Abstract The epidermal growth factor receptor (EGFR) is a central regulator of cell proliferation and tumor progression in squamous cell carcinomas of the head and neck (HNSCC). Cetuximab, a monoclonal blocking antibody against EGFR, gained FDA approval in 2006 for use in combination with radiation therapy in HNSCC or as a single agent in patients who have had prior platinum-based therapy. However, long-term response to cetuximab treatment is only observed in a limited number of patients, which prompted the search for the mechanisms underlying cetuximab resistance and the exploration of new therapeutic options to enhance cetuximab effectiveness in HNSCC. Here, we identified cetuximab-sensitive HNSCC cells lines and retro-engineered them by introducing additional pathway alterations to define potential compensatory mechanisms that might exist in cetuximab-treated HNSCC patients. These include mutations and amplification of PI3K and activating Ras mutations, both of which occur frequently in HNSCC and can bypass the requirement of EGFR signaling for cell proliferation. Indeed, active PI3K- and Ras-expressing tumors were cetuximab-resistant; they exhibited an initial anti-tumor response but relapsed within few weeks of treatment initiation. Interestingly, in these resistant tumors cetuximab did not decrease the activity of mTOR, a pathway acting downstream of EGFR, PI3K, and Ras that is persistently activated in HNSCC. However, these tumors were highly sensitive to mTOR inhibition with rapamycin and RAD001, which caused rapid tumor regression and decreased mTOR activity in HNSCC tissues. Furthermore, multiple cancer-associated cytokines declined after cetuximab treatment in responding HNSCC xenografts but not in resistant tumors; the circulating levels of these cancer-associated cytokines were reduced rapidly by mTOR inhibition. We provide evidence that mTOR inhibition can cooperate with cetuximab treatment to prevent tumor recurrence, and thus mTOR inhibitors may represent a suitable treatment option for patients acquiring cetuximab resistance, such as in patients exhibiting genetic or epigenetic alterations causing cetuximab-insensitive mTOR activity. In addition, we provide evidence that cancer-associated cytokine expression levels may serve as biomarkers to monitor the clinical response to cetuximab and mTOR inhibitors. Ultimately, these efforts may contribute to identify potential predictors of cetuximab response and resistance, and novel molecular targeted therapeutic options affording a better ‘personalized’ treatment of HNSCC patients. Citation Format: Zhiyong Wang, Daniel Martin, Vyomesh Patel, Alfredo Alberto Molinolo, Qianming Chen, J.Silvio Gutkind. mTOR inhibition can overcome cetuximab resistance in head and neck cancer. [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 LB-224. doi:10.1158/1538-7445.AM2013-LB-224