Abstract 5759: Genome-wide CRISPR screen identifies GPX4 as a potential vulnerability in cells treated with PI3Kα-mutant selective inhibitor RLY-2608

Abstract Background/Objectives: Approximately 40% of HR+ breast cancers harbor PIK3CA mutations. The clinical efficacy of PI3Kα inhibitors has been limited by toxicity and a narrow therapeutic window. Recently, mutant-selective PI3Kα inhibitors, such as RLY-2608 (zovegalisib), have shown improved efficacy and tolerability. Here, we aimed to identify genetic modulators of response to RLY-2608 in PIK3CA-mutant breast cancer cells. Methods: A genome-wide CRISPR-knockout (KO) screen (80,000 sgRNAs targeting ∼20,000 genes) was performed in T47D cells to identify genes whose loss sensitizes to PI3Kα inhibition. Key candidates were validated with CRISPR-Cas9, and RNA-seq was used to assess transcriptomic changes upon treatment. Results: Among enriched sgRNAs in vehicle-treated controls, PTEN, NF2, TSC2, and TSC1 showed the highest differential scores, consistent with known resistance mechanisms to PI3K inhibitors, therefore validating the screening’s robustness. Among the top depleted genes in RLY-2608-treated cells - potentially associated with increased sensitivity to PI3Kα inhibition - was GPX4, encoding the antioxidant enzyme glutathione peroxidase 4. GPX4 protects cells from lipid peroxidation and ferroptosis (iron-dependent cell death). Stable GPX4-KO MCF7 and T47D cells, established via CRISPR-Cas9, displayed a 2.5-3-fold increased sensitivity to RLY-2608, respectively. Bliss independence analysis revealed strong synergy between RLY-2608 and the GPX4 inhibitor RSL3. In T47D cells treated with RLY-2608, transcriptome profiling revealed broad upregulation of lipid-associated genes, including enzymes involved in fatty acid β-oxidation, lipid mobilization and membrane lipid metabolism. These data suggest a shift toward enhanced lipid turnover and oxidation. Ongoing BODIPY-C11 and lipidomic analyses aim to quantify resulting lipid peroxidation. Compared to cancer cells without PI3K pathway mutations, PIK3CA-mutant cells exhibit suppressed xCT-mediated cystine uptake to preserve NADPH for lipid synthesis. PI3Kα inhibition relieves this suppression, increasing cystine availability; however, the accompanying induction of lipid metabolic and oxidative pathways increases lipid peroxide burden. Thus, we speculated that treatment with a PIK3CA-mutant inhibitor increases dependence on the GPX4-mediated antioxidant system, creating a therapeutically exploitable vulnerability via combination with GPX4 inhibitors. Conclusions: Genome-wide CRISPR KO screen identified GPX4 inhibition as a vulnerability in HR+/PIK3CA-mutant breast cancer cells treated with the PI3Kα-mutant selective inhibitor RLY-2608. These data support a mechanistic link between PI3Kα inhibition, lipid peroxidation, and ferroptosis, providing a rationale for clinical trials with the combination of PI3K pathway and GPX4 inhibitors. Citation Format: Fabiana Napolitano, Yuan Wang, Dan Ye, Jingxuan Lu, JINGXUAN CHEN, Khushi Ahuja, Pamela Luna, Yuki Matsunaga, Dylan Calhoon, María Rosario Chica-Parrado, Yasuaki Uemoto, Javier Garcia Bermudez, Jeon Lee, Chang-Ching A. Lin, Ariella B. Hanker, Carlos L. Arteaga. Genome-wide CRISPR screen identifies GPX4 as a potential vulnerability in cells treated with PI3Kα-mutant selective inhibitor RLY-2608 abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5759.