Abstract
Epidermal growth factor receptor (EGFR) is a critical 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. To investigate cetuximab resistance mechanisms, we cultured the cetuximab-sensitive CRC cell line DiFi in 3D in type I collagen with cetuximab, generating cetuximab-resistant derivatives (DiFi-CR). Genomic and transcriptomic profiling revealed that DiFi-CR cells have a mutation of the S442 residue in the EGFR extracellular domain. Patient samples revealed EGFR S442 mutations following anti-EGFR therapy, indicating S442 as a potential resistance hotspot. For an in-depth mechanistic analysis, 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 (2D and 3D) cultures and in vivo mouse experiments. In silico structural 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. Importantly, we found that mutant EGFR-driven cetuximab resistance could be overcome by targeting EGFR family member ERBB2 with trastuzumab-deruxtecan. Notably, this combinatorial response was contingent upon a physical interaction between EGFR and ERBB2, as determined by co-immunoprecipitation. Overall, our study establishes EGFR S442 mutations as drivers of cetuximab resistance and highlights co-targeting ERBB2 as a promising therapeutic strategy to restore anti-EGFR efficacy.

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