carcinomas are summarized in Table 1. PAK1 staining intensity varied between tumor tissues from no or low staining to very strong staining in the cytoplasm and / or nucleus.

  RNA was purified from 88 primary breast cancer samples and cytoplasmic PAK1 IHC staining correlated with increased mRNA expression. These data indicate that PAK1 expression is broadly up-regulated in breast cancer and that high expression correlates with disease aggressiveness.

  Strong nuclear and cytoplasmic PAK1 expression was also prevalent in squamous non-small cell lungs, and selective PAK1 inhibition correlated with delayed cell cycle in vitro and in vivo.

頭頸部腫瘍において、上昇ＰＡＫ１発現は、頭頸部腫瘍、扁平上皮癌の他の徴候においても蔓延した(７９／１３０；６１％)(表１)。 Expression of PAK1 protein was analyzed in 27 SCLC and 97 NSCLC tissue microarrays, the latter consisting of 30 adenocarcinomas and 67 SCCs. 43/67 (64%) squamous NSCLC samples were positive for PAK1 expression and 52% of all cases showed moderate (2+) or strong (3+) intensity staining in malignant cells. Nuclear localization of PAK1 was also evident in a significant proportion of squamous NSCLC tumors (17/67; 25%). In contrast to squamous cell carcinoma, NSCLC adenocarcinoma (p = 0.0008) and SCLC (p = 0.003) tumors expressed only weak to moderate levels of PAK1 in the cytoplasm alone. Adjacent normal lung tissue did not express a clearly identifiable level of PAK1. Elevated PAK1 expression was also prevalent in other signs of head and neck tumor, squamous cell carcinoma (79/130; 61%).

In head and neck tumors, elevated PAK1 expression was also prevalent in other signs of head and neck tumor, squamous cell carcinoma (79/130; 61%) (Table 1).

Antitumor effects of PAK1 inhibition in a preclinical tumor model of squamous NSCLC—NCI-H520. Inhibition of PAK1 expression using shRNA in the X1 and EBC-1 xenograft models resulted in significant inhibition of tumor growth (FIGS. 4A and B). After tumor establishment (200-250 mm < ³ >), animals were administered Dox in sucrose drinking water and tumor growth was monitored for 21-24 days. NCI-H520. In animals bearing X1 tumors, inhibition of PAK1 but not PAK2 significantly reduced tumor growth compared to control shLacZ mice and was measured on the last day of dosing (Dunnett's t-test, p <0.0001; FIG. 4A ). Combined knockdown of PAK1 and PAK2 resulted in inhibition of tumor growth comparable to that of PAK1 inhibition alone (63.7% and 59.7%, respectively). Equal results were obtained using EBC-1 tumor xenografts and 66.8% inhibition of tumor growth was observed after in vivo knockdown of PAK1 (FIG. 4B). Finally, tumor progression data for both xenograft models were analyzed for the time required for tumor size to double from the start of treatment. In this metric, PAK1 inhibition also resulted in significant anti-tumor effects compared to other cohorts (p <0.0001). Dox treatment was well tolerated and none of the animals showed obvious weight loss. Analysis of xenograft tumors by immunohistochemistry showed a significant reduction in Ki-67 positive tumor cells in Dox-treated tumors expressing shPAK1 compared to shLacZ control. The proportion of Ki-67 positive nuclei was quantified and the antiproliferative effect of PAK1 knockdown in vivo was shown to be statistically significant (p <0.01; NCI-H520.X1 shLacZ 69 ± 6%; NCI− H520.X1 shPAK1 50 ± 4%; EBC-1 shLacZ 91 ± 3%; EBC-1 shPAK1 75 ± 11%). PAK1 and PAK2 were reduced by over 80% in Dox-treated tumors, and PAK1 knockdown was not associated with reduced AKT activation as suggested. (TC Hallstrom and JR Nevins, Cell Cycle 2009 8: 532-535.)

  Analysis of cell lines with PAK1 genomic copy number gain showed a dependence on PAK1 expression and activity for cell survival. Inhibition of PAK1 catalytic activity using IPA-3, an allosteric inhibitor that prevents PAK1-3 activation by Rho family GTPase (J. Viaud, J. and JR Peterson, Mol. Cancer Ther. 2009 8: 2559-2565 ), Resulting in a clear induction of apoptosis, determined by fluorescence-activated cell sorting analysis for Annexin V / propidium iodide staining of dead cells (7-fold increase; FIG. 1D). This phenotype was evident within 24-48 hours and was confirmed using selective siRNA-mediated knockdown of PAK1 expression (2-6 fold increase in Annexin V uptake; FIG. 1C). Cell death induced by PAK1 inhibition was also associated with caspase activation, PARP cleavage and attenuated phosphorylation of MEK1-S298 and ERK1 / 2 (FIG. 8). Thus, the strong introduction of cell death resulting from PAK1 inhibition in breast cancer cells with PAK1 amplification suggests that this kinase contributes at least in part to the oncogenic phenotype by suppressing tumor cell apoptosis.

