In the context of myelodysplasias (MDS) and acute mydeloid leukemias (AML), the discovery in hematopoietic cells of aberrant DNA methylation profiles and mutations in genes associated with DNA methylation regulation has allowed for the development of demethylating agents. However, such treatments affect the whole bone marrow (BM), including mesenchymal stromal cells (MSCs), the epigenetic landscape of which is poorly known.
Considering the importance of BM-MSCs in the pathophysiology of MDS and AML, the aim of this study was to compare the methylation profiles of MSCs from MDS and AML patients to those of healthy donors (HD).
The AML-FILOtheque (#BB-0033–00073, Cochin hospital, Paris, France) provided samples from AML patients. BM from MDS patients was collected in the course of the MILESYM clinical study (authorized by the French Ministry of Research, #DC-2016–2739). BM samples were collected after informed consent from age-matched HD during hip surgery (Tours University Hospital). All BM samples were collected prior to treatment and patients with a history of chemotherapy were excluded. The study cohort comprised 32 HD, 19 patients newly diagnosed for MDS and 21 de novo AML. Targeted next-generation sequencing (NGS) was used to determine the genetic landscape of the AML samples. MSCs were isolated from whole BM samples during a single culture passage and cryopreserved until use; thawed cells were cultured for two further passages (P1 and P2) and all experiments were performed with P2-MSCs. Global DNA methylation analysis was performed via 5-methylcytosine quantification (dot-plots); regarding methylome beadchip analysis, bisulfite-converted DNA samples were analyzed with an Infinium® MethylationEPIC BeadChip array (over 850,000 methylation sites, Illumina); initial quality control was performed with the GenomeStudio software (Illumina); the Bioconductor's ChAMP package was used with R software to conduct bio-informatics analyses; after filtering, beta values for the remaining sites were calculated and normalized using the Beta Mixture Quantile dilatation (BMIQ) algorithm; comparisons were performed to identify differentially methylated probes (DMPs) and regions (DMRs); DMRs were detected at a threshold of 7 sites per region using the DMRcate algorithm; gene-set enrichment analysis (GSEA) was computed with DMRs-coupled genes. Specific gene expression (key regulators of DNA methylation, HOX gene family) was quantified by RT-qPCR (LightCycler® 480) on total mRNA previously tested on a 2100 Bioanalyzer (Agilent Technologies).
MDS and AML-MSCs displayed a global DNA hypomethylation and under-expression of DNMT1 and UHRF1. Methylome analysis revealed an aberrant methylation profile in all MDS and in a subgroup of AML-MSCs. The latter was preferentially found in the sequence of homeobox genes, especially from the HOX family (HOXA1, HOXA4, HOXA5, HOXA9, HOXA10, HOXA11, HOXB5, HOXC4 and HOXC6), and impacted their expression.
This study highlight the fact that modifications of DNA methylation occur in MDS/AML-MSCs, both globally and at focal levels, specifically dysregulating the expression of HOX genes known for their involvement in leukemogenesis. Such DNA methylation alterations in MSCs could be the consequence of the malignant disease or could participate in its development.