Hypoxia-Inducible Factors and Neutrophil Heterogeneity in Myeloproliferative Neoplasm-Associated Venous Thrombosis

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PROJECT SUMMARY/ABSTRACT This K08 Career Development Award details a 5-year training program to advance my career goal of becoming an R01-funded physician-scientist focused on elucidating thrombotic mechanisms in myeloproliferative neoplasms (MPN) in order to find new treatment targets and improve care of MPN patients. During the award period, I will continue developing expertise in mechanistic understanding of and models of thrombosis, acquire new scientific skills, advance translational capabilities, and generate publications and data that will serve as the foundation for a successful R01 application. Under the guidance of my primary mentor, Dr. Rafal Pawlinski, and co-mentors, Drs. Jonathan Serody and Alison Moliterno, these training objectives will be met by a combination of didactic course work, participation in seminar series, research experience, and mentoring by my advisory committee. Dr. Nigel Key and Dr. Josef Prchal will serve as advisors on the proposal. The scientific proposal is aimed at identifying thrombotic mechanisms in JAK2V617F-positive MPN. Thrombosis remains the leading cause of mortality in the major MPN subtypes polycythemia vera and essential thrombocythemia. Circulating blood cells are both increased in quantity and qualitatively abnormal in MPN. Current treatment paradigms aim to normalize the number of circulating blood cells to improve blood rheology. However, despite current therapies 25% of MPN patients will experience a thrombotic event after diagnosis. Understanding the basis of thrombosis and developing primary and secondary prevention strategies are key unmet needs in this field. Dr. Prchal and I recently reported that upregulation of hypoxia-inducible factor (HIF)- mediated gene expression in neutrophils of MPN patients associated with thrombosis history. Importantly, we also found increased neutrophil gene expression of tissue factor (TF), the primary initiator of the extrinsic pathway of coagulation. We extended this finding, demonstrating that MPN neutrophils possess TF procoagulant activity. Plasminogen activator inhibitor-1 (PAI-1) is the primary physiologic inhibitor of fibrinolysis; its gene expression was also increased in MPN patients. I therefore hypothesize that JAK2V617F-driven increases of HIF activity leads to upregulation of TF and PAI-1, thereby increasing the risk of MPN thrombosis. In Aim 1, I will use a mouse model of venous thrombosis (VT) to evaluate (1) the functional consequence of increasing JAK2V617F transgene copies to VT, (2) the effect of HIF-1α or HIF-2α inhibition to VT, (3) the contribution of JAK2V617F mutation in hematopoietic versus endothelial cells to VT, and (4) the effect of neutrophil TF to VT. In Aim 2, I will evaluate the effect of 0, 1, or 2 JAK2V617F-mutated alleles on heterogeneity of neutrophil gene expression in MPN patients using single cell RNA sequencing with overlayed single cell genotyping. The 2 Aims are complementary but independent and will identify new targets for the prevention of MPN thrombosis and set the stage for additional investigations.