  Dependence on PAK1 suggests that it may be a “weakness” for breast cancer subpopulations, and oncogene-dependent evidence (IB Weinstein and A. Joe, Cancer Res. 2008 68: 3077-3080) and Provides the basis for PAK1 targeted therapy in this disease symptom.

  Abnormal cytoplasmic expression of PAK1 in more than 50% squamous non-small cell lung cancer and squamous cell carcinoma of the head and neck further suggests that they may also depend on PAK1 expression for sustained growth and survival.

Currently, further known genetic abnormalities in squamous NSCLC include p53, p16 ^Ink4a , PTEN and LKB1 loss of function due to mutation or methylation, and mutation or amplification activation of protein kinases such as EGFR, MET, HER2 and PIK3CA . (RS Herbst et al., N. Engl. J. Med. 2008 359: 1367-1380) Thus, in order to increase the antitumor effect and tumor cell death in tumor cells over-amplifying or over-expressing PAK1, Inhibition of scaffold function can be combined synergistically with therapeutic agents that target these critical growth and survival pathways. Such tumor cells include, but are not limited to, DCIS, flat NSCLC, and head and neck SCC.

  Inhibitors of PAK kinase have been described. (WO2006072831 published on 7/13/2006 by D. Bouzida et al .; WO2007023382 published on 3/1/2007 by C. Guo et al; WO2007072153 published on 6/30/2007 by WO / 200707153; WO 2010/071846 published on 6/24/2010; US 20090275570 published on K. Daly et al., 11/5/2009).

  A combination of shRNA-induced PAK1 knockdown and molecular targeted therapy induces apoptosis of NSCLC cells-a small molecule library was screened to identify strong synergistic interactions between PAK1 antagonists and other anti-hyperproliferative agents ( Table 1).

Cell viability screening was performed using a panel of 200 small molecule compounds including EBC-1-shPAK1 isogenic cells and Food and Drug Administration (FDA) approved oncology drugs, signaling pathway inhibitors and DNA damaging agents. . Among the test compounds, antagonists of inhibitors of apoptotic proteins (IAP; 12 and 57-fold), epidermal growth factor receptor (EGFR; 2.9, 7.4, 12.8 and 15-fold), MAPK / ERK kinase-1 / 2 (MEK1 / 2; 8.5-fold) and Src family kinase (5.4-fold) showed a dramatic enhancement effect in combination with PF-3758309 (Table 1). None of these agents showed a strong single-agent effect on EBC-1 cell proliferation and survival in the absence of PAK1 inhibition (EC ₅₀ > 6 μM). Thus, PAK1 inhibition can greatly enhance the effects of several classes of well-characterized molecular targeted therapies.

  Inhibitors of MEK kinase (S. Price, Expert Opin.Ther.Patents 2008 18 (6): 603-626; EM Wallace et al., Curr.Topics Med.Chem. 2005 5 (2): 215), Akt (C. Lindsley, Curr. Top. Med. Chem. 2010 10: 458-477; SEGhayad and PACohen, Rec. Pat. Anti-Cancer Drug Discov. 2010 5: 29-57), Src (X.Cao et al., Mini- Rev. Med. Chem. 2008 8: 1053-1063) and Inibitor of Apoptosis Proteins (IAP) (D. Vucic and WJFairbrother, Clin. Cancer Res. 2007 13 (20) 5995; ADSchimmer and S. Dalili, Hematology 2005 215) is reviewed.

