Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell neoplasms including polycythemia vera (PV), essential thrombocytemia (ET) and primary myelofibrosis (PMF), spontaneously evolving in leukemia. MPN are driven by somatic mutations of JAK2, calreticulin (CALR) and MPL genes, all activating the JAK2/STAT signaling pathway and uncontrolled production of myeloid cells. JAK2 and MPL mutations insist directly to essential pathways of myelopoiesis. In contrast, CALR mutations, exclusively found in ET or PMF, affect a multifunctional protein, apparently not connected with hematopoiesis. CALR mutations consist in various CALR exon 9 deletions and/or insertions, all causing a 1-bp frameshift, removing the ER retrieval motif KDEL and converting into coding sequence the first 31 bases of 3¿UTR mRNA. The biological consequences of the conversion of noncoding mRNA into coding region have not been investigated so far in MPNs. Recently, we detected a novel CALR deletion (c.1254+10_+33del24,) in two siblings diagnosed with JAK2 V617F-negative PV. CALRdel24 is located 10 bp downstream the stop codon in a highly conserved region of 3¿UTR mRNA. It does not alter the coding sequence, but leads to CALR overexpression and biological features of JAK2-driven PV, including erythrocytosis. This project is aimed at identifying, in normal myelopoiesis and MPNs, the epigenetic interactors acting on this CALR 3'UTR mRNA region and the biological role of consequent RNA modifications. We plan to identify RNA binding proteins and miRNAs targeting this CALR 3'UTR site and their relevance in complex regulative functions including mRNA modification, translation efficiency and protein localization, to be studied in relation to CALR hematopoietic functions. By exploring these new aspects of MPNs in primary samples from normal donors and MPN patients, we may identify new physiological mechanisms of hematopoiesis, which are deregulated in MPNs and that can be targeted by treatments.
Research on hematological disorders has served as a pioneer for all branches of cancer research. Advances obtained in these diseases, such as chemotherapy, transplantation and targeted therapies were extended to other malignancies. MPNs reflect an early stage of tumorigenesis, which is inaccessible in most cancers. In addition to phenotypic mimicry, each type of myeloproliferative neoplasm is capable of evolving into another type, making diagnosis, risk assessment, and therapeutic choices difficult. Indeed, myeloproliferation masks a clone of transformed hematopoietic stem cells capable of expansion and transformation to an aggressive form of bone marrow failure or acute leukemia[1].
Understanding the involvement of CALR 3'UTR mRNA proximal region in physiological hematopoiesis and MPNs could be helpful in deciphering the mechanisms linked to onset of MPNs and their progression to acute myeloid leukemia.
Identifying RNA epigenetic interactors and their role in inducing specific modifications of mRNA in the context of MPNs could contribute to depict a novel scenario, in which post-transcriptional modifications of mRNA and their possible variants due to genetic alterations could orchestrate a different regulation of gene expression in MPNs. Particularly the alteration of 3'UTR mRNA sequences due to frameshift mutations could explain aberrant regulative state involving specific RBPs or microRNAs that could become candidate for target therapy; on the other hand, mRNA modifications in MPNs could influences the recognition of consensus sequences for RBPs.
Studying the biological role of 3'UTR mRNA modifications under "natural disease" conditions in our MPN patients¿ cohort may clarify further relevant aspects of the intricate pathogenic mechanisms of MPNs, revealing a higher regulation level that could determine the phenotypic differences between PV, ET and PMF and disease progression. In parallel, "in vitro" experiments will provide a detailed view of how mutations involving CALR 3'UTR interfere with molecular mechanisms underlying hematopoiesis, which are altered in myeloid neoplasia. This complex network of regulation, which may act at physiological state, could be the hidden part of the iceberg, determining the differences between diverse clinics, although operating on the same genetic lesion. Moreover, this new area of research has immense potential with regards to targeted pharmacological interventions.