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The hallmark of chronic myeloid leukemia (CML) is the presence of a BCR-ABL fusion gene that originates in hematopoietic stem cells. The BCR-ABL oncoprotein has constitutively elevated tyrosine kinase (TK) activity and drives CML pathogenesis. Introduction of Imatinib Mesylate (IM) and other tyrosine kinase inhibitor (TKI) therapies has had a major impact on the treatment of chronic phase CML, but early relapses and persistence of leukemic stem cells remain problematic. We have recently identified an AHI-1–BCR-ABL–JAK2 protein complex that contributes to the transforming activity of BCR-ABL and to IM-resistance of CML stem/progenitor cells. We therefore hypothesized that combined suppression of BCR-ABL and JAK2 activities might more effectively eliminate CML stem/progenitor cells in vitro and in vivo. Several JAK2 inhibitors are currently in various stages of clinical trials, but their off-target effects on normal primitive hematopoietic cells remain a concern. We have now examined the biological effects of an orally bioavailable, selective JAK2 inhibitor (BMS-911543) in combination with TKIs, including IM, dasatinib (DA) and nilotinib, both on CML cells lines and CD34+ treatment-naïve IM-nonresponder cells, which were obtained at diagnosis from CML patients who were classified subsequently, after initiation of IM therapy, as IM-nonresponders. In cell line studies, Western blot analysis showed that combination treatment was more effective at reducing pSTAT5 levels in K562 cells and IM-resistant K562 cells than single agents. In colony-forming cell (CFC) assays, combination treatment resulted in a greater reduction in colonies produced from these cells compared to single agents (2-3 fold, p Disclosures: No relevant conflicts of interest to declare.

Background BMS-911543 is an oral, selective small-molecule inhibitor of JAK2 (IC50=1.1nM) with potent anti-proliferative and pharmacodynamic effects in cell lines and in vivo models dependent upon JAK2 signaling. In vitro, BMS-911543 demonstrates high selectivity within the JAK family (JAK1 [IC50=356nM], JAK3 [IC50=73nM] or TYK2 [IC50=66nM]) and across a large kinase panel (at 1 µM concentration of BMS-911543, only 8 of 451 kinases were markedly inhibited [≥67%]) It also has little activity in cells dependent on other JAK family pathways. Anti-proliferative responses were observed with BMS-911543 in colony growth assays using primary progenitor cells isolated from patients with JAK2V617F-positive polycythemia vera (PV) (Leukemia 2012;26:280). Methods In a multicenter phase 1/2a study, BMS-911543 was administered orally using a BID dosing schedule, in patients with high-, intermediate-2 or intermediate-1 risk symptomatic primary or secondary MF (JAK2 WT or mutant) with platelet counts ≥50,000/mm3. Patients were not required to have palpable splenomegaly. The aim of the study is to determine safety, maximum tolerated dose (MTD), pharmacokinetics, pharmacodynamics, and preliminary efficacy of BMS-911543. Safety was assessed using NCI-CTCAE version 4.0. Treatment effect was assessed using the International Working Group (IWG) response criteria, MF-SAF, BFI, physical examination, spleen and liver volume assessment by MRI, bone marrow morphology, karyotype, V617F mutant allele burden, plasma cytokine analysis, circulating CD34+ cells and other molecular parameters. Current status To date, 84 patients have been treated (dose escalation [n=42]; dose expansion [n=42]) and the maximum tolerated dose (MTD) was defined as 200mg BID. The summary below represents an interim data analysis of 36 patients from the dose escalation phase dosed at 5, 10, 20, 40, 80, 120, 160, and 200mg BID. Fifteen of 36 (42%) patients had received prior Jak inhibitors (Jaki) including 3 who had received ≥2 Jak inhibitors. PK, PD, Safety, Tolerability and Efficacy After repeat dosing, greater than dose-proportional increase in the Cmax and AUC, and drug accumulation was observed at doses ≥40mg BID. PD analysis indicated inhibition of JAK/STAT pathway signaling activity in peripheral blood mononuclear cells, and modulation of several soluble peripheral blood proteins including IL-6, EPO and Leptin. Eleven patients (31%) had drug-related AEs. The nine hematological AEs included 3 anemia (1 grade 3 and 2 grade 2); 3 neutropenia (grade 4); and single leukopenia (grade 4), thrombocytopenia (grade 4), and reduction in platelet count (grade 2). Nine non-hematological AEs included 3 nausea (grade1/2); amylase increased (grade 3) and lipase increased (grade 3) one each; and grade 1 fatigue, muscle spasms, insomnia, erectile dysfunction one each. Ten patients (28%) discontinued treatment: 4 were attributed to disease progression, 2 to lack of efficacy, 2 at patient’s request, 1 to poor compliance, and 1 to unrelated AE. Spleen volume reductions (SVR) by MRI were