The pro-survival activity of the IAP protein is antagonized by caspase second mitochondrial activator (SMAC) (C. Du et al., Cell 2000 102: 33-42; AM Verhagen, et al .. Cell 2000 102: 43-53) Many antagonists have been described that mimic the SMAC amino-terminal peptide and disrupt the association of IAP with SMAC and activated caspase-9 (K. Zobel et al., ACS Chem. Biol. 2006 1: 525-533). E. Varfolomeev et al., Cell 2007 131: 669-681). In particular, BV6 (C. Ndubaku et al., Future Med. Chem. 2009 1 (8): 1509) represents one such class of small molecule antagonists that bind to the baculovirus IAP repeat (BIR) domain, Promotes rapid auto-ubiquitination and proteasome degradation of c-IAP1 and cIAP-2 (Zobel supra). Consistent with the small molecule screening data, strong combinatorial activity was confirmed for dual inhibition of PAK1 and IAP in EBC-1 cells. Dramatic increase in BV6 potency to EBC-1-shPAK1 cells when co-treated with Dox (EC ₅₀ = 4.1 × 10 ⁻⁷ μM) compared to control (EC ₅₀ = 3.0 × 10 ⁻³ μM) Is reflected in the strong induction of cell apoptosis determined by annexin-V flow cytometry assay (FIG. 5B) and immunoblotting of cleaved caspase-3, 6, 7 and 9 (FIG. 9). Importantly, evidence of enhanced cell killing was also observed using pharmacological inhibitors of PAK (IPA-3, PF-3758309) or Rac1 (NSC23766) catalytic activity. To further explore this clear synergy, we investigated possible molecular mechanisms of PAK1 and IAP inhibition on the induction of apoptosis. Combined PAK1 and IAP inhibition did not include modified kinetics of IAP proteolysis induced by BV6 or increased autocrine signaling by TNFα. Finally, additional NSCLC cell lines were investigated for anti-tumor effects resulting from combined inhibition of PAK1 and IAP, including those minimally involved in the activity of either single agent. Transient siRNA-mediated knockdown of PAK1 expression followed by BV6 treatment resulted in a significant decrease in SK-MES-1 and NCI-H441 cell viability (FIG. 7B).

  The combined inhibition of PAK1 and PAK2 by inhibition of the MEK (GDC-0623) and PI3K (GDC-0941; A.J. Folkes et al., J. Med. Chem. 2008 57: 5522-5532) pathways was also investigated. A combined effect determined by reduced cell viability (FIG. 11) and induction of apoptosis biomarkers (FIG. 12) was observed after inhibition of PAK, MEK and PI3K signaling. Taken together, these studies provide strong preclinical support where breast and NSCLC may provide some opportunities for rational combination therapy with PAK1 inhibitors. In one embodiment of the invention there is provided a method of treating a tumor comprising contacting the tumor with a PAK1 inhibitor and a second anti-hyperproliferative compound.

  In another embodiment of the invention, a method of treating a tumor, comprising treating a tumor with a PAK1 inhibitor and an EGFR, Raf / MEK / ERK pathway, Src, PI3K / AKT / mTOR pathway or an inhibitor of an apoptotic protein Or a method comprising contacting with an antagonist is provided.

  In another embodiment of the invention, a method of treating a tumor comprising the step of contacting the tumor with a PAK1 inhibitor and a second anti-hyperproliferative compound, wherein the tumor exhibits elevated levels of PAK1. Provided.

  In another embodiment of the invention, a method of treating a tumor, wherein the tumor exhibits elevated levels of PAK1, wherein the tumor is treated with a PAK1 inhibitor and EGFR, Raf / MEK / ERK pathway, Src, PI3K / AKT / mTOR. There is provided a method comprising contacting with an inhibitor or antagonist of a pathway or inhibitor of an apoptotic protein.

  In one embodiment of the invention, a method of treating a tumor, wherein said tumor is a breast tumor, a squamous non-small cell lung tumor or a flat head and neck tumor, said tumor being treated with a PAK1 inhibitor and a second anti-hyperproliferative compound A method is provided comprising the step of contacting.

  In another embodiment of the present invention, a method of treating a tumor, wherein the tumor is a breast tumor, a squamous non-small cell lung tumor or a flat head and neck tumor, and the tumor is a PAK1 inhibitor and EGFR, Raf / MEK / There is provided a method comprising contacting with an inhibitor or antagonist of an inhibitor of an ERK pathway, Src, Akt or an apoptotic protein.

  In another embodiment of the invention, a method of treating a tumor, wherein the tumor is a breast tumor, a squamous non-small cell lung tumor or a flat head and neck tumor that exhibits elevated levels of PAK1, wherein the tumor is a PAK1 inhibitor and A method is provided comprising the step of contacting with a second anti-hyperproliferative compound.

In another embodiment of the invention, a method of treating a tumor, wherein the tumor is a breast tumor, a squamous non-small cell lung tumor or a flat head and neck tumor that exhibits elevated levels of PAK1, wherein the tumor is a PAK1 inhibitor and There is provided a method comprising contacting with an inhibitor or antagonist of an inhibitor of EGFR, Raf / MEK / ERK pathway, Src, Akt or an apoptotic protein.

  In one embodiment of the present invention, there is provided a method of treating a tumor comprising the step of contacting the tumor with a compound of formula I (PF-3758309) and a second anti-hyperproliferative compound.

  In another embodiment of the invention, a method of treating a tumor, wherein the tumor is contacted with a compound of formula I and an inhibitor or antagonist of an inhibitor of EGFR, Raf / MEK / ERK pathway, Src, Akt or an apoptotic protein. There is provided a method comprising.

