The Fuction And Mechanism Study Of STAT1 And FAXDC2 In Megakaryopoiesis

Platelet serves as an important component of hematologic system, which play a critical role in maintaining hemostasis. Megakaryocytes maturation and formation of platelet are orchestrated by transcription factors and signaling that form a widespread regulation of network associated with other molecules. Aberrant megakaryopoiesis and hyperproliferative megakaryocytes are the key characteristic of myeloproliferative neoplasms (MPN) and acute megakaryoblastic leukemia (AMKL). Fuction study on normal megakaryopoiesis will not only reveal the molecular mechanism of this biological process but also help us understand the pathogenesis of related disease, which provides theoretical and scientific fundermental for the treatment of disease.Constitutive activation of MPL (MPLW515L) mutation induce Primary myelofibrosis(PMF) patient to produce large numbers of atypical megakaryocytes in bone marrow and spleen that may lead to the PMF. MPL (MPLW515L) will constitutively activate a lot of downstream pathways including JAK-STAT, PI3K-AKT and MAPK-ERK. Previous study shows the main effector of MPLW515L are STAT3 and STAT5. MPLW515Lalso activate STAT1. STAT1 signaling plays an important role in the normal megakaryopoiesis, but the mechanism and contributions in the pathogenesis of PMF remain unclear. In an effort to study the function of STAT1 contributing to the pathogenesis of PMF, we first detect the downstream signaling of MPLW515L displaying accumulative phosphorylated STAT3 and STAT5 but declining total STAT1 and phosphorylated STAT1(Tyr701 site) associated with one of the pivotal megakaryocytic transcriptional factor, GATA1. Moreover, the diminishing proportion of apoptosis cells and no significant changes in cell cycle were observed. These evidence indicated that STAT1 is relevant to GATA1 expression and apoptosis in the context of constitutive activation of MPL signaling. One the other hand, we observed a conservative binding site of STAT1 at RUNX1 promoter. Dural luciferase assay and chromatin immunoprecipitation assay verified that STAT1 regulated RUNX1 expression through binding at the promoter of RUNX1. Meanwhile, we found overexpression of dominant negative of RUXN1 partially precluded the normal megakaryopoiesis mediated by STAT1. These indicates that STAT1 may promote normal megakaryopoiesis through regulation of RUNX1. Finally, we transduced progenitors isolated from STAT1 deficiency mice with MPLW515L and transplanted into recipient mice to establish mouse model of PMF in vivo. Then, we found that STAT1 deficiency group showed short disease latency:higher WBC and platelet counts, more severe fibrosis and aberrant megakaryopoiesis. Together, we demonstrate that dysregulation of STAT1 signaling is one of pivotal elements resulting in the aberrant megakaryopiesis pertaining to the primary fibrosis.AMKL serves as a rare type of leukemia which is characterized by a differentiation block with accumulative of immature megakaryocytic cells resulting in decrease of platelet. The treatment of AMKL lacks of efficient target and the poor efficacy of prognosis on chemoradiotherapy are observed. These lead us to investigate new method for AMKL. Previous study shows that forced differentiation has long been proposed to be a rational method to cure leukemia. Extensive study and identification of gene in megakaryopoiesis may be critical for the development of rational differentiation therapy. Fatty Acid Hydroxylase Domain Containing 2 (FAXDC2) is a member of the fatty acid hydroxylase superfamily. Given the important role of fatty acids in megakaryocytes, we have studied the role of this gene in the development of this lineage. Here we show that the expression of FAXDC2 is constantly elevated during megakaryocyte maturation. In contrast, FAXDC2 is significantly down-regulated in acute myeloid leukemia and acute megakaryoblastic leukemia. Moreover, FAXDC2 overexpression promotes the differentiation of primary cells. These indicates that FAXDC2 play a positive role on megakaryopoiesis. Mechanism study shows that FAXDC2 overexpression increases RUNX1 expression. FAXDC2 also restores megakaryocytic differentiation in cells expressing a dominant negative form of RUNX1. Finally, FAXDC2 overexpression leads to an increase in sphingolipid GM3 synthase, suggesting a potential role of FAXDC2 in lipid metabolism that facilitates megakaryocyte differentiation.In this study, we demonstrate the function of STAT1 in the pathogenesis of PMF showing the downregulation STAT1 plays a critical role in MPLW515L induced PMF. Meanwhile, we explore FAXDC2 functioning in the normal megakaryopoiesis and tightly related with AMKL. Function study shows that FAXDC2 plays a novel role in development of megakaryocytes, while its dysregulation may contribute to abnormal hematopoietic cell development in leukemia. Our study reveals the pathogenesis of AMKL and PMF in depth and provide new thought for the treatment of these disease.