Genome-wide analysis of enhancer-related DNA methylation in myelofibrosis reveals ZFP36L1 as a potential tumor suppressor gene

In this study we have interrogated the DNA methylome of myelofibrosis (MF) patients using high-density DNA methylation arrays. Interestingly, primary and secondary MF showed no differences in DNA methylation profiles, whereas a total of 35,238 differentially methylated CpGs (DMC) corresponding to 10,904 genes were detected between MF patients and healthy controls. Remarkably, the majority of DMCs were located outside gene promoter regions and significantly associated with enhancer regions. This enhancer aberrant hypermethylation showed a negative correlation with the expression of 27 genes in the MF patient cohort. Of these, we focused on ZFP36L1 gene and validated its decreased expression and enhancer DNA hypermethylation in an independent cohort of MF patients and myeloid cell-lines. In vitro reporter assay and 5’ azacitidine treatment confirmed the functional relevance of the intronic-enhancer hypermethylation of ZFP36L1. Finally, in vitro rescue of ZFP36L1 expression had an impact in cell proliferation and induced apoptosis in SET-2 cell line indicating a possible role of ZFP36L1 as a tumor suppressor gene in MF. Overall, we describe for the first time the MF DNA methylation profile, showing enhancer DNA hypermethylation as a potential functional mechanism playing an important role in silencing of a tumor suppressor gene in this disease. Samples and patient data were provided by the Biobank of the University of Navarra and were processed following standard operating procedures approved by the local Ethics & Scientific Committee. All patients consented prior to sample extraction and for the use of stored material for research purposes. MF patient samples cohort (n=39) was composed of either bone marrow (BM) or peripheral blood (PB) total mononuclear cells. Among the MF cohort there were primary MF (n=22), MF post-ET (n=7) or MF post-PV (n=10). PB total mononuclear cells from healthy donors (n=6) were used as control samples in this study. All patients were diagnosed using the 2008 version of the World Health Organization (WHO) classification system of hematological malignancies.