Search

Supplementary Methods

HER2 is a transmembrane tyrosine kinase receptor that mediates cell growth, differentiation, and survival. HER2 is overexpressed in approximately 20% of breast cancers and in subsets of gastric, colorectal, and esophageal cancers. Both antibody and small-molecule drugs that target HER2 and block its tyrosine kinase activity are effective in treating HER2-driven cancers. In this article, we describe the preclinical properties of tucatinib, an orally available, reversible HER2-targeted small-molecule tyrosine kinase inhibitor. In both biochemical and cell signaling experiments, tucatinib inhibits HER2 kinase activity with single-digit nanomolar potency and provides exceptional selectivity for HER2 compared with the related receptor tyrosine kinase EGFR, with a >1,000-fold enhancement in potency for HER2 in cell signaling assays. Tucatinib potently inhibits signal transduction downstream of HER2 and HER3 through the MAPK and PI3K/AKT pathways and is selectively cytotoxic in HER2-amplified breast cancer cell lines in vitro. In vivo, tucatinib is active in multiple HER2+ tumor models as a single agent and shows enhanced antitumor activity in combination with trastuzumab or docetaxel, resulting in improved rates of partial and complete tumor regression. These preclinical data, taken together with the phase-I tucatinib clinical trial results demonstrating preliminary safety and activity, establish the unique pharmacologic properties of tucatinib and underscore the rationale for investigating its utility in HER2+ cancers.

Supplementary Figure S1: Comparative potency of tucatinib, neratinib, and lapatinib in cellular HER2 and EGFR phosphorylation assays; Supplementary Figure S2: Effect of tucatinib, trastuzumab, or combination treatment on mouse body weight in HER2+ PDX models; Supplementary Table S1: Tucatinib activity correlates with HER2 surface receptor density; Supplementary Table S2. HER2 copy number variation in PDX models

Supplementary Table from ARRY-382 in Combination with Pembrolizumab in Patients with Advanced Solid Tumors: Results from a Phase 1b/2 Study

Supplementary Figure from ARRY-382 in Combination with Pembrolizumab in Patients with Advanced Solid Tumors: Results from a Phase 1b/2 Study

Purpose: ARRY-382 (PF-07265804) is a selective inhibitor of colony-stimulating factor-1 receptor. We evaluated the safety and preliminary efficacy of ARRY-382 plus pembrolizumab in patients with advanced solid tumors. Patients and Methods: This was an open-label, multicenter, Phase 1b/2 study (NCT02880371) performed over September 1, 2016 to October 24, 2019. In the Phase 1b dose-escalation, patients with selected advanced solid tumors received ARRY-382 [starting dose 200 mg once daily (QD) orally] plus pembrolizumab [2 mg/kg intravenously (IV) every 3 weeks (Q3W)]. Phase 2 patients had: Pancreatic ductal adenocarcinoma (PDA); programmed cell death protein-1 (PD-1)/PD-ligand 1 (PD-L1) inhibitor-refractory (PD-1/PD-L1 IR) advanced solid tumors; or platinum-resistant ovarian cancer (prOVCA). Patients received ARRY-382 at the maximum tolerated dose (MTD) of 300 mg QD plus pembrolizumab 200 mg IV Q3W. Results: Primary endpoints of dose-limiting toxicities (DLT; Phase 1b) and objective response rate (Phase 2) were met. In Phase 1b, 19 patients received ARRY-382 200–400 mg. Three patients reported DLTs. The MTD of ARRY-382 (plus pembrolizumab) was 300 mg QD. In Phase 1b, 2 patients (10.5%) had confirmed partial response (PR): 1 with PDA and 1 with ovarian cancer, lasting 29.2 and 3.1 months, respectively. In Phase 2, there were 27, 19, and 11 patients in the PDA, PD-1/PD-L1 IR, and prOVCA cohorts, respectively. One patient (3.7%) with PDA had a PR lasting 2.4 months. The most frequent ARRY-382–related adverse events were increased transaminases (10.5%–83.3%) and increased creatine phosphokinase (18.2%–50.0%). Conclusions: Although limited clinical benefit was observed, ARRY-382 plus pembrolizumab was well tolerated.

Activating RAS pathway mutations are found in >30% of human tumors, and often associated with poor outcomes. While RAS-pathway targeted drugs have been approved, their activity as single-agents remains modest, supporting the need for rational targeted combinations to improve patient outcomes. Multiple studies have proposed a chemical synthetic lethality between Class I histone deacetylase inhibitors (HDACi), and RAS-pathway inhibitors in RAS-pathway mutated models. This synthetic lethality occurs due to a combined effect to inhibit double-stranded (ds) DNA repair and other survival pathways to drive apoptosis and tumor regressions. The use of HDACi in solid tumors and combinations has been challenged by poor potency, selectivity, safety and convenience, highlighting the need for a better HDACi in the clinic. OKI-179, a novel largazole derivative, is a potent, Class I-selective, oral HDACi that has completed Ph 1 clinical trials in patients with solid tumors. The clinical profile shows the potential to achieve exposure consistent with preclinical activity with strong pharmacodynamic activity at tolerated doses, supporting the development of OKI-179 in solid tumor combinations. Here we show synergistic activity of combining OKI-179 with the MEK inhibitor binimetinib (bini) or bini + RAF-inhibitor encorafenib (enco/bini) in both CDX and PDX models of NRAS-mutated melanoma and BRAF-mutated colorectal cancer (CRC). In vitro, combining OKI-179 with bini in SKMEL-2 NRASMT melanoma cells shows strong synergy, inducing a significant increase in cell death compared to single agents at clinically achievable drug concentrations. This activity was associated with a significant increase in gH2AX, a marker of ds DNA breaks, and apoptosis as measured by cleaved PARP. The activity of this combination was further investigated in vivo in established PDx models of NRAS-mutant melanoma, MEL278 and MM415. As single agents, both bini (3.5 mg/kg PO bid,21 d) and OKI-179 (80 mg/kg PO; 5/wk x 3 wk), doses that mimic the clinical exposure of these molecules, showed modest tumor growth inhibition (TGI). Combining bini and OKI-179 induced significantly greater TGI, including regression in 28-50% of the tumors, as compared to 0-6% for bini alone. A similar outcome was also observed in HT29 tumors (BRAFmt CRC) where the combination of OKI-179 and enco/bini showed regression in 50% of tumors, compared to 0% for OKI-179 or enco/bini alone. This supports the potential for additive efficacy of HDACi combined with RAS-pathway inhibitors across RAS-pathway mutations and tumor types. No additivity was observed in only one model (CRC563 - BRAFmt). These data show the potential for OKI-179 to synergize with RAS-pathway inhibitors across multiple indications. To investigate, a Phase1b/2 trial of OKI-179 with binimetinib in NRAS melanoma will initiate in early 2022. Citation Format: Richard Woessner, Patrice Lee, Eric Brown, Duncan H. Walker, James Winkler, Tony Piscopio. Theclass Iselective, oral HDAC inhibitor OKI-179 increases tumor regressions when combined with the MEK inhibitor binimetinib inmodels of NRAS melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB204.

// David K. Edwards V 1,* , David Tyler Sweeney 2,* , Hibery Ho 2 , Christopher A. Eide 2 , Angela Rofelty 2 , Anupriya Agarwal 2 , Selina Qiuying Liu 2 , Alexey V. Danilov 2 , Patrice Lee 3 , David Chantry 3 , Shannon K. McWeeney 4 , Brian J. Druker 2,5 , Jeffrey W. Tyner 1 , Stephen E. Spurgeon 2,** and Marc M. Loriaux 2,** 1 Department of Cell, Developmental and Cancer Biology, Oregon Health & Science University, Portland, OR, USA 2 Division of Hematology & Medical Oncology, Oregon Health & Science University Knight Cancer Institute, Portland, OR, USA 3 Array BioPharma, Boulder, CO, USA 4 Department of Bioinformatics and Computational Biology, Oregon Health & Science University, Portland, OR, USA 5 Howard Hughes Medical Institute, Oregon Health & Science University, Knight Cancer Institute, Portland, OR, USA * These authors have contributed equally to this work ** These authors have contributed equally to this work Correspondence to: Marc M. Loriaux, email: // Keywords : colony-stimulating factor 1 receptor; chronic lymphocytic leukemia; tumor-associated macrophages; tumor microenvironment; small-molecule inhibitors Received: September 26, 2017 Accepted: March 01, 2018 Published: May 15, 2018 Abstract In many malignancies, the tumor microenvironment includes CSF1R-expressing supportive monocyte/macrophages that promote tumor cell survival. For chronic lymphocytic leukemia (CLL), these supportive monocyte/macrophages are known as nurse-like cells (NLCs), although the potential effectiveness of selective small-molecule inhibitors of CSF1R against CLL is understudied. Here, we demonstrate the preclinical activity of two inhibitors of CSF1R, GW-2580 and ARRY-382, in primary CLL patient samples. We observed at least 25% of CLL samples showed sub-micromolar sensitivity to CSF1R inhibitors. This sensitivity was observed in samples with varying genetic and clinical backgrounds, although higher white cell count and monocyte cell percentage was associated with increased sensitivity. Depleting CD14-expressing monocytes preferentially decreased viability in samples sensitive to CSF1R inhibitors, and treating samples with CSF1R inhibitors eliminated the presence of NLCs in long-term culture conditions. These results indicate that CSF1R small-molecule inhibitors target CD14-expressing monocytes in the CLL microenvironment, thereby depriving leukemia cells of extrinsic support signals. In addition, significant synergy was observed combining CSF1R inhibitors with idelalisib or ibrutinib, two current CLL therapies that disrupt tumor cell intrinsic B-cell receptor signaling. These findings support the concept of simultaneously targeting supportive NLCs and CLL cells and demonstrate the potential clinical utility of this combination.

