Abstract O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that protects cells from carcinogenic effects of alkylating agents; however, MGMT is silenced by promoter hypermethylation during carcinogenesis. Concomitant methylation of a panel of cancer-relevant genes including MGMT detected in lung epithelial cells present in sputum predicts risk for subsequent lung cancer incidence in moderate and heavy smokers. In addition, MGMT methylation is also a prognostic biomarker for response of glioblastoma patients to the alkylating agent temozolomide. A single nucleotide polymorphism (SNP, rs16906252, G/A) in an enhancer in the MGMT promoter was previously identified to be highly significantly associated with risk for MGMT methylation in lung cancer and sputum from smokers. The predisposition of the “A” allele for methylation in heterozygotes was due to the reduced gene transcription in luciferase reporter assay. In silico bioinformatics analysis suggested that rs16906252 was the causal cis-acting methylation quantitative trait locus for MGMT. We are current applying a cutting edge technology “Hybridization Capture of Chromatin-Associated Proteins for Proteomics” to identify the transcriptional factors whose binding was disrupted by rs16906252. To further genetic investigations, a genome-wide association and replication study was conducted in two smoker cohorts to identify novel loci for MGMT methylation in sputum that were independent of the MGMT enhancer polymorphism. Two novel trans-acting loci (15q15.2 and 17q24.3) that were identified acted together with the enhancer SNP to empower risk prediction for MGMT methylation. We found that the predisposition to MGMT methylation arising from the 15q15.2 locus involved regulation of the ubiquitin protein ligase E3 component UBR1. UBR1 attenuation reduced turnover of MGMT protein and increased repair of O6-methylguanine in nitrosomethylurea-treated human bronchial epithelial cells, while also reducing MGMT promoter activity and abolishing MGMT induction. Overall, our results substantiate reduced gene transcription as a major mechanism for predisposition to MGMT methylation in the lungs of smokers, and support the importance of UBR1 in regulating MGMT homeostasis and DNA repair of alkylated DNA adducts in cells. In addition, genetic polymorphisms that affect DNA methylation of the DNA repair gene MGMT have strong clinical relevance in smokers, not only for cancer risk assessment but also for stratification of lung cancer patients for alkylating agent chemotherapy. (Supported by NIH grant R01 CA097356 and NIH/NCI grant P30 CA118100). Citation Format: Shuguang Leng, Hector Guillen, Carmen Tellez, Maria Picchi, James Swenberg, Frank Gilliland, Steven Belinsky. Cis and trans quantitative trait loci for MGMT promoter hypermethylation detected in lung exfoliated cells collected in sputum from smokers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2564.