Discovery Of Genome Wide Epigenetic Programming In t(4;14) Multiple Myeloma and In The Progression From Myeloma To Plasma Cell Leukemia Via Methyl Binding Domain Protein Capture and Sequencing

The t(4;14) translocation in multiple myeloma is associated with aggressive disease behavior and shorter survival despite recent advances in therapy. The histone methyltransferase MMSET/WHSC1 is overexpressed as a consequence of the translocation, but the detailed epigenetic downstream effects of this overexpression are unknown. Our group identified a specific DNA hypermethylation pattern in t(4;14) cases, indicating a strong contribution of epigenetic changes to the aggressive biology of this subtype. We have here performed genome wide methyl-binding domain protein capture and next generation sequencing (MBD-seq) to characterize the epigenetic perturbations in t(4;14) myeloma in contrast to t(11;14) cases, a subgroup with favorable clinical behavior. Patient bone marrow myeloma cells at presentation and at progression to plasma cell leukemia were analysed to identify epigenetic mechanisms characteristic for early and late stage disease.

MBD-seq data were generated from bone marrow myeloma samples of 3 t(4;14) and 3 t(11;14) cases at both presentation and plasma cell leukemia (PCL) stages. Data were analyzed with the MEDIPS package and RPM values for 500 bp bins were calculated. Due to copy number sensitivity of the method, matched Affymetrix SNP genotyping array data were used to correct for focal genomic gains and losses. Pairwise contrasts were calculated and results were filtered for statistical significance (p<0.01) and RPM value differences (>20) to generate lists of differentially methylated regions (DMRs). Publicly available ENCODE datasets such as chromatin state segmentation for the B-lymphocytic GM12878 cell line were used to identify epigenetic mechanisms involved in MMSET activity and disease progression. Matched gene expression data have been generated for presentation myeloma cases and exome sequencing has been performed for a t(4;14) and a t(11;14) myeloma case.

Copy number corrected MBD-seq results confirmed the previous observation of a specific DNA hypermethylation pattern in t(4;14) versus t(11;14) cases (2729 hyper vs. 582 hypomethylated DMRs). Comparison of t(4;14) specific DMRs with publicly available datasets (GREAT, Bejerano Lab, Stanford) revealed genome wide significant overlap with the top 50 overexpressed genes of the “MS” cluster consisting of t(4;14) cases (binomial FDR q-value: 1.28e-9), confirming the functional epigenetic involvement in t(4;14) biology. There was also significant overlap with the module Genes in the AKT1 Pathway (q-value: 6.62e-8). Comparison of results with ENCODE Chromatin states data for B lymphocytes (GM12878) demonstrated significant overrepresentation of “weak enhancer” and “weak promoter” regions in t(4;14) hypermethylated DMRs. In contrast, hypomethylated DMRs in t(4;14) were significantly enriched for “transcriptional transition” and “transcriptional elongation” (MCFDR p-value=0.0 for both), with overrepresentation of histone marks H3K20me1 and H3K36me3 in GM12878. Hypomethylated regions mapped to MUM1/IRF4 and to several epigenetic modifiers such as CTCF, highlighting a functionally important, specific set of hypomethylated genes in t(4;14).

Progression to PCL was accompanied by significant preferential hypermethylation of regulatory enhancers and promoter regions and of the “polycomb group repressed” chromatin state both in t(4;14) and t(11;14) groups. Hypomethylation occurred in regions of “heterochromatin” state in t(4;14) MM to PCL progression and significantly more than expected in repetitive regions accompanying t(11;14) progression. Comparing t(4;14) and t(11;14) PCL demonstrated significant t(4;14) hypermethylation of regulatory regions (enhancers, promoters) and significant relative preferential t(4;14) hypomethylation of the “transcriptional elongation” chromatin state, indicating some maintained functional epigenetic programming by MMSET in t(4;14) PCL.

We have characterized the genome wide epigenetic programming of DNA methylation in MMSET overexpressing t(4;14) myeloma and PCL identifying key regulatory pathways involved in t(4;14) biology. We have also identified focal regulatory sequences, enhancers and promoters, which are affected specifically in the t(4;14) which may be linked to the biology of the this disease subtype.

No relevant conflicts of interest to declare.