Although approved BRAF inhibitors have transformed the treatment of patients with certain BRAF V600 mutant cancers, their long-term efficacy is thought to be limited by poor brain penetration. As a result, disease progression in the brain is a significant cause of morbidity and mortality. PF-07284890 (ARRY-461) is an orally bioavailable, brain penetrant, potent, small molecule inhibitor targeting BRAF V600 mutant tumors and is in clinical development in patients with BRAF V600 metastatic melanoma with progression to the brain. In biochemical in vitro studies, PF-07284890 inhibits BRAF and CRAF with IC50's of 5.8 and 4.1 nM, respectively. Additionally, PF-07284890 inhibits the clinically relevant kinase domain BRAF V600 mutants (V600E and V600K) (IC50 = 24-25 nM). In cell-based systems, PF-07284890 potently inhibits phosphorylation of ERK, a downstream marker of BRAF inhibition, and potently inhibits proliferation of BRAF V600E/K mutant melanoma cell lines (IC50 18-38 nM). PF-07284890 was designed to distribute to the brain and, as such, in vitro experiments indicate that PF-07284890 has high cellular membrane permeability and is not a substrate for human P glycoprotein (P-gp). After oral administration of PF-07284890 to mice and rats, PF-07284890 distributes to the brain where the free fraction adjusted exposure in the brain was approximately proportional to the free fraction adjusted exposure in plasma (i.e., Cbrain,u/Cplasma,u approaches 1.0). In vivo, PF-07284890 inhibits phosphorylation of ERK in A375 BRAF V600E tumors, achieving maximal target inhibitions at a dose of 10 mg/kg. PF-07284890 has been evaluated for its ability to control BRAF V600E cell line and patient-derived melanoma xenograft tumor growth in nude mice when implanted both subcutaneously and intracranially. Dose-related tumor growth inhibition was demonstrated at dose levels ranging from 1 to 30 mg/kg, BID in both subcutaneous and intracranial xenograft models. In all models, regardless of tumor location, maximal efficacy was observed in dose ranges of 10-30 mg/kg BID. In the intracranial A375-luc BRAF V600E melanoma xenograft model, significant and durable tumor regressions were seen at doses of 10 and 30 mg/kg/day which translated to a profound survival benefit in animals receiving PF-07284890 (median survival > 55 days post-implantation) compared to control animals (median survival = 15 days). GLP safety studies demonstrated a good safety profile for PF-07284890. PF-07284890 is fully brain penetrant, with the potential to address this key unmet medical need and thereby defines a new class of brain penetrant, potent and selective BRAF inhibitors. Citation Format: Karyn Bouhana, Deborah Anderson, Walter DeWolf, Suzy Brown, Lance Williams, Li Ren, David Moreno, Ross Wallace, Jay Brad Fell, Dylan Hartley, Patrice Lee. Nonclinical development of PF-07284890 (ARRY-461), a potent, brain-penetrant, small molecule inhibitor of BRAF V600-mutation-driven tumors in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1473.

HER2 is a transmembrane tyrosine kinase receptor that mediates cell growth, differentiation, and survival. HER2 is overexpressed in approximately 20% of breast cancers and in subsets of gastric, colorectal, and esophageal cancers. Both antibody and small-molecule drugs that target HER2 and block its tyrosine kinase activity are effective in treating HER2-driven cancers. In this article, we describe the preclinical properties of tucatinib, an orally available, reversible HER2-targeted small-molecule tyrosine kinase inhibitor. In both biochemical and cell signaling experiments, tucatinib inhibits HER2 kinase activity with single-digit nanomolar potency and provides exceptional selectivity for HER2 compared with the related receptor tyrosine kinase EGFR, with a >1,000-fold enhancement in potency for HER2 in cell signaling assays. Tucatinib potently inhibits signal transduction downstream of HER2 and HER3 through the MAPK and PI3K/AKT pathways and is selectively cytotoxic in HER2-amplified breast cancer cell lines in vitro. In vivo, tucatinib is active in multiple HER2+ tumor models as a single agent and shows enhanced antitumor activity in combination with trastuzumab or docetaxel, resulting in improved rates of partial and complete tumor regression. These preclinical data, taken together with the phase-I tucatinib clinical trial results demonstrating preliminary safety and activity, establish the unique pharmacologic properties of tucatinib and underscore the rationale for investigating its utility in HER2+ cancers.

Glucose flux through glucokinase (GK) controls insulin release from the pancreas in response to high levels of glucose. Flux through GK is also responsible for reducing hepatic glucose output. Since many individuals with type 2 diabetes appear to have an inadequacy or defect in one or both of these processes, identifying compounds that can activate GK could provide a therapeutic benefit. Herein we report the further structure activity studies of a novel series of glucokinase activators (GKA). These studies led to the identification of pyridine 72 as a potent GKA that lowered post-prandial glucose in normal C57BL/6J mice, and after 14d dosing in ob/ob mice.

Glucose flux through glucokinase (GK) controls insulin release from the pancreas in response to high glucose concentrations. Glucose flux through GK also contributes to reducing hepatic glucose output. Because many individuals with type 2 diabetes appear to have an inadequacy or defect in one or both of these processes, compounds that can activate GK may serve as effective treatments for type 2 diabetes. Herein we report the identification and initial optimization of a novel series of allosteric glucokinase activators (GKAs). We discovered an initial thiazolylamino pyridine-based hit that was optimized using a structure-based design strategy and identified 26 as an early lead. Compound 26 demonstrated a good balance of in vitro potency and enzyme kinetic parameters and demonstrated blood glucose reductions in oral glucose tolerance tests in both C57BL/6J mice and high-fat fed Zucker diabetic fatty rats.

Current therapies for treating skeletal pain have significant limitations as available drugs (non-steroidal anti-inflammatory drugs and opiates) have significant unwanted side effects. Targeting nerve growth factor (NGF) or its cognate receptor tropomysin receptor kinase A (TrkA) has recently become an attractive target for inhibition of adult skeletal pain. Here we explore whether sustained administration of a selective small molecule Trk inhibitor that blocks TrkA, TrkB and TrkC kinase activity with nanomolar affinity reduces skeletal pain while allowing the maintenance of sensory and sympathetic neurons in the adult mouse. Twice-daily administration of a Trk inhibitor was begun 1 day post fracture and within 8 h of acute administration fracture pain-related behaviors were reduced by 50% without significant sedation, weight gain or inhibition of fracture healing. Following administration of the Trk inhibitor for 7 weeks, there was no significant decline in the density of unmyelinated or myelinated sensory nerve fibers, sympathetic nerve fibers, measures of acute thermal pain, acute mechanical pain, or general neuromuscular function. The present results suggest that sustained administration of a peripherally selective TrkA, B and C inhibitor significantly reduces skeletal pain without having any obvious detrimental effects on adult sensory and sympathetic nerve fibers or early fracture healing. As with any potential therapeutic advance, understanding whether the benefits of Trk blockade are associated with any risks or unexpected effects will be required to fully appreciate the patient populations that may benefit from this therapeutic approach.

Tumor cells extensively utilize the pentose phosphate pathway for the synthesis of ribose. Transketolase is a key enzyme in this pathway and has been suggested as a target for inhibition in the treatment of cancer. In a pharmacodynamic study, nude mice with xenografted HCT-116 tumors were dosed with 1 ('N3'-pyridyl thiamine'; 3-(6-methyl-2-amino-pyridin-3-ylmethyl)-5-(2-hydroxy-ethyl)-4-methyl-thiazol-3-ium chloride hydrochloride), an analog of thiamine, the co-factor of transketolase. Transketolase activity was almost completely suppressed in blood, spleen, and tumor cells, but there was little effect on the activity of the other thiamine-utilizing enzymes alpha-ketoglutarate dehydrogenase or glucose-6-phosphate dehydrogenase. Synthesis and SAR of transketolase inhibitors is described.

