Introduction:

Urothelial bladder cancer exhibits extensive transcriptional and phenotypic heterogeneity, with frequent occurrence of histologic variants that deviate from conventional urothelial differentiation. Common histologic subtypes include squamous, glandular, micropapillary, plasmacytoid, small cell/neuroendocrine, sarcomatoid, and nested urothelial carcinoma, many of which are associated with more aggressive clinical behavior and therapy resistance. Despite their clinical significance, prior genomic sequencing efforts have not fully elucidated the mechanisms underlying this phenotypic plasticity, underscoring the need for integrative, multi-omics approaches to identify the regulatory mechanisms driving lineage plasticity in bladder cancer. Antibody-drug conjugates (ADCs) have transformed the treatment landscape for locally advanced and metastatic bladder cancer, whereas the molecular determinants of ADC target expression and sensitivity remain incompletely understood.

Methods:

We performed integrative multi-omics analysis on macrodissected tumor regions from 341 bladder cancer specimens representing diverse histologic variants. Comparative analyses of genomic and transcriptomic landscapes were conducted across these histologic subtypes. Spatial transcriptomic profiling using the 10X Genomics Visium platform was applied to selected tumors with mixed histology. Whole-transcriptome sequencing data were used to infer transcription factor activity and candidate lineage regulators were functionally validated in bladder cancer cell lines and patient-derived organoids using CRISPR-based perturbation and overexpression assays.

Results:

Bulk RNA-seq analysis revealed two major transcriptionally defined types associated with histologic variants in bladder cancer. Tumors with micropapillary, plasmacytoid or nested histology retained a urothelial-like luminal signature and expressed ADC targets such as NECTIN4, TACSTD2, and ERBB2. In contrast, tumors with squamous, neuroendocrine, or sarcomatoid features exhibited downregulation or loss of these therapeutic targets. Spatial transcriptomic profiling of mixed-histology tumors further confirmed intratumoral heterogeneity, revealing discordant expression of ADC targets between urothelial and variant histology components. Integrative analysis of clinical treatment data demonstrated that variant histology tumors with reduced Nectin-4 expression were associated with a shorter duration of response to the Nectin-4-targeting ADC enfortumab vedotin (EV), suggesting that lineage plasticity contributes to intrinsic ADC resistance. To investigate underlying mechanisms, master regulator analysis was used to infer transcription factor activity and identify candidate regulators driving the divergent transcriptional programs observed in variant histology tumors. Urothelial-like tumors demonstrated enriched activity of luminal transcription factors FOXA1, GATA3, and PPARG, whereas tumors that had lost urothelial identity showed activation of alternative lineage-specific regulons, such as ASCL1 and NEUROD1, indicative of neuroendocrine reprogramming. To functionally assess these regulators, we performed CRISPR/Cas9-mediated knockout and overexpression of selected candidates in bladder cancer models, including established cell lines and patient-derived organoids. Perturbation of these regulons induced transcriptional reprogramming and altered Nectin-4 expression, leading to corresponding changes in sensitivity to EV in vitro. These findings highlight lineage plasticity as a modifiable determinant of ADC response and support the development of combination strategies to overcome therapeutic resistance.

Conclusion:

This study reveals substantial transcriptional heterogeneity across bladder cancer histologic subtypes, including distinct regulon activity and variable expression of clinically relevant ADC targets. These findings highlight the challenges of implementing ADC therapies in the context of lineage plasticity. Through integrative multi-omics and functional validation, we identify transcriptional reprogramming as a key driver of lineage transitions that modulate ADC target expression and therapeutic sensitivity. These results establish lineage plasticity as a modifiable mechanism of resistance and support the development of rational combination strategies to improve the efficacy of ADC-based therapies in bladder cancer. Ongoing studies leveraging patient-derived organoids and xenograft models aim to further dissect the mechanisms by which lineage regulators drive cell state transition and to evaluate combination therapeutic strategies.

Funding: NIH/NCI Cancer Center Support Grant P30 CA008748, R01-CA233899, SPORE in Bladder Cancer P50-CA221745, and P01-CA221757