Abstract C035: Arrayed CRISPR Screening Reveals EFNA1 as a Non-Cell-Autonomous Essential Gene in Pancreatic Cancer Cells

Abstract Traditional pooled genetic dependency screens, such as DepMap, have proven powerful for identifying essential genes in cancer cell lines. However, these pooled formats are prone to the bystander effect, where secreted factors from wild-type cells support the survival of knockout (KO) cells within the same culture, potentially masking dependencies on specific secreted proteins. To address this limitation, we performed a well-by-well CRISPR screen targeting genes encoding secreted factors in the pancreatic ductal adenocarcinoma (PDAC) cell line HPAFII. This arrayed format eliminates cross-feeding and enables precise assessment of cell-intrinsic dependencies on targeted proteins and could thus expose hidden vulnerabilities. CellTiter-Glo (CTG) viability assays were used to assess gene lethality following target gene KOs. Furthermore, RNA sequencing verified effective disruption of target genes, establishing the specificity and reliability of the screening workflow. We observed strong growth inhibition in EFNA1 (Ephrin-A1) KO samples compared to lipofectamine-treated controls. However, this inhibitory effect was missed and underrepresented in the conventional pooled format. EFNA1 is a ligand for EPH receptors, involved in cell-cell communication, migration, and proliferation signaling. Differential gene expression analysis comparing EFNA1 KO to lipofectamine-treated controls revealed enriched pathways of oxidative phosphorylation, E2F targets, MYC targets, and DNA repair in EFNA1 KO samples. This result supports our observation of lethality in EFNA1 KO samples. It suggests that EFNA1 contributes to cell-cell communication and is critical for HPAFII cell survival. Its KO triggers stress pathways, including E2F, MYC, OXPHOS, and DNA repair, and acts as a suppressor of excessive proliferative programs. In conclusion, we identified EFNA1 as a critical non-cell-autonomous essential gene through an arrayed CRISPR screen, with its loss showing lethality to HPAFII cells exclusively in isolated settings, likely as a result of disrupted cell-cell interactions. Defining such dependencies may facilitate the discovery of directly druggable and more tumor-selective therapeutic strategies. Citation Format: Xingyi Shi, David Ruddy, Millicent Gabriel, Michelle Piquet, Kris Muskiewicz, Joel Wagner, Joshua Korn, Cory Johannessen. Arrayed CRISPR Screening Reveals EFNA1 as a Non-Cell-Autonomous Essential Gene in Pancreatic Cancer Cells [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 C035.