Transketolase, a key enzyme in the pentose phosphate pathway, has been suggested as a target for inhibition in the treatment of cancer. Compound 5a ('N3'-pyridyl thiamine'; 3-(6-methyl-2-amino-pyridin-3-ylmethyl)-5-(2-hydroxy-ethyl)-4-methyl-thiazol-3-ium chloride hydrochloride), an analog of the transketolase cofactor thiamine, is a potent transketolase inhibitor but suffers from poor pharmacokinetics due to high clearance and C(max) linked toxicity. An efficient way of improving the pharmacokinetic profile of 5a is to prepare oxidized prodrugs which are slowly reduced in vivo yielding longer, sustained blood levels of the drug. The synthesis of such prodrugs and their evaluation in rodent models is reported.

A series of tetrazole amide derivatives of (+/-)-2-dodecyl-alpha-phenyl-N-(2,4,6-trimethoxyphenyl)-2H-tetrazole-5- acetamide (1) was prepared and evaluated for their ability to inhibit acyl-CoA: cholesterol O-acyltransferase (ACAT) in vitro and to lower plasma total cholesterol in vivo. For this series of compounds, our objective was to systematically replace substituents appended to the amide and tetrazole moieties of 1 with structurally diverse functionalities and assess the effect that these changes have on biological activity. The ensuing structure-activity relationship (SAR) studies identified aryl (7b) and heteroaryl (7f,g) replacements for 2,4,6-trimethoxyphenyl that potently inhibit liver microsomal and macrophage ACAT in vitro and exhibit good cholesterol lowering activity (56-66% decreases in plasma total cholesterol at 30 mg/kg), relative to 1, when compared in the acute rat model of hypercholesterolemia. Replacement of the alpha-phenyl moiety with electron-withdrawing substituents (13e-h), however, significantly reduced liver microsomal ACAT inhibitory activity (IC50 > 1 microM). This is in contrast to electron-donating substituents (13ij,m-q), which produce IC50 values ranging from 5 to 75 nM in the hepatic microsomal assay. For selected tetrazole amides (1, 7b, 13n,o), reversing the order of substituents appended to the 2- and 5-positions in the tetrazole ring (36a-d), in general, improved macrophage ACAT inhibitory activity and provided excellent cholesterol-lowering activity (ranging from 65% to 77% decreases in plasma total cholesterol at 30 mg/kg) in the acute rat screen. The most potent isomeric pair in this set of unsubstituted methylene derivatives (13n and 36a) caused adrenocortical cell degeneration in guinea pigs treated with these inhibitors. In contrast, adrenal glands taken from guinea pigs treated with the corresponding alpha-phenyl-substituted analogs (7b and 36c) were essentially unchanged compared to untreated controls. Subsequent evaluation of 7b and 36c in a rabbit bioassay showed that both compounds and/or their metabolities were present in plasma after oral dosing. Unlike 7b and 36c, compound 1 and related 2,4,6-trimethoxyanilides (13j, 30c,d) showed poor oral activity in the rabbit bioassay. Nevertheless, in cholesterol-fed rabbits, both systemically available (7b, 36c) and poorly absorbed inhibitors (1, 36d) were more effective in lowering plasma total cholesterol than the fatty acid amide CI-976.

Introduction: CSF1R (M-CSFR) is a receptor tyrosine kinase that, upon binding with CSF1 ligand (M-CSF), activates downstream survival, proliferation, and differentiation pathways in myeloid lineage cells. Expression and activity of CSF1R has been shown to be important for sustaining tumor-associated macrophages (TAMs) in a variety of solid tumor types, and these TAMs have been shown to provide a supportive microenvironment that maintains tumor cell viability and growth. Monocyte and macrophage lineage cells have also been implicated in providing a supportive microenvironment in certain hematologic malignancies, such as nurse-like cells in CLL, but have not been extensively explored in AML. Using an ex vivo functional screening platform applied directly to primary specimens from AML patients, we have identified recurrent sensitivity to CSF1R inhibition or silencing due to a depletion of CSF1R-expressing monocytic-lineage cells in the AML microenvironment. Methods: To identify new therapies for AML patients, our lab has screened hundreds of AML patient specimens against panels of targeted small-molecule inhibitors or siRNA and evaluated the impact on cell viability. These panels have included two small-molecule inhibitors with exquisite specificity for CSF1R (GW-2580 and ARRY-382). We have also used a panel of recombinant growth factors and cytokines, including CSF1, to measure their impact on stimulating AML cell growth. Results: We found that 20-30% of AML specimens showed sensitivity to inhibition or silencing of CSF1R, with good concordance between CSF1R siRNA, GW-2580, and ARRY-382. Exhaustive analysis of the clinical, demographic, and genetic features of these patients failed to reveal correlation between CSF1R and any prominent disease subsets. Samples sensitive to CSF1R silencing or inhibition are significantly more likely than non-sensitive samples to respond to CSF1 growth stimulation, indicating a ligand-dependent mechanism of CSF1R signaling. Additionally, we examined CSF1R and CSF1 expression in discrete hematopoietic cell populations by flow cytometry in our specimens, both with and without CSF1R inhibition. Interestingly, we found that CSF1R was predominantly expressed a subpopulation of CD14-expressing monocytes, not tumor cells, and this population disappeared after exposure to the CSF1R inhibitor. Moreover, we identified a correlation between sensitivity to CSF1R inhibition and the presence of this CD14/CSF1R-expressing cell population. Conclusions: These results suggest that CD14/CSF1R-expressing cells support AML blasts through a ligand-dependent mechanism, and depleting these cells eliminates the supportive microenvironment, resulting in leukemia cell death. Therefore, we propose using CSF1R inhibitors, such as ARRY-382, as a promising new line of therapy to target AML by disrupting the tumor microenvironment. Disclosures Agarwal: CTI BioPharma: Research Funding. Lee:Array Biopharma: Employment. Chantry:Array Biopharma: Employment. Druker:Sage Bionetworks: Research Funding; Millipore: Patents & Royalties; Molecular MD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; McGraw Hill: Patents & Royalties; CTI Biosciences, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Fred Hutchinson Cancer Research Center: Research Funding; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; AstraZeneca: Consultancy; Oregon Health and Science University: Patents & Royalties; Roche TCRC, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncotide Pharmaceuticals: Research Funding; Novartis Pharamceuticals: Research Funding; ARIAD: Research Funding; Aptose Therapeutics Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Tyner:Incyte: Research Funding; Janssen Pharmaceuticals: Research Funding; Constellation Pharmaceuticals: Research Funding; Aptose Biosciences: Research Funding; Array Biopharma: Research Funding.