  In another embodiment of the present invention, a method of treating a tumor comprising the step of contacting the tumor with a compound of formula I and a second anti-hyperproliferative compound, wherein the tumor exhibits elevated levels of PAK1. Is provided.

  In another embodiment of the present invention, a method of treating a tumor, wherein said tumor exhibits elevated levels of PAK1, and said tumor is a compound of formula I and EGFR, Raf / MEK / ERK pathway, Src, Akt or apoptotic protein There is provided a method comprising contacting with an inhibitor or antagonist of said inhibitor.

  In another embodiment of the invention, a method of treating a tumor, wherein the tumor is a breast tumor, a squamous non-small cell lung tumor or a flat head and neck tumor that exhibits elevated levels of PAK1, wherein the tumor is a compound of formula I And a step comprising contacting with a second anti-hyperproliferative compound.

  In another embodiment of the invention, a method of treating a tumor, wherein the tumor is a breast tumor, a squamous non-small cell lung tumor or a flat head and neck tumor that exhibits elevated levels of PAK1, wherein the tumor is a compound of formula I And contacting with an inhibitor or antagonist of an inhibitor of EGFR, Raf / MEK / ERK pathway, Src, Akt or an apoptotic protein.

  In one embodiment of the present invention there is provided a method of treating a tumor comprising contacting the tumor with a compound of formula I and an inhibitor of an inhibitor of apoptosis protein.

  In another embodiment of the invention, a method of treating a tumor, comprising the step of contacting the tumor with an inhibitor of a compound of formula I and an inhibitor of an apoptotic protein, wherein the tumor exhibits elevated levels of PAK1. A method is provided.

  In one embodiment of the invention there is provided a method of treating a tumor comprising contacting the tumor with a compound of formula I and BV6 or G24416.

  In another embodiment of the invention, there is provided a method of treating a tumor, comprising the step of contacting the tumor with a compound of formula I and BV6 or G24416, wherein the tumor exhibits elevated levels of PAK1. The

  In one embodiment of the invention, there is provided a method of treating a tumor, comprising the step of contacting the tumor with a compound of formula I and an EGFR inhibitor antagonist.

  In another embodiment of the invention, a method of treating a tumor comprising the step of contacting the tumor with a compound of formula I and an EGFR inhibitor or antagonist, wherein the tumor exhibits elevated levels of PAK1. Provided.

  In one embodiment of the invention there is provided a method of treating a tumor comprising contacting the tumor with a compound of formula I and erlotinib, gefitinib or lapatinib.

  In another embodiment of the invention, a method of treating a tumor comprising the step of contacting the tumor with a compound of formula I and erlotinib, gefitinib or lapatinib, wherein the tumor exhibits elevated levels of PAK1. Provided.

  In one embodiment of the invention, there is provided a method of treating a tumor, comprising contacting the tumor with a compound of formula I and an inhibitor of Ras / Raf / MEK / Erk signaling cascade. .

  In another embodiment of the invention, a method of treating a tumor, wherein the tumor exhibits elevated levels of PAK1 and the tumor is contacted with a compound of formula I and an inhibitor of Ras / Raf / MEK / Erk signaling cascade There is provided a method comprising the step of:

  In one embodiment of the invention there is provided a method of treating a tumor comprising contacting the tumor with a compound of formula I and an inhibitor of Akt kinase.

  In another embodiment of the invention, a method of treating a tumor, comprising the step of contacting the tumor with a compound of formula I and an inhibitor of Akt kinase, wherein the tumor exhibits elevated levels of PAK1. Provided.

  In one embodiment of the present invention there is provided a method of treating a tumor comprising contacting the tumor with a compound of formula I and an inhibitor of Src kinase.

  In another embodiment of the invention, a method of treating a tumor, comprising the step of contacting the tumor with a compound of formula I and an inhibitor of Src kinase, wherein the tumor exhibits elevated levels of PAK1. Provided.

  In another embodiment of the invention, a method of treating a patient suffering from cancer or a hyperproliferative disorder comprising co-administering a PAK1 inhibitor and a second anti-hyperproliferative agent to a patient in need thereof. A method comprising is provided.

  In another embodiment of the invention, a method of treating a patient suffering from cancer or a hyperproliferative disorder, wherein a patient in need thereof is treated with a PAK1 inhibitor and EGFR, Raf / MEK / ERK pathway, Src, There is provided a method comprising co-administering an inhibitor or antagonist of an inhibitor of Akt or an apoptotic protein.

  In another embodiment, the present invention provides a combination of a PAK1 inhibitor and a second anti-hyperproliferative compound for the treatment of tumors.

