Hotspot mutations in HER2 interfaces destabilize structure, causing breast cancer treatment failure.

Many HER2-positive breast cancer (BC) patients relapse within a year of trastuzumab or neratinib treatment. We identified specific pathogenic mutations in the dimerization domains II and IV of the HER2 receptor that contribute to treatment resistance. Mutations G309A, S310Y, and P523S induce significant structural alterations, disrupting crucial HER2:HER2 binding pockets. HER3-preferring mutants exhibited increased HER2:HER3 interactions, as confirmed by proximity ligation assay in HER2-low and HER2-high cell lines. G309A, S310Y, and P523S mutations induced a receptor switch, altering downstream signaling from ERK to AKT activation, leading to high insensitivity to trastuzumab or neratinib in cell survival and migration assays, which was further confirmed by bioluminescence imaging of orthotopic tumors expressing the P523S mutation. This study identifies new hotspot mutations in HER2 domains II and IV causing trastuzumab resistance. Notably, cells with either wild-type or the examined dimerization domain mutations retained sensitivity to the FDA-approved HER2 kinase inhibitor, tucatinib.