Introduction: Monocyte/macrophage lineage cells have been reported to provide a supportive signal in a variety of neoplastic settings. Tumor-associated macrophages (TAMs) have been shown to provide microenvironmental support that maintains tumor cell viability and growth for a variety of solid tumor types, and these TAMs have been shown to depend on the receptor tyrosine kinase, CSF1R (M-CSFR). Monocyte/macrophage lineage cells have also been implicated in the microenvironment of CLL and are termed nurse-like cells in this setting. However, the role of CSF1R in CLL including potential maintenance of these nurse-like cells has not been explored. Using an ex vivo, functional screening platform applied directly to primary specimens from CLL patients, we have identified recurrent sensitivity to CSF1R inhibitors as well as decreased viability after depletion of CSF1R-expressing monocytes, thereby depriving the CLL cells of an important, microenvironmental growth/survival signal. We also have seen synergistic anti-tumor activity when combining CSF1R inhibitors with idelaisib and ibrutinib which inhibit b-cell receptor (BCR) activated pathways. Methods: We evaluated the impact on cell viability of hundreds of CLL patient specimens against panels of targeted small molecule inhibitors. These panels include two small-molecules with exquisite specificity for CSF1R (GW-2580; ARRY-382). In addition, we evaluated the impact of antibody depletion of monocytes (CD14-depletion) on ex vivo CLL cell viability as well as the effect of monocyte cell depletion on response to CSF1R inhibitors. We also evaluated the combination of GW-2580 or ARRY-382 with idelalisib (PI3kδ inhibitor) and ibrutinib (BTK inhibitor). Results: We found that 20-30% of CLL specimens showed sensitivity to inhibition of CSF1R with good concordance between GW-2580 and ARRY-382. Analysis of clinical and demographic features of these patients failed to reveal correlation of CSF1R with any prominent disease subsets. Flow cytometry analysis revealed that CSF1R was not expressed on CLL cells but only on a subpopulation of CD14-expressing monocytes. Depletion of these monocytes with CD14 antibody had little to no impact on samples not exhibiting ex vivo sensitivity to CSF1R inhibitors, however, samples showing strong sensitivity to CSF1R inhibitors were also quite sensitive to depletion of this CD14-positive monocyte population. After CD14 depletion, the remaining CLL cells showed no further sensitivity to CSF1R inhibitors. Finally, when combined with ibrutinib or idelalisib synergy was seen. Conclusions: These results show that CSF1R is a potential therapeutic target in CLL and suggest that CLL supporting monocytes (nurse-like cells) express CSF1R and depend on CSF1R for viability in a similar manner as TAMs depend on CSF1R in a variety of solid tumor settings. As such, CSF1R inhibitors may deprive CLL cells of this supportive microenvironmental signal resulting in CLL cell death. Therefore, we propose that CSF1R inhibitors, such as ARRY-382, possibly in combination with ibrutinib, idelalisib, or other approved agents, may be a promising new line of therapy to target CLL cells by impacting the tumor microenvironment. Disclosures Spurgeon: Genentech: Honoraria; Acerta Pharma: Research Funding; Bristol Meyers Squibb: Research Funding; Gilead sciences: Honoraria, Research Funding; Janssen: Research Funding; Pharmacyclics: Honoraria. Tyner:Janssen Pharmaceuticals: Research Funding; Incyte: Research Funding; Aptose Biosciences: Research Funding; Constellation Pharmaceuticals: Research Funding; Array Biopharma: Research Funding. Agarwal:CTI BioPharma: Research Funding. Lee:Array Biopharma: Employment. Chantry:Array Biopharma: Employment. Druker:Fred Hutchinson Cancer Research Center: Research Funding; Bristol-Myers Squibb: Research Funding; Henry Stewart Talks: Patents & Royalties; Millipore: Patents & Royalties; Sage Bionetworks: Research Funding; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy; Novartis Pharmaceuticals: Research Funding; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; CTI Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncotide Pharmaceuticals: Research Funding; Roche TCRC, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties; ARIAD: Research Funding; Aptose Therapeutics, Inc (formerly Lorus): Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Profiling the efficacy and pharmacodynamic activity of the kinesin spindle protein (KSP) inhibitor ARRY-520 will aid the identification of responsive tumor types and pharmacodynamic profiles that correlate with activity.In vivo activity was evaluated in a diverse panel of 16 different tumor xenograft models. Pharmacodynamic activity was evaluated in selected models.ARRY-520 had low nanomolar antiproliferative activity in tumor cell lines. Monopolar spindles were formed at active potencies. Partial or complete responses were observed in 13/16 xenograft models. Hematological tumors were particularly sensitive, with a 100% complete response rate in some models. Maintenance of mitotic block for a sufficient length of time for cells to lose survival signals and progress to apoptosis was a key component of the mechanism of activity. ARRY-520 was also active in several taxane resistant models.The data provide a rationale for clinical evaluation of the activity of ARRY-520 in hematological carcinomas and taxane-resistant tumors.

Purpose: The Ras-Raf-mitogen-activated protein kinase kinase (MEK) pathway is overactive in many human cancers and is thus a target for novel therapeutics. We have developed a highly potent and selective inhibitor of MEK1/2. The purpose of these studies has been to show the biological efficacy of ARRY-142886 (AZD6244) in enzymatic, cellular, and animal models. Experimental Design: The ability of ARRY-142886 to inhibit purified MEK1 as well as other kinases was evaluated. Its effects on extracellular signal-regulated kinase (ERK) phosphorylation and proliferation in several cell lines were also determined. Finally, the inhibitor was tested in HT-29 (colorectal) and BxPC3 (pancreatic) xenograft tumor models. Results: The IC50 of ARRY-142886 was determined to be 14 nmol/L against purified MEK1. This activity is not competitive with ATP, which is consistent with the high specificity of compound for MEK1/2. Basal and epidermal growth factor–induced ERK1/2 phosphorylation was inhibited in several cell lines as well as 12-O-tetradecanoylphorbol-13-acetate–induced ERK1/2 phosphorylation in isolated peripheral blood mononuclear cells. Treatment with ARRY-142886 resulted in the growth inhibition of several cell lines containing B-Raf and Ras mutations but had no effect on a normal fibroblast cell line. When dosed orally, ARRY-142886 was capable of inhibiting both ERK1/2 phosphorylation and growth of HT-29 xenograft tumors in nude mice. Tumor regressions were also seen in a BxPC3 xenograft model. In addition, tumors remained responsive to growth inhibition after a 7-day dosing holiday. Conclusions: ARRY-142886 is a potent and selective MEK1/2 inhibitor that is highly active in both in vitro and in vivo tumor models. This compound is currently being investigated in clinical studies.

High-grade serous ovarian carcinoma (HGSOC) has the poorest prognosis amongst all gynecological cancers with a median survival of approximately 5 years. While most patients (pts) initially respond to therapy, few achieve long-term remission or cure; thus, new treatment strategies are needed. Paclitaxel (Pac), an agent commonly used in the treatment of HGSOC, induces the Ras/Raf/MEK/ERK pathway and resistance may be partially mediated through activation of MEK/ERK, suggesting a rationale for combining a MEK inhibitor with Pac. Binimetinib (MEK162) is an oral, potent, selective, allosteric small-molecule MEK1/2 inhibitor. The combination of binimetinib and Pac was studied in a panel of in vivo ovarian carcinoma xenograft models derived from either cell lines (A2780, SK-OV-3) or primary patient tumor resections pathologically confirmed to be HGSOC (OVC38B1, OVC604, OVC629, OVC104) and propagated solely in mice (pt-derived xenograft [PDX]). Three of these PDX models were from newly diagnosed pts, while 1 (OVC38B1) was from a pt who had relapsed following a prior response to a platinum/taxane regimen. All models were sequenced for cancer-specific genes by next-generation sequencing; no mutations were found in Ras, Raf, MEK or ERK. To evaluate single-agent and combination activity, tumor-bearing mice received 25 mg/kg Pac IP (days 1,5,9) and/or 30 mg/kg binimetinib PO (days 2-15). Single-agent binimetinib was modestly effective in 3/6 models (tumor growth inhibition [TGI] ≥ 50%), which is consistent with the lack of MAPK pathway mutations in these models. Single-agent Pac was highly effective in 3/6 models (TGI ≥ 90%) including OVC38B1 and 2 PDX models from newly diagnosed pts; the remaining models were taxane-resistant. This is consistent with expected high rates of response in the setting of newly diagnosed pts and/or prior responders. Binimetinib plus Pac had superior efficacy to either agent alone in 4/6 models including all taxane-resistant models. In 2 PDX models from newly diagnosed pts, combination activity was not improved, but Pac was highly effective as a single agent (72-82% regression) leaving little room for enhancement. Encouragingly, the combination induced tumor regressions in 2 taxane-resistant models in which no single-agent regressions were observed. In conclusion, in vivo drug sensitivity studies with binimetinib and Pac recapitulate the response expected in HGSOC based on stage, genetic background and clinical history. In models of taxane-resistant disease, combination with binimetinib had improved activity over taxane alone warranting further study of this approach in HGSOC. Citation Format: Shannon L. Winski, Karyn Bouhana, Susan Rhodes, LouAnn Cable, Deborah Anderson, Lance Williams, Brian Tunquest, Tiffany Logan, Guy Vigers, Patrice Lee. Activity of the MEK inhibitor Binimetinib (MEK162) in combination with paclitaxel in patient-derived xenograft models of high-grade serous ovarian carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 666. doi:10.1158/1538-7445.AM2015-666