  In another embodiment, the present invention provides co-administration of a PAK1 inhibitor and a second anti-hyperproliferative agent for the treatment of cancer or hyperproliferative disorders.

  In another embodiment, the present invention provides the use of a combination of a PAK1 inhibitor and a second anti-hyperproliferative compound for the preparation of a medicament for the treatment of tumors.

  In another embodiment, the present invention provides the use of a PAK1 inhibitor and a second anti-hyperproliferative agent for the preparation of a medicament for the treatment of cancer or a hyperproliferative disorder.

  The following examples illustrate the biological evaluation of compounds within the scope of the invention. These examples below are provided to enable those skilled in the art to more clearly understand and to practice the present invention. They cannot be considered as limiting the scope of the invention, but are merely illustrative and exemplary.

Example 1
Tissue Samples Formalin-fixed paraffin-embedded tissue masses and corresponding pathology reports were analyzed for 97 sequential NSCLC and 27 sequential SCLC, 130 head and neck SCC, 15 DCIS, 226 primary breast cancer and 32 breast cancer lymph nodes. Obtained from metastasis (John Radcliffe Hospital, Oxford, UK). Tissue microarrays (TMA) were assembled as previously described (L. Bubendorf, et al., J. Pathol. 2001 195: 72-79.).

  In sequential patients with breast cancer, surgery was performed between 1989 and 1998, and the patient underwent extensive local excision and postoperative radiotherapy or mastectomy (with or without postoperative radiotherapy). Patients received adjuvant chemotherapy and / or adjuvant hormone therapy or did not receive adjuvant treatment. Tamoxifen has been used as endocrine therapy for 5 years in estrogen receptor (ER) positive patients. Patients <50 years old with lymph node positive tumors, or ER- and / or primary tumors> 3 cm (diameter), 6 cycles in 3 weekly intravenous regimens, adjuvant cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) ) A> 50 year old patient with ER-, lymph node positive tumor also received CMF. The amount of estrogen receptor (ER) was determined using enzyme-linked immunosorbent assay technique (Abbott Laboratories, Abbott Park, IL). A tumor was considered positive when the cytosol ER level was> 10 fmol / mg of total cytosolic protein. HER2 status was evaluated with HercepTest (DAKO, Carpinteria, CA). Receptor values were monitored by participation in the EORTC quality-control scheme.

  The operable NSCLC series included surgical excision samples from 30 adenocarcinomas and 67 squamous cell carcinomas (surgery was performed from 1984 to 2000). Clinical and pathological data were available for 75 cancers. Thirty-five cases (47%) were stage T1, and 40 cases (53%) were stage T2. 53 cases (71%) were stage N0 and 22 cases (29%) were stage N1. The patient did not receive adjuvant chemotherapy and no information on radiation therapy was available.

  The head and neck squamous cell carcinoma series included 11 oropharyngeal cancers, 27 cancers arising in the oral cavity, 17 laryngeal cancers and 75 hypopharyngeal cancers (curative surgery was performed from 1995 to 2005). Nine cancers were UICC stage 1, 16 were stage 2, 29 were stage 3, and 76 were stage 4. Postoperatively 108 patients (83%) received radiation therapy.

Example 2
Genome copy number and expression analysis
For Affymetrix 500K SNP array analysis, genomic DNA preparation, chip processing and data analysis were performed as previously published. (PM Haverty et al., Genes Chromosomes Cancer 2008 47: 530-542) Significant gain or loss regions were determined using the GISTIC (Genomic Identification of Significant Targets in Cancer) algorithm (R. Beroukhim, et al., Proc Natl Acad Sci USA 2007 104: 20007. -20012). To collect expression array data of matched tumor samples, RNA was extracted from frozen tissue from 88 cases of primary breast cancer and applied to an Affymetrix (Santa Clara, CA) HGU133 gene expression microarray. Gene probe intensity data was used to reclassify tumors into basal, luminal-A, luminal-B, Her2 and normal types according to published standards (CM Perou et al., 2000 Nature 2000 406: 747-752). The 226507_at probe set was selected to represent PAK1 mRNA expression.

