Abstract PS5-01-19: Mechanisms of resistance to anti-HER2 therapies in brain metastatic derivatives of inflammatory HER2-positive breast cancer models

Abstract Background: HER2-positive (HER2+) breast cancer (BC) is an aggressive subtype with 30-50% incidence of brain metastases in metastatic patients. The HER2-selective tyrosine kinase inhibitor (TKI) tucatinib (Tuca) and the pan-HER TKI neratinib (Nrb), mostly used in the late line setting, are effective, including in treating brain metastases. The new antibody drug conjugate T-DXd is also highly effective in the brain metastatic setting. It is crucial to understand the mechanisms of resistance to these brain permeable anti-HER2 therapies. Materials and Methods: GFP/Luc tagged SUM190-BR3 (SUM190Br), a brain-tropic derivative of the HER2+/ER- inflammatory SUM190 cell model (harboring the PIK3CA H1047R mutation), was used (PMID: 27245829). A long-term highly Tuca resistant (TucaR) derivative of this line was developed through exposure to increasing doses of Tuca up to 1uM. Characterization of these resistant and naïve cells was performed by Western blot (WB), DNA-seq, and RNA-seq. Drug efficacy studies involved methylene blue-based cell growth and IC50 assays, and included the Akt inhibitor (i), capivasertib (Capi, 1uM), T-DXd (5ug/mL), and the EGFR-specific TKI gefitinib (Gef, 1uM) or monoclonal antibody cetuximab (Cetux, 10ug/mL). To characterize the in vivo tumorigenicity and metastatic capacity of our resistant model, immunocompromised mice were injected with cells via subcutaneous or intracardiac routes and monitored for primary tumor growth and metastasis by bioluminescence imaging. Results: Interestingly, the naïve SUM190Br cells are intrinsically resistant to Tuca (IC50>600nM), but not Nrb (IC50<60nM). Capi is effective as a single agent in the TucaR model (p<.0001). WB analysis of the TucaR model showed substantially elevated levels of total EGFR and modestly elevated HER2, as well as increased phosphorylation of EGFR and HER2, suggesting pathway reactivation at time of resistance vs. short-term Tuca treated naïve cells. Further, DNA and RNA-seq analysis showed high EGFR at the mRNA level with no change in copy number. As we previously showed in other HER2+ TucaR models such as BT474 (with acquired EGFR amplification), the SUM190Br TucaR cells remain sensitive to Nrb. Likewise, EGFRi together with Tuca was effective in overcoming resistance in this TucaR model. T-DXd is highly effective in metastatic BC, but high EGFR expression has been shown to reduce its efficacy (PMID: 39437778). Both Gef and Cetux improve the efficacy of T-DXd in our models, but Cetux is selectively effective against the TucaR model. Moreover, our preliminary in vivo results show that the highly aggressive TucaR model grows as xenografts, keeps brain tropism via intracardiac injection, and metastasizes via subcutaneous injection. Conclusions: Our findings suggest the role of high EGFR and PIK3CA mutations in resistance to Tuca, which warrants additional preclinical and clinical investigation. This underscores the importance of understanding if PIK3CA mutations are associated with reduced Tuca sensitivity and the testing of new mutant specific PIK3CAi or other PI3K/Akt pathway inhibitors. Our brain tropic TucaR cell and mouse models will be useful to understanding resistance in the brain metastatic setting, and future work will test the most promising treatments in vivo. The recent DESTINY-Breast09 trial points to the promise of T-DXd in combination with the monoclonal antibody pertuzumab as the new first line for metastatic BC. Since high-EGFR is suggested to play a potential role in reducing T-DXd efficacy, our ongoing and future work seeks to understand if the new T-DXd plus pertuzumab combination will be effective compared to T-DXd plus EGFRi in treating HER2+ BC with high EGFR. Citation Format: C. M. Sabotta, F. Liao, M. J. Shea, S. Nanda, L. Qin, P. S. Steeg, M. F. Rimawi, C. Gutierrez, C. Osborne, S. G. Hilsenbeck, J. Veeraraghavan, R. Schiff. Mechanisms of resistance to anti-HER2 therapies in brain metastatic derivatives of inflammatory 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 PS5-01-19.