PROJECT SUMMARY Prefibrotic myelofibrosis (PrePMF) was formalized in 2016 by the World Health Organization (WHO) as a new early indolent form of MF which frequently progresses to myelofibrosis (MF) as well as acute leukemia. Abnormal regulation of megakaryocyte development is a key feature of both PrePMF and MF. Under normal conditions, committed megakaryocyte progenitors proliferate to a limited extent and then give rise to small numbers of large, polyploid differentiated megakaryocytes. However, upon acquisition of mutations in key signaling molecules, such as MPL, CALR or JAK2, megakaryocyte progenitors expand and promote thrombocytosis in PrePMF and thrombocytopenia/myelofibrosis in MF. Previous studies have demonstrated that a deficiency of the critical megakaryocyte transcription factor GATA1 leads a MF phenotype in mice and that levels of GATA1 are significantly reduced in the bone marrow of MF patients. We hypothesize that the loss of GATA1 in MF leads to an aberrant gene expression program which includes increased levels of pro-fibrotic and inflammatory cytokines such as TGF-β, LNC2 and IL-8, and that these changes drive the transition from Pre-PMF to MF. In our project, we will first perform a detailed assessment of cytokine secretion by PrePMF and MF megakaryocytes from animal models and patients and determine if these alterations induce aberrant megakaryocyte growth and fibrosis (Aim 1). Then we will assess the specific contributions of TGF-β to the cell cycle of normal and MF hematopoietic stem cells (Aim 2). Finally, we will use information from Aim 1 and 2, as well as from Projects 1 and 3, to study the efficacy of drugs that target the microenvironment, malignant hematopoietic stem cells or epigenetic regulators on MF in animal models. This work is innovative in that no one has characterized the way that malignant megakaryocytes guide the progression from Pre-PMF, a relative benign phase, to MF, the final fatal stage of hematopoietic failure in MPNs. Thanks to the interactions with the other projects and cores of this PPG, we are uniquely positioned to define the mechanisms by which megakaryocytes affect the transition from PrePMF to PMF and the contributions of TGF-β, LCN2 and IL8 to disease. Our work is significant in that it will assist Project 4 in prioritizing clinical trials already in the pipeline for MF and lead to new targeted therapies for the MPNs.