Methylation in multiple myeloma

Multiple myeloma (MM) is a B-cell lymphoproliferative malignancy being characterized by accumulation of clonal plasma cells in the bone marrow, osteolytic bone lesions, and a monoclonal immunoglobulin in the serum and/or urine. Although several prognostic parameters including abnormal cytogenetics (most notably deletion of chromosome 13q) have been identified to predict the clinical course of individual patients, there is still a lack in understanding the molecular features of MM entities with distinct biology and clinical behavior. Aberrant methylation of the promoter region of tumor suppressor genes (TSG) has been identified as an alternative to the genetic loss of a TSG by deletion or mutation. Recently, it has been suggested that aberrant methylation of the genes p15 and p16 plays an important role in the pathogenesis of MM. Therefore, we hypothesize that aberrant methylation of specific genes may have biological and clinical implications in MM. To address this issue, we plan to investigate the methylation status of the following genes in a large cohort of patients with newly diagnosed MM (N = 150) by methylation-specific PCR: p16, p15, p73, MGMT, CDH1, DAPK, SOCS-1, RARbeta, TIMP-3 and RASSF1A. These genes are involved in the pathogenesis of different malignant diseases and therefore it is likely that they also play an important role in the development of MM. The methylation results will be analyzed in the context of specific chromosomal abnormalities and clinicopathological characteristics of MM patients. In addition, we also plan to investigate if aberrant methylation of these genes occurs already as an early change during the pathogenesis of monoclonal gammopathies. The results of this project will help to determine the impact of aberrant methylation of certain genes for the pathogenesis of MM and may be essential for developing new treatment strategies for patients with MM.

Gene silencing by aberrant DNA methylation (referred to as methylation) is a frequently occurring event in the pathogenesis of malignant diseases. However, little was known about methylation of tumor suppressor genes (TSGs) in monoclonal gammopathies. Thus, we investigated the methylation frequencies of the TSGs p16, TIMP3, p15, CDH1, DAPK, p73, RASSF1A, p14, MGMT and RARß in patients with monoclonal gammopathies by methylation-specific polymerase chain reaction analysis. At least 1 of these genes was methylated in 79% of patients with monoclonal gammopathy of undetermined significance (MGUS) and in 80% of patients with multiple myeloma (MM). Genes frequently methylated in MGUS patients included p16 (28%), TIMP3 (35%), DAPK (17%) and p73 (21%). MM patients were frequently methylated for p16 (36%), TIMP3 (29%), p15 (27%), CDH1 (27%) and DAPK (22%). Of note, CDH1 was the only gene which was not methylated in MGUS but in MM suggesting that CDH1 methylation is a marker for disease progression. To further define the role of epigenetic gene silencing in the pathogenesis of MM we performed a microarray-based genome-wide screen for genes affected by DNA methyltransferase inhibition by 5-aza-2-deoxycytidine (Aza-dC) and histone deacetylase inhibition by trichostatin A (TSA) in 3 MM cell lines. A substantial number of genes was found to be up-regulated in at least 1 MM cell line in response to Aza-dC (282 genes), to TSA (323 genes), or to simultaneous treatment with a combination of both drugs (470 genes). These genes are involved in immune response, cell proliferation, signal transduction, apoptosis, cell cycle and cell motility. Our approach identified several genes whose epigenetically silencing in MM has already been reported in previous studies (i.e. TIMP1, CDKN1A, SOCS1 and CDH1). However, more important, we identified a large number of epigenetically inactivated cancer-related genes which have not been analyzed in MM so far (i.e. ING1p33, TADA3L, BTG1, JUP, NGFRAP1 and CGREF1). Till now, 5 genes were selected and their methylation status was examined in MM cell lines, in patients with MGUS and in MM patients, respectively. The genes analyzed are CPEB1 (involved in cell cycle), CD9 (involved in cell adhesion), GJA1 (involved in cell- cell signaling), GADD45G (involved in DNA repair) and AKAP12a (involved in G-protein signaling). The frequency of methylation of these genes ranged between 50% and 17% in MM cell lines and between 50% and 13% in MM samples. MGUS samples were less frequently methylated for the 5 genes than MM samples (range between 17% and 0%). This difference was statistically significant in the cases of CPEB1 and GADD45G indicating that methylation of these genes may be a marker for disease progression. In conclusion, our data suggest that methylation of certain genes occurs frequently in patients with MGUS and MM. Moreover, microarray analysis in with Aza-dC and/or TSA treated cells is a useful approach to identify epigenetically silenced genes. Several genes, which are not known so far to be relevant in the pathogenesis of MM and which are frequently methylated have been identified.