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PIK3CA, encoding the active alpha isoform of PI3K, is the second most commonly mutated gene in cancer leading to aberrant PI3K/AKT/mTOR signaling and increased protein translation, glucose metabolism, cellular proliferation, survival and migration. Some 4-25% of gastric cancers (GC) display activating PIK3CA mutations including 80% of the EBV-associated GC subset. Small molecules including pan-PI3K, dual PI3K/mTOR, and pan-AKT inhibitors have shown only moderate clinical success, primarily due to high toxicity and off target effects. Even isoform specific PI3K inhibitors, such as PI3Kα inhibitors, have displayed similar clinical problems. Importantly, PI3Kα mutation-selective inhibitors are on the horizon and will be key for treatment of PIK3CA mutant tumors. However, single agent drug resistance is still an anticipated clinical problem and both intrinsic and acquired resistance will affect success of new drugs. There has been a concerted effort to define mechanisms of resistance as well as identify synergistic drug combinations. In this study we aimed to identify mechanisms of sensitivity and resistance to the PI3Kα-specific inhibitor, BYL719, in PIK3CA mutant gastric cancers by using a CRISPR/Cas9-based screening approach. We found that loss of either NEDD9 or BCL-XL was synergistic with BYL719 and led to increased cell cycle arrest and apoptosis, respectively. In addition to identifying these sensitizer genes, the screening approach allowed us to model intrinsic resistance, and we discovered that knockout of the translation and transcription factor, CBFB, conferred resistance to BYL719. To model acquired resistance, we cultured cells in increasing concentrations of BYL719 over three months and generated clones that were resistant when compared to parental cells. We found that in the clones with acquired resistance, CBFB was significantly down-regulated at the protein and RNA level suggesting a conserved mechanism of drug resistance. We found that CBFB loss led to up-regulation of the serine/threonine protein kinase PIM1, which can phosphorylate and activate several of the same downstream substrates as AKT thereby maintaining pathway activity in the presence of PI3Kα inhibition. Furthermore, a pan-PIM inhibitor re-sensitized both CBFB knockout and acquired resistant cells to BYL719. In the TCGA clinical dataset, we found that CBFB is over-expressed in gastric tumors, specifically in the EBV-associated subset, compared to normal tissue and is significantly over-expressed in PIK3CA mutant compared to wild type tumors. Consistently, we found that PIM1 expression displays the inverse phenotype. These data suggest that while CBFB loss is a key step in the development of PI3Kα inhibitor resistance, PIK3CA mutant gastric tumors may be good candidates for clinical success with BYL719 alone and resistance could be prevented with combination with a PIM kinase inhibitor. Citation Format: Lyla Stanland, Hazel Ang, Jacob Hoj, Yunqiang Chu, Patrick Tan, Kris Wood, Micah Luftig. Identification of novel regulators of response to PI3Ka inhibition in PIK3CA mutant gastric cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4949.