Cell identity relies on the cross-talk between genetics and epigenetics and their impact on gene expression. Oxidation of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) is the first step of an active DNA demethylation process occurring mainly at enhancers and gene bodies and, as such, participates in processes governing cell identity in normal and pathological conditions. Although genetic alterations are well documented in multiple myeloma (MM), epigenetic alterations associated with this disease have not yet been thoroughly analyzed. To gain insight into the biology of MM, genome-wide 5hmC profiles were obtained and showed that regions enriched in this modified base overlap with MM enhancers and super enhancers and are close to highly expressed genes. Through the definition of a MM-specific 5hmC signature, we identified FAM72D as a poor prognostic gene located on 1q21, a region amplified in high risk myeloma. We further uncovered that FAM72D functions as part of the FOXM1 transcription factor network controlling cell proliferation and survival and we evidenced an increased sensitivity of cells expressing high levels of FOXM1 and FAM72 to epigenetic drugs targeting histone deacetylases and DNA methyltransferases.

In this study we interrogated the DNA methylome of myelofibrosis patients using high-density DNA methylation arrays. We detected 35,215 differentially methylated CpG, corresponding to 10,253 genes, between myelofibrosis patients and healthy controls. These changes were present both in primary and secondary myelofibrosis, which showed no differences between them. Remarkably, most differentially methylated CpG were located outside gene promoter regions and showed significant association with enhancer regions. This aberrant enhancer hypermethylation was negatively correlated with the expression of 27 genes in the myelofibrosis cohort. Of these, we focused on the ZFP36L1 gene and validated its decreased expression and enhancer DNA hypermethylation in an independent cohort of patients and myeloid cell-lines. In vitro reporter assay and 5'-azacitidine treatment confirmed the functional relevance of hyper-methylation of ZFP36L1 enhancer. Furthermore, in vitro rescue of ZFP36L1 expression had an impact on cell proliferation and induced apoptosis in SET-2 cell line indicating a possible role of ZFP36L1 as a tumor suppressor gene in myelofibrosis. Collectively, we describe the DNA methylation profile of myelofibrosis, identifying extensive changes in enhancer elements and revealing ZFP36L1 as a novel candidate tumor suppressor gene.