ZNF74 as a novel therapeutic target in melanoma: Unveiling mechanisms and developing precision treatments.

Abstract

e21606 Background: Melanoma is the most aggressive and fatal skin cancer. Acral melanoma, the predominant subtype in East Asian populations, has a poor prognosis, undefined driver genes, and limited immunotherapy efficacy. Our previous research identified chr22q11.21 gene amplification as a predictor of poor prognosis and metastasis in acral melanoma, showing a negative correlation with immune-regulatory gene expression. Among the amplified genes, zinc finger protein 74 (ZNF74) emerged as a key player. However, its role in tumor progression and immune modulation remains unclear. Methods: We analyzed ZNF74 expression and clinical relevance in melanoma using public datasets (e.g., TCGA). ZNF74 knockdown melanoma cell lines were constructed using lentiviral systems and evaluated for proliferation, colony formation, and tumor growth in xenografts. The impact of ZNF74 on the immune microenvironment was assessed through co-culture assays, flow cytometry, and cytotoxicity assays. Mechanistic studies, including RNA-seq, RT-qPCR, Western blotting, and ELISA, identified NSUN5/CXCL11 as downstream targets of ZNF74. Molecular mechanisms were further elucidated using luciferase assays, dot blotting, and RNA immunoprecipitation. Immunofluorescence was performed on clinical samples to correlate ZNF74, NSUN5, and CXCL11 expression with clinicopathological features. Finally, AI-assisted virtual screening identified ZNF74 inhibitors, which were validated in vitro and in vivo as single agents and in combination with immune checkpoint inhibitors. Results: ZNF74 was highly expressed in melanoma and correlated with worse prognosis, reduced CD8+ T cell infiltration, and poor immunotherapy response. ZNF74 knockdown significantly inhibited melanoma growth in vitro and in vivo while enhancing CD8+ T cell infiltration, migration, and cytotoxicity. Mechanistically, ZNF74 activated NSUN5, which modified CXCL11 mRNA via m5C methylation, suppressing CXCL11 expression and reducing CD8+ T cell recruitment. Clinically, ZNF74, NSUN5, and CXCL11 expression correlated with tumor stage, Breslow thickness, overall survival (OS), progression-free survival (PFS), and immunotherapy efficacy. We identified CA1001, a small-molecule ZNF74 inhibitor, which demonstrated potent anti-tumor activity in vitro and in vivo. When combined with immune checkpoint inhibitors, CA1001 showed synergistic effects, significantly enhancing tumor regression. Conclusions: ZNF74 is a critical regulator of melanoma progression and immune evasion through the NSUN5/CXCL11 axis. We developed CA1001, a first-in-class ZNF74 inhibitor, which holds significant promise as a novel therapeutic strategy, particularly in combination with immune checkpoint inhibitors, to improve outcomes in melanoma patients.