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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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. Genotyping for Dark shade is IC50 o100 nM; Light shade is IC50 o1 mM; and no JAK2V617F mutation was performed by PCR sequencing as shading is IC5041 mM). Bold indicates the JAK family kinases. described earlier.14

Results inhibition at 100 nM (that is, IC50 o100 nM) (Table 1). CYT387 inhibited JAK1 and JAK2 equipotently, but had an IC50 that was In vitro kinase selectivity of CYT387 approximately ninefold higher for the closely related JAK3 kinase CYT387 (N-(cyanomethyl)-4-[2-[[4-(4-morpholinyl)phenyl]amino]- as compared with JAK2 (Table 2). 4-pyrimidinyl]-benzamide) is a small-molecule, ATP-competitive inhibitor with a high degree of kinase selectivity for JAK1 and JAK2, but not for other closely related kinases (Tables 1 and CYT387 inhibits proliferation of cell lines driven by 2). In a ‘single-point’ screening assay that assesses the degree of constitutively activated JAK2 or MPL signaling enzyme inhibition at a speciﬁed CYT387 concentration (100 nM The activity of CYT387 was evaluated in a variety of cell-based or 1 mM), only eight kinases (including JAK2) showed 450% assays (Table 2). CYT387 inhibited proliferation of JAK2V617F

Leukemia Preclinical studies with CYT387, a selective JAK1/JAK2 inhibitor A Pardanani et al 1443 Table 2 In vitro effects of CYT387 on JAK enzyme activity and on cell lines harboring JAK2, JAK3 or MPL activating mutations

Assay Target IC50(nM)

