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

Many HER2-positive breast cancer (BC) patients relapse within a year of HER2-targeted therapy.Here, we identify pathogenic mutations in HER2 dimerization domains II and IV (DDMs) as a critical mechanism driving such resistance.Mining multiple cancer databases revealed recurrent hotspots (G309A, S310Y, P523S) in ~22% of HER2 + BC cases.Molecular dynamics simulations showed these mutations disrupt HER2:HER2 binding and favor HER2:HER3 dimerization.Proximity ligation assays confirmed elevated HER2:-HER3 complexes in mutant-expressing cells compared with wildtype, S305C, or S310F.This dimer switch redirected downstream signaling from ERK to AKT, conferring marked resistance to trastuzumab and neratinib.In vivo imaging further demonstrated that P523S promotes aggressive and trastuzumab refractory tumors.Importantly, both wild-type and mutant cells remained sensitive to the dual HER2/HER3 kinase inhibitor tucatinib, underscoring its therapeutic potential in trastuzumab-resistant settings.Antibody-drug conjugates (ADCs) such as T-DM1 and T-DXd also remained effective in wild-type or majority of HER2 DDM expressing lines tested.Collectively, these findings provide mechanistic evidences for specific DDM-driven resistance to HER2 targeted drugs like trastuzumab and neratinib, while newer entities like tucatinib or ADCs in use could prove to be more effective for treatment of HER2 + BC patients harboring dimerization-domain mutations.