Novel insights into the altered crosstalk between megakaryocytes and stromal cells in myelofibrosis progression

Primary Myelofibrosis (PMF) is a clonal hematological cancer that belongs to the group of Myeloproliferative Neoplasms (MPNs). Among MPNs, MF has the most aggressive clinical phenotype, characterized by bone marrow (BM) myelofibrosis, anemia and splenomegaly. Mechanistically, somatic mutations that constitutively activate the JAK-STAT pathway in hematopoietic stem cells (HSC) result in abnormal blood cell production, proliferation of dysplastic megakaryocytes (Mks) and dysregulated release of pro-inflammatory cytokines. These inflammatory mediators are then responsible for stromal activation and excessive production of extracellular matrix (ECM) components within the BM microenvironment, leading to myelofibrosis and extramedullary hematopoiesis (EMH). During the PhD program my research focused at unveiling novel potential therapeutic targets and approaches to tackle the aberrant crosstalk between Mks and stromal cells. In the first part, I describe the identification of the chemokine Platelet Factor 4 (PF4)/Cxcl4 as a novel pro-fibrotic factor that contribute to BM fibrosis exacerbation. In particular, using mass spectrometry-based proteomic data, I show that PF4/Cxcl4 is up-regulated in Mk proteome from a MF-like mouse model and that Mk-Cxcl4 gene silencing in vitro mitigated the acquisition of a pro-fibrotic phenotype by co-cultured stromal cells. Moreover, I show that PF4 is rapidly internalized by stromal cells through surface glycosaminoglycans (GAGs) to induce stromal activation in vitro and pharmacological inhibition of GAGs ameliorated in vivo fibrosis in the mouse model. Consistently, extensive stromal PF4 uptake and altered GAGs deposition are also detected in BM biopsies from patients with overt fibrosis. In the second part of this thesis, I tested the possibility of targeting ECM components to directly reduce or slow down BM fibrosis in vivo. In particular, I focused my attention on Fibronectin (FN), a major structural ECM protein, whose synthesis and organization are altered during BM fibrosis progression. In my experiments I tested the efficacy of Antisense Oligonucleotides (ASOs) targeting FN mRNA in reducing FN expression and deposition in fibrotic settings. In vitro testing of FN ASO in both mouse and human stromal cells resulted in a significant reduction of FN mRNA expression, protein synthesis and deposition in both physiological and pro-fibrotic conditions. FN knockdown also reduced the production of the Extra Domain A (EDA) isoform, which contains an additional profibrotic domain and is extensively expressed during myelofibrosis progression to sustain inflammation and fibrosis. Injection of the FN ASO in vivo revealed a significant reduction of FN expression and deposition in the BM and liver tissue of wild type mice, without the emergence of side toxic effects. Experiments on myelofibrosis mice models are still ongoing. Overall, the findings from these studies improved our knowledge on the pathological mechanisms that sustain the abnormal Mk-stroma cross-talk in myelofibrosis, and suggest that therapeutically targeting both pro-inflammatory factors and extracellular matrix proteins may represent novel strategies for the treatment of this disease.