Abstract Overexpression of human epidermal growth factor receptor 2 (ERBB2), often accompanied by ERBB2 gene amplification, is present in ∼25% of early-stage breast cancers. In a relatively small subset of breast cancer patients (4,000 cases each year in the US) ERBB2 is alternatively activated by gain-of-function mutations without gene amplification and receptor overexpression. These somatic mutations frequently occur in the kinase domain of ERBB2 and induce receptor phosphorylation, PI3K/AKT pathway hyperactivation, and malignant transformation. Breast cancer patients bearing ERBB2-mutated tumors are candidates for treatment with the irreversible kinase inhibitor neratinib. Although this therapeutic strategy is showing remarkable activity in a significant proportion of patients, emergence of acquired resistance inevitably occurs. The aim of this study is to identify the possible mechanisms of acquired resistance to neratinib therapy in ERBB2-mutated breast cancers. As part of the ongoing phase II SUMMIT basket study of neratinib in ERBB2-mutant cancers (NCT01953926), we have identified a common genomic alteration in tumors from three patients that progressed following initial benefit from neratinib treatment. Targeted exome sequencing of biopsies collected at time of disease progression revealed increased copy number of the ERBB2-mutant allele. In one case, the resistant lesions were confirmed to be avid for zirconium-89-trastuzumab and the patient responded to subsequent antibody-based anti-ERBB2 therapy. In an attempt to confirm that increased expression of the mutant allele was sufficient to limit neratinib sensitivity, we measured the antitumor activity of this agent in MCF10A cells stably expressing several activating mutations of ERBB2 compared to cells expressing equal levels of the wild type receptor. We found that higher concentrations of neratinib were needed to inhibit the proliferation of ERBB2-mutant cells compared to wild-type. Further, enhanced receptor activity in the ERBB2-mutant cells correlated with increased formation of ERBB2/ERBB3 dimers, activation of the PI3K/AKT pathway and in vivo tumorigenic potential. Combined ERBB2 and ERBB3 inhibition efficiently inhibited phosphorylation of ERBB2/ERBB3 and cell proliferation. We plan to overexpress the ERBB2-mutant allele in cell lines/organoids obtained from ERBB2-mutant patient-derived xenografts, phenocopying our results from the clinical specimens. We hypothesize that this manipulation will be sufficient to induce increase ERBB2/ERBB3 dimerization and resistance to neratinib. In summary, we believe that amplification of mutated ERBB2ERBB2 promotes increased ERBB2/ERBB3 dimerization, ERBB3 activation, and subsequent downstream signaling activation. Dual ERBB2/ERBB3 blockade may be a potential strategy to delay or prevent resistance to neratinib in ERBB2-mutant breast tumors. Citation Format: F Javier Carmona, David Hyman, Gary Ulaner, Joe Erinjeri, Nancy Bouvier, Helen Won, Richard Cutler, Al Alani, Michael Berger, José Baselga, Maurizio Scaltriti. Amplification of mutant ERBB2 drives resistance to the irreversible kinase inhibitor neratinib in ERBB2-mutated breast cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 298.