Global methylation analysis identifies prognostically important epigenetically inactivated tumor suppressor genes in multiple myeloma

Abstract

Outcome in multiple myeloma is highly variable and a better understanding of the factors that influence disease biology is essential to understand and predict behavior in individual patients. In the present study, we analyzed combined genomewide DNA methylation and gene expression data of patients treated in the Medical Research Council Myeloma IX trial. We used these data to identify epigenetically repressed tumor suppressor genes with prognostic relevance in myeloma. We identified 195 genes with changes in methylation status that were significantly associated with prognosis. Combining DNA methylation and gene expression data led to the identification of the epigenetically regulated tumor modulating genes GPX3, RBP1, SPARC, and TGFBI. Hypermethylation of these genes was associated with significantly shorter overall survival, independent of age, International Staging System score, and adverse cytogenetics. The 4 differentially methylated and expressed genes are known to mediate important tumor suppressive functions including response to chemotherapy (TGFBI), interaction with the microenvironment (SPARC), retinoic acid signaling (RBP1), and the response to oxidative stress (GPX3), which could explain the prognostic impact of their differential methylation. Assessment of the DNA methylation status of the identified genes could contribute to the molecular characterization of myeloma, which is prerequisite for an individualized treatment approach.

Figure 1

OS and PFS for pd-DMR genes according to their methylation status. Kaplan-Meier curves for OS (left column) and PFS (right column) according to methylation status of the pd-DMR array probes mapping to the genes GPX3 (probe ID cg07699362), RBP1 (cg23363832), SPARC (cg25913233), and TGFBI (cg21034676). Methylation status was categorized into “low” and high” methylation as described in the “Results” section. Log-rank P values indicate results of univariate analysis comparing low and high methylation groups.

Figure 2

Binary relation of methylation and expression for pd-DMR genes. Each circle in the scatterplot represents 1 case of the total of 115 samples with methylation and expression data. X-axis data represent methylation β values (ranging from 0 to 1.0, equivalent to 0% to 100% methylation), y-axis data represent normalized, log2 Affymetrix HG U133plus2.0 gene expression data. The area shaded in gray in each plot represents cases with high methylation values as defined per the k-means clustering algorithm. Note that cases with a low methylation status (below the individual p-DMR cutoff; white background) can be, but are not constitutionally, highly expressed (transcriptionally permissive). In contrast, cases with high methylation status (above the cutoff; gray background) are silenced (transcriptionally locked).

Figure 3

Methylation changes at pd-DMR genes accompany progression of myeloma cells to aggressive phenotypes. Methylation β values of 5 MGUS individuals, 159 MM and 31 PCL samples, and 11 myeloma cell lines (HMCL) are shown as box-whisker plots with error bars indicating 2.5 and 97.5 percentiles. Solid circles represent outlier cases. **Statistically significant (P < .01) differences in group β values per Mann-Whitney U test. Methylation values between MGUS and MM were not significantly different.

Figure 4

Loss of methylation and induction of expression of pd-DMR genes under 5-aza-2′-deoxycytidine treatment. The cell line KMS11 was treated with demethylating low doses (200 nM) of DAC for 4 consecutive days and cell material was harvested at baseline, after DAC treatment, and after additional 17 days of culture after removal of DAC. Gene expression of pd-DMR genes was assayed by RT-PCR and products were run on a 2% agarose gel with glyceraldehyde-3-phosphate dehydrogenase as a housekeeping gene control (A). DNA methylation at the genomic loci of the pd-DMR probes was assayed by bisulfite pyrosequencing for the different time points. Black areas in the pie charts represent % methylated, white area % unmethylated cytosine residues. Pyrosequencing assay design for the probe mapping to GPX3 was technically impossible due to very high CpG density at the surrounding region of the pd-DMR array probe (B).