Inhibitor-induced HER2-HER3 heterodimerisation promotes proliferation through a novel dimer interface ClausJeroen PatelGargi AutoreFlavia ColombaAudrey WeitsmanGregory SolimanTanya N RobertsSelene Zanetti-DominguesLaura C HirschMichael ColluFrancesca GeorgeRoger Ortiz-ZapaterElena BarberPaul R VojnovicBoris YardenYosef Martin-FernandezMarisa L CameronAngus FraternaliFranca NgTony ParkerPeter J 2020 While targeted therapy against HER2 is an effective first-line treatment in HER2+ breast cancer, acquired resistance remains a clinical challenge. The pseudokinase HER3, heterodimerisation partner of HER2, is widely implicated in the resistance to HER2-mediated therapy. Here, we show that lapatinib, an ATP-competitive inhibitor of HER2, is able to induce proliferation cooperatively with the HER3 ligand neuregulin. This counterintuitive synergy between inhibitor and growth factor depends on their ability to promote atypical HER2-HER3 heterodimerisation. By stabilising a particular HER2 conformer, lapatinib drives HER2-HER3 kinase domain heterocomplex formation. This dimer exists in a head-to-head orientation distinct from the canonical asymmetric active dimer. The associated clustering observed for these dimers predisposes to neuregulin responses, affording a proliferative outcome. Our findings provide mechanistic insights into the liabilities involved in targeting kinases with ATP-competitive inhibitors and highlight the complex role of protein conformation in acquired resistance.