Cholangiocarcinoma (CC) responds poorly to chemo- and immunotherapy and is nearly always fatal within one year. In recent years we have gained important insight into the signaling pathways that drive this cancer. These cancers are characterized by deregulated mitogen-activated protein kinase (MAPK), PI3 kinase/Akt and a dependence on the IL-6 axis of signal transduction. Importantly, we previously reported clinical responses for CC patients receiving single agent MEK inhibitors, indicating these agents have activity in this disease. We hypothesized that dual targeting of these pathways in CC using MEK162 and buparlisib would lead to potent antitumor and immunomodulatory activity, possibly circumventing resistance to single agent MEK162. In a panel of n = 7 human CC cell lines with diverse genetic profiles, constitutive phosphorylation of ERK (7/7) and Akt (2/7) was observed. Human CC cell lines displayed variable sensitivity to the growth inhibitory and pro-apoptotic effects of single agent MEK162 or buparlisib. CC cell lines with basal AKT phosphorylation (WITT, Mz-Cha-1) showed greater sensitivity to growth inhibition by buparlisib (IC50 10-20 μM), as compared to CC lines lacking pAKT (HuCCT1, HuH28, IC50 > 20μM). Immunoblot analysis confirmed decreased phosphorylated ERK (pERK) in the HuCCT1 and SNU-478 CC cell lines following treatment with MEK162. Culture supernatants from four separate human CC cell lines displayed significant reductions in IL-6, VEGF, and GM CSF in a concentration-dependent manner after treatment with MEK162. Immunomodulatory effects of MEK162 were also evident, independent of its tumor-intrinsic effects upon CC cell lines. Namely, it significantly reduced IL 6/GM-CSF driven MDSC differentiation (HLA-DRlo CD11b+ CD33+) from healthy normal donor PBMC in vitro. These observations were not due to cytotoxic activity as treatment with MEK162 for 72 hours did not reduce viability of bulk human PBMCs. Finally, to evaluate the effect of this treatment combination, in vivo studies were conducted in athymic mice bearing Mz-Cha1 or SNU-478 xenografts. Tumor bearing mice received daily oral administration of MEK162 (30 mg/kg), buparlisib (25 mg/kg), or both agents combined. Vehicle treated animals served as negative controls. Inhibition of tumor growth was observed following administration of single agent MEK162 or buparlisib as compared to control animals. This effect was further enhanced in the combination treated animals. Body weight of animals indicated this regimen was well-tolerated. Together, these data suggest that dual PI3K and MEK inhibition can target CC with varying genotypes and represents a promising therapeutic regimen with potential for direct antitumor activity and immunomodulation in CC. Citation Format: Jennifer Yang, Omar Elnaggar, Thomas Mace, Matthew Farren, Gregory Young, Patrice Lee, Kaitlin Keenan, Zheng Che, Jacob Kaufman, Denis Guttridge, David Carbone, Cynthia Timmers, Tanios Bekaii-Saab, Gregory Lesinski. Combined inhibition of MEK and PI3K elicits anti-tumor activity in human cholangiocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2656. doi:10.1158/1538-7445.AM2015-2656

The role of MEK 1,2 in cancer tumorgenesis has been clearly demonstrated preclinically, and two selective inhibitors are currently undergoing clinical evaluation to determine their role in the human disease. We have discovered 4-(4-bromo-2-fluorophenylamino)-1-methylpyridin-2(1H)-ones as a new class of ATP noncompetitive MEK inhibitors. These inhibitors exhibit excellent cellular potency and good pharmacokinetic properties and have demonstrated the ability to inhibit ERK phosphorylation in HT-29 tumors from mouse xenograft studies.

A nuclease resistant ribozyme targeting the 5′ untranslated region (5′ UTR) of hepatitis C virus (HCV) at site 195 has been identified. To investigate the therapeutic utility of this ribozyme, we evaluated the pharmacokinetics and tissue distribution with two labeled forms of this ribozyme. [32P]-labeled ribozyme was administered as a single subcutaneous (SC) or intravenous (IV) bolus at a dose of 10 mg/kg or 30 mg/kg in C57Bl/6 mice. Regardless of route of administration, peak liver concentrations achieved were greater than the concentration necessary to inhibit HCV-IRES-luciferase expression in cell culture. The ribozyme was well absorbed after SC administration (89%) and had an elimination half-life of 23 minutes. To show intracellular localization of the ribozyme in target tissue, a tetramethyl rhodamine (TMR)–labeled ribozyme was administered as a single SC or IV bolus at a dose of 30 mg/kg in C57Bl/6 mice. Mice treated SC or IV with TMR–labeled ribozyme had positive fluorescence in the liver from 15 minutes to 48 hours after dosing. Definite positive fluorescence was still present at 72 hours in the mice dosed via the IV route. At early time points (15 and 30 minutes postinjection), nuclear and possibly cytoplasmic fluorescence was present in the hepatocytes, and sinusoidal fluorescence was intense. At the later time points, fluorescence became more punctate. Abundant staining was often present in Kupffer cells. This study confirms the retention of ribozyme in liver cells and supports the potential of an anti-HCV ribozyme as a therapeutic agent for treatment of chronic hepatitis C. (Hepatology 2000;32:640-646.)

Biliary cancer (BC), or cholangiocarcinoma, is derived from the malignant transformation of epithelial cells in the bile ducts. The disease usually presents at advanced stages and is almost uniformly deadly. Current therapeutic regimens have limited efficacy for treatment of BC, thus the need for developing novel and effective targeted strategies is a priority. This malignancy is characterized by deregulated PI3 kinase/Akt signaling, upregulated mitogen-activated protein kinase (MAPK) pathway and an autocrine IL-6/STAT3 signal transduction feedback loop, which can also drive immunosuppression. A recent report showed clinical responses for BC patients receiving the MEK inhibitor AZD244, which promoted more extensive pre-clinical studies to validate the effect of MEK inhibition and importance of this pathway in BC. In the present study, MEK162, a MEK inhibitor, was used to assess the growth inhibitory, pro-apoptotic, and immunomodulatory effects in BC. In vitro studies in a panel of human BC cell lines (HuCCT1, HuH28, MzCha1) revealed that MEK162 significantly inhibited cell growth and induced apoptosis in a concentration-dependent manner in all three cell lines. Immunoblot data confirmed decreased phosphorylated ERK (pERK) in the HuCCT1 BC cell line following a 24 hour treatment with MEK162. MEK inhibition was also associated with clinically-relevant immunomodulatory effects. First, culture supernatants from human BC cells displayed significant reductions in IL-6 protein by ELISA after treatment with MEK162 at varying concentrations. Second, MEK162 inhibited in vitro generation of human myeloid derived suppressor cells from peripheral blood mononuclear cells cultured with IL-6 and GM-CSF. Finally, in vivo studies in athymic mice bearing HuCCT1 xenografts showed that daily oral administration of MEK162 (30 mg/kg in 1% (w/v) carboxymethylcellulose, 0.5% (v/v) Tween 80) was well-tolerated and resulted in significant growth inhibition as compared to vehicle-treated controls (p < 0.0001). Immunohistochemical analysis of tumor xenografts revealed significant decreases in both Ki67 and pERK staining in the MEK162-treated mice as compared to controls (p = 0.0163 and 0.0132,respectively). Together, these data suggest that MEK inhibition represents a promising therapeutic approach with potential for direct antitumor activity and immunomodulation in BC. Citation Format: Jennifer Yang, Thomas A. Mace, Gergory S. Young, Patrice A. Lee, Kaitlin E. Keenan, Zheng Che, Tonios Bekaii-saab, Gregory B. Lesinski. MEK162, an allosteric MEK inhibitor promotes apoptosis and in vivo antitumor activity against human biliary cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 934. doi:10.1158/1538-7445.AM2013-934

ARRY-380 is an orally active, potent small molecule targeting ErbB2 inhibitor currently in clinical development in patients with ErbB2+ metastatic breast cancer (MBC). This compound has shown excellent activity in numerous SC mouse tumor models including breast (BT-474, MDA-MB-453), ovarian (SK-OV-3) and gastric (N87) carcinoma models. In breast cancer patients, brain metastases are a serious unmet medical need. Patients with ErbB2+ breast cancer have a significantly increased incidence of brain metastases following trastuzumab therapy. Here we demonstrate significant single agent activity of ARRY-380 in two ErbB2+ intracranial mouse xenograft models. For these studies, female nude mice received intracranial implantations of tumor cells (either NCI-N87 or BT-474) by direct injection into the brain parenchyma (via the sagittal suture). In pilot studies, we demonstrated that the blood brain barrier was not disrupted by mechanical injections and that increasing tumor burden correlates negatively with neurologic outcome, body weight and survival. In the N87 studies, animals received treatments beginning on Day 2 post-implantation and continuing for up to 6 weeks. Dose groups (n=12/group) were vehicle, ARRY-380 at MTD (75 mg/kg, PO, BID) or lapatinib at MTD (50 mg/kg, PO, BID). All animals in the vehicle- or lapatinib-treated groups did not survive beyond Day 22. In the ARRY-380-treated-group, 75% of the animals were alive on Day 43. Brain PK/PD was also evaluated in the N87 model. ARRY-380 and its active metabolite caused a significant reduction in brain pErbB2 (80%). In the BT-474 model, animals received treatments beginning on Day 2 post-implantation and continuing for up to 8 weeks. Dose groups (n=13/group) were vehicle, ARRY-380 at MTD (75 mg/kg, PO, BID), lapatinib at MTD (50 mg/kg, PO, BID) or neratinib at MTD (40 mg/kg, PO, QD). On Day 56, survival in the ARRY-380 group was 69% while survival rates in the vehicle, lapatinib or neratinib-treated groups were 23%, 8% and 23%, respectively. Thus ARRY-380 treatment significantly enhances survival in two ErbB2 driven intracranial tumor xenograft models, with superior activity compared to other ErbB2 agents in these studies. Additionally, ARRY-380 has demonstrated durable clinical activity in heavily pre-treated patients with ErbB2+ MBC. These preclinical and clinical data suggest that ARRY-380 may provide benefit to patients with ErbB2+ MBC with brain metastases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 852. doi:1538-7445.AM2012-852

