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Head and neck squamous cell carcinoma (HNSCC) lacks predictive biomarkers for drug responses. By targeted sequencing, we identified two  MAPK1  mutations in recurrent HNSCC,  MAPK1 p.D321N, and p.R135K. We previously reported an exceptional erlotinib responder with  MAPK1 p.E322K. Here, by in silico and drug studies, we determined functions of these two recurrence-associated  MAPK1  mutations. Residues D321, R135, and E322 are in 3D proximity.  MAPK1 p.D321N drives marked in vivo erlotinib sensitivity, while p.R135K’s effect is moderate.

Erlotinib sensitivity of MAPK1p.D321N mutation in head and neck squamous cell carcinoma