Metabolic Reprogramming of NSUN2-Mediated m5C Modification Promotes the Progression of Hepatocellular Carcinoma by Restricting Natural Killer Cell-Mediated Cytotoxicity
Background: Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality, with resistance to immunotherapy posing a major clinical challenge. Natural killer (NK) cells exhibit impaired infiltration and cytotoxicity in HCC; however, the mechanisms underlying NK cell-mediated immune evasion are still poorly understood. This study investigated how NOP2/Sun RNA methyltransferase 2 (NSUN2), a 5-methylcytosine (m5C) RNA methyltransferase, induces metabolic reprogramming and immunosuppression to drive HCC progression. Methods: We conducted a genome-wide CRISPR screen in HCC cells cocultured with NK cells. To delineate the downstream mechanisms, we integrated profiling of the m5C epitranscriptome, transcriptome, and chromatin landscape with metabolic characterization. The impact of NSUN2 on histone lactylation and programmed cell death 1 ligand 1 (PD-L1) transcription was further investigated. Functional assays in vitro and in vivo using syngeneic murine models and pharmacological inhibition validated these findings. Clinical relevance was assessed using patient tissues, The Cancer Genome Atlas dataset, and immunotherapy cohorts. Results: Genome-wide CRISPR screening in HCC cell–NK cell coculture models identified NSUN2 as a key suppressor of NK cell-mediated cytotoxicity. Mechanistically, NSUN2-mediated RNA m5C modification enhanced the messenger RNA stability and expression of glycolytic enzymes, including enolase 1 (ENO1), pyruvate kinase M1/2 (PKM), and lactate dehydrogenase A (LDHA), thereby increasing lactate production. Accumulated lactate promoted histone H3 lysine 18 lactylation (H3K18la), which enhanced chromatin accessibility at the CD274 (encoding PD-L1) promoter and recruited signal transducer and activator of transcription 3 (STAT3) to drive PD-L1 expression, ultimately inhibiting NK cell-mediated cytotoxicity. Clinically, high NSUN2 expression was associated with elevated PD-L1 levels, poor prognosis, and immunotherapy resistance in patients with HCC. In vivo, NSUN2 knockout increased NK cell infiltration and suppressed tumor growth, while the STAT3 inhibitor TTI-101 combined with anti-PD-L1 therapy enhanced NK cell cytotoxicity and inhibited HCC progression. Conclusions: Our data demonstrated that NSUN2 drove immune evasion in HCC by coupling m5C-dependent glycolytic reprogramming with H3K18la-mediated epigenetic activation of PD-L1. These findings suggest that NSUN2 could represent a critical nexus between m5C RNA methylation and immunosuppression, providing a therapeutic rationale for combination immunotherapy in HCC.