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Characterization of BMS-911543, a Functionally Selective Small-Molecule Inhibitor of JAK2

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Characterization of BMS-911543, a Functionally Selective Small-Molecule Inhibitor of JAK2 Leukemia (2012) 26, 280–288 & 2012 Macmillan Publishers Limited All rights reserved 0887-6924/12 www.nature.com/leu ORIGINAL ARTICLE Characterization of BMS-911543, a functionally selective small-molecule inhibitor of JAK2 AV Purandare1, TM McDevitt1, H Wan1, D You1, B Penhallow1, X Han1, R Vuppugalla1, Y Zhang1, SU Ruepp1, GL Trainor1, L Lombardo1, D Pedicord1, MM Gottardis1, P Ross-Macdonald1, H de Silva1, J Hosbach1, SL Emanuel1, Y Blat1, E Fitzpatrick1, TL Taylor1, KW McIntyre1, E Michaud1, C Mulligan1, FY Lee1, A Woolfson1, TL Lasho2, A Pardanani2, A Tefferi2 and MV Lorenzi1 1Research and Development, Bristol-Myers Squibb, Princeton, NJ, USA and 2Department of Medicine, Mayo Clinic, Rochester, MN, USA We report the characterization of BMS-911543, a potent and with decreased lymphocyte development through defects in selective small-molecule inhibitor of the Janus kinase (JAK) type I (IFN-a) and type II (IL-2) cytokine signaling, whereas family member, JAK2. Functionally, BMS-911543 displayed human TYK2 deficiency is associated with impaired anti- potent anti-proliferative and pharmacodynamic (PD) effects in cell lines dependent upon JAK2 signaling, and had little activity microbial and allergic responses indicating important roles of in cell types dependent upon other pathways, such as JAK1 these family members in the regulation of key aspects of and JAK3. BMS-911543 also displayed anti-proliferative re- immune cell development and maintenance.6,7 In contrast, sponses in colony growth assays using primary progenitor JAK2 has been implicated in erythropoiesis through its associa- V617F cells isolated from patients with JAK2 -positive myelopro- tion with the selective association with the single chain cytokine liferative neoplasms (MPNs). Similar to these in vitro observa- receptor, erythropoietin receptor (EPO-R).8 tions, BMS-911543 was also highly active in in vivo models of JAK2 signaling, with sustained pathway suppression being Myeloproliferative neoplasms (MPNs) are clonal malignan- observed after a single oral dose. At low dose levels active in cies characterized by the uncontrolled expansion of multipotent JAK2-dependent PD models, no effects were observed in an in hematopoeitc progenitor cells.9 The most common BCR–ABL- vivo model of immunosuppression monitoring antigen-induced negative MPNs are polycythemia vera (PV), essential thrombo- V617F IgG and IgM production. Expression profiling of JAK2 - cytosis (ET) and primary myelofibrosis. Each MPN is associated expressing cells treated with diverse JAK2 inhibitors revealed a with clinically distinct features with PV, ET and primary shared set of transcriptional changes underlying pharmacolo- gical effects of JAK2 inhibition, including many STAT1- myelofibrosis being associated with increased erythrocytes, regulated genes and STAT1 itself. Collectively, our results platelets or bone marrow fibrosis, respectively, and all MPNs highlight BMS-911543 as a functionally selective JAK2 inhibitor are associated with progression to acute myeloid leukemia.10,11 and support the therapeutic rationale for its further character- Several laboratories identified JAK2V617F-activating mutations at ization in patients with MPN or in other disorders characterized high prevalence in MPNs with frequencies of mutation by constitutively active JAK2 signaling. estimated at B90% in PV and B50% in primary myelofibrosis Leukemia (2012) 26, 280–288; doi:10.1038/leu.2011.292; 12–14 V617F published online 21 October 2011 and ET. In JAK2 -negative MPN, mutually exclusive Keywords: tyrosine kinase inhibitor; immunosuppression; STAT1; activating mutations in the JAK2 at exon 12 have also been JAK2V617F observed.15,16 Lastly, in B5–10% of JAK2V617F-negative ET and primary myelofibrosis, mutations that result in the ligand- independent activation of the thrombopoietin receptor have been identified which produce constitutive downstream activa- Introduction tion of JAK2-STAT signaling.17,18 Transgenic expression of these mutations into hematopoietic progenitor cells in mice is able to The Janus kinases (JAK) are a family of non-receptor tyrosine 1 reconstitute many of the features of human MPN including kinases comprised of JAK1, JAK2, JAK3 and TYK2. JAKs increased red cell mass, splenomegaly and bone marrow integrate cytokine signal transduction cascades in a variety of fibrosis.19,20 Collectively, these findings underscore the critical diverse cell types through the phosphorylation and subsequent role of aberrant JAK2 signaling in MPNs and highlight JAK2 as activation of the family of signal transducer and activator of an attractive molecular target for the therapeutic intervention