Epidermal Growth Factor Receptor (EGFR) expression is upregulated in the majority of head and neck squamous cell carcinomas (HNSCC) and is associated with poor clinical outcomes. Unfortunately, the incorporation of EGFR inhibitors (EGFRIs) into the management of HNSCC has not improved long term survival rates in HNSCC patients despite high EGFR expression. Additionally, EGFR tyrosine kinase inhibitors (TKIs) in particular, have failed to show clinical benefit for HNSCC patients in clinical trials. Therefore, the identification and understanding of strategies that will improve the efficacy of EGFRIs may improve patient treatment and survival. Previous work in our lab has shown that HNSCC cells treated with erlotinib, a first-generation EGFR TKI, increased expression and release of the proinflammatory cytokine interleukin-1 alpha (IL-1alpha) resulting in inflammation and the development of drug resistance. The goal of this study is to determine if IL-1alpha-induced drug resistance is maintained across second, third and fourth generation EGFR TKIs.EGFR wildtype-positive HNSCC cells (FaDu, Cal-27 and SQ20B) were treated with the IC 50 doses of erlotinib (first generation), afatinib (second generation), osimertinib (third generation), and silevertinib (fourth generation) EGFR TKIs for 48 h. Cell viability responses to EGFR TKIs were assessed using MTT assays. IL-1alpha and IL-6 cytokine expression was detected by ELISA and Western blot. Manipulation of IL-1 expression and signaling was performed using neutralizing antibodies to human IL-1alpha antibody (nIL-1alpha) and IL-1beta (nIL-1beta), and the human IL-1 receptor antagonist - anakinra. Results showed that all four EGFR TKIs triggered the release of IL-1alpha but not IL-1beta from all 3 HNSCC cell lines. Additionally, all four EGFR TKIs increased IL-6 (IL-1alpha signaling endpoint) release, which was suppressed by nIL-1alpha and anakinra (but not nIL-1beta) implying that IL-1alpha, rather than IL-1beta, acts upstream of IL-6 activation. Overexpression of IL-1alpha in Cal-27 cells resulted in resistance to all four generations of EGFR TKIs. Lastly, erlotinib-resistant Cal 27 and SQ20B cells also demonstrated resistance to all other generations of EGFR TKIs. Collectively, these findings identify IL-1alpha expression as a central mediator of poor response and resistance to all EGFR TKIs via an EGFR mutation-independent mechanism. Hence, targeting IL-1alpha expression/signaling could be a promising strategy to improve long term tumor response to EGFR TKIs in HNSCC.