P1002: PROTEOMIC SCREENING IDENTIFIES MEGAKARYOCYTE DERIVED PF4/CXCL4 AS A CRITICAL DRIVER OF MYELOFIBROSIS

Topic: 15. Myeloproliferative neoplasms - Biology & Translational Research Background: Despite the increasing knowledge of the genomic landscape of Myeloproliferative Neoplasms (MPNs), the precise pathological mechanisms underlying fibrotic progression in MPN are not entirely understood. Megakaryocytes (Mks) have been shown to contribute to MPN pathology by promoting inflammation and extracellular matrix (ECM) deposition by activated stromal cells. However, the specific Mk-derived factors that determine the inflammatory milieu and myelofibrosis progression are poorly defined. Aims: To evaluate the events that drive bone marrow (BM) fibrosis by abnormal megakaryocyte-stromal crosstalk, we applied mass spectrometry-based proteomics in a myelofibrosis-like mouse model and identified differentially expressed proteins featuring Mks, platelets and BM cells in fibrotic versus non-fibrotic conditions. Methods: Myelofibrosis was induced in mice through repeated injections of supra-pharmacological doses of the thrombopoietin-mimetic romiplostim (TPOhigh). Mks, BM cells and platelets were isolated from TPOhigh and control mice (injected with saline) and protein extracts analyzed by LC-MS/MS assay. Cytokines levels in plasma and BM cell-free fluids of treated mice were assessed by cytokine arrays. Expression of myofibroblasts markers (α-SMA, Vimentin) and ECM synthesis (Fibronectin, Type III Collagen) were analyzed in mouse embryonic fibroblasts (mEFs) and human BM mesenchymal stromal cells upon stimulation with recombinant PF4 by western blot. mEFs were co-cultured with BM-derived Mks in direct and indirect co-culture systems in the presence of growing TPO concentrations. In some experiments, Mks were transfected with siRNA that specifically targets CXCL4 gene or control siRNA before co-culture with mEFs. Plasma PF4/Cxcl4 levels were quantified by ELISA assay in a large cohort of MPN patients and normal controls. Results: We found that the signaling pathways related to cytoskeletal reorganization, cell adhesion and inflammation, were commonly activated in Mks, platelets and BM cells of TPOhigh mice with respect to control mice. MSP-RON signaling pathway and the Unfolded Protein Response were specifically activated in Mks and platelets, respectively. Among the differentially expressed proteins, the chemokine PF4/Cxcl4 was up regulated in all the proteasomes exclusively in the TPOhigh mice. Increased levels of PF4/Cxcl4 in plasma and BM cell-free fluids of fibrotic mice were confirmed by cytokine arrays. In vitro stimulation of BM-derived Mks with high TPO concentrations resulted in increased synthesis and secretion of PF4/Cxcl4. Recombinant PF4/Cxcl4 was rapidly internalized through surface glycosaminoglycans by mEFs and human BM mesenchymal stromal cells to induce myofibroblast differentiation. Importantly, we proved that these mechanisms are intertwined when Mk-mEF cross-talk is established in co-culture systems in the presence of high TPO concentrations. Genetic down-regulation of CXCL4 in Mks, before co-culture in TPO-saturated conditions, mitigated the profibrotic phenotype of mEFs in terms of ECM synthesis and myofibroblast markers expression. Finally, higher levels of circulating PF4/Cxcl4 were also detected in the plasma of patients with MPNs. Summary/Conclusion: Our findings identify a pivotal role of megakaryocyte derived PF4 in fibrosis progression of MPNs, further substantiating the potential role of neutralizing PF4 as a therapeutic strategy. Keywords: Platelet factor 4, Myelofibrosis, Megakaryocyte, Myeloproliferative disorder