Abstract 3620: Investigating the role of altered replication timing during tumor evolution in lung and breast cancer

Introduction: Faithful DNA replication is a key requirement for normal cell division which initiates from replication origins distributed throughout the genome. Not all origins initiate replication at the same time, generating a conserved temporal order of DNA replication (the replication timing program) which is cell and tissue type specific. The extent to which replication timing alters during malignant transformation and the resultant impact on the genomic landscape in cancer remains unclear. Here, we explored the landscape of altered replication timing in lung and breast cancer and the interplay between altered replication timing and genomic cancer evolution. Methods: To characterize replication timing profiles, the Repli-Seq protocol was applied to 4 lung adenocarcinoma, 3 lung squamous cell carcinoma, and 4 breast cancer cell lines, as well as the cell-of-origin of each cancer type. Replication timing was measured as the log2-transformed ratio between early and late replicated reads in 50kb windows. To identify altered replication timing regions the log2-ratio of each cancer cell line was compared to its matched normal. Publicly available WGS and RNA-seq data from patients were used to investigate the relationship between replication timing and genomic alterations. Results: We identified a systematic shift in replication timing from normal to cancer cell lines in 6%-17% of the genome, half of which were classified as late-to-early and half early-to-late replicated. We identified an increase in mutation load in early-to-late replicated regions in comparison to non-altered early replicated regions, whereas the opposite was true for late-to-early replicated regions. We observed a more prevalent shift in the mutational landscape in breast compared to lung cancer, suggesting that most mutations in breast cancer were accumulated after the alteration in replication timing. In addition, we identified known and novel mutational processes active in differently replicated parts of the genome. Furthermore, we observed an enrichment of APOBEC3-associated mutation clusters (omikli events) in early and late-to-early replicated regions. Recurrently gained copy number segments showed an enrichment in not-altered early replicated regions. Also, late-to-early replicated genes exhibited an increase in expression in cancer relative to normal, whereas early-to-late replicated genes exhibited a decrease in expression. Cancer type-specific cancer genes were enriched in late-to-early replicated regions that became up-regulated in cancer, whereas essential genes tended not to change their replication timing during malignant transformation. Conclusion: We identified significant alterations in the replication timing program during malignant transformation which influence the genomic and transcriptomic landscape during tumor evolution. Citation Format: Michelle Dietzen, Haoran Zhai, Olivia Lucas, Sophia Ward, Yanping Guo, Wei Ting-Lu, Oriol Pich, Simone Zaccaria, Charles Swanton, Nicholas McGranahan, Nnennaya Kanu. Investigating the role of altered replication timing during tumor evolution in lung and breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3620.