JAK2 enzyme 18 JAK1 enzyme 11 JAK3 enzyme 155 Ba/F3-wt (+IL-3, proliferation) JAK2 wt 1424 Ba/F3-JAK2V617F (proliferation) JAK2 const. active 1500 CHRF-288-11 (JAK2T875N) JAK2 const. active 1 (proliferation) CMK (JAK3A572V) (proliferation) JAK3 const. active 700 Ba/F3-TEL-JAK2 (proliferation) JAK2 const. active 724 Ba/F3-TEL-JAK3 (proliferation) JAK3 const. active 2348 Ba/F3-MPLW515L (proliferation) MPL const. active 200 K562 (proliferation) BCR–ABL 58 000 MV4-11 (proliferation) FLT3 const. active 3000 Abbreviations: Ba/F3, an IL-3 dependent murine pro B cell line; const, constitutively; HEL, human erythroleukemia cell line harboring the JAK2V617F mutation; IL, interleukin; nM, nanomolar; wt, wild type. Figure 1 Effect of increasing concentration of CYT387 on STAT5 CHRF-288-11 and CMK cells are acute megakaryoblastic leukemia cell phosphorylation (a) and STAT3 phosphorylation (b), in human lines that carry JAK2T875N and JAK3A572V mutations, respectively. erythroleukemia (HEL) cells. Western blots were performed using whole-cell lysates from HEL cells, and were visualized with antibodies that detect the phosphorylated isoform of each protein. Total STAT-5 served as the loading control in (a) and b-actin as the loading control in (b). mutation harboring human erythroleukemia (HEL) cells as well as Ba/F3-JAK2V617F cells with an IC50 of approximately 1500 nM. Proliferation of parental Ba/F3 cells (Ba/F3-wt) stimu- Table 3a CYT387 effects on erythroid colony growth in PV patients B lated with IL-3 was inhibited with an IC50 value of 1400 nM, consistent with the established role of IL-3-dependent signaling Patient Sex/age JAK2 status IC50 (mM) in the parental cell line. Similarly, proliferation of Ba/F3- MPLW515L cells was inhibited at nanomolar concentrations Epo No Epo (IC50 ¼ 200 nM). In contrast, CYT387 was considerably less PV1 M/77 VF 4 2 potent at inhibiting growth of BCR–ABL harboring K562 cells PV2 F/72 VF 2–4 0.5 (IC50 ¼ 58 000 nM) or MV4-11 cells harboring a FLT3 mutation PV3 M/39 VF 1 0.5 (IC50 ¼ 3000 nM). Cell lines harboring mutated JAK2 alleles PV4 M/47 VF 444 (CHRF or Ba/F3-TEL-JAK2 cells) were inhibited more potently PV5 F/77 VF 2 o0.5 than the corresponding cell lines harboring mutated JAK3 alleles PV6 F/43 VF 2 1 (CMK or Ba/F3-TEL-JAK3 cells), thereby confirming the relative Abbreviations: Epo, erythropoietin; F, female; M, male; mM, micro- specificity of CYT387 for JAK2 as compared with JAK3 kinase molar; PV, polycythemia vera; VF, JAK2V617F. (Table 2). These data indicate that CYT387 is a potent and selective inhibitor of JAK2 kinase in cell-based assays of transformation. colonies. CYT387 inhibited EEC growth more potently, with IC50 values that were two- to fourfold lower as compared with those obtained with erythropoietin (Table 3a). JAK2V617F incidence CYT387 inhibits phosphorylation of STAT-5 and STAT-3 was consistently higher in EEC as compared with erythropoietin- in human erythroleukemia (HEL) cells supported colonies, which may underpin the differential Exposure of HEL cells to CYT387 resulted in dose-dependent inhibitory activity of CYT387 against the two types of colonies decreases in STAT-5 and STAT-3 phosphorylation, with IC50 ¼ (Table 3b). Genotyping of individual colonies obtained in the 400 and 2500 nM, respectively (Figure 1). These data confirm presence of CYT387 showed a dose-dependent, relatively downregulation of key signaling intermediates downstream of selective suppression of JAK2V617F-harboring EEC in all six activated JAK2. PV patients studied (Table 3b). A similar effect, that is, selective suppression of mutation-harboring erythroid colonies, was not observed in the presence of erythropoietin in vitro (data not CYT387 inhibits in vitro erythroid colony formation in shown). Fewer myeloid colonies were found to be mutated at polycythemia vera patients: exogenous erythropoietin baseline as compared with EECs for the six PV patients (data attenuates this effect not shown). The growth of myeloid colonies, however, was CYT387 decreased the number and size of erythroid colonies relatively sparse, and consequently, genotyping data were obtained in vitro. For six normal controls, erythroid colony inconclusive given the small number of myeloid colonies B growth was inhibited with an IC50 of 2-4 mM (data not shown). available for analysis. In the presence of erythropoietin, CYT387 inhibited erythroid colony growth from JAK2V617F-positive PV patients with B similar potency (IC50 2–4 mM; Table 3a). As JAK2V617F- Discussion positive PV is characterized by growth of erythropoietin- independent erythroid colony formation (EEC) in vitro, we also CYT387 is a small-molecule, ATP-competitive inhibitor that was assessed the efficacy of CYT387 in inhibiting growth of these developed by Cytopia, using a structure-based drug design

Leukemia Preclinical studies with CYT387, a selective JAK1/JAK2 inhibitor A Pardanani et al 1444 Table 3b CYT387 effects on erythroid colonies harboring JAK2V617F as assessed by single-colony genotyping

Patient Total number of colonies Percentage of mutation-positive Percentage of mutation-positive genotyped colonies (no drug) colonies (with drug)

Epo No Epo No Epo

Drug conc. 0 mM 0 mM 0.5 mM 1 mM 2 mM 4 mM

PV1 62 10 20 40 20 0 nc PV2 55 22 100 25 50 gf 0 PV3 42 0 40 20 0 nc nc PV4 55 45 67 50 0 0 0 PV5 47 20 20 80 25 nc nc PV6 53 40 100 80 100 33 20

Abbreviations: conc., concentration; Epo, erythropoietin; gf, genotyping failed; mM, micromolar; nc, no colony growth; PV, polycythemia vera.

