Role of Mesenchymal Stromal Cell Derived Extracellular Vesicles of Primary Myelofibrosis Patients on CD34+ Cells

Primary myelofibrosis (PMF) is a chronic myeloproliferative neoplasm characterized by the clonal proliferation of CD34+ progenitor cells. A constitutive activation of the JAK-STAT pathway, due to an acquired gain of function mutation either in JAK2, MPL or CALR genes, plays a relevant pathogenetic role. Moreover, a state of chronic inflammation affects the disease course. Currently, the therapy with the JAK inhibitor Ruxolitinib ameliorates the symptoms of the disease, with no effects on the pathological cell clone; hematopoietic progenitor cell (HPC) transplantation is the only curative option. The investigators previously published that mesenchymal stromal cells (MSCs) from patients with PMF display functional and genetic abnormalities (low proliferative potential, precocious senescence, reduced differentiation potential). Regarding Ruxolitinib, direct effects on MSCs have been described, such as the inhibition of the JAK-STAT signaling, the growth of both healthy donor (HD)- and PMF-MSCs, and the decrease of fibrosis related gene expression. Ruxolitinib has also been reported to reduce MSC secretion of inflammatory cytokines MCP-1 and IL-6, suggesting a role of this drug in modifying the bone marrow (BM) microenvironment. In PMF, as in other hematological malignancies, MSCs participate in the communication between microenvironment and CD34+ cells. Recent studies demonstrated in other pathologies that MSC-derived extracellular vesicles (EVs) and parental MSCs have similar effects. In fact EVs, containing mRNA, microRNA, lipids, and proteins can be incorporated into recipient cells and modify their functions. In normal hematopoiesis, MSC-EV incorporation in CD34+ cells has been shown to induce an activation of the JAK-STAT pathway through increased levels of phospho-STAT5; in addition, a significant decrease in apoptosis and a higher colony-forming unit granulocyte/macrophage clonogenic potential have been reported.

The objectives of this study are:

* to isolate MSCs from BM of PMF patients and HDs. MSCs will be incubated with/out Ruxolitinib. EVs will be obtained following MSC starvation and ultracentrifugations, and stored at -80°C * to assess EV effects on HD-CD34+ or JAK2V617F+ SET2 cells by evaluation of apoptotic and activation pathways, ROS production and clonogenic capacity. To this regard, confirmatory experiments will be performed with JAK2-mutated CD34+ cells from patients receiving or not Ruxolitinib. * to perform gene expression profile on CD34+ and SET2 cells after incubation with EVs. * to investigate EV biocargoes both qRT-PCR and proteomics will be performed on MSC-derived EVs, before and after incubation with Ruxolitinib.

The expected impact will include the identification of modifications in functional activities of CD34+ or SET2 cells following incubation with PMF-MSC-EVs with respect to HD-MSC-EVs, reflecting the effect of an altered microenvironment on HPCs. The investigators also expect the recognition of important targets of severity/progression of the disease by assessing the gene expression profile in CD34+ or SET2 cells that incorporate EVs from different sources.

In addition, the identification of the Ruxolitinib effects on PMF BM microenvironment may be clarified.

This study may allow to act on new targets of disease progression or to support the future use of HD-EVs as an acellular therapy that favours the survival of healthy CD34+ cells or acts against their clonal counterpart.

• Components of EV biocargoes [Срок оценки: Through study completion, an average of 1 year]