In vivo CRISPR screens identify dual functions of DNMT3A in mediating tumor immune evasion

Cancer immunotherapy has achieved remarkable clinical efficacy across multiple cancers, but it remains ineffective for most patients and the resistance mechanisms remain elusive. Epigenetic regulators play pivotal roles in both tumorigenesis and the modulation of antitumor immunity. Here, we performed in vivo CRISPR-Cas9 screens targeting epigenetic regulators and identified DNMT3A as a key driver of tumor immune evasion, exerting its effects through both methyltransferase activity-dependent and -independent mechanisms. Mechanistically, DNMT3A repressed the expression of TNFRSF1A and TNFRSF1B in a methyltransferase activity-dependent manner, enabling tumor cells to evade TNFα-induced killing. Importantly, DNMT3A exerted an additional noncanonical function by interacting with NFYA to activate cholesterol biosynthesis-related genes. This led to the accumulation of zymosterol in the tumor interstitial fluid, which directly impaired SREBP2 cleavage and enhanced HMGCR degradation in CD8⁺ T cells. Consequently, zymosterol-mediated suppression of cholesterol biosynthesis in CD8⁺ T cells compromised their activation and antitumor effector functions. Targeting DNMT3A’s multifaceted functions with antisense oligonucleotides (ASOs) effectively overcame resistance to immune checkpoint blockade (ICB) therapy. Collectively, our findings uncover a dual role of DNMT3A in tumor immune evasion and establish it as an integrated epigenetic and metabolic checkpoint, thus providing a potential target for immunotherapy.