MAF Induces Inflammatory Mediators Involved in the Pathogenesis of Primary Myelofibrosis

Abstract

Primary Myelofibrosis (PMF) is a Philadelphia-negative myeloproliferative neoplasm characterized by the presence of hyperplastic/dysplastic megakaryocytes and the development of myelofibrosis and osteosclerosis. This disruption of the bone marrow (BM) structure results from a deregulated release of growth factors by malignant hematopoietic cells, particularly monocytes and megakaryocytes, that activate BM stromal cells (Desterke-C, Mediators Inflamm, 2015). Therefore, it remains of primary importance to identify novel therapeutic strategies to restore BM microenvironment homeostasis in PMF patients.In order to better characterize the molecular mechanisms underlying PMF pathogenesis, we have recently compared the gene expression profile (GEP) of CD34+ hematopoietic progenitor cells (HPCs) from PMF patients and healthy donors, and we observed the upregulation of the transcription factor MAF (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog) (Norfo-R, Blood, 2014).With the aim to investigate the role of MAF in PMF pathogenesis and to reproduce the condition observed in PMF CD34+ cells, we studied the effects of MAF overexpression on HPCs differentiation. Human cord blood-derived CD34+ cells were transduced with a retroviral vector carrying the coding sequences for the two MAF transcript variants (LMAFVAR1IDN and LMAFVAR2IDN). The megakaryocyte lineage-committed CD41+ population was increased in LMAFVAR1IDN (38.2±2.3%) and LMAFVAR2IDN (39.4±2.3%) samples compared with the empty vector LXIDN (16.1±2.7%) at day 3 after NGFR+ cells purification. Values are reported as mean ± SEM from 3 independent experiments, p<.05. Moreover, MAF overexpression determined a remarkable increase of the CD14+ monocyte population in LMAFVAR1IDN (45.5±5.7%) and LMAFVAR2IDN (43.1±8.7%) compared to LXIDN control (8.0±0.4%) at t0 post-purification. Data represent mean ± SEM from 3 independent experiments, p<.05.Next, we analyzed the GEP of MAF-overexpressing CD34+ cells and observed the upregulation of genes involved in megakaryopoiesis, such as MERTK and RCAN1, and in monocyte/macrophage differentiation (e.g. EGR2 and KLF4). In particular, GEP revealed the upregulation of genes related to processes deregulated in PMF, such as fibrosis (i.e. ENPP2, ADAM9) and inflammation (i.e. TNF, CCL2 and CCL3). Noteworthy, several upregulated transcripts encoded for secreted proteins.Based on their role in inflammation and fibrosis, we selected a set of secreted molecules (IL8, MMP9, CCL2, SPP1, LGALS3 and PLAUR) and assessed them by quantitative enzyme-linked immunoassay (ELISA) in the culture supernatants from LMAFVAR1IDN-, LMAFVAR2IDN- and LXIDN-transduced cells. The levels in culture supernatants of all these molecules were increased in MAF-overexpressing samples compared to control. In order to correlate the expression of these molecules with MAF upregulation in PMF, we assessed them in plasma samples of PMF patients (n=30) and healthy donors (n=10). All the selected molecules displayed higher plasma levels in PMF patients than in healthy controls (Figure 1).Since the increase of SPP1 and LGALS3 has never been described before in PMF, we investigated which cell type could be responsible for SPP1 and LGALS3 production. Real Time qRTPCR performed on healthy donors peripheral blood-derived cells showed that the highest SPP1 expression levels were observed in CD34+ cells-derived megakaryocytes and granulocytes, whereas LGALS3 was expressed at higher levels in monocytes compared to the other cell types.Because the increased proliferation of fibroblasts is involved in BM fibrosis and is a PMF feature, we investigated the effects of LGALS3 and SPP1 on normal human primary dermal fibroblasts. The treatment with recombinant human LGALS3 (1000 ng/ml) or SPP1 (500 ng/ml) caused an increase in fibroblasts cell count of 20.9±2.7% (p<.001) and 14.9±4.1% (p<.01) compared to the untreated control, respectively. Values are reported as mean ± SEM from 7 independent experiments.Finally, we observed that increased SPP1 plasma levels in PMF patients correlate with a more severe fibrosis and lower overall survival (p<.05).In conclusion, our data unveil the involvement of MAF in PMF pathogenesis through the induction of a pro-fibrotic and pro-inflammatory phenotype in the malignant myeloid differentiated progeny. [Display omitted] DisclosuresVannucchi:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.