Abstract 2776: Leveraging HiChIP to assess chromatin interactions of lung cells upon smoking-related BaP exposure and identify target genes of lung cancer risk-associated variants

Lung cancer is the leading cause of cancer-related deaths in the United States, with lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) as the most frequent subtypes. These subtypes have distinct biological origins and risk loci, as identified through genome-wide association studies (GWAS), which are influenced by environmental exposures such as smoking. GWAS variants, largely found in non-coding regions, are hypothesized to function as cis-regulatory elements (CREs) that influence gene expression. However, CREs do not always regulate the nearest gene and can act over megabase distances. Chromatin conformation capture (Hi-C) assays can link GWAS variants to their target genes over long ranges, but these datasets are scarce and often derived from lung cancer cell lines, which may not recapitulate early tumor development. To address this gap, we performed HiChIP, which combines Hi-C and chromatin immunoprecipitation (ChIP) to profile chromatin interactions at active enhancer regions (marked by H3K27ac) in two non-tumorigenic lung cell lines: HPL1-D and BEAS-2B. These cell lines model the alveolar and bronchial tissues where LUAD and LUSC originate, respectively. To determine molecular effects of smoking on chromatin interactions, we exposed cells to the tobacco smoke carcinogen benzo[a]pyrene (BaP). We tested multiple time points to establish acute (48-hour) and chronic (10-day) BaP protocols, evaluating transcriptional responses, DNA damage, and viability. Acute exposure induced DNA damage marked by γ-H2AX and dose-dependent CYP1A1/CYP1B1 responses by 48 hours, while chronic exposure elicited stronger nuclear metabolic and inflammatory responses measured by RRM2 and IL1R1 expression, respectively. The IC20 dose of BaP, determined as 0.10 µM after 10- and 20-day treatments, guided the selection of 0.10 µM and 0.25 µM BaP for chronic exposure and 0.25 µM and 0.50 µM for acute exposure, with DMSO as a control. Notably, BEAS-2B cells showed significant responses to BaP, while HPL1-D did not. Critical optimization steps in HiChIP to ensure ChIP efficiency, sensitivity, and specificity include complete cell lysis and efficient DNA shearing by sonication. Incorporating a FACS filter successfully reduced clumped cells and improved cell lysis. Optimizing lysis time and sonication power achieved the desired chromatin fragment distribution range. With an optimized workflow, we use the Arima-adapted MAPS and FitHiChIP pipelines to perform quality control, identify interacting regions, and analyze differential loops between conditions. By integrating GWAS data with chromatin interaction profiles, we aim to pinpoint functional variants and their regulatory genes influenced by cell type and BaP exposure. This work will further our understanding of lung cancer etiology while providing a unique dataset for the public. Citation Format: Elelta Sisay, Thong Luong, Chia-Han Lee, Mai Xu, Jinhu Yin, Jiyeon Choi. Leveraging HiChIP to assess chromatin interactions of lung cells upon smoking-related BaP exposure and identify target genes of lung cancer risk-associated variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2776.