Abstract PS3-06-12: Pre-existing TOP2A-High Cells Drive Resistance to Trastuzumab Deruxtecan in HER2+ Breast Cancer

Abstract Background: Trastuzumab deruxtecan (T-DXd) has significantly improved outcomes for patients with HER2-positive (HER2+) breast cancer. However, acquired resistance remains a critical clinical challenge. The underlying mechanisms driving this resistance are not fully understood. Emerging evidence suggests that resistance may arise from pre-existing tumor cell heterogeneity rather than being acquired de novo. Among candidate pathways, dysregulation of DNA replication and repair has been implicated in therapeutic resistance. Topoisomerase II alpha (TOP2A), a key enzyme in DNA topology and replication, is frequently overexpressed in aggressive breast cancers and associated with poor prognosis and treatment resistance. Building on this concept, we hypothesized that a subpopulation of resistant-like cells exists within tumors prior to treatment with T-DXd and is characterized by a distinct molecular signature involving TOP2A. Methods: We performed whole transcriptome analysis to compare gene expression profiles of T-DXd-resistant (TDXd-R) cells against their parental counterparts. We then analyzed public single-cell RNA sequencing (scRNA-seq) datasets from 17 HER2+ breast cancer cell lines and 8 HER2+ patient tumors to identify pre-existing cellular populations. Bioinformatics analyses included differential gene expression, gene set enrichment analysis (GSEA), gene regulatory network (GRN) analysis, and drug-gene database (DsigDB) screening to predict synergistic therapies. Results: Transcriptomic analysis revealed that HER2+ TDXd-R cells consistently exhibited significant upregulation of TOP2A expression and E2F signaling. GSEA confirmed activation of pathways related to the cell cycle, DNA replication, and genomic stability in the TDXd-R cell lines. Notably, scRNA-seq analysis demonstrated that a distinct TOP2A+ HER2+ cell cluster was present in all examined HER2+ cell lines and patient tumors prior to T-DXd exposure. With GRN analysis, we found these pre-existing putative resistant cells were characterized by high activity of E2F signaling pathways, involved in S/G2-M cell cycle progression and DNA synthesis. Based on this molecular signature, drug prediction analysis identified potential synergistic agents, including the topoisomerase inhibitor lucanthone, as candidates to target T-DXd resistant population. Conclusion: Our findings suggest that resistance to T-DXd in HER2+ breast cancer is driven by the selection and expansion of a pre-existing cancer cell subpopulation defined by high TOP2A expression and its elevated E2F-associated regulon activity. This molecular signature may serve as a biomarker for innate resistance. Furthermore, targeting this population with T-DXd and synergistic agents such as lucanthone could be a promising therapeutic strategy to overcome or prevent resistance to T-DXd and improve patient outcomes. Citation Format: S. Han, M. Jung, J. Lee, S. Rhie. Pre-existing TOP2A-High Cells Drive Resistance to Trastuzumab Deruxtecan in HER2+ Breast Cancer [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 PS3-06-12.