Abstract RF4-05: <i>PIK3R1</i> (p85α) alterations define a targetable subset of breast cancer with broad sensitivity to PI3K and AKT inhibitors

Abstract Background: PI3K pathway alterations occur in ∼40% of estrogen receptor-positive metastatic breast cancer (ER+ MBC). These form the basis for the approved PI3Kα inhibitors alpelisib and inavolisib in patients with PIK3CA-altered tumors, and the AKT inhibitor capivasertib in patients with PIK3CA, AKT1, or PTEN-altered tumors. On-target toxicities such as hyperglycemia and rash limit PI3Kα and AKT inhibitor use, and next-generation mutant-selective PI3K inhibitors (e.g., STX-478, RLY-2608) are showing promising clinical activity with reduced metabolic toxicity. We previously demonstrated that PIK3CA double mutations confer heightened PI3Kα inhibitor sensitivity (Vasan et al., Science 2019), underscoring the predictive potential of novel genomic alterations. PIK3R1 encodes the p85α regulatory subunit of PI3Kα, which restrains p110α activity, yet its role in breast cancer is poorly defined. Notably, patients with PIK3R1-altered ER+ MBC were excluded from registrational trials of PI3K and AKT inhibitors and are currently ineligible for these therapies. We sought to define the biological and therapeutic relevance of PIK3R1 mutations in breast cancer. Methods and Results: We analyzed targeted sequencing data from the largest cohort of breast cancer patients to undergo comprehensive genomic profiling in routine clinical care (n = 51,574) and identified PIK3R1 alterations in 3.2% of tumors (n=1,662), evenly distributed between ER+ and ER- disease. Nearly half (49%) of these alterations were in-frame multi-residue insertions and deletions (indels), primarily clustered within the inter-SH2 (iSH2) region, which directly interacts with the adaptor binding domain and C2 domain of p110α. Truncating (20%) and missense (7%) mutations were also frequent. In MCF10A breast epithelial cells, PIK3R1 mutations induced two tiers of increased growth relative to wild-type: a moderate effect from single amino acid substitutions and a stronger effect from multi-residue indels. These two tiers of increased growth were paralleled by similarly graded increases in downstream PI3K signaling. Across cancer cell lines (Cancer Cell Line Encyclopedia) and patient tumors (The Cancer Genome Atlas), PIK3R1 alterations were associated with elevated PI3K pathway activity by phosphoproteomic analysis. Biochemically, PIK3R1 iSH2 alterations increased lipid kinase activity and membrane binding. Phosphopeptides did not appreciably enhance lipid kinase activity, mimicking the activation mechanism of p110α adaptor binding domain and C2 domain mutations, which relieve p85α-mediated inhibition. Strikingly, PIK3R1 mutations conferred pan-sensitivity to active-site PI3Ki (alpelisib, inavolisib), AKTi (capivasertib), and mutant-selective PI3Ki (STX-478, RLY-2608) in vitro. Additional biochemical data, structural studies using hydrogen-deuterium exchange mass spectrometry (HDX-MS), and in vivo data will be presented. Conclusion: This is the first comprehensive study of PIK3R1 alterations in breast cancer. Our findings establish PIK3R1 as a functional driver of oncogenic PI3K signaling and show that PIK3R1 alterations confer sensitivity to both established and investigational PI3K and AKT inhibitors. These findings nominate PIK3R1 alterations as an actionable genomic biomarker and support the inclusion of patients with PIK3R1-altered breast cancer in clinical trials testing PI3K and AKT inhibitors. Citation Format: S. Fernando, S. Sisoudiya, I. Barlow-Busch, J. Tao, M. Fine, M. Cohen, S. Sivakumar, E. Sokol, J. Burke, N. Vasan. PIK3R1 (p85α) alterations define a targetable subset of breast cancer with broad sensitivity to PI3K and AKT inhibitors [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr RF4-05.