The Trk family of genes, which includes trkA/NTRK1, trkB/NTRK2 and trkC/NTRK3, encode the tyrosine kinase receptors for the neurotrophin family of nerve growth factors. Deregulated kinase activity of trk family members is associated with human cancer. Oncogenic translocations involving trkC kinase domain have been identified in AML, salivary gland carcinoma, adult secretory breast cancer, congenital fibrosarcoma, and pediatric nephroma. Oncogenic trkA translocations have been reported in papillary thyroid and colorectal cancers. We have identified orally bioavailable, potent and selective ATP-competitive inhibitors of the trk family of receptor tyrosine kinases and are developing these for the treatment of Trk-driven cancers. AR523 is a pan-Trk inhibitor which demonstrates similar activity against TrkA, TrkB and TrkC receptors in a cell based assay (IC50 ∼10 nM). In a screen at 1 µM against a panel of 230 kinases, AR523 inhibited only three additional kinases more than 50% (TNK2, Bmx and Txk). In cell culture AR523 was found to exhibit selective anti-proliferative activity toward the cell line KM12, which harbors the oncogenic TPM-TrkA translocation, while exhibiting no activity toward HT29, a cell line with no trk gene rearrangements. Studies in mice engrafted with KM12 tumors revealed significant anti-tumor and pharmacodynamic activity of AR523. Administration of a single oral dose of AR523 reduced tyrosine phosphorylation of TrkA by nearly 80% at twelve hours after dosing. Parallel analysis of Akt and Erk revealed reduced phosphorylation of these downstream effectors. Administration of 100 mg AR523 daily for two weeks to mice with KM12 xenografts produced mean tumor growth inhibition of 86% and a mean of 42% tumor regression. AR523 exhibits dose dependent inhibition of tumor growth at 10 and 30 mg/kg in KM12 xenografts with 54 and 72% TGI respectively. AR523 was well-tolerated, causing no weight loss or deaths compared with vehicle control. Increasingly Trk mutations have been shown to be activating and the use of Trk inhibitors may provide a new therapeutic strategy for targeted treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1798. doi:1538-7445.AM2012-1798

Increasing evidence suggests that interactions between tumor cells, stromal cells, macrophages and the extracellular matrix are pivotal to the processes of tumorigenesis, metastasis, and neovascularization. Macrophages within the tumor microenvironment are thought to facilitate cancer progression, making them intriguing targets for therapy. Colony stimulating factor 1 (CSF-1) and its receptor, cFMS, play a central role in the development of mononuclear phagocytes, recruitment of macrophages to tumors, and differentiation and function of osteoclasts. We have developed an orally active, selective small-molecule cFMS inhibitor for cFMS. This molecule inhibits cFMS cellular activity (IC50 = 9 nM) in vitro and inhibits cFMS phosphorylation in a transfected cell line grown in nude mice (ED50 = 3 mg/kg). Our compound also inhibits CSF-1-mediated osteoclast differentiation and function (IC50 values of = 4 nM and 58 nM, respectively). To further explore the potential of our selective inhibitor for the treatment of cancer, we evaluated anti-tumor activity in several preclinical models. We first explored the effect on the murine ovarian cancer cell line, ID8. ID8 cells injected intraperitoneally into nude mice form multiple peritoneal tumor deposits and abundant ascites. Macrophage infiltration in the ID8 ascites was markedly lowered in mice treated with a cFMS inhibitor. Using MCF-7, a human breast adenocarcinoma cell line that has been shown to produce M-CSF, a daily oral dose with 100 mg/kg of our inhibitor for 21 days significantly reduced tumor growth and was accompanied by a marked reduction in tumor-associated macrophages. These findings support the potential of a selective inhibitor of cFMS to favorably impact human cancers by modulating tumor-associated macrophage functions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 551. doi:10.1158/1538-7445.AM2011-551

The RAS/RAF/MEK/ERK and PI3K signaling pathways are known to play key roles in cell growth, proliferation and survival. Targeted agents against factors in these pathways have been shown to provide therapeutic benefit in the treatment of cancer. However, there exists a considerable degree of cross-talk between these pathways such that when one pathway is inhibited the other is upregulated to sustain growth and survival signaling. Therefore, it would be expected that the concomitant use of targeted agents against both of these pathways would overcome the negative effects of this crosstalk, and enhance the ability of these agents to block cancer cell growth. We sought to understand whether or not the combination of the MEK inhibitor ARRY-438162 and the TORC1 inhibitor Everolimus would combine to enhance cell killing compared to the activity of the single agents. It has been established that in the majority of cancers, constituents of one or both of these pathways are mutated conferring constitutive activation to the pathway. Thus, we investigated whether key common mutations in either of these pathways conferred particular sensitivity or resistance to the combination. The growth inhibitory activities of ARRY-438162 and Everolimus were assessed alone and in combination in 26 different cell lines from a variety of human cancers with documented mutations in the KRAS, PTEN, PI3K and BRAF genes. In this regard, we report the identification of key genotypes that are associated with antagonistic, additive, and synergistic responses to the combination. In particular, treatment of the NCI-H460 NSCLC line [KRASG12D/PIK3CA] was only modestly responsive to either ARRY-162 or Everolimus as single agents (10% and 20% growth inhibition, respectively). However, the combination significantly inhibited the growth of this line, a result that was confirmed as synergistic by Bliss analysis. In order to validate the relevance of our in vitro data, an in vivo study was performed using NCI-H460 xenografts in nude mice. Everolimus alone showed no tumor growth inhibition while ARRY-438162 at doses of 30, 100 and 300 mg/kg, BID produced dose-related modest tumor growth inhibition (up to ∼50%). Combining the agents produced enhanced activity at all dose levels of ARRY-438162 without altering the exposure of either agent. Finally, in addition to growth inhibition, the biochemical effects of the single agent and the combination treatment on S6 protein, Erk, Akt, and various apoptosis regulator proteins were assessed as well as their effects on apoptosis and autophagy. ARRY-438162 having demonstrated significant tumor growth inhibitory activity in numerous pre-clinical models and acceptable preclinical and clinical safety profiles has advanced into Phase 1 clinical development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3855.

Activation of redundant receptor tyrosine kinases (RTKs) in tumors can confer resistance to selective RTK inhibitors. In particular, activation of multiple ErbB-family kinases is associated with diminished activity of selective EGFR or ErbB2 inhibitors. ErbB-family ligands are commonly expressed in cancers and can activate multiple redundant ErbB dimers. We hypothesize that ligand activation of multiple ErbB-family kinases in tumors can confer resistance to selective ErbB inhibitors, but not pan-ErbB inhibitors, suggesting a setting where pan-ErbB inhibitors may be advantageous over selective inhibitors. One such candidate currently in Phase 1/2 clinical trials is ARRY-334543, an oral, reversible pan-ErbB inhibitor with potent in vivo activity in both EGFR- and ErbB2-dependent human tumor xenografts. We tested this hypothesis in vitro with N87 gastric carcinoma cells that express high levels of constitutively active ErbB2 and low levels of EGFR. In the absence of EGF, ErbB2 homodimers are predominant and cells are highly sensitive to ErbB2 inhibition. ARRY-334543 and the ErbB2-selective inhibitor, ARRY-380, strongly inhibited N87 proliferation (IC50 = 60 nM and 5 nM, respectively) while the EGFR-selective inhibitor erlotinib was active only at high concentrations (IC50 = 860 nM). No benefit was observed by combining ARRY-380 and erlotinib. EGF-stimulation of N87 cells caused the formation of EGFR homodimers and EGFR/ErbB2 heterodimers which significantly alters cellular responses to selective ErbB inhibitors. The cellular IC50 for ARRY-380 increased 57-fold while erlotinib activity was relatively unchanged, suggesting a loss of dependence on ErbB2 signaling alone. The dual inhibition of EGFR and ErbB2, either by ARRY-334543 or the combination of ARRY-380 and erlotinib, largely maintained potency with only a modest increase in IC50 (9- to 10-fold). These data suggest that activation of EGFR can significantly diminish the activity of selective ErbB inhibitors, but not pan-ErbB inhibitors. Dual-responsiveness was also observed in vivo where both EGFR and ErbB2 homodimers were detected in N87 xenografts and dual EGFR/ErbB2 treatment resulted in superior tumor growth inhibition (TGI). ARRY-334543 was highly active with 90% TGI (100 mg/kg BID). Treatment with the combination of ARRY-380 and erlotinib (50 mg/kg QD each, MTD when given in combination) had similar activity with 82% TGI. Treatment with either erlotinib (100 mg/kg QD) or ARRY-380 (50 mg/kg QD) alone was less efficacious. These results suggest that the pan-ErbB inhibitor, ARRY-334543, can be differentiated from selective ErbB inhibitors by targeting tumors that signal through multiple ErbB-family receptors. A wide range of tumor types has been shown to activate multiple ErbB-family members and clinical evaluation of this hypothesis is ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3610.

