Abstract 3003: Targeting mechanisms of adaptive resistance to the PI3Kαmutant selective inhibitor RLY-2608 in HR+/PIK3CA mutant breast cancer

Abstract The PIK3CA gene is mutated in ∼40% of HR+/HER2- breast cancers. Alpelisib and inavolisib are currently FDA approved for treatment of PIK3CA-mutant breast cancers, although neither is selective for mutant PIK3CA. Inhibition of wild-type PI3K results in multiple adverse events, including hyperglycemia, which limit the dose and duration of treatment. Further, inhibition of PI3K/AKT relieves feedback suppression of receptor tyrosine kinase (RTK) expression and activity, thus limiting complete inhibition of PI3K/AKT signaling in cancer cells and resulting in adaptive drug resistance. Whether PI3K mutant-selective inhibitors can more potently block this compensatory adaptive resistance is unclear. In this study, we interrogated early adaptive resistance mechanisms to the PI3Kα mutant-selective inhibitor RLY-2608 in HR+ breast cancer cells with PIK3CA mutations. Phospho-RTK arrays of MCF7 and T47D cells treated with the IC75 of RLY-2608 for 0-48h revealed feedback activation of the ERBB family of RTKs (EGFR, HER2, HER3, HER4) at 24 h. These results were confirmed by immunoblot analysis with phospho-specific antibodies. After an initial inhibition of MAPK and PI3K/AKT signaling at 6 h, we found rebound activation of pERK1/2, pAKT1, and pS6 at 24 h, which was maintained for 48 h, suggesting a potential mechanism of adaptive resistance. To identify the ERBB RTKs causing this adaptation, we tested the TKIs erlotinib, neratinib, and tucatinib, and the HER3 antibodies patritumab and zenocutuzumab, each in combination with RLY-2608. RLY-2608 combined with the pan-HER TKI neratinib and the HER2-selective TKI tucatinib most effectively reduced pERK1/2, pAKT1 S473, and pS6 at 48 h, suggesting inhibition of HER2 could block this adaptive mechanism. In short and long-term proliferation assays, neratinib and tucatinib also synergized better with RLY-2608 compared to the other inhibitors. GSEA of RNA-seq data from MCF7 and T47D cells treated with RLY-2608 for 48 h revealed upregulation of pathways related to tyrosine kinase signaling, extracellular matrix, and redox signaling. FoxO signaling was one of the top upregulated KEGG and Reactome pathways, suggesting that activation of FoxO transcriptional activity may compensate for PI3K/AKT inhibition. The Hallmark Estrogen signaling pathway was also upregulated following RLY-2608 treatment; this was validated by immunoblot analysis, showing that ERα levels increased in T47D and MCF7 cells treated with RLY-2608 over a time course. Other upregulated Hallmark pathways in both cell lines included Myogenesis, Hypoxia, and KRAS signaling down. These findings suggest that, in addition to RTK activation, adaptive resistance to RLY-2608 may involve metabolic reprogramming, increased oxidative stress, and hypoxia signaling, highlighting the need for combination strategies to overcome resistance and enhance treatment efficacy. Citation Format: Fabiana Napolitano, Chang-Ching Lin, Khushi Ahuja, Dan Ye, María Rosario Chica-Parrado, Yasuaki Uemoto, Pamela Luna, Yuki Matsunaga, Saurabh Mendiratta, Nisha Unni, Jeon Lee, Ariella B. Hanker, Carlos L. Arteaga. Targeting mechanisms of adaptive resistance to the PI3Kαmutant selective inhibitor RLY-2608 in HR+/PIK3CA mutant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3003.