Abstract 4036: Deciphering regulatory drivers of lineage plasticity and resistance to antibody-drug conjugates in urothelial bladder cancer

Abstract Urothelial bladder cancer (UC) displays significant phenotypic heterogeneity, with ∼ 30% of tumors exhibiting variant histologic features such as micropapillary, plasmacytoid, squamous, neuroendocrine, and sarcomatoid differentiation. Many of these histologic variants are often associated with aggressive behavior and reduced response to targeted therapies. Emerging clinical data suggest that lineage plasticity may underlie resistance to antibody-drug conjugates (ADCs), including enfortumab vedotin (EV), a Nectin-4-targeting ADC now standard in advanced UC. However, the mechanistic link between lineage transitions, ADC target expression, and ADC sensitivity remains incompletely understood. To study the regulatory mechanism, we conducted integrative genomic and transcriptomic profiling on 341 macrodissected bladder tumor specimens encompassing conventional UC and diverse histologic variants. A subset of mixed histology tumors was further profiled using spatial transcriptomics (10x Genomics Visium) to evaluate intratumoral heterogeneity. Regulon activity was inferred using transcriptomic data. Functional validation of candidate regulators was conducted in bladder cancer cell lines and patient-derived organoids using CRISPR/Cas9-mediated knockout and overexpression.We identified two dominant transcriptional categories across the histologic subtypes. Tumors with plasmacytoid, micropapillary, or nested histology retained a luminal urothelial signature and consistently expressed ADC targets such as NECTIN4, TACSTD2 (Trop-2), and ERBB2 (HER2). In contrast, tumors with squamous, sarcomatoid, or neuroendocrine features showed loss of urothelial identity, downregulation of these ADC targets, and activation of lineage-alternative transcriptional programs. Spatial transcriptomics confirmed marked intratumoral heterogeneity, showing spatially differential lineage program and ADC target expression. Master regulator analysis discovered a core luminal transcriptional program governed by FOXA1, GATA3, and PPARG, which was progressively silenced in variant histologies. These tumors concurrently activated alternative lineage-specific regulators, including ASCL1 and NEUROD1, consistent with neuroendocrine reprogramming. Perturbation of these regulators in organoid and cell line models modulated Nectin-4 expression and altered sensitivity to EV in vitro. This study establishes lineage reprogramming as a central mechanism regulating ADC target expression and ADC response. Lineage regulators represent tractable modulators of ADC response and support the development of rational combination strategies to overcome plasticity-associated resistance. Ongoing studies using multi-omics and functional modeling aim to further define lineage regulatory networks and inform targeted therapeutic strategies. Citation Format: Jiaqian Luo, Sizhi P. Gao, Jacob E. Tallman, Fengshen Kuo, Merve Basar, Cansu Yol, Syed M. Alam, Hui Jiang, Xinran Tang, Doris X. Zheng, Jordan E. Eichholz, Alejandra Lopez Rojas, Ecenur Turkay, Jonathan E. Rosenberg, Gopakumar Iyer, Eugene J. Pietzak, David B. Solit, Hikmat Al-Ahmadie. Deciphering regulatory drivers of lineage plasticity and resistance to antibody-drug conjugates in urothelial bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4036.