Abstract 4188: The role of NHEJ/HR mediated DSB repair in cancer cell sensitivity to payload of T-DXd ADC

Abstract Introduction: Trastuzumab deruxtecan (T-DXd) is an innovative HER2-targeting antibody-drug conjugate comprising a HER2-specific antibody linked to an exatecan derivative (DXd) payload. This therapeutic agent has demonstrated substantial clinical efficacy across various HER2-expressing malignancies. The DXd payload functions as a topoisomerase I inhibitor, stabilizing the topoisomerase-DNA cleavage complex. This stabilization inhibits the re-ligation of DNA, leading to the accumulation of DNA double-strand breaks (DSBs) and ultimately inducing apoptotic cell death. DNA DSBs are primarily repaired through two distinct mechanisms: Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). However, the contributions of these DNA damage repair pathways to the sensitivity of cancer cells to T-DXd/DXd remain poorly understood. Methods: In this in vitro study, we investigated the roles of NHEJ and HR in modulating the sensitivity of the HER2-expressing breast cancer cell line, MDA-MB-453, to T-DXd/DXd. This was achieved through siRNA-mediated knockdown of key components of the respective repair pathways, specifically TP53BP1 for NHEJ and BRCA1 for HR. Drug sensitivity was assessed through cell growth inhibition assays and caspase-3/7 activity assays. DNA repair machinery was evaluated using immunofluorescence to quantify RAD51 foci formation. Additionally, cell cycle dynamics in response to DXd treatment and the modulation of NHEJ/HR-mediated repair pathways were analyzed using flow cytometry and live cell imaging of MDA-MB-453 cells transduced with a Fluorescent Ubiquitination-based Cell Cycle Indicator. Results: Our results demonstrated that the suppression of TP53BP1 led to a decrease in T-DXd/DXd sensitivity, which was associated with an increase in HR-mediated DSB repair, as evidenced by a higher incidence of RAD51 foci formation in the nuclei. Conversely, knockdown of BRCA1, which inhibits HR, substantially enhanced sensitivity to T-DXd/DXd. Notably, the alteration in DNA repair pathway balance resulted in distinct cell cycle phase shifts upon T-DXd/DXd treatment; specifically, BRCA1 knockdown increased the G2/M phase population while concurrently reducing DNA damage response (DDR)-related cell cycle arrest. Furthermore, the combination of Wee1 inhibitor with T-DXd and DXd enhanced cytotoxicity via aberrant cell cycle arrest. Conclusions: Collectively, these findings point to the role of the mode of DNA damage repair pathways in modulating the activity of the DXd payload, suggesting the possibility of improved efficacy by the combination with DDR inhibitors. Further investigations are warranted to elucidate the contextual factors influencing these outcomes and to validate our findings in clinical samples. Citation Format: Len Katsumata, Manabu Abe, Misa Ikuta, Fumitaka Suto, Kumiko Koyama. The role of NHEJ/HR mediated DSB repair in cancer cell sensitivity to payload of T-DXd ADC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4188.