Table 4 Comparison of CYT387 with other JAK inhibitors currently in clinical trials for treatment of myeloproliferative neoplasms (in vitro kinase inhibitory data)

Compound JAK2 IC50 (nM) JAK family selectivity proﬁle (X-fold selectivity)

JAK2 vs JAK3 JAK2 vs JAK1 JAK2 vs TYK2

CYT387 18 9 Â 0.6 Â NA TG101348a 3 332 Â 35 Â 135 Â INCB18424b 4.5 72 Â 0.6 Â 4 Â XL019c 298Â 67 Â 172 Â CEP701d 13Â NA NA Abbreviation: NA, not available. aLasho et al.15 bFridman et al.16 cPaquette et al.17 dDobrzanski et al.18

process. Our data indicate that CYT387 has activity against within the context of the CHRF/CMK and Ba/F3-TEL-JAK2/Ba/ mutant-activated JAK2 and MPL alleles; it is anticipated that a F3-TEL-JAK3 cell line pairs. Phase-I clinical trial to test the safety and preliminary efficacy CYT387 inhibited the in vitro growth of erythroid colonies of CYT387 for the treatment of myelofibrosis will begin in the from PV patients at low micromolar concentrations; in these near future. experiments, addition of exogenous erythropoietin to the culture CYT387 has a restricted kinase inhibitory profileFonly 8 medium significantly attenuated the inhibitory effect (2–4 fold). of 150 kinases including JAK2 were significantly inhibited in an At baseline (that is, in the absence of drug), in every case in vitro enzyme assay. Within the JAK family, JAK1 and JAK2 studied, a majority of erythroid colonies (450%) was found to were inhibited equipotently, almost ninefold more potently than harbor wild-type JAK2 when exogenous erythropoietin was JAK3. This JAK inhibitory profile resembles some (INCB018424, added to the cultureFthese data suggest that erythropoietin CEP-701), but not other (TG101348, XL019), small-molecule preferentially supports growth of PV progenitor cells harboring JAK inhibitors that are currently being tested in clinical trials wild-type JAK2. One may infer from these observations that for the treatment of MPN (Table 4); however, the clinical CYT387 preferentially inhibits growth of JAK2V617F-harboring implications of the distinct profiles remain to be determined. PV progenitor cells as compared with those harboring wild-type CYT387 inhibits the growth of cell lines harboring mutant- JAK2. Confirming this, and consistent with our observations activated JAK2 or MPL, at nanomolar or low micromolar with other inhibitors,10,11 single-colony genotyping analysis concentrations. There was a 3-log variation in CYT387’s showed that CYT387 selectively suppressed the growth of EECs inhibitory activity against mutant alleles (that is, JAK2T875N4 harboring JAK2V617F, in a dose-dependent manner. A similar MPLW515L4TEL–JAK2/JAK3A572V4JAK2V617F); in the selective effect on mutation-harboring myeloid colonies could absence of high-resolution X-ray crystallography or nuclear not be confirmed because of inadequate colony growth magnetic resonance spectroscopy data, however, the structural for single-colony genotyping analysis. If the aforementioned basis for this variation in inhibitory activity is not known. ‘therapeutic index’ (that is, selective suppression of mutated Regardless, these data indicate that the allelic context may be an erythroid colonies) persists in vivo, it may limit the potential important factor in determining the drug’s potency in vivo; deleterious effects of CYT387 on normal (non-clonal) erythro- consequently, it will be important to compare clinical responses poiesisFthis effect could be quantified by measuring the ratio across molecular subgroups in future clinical trials, and of mutant to wild-type JAK2 allele burden in peripheral blood correlate these responses with pharmacokinetic and pharmaco- during the course of study drug treatment. dynamic data, to confirm the in vitro observations. The cell- Overall, our data indicate that the JAK1/JAK2 selective based assays also suggest that overall CYT387 has greater inhibitor CYT387 has potential for efficacious treatment of activity against mutant JAK2 versus mutant JAK3 alleles, at least JAK2V617F-associated MPN.

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