Example 3
Cell culture and viability assays Cell lines were obtained from Health Science Research Resources Bank (HSRRB, Japan) or American Type Culture Collection (ATCC; Manassas, VA) and Roswell Park with 10% fetal calf serum and 4 mM L-glutamine. Maintained at 37 ° C. and 5% CO ₂ in Memorial Institute 1640 (RPMI 1640) medium or Dulbecco's Modified Eagle Medium (DMEM). For analysis of cell proliferation by thymidine incorporation into DNA, cells were incubated with 1 μCi / well [ ³ H] thymidine for 18 hours and Unifilter® using a Filtermate ^™ 196 harvester (Perkin Elmer, Waltham, Mass.). Collected on GF / C plates. MicroScint ^™ 20 liquid scintillation cocktail was added to the dry filter plate followed by sealing and counting in a Topcount ^™ (Perkin Elmer, Waltham, MA). For cell cycle analysis by flow cytometry, cells at a density of 1 × 10 ⁶ were fixed in 70% ice-cold ethanol for 1 hour, then washed with PBS, propidium iodide (PI) solution (0.05 mg / ml in PBS). Incubation was performed at 4 ° C. for 3 hours in ml RNase solution (Sigma, St. Louis, MO), 0.05 mg / ml PI (Sigma, St. Louis, MO)). Cells were analyzed immediately on a FacScan flow cytometer (Becton Dickinson, San Jose, CA).

  To confirm the role of PAK1 in cell survival, the amount of cytoplasmic histone-related DNA fragments was quantified using the Cell Death Detection ELISA Plus kit from Roche (Mannheim, Germany). For cell death analysis by flow cytometry, cells were collected by centrifugation and stained with annexin V-FITC and PI solution (BD Biosciences, San Jose) according to the manufacturer's instructions.

In the pharmarray viability screen using a 200 compound library, EBC1-shPAK1 cells were cultured in complete growth medium and either untreated or treated with 300 ng / mL doxycycline for 3 days prior to compound addition. Cells were then replated at a suitable density in a 384 well plate and treated with 6 concentrations of each compound (4-fold serial dilution from 10 μM) for 72 hours. Cell viability was assessed by ATP content using CellTiter-Glo® Luminescent Assay (Promega, Madison, Wis.). Cell growth inhibition and EC ₅₀ differences were determined for PAK1 knockdown and wild type cells.

Example 4
Generation of RNA interference and inducible shRNA cell pools PAK1 and PAK2 small interfering RNA (siRNA) oligonucleotides were obtained from Dharmacon RNAi Technologies (Chicago, IL). The short hairpin RNA oligonucleotides used in this study are as follows: LacZ shRNA (sense) 5'-CTT ATA AGT TCC CTA TCA GTG ATA GAG ATC CCC AAT AAG CGT TGG CAA TTT ATT CAA GAG ATA TGC CAA CGC TTA TTT TTT TTG GAA-3 ′, LacZ shRNA TGA TGA TAT CATG ATC TGA TGA TGA TGA TGA TGA TGA AAG-3 ′, PAK1 shRNA-1 (sense) 5′-GAT CCC CGA AGA GAG GTT CAG CTA AAT TCA AGA G T TTA GCT GAA CCT CTC TTC TTT TTT GGA AA-3 ', PAK1 shRNA-1 (antisense) 5'-AGC TTT TCC AAA AAA GAA GAG AGG TTC AGC TAA ATC TCT ', PAK2 shRNA-3 (sense) 5'-GAT CCC CCT GCA TAA CCT GAA TGA AAT TCA AGA GAT TTC ATT CAG GTT ATG CAG TTT TTT GGA AA-3', PAK2 shRNA-5 (antisense) TTT TCC AAA AAA CTG CAT AAC CTG AAT GAA ATC TCT TGA ATT TCA TTC AGG TTA TGC AGG GG-3 ′. Inducible shRNA-carrying lentiviral constructs were prepared according to the method described previously (KP Hoeflich et al., Cancer Res. 2006 66: 999-1006; J. Climent et al., Biochem. Cell Biol. 2007 85: 497-508.) And Lipofectamine ^™ (Invitrogen, Carlsbad, Calif.) Have the desired shRNA using plasmids expressing vesicular stomatitis virus (VSV-G) envelope glycoprotein and HIV-1 packaging protein (GAG-POL) in HEK293T cells Made by co-transfecting pHUSH-Lenti-GFP and pHUSH-Lenti-dsRed constructs. Target cells were transduced with these viruses and sterilized by flow cytometry for the presence of dsRed or GFP or both (top 2-5%). Cells were characterized for doxycycline-induced protein knockdown by Western blot analysis.

