Abstract 5646: Altered intronic polyadenylation by mutant p53 impairs transcription of DNA repair genes in lung cancer

Abstract

Abstract Introduction: Changes in alternative pre-mRNA processing have been found in many cancers. Aberrant intronic polyadenylation (IPA) causes a stop of normal transcription resulting in the production of truncated mRNA isoform or lncRNA. We sought to examine the role of mutant p53 in pre-mRNA processing, especially in IPA regulation in lung cancer. Methods: We established stable cell lines transfected with mutant p53 (R273H, R175H, H179Q, C238Y, and C242F) or vector control using the NSCLC cell line H1299 with a homozygous deletion of the endogenous p53. Total RNA was used to build libraries enriched in sequences near polyadenylation sites (PASs) using the 3’mRNA-Seq REV library kit (Lexogen; Greenland, NH). Sequencing reads were mapped to the human genome (GRCh37), and PASs with ≥2 supporting reads and ≥0.05 usage in any experiment were identified. Counts for each Intronic Polyadenylation (IPA) and total reads in the terminal exon were compiled into matrices for each condition. DEXseq was applied to detect changes in the relative abundance of each exon part normalized to all exons within the gene. Gene Ontology (GO) analysis was implemented using the enrichR package. Results: We identified 32,752 IPA sites in 5,718 genes, and 26,416 distal PASs (dPASs) in the 3’UTRs of 10,298 genes. Across all five cell lines, when compared individually to the vector control, a comprehensive shift in IPA events was observed, with a concurrent decrease in dPASs. Many of IPA isoforms exhibited consistent patterns of increased or decreased abundance changes. Upon closer examination of each individual cell line, such as the p53-R273H cell line (vs. vector), significant usage changes were identified for 2,389 IPA sites, of which 1,210 were increased and 1,128 terminal exons were altered, with 650 being suppressed. At the gene level, a total of 1,183 genes had at least one significantly increased IPA isoform, a significantly decreased distal isoform, or both. GO analysis showed that these genes were highly enriched in DNA repair-related functional terms such as DNA repair, double-strand break repair, and nucleotide excision repair. The observed IPA changes in DNA-repair genes were consistently observed across the other 4 mutant lines (vs. vector). This observation was further supported by analyzing data from patients with lung adenocarcinoma (LUAD) in the TCGA cohort, where we selected samples carrying nonsense/frameshift mutations, and/or a deep deletion as the p53-null control group, and those carrying missense mutations as the p53-mutant group. Conclusions: Our study illustrated a novel role of p53 mutants in the regulation of intronic polyadenylation processing that contributes to deficient DNA repair. These observations offer a fresh perspective on the multifaceted roles of p53 in cellular homeostasis. Acknowledgment: This work was supported by NCI Cancer Center Support Grant P30CA012197 and NIH/NCI grant R03CA256100. Citation Format: Liang Liu, Elizabeth Forbes, Wei Zhang. Altered intronic polyadenylation by mutant p53 impairs transcription of DNA repair genes in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5646.