STUDIO DELLA FUNZIONE E DEL PROFILO DEI MICRORNA IN MICROPARTICELLE CIRCOLANTI ISOLATE DA PAZIENTI ‘TRIPLI NEGATIVI’ E JAK2V617F MUTATI CON MIELOFIBROSI

Introduction: Myelofibrosis (MF) is a myeloproliferative neoplasm (MPN) characterized by clonal hemopoiesis, inflammatory microenvironment and mutations in JAK2, MPL, or CALR genes. Around 10% of patients (pts) do not carry the 3 mutations (Triple negative (TN)). Microparticles (MPs; 0.1-1 μm) are small vesicles deriving from plasma membrane during homeostasis and cell activation of a wide variety of cells with a role in intercellular signalling. Circulating MPs are increased in inflammation and cancer including MPN. However, their role in the pathogenesis of MF is still elusive. Here we compared phenotype, function and microRNAs (miRs) expression of isolated MPs of JAK2V617F mutated and TN pts. Methods: Peripheral blood was collected from 10 MF pts (at diagnosis or out of cytotoxic treatment for at least 3 months) and 10 age/sex-matched healthy donors (HD). MF pts were JAK2V617F mutated (n=5) and TN n=5). MPs were purified from 2 mL of platelet poor plasma by ultracentrifugation and quantified using Nanosight technology. Isolated MPs were characterized by flow cytometry (CytoFLEX, Beckman Coulter) for the expression of platelets (PLT:CD61+CD62P+) and megakaryocytes (MK:CD61+CD62P-) markers. MPs were then analyzed for their ability to modulate the in vitro viability of immunomagnetically isolated CD34+ cells from MF pts and cord blood (CB). MiRs expression of isolated MPs (10e9) from 3 JAK2V617F mutated, 3 TN pts and 3 HD was investigated using TaqMan™ Array Human MicroRNA A Cards (ThermoFisher). Results: The mean number of plasma MPs isolated from MF pts was similar to HD (5*10e10/mL). However, flow cytometry analysis revealed that the mean percentage of PLT-MPs was significantly increased (p<0.05) only in JAK2V617F mutated pts. On the contrary, MK-MPs were significantly decreased in both pts groups (p<0.01) as compared to HD. Co-culture with HD-MPs significantly increased the survival of CB CD34+ cells (p<0.05). Concomitantly, we observed that the survival of CB CD34+ cells was significantly reduced in co-cultures with TN-MPs (p<0.05). Of note, TN CD34+ cells survival was strongly enhanced by autologous MPs as compared to untreated cells (p<0.05). CD34+ cells from JAK2V617F mutated pts showed only a slightly promotion in the presence of MPs from TN pts. Comparing the miRs profile of pts-MPs with those of HD, various pro-apoptotic (+) and anti-apoptotic (-) miRs were upregulated. Specifically, miR-155(-), 24(-), 222(-) and 744(+) were upregulated in TN MPs only; miR-21(-), 19a(-), 221(-), 15b(-), 127(+), 34a-5p(+), 423-5p(+) were upregulated in both JAK2V617F mutated and TN MPs. Let-7b(-) miRNA expression was increased in JAK2V617F mutated-derived MPs only. RT-PCR validation assays are under way. Conclusions: Circulating MPs from TN MF pts show distinct phenotype, function and apoptosis-related miRs signature as compared with the JAK2V617F mutated counterparts.