Physiological Levels of Jak2 V617F Result in Enhanced Megakaryocyte Differentiation, Proplatelet Formation and Platelet Reactivity.

Abstract 226

The major clinical challenge in treating patients with myeloproliferative diseases (MPD) such as essential thrombocythaemia (ET) and polycythaemia vera (PV) is to prevent thrombotic events. Despite adequate red cell and platelet count control by means of venesection or cytoreductive therapy and the use of anti-platelet agents, patients with ET and PV have a three-fold risk-increase of cardiovascular and cerebral ischaemic events. A V617F mutation in the Janus kinase 2 (Jak2) that causes constitutive activation of Jak2 has been shown to be present in >90% and ∼50% of PV and ET patients, respectively. The effect of this activation on the biology of primary megakaryocytic cells is not known and neither is it clear whether the thrombotic risk conferred by this mutation reflects the increase in red cell/platelet counts, an intrinsic increase in platelet reactivity or both. Studies in humans are hampered by the co-existence of normal and clonal haematopoiesis with significant clonal and phenotypic heterogeneity between patients. Chimaeric mouse models based on retroviral transduction of Jak2 V617F have marked overexpression of the mutant Jak2 and show a PV phenotype with normal platelet counts. Recently, Jak2 V617F transgenic mice models have been published but the transgenes have multiple (and variable) insertions and are therefore subject to position effects with varying degrees of expression of the mutant Jak2 compared to the wild-type allele. We have generated a Cre-inducible mouse model where the human Jak2 V617F gene has been knocked into one allele of the mouse endogenous Jak2 gene. Upon Cre induction, the mice exhibit an ET phenotype (platelet count increased by approximately 30% but with a normal haematocrit) that is stable for over 26 weeks. Bone marrow histology shows classical features of ET with increased numbers of megakaryocytes (MKs) and clusters with no fibrosis. Quantitative RT-PCR shows stable expression of Jak2 V617F in the MKs at a similar level to that of endogenous mouse Jak2 therefore reflecting the physiological situation found in human ET patients. In liquid cultures, bone marrow-derived MKs show increased ploidy in response to suboptimal concentrations of thrombopoietin (TPO) in keeping with the increased number of MKs found in the bone marrow histology. Crucially, in vitro proplatelet formation in mutant MKs was increased two-fold compared to MKs derived from litter-match control animals showing that platelet production may not only relate to the increased number of MKs but to intrinsic differences in MK biology and platelet production. Platelet aggregation studies and P-selectin expression in response to an array of agonists was not significantly different between mutant and controls but in vitro laminar flow assays show increased thrombus formation on collagen. Although Jak2V617F expression was down-regulated at protein level in mature MKs and platelets, analysis of downstream signalling pathways showed alteration of the phosphorylation status of Src kinases. This mouse model therefore provides a unique opportunity to understand the biological mechanism of increased platelet production and thrombotic risk in ET patients as well as unravelling the signalling pathways downstream of the Jak2 V617F in primary cells, which will be crucial in the context of specific therapeutic Jak2 inhibitors currently in clinical development.

This work was supported by the British Heart Foundation