Myeloproliferative Neoplasms (MPNs) are distinguished in Polycythemia Vera (PV), Essential Thrombocytemia (ET) and Primary Myelofibrosis (PMF). MPNs are associated to recurrent driver mutations of JAK2, MPL and CALR genes, all causing deregulation of JAK/STAT signaling. Whereas JAK2 and MPL mutations insist directly on JAK/STAT pathway, CALR mutants, associated almost exclusively with ET and PMF, activate the pathway through the interaction with TPO-receptor (MPL). All CALR mutations generate the same common novel peptide, which abolishes the KDEL-ER-retention domain. Such novel peptide mediates the interaction with TPO receptor and arises from a +1 frameshift that converts the first 31 bases of CALR 3'UTR into coding sequence. However, no consideration has been reserved to this 3'UTR region so far. Recently, in two JAK2V617F-negative patients resembling PV, we identified a novel CALR deletion (CALR del24) within such 3'UTR region, not altering the coding sequence. CALR del24 induced properties ascribable to PV in vitro. Importantly, a second CALR deletion of coding sequence, CALR del12, not altering KDEL, was identified in a PV case, but no functional data are yet available about this mutant. Both CALR del24 and del12 are predicted to alter CALR 3'UTR secondary structure and occur in evolutionary conserved regions.
The present project aims to study the role of CALR 3`UTR in normal myelopoiesis and MPNs. We will focus on miRNA regulation and complex regulative functions of 3`UTR, such transcript stability and interactions with proteins. As additional degree of complexity, RNA editing of CALR mRNA will be studied in relationship with 3`UTR functions. All this aspects will be related to the functional characterization of CALR mutants. By exploring these unknown aspects of MPNs in primary samples from normal donors and patients, we propose to shed light on the biology and pathogenesis of these diseases, revealing useful cues for the design of novel targeted treatments.
Currently the study of molecular mechanisms that originate and sustain MPNs lacks of links that explain how the same mutation could originate different phenotypes of diseases, and therapies are limited to JAK2 inhibitors, phlebotomy and hydroxyurea.
The present project proposes to analyze some aspect related to CALR 3'UTR functions within the context of hematopoiesis and MPNs.
By focusing our investigations on CALR 3'UTR functions, we aim to shed lights on additional mechanisms, unknown at the present, that are involved in normal hematopoiesis and might influence the etiology of MPNs.
The first objective of the project is to study a possible regulative correlation between CALR mRNA and miR-1972. This investigation could identify a novel network, which, if validated, might lead to the discovery of new regulators implied in the pathogenesis of MPNs, possibly furnishing molecular information about diagnosis and prognosis of such patients.
The second objective of the project is the analysis of RNA-editing profiles of CALR 3'UTR, focusing on A-to-I and m6A editing. If the protein complexes operating these enzymatic modifications would differently operate in MPNs respect to the physiological conditions, possibilities of new therapy targeting such molecular actors might be opened. The study of CALR 3'UTR mRNA stability could be helpful in understanding how 3'UTR sequences could regulate mRNA/protein expression in a pathological context, highlighting if different mutations could differently modify the transcriptional rate or decay of the relative transcript, and indicating how such different mutations impact on MPNs expression profiles.
Lastly, the generation of in vitro models to study CALR novel and canonical mutations would allow to compare functional properties of the various CALR mutation respect to CALR 3'UTR functions, analyzing which effects might be implied by the direct removal of 3'UTR nucleotides (CALR del24) or by the alteration that compromises exclusively its secondary structure (CALR del12).
The study of CALR 3'UTR might lead to new cues that expand the alternatives for new therapies of MPNs, targeting the interactors of 3'UTR-dependent complexes, containing the evolution of these pathologies towards negative outcome and preventing the onset of acute leukemia. Furthermore, 3'UTR functions are an emerging topic, whose implications are still evolving and not yet fully understood. We are convinced that gaining novel insights about this field could be crucial for the comprehension of advanced mechanism regarding human differentiation and hematopoiesis, paving the way for solving currently unexplained issues of cancers.