An Acquired Gatekeeper Mutation Induces Resistance to Neratinib in a Patient with HER2 Mutant-Driven Breast Cancer. Hanker Ariella B,Brewer Monica Red,Sheehan Jonathan H,Koch James P,Sliwoski Gregory R,Nagy Rebecca,Lanman Richard,Berger Michael F,Hyman David M,Solit David B,He Jie,Miller Vincent,Cutler Richard E,Lalani Alshad S,Cross Darren,Lovly Christine M,Meiler Jens,Arteaga Carlos L Cancer discovery We report a gatekeeper mutation in a patient with -mutant breast cancer with acquired resistance to neratinib. Laboratory studies suggested that is a neratinib-sensitive, gain-of-function mutation that upon dimerization with mutant HER3, also present in the breast cancer, amplifies HER2 signaling. The patient was treated with neratinib and exhibited a sustained partial response. Upon clinical progression, was detected in plasma tumor cell-free DNA. Structural modeling of this acquired mutation suggested that the increased bulk of isoleucine in HER2 reduces neratinib binding. Neratinib blocked HER2-mediated signaling and growth in cells expressing HER2 but not HER2 In contrast, afatinib and the osimertinib metabolite AZ5104 strongly suppressed HER2-induced signaling and cell growth. Acquisition of HER2 upon development of resistance to neratinib in a breast cancer with an initial activating mutation suggests is a driver mutation. HER2-mediated neratinib resistance may be overcome by other irreversible HER2 inhibitors like afatinib. We found an acquired gatekeeper mutation in a patient with -mutant breast cancer upon clinical progression on neratinib. We speculate that may arise as a secondary mutation following response to effective HER2 tyrosine kinase inhibitors (TKI) in other cancers with -activating mutations. This resistance may be overcome by other irreversible HER2 TKIs, such as afatinib. . 10.1158/2159-8290.CD-16-1431