Extent and Patterns ofMGMTPromoter Methylation in Glioblastoma- and Respective Glioblastoma-Derived Spheres

Abstract

Purpose: Quantitative methylation-specific tests suggest that not all cells in a glioblastoma with detectable promoter methylation of the O6-methylguanine DNA methyltransferase (MGMT) gene carry a methylated MGMT allele. This observation may indicate cell subpopulations with distinct MGMT status, raising the question of the clinically relevant cutoff of MGMT methylation therapy. Epigenetic silencing of the MGMT gene by promoter methylation blunts repair of O6-methyl guanine and has been shown to be a predictive factor for benefit from alkylating agent therapy in glioblastoma.Experimental Design: Ten paired samples of glioblastoma and respective glioblastoma-derived spheres (GS), cultured under stem cell conditions, were analyzed for the degree and pattern of MGMT promoter methylation by methylation-specific clone sequencing, MGMT gene dosage, chromatin status, and respective effects on MGMT expression and MGMT activity.Results: In glioblastoma, MGMT-methylated alleles ranged from 10% to 90%. In contrast, methylated alleles were highly enriched (100% of clones) in respective GS, even when 2 MGMT alleles were present, with 1 exception (<50%). The CpG methylation patterns were characteristic for each glioblastoma exhibiting 25% to 90% methylated CpGs of 28 sites interrogated. Furthermore, MGMT promoter methylation was associated with a nonpermissive chromatin status in accordance with very low MGMT transcript levels and undetectable MGMT activity.Conclusions: In MGMT-methylated glioblastoma, MGMT promoter methylation is highly enriched in GS that supposedly comprise glioma-initiating cells. Thus, even a low percentage of MGMT methylation measured in a glioblastoma sample may be relevant and predict benefit from an alkylating agent therapy.

MGMT testing—the challenges for biomarker-based glioma treatment

“…However, it is still not completely clear yet how many and which particular CpG sites in the MGMT CGI have to be methylated in order to (i) cause transcriptional silencing in primary tumor cells and (ii) to demonstrate the best association with treatment outcome and patient survival. In fact, each diagnostic methylation-specific assay -whether qualitative, semi-quantitative or quantitative -interrogates distinct sets of CpG sites within the MGMT CGI [35,36] (Fig. 1).…”

“…SP1 [35] NFκB [36] CBP/p300 [37] AP1 [38] HIF1α [40] Promoter miR-181b [43,44], miR-181d [44], mir221/222 [45], miR-767-3p, miR-648 [42] + NDRG1 [42] mRNA protein *50% events (immature data) Abbreviations: Karnofsky Performance Status (KPS), radiotherapy (RT), O6-methylguanine DNA-methyltransferase (MGMT), temozolomide (TMZ), overall survival (OS)…”

DNA damage responses in cancer stem cells: Implications for cancer therapeutic strategies

“…The epigenetically silenced MGMT might be responsible for the resistance of GICs to temozolmide [38] due to MGMT's ability to reverse alkylation at the O 6 positon of guanine and neutralize the cytotoxicity of alkylating agents such as temozolmide [39] . In addition to glioma stem cells, enhanced DNA repair capacity was also found in other CSCs.…”

Methylation of O6-Methyl Guanine Methyltransferase Gene Promoter in Meningiomas - Comparison between Tumor Grades I, II, and III

“…It is worthy of note that the status of MGMT methylation can vary for a particular tumor. For example in glioblastoma patients, previous studies have reported rates of 24%,33%,34%,35%, 40%,45%,47.5%,53%,68%, and 70% for MGMT promoter methylation (Esteller et al, 2000;Hegi et al, 2004;Hegi et al, 2005;Brandes et al, 2008;Dunn et al, 2009;Costa et al, 2010;Rivera et al, 2010;Sciuscio et al, 2011;Havik et al, 2012;Tang et al, 2012). It is possible that variability in the rates of promoter-region gene hypermethylation in meningioma tumors may be influenced by race, high fat food diet, polymorphism, smoking, dietary variables, and other environmental exposures.…”