Example 5
Immunoblotting and immunofluorescence Frozen tumors were ground on dry ice using a small Bessman tissue grinder (Spectrum Laboratories, Rancho Dominguez, Calif.) And the protein extract was clarified with cell extraction buffer (Invitrogen, Carlsbad, Calif.), 1 mM fluoride. 4 ° C. with phenylmethylsulfonyl bromide (PMSF), phosphatase inhibitor cocktail 1/2 (Sigma-Aldrich, St. Louis, MO), and 1 tablet Complete EDTA-free Mini ^™ protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) It was prepared with. Lysates were centrifuged for 15 minutes at 16,100 g and protein concentration was determined using the BCA protein assay (Pierce Biotechnology, Rockford, IL). For Western blot analysis, proteins were separated by 4-12% SDS-PAGE and transferred to nitrocellulose membrane (Millipore Corporation, Billerica, Mass.). Immunoblotting was performed using primary antibodies for PAK1, p27, E2F1 (Cell Signaling Technology, Danvers, Mass.) And anti-β-actin (Sigma-Aldrich, St. Louis, MO). Secondary antibody was obtained from Pierce Biotechnology (Rockford, IL).

Immunofluorescence imaging was performed using a primary antibody for p27 ^Kip1 (Becton Dickinson, San Jose, CA). Secondary antibodies were obtained from Millipore Corporation (Billerica, MA). Images were analyzed in Metamorph (version 7.5.3.0, MDS Analytical; Sunnyvale, CA) using an automated analysis routine. Briefly, a smoothing filter was applied to the DAPI channel to make the nuclear staining pattern uniform. The MWCS application at Metamorph was then used to identify and count DAPI stained nuclei and classify them as positive or negative for p27 in the Cy3 channel.

Example 6
Tumor xenograft model Cultured NCI-H520. X1 and EBC1 cells are taken from the culture, suspended in Hank's balanced salt solution (HBSS), mixed 1: 1 with Matrigel (BD Biosciences, USA) and then naive female NCR nude mice (Taconic Farms, Hudson, NY). Transplanted on the right flank. Mice with an average volume of tumor of approximately 250 mm ³ were grouped into treatment cohorts of 10 mice each. Mice received 5% sucrose alone or 5% sucrose + 1 mg / ml doxycycline (Clontech, Mountain View, Calif.) In the control and knockdown cohorts, respectively. All water bottles were changed 3 times a week. Body weight and tumor volume measurements (obtained from caliper length and width) were measured twice weekly during the study. All experimental procedures follow the guidelines of the American Physiology Society, and Genentech's Institutional Animal Care and Use Approved by the Committee. Tumor volume was calculated by the following formula: tumor volume = 0.5 * (a * b ² ), where “a” is the maximum tumor diameter and “b” is the vertical tumor diameter. Tumor volume results are expressed as mean tumor volume ± standard error of the mean (SEM). Percent growth inhibition at the end of the study (EOS) was calculated as% INH = 100 [(EOS vehicle-EOS treatment) / (EOS vehicle)]. Data analysis using Dunnet's t-test and p-value generation were performed using JMP software (SAS Institute, Cary, NC).

Xenograft tissue was fixed in 10% neutral buffered formalin for 24 hours, then processed and paraffin embedded. Sections were cut at a thickness of 3 μm and samples with sufficient live tumor (assessed on H & E-stained slides) were further evaluated by immunohistochemistry. Anti-Ki-67 (clone MIB-1, mouse anti-human) was used with DAKO ARK Kit for detection. Tissues were counterstained with hematoxylin, dehydrated and mounted. DAKO for antigen recovery according to manufacturer's instructions This was performed using a Target Retrieval Kit. Images were acquired with an Ariol SL-50 automated slide scanning platform (Genetix Ltd.) at a final magnification of x100 for quantification of immunohistochemical Ki-67 positive cells. Tumor specific areas were manually identified for analysis in Ariol software. The 3,3'-diaminobenzidine specific color range was identified using hue, saturation, and intensity color space, the area of staining was quantified, and the output was total Ki-67 positive cells relative to the total cell count .

  The characteristics disclosed in the foregoing description or in the following claims are expressed in their specific form or by means for performing the disclosed function or method or process for obtaining the disclosed results. In order to implement the invention in various forms, it may be utilized separately as appropriate or in any combination of such characteristics.

  The foregoing invention has been described in some detail by way of illustration and example, for clarity of understanding, for example. It will be apparent to those skilled in the art that changes and modifications can be practiced within the scope of the appended claims. Accordingly, it is understood that the above description is intended to be illustrative and not restrictive. Accordingly, the scope of the invention should not be determined with reference to the above description, but should be determined with reference to the following appended claims, along with equivalents to the full scope of such claims. .

  The patents, published applications, and scientific literature cited herein establish the knowledge of those skilled in the art, and to their full extent to the extent that each is specifically and individually described to be incorporated by reference. This is incorporated herein by reference. Any inconsistencies between any reference cited herein and the specific teachings of this specification are resolved by the latter. Similarly, definitions understood in the field of words or expressions and definitions of words or expressions specifically indicated in this specification are overridden by the latter.

