Dottorato in MORFOGENESI E INGEGNERIA TISSUTALE

Titolo della tesi: Epigenetic role of microRNAs in normal and neoplastic hematopoiesis

Epigenetic signals and microRNA (miRNA) are key regulators of developmental cell programs, including hematopoietic cells lineage-specification and fate. Alteration of their function is at the basis of neoplastic transformation. miRNAs mainly mediate the post-transcriptional gene silencing of target mRNAs. However, miRNAs also bind complementary DNA sequences at specific gene sites and participate in the epigenetic regulation of chromatin structure. The general aim of the PhD research project was the identification of novel epigenetic mechanisms involving miRNAs and/or their targets and their relevance in normal and neoplastic hematopoiesis. To this end we: i) analysed the genomic seeding and DNA sequences bound by the myelopoiesis regulator miRNA-223 during the maturation/differentiation hematopoietic progenitors (HPCs); ii) addressed the epigenetic mechanisms of transcriptional regulation of target genes by miR-223; iii) studied the expression patterns and biological relevance of identified miR-223 bound genes in normal and leukemic hematopoietic cells; iv) identified and functionally validated new miRNAs and pathways that are relevant for myeloproliferative disorders. In chromatin isolated from myeloid cells undergoing granulocytic differentiation by retinoic acid (RA) treatment we identified at the whole genome level (ChIP-Seq) the promoter regions targeted by miR-223, presenting a spread overlap with activating (H3K4me3) and/or or repressing (H3K27me3) histone marks. Among the genomic loci bound by miR-223, we identified the Flotillin 1 (FLOT1) gene, which is associated with transcriptionally active H3K4me3 marks. FLOT1 encodes for a lipid-rafts associated protein, whose role in normal and neoplastic hematopoiesis is mostly unknown. We validated the expression levels of FLOT1 in myeloid cell lines and confirmed its induction by RA treatment, which is inhibited by miR-223 silencing. FLOT1 is highly induced in HPCs isolated from normal donors’ cord blood and peripheral blood undergoing granulocytic differentiation; its expression levels are significantly altered in bone marrow samples from acute myeloid leukemia (AML) patients. Overall, these data suggest FLOT1 as a transcriptional target of miR-223, playing a role in normal and leukemic differentiation. We also proceeded to the identification of miRNAs involved in the pathogenesis of Myeloproliferative disorders (MPNs), by using as a model system a novel CALR non-canonical mutation (del24), deleting 24nt of the 3’UTR mRNA proximal region, associated to increased erythropoiesis and a Polycytemia Vera (PV) phenotype. Bioinformatic analysis predicted that miR-1972 and miR-455-3p target sites are lost in CALR del24 mRNA, thus suggesting a role for these miRNAs in the differentiation program of myeloid progenitors and in the control of CALR expression levels. Indeed, we found a significant correlation between the expression levels of miR-1972 and CALR mRNA/ protein levels in myeloid cell lines, in HPCs undergoing unilineage maturation/differentiation and in a large series of MPNs patients. We addressed the biological relevance of CALR-3’UTR targeting by miR-1972 in a K562 CRISPR/CAS9 cell model carrying or not CALR del24. In wild type and mock K562 cells the inhibition of miR-1972 activity increased erythroid differentiation, whereas K562 clones with CALR del24 were not responsive, thus suggesting a role for the post-transcriptional CALR gene silencing by miR-1972 in the erythroid differentiation program of myeloid cells.