MAPK kinase pathway-activation is implicated in uncontrolled cell proliferation and tumor growth in numerous tumor types. Targeting MEK may inhibit cancer cell signaling mediated by a wide variety of signals, making MEK an attractive target for the treatment of cancer. Recent data suggests that some cancers are resistant/refractory to MEK inhibitors due to activation of alternate pathways that optimal efficacy may require inhibition of additional pathways. We report activity of the MEK inhibitor, ARRY-162, in combination with inhibitors of mTOR and the ErbB receptor family, as well as standard-of-care chemotherapeutics in various tumor xenograft models. ARRY-162 is a novel, potent and selective allosteric MEK inhibitor that has entered clinical development for the treatment of cancer. In vivo, ARRY-162 is efficacious in numerous tumor xenograft models that harbor b-Raf or Ras mutations. ARRY-162 activity, alone and in combination with, an mTOR inhibitor (ARR-mTOR-1) was evaluated in A549 (KRas mutant) and in NCI-H460 (KRas mutant and constitutively active PI3K) models. In A549, both ARRY-162 and ARR-mTOR-1, as single agents, produced significant tumor growth inhibition (TGI; 71 and 82%, respectively). Enhanced inhibition (89% TGI) and regressions were seen when these agents were given in combination. In NCI-H460, ARRY-162 alone was inactive while ARR-mTOR-1 showed moderate activity (64 %TGI). Combination of these treatments enhanced TGI and produced significant tumor growth delay confirming recent reports that mTor pathway activation confers resistance to MEK inhibitors. The LoVo CRC model (KRas mut and pEGFR overexpression) has demonstrated resistance to EGFR-targeted therapies (i.e., cetuximab). In LoVo xenografts, ARRY-162 produced modest TGI (50%) as did ARRY-543, a pan-ErbB KI (57% TGI), with no tumor regressions in either single agent group. Combination treatment produced 83% TGI with 3 partial responses (>50% tumor regression). Thus, combining ARRY-162 with agents that inhibit signaling through the ErbB pathway produced additive efficacy and significant regressions. Lastly, the activity of ARRY-162 in combination with gemcitabine or paclitaxel was determined. ARRY-162 alone produced 40 and 79% TGI while gemcitabine or paclitaxel alone achieved 16% and 43% TGI in MiaPaCa or COLO 205 xenografts, respectively. When dosed as combinations in either model, TGI was enhanced and regressions were achieved. Thus, ARRY-162 has demonstrated significant single agent activity as well as promising additivity with anti-cancer agents. The added activity in these wide-ranging models with many different chemotherapeutics suggests a large versatility may be expected when this drug is used in combination in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2514.

Activation of the Ras/Raf/MEK/MAP kinase pathway is implicated in uncontrolled cell proliferation and tumor growth. Inappropriate activation of the RAS pathway can occur through several distinct mechanisms, including activating mutations in Ras and B-raf, or activated growth factor-signaling, cytokines and stress responses. Mutated, oncogenic forms of Ras are found in 50% of colon, 90% of pancreatic, and 30% of lung cancers. Also, B-Raf mutations have been identified in more than 60% of malignant melanomas and from 40-70% of papillary thyroid cancers. MEK, a dual specific kinase, is a key player in this pathway; it is downstream of both Ras and Raf and activates ERK1/2 through phosphorylation of key tyrosine and threonine residues. These data suggest that targeting MEK can inhibit cancer cell signaling mediated by a wide variety of signals, making MEK an attractive target for the treatment of cancer. We have discovered ARRY-162, a novel ATP-uncompetitive inhibitor of MEK 1/2, which is un-competitive with respect to ATP. ARRY-162 has nanomolar activity against purified MEK enzyme (IC50 = 12 nM) and is highly selective. It has been evaluated against 220 serine/threonine and tyrosine kinases with no inhibitory activity observed up to 20 μM. ARRY-162 inhibits both basal and induced levels of ERK phosphorylation in numerous cancer cell lines with IC50s as low as 5 nM. ARRY-162 is especially potent at inhibiting the cell proliferation of mutant B-Raf and Ras cell lines such as HT29, Malme-3M, SK-MEL-2, COLO 205, SK-MEL-28 and A375 (IC50s from 30-250 nM). In vivo, ARRY-162 has demonstrated efficacy in several xenograft tumor models in mice, including HT29, BxPC3, MIA PaCa2, A549, LoVo, Calu6, DU145 and COLO 205. In the HT29 and in the COLO 205 colon carcinoma models, dose-dependent inhibition of tumor growth (up to 75% TGI) was observed at doses ranging from 3 to 30 mg/kg, QD, PO for 21 days. In the Colo-205 colon carcinoma model, significant tumor regressions were observed with 50% partial responses and 13% complete responses at 30 mg/kg, PO, QD. In the BxPC3 pancreatic carcinoma model (which does not harbor either Ras or Raf mutations), tumor growth inhibition (∼70% TGI) and 13% partial responses were seen at doses of 30 mg/kg, QD, PO for 21 days. Consistent with ARRY-162's mechanism of action, tumor growth inhibition correlates with decreased phospho-ERK levels in tumor xenografts. In addition to its potency against MEK, this compound demonstrates other desirable attributes for development including good physical chemical characteristics, low clearance, medium-to-high Caco-2 permeability and minimal predicted drug-drug interactions. With preclinical efficacy and safety studies on ARRY-162 completed, this compound has entered clinical development for treatment of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2515.

ARRY-380 is an orally active, potent small molecule tyrosine kinase inhibitor targeting ErbB2. The compound is a reversible, ATP-competitive inhibitor with nanomolar potency against ErbB2 in both in vitro and in cell-based assays. This compound has very good in vivo and in vitro PK/ADME properties and has shown excellent activity in numerous mouse tumor models including breast (BT-474, MDA-MB-453), ovarian (SKOV-3) and gastric (N87) carcinoma models. Here we demonstrate excellent single agent activity and combinability with trastuzumab, docetaxel or bevacizumab in breast and ovarian carcinoma models. For the BT-474 studies, female SCID beige mice were implanted with tumor fragments. For the SKOV-3 tumor studies, female nude mice were inoculated with cells subcutaneously in the flank. Animals received: doses of ARRY-380 ranging up to 200 mg/kg/d, PO; and/or trastuzumab at 20 mg/kg, IP, Q3D or QW; and/or docetaxel at 10 mg/kg, IV, Q3D; and/or bevacizumab at 10 mg/kg, IP, Q4D x3. Tumor size was measured at regular intervals and subsets of animals were monitored for up to 90 days to determine tumor-free survival. In the BT-474 model, ARRY-380 demonstrated significant dose-related tumor growth inhibition (TGI; 50% at 50 mg/kg/d and 96% at 100 mg/kg/d) with numerous partial regressions (>50% reduction from baseline size) at the higher dose level in 9/12 animals. One complete response was observed at the higher dose. Trastuzumab alone provided a 45% TGI with no regressions. ARRY-380 (50 mg/kg/d) in combination with trastuzumab showed a 98% TGI with complete regressions in 9/12 animals and two partial regressions. At dose of 100 mg/kg/d of ARRY-380 in combination with trastuzumab, there was 100% TGI and all animals had complete responses. Docetaxel as a single agent produced a 55% TGI with no regressions. In combination with ARRY-380 (50 mg/kg/d), there was an 81% TGI and five partial regressions. In the SKOV-3 model, ARRY-380 demonstrated significant dose-related tumor growth inhibition (TGI; 39% at 50 mg/kg, BID and 96% at 100 mg/kg, BID) with partial regressions (>50% reduction from baseline size) at the higher dose level in all animals. Bevacizumab alone provided a 55% TGI with no regressions. ARRY-380 (50 mg/kg, BID) in combination with bevacizumab showed 80% TGI with partial responses in 7/8 animals and one stable disease. From this work we have demonstrated superb single agent activity for ARRY-380 in the BT-474 human breast carcinoma xenograft model and the SKOV-3 human ovarian carcinoma model. In addition, ARRY-380 has shown additive activity and tolerability with trastuzumab, docetaxel and bevacizumab. ARRY-380 has entered Phase I clinical trials in patients with advanced cancers. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5104.

