DNA hydroxymethylation is associated with disease severity and persists at enhancers of oncogenic regions in multiple myeloma

Abstract

Background: Multiple myeloma (MM) is a heterogeneous plasma cell malignancy that remains challenging to cure. Global hypomethylation correlates with an aggressive phenotype of the disease, while hypermethylation is observed at particular regions of myeloma such as B cell-specific enhancers. The recently discovered active epigenetic mark 5-hydroxymethylCytosine (5hmC) may also play a role in tumor biology; however, little is known about its level and distribution in myeloma. In this study, we investigated the global level and the genomic localization of 5hmC in myeloma cells from 40 newly diagnosed patients, including paired relapses, and of control individuals.

Results: Compared to normal plasma cells, we found global 5hmC levels to be lower in myeloma (P < 0.001). Higher levels of 5hmC were found in lower grades of the International Staging System prognostic index (P < 0.05) and tend to associate with a longer overall survival (P < 0.1). From the hydroxymethylome data, we observed that the remaining 5hmC is organized in large domains overlapping with active chromatin marks and chromatin opening. We discovered that 5hmC strongly persists at key oncogenic genes such as CCND1, CCND2 and MMSET and characterized domains that are specifically hydroxymethylated in myeloma subgroups. Novel 5hmC-enriched domains were found at putative enhancers of CCND2 and MYC in newly diagnosed patients.

Conclusions: 5hmC level is associated with clinical aspects of MM. Mapping 5hmC at a genome-wide level provides insights into the disease biology directly from genomic DNA, which makes it a potent mark to study epigenetics on large patient cohorts.

Keywords: DNA modifications; Epigenetics; Hydroxymethylation; Multiple myeloma.

Fig. 1

DNA hydroxymethylation is depleted in NDMM and remains locally at active chromatin. a Dot plot of 5hmC global quantification by MS in normal plasma cells from healthy donors (n = 5), and of myeloma cells of patients at diagnosis (n = 39) and b at disease stage (ISS I n = 9; ISS II n = 17; ISS III n = 12; NA n = 1). c 5hmC level-based survival analysis with separation of two groups of NDMM (n = 20 and 19, 1 measurement failure) d Distribution of the 5hmC signal at the different ChromHMM chromatin states in multiple myeloma (n = 40, each left-side boxplot) versus normal plasma cells (n = 5, right sides). e Correlation matrix between 5hmC signal (this study) and the histone marks H3K36me3, H3K27ac, H3K4me3, H3K9me3 and H3K27me3 (ChIP-seq data from the Blueprint Consortium) in MM patients. f Average signal and signal matrix at 5hmC peaks of 5hmC (this study), histone marks (Blueprint) and ATAC-seq [33] in MM patients. ISS, International Staging System, NDMM, Newly Diagnosed Multiple Myeloma

Fig. 2:

5hmC peaks are organized in large domains and correlate with RNA expression in MM. a Example of two 5hmC-enriched domains at the locus DUSP22-IRF4 and Venn diagram showing the overlap between the top 500 5hmC-enriched domains (patient MM27) and H3K27ac super-enhancers (patient Jin_MM2). b ROSE plot (rank of super-enhancers) of the top 500 H3K27ac super-enhancers (patient Jin_MM2) and 5hmC-enriched domains (patient MM27). c 5hmC signal at 5hmC-enriched domains correlates with RNA expression of their neighbor gene. d 5hmC (this study), ATAC [33], H3K27ac [33] signals and ChromHMM states at the CCND2 genomic locus. The 5hmC signal correlation between the CCND2 gene and its putative enhancer is indicated

Fig. 3:

5hmC persists at specific oncogenic regions of myeloma subgroups. a MS quantification of 5hmC in myeloma cells of patients belonging to the MMSET (red), CCND1 (blue), hyperdiploid (HD, green) groups and the patients not related to any of the 3 groups (other, gray). b Heatmap of 5hmC-enriched domains specificity among the 40 MM patients and the 5 healthy donors (normal, pink). Color code for patients (in rows) corresponds to that of panel a. Asterisks stand for proximal non-genic loci. c Plot showing the 5hmC-enriched domains that are specifically enriched in patient groups MMSET, CCND1, hyperdiploid and normal plasma cells. Significant domains are colored. d 5hmC, ATAC and H3K27ac signals at the FGFR3-MMSET locus in the MMSET patient group and the non-MMSET patients

Fig. 4:

5hmC is dynamic and heterogeneous in MM between diagnosis and relapse. a MS quantification of 5hmC at diagnosis and relapse in patients MM02, MM05, MM07 and MM21 (average shown). b Plot showing the differential 5hmC-enriched domains between diagnosis and relapse in patients MM02, MM05, MM07 and MM21. c 5hmC signal and RNA expression levels of the MDM2 gene at diagnosis and relapse in patient MM05. At relapse, myeloma cells harbor one copy of TP53 locus (del17p FISH)