Integrated transcriptomic and epigenomic analysis to study the mechanism by which smoke-induced hypomethylation of cg05575921 increases lung cancer risk

Lung cancer is the leading cause of cancer death in the US. The most common subtype is lung adenocarcinoma (LUAD), arising out of the alveolar epithelium. Tobacco smoke and pollution play a key role in LUAD risk and have been associated with changes in DNA methylation in numerous epigenome-wide association studies. Hypomethylation of cg05575921 is one of the most significant changes, predicting over 30% of the increased risk of lung cancer seen in smokers. However, the mechanism by which this methylation loss is linked to lung cancer risk remains unknown. We previously showed that cg05575921, located in intron 3 of the gene aryl hydrocarbon receptor repressor ( AHRR ), borders a tobacco smoke-inducible enhancer. Upon smoke exposure AHRR is the only gene within a two megabase window whose expression increases. AHRR is a negative feedback regulator of aryl hydrocarbon receptor (AHR) induced detoxification responses. Like AHR, AHRR binds to the partner protein ARNT. However, unlike the AHR/ARNT heterodimer, which turns on detoxification genes, the AHRR/ARNT heterodimer downregulates the detoxification response. We hypothesize that prolonged tobacco smoke exposure triggers maladaptive expression of AHRR , that hypomethylation of cg05575921 is a byproduct of the adjacent enhancer activation, and that constitutive expression of AHRR prevents protective xenobiotic detoxification responses, thereby increasing lung cancer risk. Using our unique immortalized human alveolar epithelial cells, we are determining what role the enhancer and cg05575921 play in AHRR expression by i) using Oxford Nanopore long-read sequencing to study DNA methylation changes in the region over time during tobacco smoke exposure, ii) using CRISPR/Cas 9 to heterozygously delete the known enhancer and/or the CpG to determine the effects on the expression of AHRR from the enhancer/CpG-deleted and control alleles, iii) using an inducible AHRR gene to test the effects of constitutive expression of AHRR on the xenobiotic response genes induced by cigarette smoke. Understanding why cg05575921 hypomethylation is strongly associated with lung cancer risk may help devise strategies to mitigate the effects of smoke exposure. Supported T31IP1913 from the Tobacco-Related Disease Research Program, an Epigenomic Regulation in Cancer Pilot ward from the USC/Norris Comprehensive Cancer Center, the Norris Comprehensive Cancer Center core grant NIH/NCI P30CA014089, and a Keck School of Medicine Dean's Fellowship. MAG and IAO are members of CaRE 2 , the Cancer Research Education and Engagement Health Center, supported by NIH/NCI grants U54CA233396, U54CA233444, and U54233465.