observed in Jaki failure and in Jaki naive patients. Of 11 patients treated at 160 and 200mg, 8 patients (73%) had SVR greater than 35% at 3 months. In contrast, 1 of 4 patients treated at 120mg and none treated below 120mg had SVR greater than 35%. Of the patients treated at 160mg and 200mg, more than 50% had MF-SAF reductions > 50%. Overall, 3 patients (8.3%; 2 treated at 10mg BID and 1 at 200mg BID) had an increase in hemoglobin of ≥2g/dL;. Other signs of clinical activity included reductions in V617F mutant allele burden, leukocytosis, leukoerythroblastosis, circulating CD34+ cells, and LDH. Conclusion BMS-911543 was generally well tolerated and has an acceptable safety and efficacy profile. BMS-911543 induced rapid control of constitutional symptoms, and splenomegaly in patients with symptomatic MF, including those previously treated with one or more Jak inhibitors. Updated data from all 84 patients who have been treated in the dose escalation and ongoing expansion phases, including MRI assessments of spleen and liver volume, V617F mutant allele burden and levels of select plasma cytokines, will be presented. Disclosures: Pardanani: JW Pharmaceutical Corp.: Clinical trial support, Clinical trial support Other; PharmaMar: Clinical trial support, Clinical trial support Other; Sanofi: Clinical trial support, Publication support services, Clinical trial support, Publication support services Other; Bristol Myers Squibb: Clinical trial support Other. Off Label Use: Use of BMS-911543, Ruxolitinib, Momelotinib, Fedratinib and Pomalidomide for treatment of Myelofibrosis in Clinical Trial setting. Roberts:AbbVie, Inc.: Research Funding; Genentech: Research Funding; Walter and Eliza Hall Institute of Medical Research: Employee, recieves commercial income related to ABT-199, Employee, recieves commercial income related to ABT-199 Other, Employment. Seymour:Genentech: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Verstovsek:Incyte Corporation: Research Funding. Yoshitsugu:Bristol-Myers Squibb: Employment, Equity Ownership. Gestone:Bristol-Myers Squibb: Employment, Equity Ownership. Phillips:Bristol-Myers Squibb: Employment, Equity Ownership. Xing:Bristol-Myers Squibb: Employment, Equity Ownership. Peltz:Bristol-Myers Squibb: Employment, Equity Ownership. Lorenzi:Bristol-Myers Squibb: Equity Ownership, Former employment Other. Alland:Bristol-Myers Squibb: Employment, Equity Ownership. Woolfson:Bristol-Myers Squibb: Equity Ownership, Former employment Other.

Abstract 1339 Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder characterized by a BCR-ABL fusion gene with constitutive tyrosine kinase activity that drives the pathogenesis of the disease. Deregulated tyrosine kinase activity of BCR-ABL perturbs several intracellular signaling pathways, including the JAK2/STAT5 pathway. In particular, JAK2 physically interacts with the C-terminal region of BCR-ABL and is one of the most prominent targets of BCR-ABL. Recent introduction of imatinib mesylate (IM) and other tyrosine kinase inhibitor (TKI) therapies have had a major impact on treatment of chronic phase CML. However, early relapse, acquired drug resistance and ineffectiveness in eliminating leukemic stem cells remain impediments to successful treatment for many patients. Improved treatment approaches to prevent development of resistant subclones, by combined targeting of other key molecular elements active in CML stem cells, are thus clearly needed. One candidate is a complex between the oncoproteins encoded by Abelson helper integration site-1 (AHI-1), BCR-ABL and JAK2 that we recently identified. This complex contributes to the transforming activity of BCR-ABL and also plays a key role in the IM response/resistance of primary CML stem/progenitor cells. We have therefore hypothesized that combined suppression of BCR-ABL and JAK2 activities might be more effective in eliminating treatment-naïve CML stem/progenitor cells in patients destined to develop resistance to TKI monotherapy. Several JAK2 inhibitors are currently in various stages of clinical trials, but their nonspecific, off-target effects on normal primitive hematopoietic cells remain a concern. We have now examined the biological effects of a new, highly specific JAK2 inhibitor (BMS-911543) in combination with a number of TKIs, including IM, dasatinib and nilotinib. First, Western blot analysis showed that combination treatment was more effective at reducing pSTAT5 levels in K562 cells and IM-resistant K562 cells than single agents. Also, in colony-forming cell (CFC) assays, combination treatment resulted in a greater reduction in colonies produced from these cells compared to single agents, especially in medium-sized colonies (50–500 cells per colony, 2–3 fold, p Disclosures: Lorenzi: Bristol-Myers Squibb: Employment, Equity Ownership. Woolfson:Bristol-Myers Squibb: Employment, Equity Ownership.

