Recent single cell studies have shown that the megakaryocyte (MK) lineage is heterogeneous with multiple sub-populations such as immune-MKs, proliferating MKs, niche MKs, and platelet producing MKs. The heterogeneity in megakaryocyte populations in the Myeloproliferative Neoplasms (MPN), a disease that is characterized by aberrant megakaryopoiesis has not been described. We leveraged single-cell RNA sequencing (scRNAseq) of enriched MKs in two animal models of the MPNs to dissect the heterogeneity of megakaryocytes in this disease with the goal of unravelling the molecular underpinnings of MPN progression. To this end, we isolated megakaryocytes from bone marrow by lineage depletion and sorting for CD41+ cells at low pressure. Individual cells were isolated, and full length scRNAseq libraries were generated using the Takara ICell8 Smartseq platform. We first isolated MKs from WT (n=3) and JAK2V617F (n=3) mice, which present with polycythemia and atypical megakaryopoiesis, at 12 and 24 weeks post-transplant, when the mice have little to moderate degree of bone marrow fibrosis. Our analysis revealed 6 clusters of distinct megakaryocyte subpopulations based on the gene expression profiles. These included clusters that were enriched for gene signatures of platelets, megakaryocyte progenitor cells (MKP), erythroid progenitors, HSPCs, immune megakaryocytes, and cycling cells. All the clusters expressed common megakaryocytes markers including Itga2b, Pleck and Nfe2. Neutrophils clusters, positive for Mpo expression were removed as these were likely contaminants of the isolation procedure. We next compared the composition of the various clusters between WT and JAK2V617F groups and saw an 8-fold increase in the platelet cluster marked by expression of mature megakaryocyte markers, including , Tubb1, and Myl9. The erythroid progenitor population characterized by expression of Hbb-bt, Snca and Gypa, but low levels of Itga2b, increased by 2.5 fold. This is consistent with previous observations that JAK2V617F expression leads to increased erythropoiesis. scRNA sequencing of cells isolated from mice at the fibrotic stage (24 week post-transplant), revealed the increase in platelet population to be diminished to 2 fold. This decline in platelet population with advanced stages of fibrosis suggests a shift in hematopoiesis from bone marrow to extramedullary hematopoiesis in the spleen.

To assess the megakaryocyte heterogeneity in the more aggressive MPL W515L mouse model, which develops leukocytosis, thrombocytosis, and profound bone marrow fibrosis, we analyzed MKs at 3 weeks (n=3) and 5 weeks (n=3) post-transplantation. We observed an increase in platelet population (4 fold) and erythroid progenitor population (2 fold). Follow up analysis at the advanced stage of fibrosis revealed, erythroid population to be increased to 13 fold, however, with a decrease in platelet population to the wild-type level. This indicates a strong bias towards the erythroid lineage in the MPL driven myelofibrosis model. A comparison of the two MPN models, indicates a divergence in megakaryocyte differentiation, leading to a significant increase in erythroid progenitor population in the MPL W515L mutation driven MPN.

We next assayed the effect of the JAK inhibitor ruxolitinib on enriched MK populations. Treatment of the MPLW515L mice (n=3) with ruxolitinib significantly reduced the spleen weight, normalized peripheral blood counts and reduced the degree of bone marrow fibrosis. scRNA seq revealed that the most prominent effects of ruxolitinib were a significant reduction (8 fold) in the erythroid-progenitor population. Moreover, a 3 fold reduction both in CD41+ HSPC population and platelet population was observed. This reduction in the CD41+ HSPC, erythroid-progenitor and platelet population may significantly affect hematopoiesis following ruxolitinib treatment and may provide an explanation for the cause of anemia and thrombocytopenia observed in some ruxolitinib treated myelofibrosis patients. These observations suggest that new therapeutic approaches targeting the megakaryocyte populations more precisely may be more beneficial. We are currently assessing the effects of other investigational agents on megakaryocytes in the MPN setting.

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2025
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