The overarching goal of this proposal is to develop precision therapy for PIK3CA-mutant colorectal cancer (CRC). Phosphatidylinositol 3-kinases (PI3K) are heterodimers consisting of a p110 catalytic subunit and a p85 regulatory subunit. The PI of this application co-discovered that PIK3CA, which encodes p110α, is frequently mutated in a variety of human cancers, including ~30% of CRC. Most PIK3CA/p110α oncogenic mutations occur at two hot spot regions, one in the helical domain and the other in the kinase domain. Nearly half of all p110α mutations are located in the helical domain. Increasing evidence suggests that the helical domain and kinase mutations exert their oncogenic function through distinct mechanisms. For the helical domain mutations, we discovered that the oncogenic signal is transduced by two unique pathways. We previously found that the p110α helical domain mutant protein directly associates with IRS1 independent of p85 to activate PI3Kα-AKT. Now, our preliminary studies demonstrate that p85β disassociates from the p110α helical domain mutant protein complexes and translocates into the nucleus. The nuclear p85β stabilizes EZH1 and EZH2, two enzymes that catalyze histone H3K27 trimethylation. Remarkably, we found that a combination of EZH inhibitor GSK126 with a p110α-specific inhibitor Alpelisib induced tumor regression of CRCs harboring a PIK3CA helical domain mutation. Thus, we hypothesize that nuclear p85β promotes oncogenic functions of p110α helical domain mutations and that simultaneous inhibition of both nuclear p85β function and p110α kinase will be an effective approach to treat tumors with a helical domain mutation. Two aims are proposed to test the central hypothesis: 1) elucidate the mechanisms by which nuclear p85β promotes oncogenic functions of PIK3CA helical domain mutations; and 2) determine the efficacy of the combination of GSK126 and Alpelisib using a panel of CRC patient-derived xenografts with a PIK3CA helical domain mutation. Successful completion of our studies will demonstrate the combination of GSK126 and Alpelisib as an effective treatment for CRCs with PIK3CA helical domain mutations in preclinical models and lay a solid foundation for future clinical trials. Moreover, our studies uncover p85β nuclear translocation as a novel mechanism by which PIK3CA helical domain mutations exert their oncogenic functions.