Abstract A040: Resistance to trastuzumab deruxtecan acquired through multifaceted pathway alterations in preclinical models

Abstract Background: Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate (ADC) composed of an anti-HER2 antibody, a tetrapeptide-based cleavable linker, and a topoisomerase I inhibitor payload. While T-DXd demonstrates clinical efficacy across HER2-expressing tumors, drug resistance may emerge with time. Given the complexity of ADCs, resistance can arise through diverse mechanisms, including modifications in target expression, alterations in ADC internalization and trafficking, changes in payload sensitivity, or disruptions in downstream processing. Here, we investigate mechanisms of acquired T-DXd resistance in preclinical models. Methods: Acquired resistance models were generated via high-dose pulsatile T-DXd treatment in HER2 expressing cell lines (NCI-H2170, HCC1954, KPL-4). Resistant models were profiled (Whole exome sequencing, RNA-sequencing, single-cell RNA-sequencing, proteomics) and functionally characterized to evaluate T-DXd activity and validate resistance mechanisms. Mechanisms of resistance were validated using pharmacologic and genetic tools. Results: Resistant cells showed substantial loss of T-DXd sensitivity compared with parental lines, with substantial GI50 shifts – H2170-R, 200-fold; HCC1954-R, 1640-fold. In contrast, less pronounced shifts in topoisomerase I inhibitor sensitivity were seen (H2170-R, 1.9-fold; HCC1954-R, 3.5-fold). Transcriptome analysis revealed marked changes in expression of HER2, drug efflux pumps, and DNA repair genes. HER2 surface expression varied between models, with no change in KPL-4-R, 60% reduction in H2170-R, and 92% reduction in HCC1954-R as measured by flow cytometry. Notably, a decrease in T-DXd internalization and reduced intracellular payload levels was observed (by 50% in H2170-R), confirming reduced ADC delivery in resistant models. RNA expression of drug efflux pumps, particularly ABCC1 (7-fold increase) and ABCG2 (slightly increased), were upregulated in resistant lines, corroborated by protein analysis. Pharmacological inhibition of efflux (KO-143, Reversan) and genetic knock out of efflux pumps partially restored T-DXd sensitivity in vitro, supporting the contribution of efflux pumps to T-DXd resistance. Resistance lines also displayed altered DNA repair pathways, including upregulated TOP2A. DNA damage response modulation by ATR inhibition (AZD6738) and PARP inhibition (AZD5305) partially restored T-DXd activity in resistant models. Single-cell RNA sequencing revealed population heterogeneity within resistance models, and notably, co-occurrence of multiple altered resistance pathways was observed. Conclusions: These preclinical studies demonstrate that T-DXd resistance in HER2-expressing cell lines emerges via multifaceted mechanisms, including reduced HER2 expression, altered ADC trafficking/internalization, increased efflux, and changes in payload sensitivity. Citation Format: Deepa Bhavsar, Eylem Kulkoyluoglu Cotul, Anders Nelson, Richard Tourdot, Xiaochuan Liu, Alan Rosen, Michael Caprio, Laura Kazlauskas, Catherine Rhee, Karen Niss, Eric Gosselin, Wenyan Zhong, Jerome Mettetal, Emily Mason-Osann. Resistance to trastuzumab deruxtecan acquired through multifaceted pathway alterations in preclinical models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A040.