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JAK2 Inhibitor: in Vitro Assessment of Kinase Selectivity and Preclinical Studies Using Cell Lines and Primary Cells from Polycythemia Vera Patients

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JAK2 Inhibitor: in Vitro Assessment of Kinase Selectivity and Preclinical Studies Using Cell Lines and Primary Cells from Polycythemia Vera Patients Leukemia (2009) 23, 1441–1445 & 2009 Macmillan Publishers Limited All rights reserved 0887-6924/09 $32.00 www.nature.com/leu ORIGINAL ARTICLE CYT387, a selective JAK1/JAK2 inhibitor: in vitro assessment of kinase selectivity and preclinical studies using cell lines and primary cells from polycythemia vera patients A Pardanani1, T Lasho1, G Smith2, CJ Burns2, E Fantino2 and A Tefferi1 1Hematology Division, Mayo Clinic, Rochester, MN, USA and 2Cytopia Research Pty Ltd, Melbourne, Australia Somatic mutations in Janus kinase 2 (JAK2), including elevated hemoglobin/hematocrit levels. Recent data from a JAK2V617F, result in dysregulated JAK-signal transducer and transgenic mouse model suggest that the ratio of mutant activator transcription (STAT) signaling, which is implicated in myeloproliferative neoplasm (MPN) pathogenesis. CYT387 is an JAK2V617F to wild-type JAK2 determines the MPN phenotype, ATP-competitive small molecule that potently inhibits JAK1/ with a lower ratio favoring an ET-like phenotype, and a higher 5 JAK2 kinases (IC50 ¼ 11 and 18 nM, respectively), with signifi- ratio, a PV-like phenotype. cantly less activity against other kinases, including JAK3 Given the remarkable clinical utility of imatinib for the (IC50 ¼ 155 nM). CYT387 inhibits growth of Ba/F3-JAK2V617F treatment of BCR–ABL-positive chronic myelogenous leukemia B and human erythroleukemia (HEL) cells (IC50 1500 nM)or (CML),6 FIP1L1–PDGFRA-positive CEL7 and PDGFRB-rear- Ba/F3-MPLW515L cells (IC50 ¼ 200 nM), but has considerably 8 less activity against BCR–ABL harboring K562 cells ranged MPN, several groups have begun to develop specific, (IC ¼ 58 000 nM). Cell lines harboring mutated JAK2 alleles potent, orally bioavailable inhibitors of JAK2 for the treatment of 9 (CHRF-288-11 or Ba/F3-TEL-JAK2) were inhibited more potently MPN. The agents currently undergoing preclinical and clinical than the corresponding pair harboring mutated JAK3 alleles testing inhibit both wild-type and mutant JAK2 allelesFthese (CMK or Ba/F3-TEL-JAK3), and STAT-5 phosphorylation was compounds inhibit the growth of cell lines harboring mutant inhibited in HEL cells with an IC50 ¼ 400 nM. Furthermore, JAK2, and the in vitro growth of hematopoietic colonies from CYT387 selectively suppressed the in vitro growth of erythroid 10,11 colonies harboring JAK2V617F from polycythemia vera (PV) MPN patients at pharmacological concentrations. In a patients, an effect that was attenuated by exogenous erythro- murine model of JAK2V617F-induced PV, one such compound poietin. Overall, our data indicate that the JAK1/JAK2 selective produced a therapeutic response that was associated with inhibitor CYT387 has potential for efficacious treatment of MPN favorable histopathological changes and a quantitative decrease harboring mutated JAK2 and MPL alleles. in the mutant genomic copy burden.12 Leukemia (2009) 23, 1441–1445; doi:10.1038/leu.2009.50; Here, we report our findings pertaining to the small-molecule published online 19 March 2009 Keywords: myeloproliferative neoplasm; myelofibrosis; Janus JAK1/JAK2 inhibitor, CYT387, including its