DNA Methylation Analysis Determines the High Frequency of Genic Hypomethylation and Low Frequency of Hypermethylation Events in Plasma Cell Tumors

The Translational Genomics Research Institute, Phoenix, AZ, USA.
Cancer Research (Impact Factor: 9.33). 09/2010; 70(17):6934-44. DOI: 10.1158/0008-5472.CAN-10-0282
Source: PubMed


Multiple myeloma (MM) is a plasma cell malignancy of the bone marrow, which evolves from a premalignant stage called monoclonal gammopathy of undetermined significance (MGUS). In some patients, an intermediate stage referred to as smoldering multiple myeloma (SMM) is clinically recognized, with the full-bore malignancy termed MM. We conducted a study to assess differential CpG methylation at 1,500 genic loci during MM progression and profiled CD138(+) plasma cells from MGUS, SMM, and MM specimens; human myeloma cell lines; and normal plasma cell (NPC) samples. We showed that the number of differentially methylated loci (DML) increased with tumor grade, and the vast majority were due to hypomethylation. Hierarchical clustering analysis revealed samples that coclustered tightly with NPC. These cases, referred to as "normal-like," contained significantly fewer DML when compared with their non-normal-like counterparts and displayed overall methylation levels resembling NPC. This study represents one of the first methylome interrogation studies in MM and points toward global hypomethylation at genic CpG loci as an important and early mechanism driving myelomagenesis. Determining the set of critical genes and pathways based on the myeloma methylome is expected to lead to an improved understanding of biological mechanisms involved in myelomagenesis.

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Available from: Bodour Salhia, Mar 19, 2015
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    • "In spite of these significant findings, the high-throughput DNA methylation reports published so far in MM were restricted to the study of promoter regions (Heuck et al. 2013; Kaiser et al. 2013; Nojima et al. 2009; Salhia et al. 2010; Walker et al. 2011). Hence, the purpose of our study was to adopt a more extensive and unbiased analysis of the DNA methylome, including promoters, gene bodies and intergenic regions in normal plasma cells (NPC) and plasma cells from MGUS and MM patient samples. "
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    ABSTRACT: While analyzing the DNA methylome of multiple myeloma (MM), a plasma cell neoplasm, by whole-genome bisulfite sequencing and high-density arrays, we observed a highly heterogeneous pattern globally characterized by regional DNA hypermethylation embedded in extensive hypomethylation. In contrast to the widely reported DNA hypermethylation of promoter-associated CpG islands (CGIs) in cancer, hypermethylated sites in MM, as opposed to normal plasma cells, were located outside CpG islands and were unexpectedly associated with intronic enhancer regions defined in normal B cells and plasma cells. Both RNA-seq and in vitro reporter assays indicated that enhancer hypermethylation is globally associated with downregulation of its host genes. ChIP-seq and DNase-seq further revealed that DNA hypermethylation in these regions is related to enhancer decommissioning. Hypermethylated enhancer regions overlapped with binding sites of B cell-specific transcription factors (TFs) and the degree of enhancer methylation inversely correlated with expression levels of these TFs in MM. Furthermore, hypermethylated regions in MM were methylated in stem cells and gradually became demethylated during normal B-cell differentiation, suggesting that MM cells either reacquire epigenetic features of undifferentiated cells or maintain an epigenetic signature of a putative myeloma stem cell progenitor. Overall, we have identified DNA hypermethylation of developmentally-regulated enhancers as a new type of epigenetic modification associated with the pathogenesis of MM. Published by Cold Spring Harbor Laboratory Press.
    Full-text · Article · Feb 2015 · Genome Research
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    • "Development of new therapeutic interventions is strikingly needed for increasing patient survival rate. It has been shown that abnormal methylation of tumor suppressor genes is a common event in malignant plasma cell disorders [2-4] and aberrant global methylation patterns also affect the molecular pathogenesis of myeloma [3]. Azacytidine, a ring analog of the naturally occurring pyrimidine nucleoside cytidine, is an inhibitor of DNA methyltransferase. "
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    ABSTRACT: Azacytidine is an inhibitor of DNA methyltransferase and is known to be an anti-leukemic agent to induce cancer cell apoptosis. In the present study, multiple myeloma cells were treated with azacytidine at clinically relevant concentrations to induce necrosis through oxidative stress. Necrotic myeloma cells exhibit unique characteristics, including enrichment of the cell-bound albumin and overexpression of endoplasmic reticulum (ER)- and mitochondrial-specific chaperones, which were not observed in other necrotic cells, including HUH-7, A2780, A549, and Hoc1a. Proteomic analysis shows that HSP60 is the most abundant up-regulated mitochondrial specific chaperone, and azacytidine-induced overexpression of HSP60 is confirmed by western blot analysis. In contrast, expression levels of cytosolic chaperones such as HSP90 and HSP71 were down-regulated in azacytidine-treated myeloma cells, concomitant with an increase of these chaperones in the cell culture medium, suggesting that mitochondrial chaperones and cytosolic chaperones behave differently in necrotic myeloma cells; ER- and mitochondrial-chaperones being retained, and cytosolic chaperones being released into the cell culture medium through the ruptured cell membrane. Our data suggest that HSP60 is potentially a new target for multiple myeloma chemotherapy.
    Full-text · Article · Jun 2013 · Proteome Science
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    • "Tumor-specific hypermethylation has also been found in the promoter regions of various tumor suppressors and other tumor-related genes, including BNIP3, DAPK and RASD1, which are associated with prognosis and drug resistance in MM [11-14]. Unexpectedly, however, recent advances in genome-wide analysis revealed that the number of methylated genes declines markedly with the progression of malignant transformation of plasma cells [15,16]. In addition, histone modifications are also involved in the pathogenesis of MM, and are associated with aberrant gene expression or important translocations such as t(4;14) [17,18]. "
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    ABSTRACT: Background The aim of this study was to clarify the role of global hypomethylation of repetitive elements in determining the genetic and clinical features of multiple myeloma (MM). Methods We assessed global methylation levels using four repetitive elements (long interspersed nuclear element-1 (LINE-1), Alu Ya5, Alu Yb8, and Satellite-α) in clinical samples comprising 74 MM samples and 11 benign control samples (7 cases of monoclonal gammopathy of undetermined significance (MGUS) and 4 samples of normal plasma cells (NPC)). We also evaluated copy-number alterations using array-based comparative genomic hybridization, and performed methyl-CpG binding domain sequencing (MBD-seq). Results Global levels of the repetitive-element methylation declined with the degree of malignancy of plasma cells (NPC>MGUS>MM), and there was a significant inverse correlation between the degree of genomic loss and the LINE-1 methylation levels. We identified 80 genomic loci as common breakpoints (CBPs) around commonly lost regions, which were significantly associated with increased LINE-1 densities. MBD-seq analysis revealed that average DNA-methylation levels at the CBP loci and relative methylation levels in regions with higher LINE-1 densities also declined during the development of MM. We confirmed that levels of methylation of the 5' untranslated region of respective LINE-1 loci correlated strongly with global LINE-1 methylation levels. Finally, there was a significant association between LINE-1 hypomethylation and poorer overall survival (hazard ratio 2.8, P = 0.015). Conclusion Global hypomethylation of LINE-1 is associated with the progression of and poorer prognosis for MM, possibly due to frequent copy-number loss.
    Full-text · Article · Dec 2012 · Genome Medicine
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