Somatic mutations in Janus kinase 2 (JAK2), including JAK2V617F, result in dysregulated JAK-signal transducer and activator transcription (STAT) signaling, which is implicated in myeloproliferative neoplasm (MPN) pathogenesis. CYT387 is an ATP-competitive small molecule that potently inhibits JAK1/JAK2 kinases (IC50=11 and 18 nM, respectively), with significantly less activity against other kinases, including JAK3 (IC50=155 nM). CYT387 inhibits growth of Ba/F3-JAK2V617F and human erythroleukemia (HEL) cells (IC50 ∼1500 nM) or Ba/F3-MPLW515L cells (IC50=200 nM), but has considerably less activity against BCR–ABL harboring K562 cells (IC=58 000 nM). Cell lines harboring mutated JAK2 alleles (CHRF-288-11 or Ba/F3-TEL-JAK2) were inhibited more potently than the corresponding pair harboring mutated JAK3 alleles (CMK or Ba/F3-TEL-JAK3), and STAT-5 phosphorylation was inhibited in HEL cells with an IC50=400 nM. Furthermore, CYT387 selectively suppressed the in vitro growth of erythroid colonies harboring JAK2V617F from polycythemia vera (PV) patients, an effect that was attenuated by exogenous erythropoietin. Overall, our data indicate that the JAK1/JAK2 selective inhibitor CYT387 has potential for efficacious treatment of MPN harboring mutated JAK2 and MPL alleles.
The discovery of somatic mutations in Janus kinase 2 (JAK2), particularly JAK2V617F, in chronic myeloproliferative neoplasms (MPNs) marks an important milestone in our understanding of the pathogenesis of these disorders.1, 2 The annual 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 States.3 Currently, conventional therapy of MPN is not curative and does not prevent clonal evolution, and the goal of therapy is to prevent and/or treat thrombohemorrhagic events, as well as to palliate the symptoms.
Although JAK2V617F occurs frequently in MPNs (that is, in >95% of PV patients, and ∼50% of ET and PMF patients), the issue as to whether this mutation is sufficient for MPN development remains under active study. Overexpression of mutant JAK2 (including the JAK2 exon 12 mutant alleles)4 constitutively activates JAK–STAT signaling and is associated with cytokine-independent cell growth in vitro; in a murine model, these mutant alleles produce a MPN resembling PV, with elevated hemoglobin/hematocrit levels. Recent data from a transgenic mouse model suggest that the ratio of mutant JAK2V617F to wild-type JAK2 determines the MPN phenotype, with a lower ratio favoring an ET-like phenotype, and a higher ratio, a PV-like phenotype.5
Given the remarkable clinical utility of imatinib for the treatment of BCR–ABL-positive chronic myelogenous leukemia (CML),6 FIP1L1–PDGFRA-positive CEL7 and PDGFRB-rearranged MPN,8 several groups have begun to develop specific, potent, orally bioavailable inhibitors of JAK2 for the treatment of MPN.9 The agents currently undergoing preclinical and clinical testing inhibit both wild-type and mutant JAK2 alleles—these compounds inhibit the growth of cell lines harboring mutant JAK2, and the in vitro growth of hematopoietic colonies from MPN patients at pharmacological concentrations.10, 11 In a murine model of JAK2V617F-induced PV, one such compound produced a therapeutic response that was associated with favorable histopathological changes and a quantitative decrease in the mutant genomic copy burden.12
Here, we report our findings pertaining to the small-molecule JAK1/JAK2 inhibitor, CYT387, including its kinase selectivity characteristics and efficacy in inhibiting growth of cell lines and primary cells harboring activating JAK2 and MPL mutations.
Materials and methods
Patient accrual and sample collection
This study was approved by the Mayo Clinic institutional review board. All patients provided verbal and written informed consent, and research was carried out according to the principles of the Declaration of Helsinki.
CYT387 was discovered and synthesized by Cytopia Research Pty Ltd (Melbourne, Australia). Stock solutions were made in dimethyl sulfoxide (DMSO) and subsequently diluted in culture medium for use.
IC50 determinations by cell-free kinase activity assays
Glutathione-S-transferase (GST)-tagged JAK kinase domains expressed in insect cells were purified before use in a peptide substrate phosphorylation assay. Assays were carried out in 384-well optiplates using an Alphascreen Protein Tyrosine Kinase P100 detection kit (Perkin-Elmer, Waltham, MA, USA) and a PerkinElmer Fusion Alpha instrument.
