Abstract 5155: Gain of direct interaction with IRS1 by the helical domain mutations of p110 alpha is crucial for their oncogenic functions.

PIK3CA, which encodes the catalytic subunit of phosphoinositide 3-kinase PI3Kα (p110α), is frequently mutated in human cancers. PI3Kα consists of a catalytic subunit p110α and one of several regulatory subunits (a major one being p85α). The p110α subunit contains an N-terminal adaptor-binding domain (ABD), a Ras-binding domain (RBD), a C2 domain, a helical domain and a catalytic domain. In the basal state, the regulatory p85 subunit stabilizes the catalytic p110α subunit and inhibits its enzymatic activity. Upon growth factor stimulation, the SH2 domains of p85 bind to the phospho-tyrosine residues on the receptor protein kinases or adaptor proteins such as insulin receptor substrate 1 (IRS1), thereby activating the lipid kinase activity of PI3Kα. Activated PI3Kα converts phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-triphosphate (PIP3). The second messenger PIP3 then activates downstream AKT signaling. The majority of these mutations occur at two hot-spots located in the helical (E545K) and the kinase (H1047R) domains. Here, we report that p110α E545K mutant proteins, but not the wild-type and H1047R mutant proteins, directly associate with IRS1 and that this interaction does not require the p85 regulatory subunit of PI3Kα. Disruption of IRS1-p110α E545K specific interaction results in reduction of AKT phosphorylation and slower xenograft tumor growth of a colon cancer cell line harboring this mutation. Moreover, IRS1- p110α E545K interaction stabilizes p110α proteins and brings it cytoplasmic membrane. Our study establishes a new paradigm whereby a mutant protein exerts its oncogenic functions through rewiring an oncogenic signaling pathway. Moreover, we designed an 18 amino acid hydrocarbon-stapled peptide encompassing the p110α E545K mutation that disrupts IRS1-p110α E545K interaction. The stapled mutant peptide reduces AKT phosphorylation in cancer cell lines with a p110α E545K mutation, whereas it has no effect on those cancer cell lines harboring a p110α H1047R mutation. When injected into xenograft tumors, the stapled peptide specifically inhibits growth of tumors with a p110α E545K mutation. Therefore, our data suggest that interference of IRS1-p110α E545K interaction may be exploited for targeted therapy of cancer patients harboring this mutation. The discovery of frequent mutations of PIK3CA in human cancer provides a strong rationale for inhibition of mutated p110α activities for targeted cancer therapy. However, it has remained a challenge to develop p110α isoform-specific inhibitors. Our data suggest that disruption of the interactions between helical domain mutations of p110α and IRS1 may be exploited as a more accessible targeted therapy approach. Citation Format: Yujun Hao, Chao Wang, Weiping Zheng, David Sedwick, Sanford Markowitz, Rob Ewing, Lili Liu, Zhenghe Wang. Gain of direct interaction with IRS1 by the helical domain mutations of p110 alpha is crucial for their oncogenic functions. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5155. doi:10.1158/1538-7445.AM2013-5155