Tumor-intrinsic RNF128 drives a non-inflamed microenvironment and immune checkpoint blockade resistance in bladder cancer

Immune checkpoint blockade (ICB), particularly anti-PD-1/PD-L1 therapy, has emerged as an important treatment option for bladder cancer (BLCA). However, a substantial proportion of patients fail to achieve durable clinical benefit due to primary or acquired resistance. Accumulating evidence indicates that an immunosuppressive, non-inflamed tumor microenvironment (TME) is a major determinant of resistance to immunotherapy. Nevertheless, the key tumor-intrinsic regulators that shape the immune landscape and limit ICB efficacy in BLCA remain poorly defined. Integrated multi-omics analyses were performed using bulk RNA sequencing, single-cell RNA sequencing, and publicly available and in-house BLCA cohorts to characterize the immunological role of RNF128. Bioinformatic analyses were applied to assess immune cell infiltration, immune signaling pathways, and molecular subtypes. Functional validation was conducted using RNF128 knockdown and overexpression in human and murine bladder cancer cell lines, chemotaxis assays, T cell–mediated cytotoxicity assays, and syngeneic mouse tumor models combined with anti-PD-1 therapy. The association between RNF128 expression and ICB response was further evaluated in independent immunotherapy cohorts. RNF128 was highly expressed in bladder cancer and was associated with an immunosuppressive TME. Elevated RNF128 expression was associated with reduced CD8⁺ T-cell infiltration, lower expression of T-cell-recruiting chemokines, including CCL3, CCL4, CCL5, CXCL9, and CXCL10, and lower T cell–inflamed signature scores. Single-cell analyses revealed that RNF128 was predominantly expressed in malignant urothelial cells and was associated with lower enrichment of cytokine- and chemokine-related transcriptional signatures. Functional experiments showed that manipulation of RNF128 affected CD8⁺ T-cell recruitment and cytotoxic activity, whereas RNF128 knockdown enhanced the antitumor efficacy of PD-1 blockade in a syngeneic mouse model. Clinically, lower RNF128 expression was associated with more favorable ICB responses and improved survival outcomes in the evaluated immunotherapy cohorts. RNF128 is associated with a non-inflamed immune microenvironment and reduced ICB responsiveness in BLCA, while functional studies support a role for tumor-cell RNF128 in limiting CD8⁺ T-cell recruitment and antitumor activity. RNF128 may therefore represent a candidate biomarker and potential therapeutic target for improving immunotherapy response in BLCA.