Cancer Biology and Signal Transduction The Role of Gene Body Cytosine Modi fi cations in MGMT Expression and Sensitivity to Temozolomide

The DNA repair protein O-methylguanine-DNA methyltransferase (MGMT) is known to play a role in sensitivity to temozolomide. Promoter hypermethylation of MGMT is commonly used to predict low expression levels of MGMT in gliomas, despite observed discordance between promoter methylation and protein levels. Here, we investigated the functional role of gene body cytosinemodification in regulating levels ofMGMT gene expression and sensitivity to temozolomide. In 91 human glioblastoma samples, we observed significant variation inMGMT expression levels in patientswith anunmethylated promoter,with higher levels of gene body cytosine modification correlating with higher gene expression levels. Furthermore, inducing hypomethylation across theMGMT gene body with decitabine corresponded with decreased levels ofMGMT gene expression in lymphoblastoid and glioblastoma cell lines, indicating an important functional role for gene body cytosine modifications in maintaining gene expression. We reasoned that the decrease in MGMT expression induced by decitabinemay render resistant glioblastoma cell linesmore sensitive to temozolomide. Consistent with this reasoning, we found that the MGMT-expressing glioblastoma cell lines exhibiting an unmethylated MGMT promoter that were pretreated with decitabine became significantly more sensitive to temozolomide. Overall, our results suggest a functional role for gene body cytosine modification in regulating gene expression ofMGMT and indicate that pretreating patients whose tumors have an unmethylatedMGMT promoter with decitabine before temozolomide treatment may increase their response to therapy.Mol Cancer Ther; 13(5); 1334–44. 2014 AACR. Introduction The O-methylguanine-DNA methyltransferase (MGMT) gene encodes for a DNA repair protein that repairs Oalkylguanine-DNA adducts (1). This type of DNA damage can occur after certain environmental exposures, such as tobacco-specific nitrosamines, and from DNA alkylating agents used for chemotherapy (2, 3). Temozolomide is an alkylating agent that was FDA approved to treat brain tumors in 2005, including anaplastic astrocytoma and glioblastoma multiforme, the most aggressive form of brain cancer (4). Only 5% to 10% of the methylation adducts on DNA resulting from temozolomide treatment are O-methylguanine, but if MGMT is not present to repair the damage, these adducts trigger DNA mismatch repair and are highly cytotoxic (5, 6). Upon repair, MGMT is irreversibly inactivated, thereby requiring new protein synthesis for additional repair (7). Consequently, epigenetic alterations that affect transcriptional activity of MGMT, such as promoter hypermethylation, can have a significant effect on the number ofO-guanine lesions that are repaired (8, 9). Patientswith gliomaswith an aberrantly hypermethylated MGMT promoter respond better to temozolomide treatment compared with patients with an unmethylatedMGMT promoter, because they lackMGMT protein expression (10, 11). Although approximately 40% to 70% of patients with gliomas have a methylatedMGMT promoter (9, 12–14), promotermethylationdoesnot always show a strong correlation with MGMT protein levels (13, 15). For example, one study investigating the correlation between MGMT promoter methylation and protein levels showed that 7 of 40 gliomas had an unmethylated promoter yet no detectable MGMT protein, and 16 of 40 gliomas had aberrant methylation at the promoter yet still had detectable MGMT protein (13). Therefore, additional mechanisms of transcriptional and translational regulation are likely affecting expression of MGMT. The role of gene body cytosine methylation and hydroxymethylation in regulating gene expression levels is not fully understood, but several studies have observed a positive correlation between gene body cytosine modifications and gene expression levels (16–18). ForMGMT in particular, gene body methylation was first shown to correlate with gene expression levels in 1992 (19). Two Authors' Affiliations: Committee on Cancer Biology, Department of Medicine, and Comprehensive Cancer Center, The University of Chicago; and Institute of Human Genetics, University of Illinois, Chicago, Illinois Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Corresponding Authors: Lucy Godley, Room 7124, 900 E. 57th St., Chicago, IL 60637. Phone: 773-702-4140; Fax: 773-702-9268; E-mail: lgodley@medicine.bsd.uchicago.edu; and M. Eileen Dolan, edolan@medicine.bsd.uchicago.edu doi: 10.1158/1535-7163.MCT-13-0924 2014 American Association for Cancer Research. Molecular Cancer Therapeutics Mol Cancer Ther; 13(5) May 2014 1334 on June 21, 2017. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Published OnlineFirst February 25, 2014; DOI: 10.1158/1535-7163.MCT-13-0924