Head and neck squamous cell carcinoma (HNSCC) is a morbid and lethal malignancy where increased understanding of the genetic alterations that characterize this cancer has yet to identify predictive biomarkers to guide therapy.PIK3CA is the most commonly altered oncogene in both human papillomavirus (HPV)- negative (34% of cases) and HPV-positive (56% of cases) HNSCC. In the current funding period we found that PIK3CA mutation or amplification is a biomarker of poor prognosis in HNSCC and that only a subset of PIK3CA- mutated HNSCC tumors are sensitive to PI3K pathway inhibition. Further investigation suggested that HPV oncoproteins regulate the antitumor effects of PI3K inhibitors. We hypothesize that elucidation of the biologic impact of individual PIK3CA alterations and mechanisms of PI3K inhibitor resistance will guide therapeutic strategies to improve clinical outcomes for HNSCC patients whose tumors contain genetic alterations that activate PI3K signaling. To test this hypothesis we propose three Specific Aims. Specific Aim 1 will elucidate the protein interactome and synthetic lethal dependencies for each mutant p110α demonstrated to “drive” HNSCC survival using: a) affinity purification- mass spectrometry (AP-MS) in HPV+ and HPV- HNSCC models; and b) genetic interaction CRISPR screening. In Aim 2 we will determine the impact of targeting PI3K alone and in combination with inhibition of individual mutant p110α−interacting proteins in both immunocompetent and immunodeficient HNSCC preclinical models. Aim 3 will examine biomarkers of PI3K inhibitor resistance by analyzing paired biospecimens and PDXs developed from HNSCC patients enrolled on a window-of-opportunity trial of the p110α PI3K inhibitor BYL719. Successful completion of these studies has the potential to change clinical practice in HNSCC by providing effective treatment strategies for patients based on the specific PIK3CA mutational status of their tumor.