Study to understand the role of circular RNAs in CD34+ cells of myelofibrosis

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic disorders characterized by an overproduction of differentiated hematopoietic cells all derived from the same progenitor cell that has acquired one or more genetic mutations that give it a proliferative advantage. To this disorders belong polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) and genetic lesions include somatic driver mutations, so called because of their role in driving the myeloproliferative phenotype, are mutually exclusive in MPNs and take place in JAK2, CALR o MPL. The mutational landscape of MPN is much more complex and there are other mutations, with different function that can play a central role in clonal evolution and influence the clinical course. Moreover, exists a not negligible category of patients, not affected by the three mutations drivers and called “triple negative” (TN), for whom known molecular marker are lacking. Thus, the aim of this project is to explore new routes to possible novel prognostic treatments and alternative strategies for MPN patients and circularRNAs (circRNAs), non-coding RNA molecules, could be interesting for this purpose. From an RNA-seq analysis we detected 11 circRNAs disregulated in MPNs. Granulocytes and CD34+ cells were isolated from healthy donors’ (n=12 and n=8, respectively) and from bone marrow or peripheral blood of MPN patients (n=29 and n= 47, respectively) to perform qualitative analysis. We have identified n=6/11 circRNA because backsplicing validation frequencies result more markedly different in MPN compared to controls. Thus we selected one, circPLOD2, that results significantly overexpressed in MPN patients. We divided patients for driver mutations and focused the interest on JAK2- mutated patients. We evaluated the expression levels of circPLOD2 in patients before and after the treatment with JAK2 inhibitor and we observed a downregulation after the treatment compared to control. Moreover, CD34+ cells from healthy donors were transfected with the circPLOD2 overexpressing plasmid, and showed a significant increase of the megakaryotic colonies upon circPLOD2 overexpression. This study showed that circPLOD2 expression is dependent on the JAK-STAT pathway activation. Of interest, enforced circPLOD2 expression significantly boosted differentiation of megakaryocytes. Further studies are needed to understand the role of circular RNAs in the context of myeloproliferative neoplasms, the data presented suggest that circular RNAs can be a useful tool for developing new diagnostic and therapeutic strategies in MPN.