- The phosphatidylinositol 3-kinase (PI3K) pathway is deregulated across a broad spectrum of human cancers. Therefore, there have been strong efforts to inhibit this pathway, with many novel targeted agents, including isoform-specific PI3K inhibitors, pan-PI3K inhibitors, and dual PI3K/mTOR inhibitors, entering the clinic. Although initial responses have been observed in PIK3CA and HER2 amplified cancers, resistance frequently emerges, thereby limiting the benefit from these drugs. We developed several PI3K inhibitor resistant cell lines by culturing sensitive PIK3CA mutant cells in gradually escalating doses of PI3K inhibitor until resistance emerges, typically over a period of several months. We then used a drug screen utilizing ~40 targeted agents to identify drugs that synergize with PI3K inhibitors in the resistant cells. Subsequently, to elucidate mechanisms of resistance to PI3K inhibitors, we used a pooled shRNA screen to identify hairpins that sensitize the resistant cells to PI3K inhibition. These two complementary screens have identified mTORC as a likely mediator of resistance to PI3K inhibition: mTORC inhibitors synergize with PI3K inhibitors in the resistant cells, and furthermore, hairpins directed against mTORC (FRAP1) sensitize resistant cells to PI3K inhibition. Other promising therapeutic targets identified by these screens that may contribute to resistance to PI3K inhibitors include IGF1R and AKT. This abstract is also presented as Poster B069. Citation Format: Sadhna Vora, Moshe Elkabets, Nayoon Kim, Carlotta Costa, Manway Liu, Li Xiaoyan, Alex Cao, Robert Schlegel, Maurizio Scaltriti, Jose Baselga, Alan Huang, Jeffrey A. Engelman. Overcoming resistance to PI3K inhibitors in breast cancer: Results of a pooled shRNA screen and combinatorial drug screen. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr PR06.