Mechanism of thrombosis in two myeloproliferative neoplasms (MPNs), polycythemia vera and essential thrombocythemia

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Project Summary/Abstract Arterial and venous thromboses are the major causes of morbidity and mortality in in two myeloproliferative neoplasms (MPNs): polycythemia vera (PV) and essential thrombocythemia (ET), associated with JAK2, and in ET in addition to JAK2 also calreticulin (CALR), and infrequently thrombopoietin receptor (cMPL) somatic mutations. However, the molecular mechanism of thrombosis is largely unknown. In multivariate analyses, the leukocyte count independently correlates with their risk of thromboses. Our published work demonstrates that the hypoxia inducible transcription factors (HIF-1 and HIF-2), which are upregulated in both granulocytes and platelets in PV and ET, promote the transcription of prothrombotic and proinflammatory genes. Leukocytes are the only source of tissue factor (TF) in the blood, and we also showed that PV neutrophils constitutively express TF activity. Transcripts of Krüppel-like Factor 2 (KLF2) - a zinc finger transcription factor, are down regulated in granulocytes and platelets from PV and ET patients and correlate inversely with the transcripts of prothrombotic genes, suggesting that KLF2 might be a suppressor of thrombotic gene expression in these conditions. In Chuvash erythrocytosis/polycythemia (CE) - a congenital disorder associated with increased HIF-1 and HIF-2 due to a hypomorphic R200W mutation of the Von Hippel- Lindau (VHL) gene, the incidence of thrombosis is higher than in PV and phlebotomy did not prevent thrombosis but instead appeared to have facilitated it. Repeated phlebotomies induced iron deficiency (ID) which further increased the level of HIF-1 and HIF-2 by inhibiting the negative HIFs regulator prolyl hydroxylase domain 2 (PHD2) enzyme, which requires iron as a co-factor. Therefore, we hypothesize that the up-regulation of HIF signaling in ET and PV granulocytes and platelets, perhaps with an additional contribution of augmented inflammation, plays a central role in the development of thrombosis. In order to achieve our objective, we propose to follow these specific aims (SA): SA 1a. Determine whether the correction of ID decreases HIF signaling and ameliorates the thrombotic milieu in PV and ET. SA 1b. Determine the effect of HIFs in PV and ET thrombosis using inhibitors of HIF-1, HIF2 and JAKs. SA 2. Extend our observations from mRNA transcripts of HIF-regulated, prothrombotic and antithrombotic genes and inflammatory genes to the activity of TF in all blood lineages. SA 3. Determine the role of KLF2 in increasing the risk of thrombosis in PV and ET and its regulation and elucidate in more detail the interaction of KLF2 and HIFs. We submit that proving our hypothesis will uncover novel mechanisms of thrombosis in PV and ET and open new therapeutic targets for prevention of thrombosis in PV and ET.