Abstract 2743 The hallmark of chronic myeloid leukemia (CML) is the BCR-ABL fusion gene that originates in hematopoietic stem cells. The BCR-ABL oncoprotein has constitutively elevated tyrosine kinase (TK) activity and drives CML pathogenesis. The first specific BCR-ABL inhibitor, Imatinib Mesylate (IM), has had a major impact on the treatment of chronic phase CML. However, early relapse and molecular evidence of residual disease remain a significant issue in some IM-treated patients. We and others have recently demonstrated that CML stem/progenitor cells are insensitive to IM, genetically unstable, and characterized by elevated BCR-ABL expression and TK activity. The prevention of the development of resistant subclones through the specific targeting of CML stem/progenitor cells consequently represents a key unmet medical need in CML therapy. We recently identified the presence of a physical complex between oncoproteins encoded by AHI-1 (Abelson helper integration site 1), BCR-ABL and JAK2. This molecular complex contributes to the transforming activity of BCR-ABL and plays a key role in the IM responsivity/resistance of primary CML stem/progenitor cells. We therefore hypothesized that a combination treatment strategy designed to destabilize the complex might provide a rational approach to the elimination of CML stem/progenitor cells. Several JAK2 inhibitors are currently in various stages of clinical development, but myelosuppressive effects of these inhibitors on normal hematopoietic stem/progenitor cells remains a concern. We have examined a new and selective JAK2 inhibitor (BMS-911543) in combination with a number of different tyrosine kinase inhibitors (TKIs), including IM, dasatinib (DA), and nilotinib (NL) in CML cells. First, Western blot analysis showed that combination treatment with IM and BMS-911543 was more effective at reducing pSTAT5 levels in K562 cells and IM-resistant K562 cells (K562R) than in cells treated with BMS-911543 or IM alone. BMS-911543 treatment alone was not though effective in reducing pSTAT5 levels. Consistent with the Western blot analysis, in colony-forming cell (CFC) assays CML cell lines derived from myeloid or B-lymphoid blast crisis patient samples that expressed high levels of BCR-ABL (K562, K562R, BV173, and UT7 transduced with BCR-ABL) demonstrated reduced colony numbers, especially in medium-sized colonies (50–500 cells per colony) following combination treatment (BMS-911543 and IM) as compared with cells treated with BMS-911543 or IM alone (∼2–3 fold for all cell lines, p Disclosures: No relevant conflicts of interest to declare.

Abstract 4112 We report the characterization of BMS-911543, a potent and functionally selective small molecule inhibitor of the Janus kinase family (JAK) member, JAK2. BMS-911543 is a reversible inhibitor of JAK2 with a biochemical IC50 of 0.001 μ M and Ki of 0.48 nM. It has over 74- and 350-fold selectivity against the other JAK family members, JAK3 and JAK1, respectively. Further, examination of > 450 other kinases did not reveal significant inhibitory activity for this JAK2 inhibitor. Functionally, BMS-911543 displayed potent anti-proliferative and pharmacodynamic (PD) effects in mutated JAK2-expressing cell lines dependent upon JAK2-STAT signaling and had little activity in cell types dependent upon other pathways such as JAK1 and JAK3. BMS-911543 was evaluated in colony growth assays using primary progenitor cells isolated from patients with JAK2V617F-positive myeloproliferative disease (MPD) and resulted in an increased anti-proliferative response in MPD cells as compared with those from healthy volunteers. Similar to these in vitro observations, BMS-911543 was also highly active in in vivo models of JAK2-pSTAT signaling in multiple species (mouse, rat, dog and monkey) with sustained pathway suppression being observed after a single oral dose. Additionally, BMS-911543 was evaluated for effects in a JAK2V617F-expressing SET-2 xenograft model system and displayed a minimally effective dose of Disclosures: Purandare: Bristol-Myers Squibb: Employment. McDevitt:Bristol-Myers Squibb: Employment. Gottardis:Bristol-Myers Squibb: Employment. You:Bristol-Myers Squibb: Employment. Lombardo:Bristol_Myers Squibb: Employment. Penhallow:Bristol-Myers Squibb: Employment. Vuppugalla:Bristol-Myers Squibb: Employment. Trainor:Bristol-Myers Squibb: Employment. Lorenzi:Bristol-Myers Squibb: Employment.