Mechanistic insights of mutated Calreticulin in chronic myeloproliferative neoplasms

Chronic myeloproliferative neoplasms (MPN) originate from hematopoietic stem cell (HSC) and include polycythemia vera, essential thrombocythemia and primary myelofibrosis. They are characterized by mutations in JAK2, MPL and CALR. This project focused on the most recently described driver mutations in Calreticulin , whose functions and downstream targets are still largely unknown. There are several types of CALR mutations, the most frequent are a 52 bp (Type1) deletion and a 5 bp (Type2) insertion in exon 9. All described abnormalities cause a frameshift with generation of a novel C-terminal domain with a common novel sequence of 36 aminoacidic. In order to create a novel tool allowing mechanistic analysis of mutated CALR in a hematopoietic setting, I used CRISPR/Cas9 technology to perform targeted site-specific genome editing in K562 and UT7 cell line. I was successful in generating CRISPR/Cas9 K562 and UT7 CALR Knock-Out (KO) and CALR mutated with 52 bd delection (T1), that is an unique model where changes are to be entirely ascribed to CALR mutation and are not affected by endogenous CALR. Further characterization of K562 and UT7 CALR KO and CALR T1 showed no significant abnormalities in proliferation and cell cycle compartments compared to parental cells, while mutant cells had increased resistance to apoptosis. I showed that mutant CALR protein remained largely in the cytosol and it had a lower stability compared to wild-type counterpart. Since CALR mutations are restricted to MPN subtypes displaying aberrant megakaryopoiesis I also evaluated the effects of the mutations on the megakaryocytic commitment by culturing K562 with 10 nM PMA, a known inducer of megakaryocyte differentiation. I found that both CALR KO and T1 cell lines showed accelerated and enhanced expression of CD41/CD61 differentiation markers compared to CALR wild-type cells; this was also confirmed in K562 KO cells stably expressing CALR Type1 and Type2 by lentiviral transduction and in UT7 CALR KO and T1 culturing with PMA. Moreover clonogenic assay with CRISPR/Cas9 genome edited CD34+ cells showed that the knock-out of the protein resulted in promotion of megakaryocytopoiesis, mimicking the effects of CALR mutations. To reinforce these observations, the studies with inhibitor drugs in K562 CALR KO, CALR T1 and in CALR KO CD34+ cells, confirmed that PI3K and Erk pathways are involved in calr-mediated Mk commitment. These data were in line with results obtained with the phosphoproteomic assay in K562 CALR T1 and KO cells. Finally we assessed MPO expression in our cell models and we found that K562 CALR KO cells had a reduced expression of MPO, mimicking the observation in CALR T1 cells. Further, following the re-expression of CALR WT in CALR KO cells, we observed that MPO expression was restored, demonstrating that both mutated CALR and the absence of CALR (KO cells) lead to MPO degradation. Overall, these data indicate that the generated CALR-mutated and KO models represent useful tools to study the pathogenic and phisiologic role of CALR in MPN and could be useful to develop new diagnostic and therapeutic strategies for future clinical applications.