Abstract NG16: Double PIK3CA mutations in cis increase oncogenicity and sensitivity to PI3Kα inhibitors

PIK3CA is the most frequently mutated oncogene across all human cancers, and codes for p110α, the catalytic subunit of the PI3K complex. PI3K catalyzes the phosphorylation of the lipid PIP2 to PIP3, which initiates a downstream signaling cascade involving the activation of AKT and mTOR. Hotspot E545K and H1047R mutations constitutively activate PI3K and are oncogenic in multiple cancer histologies including breast cancer, where PIK3CA mutations are present in 40% of tumors and are a target for cancer therapy. Recently, the PI3Kα inhibitor alpelisib has demonstrated improved PFS in patients with ER+ PIK3CA mutant metastatic breast cancer on a phase 3 randomized clinical trial, resulting in its FDA approval. In early clinical trials, we observed a population of patients with displayed prolonged clinical benefit to alpelisib, with double PIK3CA mutant tumors. This prompted us to undertake a comprehensive analysis of the prevalence of multiple PIK3CA mutations and to investigate their potential biological relevance and correlation with sensitivity to PI3Kα inhibitors.We analyzed multiple independent primary and metastatic tumor cohorts and found that 10-15% of all PIK3CA mutant tumors across all cancer histologies contain multiple PIK3CA mutations, the vast majority of which carry exactly two mutations. Double mutations occur at specific amino acid positions by codon enrichment analysis, where the most frequent combinations in breast cancer are comprised of a canonical “major mutant” hotspot (involving either E542, E545, or H1047) combined with a second “minor mutant” site (involving either E453, E726, or M1043). Double PIK3CA mutations are enriched in ER+/HER2- breast cancers and occur at similar frequencies in therapy-naïve primary tumors and metastatic tumors. To study the allelic configuration of double mutations, we leveraged single molecule real time sequencing (SMRT-seq) on fresh breast tumor samples from patients known to carry two PIK3CA mutations in their tumors by NGS. Six tumors representative of the most frequent double mutants in breast cancer were obtained from patients, and were analyzed by SMRT-seq. All contained double mutations in cis. We overexpressed double cis mutants and constituent single mutants in nontransformed and ER+ breast cancer cells. Double PIK3CA mutations in cis increased downstream PI3K pathway signaling and cell/tumor proliferation in vitro and in vivo, when compared to single mutations. In contrast, mutations in trans do n