Abstract
Background: The V617F mutation of JAK2 (JAK2-V617F) is present in almost all patients with polycythemia vera (PV), and approximately 50% of patients with essential thrombocythemia and idiopathic myelofibrosis. JAK2-V617F leads to constitutive activation of the JAK2 kinase, causing cytokine independent growth in cell lines and development of a PV-like MPD in mice. Targeting JAK2-V617F may be therapeutically useful. We tested CYT387, a novel small molecule inhibitor of JAK2, in a variety of cells, and in a murine MPD model.
Methods: Specificity profiling was performed using in vitro kinase assays on the Upstate and Invitrogen platforms. Cell lines (n=26) were cultured in graded concentrations of CYT387 and cell proliferation was measured by MTT assay after 72 hours. For in vivo experiments bone marrow from 5-FU treated BalB/C mice was infected with a retrovirus expressing JAK2-V617F and GFP and transplanted into lethally irradiated recipients. Upon development of MPD CYT387 was initiated at doses of 25mg/kg (low dose) and 50mg/kg (high dose) twice daily by oral gavage. Mice were followed by blood counts and inspection. At 60 days treatment was stopped and a subset of mice was euthanized for detailed autopsy, histopathology and FACS analysis of hematopoietic cells. Accompanying experiments included pharmacokinetic and pharmacodynamic studies.
Results: In kinase assays CYT387 inhibited JAK1 and JAK2 with an IC50 of 11 and 18nM, while JAK3 was much less sensitive (IC50:155nM). Upon screening >100 additional kinases significant activity (IC50<100nM) was seen only for CDK1 and TBK1. CYT387 inhibited the growth of hematopoietic cell lines expressing JAK2-V617F with IC50 values between 0.5 and 1.5 microM. Similar values were seen in IL3, GM-CSF and IL-6-dependent lines, CMK cells (which carry A572V of JAK3), Molm14 cells (FLT3-ITD-positive) and several lines engineered to express BCR-ABL. In contrast, IC50 for most other hematopoietic and non-hematopoietic lines was >5 microM. In mice treated with high dose CYT387 hematocrit and white cell counts normalized, while they remained mildly elevated in the low dose group. On autopsy high dose mice showed almost complete resolution of splenomegaly with partially restored architecture, and reduced bone marrow fibrosis (but not cellularity), while the effects of low dose CY387 were less pronounced. FACS analysis of peripheral blood cells confirmed dose- and time-dependent inhibition of JAK2 signal transduction. A detailed analysis of spleen and bone marrow revealed a reduction (but not elimination) of MPD cells (GFP+), with partial normalization of progenitor cell distribution and differentiation. No significant weight loss or other toxicity was observed. Drug was stopped in two mice each from the low and high dose groups, with normal blood counts after 60 days of treatment. All 4 animals relapsed with MPD within 2 weeks.
Conclusions:
CYT387, which structure will be presented, is a pyrimidine derivative with low nanomolar activity against JAK1/2 in biochemical assays and a very narrow scope of additional targets. CYT387 inhibits the growth of JAK2-dependent cell lines in the high nanomolar – low micromolar dose range. CYT387 is active in a murine model of JAK2-V617F-positive MPD, with normalization of blood counts and spleen size, without apparent toxicity.
However, residual disease remained detectable and relapse occurred upon discontinuation of drug, reminiscent of preliminary data from studies of JAK2 inhibitors in humans. Studies to evaluate cytokine profiles are ongoing and will be presented.
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