EZH2 Inhibition Remodels Cell States and Enhances EGFRi Sensitization in Bladder Cancer

BACKGROUND: Bladder cancer (BCa) displays interpatient heterogeneity and epigenetic plasticity that may limit treatment efficacy. OBJECTIVE: To investigate whether inhibition of Enhancer of zeste homolog 2 (EZH2) remodels tumor cell states and creates therapeutic vulnerabilities in BCa. DESIGN, SETTING, AND PARTICIPANTS: We integrated single-nucleus ribonucleic acid sequencing (snRNA-seq) of formalin-fixed paraffin-embedded (FFPE) primary tumors with single-cell ribonucleic acid sequencing (scRNA-seq) of matched patient-derived organoids (PDOs) from four BCas spanning stages Ta-T3. INTERVENTION: PDOs were treated with the EZH2 inhibitor tazemetostat. Sequential tazemetostat pretreatment followed by afatinib treatment was used to evaluate whether EZH2 inhibition enhanced sensitivity to epidermal growth factor receptor (EGFR)-targeted therapy. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Transcriptional responses to EZH2 inhibition were profiled by scRNA-seq. Trimethylation of histone H3 lysine 27 (H3K27me3) chromatin immunoprecipitation sequencing (ChIP-seq) was performed in PDOs and EZH2 ChIP-seq in HT1376 cells to assess epigenomic changes associated with treatment. Gene set enrichment analysis was used to evaluate treatment-associated pathway alterations, including EGFR tyrosine kinase inhibitor resistance-related signatures. Functional relevance was assessed using afatinib sensitivity assays after sequential tazemetostat pretreatment. RESULTS AND LIMITATIONS: Native tumors showed heterogeneity in epithelial, stromal, and immune composition, whereas matched PDOs selectively retained and expanded tumor-intrinsic stress- and plasticity-associated epithelial states. EZH2 inhibition induced model-specific reprogramming, including inflammatory, proliferative, hypoxic/glycolytic, and neural/neuroendocrine (NE)-like programs. EZH2-bound regions were preferentially associated with treatment-related H3K27me3 loss, and loci with both EZH2 occupancy and reduced H3K27me3 were enriched for neural and NE developmental processes. Despite these heterogeneous responses, gene set enrichment analysis showed downregulation of EGFR tyrosine kinase inhibitor resistance-related signatures across all PDOs, and sequential tazemetostat pretreatment increased afatinib sensitivity in all four models. Limitations include the small cohort, ex vivo design, and use of a cell line for EZH2 ChIP-seq. CONCLUSIONS: EZH2 is a context-dependent regulator of cell-state plasticity in BCa. Although EZH2 inhibition alone induced heterogeneous adaptive responses, it also suppressed EGFR inhibitor resistance-related programs and enhanced subsequent afatinib sensitivity, supporting further evaluation of sequential epigenetic priming before EGFR-targeted therapy in BCa.