The role of NECTIN4 in shaping the tumor microenvironment and potentiating the efficacy of immunotherapy in bladder cancer.

4574 Background: The EV-302 trial established enfortumab vedotin plus pembrolizumab (EV/P) as first-line therapy for metastatic urothelial carcinoma (UC). Although EV/P is administered without NECTIN4 testing based on the assumption of uniform NECTIN4 expression, real-world data have identified NECTIN4-low tumors associated with inferior responses to EV, highlighting the need for NECTIN4-based precision therapy. We aimed to define the molecular and immunologic roles of NECTIN4, identify predictive biomarkers of EV/P outcomes, and explore rational therapeutic strategies for NECTIN4-low UCs. Methods: We performed multi-omics analyses of 817 UC cases integrating whole-exome sequencing, RNA-seq, multiplex immunofluorescence, and spatial single-cell profiling (CODEX). We evaluated associations among NECTIN4 status, molecular subtype, tumor microenvironment (TME), survival, and response to immune checkpoint inhibitors. Basal bladder cancer (BC) cell lines and orthotopic MB49 bladder cancer models with NECTIN4 overexpression (OE) or control were used to assess tumor biology and therapeutic response. Results: Clustering by NECTIN4 H-score and molecular subtype revealed that basal/squamous (Ba/Sq) muscle-invasive BCs with low NECTIN4 had significantly worse survival outcomes than NECTIN4-high tumors in both our cohort (P=0.001) and the IMvigor210 trial (P=0.019). In vitro, NECTIN4 OE in basal BC cells suppressed proliferation and migration through downregulation of inflammation-related signaling pathways. CIBERSORTx and EcoTyper analysis of RNA-seq data indicated a more immune-inflamed TME in NECTIN4-high Ba/Sq tumors. Spatial single-cell profiling showed increased CD8+ T cells, particularly CD8+/TIGIT+ cells, in NECTIN4-high Ba/Sq UCs. Moreover, in orthotopic mouse models, NECTIN4-OE tumors exhibited reduced growth and increased CD8+ T-cell infiltration. Clinically, NECTIN4-high Ba/Sq tumors showed higher objective response rate (ORR) to pembrolizumab/avelumab than NECTIN4-low tumors (55.5% vs 0%, P=0.002) in our cohort; in IMvigor210, NECTIN4-high Ba/Sq patients also had superior ORR (38.5% vs 15.6%, P=0.025). Additionally, in orthotopic mouse models, EV/P showed limited efficacy in NECTIN4-low tumors, whereas NECTIN4-OE tumors responded well. Transcriptomic analyses identified histone deacetylases (HDAC) as potential targets in NECTIN4-low tumors; accordingly, HDAC inhibitors demonstrated greater efficacy in NECTIN4-low basal BCs in vitro and vivo. Conclusions: NECTIN4 influences tumor progression and shapes the TME, thereby impacting immunotherapy efficacy. NECTIN4 is a potential predictive biomarker for survival and for outcomes with immunotherapy and EV/P in Ba/Sq UCs. NECTIN4-low Ba/Sq tumors may require alternative strategies, and HDAC inhibition represents a rational therapeutic approach for NECTIN4-low Ba/Sq UC patients.