Myeloproliferative neoplasms (MPN) are a group of diseases characterized by aberrant proliferation of the erythroid, megakaryocytic and myeloid lineages. They represent clonal disorders of the hematopoietic stem cell with an inherent tendency towards leukemic transformation. MPN are subdivided into three disease entities: polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Somatic gain of function mutations in the genes JAK2, CALR, or MPL can be detected in blood cells of close to 90% of MPN patients. These gene mutations are known to act as drivers that can cause MPN phenotypes in patients and mouse models. MPN is a clonal disease of the hematopoietic stem cells. Disease manifestation and prognosis is in part influenced by additional somatic mutation that synergize with the driver mutations, but alone do not cause disease. These additional mutations most frequently occur in genes encoding epigenetic regulators, such as TET2, DNMT3a or EZH2. JAK2-V617F is the most frequent driver gene mutation occurring in 70-80% of MPN patients. Why some patients with JAK2-V617F, develop ET while others manifest as PV remains an open question. In contrast, JAK2 exon 12 mutations are tightly associated with PV and in many cases present as pure erythrocytosis. Both types of JAK2 mutations signal through the same C-terminal tyrosine kinase of JAK2, but result in very different phenotypic readouts. Furthermore, various knockin and transgenic mouse models of JAK2-V617F substantially differ in phenotypes, despite existing on the same genetically defined inbred background. We derived hypotheses and found factors that are likely to influence the phenotypic expression of MPN, which we will study in this proposal. We also derived preliminary data suggesting that the MPN hematopoietic stem cells are profoundly altered in their metabolism. Finally, we identified potential target genes that mediate the synergistic effects of JAK2-V617F and the loss of the epigenetic regulator EZH2. The proposed experiments will address the following specific aims:1. To examine genotype-phenotype correlations in MPN and define factors that determine ET versus PV phenotypes in MPN with mutated JAK2.2. To characterize altered characteristics of MPN stem cells, in particular the changes in transcriptome, metabolism, autophagy and lysosomal integrity.3. To study synergism between JAK2-V617F and mutations in epigenetic regulators in MPN pathogenesis and functionally test and validate candidate targets that mediate loss of Ezh2 in MPN.Significance: Our studies address important unsolved questions in the pathogenesis of MPN. We aim to test new hypotheses that may explain why one mutation (JAK2-V617F) can manifest as different clinical entities (ET or PV) with large inter-individual variation in the involvement of peripheral blood lineages. We also focus on poorly studied aspects of MPN stem cell biology, in particular metabolism, that could have major impact on the biology of MPN and may represent target for specific intervention. Our studies of synergism between JAK2-V617F and mutations in epigenetic regulator genes are likely to yield new insight into MPN progression to myelofibrosis and acute leukemia.