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

作者：Fabiana Napolitano, Y Wang, Dan Ye, Jingxuan Lu, Jingxuan Chen, Khushi Ahuja, Pamela Luna, Yuki Matsunaga, Dylan Calhoon, M Rosario Chica-Parrado, Yasuaki Uemoto, Javier García Bermúdez, J. J. Lee, Chang-Ching A. Lin, Ariella B. Hanker, Carlos L. Arteaga · 发表于：Cancer Research · 年份：2026 · DOI：10.1158/1538-7445.am2026-5759 · 研究领域：PI3K/AKT/mTOR signaling in cancer、Cancer Genomics and Diagnostics、CRISPR and Genetic Engineering

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 c...