Abstract 1169: <i>FAT1</i> alterations contribute to chromosomal instability in cancer cells

Abstract Background Chromosomal instability (CIN) is pervasive during cancer evolution, particularly in non-small cell lung cancer (NSCLC) where it is associated with poor recurrence-free survival and correlates with the frequency of whole genome doubling events (WGD). By duplicating the complete set of chromosomes, WGD is a key event during cancer evolution and correlates with poor prognosis and targeted therapy resistance. Despite the importance of these events, genetic events responsible for the initiation and maintenance of CIN and WGD in NSCLC have not been systematically investigated. Methods TRAcking Cancer Evolution through Therapy/Rx (TRACERx) is a multicancer longitudinal study which utilizes multiregional sampling and whole exome sequencing (WES) to elucidate potential causes of tumor heterogeneity and cancer progression. TRACERx enables us to investigate genetic alterations and their relationship with WGD and CIN, and proceed to functionally characterize them. Results We identified that genetic alterations in 6 genes, including FAT1, results in homologous recombination (HR) repair deficiencies and CIN. Mutations in FAT1 are further associated with HR deficiency in patient cohorts. Using orthogonal genetic and experimental approaches, we demonstrate that FAT1 alterations are positively selected before WGD in lung cancer evolution. FAT1 ablation causes persistent replication stress, an elevated mitotic failure rate, nuclear deformation, and elevated structural CIN including chromosome translocations and radial chromosomes. Furthermore, FAT1 loss contributes to WGD, which we attribute to the dysregulation of YAP1. Co-depletion of the oncogene YAP1 partially reduces the elevated numerical CIN caused by FAT1 loss but does not relieve HR deficiencies nor structural CIN. Importantly, overexpression of constitutively active YAP15SA is sufficient to induce numerical CIN. HR deficiencies and high CIN caused by FAT1 loss synergizes with WGD to fuel tumor evolution, a prominent factor underpinning therapy resistance. Conclusions These data suggest that FAT1 loss in lung cancer attenuates HR repair and exacerbates structural and numerical CIN through two distinct downstream mechanisms, leading to increased tumor heterogeneity. Citation Format: Wei-ting Lu, Lykourgos-Panagiotis Zalmas, Chris Bailey, James R. Black, Carlos Martinez-Ruiz, Oriol Pich, Francisco Gimeno-Valiente, Ieva Usaite, Mariam Jamal-Hanjani, Nicolas Tapon, Kasper Fugger, Nicholas McGranahan, Kevin Litchfield, Nicolai J. Birkbak, Jiri Bartek, Nnennaya Kanu, Charles Swanton. FAT1 alterations contribute to chromosomal instability in cancer cells [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 1169.