Myeloma is characterized by stage-specific alterations in DNA methylation that occur early during myelomagenesis

Abstract

Epigenetic changes play important roles in carcinogenesis and influence initial steps in neoplastic transformation by altering genome stability and regulating gene expression. To characterize epigenomic changes during the transformation of normal plasma cells to myeloma, we modified the HpaII tiny fragment enrichment by ligation-mediated PCR assay to work with small numbers of purified primary marrow plasma cells. The nano-HpaII tiny fragment enrichment by ligation-mediated PCR assay was used to analyze the methylome of CD138(+) cells from 56 subjects representing premalignant (monoclonal gammopathy of uncertain significance), early, and advanced stages of myeloma, as well as healthy controls. Plasma cells from premalignant and early stages of myeloma were characterized by striking, widespread hypomethylation. Gene-specific hypermethylation was seen to occur in the advanced stages, and cell lines representative of relapsed cases were found to be sensitive to decitabine. Aberrant demethylation in monoclonal gammopathy of uncertain significance occurred primarily in CpG islands, whereas differentially methylated loci in cases of myeloma occurred predominantly outside of CpG islands and affected distinct sets of gene pathways, demonstrating qualitative epigenetic differences between premalignant and malignant stages. Examination of the methylation machinery revealed that the methyltransferase, DNMT3A, was aberrantly hypermethylated and underexpressed, but not mutated in myeloma. DNMT3A underexpression was also associated with adverse overall survival in a large cohort of patients, providing insights into genesis of hypomethylation in myeloma. These results demonstrate widespread, stage-specific epigenetic changes during myelomagenesis and suggest that early demethylation can be a potential contributor to genome instability seen in myeloma. We also identify DNMT3A expression as a novel prognostic biomarker and suggest that relapsed cases can be therapeutically targeted by hypomethylating agents.

Figure 1. DNA methylation patterns can differentiate between different stages of plasma cell dyscrasias

A) Unsupervised hierarchical clustering of using methylation profiles generated by the HELP assay separate the samples in two major clusters containing the majority of NEWMM (orange) and REL (red) samples or the majority of NL (green) and MGUS (light blue) samples respectively. These two clusters were also identified by the presence (dark green) or absence (dark blue) of abnormalities detected by FISH or conventional cytogenetics. The clusters are each further separated into two subgroups resulting in a total of four cohorts representing the majority of NL, MGUS, NEWMM and REL. Green = NL, blue = MGUS, yellow = SMM, orange = NEWMM, red = REL, purple = REM. The bottom 6 lines represent FISH data, green = not detected by FISH, pink = detected by FISH, gray = not done. ◆ or ● indicate paired samples. B) Unsupervised 3D clustering based on nearest shrunken centroid algorithm using methylation profiles also shows distinction between normal and myeloma samples. Among the myeloma samples, clustering of MGUS samples is distinct from New and Relapsed cases. Green = NL, blue = MGUS, yellow = SMM, orange = NEWMM, red = REL, purple = REM

Figure 2. MGUS and NEWMM show predominant hypomethylation, whereas REL are predominantly hypermethylated

Volcano Plots showing difference of mean methylation (X Axis) and significance of the difference (Y Axis) demonstrate aberrant hypomethylation in MGUS (A) and both hypo and hypermethylation in New (B) and relapsed (C) cases of myeloma. The number of differentially methylated HpaII amplifiable fragments (HAFs) are indicated above each volcano plot. Numbers to the left indicate hypomethylated HAFs and numbers to the right indicate hypermethylated HAFs. Aberrant hypermethylation is the predominant change in Relapsed cases (D).

Figure 3. Differential methylation in MGUS occurs mainly within CpG islands

The genomic location of differentially methylated regions was mapped to CpG islands for each subcategory of myeloma. DMRs in MGUS were significantly enriched within CPG islands (dark gray) compared to the percentage of probes in the whole array (Proportions Test, P Value<0.01). DMRs in NEWMM and REL were significantly located outside of CpG islands (Proportions Test, P Value<0.01).

Figure 4. DNMT3A is underexpressed and hypermethylated in myeloma

A) Box plots showing gene expression values in CD138+ cells from NL (n=22), MGUS (n= XX) and MM (n= 559) from Arkansas datasets GSE5900 and GSE2658 shows significantly reduced expression levels in myeloma (TTest, P Value< 0.05). The MM samples included in these datasets contained untreated samples. B) Boxplots representing the methylation of the DNMT3A promoter in normal PCs (NL), MGUS and myeloma samples (MM) shows hypermethylation in MM compared to NL (TTest, P Value <0.05). C) Low DNMT3A expression is associated with worse overall survival in TT2. Differences between groups with top and bottom quartile gene expression are shown with Kaplan Meier graphs.

Figure 5. Decitabine treatment leads to growth inhibition in myeloma cell lines

Cell lines were treated with different doses of decitabine for 5 days and proliferation was assessed by the MTS assay. Significant inhibition of growth was seen after treatment even with low doses of Decitabine (TTest, P Value <0.05). Shown is one representative of three experiments.