Claims (36)

  Combination of a PAK1 inhibitor and a second anti-hyperproliferative compound for the treatment of tumors.   The combination of claim 1, wherein the tumor exhibits elevated levels of PAK1 protein.   The combination according to claim 1 or 2, wherein the tumor is a breast tumor, a squamous non-small cell lung tumor or a flat head and neck tumor. The combination according to any one of claims 1 to 3, wherein the PAK1 inhibitor is a compound of formula I:

  The combination according to any one of claims 1 to 4, wherein the second anti-hyperproliferative compound is an inhibitor of an inhibitor of apoptosis protein.   6. The combination according to claim 5, wherein said inhibitor of apoptosis protein inhibitors is BV6 or G24416.   The combination according to any one of claims 1 to 6, wherein the second anti-hyperproliferative compound is an EGFR inhibitor or antagonist.   The combination according to claim 7, wherein the EGFR inhibitor is erlotinib, gefitinib or lapatinib.   The combination according to any one of claims 1 to 4, wherein the second anti-hyperproliferative compound is an inhibitor of the Ras / Raf / MEK / Erk signaling cascade.   The combination according to any one of claims 1 to 4, wherein the second anti-hyperproliferative compound is an inhibitor of the PI3K / AKT / mTOR signaling cascade.   The combination according to any one of claims 1 to 4, wherein the second anti-hyperproliferative compound is an inhibitor of Src kinase.   Co-administration of a PAK1 inhibitor and a second anti-hyperproliferative agent for the treatment of cancer or hyperproliferative disorders.   Use of a combination of a PAK1 inhibitor and a second anti-hyperproliferative compound for the preparation of a medicament for the treatment of tumors.   14. Use according to claim 13, wherein the tumor exhibits elevated levels of PAK1 protein.   15. Use according to claim 13 or 14, wherein the tumor is a breast tumor, a squamous non-small cell lung tumor or a flat head and neck tumor. 16. Use according to any one of claims 13 to 15, wherein the PAK1 inhibitor is a compound of formula I:

  The use according to any one of claims 13 to 16, wherein the second anti-hyperproliferative compound is an inhibitor of an inhibitor of apoptosis protein.   18. Use according to claim 17, wherein the inhibitor of apoptosis protein inhibitor is BV6 or G24416.   17. Use according to any one of claims 13 to 16, wherein the second anti-hyperproliferative compound is an EGFR inhibitor or antagonist.   20. Use according to claim 19, wherein the EGFR inhibitor is erlotinib, gefitinib or lapatinib.   17. Use according to any one of claims 13 to 16, wherein the second anti-hyperproliferative compound is an inhibitor of the Ras / Raf / MEK / Erk signaling cascade.   17. Use according to any one of claims 13 to 16, wherein the second anti-hyperproliferative compound is an inhibitor of the PI3K / AKT / mTOR signaling cascade.   The use according to any one of claims 13 to 16, wherein the second anti-hyperproliferative compound is an inhibitor of Src kinase.   Use of a PAK1 inhibitor and a second anti-hyperproliferative agent for the preparation of a medicament for the treatment of cancer or hyperproliferative disorders.   A method for treating a tumor comprising the step of contacting said tumor with a PAK1 inhibitor in combination with a second anti-hyperproliferative compound.   26. The method of claim 25, wherein the tumor exhibits elevated levels of PAK1 protein.   27. A method according to claim 25 or 26, wherein the tumor is a breast tumor, a squamous non-small cell lung tumor or a flat head and neck tumor. 28. The method of claim 27, wherein the PAK1 inhibitor is a compound of formula I:

  29. A method according to any of claims 25-28, wherein the second anti-hyperproliferative compound is an inhibitor of an inhibitor of apoptosis protein.   30. The method of claim 29, wherein the inhibitor of apoptosis protein inhibitor is BV6 or G24416.   29. A method according to any of claims 25-28, wherein the anti-hyperproliferative compound is an EGFR inhibitor or antagonist.   32. The method of claim 31, wherein the EGFR inhibitor is erlotinib, gefitinib or lapatinib.   29. A method according to any of claims 25-28, wherein the second anti-hyperproliferative compound is an inhibitor of the Ras / Raf / MEK / Erk signaling cascade.   29. A method according to any of claims 25-28, wherein the second anti-hyperproliferative compound is an inhibitor of the PI3K / AKT / mTOR signaling cascade.   29. A method according to any of claims 25-28, wherein the second anti-hyperproliferative compound is an inhibitor of Src kinase.   A method of treating a patient suffering from cancer or a hyperproliferative disorder, comprising co-administering a PAK1 inhibitor and a second anti-hyperproliferative agent to a patient in need thereof.

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