Mutations in the PI3K pathway are prevalently observed in luminal A-type breast cancer, characterized by HR positivity and HER2 negativity. While PI3K inhibitors, such as alpelisib, are FDA-approved for the treatment of PIK3CA-mutant breast cancers, PI3K pathway inhibition can activate compensatory signaling, leading to drug resistance. The objective of this study is to identify actionable resistance mechanisms associated with the FDA-approved combination of alpelisib and fulvestrant in the treatment of advanced-stage PIK3CA-mutant breast cancer. Transcriptomic analysis reveals activation of FGFR signaling as a key mechanism of resistance. We demonstrate that FGFR inhibition synergizes with alpelisib to inhibit tumor growth in alpelisib/fulvestrant-resistant cell lines, 3D tumor spheroids, in vivo xenografts, and patient-derived xenograft models.

MCF7 and T47D cell lines with acquired resistance to the alpelisib/fulvestrant combination were generated and subjected to comparative transcriptome analysis to identify potential resistance networks. Biochemical analysis was used to validate key components of the resistance-associated signaling pathways. To elucidate the functional impact of FGFR-dependent resistance mechanisms, resistant cells, 3D tumor spheroids, in vivo xenografts, and PDX models were treated with combinations of alpelisib and FDA-approved pan-FGFR inhibitors, erdafitinib and pemigatinib, or the FGFR2-specific inhibitor lirafugratinib.

Transcriptomic analysis revealed overactivation of FGFR signaling as a key bypass mechanism of resistance. Immunoblotting confirmed the activation of FGFR and downstream MAPK pathways in resistant cells. Functional studies demonstrated synergistic growth-inhibitory effects of pemigatinib and lirafugratinib, when combined with alpelisib in alpelisib/fulvestrant-resistant cell lines and 3D tumor spheroids. Concurrent inhibition of the FGFR pathway and p110α led to sustained tumor regression in drug-resistant tumor xenografts and PDX models in vivo.

Overall, our study identifies FGFR signaling as a key mediator of resistance to combined alpelisib and fulvestrant treatment in PIK3CA-mutant luminal A breast cancer. These findings suggest that targeting the FGFR pathway in combination with PI3K inhibition may represent a potential strategy to overcome drug resistance in advanced-stage refractory luminal A breast cancer.