Pseudouridine Synthesis Targeted Therapy Activates Antiviral Immunity to Boost Cancer Immunotherapy

Epigenetic plasticity is critical for tumor transformation, progression, and response to immunotherapy. Although pseudouridine is the first discovered and most abundant epitranscriptomic modification, its cellular functions remain poorly understood. In this study, we identified pseudouridine synthase (PUS) as a key driver of tumor immune evasion. Specifically, we found that PUS was aberrantly overexpressed in tumors and correlated with tumor malignant progression as well as poor patient prognosis, particularly in patients with TP53 mutations. Notably, genetic ablation of PUS effectively suppressed tumor progression, increased T cell infiltration, and boosted T cell function in a de novo MYC/Trp53-/- mouse liver cancer, as well as in chemically induced liver cancer models. Mechanistically, PUS loss induced the expression of retrotransposon sequences, resulting in elevated levels of double-stranded RNA and subsequent activation of innate antiviral immune signaling. We further demonstrated the role of PUS1 in tumor immune evasion was dependent on its enzymatic activity. Importantly, inhibition of PUS1 could sensitize tumors to anti-PD-1 therapy in a MYC/Trp53-/- mouse liver cancer model. Similarly, we revealed that 5-fluorouracil could inhibit pseudouridine synthesis and significantly enhance the efficacy of PD-1 inhibition. Clinically, low PUS level showed a higher response rate and better clinical outcome to immune checkpoint blockade therapies. Overall, our findings demonstrate PUS1 as a critical regulator of immune evasion and targeting pseudouridine synthesis may a novel strategy to enhance immunotherapy efficacy by activating dsRNA-sensing pathways. Overall design: Given the clinical relevance of PUS enzymes in hepatocellular carcinoma (HCC) development, we further explored their pro-tumorigenic role in liver cancer progression. Using a chemically induced liver cancer model, we compared liver-specific Pus1 knockout mice (Alb-cre; Pus1f/f, hereafter Pus1-cKO) with control mice (Pus1f/f, WT). To determine whether PUS1's role in tumor progression and immune evasion depends on its pseudouridine synthase activity, we generated a catalytically inactive mutant (PUS1-Mut) by substituting Ala for Asp146 (D146A). The functional impact of wild-type PUS1 (PUS1-WT) and PUS1-Mut was then evaluated in a de novo MYC/Trp53-/- mouse liver tumor model. High-throughput sequencing was performed on both mouse models.