Abstract Epidermal growth factor receptor (EGFR) is overexpressed in up to 90% of head and neck squamous cell carcinoma (HNSCC) tumors. Cetuximab is the first targeted (anti-EGFR) therapy approved for the treatment of patients with locally advanced HNSCC. However, its efficacy is limited due to primary and secondary resistance, and there is no predict biomarkers of response. New generation of EGFR inhibitors with pan HER targeting and irreversible action, such as afatinib and allitinib represents a significant therapeutic promise in HNSCC treatment. In this study, we intend to compare the potential cytotoxicity of two irreversible anti-EGFR inhibitors (afatinib and allitinib) with cetuximab and to identify potential predictive biomarkers of response in a panel of HNSCC cell lines. The mutational analysis in hotspot regions of EGFR, KRAS, NRAS, PIK3CA and PTEN in the eight HNSCC cell lines revealed an EGFR mutation (p.H773Y) and gene amplification in the HN13 cells, and KRAS mutation in the JHU-28 cell line. According to the growth inhibition score (GI), allitinib was the most cytotoxic drug, followed by afatinib and finally cetuximab. The HN13 cell line exhibited a less responsive behavior to all drugs assessed, and interestingly, also displayed the higher levels of AKT phosphorylation. Therefore, we further performed drug combinations and found that with anti-AKT agent (MK2206), afatinib and allitinib, but not cetuximab, sensitivity was restored. Additionally, AKT1gene editing in afatinib and allitinib-treated cell lines showed that AKT1 silencing-induced cell line decreased viability and increased cytotoxicity through caspases 3/7activation.Additionally, in silico analysis of TCGA database showed that AKT1 overexpression was present in 14.7% (41/279) of HNSCC cases, and was associated with perineural invasion in advanced stage. In conclusion, allitinib presented a greater cytotoxic profile when compared to afatinib and cetuximab, and the AKT pathway constitutes a predictive marker of allitinib response. Importantly, in allitinib and afatinib-resistant cases, the pharmacological combination with AKT inhibitors could restore response and increase treatment success. Citation Format: Renato José Silva-Oliveira, Matias Melendez, Olga Martinho, Maicon Fernando Zanon, Luciano de Souza Viana, André Lopes Carvalho, Rui Manuel Reis. AKT can modulate the in vitro response of HNSCC cell to irreversible EGFR inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4091. doi:10.1158/1538-7445.AM2017-4091