Abstract 2858 Poster Board II-834 Introduction: The allosteric kinesin spindle protein (KSP) inhibitor ARRY-520 has potent antitumor activity as a single agent in xenograft models of multiple myeloma, with complete response rates as high as 100% in some models (Eur J Cancer Supp 6(12): 447, 2008). These observations led to a phase 1 clinical trial (currently in progress) in patients with advanced / refractory multiple myeloma who have failed treatment with both bortezomib and an imid. In further preclinical work, we report the activity of ARRY-520 in combination with standard agents used for the treatment of multiple myeloma, bortezomib and revlimid. Methods: RPMI8226, JJN3 and H929 multiple myeloma tumor xenografts were grown in female SCID-beige mice by implanting 0.5 –2 × 107 cells subcutaneously in the right flank. When tumors reached a volume of ∼ 150 – 250 mm3, mice were randomized into treatment groups based on tumor volume. Compounds were administered on schedules similar to those used in the clinic: bortezomib twice a week for two weeks, revlimid QD, and a single course of ARRY-520 on d1,2. ARRY-520 was formulated in normal saline and administered IP. Bortezomib was formulated following the supplier's instructions and also administered IP. Revlimid was formulated in 1% CMC / 0.5% Tween-80 and administered PO. Animal weights and tumor volumes were measured two or three times a week during the course of the experiments. Results: In mice bearing JJN3 or RPMI8226 xenografts, ARRY-520 administered alone was highly efficacious, with a 100% response rate (CR + PR) at doses of 12.5 to 20 mg/kg. Bortezomib administered alone at the maximum tolerated dose (MTD) of 1 mg/kg was only marginally efficacious (short term stable disease in JJN3 and H929), as was revlimid administered at 50 mg/kg (short term stable disease in RPMI8226). Because of the high initial response rate to ARRY-520 in these models, the best way to quantify the additional activity of combinations was to compare time to regrowth (time for tumor volume to return to the volume at initiation of treatment). As shown in the table, the combination of ARRY-520 with bortezomib or revlimid produced a significant increase in time to regrowth in several cases, compared to ARRY-520 alone. The additivity was schedule dependent, with greater efficacy observed when ARRY-520 was administered simulataneously with bortezomib, compared to ARRY-520 treatment before bortezomib. The activity of the ARRY-520 + bortezomib combination was specific for multiple myeloma models, as no additivity was observed in xenograft models of solid tumors (BxPC-3, LoVo, N87 and Hs294T). Conclusion: Combination of ARRY-520 with either bortezomib or revlimid has significantly greater activity than the single agents in xenograft models of multiple myeloma, suggesting these as rational combinations for evaluation in clinical trials. Disclosures: Woessner: Array BioPharma: Employment, Equity Ownership, Patents & Royalties. Tunquist:Array BioPharma: Employment, Patents & Royalties. Cox:Array BioPharma: Employment, Equity Ownership. Rana:Array BioPharma: Employment, Equity Ownership. Walker:Array BioPharma: Employment, Equity Ownership, Patents & Royalties. Lee:Array BioPharma: Employment, Equity Ownership, Patents & Royalties.

We report here the biological effects of inhibiting Pim-1, -2 and -3 serine/threonine protein kinases in cells and in vivo using a novel, potent and selective small molecule inhibitor directed against these kinases. In vitro enzyme assays revealed potent inhibition of all three Pim kinase isoforms while maintaining selectivity against more than 220 other protein kinases. In cells, compound treatment produced biological effects consistent with Pim inhibition, including a reduction in BAD protein phosphorylation. Moreover, this biological activity corresponds well with the compound’s anti-proliferative and pro-apoptotic activity in the IL-3 dependent mouse pro-B cell line Ba/F3 and in factor-indpendent Ba/F3 cell lines dependent upon the expression of BCR-Abl or TEL-JAK2 for growth and survival. Importantly, compared to the BCR-Abl driven Ba/F3 cell line, this compound was equally effective at blocking the growth of an Imatinib-resistant BCR-Abl[ T315I]-driven Ba/F3 cell line. Finally, this compound possesses pharmacokinetic properties that predict the potential for in vivo activity. Therefore, we assessed the effect of Pim kinase inhibition on tumor growth in a mouse subcutaneous tumor xenograft model employing a cell line driven by the TEL-JAK2 oncogene. Results demonstrated that the compound significantly inhibited tumor growth in a dose-dependent manner. In terms of potential clinical utility, these data show that Pim kinase inhibition could be an effective therapeutic strategy for numerous hematologic malignancies including chronic and acute myelogenous leukemias, particularly in those patients that have become resistant to existing therapies such as Imatinib.

Multiple Myeloma (MM) is a malignancy characterized by the clonal expansion of plasma cells within the bone marrow microenvironment. Within this compartment, the proliferative capacity of MM cells is enhanced by pro-inflammatory cytokines, and there is growing recognition that bone marrow stromal cells play an important role in the support of MM growth and chemo-resistance. We have identified a series of compounds, of which ARRY-614 is representative, that are potent inhibitors (EC50

Multiple myeloma (MM) is characterized by the expansion of malignant plasma cells within the bone marrow. Their growth, survival, and migration are mediated in part via cytokines. Interleukin 6 (IL-6) is necessary for sustaining the in vitro growth of many MM cell lines and enhancing the proliferation of explanted human myeloma cells. The mitogen-activated protein kinase family member, p38, is activated by cytokines and growth factors and plays a significant role in inflammatory diseases. However, its role in the pathogenesis of multiple myeloma is poorly understood. Specific p38 inhibitors inhibit paracrine MM cell growth which is associated with IL-6 and VEGF secretion from bone marrow stromal cells (BMSCs). Furthermore, p38 inhibition blocks TNF-alpha-induced IL-6 secretion in BMSCs, thereby further inhibiting MM cell growth and survival. Although these data suggest an important role for p38 in MM, the direct effects of p38 inhibiton on MM has not been extensively explored. Therefore, we investigated the effects of p38 inhibition on in vitro and in vivo IL-6 production and MM cell growth in vivo after lipopolysaccaride (LPS) stimulation. LPS has been shown to induce various cytokines, including TNF-alpha and IL-6, via the p38 pathway. ARRY-797, an orally bioavailable, small molecule inhibitor of p38 directly inhibited LPS-induced IL-6 production from RPMI-8226 (IC50 = 100 pM) in vitro. In SCID-beige mice, LPS (3 μg/kg) induced IL-6 (7897 ± 827 pg/mL) and TNF-alpha (1922 ± 282 pg/mL) after 2 hours and these cytokines were inhibited by oral administration of ARRY-797 (30 mg/kg) by 91% and 95%, respectively. In MM xenograft models, ARRY-797 (30 mg/kg, BID, PO) inhibited RPMI 8226 tumor growth by 72% as a single agent and by 56% when LPS was administered to stimulate growth in vivo. In addition, ARRY-797 inhibited LPS-induced phosphorylation of p38 in RPMI-8226 xenografts. Together, these data support a role for p38 in IL-6-mediated growth of multiple myelomas. To our knowledge, ARRY-797 is the first small molecule p38 inhibitor to demonstrate single agent activity in a MM xenograft model and it has been advanced into preclinical development.

Kinesins are eukaryotic microtubule-associated motor proteins. One of these, kinesin spindle protein (KSP) is a mitotic-specific kinesin that plays a key role in spindle pole separation and production of the bipolar spindle, as well as centrosome separation and maturation. As KSP is expressed predominately in proliferating cells and is absent from postmitotic neurons, inhibition of KSP has the potential to provide the antitumor activity of a mitotic inhibitor while avoiding peripheral neuropathy. ARRY-429520 is a member of a series of KSP inhibitors discovered and optimized by structure-based design. It is a potent inhibitor of human KSP (IC50 6 nM), with an EC50 of 1.5 nM for cellular phosphorylation of histone H3 (a pharmacodynamic marker for accumulation of cells in mitosis), and in vitro antiproliferative IC50s ranging from subnanomolar to low single digit nanomolar across a varity of human and murine tumor cell lines. We report here the in vivo characterization of this compound. ARRY-429520 inhibits histone (H3) phosphorylation in tumor xenografts , showing that the compound has pharmacodynamic activity in vivo. As a result, it is a highly efficacious inhibitor of the growth of tumor xenografts. At its maximally tolerated dose in mice of 25 – 30 mg/kg i.p. on a Q4Dx3 schedule, ARRY-429520 caused tumor regressions, with some complete responses, in subcutaneous mouse xenograft models of leukemia: HL-60 (acute promyelocytic leukemia) and K-562 (chronic myelogenous leukemia). The compound was also highly efficacious against other tumor types, including HT-29 (colon), HCT-116 (colon), and A2780 (ovarian). ARRY-429520 also showed activity in intravenous leukemia models. The molecule possess desirable drug-like properties, including high aqueous solubility (> 4 mg/ml at physiological pH), low CYP inhibition (> 25uM for 3A4, 2C9, 1A2, 2D6, and 2C19), and pharmacokinetics favorable for an IV-infused targeted chemotherapeutic.