EGFR S442 ectodomain mutation confers cetuximab resistance that can be overcome by ERBB2 blockade with trastuzumab-deruxtecan.

Epidermal growth factor receptor (EGFR) is an oncogenic driver in multiple cancers and a therapeutic target of tyrosine kinase inhibitors and neutralizing monoclonal antibodies. However, resistance to EGFR-targeted therapies, particularly the anti-EGFR antibody cetuximab, remains a clinical challenge in colorectal (CRC) and head and neck (HNSCC) cancers. Cetuximab exerts its antitumor activity by blocking ligand-dependent EGFR signaling and by engaging immune effector mechanisms. To investigate cetuximab resistance mechanisms, we cultured the cetuximab-sensitive CRC cell line DiFi in 3D with cetuximab, generating the cetuximab-resistant derivative (DiFi-CR). Genomic and transcriptomic profiling revealed that DiFi-CR cells harbor a mutation of the S442 residue within the EGFR ectodomain. Patient samples revealed recurrent EGFR S442 mutations following anti-EGFR therapy, suggesting S442 as a potential resistance hotspot. For mechanistic analyses, we reconstituted the EGFR S442I mutation, using a doxycycline-inducible system, and showed that it was necessary and sufficient to induce cetuximab resistance in CRC and HNSCC cells using in vitro cultures and in vivo mouse experiments. In silico studies, live-cell binding assays, and antibody enrichment in nude mice xenografts revealed that the S442I mutation leads to weaker EGFR-cetuximab binding. Weaker cetuximab binding was also predicted in silico for other S442 patient mutations. We found that mutant EGFR-driven resistance could be overcome by targeting the EGFR family member, ERBB2, with trastuzumab-deruxtecan. This combinatorial response required a physical interaction between EGFR and ERBB2, determined by co-immunoprecipitation. Our study supports EGFR S442 mutations as cetuximab resistance drivers and highlights co-targeting ERBB2 as a therapeutic strategy to restore anti-EGFR efficacy.