Megakaryocyte Ablation Ameliorates Bone Marrow Fibrosis By Reducing Myofibroblast Differentiation in a Mouse Model of Primary Myelofibrosis

Abstract

Primary Myelofibrosis (PMF) is a chronic myeloproliferative disorder characterized by excessive bone marrow (BM) fibrosis that can lead to ineffective hematopoiesis and reduced survival. PMF patients develop several megakaryocyte (MK) abnormalities including increased proliferation, abnormal morphology and upregulated expression of pro-fibrotic genes. Although several studies support the role of MK in BM fibrosis, the significance of MK in the development of BM fibrosis has not been demonstrated in vivo. Here, we investigated the in vivo role of MK in the pathogenesis of BM fibrosis. First, we reproduced an established bone marrow transplantation (BMT) mouse model of PMF. To achieve this, lethally irradiated wild-type mice were transplanted with wild-type hematopoietic stem cells (HSC) transduced with either MPLW515L gene construct (MPLW515L BMT) or MIGR1 empty vector (MIGR1 BMT). Consistent with previous studies, starting at day 14 post-BMT, MPLW515L BMT mice gradually developed increased number of leukocytes and platelets, increased proliferation of abnormal megakaryocytes in the BM and spleen, hepatosplenomegaly with extra-medullary hematopoiesis, and reticulin fibrosis in the BM. Notably, robust fibrosis was also seen in the spleen and liver. Next, to study the significance of MK in the development of BM fibrosis, we developed a MPLW515L BMT mouse model in which MK lineage could be selectively ablated. We accomplished this using PF4-Cre inducible diphtheria toxin receptor transgenic mice (iDTR+/-/PF4-Cre) as BMT donors. HSCs from iDTR+/-/PF4-Cre mice were transduced with MPLW515L or MIGR1 empty vector and transplanted as described above. Beginning day 14 post-BMT, recipient mice were injected with diphtheria toxin (DT) or water every 48 hours. At day 20 and day 29, DT injection significantly depleted MK in both MPLW515L BMT and MIGR1 BMT groups compared to water injection. Importantly, DT-injected MPLW515L BMT mice displayed attenuated fibrosis in the BM, spleen, and liver compared to water-injected MPLW515L BMT mice. In addition, DT treatment decreased the level of alpha smooth muscle actin in both BM and spleen of MPLW515L BMT mice, which suggests that MKs are critical for myofibroblast differentiation. MK ablation did not leukocytosis, thrombocytosis or hepatosplenomegaly. Together, our study show that successful MK ablation in vivo reduces fibrosis development in the BM, spleen, and liver of the MPLW515L mouse model of PMF. In summary, these results support the essential role of MK in the pathogenesis of BM fibrosis in PMF. Further studies are underway to elucidate the mechanisms by which MK contribute to fibrosis in PMF. Disclosures No relevant conflicts of interest to declare.