Identification and characterization of the role of CALR protein and 3’untranslated region in normal and neoplastic hematopoiesis

Myeloproliferative Neoplasms (MPNs) are hematopoietic stem cell-derived clonal disorders including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), with an overproduction of red blood cells, platelets and bone marrow fibrotic tissue, respectively. MPNs are triggered by JAK2, MPL and CALR (CALR) somatic mutations, all deregulating JAK/STAT signalling. CALR mutations, which are exclusively associated to ET and PMF, generally arise from a +1 frameshift converting the first 31 nucleotides of CALR-3’UTR, into coding sequence. However, the physiologic role of CALR and such CALR-3’UTR region in hematopoiesis are unknown. This thesis was aimed to: i) investigate the role of CALR in physiological hematopoiesis and the context of MPN; ii) elucidate the function of novel mutated variants of CALR 3’UTR that we identified; iii) realize a model in vitro to study the functional consequences of CALR 3’UTR disruption in myelopoiesis. Results and conclusions. We observed a hematopoietic lineage- specific regulation of CALR. Moreover, in a cohort of MPN patients, we detected two non-canonical CALR mutations associated to enhanced erythropoiesis in patients diagnosed as JAK2V617F-negative PVs. One was an in frame mutation (c.1214_1225del/del12), whereas the other occurred in the 3’UTR (c.1254+10_+33del/del24). Interestingly, the RNA folding prediction of del24, and even more of del12 indicated a structural impairment of this 3’UTR region. Primary CD34+ cells from such patients exhibited an erythroid growth push on unilineage culture and colony assays, associated with JAK/STAT activation and increase of CALR expression. Strikingly, CALR 3’UTR disruption in myeloid progenitors by CRISPR-CAS9 technology increased CALR levels and JAK/STAT activation, and, more surprisingly, induced erythropoiesis. Overall, these results suggest a novel role for CALR-3’UTR in lineage fate decision of myeloid progenitors. Its disruption might induce a PV-phenotype in vitro and in vivo.