Janus Kinase (Jak) 1 Inhibition Affects Both Megakaryopoiesis and Thrombopoiesis

JAK inhibitors are being developed to treat inflammatory, myeloproliferative and neoplastic disorders. Murine and human studies have demonstrated an essential role for JAK2 in the proliferation of hematopoietic stem/progenitor cells (HSPC) and multiple hematopoietic lineages, including erythrocytes and megakaryocytes, while Jak1 murine studies have shown a role in HSPC proliferation and myelopoiesis, but not in megakaryopoiesis. Patients enrolled in clinical studies of INCB052793, which selectively binds to JAK1, have shown thrombocytopenia occurring within 2 weeks. The aim of this study was to elucidate the basis for thrombocytopenia associated with this JAK1 inhibitor, in comparison to INCB026115, which inhibits JAK2 more so than Jak1 (Jak2/1). Knowing the precise mechanism by which Jak inhibitors induce thrombocytopenia may lead to therapeutic strategies limiting side effects, while preserving intended clinical application. We tested a broad concentration range of each of these Jak1 and Jak2/1 inhibitors from IC50 (40 and 30nM, respectively) to IC90 (400 and 300nM) to 10xIC90 (4 and 3 µM) on mobilized progenitor-derived CD34⁺ cells incubated 12-14 days under semisolid and under liquid conditions, focusing on effects on megakaryocyte (Meg) and platelet production. At IC90, the Jak1-selective inhibitor limited large Meg colony number to 47±8% of untreated control in semisolid growth conditions. Under similar concentrations in liquid growth conditions, the number of Megs seen was 45±8% of the untreated controls, but with a 139±17% higher level of ≥8N Megs. Agonist response of mature Megs to thrombin was not compromised. Total number of healthy, in vitro-released, platelet-like particles (PLPs) collected from Jak1-exposed cultures at Day 12 was reduced to 57±14% of the control, and similar to the decrease in Meg yield. At a similar level of inhibition, the Jak2/1 inhibitor was more robust at inhibiting megakaryopoiesis. At IC90, the Jak2/1 inhibitor fully inhibited development of large Meg colonies and reduced the number of small colonies to 43±14% of untreated control. Under liquid growth conditions, the number of Megs seen at Day 12 was 20±9% of the untreated controls, but with 132±28% higher % of ≥8N Megs. Agonist response of mature Megs was not compromised. Total number of healthy PLPs collected at Day 12 was insignificantly different despite much lower Meg yield. More detailed Jak2/1 inhibitor cultures analysis revealed enhanced Meg apoptosis by 209±61% at Day 7, and accelerated maturation as indicated by a 2-fold and 3-fold mpl receptor level at Days 7 and 11 and 321±217% higher number of Megs >2N at Day 7. As opposite to what might be expected, thrombopoiesis appeared not to be impaired by the Jak2/1 inhibitor. Inhibitor-treated Megs released similar or higher number of platelets per Meg as untreated controls upon their infusion into immunocompromized NSG mice, with similar high levels of young, thiazole orange-positive, low apoptotic, Annexin-V⁺ platelets. Baseline released platelet CD62p expression and PAC1 binding prior to agonist exposure were similar and increased to the same extent after thrombin (0.1-10U/ml) stimulation. In contrast, Jak1 inhibitor-treated Megs had ~50% lower number of released human platelets upon infusion into NSG mice although the released platelets were healthy and responsive to agonists. In summary, our results shed significant insight into the mechanisms of Jak1 inhibitor-associated thrombocytopenia observed in patients. We show that thrombocytopenia post the Jak2/1 inhibitor INCB026115 is due to impaired megakaryopoiesis with intact thrombopoiesis and functional, released platelets. In contrast, thrombocytopenia post the Jak1 inhibitor INCB052793 is a result of combined impairment of both megakaryopoiesis and thrombopoiesis, although the released platelets appear intact. The exact pathways blocked by the Jak1 inhibitor important for thrombopoiesis remain to be defined. Also, as liver hepatocytes together with bone marrow stromal cells are a source of thrombopoietin (TPO), and Jak1 and Jak2 are known to be involved in regulation of TPO production, studies to check the influence of Jak inhibitors on TPO production from both hepatocytes and marrow stromal cells are needed to fully understand the influence of Jak inhibitors on megakaryopoiesis/thrombopoiesis.