Abstract PS2-11-13: Resistance to tucatinib associated with hyperactive EGFR could be overcome by co-targeting HER2 and EGFR in HER2-positive breast cancer models

Abstract Background: Tucatinib (Tuca) is a highly HER2-selective small molecule tyrosine kinase inhibitor (TKI) approved for use in HER2+ metastatic breast cancer (MBC), including in patients with brain metastasis. In current practice, despite the expanding use of other anti-HER2 agents like TDXd in the 2nd and potentially the 1st line setting, Tuca continues to be a key treatment choice for HER2+ MBC, mostly after TDxd. We previously showed that EGFR amplification leading to HER pathway reactivation is associated with acquired resistance to Tuca in HER2+ BC models, which could be overcome using dual/pan-HER TKIs or the combination of potent EGFR and HER2 inhibitors (Liao et al, Can Res, 2022). Building on these key prior in vitro findings, here we sought to validate the efficacy of this treatment strategy in the in vivo setting. Materials and Methods: Our recently developed HER2+/ER+ BT474/AZ cell model with acquired resistance to Tuca (TucaR) generated through long-term exposure to gradually increasing doses of Tuca was used. NSG mice bearing GFP/Luc-tagged TucaR xenografts grown with estrogen supplementation were randomized to vehicle, Tuca (100mg/kg, once daily gavage, 7 days/week), or EGFR-specific inhibitors: 1) the TKI gefitinib (Gef 100 mg/kg, once daily gavage, 5 days/week) or 2) the monoclonal antibody cetuximab (Cetux, 30mg/kg, IP twice a week)), either alone or in combination, and monitored for primary tumor growth (caliper measurement) and spontaneous metastasis (by bioluminescence). Results: We first observed that our TucaR model grew effectively as xenografts in the presence of Tuca thereby confirming its Tuca resistant phenotype. EGFR inhibition, using Gef or Cetux, in combination with Tuca achieved complete regression of the primary TucaR tumors. Interestingly, although complete tumor regression was also observed in all mice treated with single-agent Cetux, within 10-14 days post randomization, the tumors recurred in all mice, starting at around day 55, suggesting the potential need for simultaneous HER2 blockade. Importantly, though the combination of Tuca with Gef or Cetux achieved complete primary tumor regression (i.e., no palpable/measurable tumor) within 10-14 days post randomization, bioluminescence imaging at the end of the experiment (around day 70-80) revealed minimal/microscopic residual disease in all mice at the subcutaneous injection site. This finding was further confirmed through histological analysis. Monitoring the metastatic propensity, our TucaR model was highly metastatic, including to the lungs, lymph nodes, and potentially bone. Specifically, lung metastasis was observed in almost all mice in the vehicle (6/6), Tuca (6/7), and Cetux (5/6) treatment arms. However, these lung metastatic lesions were completely abolished in all mice treated with Gef+Tuca (6/6) and Cetux+Tuca (5/5), further accentuating the promise of this strategy in the metastatic setting. Ongoing histochemical and molecular analyses of the primary tumors, metastatic lesions, and residual disease harvested at the end of the experiment are focused on gaining additional mechanistic insights. Conclusions: Our novel findings have crucial therapeutic implications and suggest that HER2+ primary and metastatic tumors with high EGFR (∼30% of HER2+ MBC) may not benefit from Tuca or EGFR inhibitors alone and need dual/pan-HER inhibitor therapy, a strategy that we intend to translate via a prospective trial. Our data also suggest the need for imaging-based endpoints in order to capture the minimal/microscopic residual disease that may otherwise be missed through routine tumor measurements, as well as the potential need for additional targeted therapies such as endocrine therapy or CDK4/6 inhibitors, beyond HER2-targeted treatment, to achieve complete tumor eradication. Citation Format: F. Liao, L. Qin, S. Nanda, M. J. Shea, C. Liu, C. M. Sabotta, A. Elkhanany, S. G. Hilsenbeck, C. Gutierrez, M. F. Rimawi, C. K. Osborne, J. Veeraraghavan, R. Schiff. Resistance to tucatinib associated with hyperactive EGFR could be overcome by co-targeting HER2 and EGFR in HER2-positive breast cancer models abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-11-13.