Abstract 1772: PIK3CA C2 domain deletions hyperactivate PI3K, generate oncogene dependence and are exquisitely sensitive to PI3Kα inhibitors
Authors: Croessmann Sarah , Sheehan Jonathan , Sliwoski Gregory , Leelatian Nalin , He Jie , Nagy Rebecca , Balko Justin M. , Mayer Ingrid A. , Lanman Richard B. , Miller Vincent , Cantley Lewis C. , Irish Jonathan M. , Meiler Jens , Arteaga Carlos L. 
Published: July 2017

Abstract
Background: A 63 year old postmenopausal woman with advanced ER+ breast cancer resistant to endocrine therapy exhibited an exceptional response to the PI3Kα inhibitor alpelisib (BYL719) and the aromatase inhibitor letrozole (Mayer et al. Clin Cancer Res 2016). Targeted capture next generation sequence (NGS) of DNA from a liver metastasis identified a P447_L455 deletion in the C2 domain of PIK3CA. About 80% of PIK3CA activating mutations are in ‘hot spots’ within the helical and kinase domains. C2 domain mutations make up ~10% of all PIK3CA mutations in breast cancer (TCGA, Foundation Medicine) and are frequently not reported by tumor and plasma cell-free DNA NGS panels. Deletions and mutations in this domain cluster in a region encompassing amino acids 446-460 of PIK3CA. We investigated herein the functional role of PIK3CA C2 domain mutations and their response to PI3K inhibitors.
Methods: V5-tagged lentiviral vectors encoding wild type, delP447-L455, and delH450-P458 were stably transduced into MCF10A non-tumorigenic human breast epithelial cells. Cell viability and acini formation in 3D Matrigel were examined in media ± EGF or insulin or alpelisib. Markers of PI3K activation were examined by immunoblot and phosphoflow analysis. The Rosetta software suite was used to construct a structural model that would predict the change in stability of the p85/p110α complex. Physical association of p85α and p110α was determined by precipitation with V5 antibodies and immunoblot analysis.
Results: MCF10A cells stably expressing V5-tagged PIK3CAdelP447-L455 and PIK3CAdelH450-P458 exhibited EGF- and insulin-independent growth and higher phosphorylation of AKT, ERK and S6 when compared to parental MCF10A cells. In 3D Matrigel, MCF10A cells with PIK3CA C2 domain deletions formed invasive acini with increased protrusions, spindling, and bridging between acini. All these changes were ablated upon the addition of 1 µM of alpelisib whereas parental MCF10A cells were unaffected by alpelisib. We hypothesized that delP447-L455 would reduce the binding affinity of p110α with the p85 regulatory subunit of PI3K. A structural model of PIK3CAdelP447-L455 in the context of the regulatory complex revealed specific favorable inter-residue contacts that would be lost as a result of the deletion, predicting a significant decrease in binding energy. Consistent with this structural analysis, coimmunoprecipitation of p85 with V5 antibodies showed reduced binding of the C2 domain deletion mutants with p85 compared to wild type p110α.
Conclusions: These data suggest that C2 domain deletions in PIK3CA are activating mutations and generate oncogene dependence. As a result, tumors expressing these mutations are exquisitely sensitive to PI3Kα inhibitors. Thus, in addition to PIK3CA ‘hot spot’ mutations, C2 domain mutations should also be considered biomarkers of sensitivity to PI3K inhibitors.
Citation Format: Sarah Croessmann, Jonathan Sheehan, Gregory Sliwoski, Nalin Leelatian, Jie He, Rebecca Nagy, Justin M. Balko, Ingrid A. Mayer, Richard B. Lanman, Vincent Miller, Lewis C. Cantley, Jonathan M. Irish, Jens Meiler, Carlos L. Arteaga. PIK3CA C2 domain deletions hyperactivate PI3K, generate oncogene dependence and are exquisitely sensitive to PI3Kα inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1772. doi:10.1158/1538-7445.AM2017-1772