transcription (STAT) proteins that in turn regulate a gene in MPN and other malignancies associated with aberrant expression program underlying survival, proliferation and 2,3 JAK2-STAT signaling. differentiation. Human genetic disorders or mouse knockout Because of these features, several small-molecule JAK studies have collectively implicated essential roles of JAK-STAT inhibitors are being optimized pre-clinically or are being tested signaling in various aspects of hematopoiesis. The importance of in clinical development for patients afflicted with MPN.21 The these family members in immune cell function is highlighted by compounds in development inhibit JAK2 tyrosine kinase activity observations that genetic inactivation of human JAK3 or its by competing for ATP binding and have varying degrees of cross cognate cytokine receptor, gc, results in a severe combined 4,5 inhibitory activity to other JAK family members, such as JAK1 immunodeficiency. Similarly, JAK1 deficiency is associated and JAK3, as well as other kinases in the kinome.22 A consequence of non-selectivity to JAK1 or JAK3 is thought Correspondence: Dr MV Lorenzi, Oncology Drug Discovery, Research to be a potential for immunosuppression given the role of these and Development, Bristol-Myers Squibb, P.O. Box 4000, K13-04, kinases in immune cell function23 and as such, compounds with Princeton, NJ 08543-4000, USA. E-mail: [email protected] such activities may be associated with additional dose-limiting Received 9 August 2011; revised 11 September 2011; accepted 15 toxicities compared with other JAK2-selective inhibitors. Here, September 2011; published online 21 October 2011 we report the characterization of BMS-911543, a reversible BMS-911543 inhibits JAK2V617F cells through STAT1 and STAT5 AV Purandare et al 281 pyrrolopyridine ATP-competitive inhibitor of JAK2. The results fluorescence-activated cell sorting (FACS) analysis using annex- of our characterization highlight the high degree of functional in V surface staining following the manufacturer’s instructions selectivity of this small-molecule inhibitor toward JAK2 in a (Sigma, St Louis, MO, USA). Percent apoptosis was expressed as variety of pre-clinical models and support the ongoing clinical the level of annexin V staining observed versus dimethylsulf- testing of BMS-91153 in MPN patients. oxide control under the same treatment conditions. Human whole-blood assays and MPN-patient colony Materials and methods growth assays JAK family functional selectivity was monitored in human whole Reagents blood ex vivo using FACS and cytokine stimulation. Briefly, The chemical characterization of BMS-911543 (N,N-dicyclopropyl- blood from healthy volunteers was drawn and added to the 4-((1,5-dimethyl-1H-pyrazol-3-yl)amino)-6-ethyl-1-methyl-1,6-dihy- 96-well plate containing varying concentrations of compound in droimidazo[4,5-d]pyrrolo[2,3b]pyridine-7-carboxamide) will be dose–response format and incubated with a fixed concentration described elsewhere (AP and MVL, unpublished data). BMS- of cytokine for 1 h. Removal of red blood cells by lysis and 911543 was prepared in dimethylsulfoxide as a 10 mM stock fixation of the remaining cells was achieved by incubation at solution or in 20% citrate/80% PEG400 vehicle for in vitro or in 37 1C for 15 min (Fix and Lyse, BD Biosciences, San Jose, CA, vivo experiments, respectively. Recombinant enzymes were USA). Surface antibodies were then incubated with the cells purchased from Invitrogen (Carlsbad, CA, USA) or produced as prior to permeabilzation and phospho-STAT antibodies addi- described previously.24 Keyhole limpet hemocyanin (KLH) used tion. Cytokine-stimulated JAK2 activity was monitored in human for immunization was purchased from Pierce (Rockford, IL, platelets selected with CD61 fluorescein isothiocyanate-con- USA). SET2 cells were obtained from DSMZ (Braunschweig, jugated antibody (BD Biosciences), with thrombopoietin (TPO) Germany). stimulation (Peprotech, Rocky Hill, NJ, USA) and pSTAT5 detection (pY695 Alexa647 conjugated, BD Biosciences). Samples were analyzed on a FACS Canto II using DIVA 6.1.1 Biochemical kinase assays software (BD Biosciences). Assays for JAK1, JAK3 and TYK2 The inhibitory activity of BMS-911543 in biochemical kinase functional selectivity responses were monitored in a similar assays using recombinant enzymes was performed as described format using CD3-selected lymphocytes stimulated with IL-2, IL- 24 previously. Briefly, incubation mixtures included: 1.1 nM 4, IL-15 and IFN-a (Peprotech). Inhibitory concentration (IC50) JAK2, 1.5 mM peptide substrate (5-FAM-KKKKEEIYFFFG-OH for determinations were calculated based on 50% inhibition on the JAK2) and 30 mM ATP. The reaction mixture was analyzed on a fold-induction between cytokine and unstimulated treatments. Caliper LabChip 3000 (Caliper LifeSciences, Hopinkton, MA, The protocol for examining the effects
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