Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer globally with more than 58,000 new cases in the United States each year. HNSCC frequently overexpress the epidermal growth factor receptor (EGFR), and this receptor tyrosine kinase mediates cell survival signaling and resistance to therapy. Therapeutic inhibition of the EGFR improves patient outcomes, and the EGFR remains the only validated molecular target for this disease. Despite the success of EGFR targeted therapies such as cetuximab, therapeutic resistance to EGFR targeting ultimately develops. A significant challenge for improving EGFR targeted therapies is to identify and clinically validate actionable mechanisms of therapeutic resistance. In this proposal, we have designed a strategy that iterates between basic science investigations, preclinical testing, and clinical specimen testing to elucidate the mechanism of cetuximab resistance in HNSCC. Using an in vitro approach for analyzing cetuximab resistance, we identified upregulation of an autocrine ligand, NRG-1, as a target mechanism of resistance. We have modeled this resistance both in cell lines and in vivo, using mouse xenograft studies, and have shown that it can be reversed therapeutically by using an ErbB3-targeted antibody therapeutic (CDX-3379) - which can restore responses to cetuximab and radiation therapy. We have also observed NRG-1-induced resistance to small molecule EGFR kinase inhibitors in cancer cells, and have studied the mechanistic origin of this resistance at a structural level. Importantly with this approach we seek to model the effects of EGFR inhibition in the setting of the relatively frequent PIK3CA mutations. We propose to exploit the knowledge gained from these studies to advance small molecule approaches for targeting or improving the targeting of EGFR family members in HNSCC. In parallel with these studies, we will study clinical specimens from an ongoing phase II HNSCC trial of afatinib plus cetuximab, plus two ECOG trials of cetuximab, to investigate resistance mechanisms in the clinic. We will also develop patient-derived xenografts (POX) models from the ongoing clinical trial to test hypotheses for resistance mechanisms and to assess effectiveness of new strategies devised to overcome it. In this post-baccalaureate training proposal, we seek to expand our studies into the mechanisms that underlie therapeutic resistance to cetuximab through modeling the impact of enhanced PIK3CA signaling. Using established CRISPR-Cas9 gene knockout models in two HNSCC cell lines, we will first define the effects of loss of PTEN (negative regulator of Pl3KCA) on activation of the EGFR receptor and its downstream signaling cascades. A second component of this proposal will evaluate altered drug sensitivities in the setting of activation Pl3KCA mutations as a therapeutic maneuver to overcome this mechanism of therapeutic resistance. Successful completion of these goals will provide a rigorous...