Accessible Chromatin Landscape Reveals a Proliferative Osteoprogenitor Transcriptional Program in Bone Marrow Mesenchymal Stromal Cells from Patients with Primary Myelofibrosis

Abstract

Primary myelofibrosis (PMF) is chronic myeloproliferative neoplasm characterized by clonal hematopoietic alterations contributing to myeloproliferation, egress of CD34+ cells from bone marrow to spleen associated with extra-medullary hematopoiesis, myelofibrosis with neo-ossification, leading to an osteomyelosclerosis as a result of increased CD146+ osteoprogenitor compartment. Recent studies from our laboratory have reported that bone marrow (BM) mesenchymal stromal cells (MSCs) from PMF patients (PMF-MSCs) showed a transcriptome and functional profile in agreement with an imprinted alteration of their osteogenic potential1&2.We performed advanced in silico analysis of accessible chromatin landscape of proliferative hFOB cells (human Fetal OsteoBlastic cell line) and identified 1183 target promoters during early phase of osteoblastic differentiation. Integration of these osteoblastic promoters in the transcriptome of PMF versus healthy donors (HD) BM-MSCs2 revealed an overexpression of 222 genes in the mesenchymal transcriptional program of patients (False discovery rate: FDR<5%), corresponding to a genomic enrichment of 5.95 (Fisher Exact test: p-value<2.2E-16) of the osteoblastic program in PMF-MSC transcriptome. Unsupervised principal component analysis significantly discriminated PMF patients from HD samples (p-value=7.92E-6). Actually, the osteoblastic transcriptional program activated in BM-MSCs from PMF patients shows an enrichment of genes coding for cell functions such as mRNA processing (-log p-value=11.39), MAPK pathway (-log p-value=6.39) and transcription start sites (TSS) bound by the ETS-domain transcription factor ELK1 (-log10 p-value=8.99). Geneset enrichment analysis (GSEA) also reveals an increase of MYC targets in the PMF-MSC transcriptome which shared 11 targets with ELK1 (NES=2.69, p-value<0.001). Interestingly, ELK1-ETS transcription factor downstream of Erk-MAPK is increased in PMF-MSC transcriptome (p-value = 0.0017) and is a target of MYC. These results suggest that the osteoblastic program is over expressed in PMF BM-MSCs and that it is likely controlled by MAPK and MYC.We also showed that a majority of transcription factors link to osteoblastic differentiation such as JUN, NFATC1, SP7, DLX5, FOS, RUNX2 were down regulated in PMF-MSC transcriptome (GSEA NES=-2.79, p-value<0.001), suggesting an imbalance in the proliferative vs. differentiation transcription program in these cells. Erk1/Erk2 was found to be hyperphosphorylated in PMF-MSCs by flow cytometry. Inhibition of the MAPK pathway by PD98059 during the early phase of in vitro MSC osteoblastic differentiation reveals an increase expression of collagen, a marker of differentiation, as shown by confocal microscopy. This is less the case with myc-inhibitor I (Calbiochem), suggesting the participation of the MAPK pathway in the proliferative vs. differentiation balance of MSCs/osteoprogenitors.In conclusion, integration of the open chromatin genomic landscape of the hFOB osteoblast fetal cells in the PMF BM-MSC transcriptome reveals a proliferative program of MSCs/osteoprogenitors associated in vitro with a repression of the differentiation in accordance with an increase proliferative osteoprogenitor priming of PMF-MSCs1 and with the presence of large size osteoblastic cells in vicinity of the neo-ossification area observed in the BM of PMF patients with osteomyelosclerosis.Martinaud C et al., Cancer Res. 2015 Nov 15;75(22):4753-65Martinaud C et al., Genom Data. 2015 Sep; 5: 1-2 DisclosuresVannucchi:Novartis: Honoraria, Speakers Bureau.