Genome-wide CRISPR screen identifies GPX4 as a potential vulnerability in cells treated with PI3Kalpha-mutant selective inhibitor RLY-2608

Background/Objectives: Approximately 40% of HR+ breast cancers harbor PIK3CA mutations. The clinical efficacy of PI3Kalpha inhibitors has been limited by toxicity and a narrow therapeutic window. Recently, mutant-selective PI3Kalpha 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 PI3Kalpha 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 PI3Kalpha 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 beta-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. PI3Kalpha 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 PI3Kalpha-mutant selective inhibitor RLY-2608. These data support a mechanistic link between PI3Kalpha inhibition, lipid peroxidation, and ferroptosis, providing a rationale for clinical trials with the combination of PI3K pathway and GPX4 inhibitors.