Abstract B062: Loss of Flotillin-2 increases Trastuzumab emtansine internalization and cytotoxicity in HER2 amplified cancers

Abstract While Trastuzumab emtansine (T-DM1) and other HER2-targeting antibody-drug conjugates (ADC’s) are used to treat breast cancer patients with HER2-amplified tumors, there is a need to improve the efficacy of these ADC’s through the understanding of their mechanism of uptake into cells. Flotillin-2 (FLOT2), a cell membrane associated scaffolding protein regulates the internalization of epidermal growth factor receptor (EGFR), which led us to investigate whether FLOT2 has similar effects on HER2. In this study we investigated the regulation of T-DM1 and HER2 internalization by FLOT2 and the effects of FLOT2 on TDM1 toxicity. Signaling was characterized by immunoblot, protein interactions by immunoprecipitation and proximity ligation assay, cytotoxicity by cell viability and dead cell assays, TDM1 uptake by confocal microscopy, and drug/protein interaction by thermal shift. Caris Life Science CODEai was used to evaluate real-world overall survival (OS) obtained from insurance claims and calculated from treatment to last contact using Kaplan-Meier estimates. Higher FLOT2 expression in several HER2 amplified cell lines (SKBR3, HCC202, HCC1954, BT474, HCC1419, HCC2218, UACC893, ARK1, ARK2) correlated with a higher TDM1 IC50 in vitro, and higher FLOT2 expression correlated with worse survival in breast cancer patients receiving either T-DXd (16.2 months (m) vs 18.2 m. HR 1.1, 95% CI 1 - 1.3, p=0.04) or T-DM1 (37.9 m vs 41.2 m, HR 1.2, 95% CI 0.96 – 1.4, p=0.1) in real-word Caris Life Sciences data. FLOT2 interacts with HER2 and knockdown of FLOT2 reduced HER2 activation and downstream signaling, while also reducing cell viability of HER2 amplified cancer cells. The loss of FLOT2 results in increased HER2 internalization upon binding of TDM1 and this internalization is mediated by ubiquitination by the Cbl family ubiquitin ligases, leading to a reduced IC50 of TDM1 in HER2 amplified cancer cells. To pharmacologically disrupt the FLOT2/HER2 interaction, we investigated the effects of various small molecules on this interaction and discovered that zoledronic acid binds to FLOT2 and disrupts the HER2/FLOT2 interaction, which enhances TDM1 cytotoxicity. In conclusion, FLOT2 mediates the internalization and cytotoxicity of TDM1 through Cbl-dependent ubiquitination of HER2. Zoledronic acid disrupts the HER2/FLOT2 interaction, therefore increasing the internalization and cytotoxicity of TDM1, providing proof of principle that a small molecule inhibitor of the HER2/FLOT2 interaction can enhance the activity of the ADC. Zoledronic acid or an optimized derivative is a candidate to combine with Trastuzumab conjugated ADC’s to improve patient outcomes. Citation Format: David J. Wisniewski, Rachel Pritz, Jake Munch, Diya Desai, Sachin Kumar Deshmukh, Sharon Wu, George W. Sledge Jr., Natalie Porat-Shliom, Stanley Lipkowitz. Loss of Flotillin-2 increases Trastuzumab emtansine internalization and cytotoxicity in HER2 amplified cancers [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 B062.