Ba/F3 cells expressing JAK2V617F (Ba/F3-JAK2V617F) and MPLW515L (Ba/F3–MPLW515L) mutants, as well as CHRF-288-11 (JAK2T875N) and CMK (JAK3A572V) cells were generously provided by D Gary Gilliland (Brigham and Women's Hospital, Boston, MA, USA). The TEL/JAK2 and TEL/JAK3 fusions were generated and introduced into Ba/F3 murine cells as described earlier.13 The TEL/JAK2- or TEL/JAK3-transfected cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS). Ba/F3 wild-type cells were cultured in RPMI containing 10% FCS supplemented with 5 ng/ml murine IL-3 (Peprotech, Rocky Hill, NJ, USA). Proliferation was measured using the Alamar Blue assay (TREK Diagnostic Systems, Cleveland, OH, USA) after incubating for 72 h at 37 °C with 5% CO2.
Western blot analysis
Human erythroleukemia cells were grown in starvation media (RPMI containing 1% FCS) overnight at 5 × 105 cells/ml, incubated with CYT387 for 2 h at 37 °C, and then lysed using RIPA buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 10% glycerol, 1.5 mM MgCl2, 1 mM EGTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)) containing protease inhibitors (Complete Protease Inhibitor Cocktail, Roche, Indianapolis, IN, USA) and phosphatase inhibitors (1 mM sodium vanadate, 1 mM NaF, 1 mM sodium pyrophosphate, 1 mM sodium tartrate, 1 mM imidazole, 1 mM sodium molybdate). The protein concentration of each lysate was determined using a Bradford protein assay (Biorad, Hercules, CA, USA). Cell lysates (50 μg/lane) were separated by electrophoresis through NuPage Novex gels (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The separated proteins were transferred to polyvinylidine fluoride (PVDH) membranes (Millipore, Billerica, MA, USA) and blocked in 3% skim milk powder in Tris buffered saline containing 0.05% Tween 20. Membranes were probed overnight with anti-phospho-STAT5 (Y694) antibodies (Upstate, Billerica, MA, USA) at 4 °C and detected with anti-rabbit horseradish peroxidase (HRP) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Western blots were developed using SuperSignal West Dura Extended Duration Substrate (Pierce Biotechnology, Rockford, IL, USA). Membranes were then stripped using Reblot Plus (Upstate) and reprobed with rabbit anti-STAT5 antibodies (Cell Signaling Technology, Danvers, MA, USA).
Peripheral blood samples were collected after informed consent and the mononuclear cell fraction was plated in duplicate at a concentration of 3 × 105 cells/plate with and without erythropoietin, as described earlier.10 CYT387 was added at the following concentrations: 0, 0.5, 1, 2 and 4 μM. Colonies were scored between days 10–14 using standard morphological criteria and isolated for individual genotyping. Genotyping for JAK2V617F mutation was performed by PCR sequencing as described earlier.14
In vitro kinase selectivity of CYT387
CYT387 (N-(cyanomethyl)-4-[2-[[4-(4-morpholinyl)phenyl]amino]-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 2). In a ‘single-point’ screening assay that assesses the degree of enzyme inhibition at a specified CYT387 concentration (100 nM or 1 μM), only eight kinases (including JAK2) showed >50% inhibition at 100 nM (that is, IC50 <100 nM) (Table 1). CYT387 inhibited JAK1 and JAK2 equipotently, but had an IC50 that was approximately ninefold higher for the closely related JAK3 kinase as compared with JAK2 (Table 2).
CYT387 inhibits proliferation of cell lines driven by constitutively activated JAK2 or MPL signaling
The activity of CYT387 was evaluated in a variety of cell-based assays (Table 2). CYT387 inhibited proliferation of JAK2V617F 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) stimulated with IL-3 was inhibited with an IC50 value of ∼1400 nM, consistent with the established role of IL-3-dependent signaling in the parental cell line. Similarly, proliferation of Ba/F3-MPLW515L cells was inhibited at nanomolar concentrations (IC50=200 nM). In contrast, CYT387 was considerably less potent at inhibiting growth of BCR–ABL harboring K562 cells (IC50=58 000 nM) or MV4-11 cells harboring a FLT3 mutation (IC50=3000 nM). Cell lines harboring mutated JAK2 alleles (CHRF or Ba/F3-TEL-JAK2 cells) were inhibited more potently than the corresponding cell lines harboring mutated JAK3 alleles (CMK or Ba/F3-TEL-JAK3 cells), thereby confirming the relative specificity of CYT387 for JAK2 as compared with JAK3 kinase (Table 2). These data indicate that CYT387 is a potent and selective inhibitor of JAK2 kinase in cell-based assays of transformation.
CYT387 inhibits phosphorylation of STAT-5 and STAT-3 in human erythroleukemia (HEL) cells
Exposure of HEL cells to CYT387 resulted in dose-dependent decreases in STAT-5 and STAT-3 phosphorylation, with IC50=400 and 2500 nM, respectively (Figure 1). These data confirm downregulation of key signaling intermediates downstream of activated JAK2.
