ARTICLE ABSTRACTPurpose: Activating mutations in the phosphoinositide-3-kinase (PI3K)/AKT/mTOR pathway are present in the majority of breast cancers and therefore are a major focus of drug development and clinical trials. Pathway mutations have been proposed as predictive biomarkers for efficacy of PI3K-targeted therapies. However, the precise contribution of distinct PI3K pathway mutations to drug sensitivity is unknown.Experimental Design: We describe the creation of a physiologic human luminal breast cancer cell line model to study the phenotype of these mutations using the MCF-7 cell line. We used somatic cell gene targeting to “correct” PIK3CA E545K-mutant alleles in MCF-7 cells to wild-type sequence. The AKT1 E17K hotspot mutation was knocked in on this wild-type background.Results: Loss of mutant PIK3CA dramatically reduced phosphorylation of AKT proteins and several known AKT targets, but other AKT target proteins and downstream effectors of mTOR were not affected. PIK3CA wild-type cells exhibited reduced proliferation in vitro and in vivo. Knockin of the AKT1 E17K hotspot mutation on this PIK3CA wild-type background restored pathway signaling, proliferation, and tumor growth in vivo. PIK3CA, but not AKT1 mutation, increased sensitivity to the PI3K inhibitor GDC-0941 and the allosteric AKT inhibitor MK-2206.Conclusions:AKT1 E17K is a bona fide oncogene in a human luminal breast cancer context. Distinct PI3K pathway mutations confer differential sensitivity to drugs targeting the pathway at different points and by distinct mechanisms. These findings have implications for the use of tumor genome sequencing to assign patients to targeted therapies. Clin Cancer Res; 19(19); 5413–22. ©2013 AACR.