Abstract 2050: The impact of cell cycle inhibitors on bladder cancer with low NECTIN-4 expression

Abstract Background: Antibody-drug conjugates (ADC) combine a cytotoxic agent with a monoclonal antibody for targeted delivery. Enfortumab-vedotin (EV) links a monoclonal antibody targeting NECTIN-4 with the cytotoxic agent monomethyl auristatin E (MMAE). Given the near-ubiquitous expression of NECTIN-4 in urothelial cancer (UC) tissues, EV has shown efficacy in UC and is approved for metastatic urothelial carcinoma (mUC). However, significant heterogeneity in NECTIN-4 expression within UC limits therapeutic efficacy in cases with low NECTIN-4 expression. This study evaluated cell cycle inhibitors' efficacy in UC cells with low NECTIN-4 expression. Methods: We measured NECTIN-4 and cell cycle-related molecules (Cyclin D1 and PLK1) in bladder cancer cell lines (253J, 253J-BV, 5637, RT4, T24, J82, HT1197, HT1376) and correlated these levels with sensitivity to the PLK1 inhibitor. Drug sensitivity was assessed by a CCK-8 assay over 72 hours following treatment with the PLK1 inhibitor (onvansertib). An ex vivo model using bladder tumor samples from patients validated the in vitro findings. NECTIN-4 levels in bladder cancer cell lines (HT1376 and RT4) were modulated by siNECTIN-4 transfection for 48 hours. Cell cycle analysis and the expression levels of cyclin D1 and PLK1 were examined in NECTIN-4 knockdown cell lines. Results: The therapeutic efficacy of the PLK1 inhibitor was significantly enhanced in bladder cancer cell lines with reduced NECTIN-4 expression (253J, 253J-BV, 5637, T24, and J82), as indicated by an IC50 <0.1 μM. In contrast, cell lines with high NECTIN-4 expression (RT4, HT1197, and HT1376) were resistant to onvansertib (IC50 >1 μM). NECTIN-4 expression correlated with cell cycle-related molecule levels. Consistently, bladder tumor samples showed a negative correlation between NECTIN-4 expression and PLK1 inhibitor sensitivity. PLK1 inhibitor-resistant tumors (IC50 >40 μM) had lower NECTIN-4 levels than sensitive samples (20 μM < IC50 < 40 μM). NECTIN-4 knockdown via siRNA increased G0/G1 phase cells and reduced cyclin D1 and PLK1 levels. Furthermore, siNECTIN-4-transfected cells were more sensitive to the PLK1 inhibitor than control siRNA-transfected cells. Conclusion: This study underscores the therapeutic potential of cell cycle inhibitors, notably PLK1 inhibitors, in bladder cancer cells with low NECTIN-4 expression, showing an inverse relationship between NECTIN-4 expression and sensitivity to PLK1 inhibitors. NECTIN-4 levels could predict PLK1 inhibitor treatment response. NECTIN-4 knockdown experiments confirmed that reduced NECTIN-4 expression disrupted cell cycle regulation and increased PLK1 inhibition efficacy. These findings advocate for further investigation of combination therapies involving ADCs and cell cycle inhibitors to counter resistance and improve outcomes in bladder cancer patients. Citation Format: Jee Soo Park, Ji Eun Lee, Myung Eun Lee, Jongchan Kim, Won Sik Ham. The impact of cell cycle inhibitors on bladder cancer with low NECTIN-4 expression [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 2050.