Triple-negative breast cancer (TNBC) is an aggressive breast cancer (BC) subtype lacking hormone receptor (HR) and HER2 expression, making it unresponsive to conventional therapies and necessitating novel targeted strategies. The PI3K/AKT signalling pathway is frequently dysregulated in TNBC, with PIK3CA mutations (~15% of cases) promoting uncontrolled proliferation. Alpelisib, a PI3K/AKT inhibitor, is approved for PIK3CA-mutant, HR-positive BC, but resistance mechanisms remain. We hypothesise that combining alpelisib with approved drugs targeting alternative PI3K/AKT activation mechanisms may benefit a TNBC cohort.

Differential expression analyses (DEAs) based on TP53 and PIK3CA mutations were performed on TCGA BC patient RNA-seq data (N=981) using R. The 5-day anti-proliferative effects of targeted drugs, alone and in combination, were assessed using acid phosphatase assays. Reverse-phase protein array (RPPA) and western blotting were performed on lysates from 24-hour-treated cells. Mutant p53-expressing cell lines were transduced with lentivirus to express mutant PIK3CA.

DEAs showed TP53 contributes to genetic dysregulation in TNBC, with distinct patterns in cases harbouring both TP53 and PIK3CA mutations. TP53 mutations downregulate PTEN, a negative regulator of PI3K, leading to aberrant PI3K/AKT activation. We hypothesised that TP53/PIK3CA co-mutations contribute to alpelisib resistance. The TP53/PIK3CA-mutant MFM-223 cell line was less sensitive to alpelisib than PIK3CA-only mutated TNBC cells but more sensitive than TP53-only mutated cells, suggesting PIK3CA mutations improve response while TP53 mutations drive resistance via sustained PI3K/AKT activation. Dual PI3K/AKT targeting could overcome this. Given frequent receptor tyrosine kinase (RTK) dysregulation in TNBC, we tested the approved RTK inhibitor crizotinib with alpelisib, identifying a synergistic effect in TP53/PIK3CA-mutated MFM-223 cells. RPPA analysis revealed dysregulated 4E-BP1 expression in cells treated with this combination, which was validated by western blotting, confirming reduced 4E-BP1 expression and phosphorylation. As a key PI3K target, 4E-BP1 is typically phosphorylated by mTORC1, promoting translation. The alpelisib-crizotinib combination may impair translation regulation, disrupting protein synthesis and proliferation. In MCF10A lines expressing different mutant p53 proteins, alpelisib sensitivity varied by mutation, with western blotting revealing differing PI3K pathway marker expression, suggesting TP53 mutations affect PI3K activation independently of PIK3CA.

TP53/PIK3CA co-mutations drive TNBC dysregulation and may confer PI3K inhibitor resistance. The alpelisib-crizotinib combination may overcome resistance in this TNBC cohort. Future in vitro and in vivo studies will further investigate therapeutic potential.