Involvement of MAF/SPP1 axis in the development of bone marrow fibrosis in PMF patients

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by hyperplastic megakaryopoiesis and myelofibrosis. We recently described the upregulation of MAF (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog) in PMF CD34+ hematopoietic progenitor cells (HPCs) compared to healthy donor. Here we demonstrated that MAF is also upregulated in PMF compared with the essential thrombocytemia (ET) and polycytemia vera (PV) HPCs. MAF overexpression and knockdown experiments shed some light into the role of MAF in PMF pathogenesis, by demonstrating that MAF favors the megakaryocyte and monocyte/macrophage commitment of HPCs and leads to the increased expression of proinflammatory and profibrotic mediators. Among them, we focused our further studies on SPP1 and LGALS3. We assessed SPP1 and LGALS3 protein levels in 115 PMF, 47 ET and 24 PV patients plasma samples and we found that SPP1 plasma levels are significantly higher in PMF compared with ET and PV patients. Furthermore, in vitro assays demonstrated that SPP1 promotes fibroblasts and mesenchymal stromal cells proliferation and collagen production. Strikingly, clinical correlation analyses uncovered that higher SPP1 plasma levels in PMF patients correlate with a more severe fibrosis degree and a shorter overall survival. Collectively our data unveil that MAF overexpression contributes to PMF pathogenesis by driving the deranged production of the profibrotic mediator SPP1

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

Tamoxifen for the treatment of myeloproliferative neoplasms: A Phase II clinical trial and exploratory analysis

Current therapies for myeloproliferative neoplasms (MPNs) improve symptoms but have limited effect on tumor size. In preclinical studies, tamoxifen restored normal apoptosis in mutated hematopoietic stem/progenitor cells (HSPCs). TAMARIN Phase-II, multicenter, single-arm clinical trial assessed tamoxifen's safety and activity in patients with stable MPNs, no prior thrombotic events and mutated JAK2(V617F), CALR(ins5) or CALR(del52) peripheral blood allele burden ≥20% (EudraCT 2015-005497-38). 38 patients were recruited over 112w and 32 completed 24w-treatment. The study's A'herns success criteria were met as the primary outcome ( ≥ 50% reduction in mutant allele burden at 24w) was observed in 3/38 patients. Secondary outcomes included ≥25% reduction at 24w (5/38), ≥50% reduction at 12w (0/38), thrombotic events (2/38), toxicities, hematological response, proportion of patients in each IWG-MRT response category and ELN response criteria. As exploratory outcomes, baseline analysis of HSPC transcriptome segregates responders and non-responders, suggesting a predictive signature. In responder HSPCs, longitudinal analysis shows high baseline expression of JAK-STAT signaling and oxidative phosphorylation genes, which are downregulated by tamoxifen. We further demonstrate in preclinical studies that in JAK2V617F+ cells, 4-hydroxytamoxifen inhibits mitochondrial complex-I, activates integrated stress response and decreases pathogenic JAK2-signaling. These results warrant further investigation of tamoxifen in MPN, with careful consideration of thrombotic risk.Published version, accepted versionThis article is freely available online. Click on the 'Additional Link' above to access the full-text via the publisher's site