CYT387 inhibits in vitro erythroid colony formation in polycythemia vera patients: exogenous erythropoietin attenuates this effect
CYT387 decreased the number and size of erythroid colonies obtained in vitro. For six normal controls, erythroid colony growth was inhibited with an IC50 of ∼2-4 μM (data not shown). In the presence of erythropoietin, CYT387 inhibited erythroid colony growth from JAK2V617F-positive PV patients with similar potency (IC50 ∼2–4 μM; Table 3a). As JAK2V617F-positive PV is characterized by growth of erythropoietin-independent erythroid colony formation (EEC) in vitro, we also assessed the efficacy of CYT387 in inhibiting growth of these 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 was consistently higher in EEC as compared with erythropoietin-supported colonies, which may underpin the differential inhibitory activity of CYT387 against the two types of colonies (Table 3b). Genotyping of individual colonies obtained in the presence of CYT387 showed a dose-dependent, relatively selective suppression of JAK2V617F-harboring EEC in all six 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 shown). Fewer myeloid colonies were found to be mutated at baseline as compared with EECs for the six PV patients (data not shown). The growth of myeloid colonies, however, was relatively sparse, and consequently, genotyping data were inconclusive given the small number of myeloid colonies available for analysis.
CYT387 is a small-molecule, ATP-competitive inhibitor that was developed by Cytopia, using a structure-based drug design process. Our data indicate that CYT387 has activity against mutant-activated JAK2 and MPL alleles; it is anticipated that a Phase-I clinical trial to test the safety and preliminary efficacy of CYT387 for the treatment of myelofibrosis will begin in the near future.
CYT387 has a restricted kinase inhibitory profile—only 8 of 150 kinases including JAK2 were significantly inhibited in an in vitro enzyme assay. Within the JAK family, JAK1 and JAK2 were inhibited equipotently, almost ninefold more potently than JAK3. This JAK inhibitory profile resembles some (INCB018424, CEP-701), but not other (TG101348, XL019), small-molecule JAK inhibitors that are currently being tested in clinical trials for the treatment of MPN (Table 4); however, the clinical implications of the distinct profiles remain to be determined. CYT387 inhibits the growth of cell lines harboring mutant- activated JAK2 or MPL, at nanomolar or low micromolar concentrations. There was a 3-log variation in CYT387's inhibitory activity against mutant alleles (that is, JAK2T875N>MPLW515L>TEL–JAK2/JAK3A572V>JAK2V617F); in the absence of high-resolution X-ray crystallography or nuclear magnetic resonance spectroscopy data, however, the structural basis for this variation in inhibitory activity is not known. Regardless, these data indicate that the allelic context may be an important factor in determining the drug's potency in vivo; consequently, it will be important to compare clinical responses across molecular subgroups in future clinical trials, and correlate these responses with pharmacokinetic and pharmacodynamic data, to confirm the in vitro observations. The cell-based assays also suggest that overall CYT387 has greater activity against mutant JAK2 versus mutant JAK3 alleles, at least within the context of the CHRF/CMK and Ba/F3-TEL-JAK2/Ba/F3-TEL-JAK3 cell line pairs.
CYT387 inhibited the in vitro growth of erythroid colonies from PV patients at low micromolar concentrations; in these experiments, addition of exogenous erythropoietin to the culture medium significantly attenuated the inhibitory effect (2–4 fold). At baseline (that is, in the absence of drug), in every case studied, a majority of erythroid colonies (>50%) was found to harbor wild-type JAK2 when exogenous erythropoietin was added to the culture—these data suggest that erythropoietin preferentially supports growth of PV progenitor cells harboring wild-type JAK2. One may infer from these observations that CYT387 preferentially inhibits growth of JAK2V617F-harboring PV progenitor cells as compared with those harboring wild-type JAK2. Confirming this, and consistent with our observations with other inhibitors,10, 11 single-colony genotyping analysis showed that CYT387 selectively suppressed the growth of EECs harboring JAK2V617F, in a dose-dependent manner. A similar selective effect on mutation-harboring myeloid colonies could not be confirmed because of inadequate colony growth for single-colony genotyping analysis. If the aforementioned ‘therapeutic index’ (that is, selective suppression of mutated erythroid colonies) persists in vivo, it may limit the potential deleterious effects of CYT387 on normal (non-clonal) erythropoiesis—this effect could be quantified by measuring the ratio of mutant to wild-type JAK2 allele burden in peripheral blood during the course of study drug treatment.
Overall, our data indicate that the JAK1/JAK2 selective inhibitor CYT387 has potential for efficacious treatment of JAK2V617F-associated MPN.
Conflict of interest
GS, CJB and EF are full-time employees of Cytopia. This project was supported by a research Grant from Cytopia to AP.
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Cite this article
Pardanani, A., Lasho, T., Smith, G. et al. 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. Leukemia 23, 1441–1445 (2009) doi:10.1038/leu.2009.50
- myeloproliferative neoplasm
- Janus kinase
- kinase inhibitor
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