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 not increase cell signaling or growth proliferation more than single mutations.The prevailing model of PI3K activation occurs through the engagement of the p85α regulatory subunit with phosphotyrosines on RTK signaling complexes, which relieves catalytic inhibition. Single oncogenic mutations recapitulate these events by weakening the interactions between p110α and p85α (“disrupters”), or by promoting binding to membrane (“binders”). We purified recombinant full length PI3Kα complexes containing single and double cis p110α mutations to dissect the biochemical mechanisms by which these double PIK3CA mutations in cis modulate kinase activity. We demonstrate that cis mutants have increased kinase activity compared to single mutants and that this activation is due to both increased p85α disruption and increased lipid binding, compared to single mutants.We tested whether cis mutant cells exhibit differential sensitivity to PI3Kα inhibitors. While in the absence of treatment, cis mutant signaling is increased compared to single mutants, treatment with the PI3Kα inhibitors alpelisib or GDC-0077 results in a similar PI3K pathway inhibition in nontransformed and ER+ breast cancer cells. With respect to cell viability, E545K and H1047R major hotspot mutants are more sensitive to alpelisib and GDC-0077 compared to minor mutants and WT. In turn, all cis mutants are synergistically more sensitive to alpelisib and GDC-0077 compared to single major and minor hotspots.We analyzed response data from SANDPIPER, a randomized phase 3 clinical trial that tested the efficacy of the ER degrader fulvestrant with or without the PI3Kα/γ/δ inhibitor taselisib in metastatic ER+ PIK3CA mutant breast cancer. We used ctDNA to detect the presence of PIK3CA mutations. PIK3CA mutant patients on the taselisib arm had an objective response rate (ORR) of 20.3% vs 9.7% compared to the placebo arm, which was statistically significant, confirming that PIK3CA mutation predicts response to PI3Kα inhibition. We then compared responses of patients with single vs multiple mutations. Single mutant patients on the taselisib arm had an ORR of 18.1% vs 10.0% compared to the placebo arm, which was not statistically significant. On the contrary, multiple mutant patients on the taselisib arm achieved an ORR of 30.2% vs 8.7% compared to the placebo arm, which was statistically significant. These findings confirm that breast cancer patients with multiple mutant tumors achieve higher clinical benefit to PI3Kα inhibition compared to single mutant tumors.In this work, we have identified double mutations in cis as a novel, and relatively frequent, genomic alteration in PIK3CA, which translates into a clinically meaningful number of patients who may derive additional benefit from targeted therapy. Our results implicate a model of oncogene addiction to double mutant PIK3CA in breast cancer, with graded levels of activation and inhibition for single vs double mutants. PI3Kα inhibitors are now a standard of care in PIK3CA mutant ER+ metastatic breast cancer and are being explored in other PIK3CA mutant tumor histologies. Our findings provide a rationale for testing whether patients with multiple PIK3CA mutant tumors are exquisitely sensitive to PI3Kα inhibitors.