Abstract B097: Structural Interpretations of High-throughput PI3K Pathway CRISPR Base-editing Data

Abstract Detailed knowledge of protein function and structure can facilitate drug discovery. Although protein active sites are the most common point of intervention by small molecule therapeutics, many targets lack a precedented, druggable site that can be selectively targeted. In these cases, it might be desirable to modulate activity by targeting a novel allosteric pocket or a region involved in a protein-protein interaction (PPI). Current structural and biochemical methods give insight into in vitro protein function, but might not reflect the activity of proteins in situ in cells, and are not readily scalable across multiple proteins. CRISPR base-editing can introduce precise base-pair C->T and A->G alterations in diverse cell types in a high-throughput manner, allowing the introduction of many protein mutations in a single assay1,2. This approach provides a route to probing protein structure-function relationships and potential points of intervention, at scale, in a physiologically relevant cell type3. In this study, we performed semi-saturating ABE and CBE mutagenesis screens on 30 proteins within the PI3K-pathway in 3 cancer cell lines to map functional residues. We show that CRISPR base-editing mutagenesis together with structural information can be used to identify relevant functional sites on PI3K-pathway proteins. We validated this approach through identification of known druggable active sites, fragment binding sites and PPIs on target proteins. Additionally, our approach supported interpretation of an intrinsically disordered conserved domain from an AlphaFold predicted structure, and we identified putative allosteric pockets in proximity to regions with no clearly identified structural function. This proof-of-concept study highlights the ability of our platform to uncover novel binding sites amenable to small molecule targeting, offering a new strategy to address traditionally undruggable proteins.CRISPR assays were performed at the Sanger Institute and computational processing, analysis and interpretation were carried out at Astex Pharmaceuticals. References 1. Coelho, M. A. et al. Base editing screens define the genetic landscape of cancer drug resistance mechanisms. Nature Genetics 1–14 (2024). 2. Coelho, M. A. et al. Base editing screens map mutations affecting interferon-γ signaling in cancer. Cancer Cell 41, 288-303.e6 (2023). 3. Picco, G. et al. Novel WRN Helicase Inhibitors Selectively Target Microsatellite-Unstable Cancer Cells. Cancer Discovery 14, 1457–1475 (2024). Citation Format: Benoit Baillif, Katrina McCarten, Barbara Abreu, Lisa Koob, Matthew Coelho, Maria Ahn, Mark Wade, John Lyons, R. Frederick Ludlow, Marcel Verdonk, Julian Esselborn, Thomas G. Davies, Mathew Garnett. Structural Interpretations of High-throughput PI3K Pathway CRISPR Base-editing Data [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B097.