The importance of megakaryocytes in myeloproliferative neoplams

Aberrant megakaryopoiesis is a hallmark of the myeloproliferative neoplasms (MPN). It is has been long known that abnormal megakaryocytes secrete elevated levels of cytokines such as TGFβ, resulting in pathologies including bone marrow fibrosis. However, the mechanism by which megakaryocytes regulate the initiation and progression of MPNs is largely unknown. To study the role of megakaryocytes in the MPNs, we analyzed the phenotype of PF4-Cre/Jak2V617F mice in which Jak2 is expressed in the megakaryocyte lineage from the endogenous locus. Selective activation of Jak2V617F was confirmed by allele-specific qPCR. CD41+ cells were positive for mutant Jak2, whereas sorted stem/progenitor cells and erythroid cells were Jak2 wild-type. Furthermore, flow cytometry showed that Stat5 activation was present in megakaryocytes, but not in erythroid or myeloid cells. Activation of JAK-STAT signaling caused an expansion of megakaryocytes in the bone marrow and spleen and a modest increase in the platelet count. Surprisingly, PF4-Cre/Jak2V617F mice also displayed a robust expansion of TER119(low)/CD71(high) and TER119(high)/CD71(high) red cells in the spleen, increased hematocrit and splenomegaly. Histological examination of the spleen revealed expansion of the erythroid lineage coupled with disrupted splenic architecture and fibrosis. This PV-like phenotype was fully penetrant. Profiling of hematopoietic progenitors by flow cytometry demonstrated that myeloid progenitor populations were expanded and skewed toward the erythroid-megakaryocyte lineage in the PF4Cre/Jak2V617F mice. In addition, LSK cells were increased in both the bone marrow and spleen. Cytokine profiling of the plasma revealed increased levels of several cytokines, including Il-6, which is known to be upregulated in human JAK2 mutant PV megakaryocytes. Real-time qPCR analysis confirmed increased expression of these cytokines/chemokines in Jak2V617F-mutant CD41+ cells. Furthermore, IL6 treatment increased EPO-dependent colony formation of wild type LSKs and MEPs, and also enhanced expression of the erythroid cell markers CD71 and Ter119. To further explore the role of megakaryocytes in the MPNs, we used a strategy in which expression of the diphtheria toxin receptor (DTR) sensitizes cells to diphtheria toxin (DT). We transduced c-Kit+ cells from PF4-Cre/iDTR+/- mice with MPLW515L and transplanted the cells to irradiated mice. As expected, both iDTR+/- and PF4-Cre/iDTR+/- mice developed a PMF–like phenotype, including leukocytosis, thrombocytosis, splenomegaly and myelofibrosis. Treatment of these animals with DT caused significant reductions in megakaryocytes in the bone marrow and spleen as well as a decrease in the platelet count of PF4-Cre/iDTR+/- mice. Of note, DT also significantly reduced the white count and spleen weight, while restoring splenic architecture. PF4Cre/iDTR+/- mice also showed significant reduction of c-Kit+ myeloid progenitor cells. Therefore, depletion of megakaryocytes