The Changes In The Megakaryocyte Lineage Contribute To MPN progression: the Immature Niche Megakaryocytes are associated with Myelofibrosis

Myelofibrosis (MF) is a disease of the elderly associated with bone marrow (BM) failure, as consequence of abnormal production of TGF-β by corrupted megakaryocytes (MK). MF is the end stage of the Philadelphia-negative myeloproliferative neoplasms driven by mutations of the MPL/JAK2 axis induced by megakaryocytes with reduced GATA-1 content. These observations suggest that abnormal TGF-β production by corrupted MKs may be responsible for the disease. We exploited recent insights into MKs differentiation and their roles in myelofibrosis. In fact, by leveraging scRNA-Seq profiling and other methods, it has been recently shown that there are four functionally distinct types of megakaryocytes: the most prevalent are Platelets producing MK, the second are HSC-supportive MK, the third are classified as immune-MK, and the fourth, present in the developing embryos but not in healthy bone marrow is the niche-poised MK which is responsible to synthesize the extracellular matrix during the organogenesis. The MK subpopulations were recognized according to the following published markers (Platelet-MK: CD42b/ARNT; immune-MK: CD41/LPS1; HSC supporting-MK: CD61/MYLK4 and niche-MK expressing Collagen I-III). At first, we identified the MK subpopulations in the Gata1low mouse model for MF. The MKs from the bone marrow of Gata1low mice were mostly immature and express reduced levels of markers of platelet-poised cells. The increased MKs observed in the BM microenvironment from Gata1low aged mice were mostly immature, according to the low GATA-1 content with the increased Niche embryo signature, expressing Collagen I and III. The Gata1low MKs also express high level of the interleukin-8 (IL-8), a cytokine which increases MK in emperipolesis. The IL-8 activity inhibition reduced the levels of fibrosis in the Gata1low BM, by decreasing the niche-MK as the Neutrophil-MK emperipolesis event and increased the content of GATA-1 in MK, providing proof that reducing IL-8 signaling is responsible of mechanism that regulate the TGF-β bioavailability in the microenvironment. We then used confocal microscopy methods to compare the GATA-1 content and the frequency of MK subpopulations in the BM from MF patients in respect to normal controls. Overall, the bone marrow from MF patients contained more MK, the majority of which negative 5 for GATA-1, than that of patients with essential thrombocythemia (ET) or with lymphoma but without fibrosis used as control. We then confirmed the MKs heterogeneity in the BM microenvironment from MF patient, which were immature and characterized by higher Collagen I-III expression. Lastly, we studied the effects of MF therapies on MK lineages. The TGF-β inhibitor AVID200 in MF patients promoted the MKs maturation, restoring the GATA-1 content and platelets production, by interrupting the TGF- β effect. These results support that great numbers of MK in the BM from Gata1low mice and MF patients, express Collagen I and III which may represent niche-MKs the differentiation of which are reactivated by the high TGF-β bioavailability in the myelofibrotic BM.