P-glycoprotein (ABCB1) overexpression confers resistance to the antibody-drug conjugate Enfortumab vedotin in EV-resistant organoid and PDX models

Background: Resistance to Enfortumab vedotin (EV) remains a major clinical challenge in bladder cancer (BC). The resistance can emerge through diverse mechanisms such as alterations in Nectin-4 expression, defects in ADC internalization and trafficking, or changes in downstream processing and payload response. We previously developed EV-resistant BC PDX and organoid models (poster 50, AACR 2025), providing validated preclinical tools to investigate the molecular basis of EV resistance. These models were designed both to elucidate resistance mechanisms and to enable direct comparison with patterns of resistance observed in the clinic. Methods: F659 PDX model was selected based on its high Nectin-4 mRNA expression and its strong response to EV treatment. Acquired resistance was generated by repeated EV administrations until tumors ceased to regress. From the resulting EV-resistant PDX (F659rP2), a matched organoid model was subsequently established. Both resistant and parental models were profiled using whole-exome sequencing, RNA-sequencing, and immunohistochemistry (IHC), and functionally characterized to assess EV activity and identify resistance mechanisms. Validation of these mechanisms was performed using pharmacological inhibition assays. Results: Resistance to EV was confirmed in both organoid and PDX models, as evidenced by a marked loss of sensitivity compared with their parental counterparts. Then, Nectin-4 expression was evaluated in resistant and parental models to investigate the underlying mechanisms, Flow cytometry and IHC analyses revealed a decreased cell surface expression of Nectin-4 in resistant models. Transcriptomic profiling identified significant changes in the expression of drug efflux transporters, FGFR3 and PPARgamma pathways. Notably, the ABCB1 gene was markedly upregulated in resistant models, and corresponding P-glycoprotein (P-gp) overexpression was confirmed by flow cytometry and IHC.Given that ABC transporters promote drug efflux, reducing intracellular drug accumulation and therapeutic efficacy, we assessed the impact of P-gp inhibition. Our results demonstrate that co-treatment with the P-gp inhibitor tariquidar (XR9576) partially restored EV sensitivity in both resistant organoid and PDX models. Conclusion: We successfully generated paired in vitro and in vivo EV resistant models. Our findings highlight the role of ABC transporter upregulation in EV resistance associated with a reduction of Nectin4 expression consistent with clinical observations. Other pathways seem to be involved and will need further investigations. Importantly, these EV-resistant preclinical models provide powerful tools for evaluating innovative therapeutic strategies against resistant tumors.