kinase selectivity kinase; JAK2V617F; kinase inhibitor characteristics and efficacy in inhibiting growth of cell lines and primary cells harboring activating JAK2 and MPL mutations. Materials and methods Introduction Patient accrual and sample collection The discovery of somatic mutations in Janus kinase 2 (JAK2), This study was approved by the Mayo Clinic institutional review particularly JAK2V617F, in chronic myeloproliferative neo- board. All patients provided verbal and written informed plasms (MPNs) marks an important milestone in our under- consent, and research was carried out according to the 1,2 standing of the pathogenesis of these disorders. The annual principles of the Declaration of Helsinki. incidence of MPNs, that is, polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), has been estimated to be 2.1 per 100 000 population in the United Reagents States.3 Currently, conventional therapy of MPN is not curative CYT387 was discovered and synthesized by Cytopia Research and does not prevent clonal evolution, and the goal of therapy Pty Ltd (Melbourne, Australia). Stock solutions were made in is to prevent and/or treat thrombohemorrhagic events, as well as dimethyl sulfoxide (DMSO) and subsequently diluted in culture to palliate the symptoms. medium for use. Although JAK2V617F occurs frequently in MPNs (that is, in 495% of PV patients, and B50% of ET and PMF patients), the issue as to whether this mutation is sufficient for MPN IC50 determinations by cell-free kinase activity assays development remains under active study. Overexpression of Glutathione-S-transferase (GST)-tagged JAK kinase domains mutant JAK2 (including the JAK2 exon 12 mutant alleles)4 expressed in insect cells were purified before use in a peptide constitutively activates JAK–STAT signaling and is associated substrate phosphorylation assay. Assays were carried out in 384- with cytokine-independent cell growth in vitro; in a murine well optiplates using an Alphascreen Protein Tyrosine Kinase model, these mutant alleles produce a MPN resembling PV, with P100 detection kit (Perkin-Elmer, Waltham, MA, USA) and a PerkinElmer Fusion Alpha instrument. Correspondence: Dr A Pardanani, Hematology and Internal Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA. E-mail: [email protected] Cell-based assays Received 28 October 2008; revised 29 January 2009; accepted 9 Ba/F3 cells expressing JAK2V617F (Ba/F3-JAK2V617F) and February 2009; published online 19 March 2009 MPLW515L (Ba/F3–MPLW515L) mutants, as well as Preclinical studies with CYT387, a selective JAK1/JAK2 inhibitor A Pardanani et al 1442 CHRF-288-11 (JAK2T875N) and CMK (JAK3A572V) cells were Table 1 Kinase selectivity profile of CYT387 generously provided by D Gary Gilliland (Brigham and ABL1 FYN PAK6 Women’s Hospital, Boston, MA, USA). The TEL/JAK2 and TEL/ ACVR1B (ALK4) GRK4 PASK JAK3 fusions were generated and introduced into Ba/F3 murine ADRBK1 (GRK2) GRK7 PDGFRA (PDGFR a) 13 cells as described earlier. The TEL/JAK2-orTEL/JAK3-trans- ADRBK2 (GRK3) GSK3A (GSK3 a) PDGFRB (PDGFR b) fected cells were cultured in Dulbecco’s modified Eagle’s AKT1 (PKB a) GSK3B (GSK3 b) PDK1 medium (DMEM) containing 10% fetal calf serum (FCS). Ba/F3 ALK HIPK1 (Myak) PHKG2 wild-type cells were cultured in RPMI containing 10% FCS AMPK A1/B1/G1 HIPK4 PIM1 AURKB (Aurora B) IGF1R PKN1 (PRK1) supplemented with 5 ng/ml murine IL-3 (Peprotech, Rocky Hill, AURKC (Aurora C) IKBKB (IKK b) PLK1 NJ, USA). Proliferation was measured using the Alamar Blue BLK INSR PLK3 assay (TREK Diagnostic Systems, Cleveland, OH, USA) after BRAF IRAK4 PRKACA (PKA) incubating for 72 h at 37 1C with 5% CO2. BRSK1 (SAD1) ITK PRKCA (PKC a) CAMK1D (CaMKI d) JAK1 PRKCB1 (PKC b I) CAMK2A (CaMKII a) JAK2 PRKCG (PKC g) CAMK4 (CaMKIV) JAK3 PRKCN (PKD3) Western blot analysis CDC42 BPA (MRCKA) KDR (VEGFR2) PRKD1 (PKC m) Human erythroleukemia cells were grown in starvation media CDK1/cyclin B LCK PRKD2 (PKD2) 5 (RPMI containing 1% FCS) overnight at 5 Â 10 cells/ml, incubated CDK2/cyclin A LTK (TYK1) PRKG1 with CYT387 for 2 h at 37 1C, and then lysed using RIPA buffer CDK5/p35 LYN A PRKX (50 mM HEPES pH 7.4, 150 mM NaCl, 10% glycerol, 1.5 mM CHEK1 (CHK1) MAP2K1 (MEK1) PTK2B (FAK2) CHEK2 (CHK2) MAP3K8 (COT) PTK6 (Brk) MgCl ,1mM EGTA, 1% Triton X-100, 1% sodium deoxycholate, 2 CLK1 MAP3K9 (MLK1) RAF1 (cRAF) Y340D 0.1% sodium dodecyl sulfate (SDS)) containing protease inhibitors Y341D (Complete Protease Inhibitor Cocktail, Roche, Indianapolis, IN, CLK2 MAP4K2 (GCK) RET USA) and phosphatase inhibitors (1 mM sodium vanadate, 1 mM CLK3 MAP4K4 (HGK) ROCK1 NaF, 1 mM sodium pyrophosphate, 1 mM sodium tartrate, 1 mM CSF1R (FMS) MAPK1 (ERK2) ROCK2 imidazole, 1 mM sodium molybdate). The protein concentration of CSK MAPK10 (JNK3) ROS1 each lysate was determined using a Bradford protein assay CSNK1G1 (CK1 g 1) MAPK11 (p38 b) RPS6KA1 (RSK1) CSNK1G2 (CK1 g 2) MAPK12 (p38 g) RPS6KA3 (RSK2) m (Biorad, Hercules, CA, USA). Cell lysates (50 g/lane) were sepa- CSNK2A1 (CK2 a 1) MAPK14 (p38 a) RPS6KA5 (MSK1) rated by electrophoresis through NuPage Novex gels (Invitrogen, DAPK3 (ZIPK) MAPK3 (ERK1) RPS6KB1 (p70S6K) Carlsbad, CA, USA) according to the manufacturer’s instructions. DCAMKL2 (DCK2) MAPK8 (JNK1) SGK (SGK1) The separated proteins were transferred to polyvinylidine fluoride DYRK1A MAPK9 (JNK2) SGKL (SGK3) (PVDH) membranes (Millipore, Billerica, MA, USA) and blocked DYRK3 MAPKAPK2 SRC in 3% skim milk powder in Tris buffered saline containing 0.05% EPHA1 MAPKAPK5 (PRAK) SRMS (Srm) EPHA2 MARK1 (MARK) SRPK1 Tween 20. Membranes were probed overnight with anti-phospho- EPHA3 MERTK (cMER) SRPK2 STAT5 (Y694) antibodies (Upstate, Billerica, MA, USA) at 4 1Cand EPHA8 MET (cMet) STK22B (TSSK2) detected with anti-rabbit horseradish peroxidase (HRP) (Santa EPHB1 MINK1 STK22D (TSSK1) Cruz Biotechnology Inc., Santa Cruz, CA, USA). Western blots EPHB3 MST1R (RON) STK23 (MSSK1) were developed using SuperSignal West Dura Extended Duration ERBB2 (HER2) MST4 STK24 (MST3) Substrate (Pierce Biotechnology, Rockford, IL, USA). Membranes FER MUSK STK25 (YSK1) FES (FPS) MYLK2 (skMLCK) STK3 (MST2) were then stripped using Reblot Plus (Upstate) and reprobed with FGFR1 NEK2 STK6 (Aurora A) rabbit anti-STAT5 antibodies (Cell Signaling Technology, Danvers, FGFR3 NEK6 SYK MA, USA). FGR NEK9 TAOK2 (TAO1) FLT1 (VEGFR1) NTRK1 (TRKA) TBK1 FLT3 NTRK2 (TRKB) TYRO3 (RSE) Colony assays FRK (PTK5) PAK2 (PAK65) YES1 Peripheral blood samples were collected after informed consent PAK4 and the mononuclear cell fraction was plated in duplicate at a concentration of 3 Â 105 cells/plate with and without erythro- % Inhibition % Inhibition % Inhibition poietin, as described earlier.10 CYT387 was added at the 450% at 100 nM o50% at 100 nM, o50% at B following concentrations: 0, 0.5, 1, 2 and 4 mM. Colonies were ( IC50 o100 nM) 450% at 1 lM 1 lM B B scored between days 10–14 using standard morphological ( IC50 o1 lM)(IC50 41 lM) criteria and isolated for individual genotyping.
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