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. 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.

表观遗传可塑性（epigenetic plasticity）对于肿瘤转化、进展以及免疫治疗响应至关重要。尽管假尿苷（pseudouridine）是首个被发现且丰度最高的表观转录组修饰（epitranscriptomic modification），但其细胞功能仍未得到充分阐释。本研究中，我们鉴定出假尿苷合酶（pseudouridine synthase, PUS）是肿瘤免疫逃逸的关键驱动因子。具体而言，我们发现PUS在肿瘤中存在异常高表达，且与肿瘤恶性进展及患者不良预后显著相关，这一关联在携带TP53突变的患者中尤为突出。值得注意的是，在MYC/Trp53-/-新发肝癌小鼠模型与化学诱导肝癌模型中，敲除PUS可有效抑制肿瘤进展、增加T细胞浸润并增强T细胞功能。机制层面，PUS缺失可诱导反转录转座子序列的表达，导致双链RNA（double-stranded RNA, dsRNA）水平升高，进而激活先天抗病毒免疫信号通路。我们进一步证实，PUS1介导肿瘤免疫逃逸的功能依赖于其酶活性。尤为重要的是，在MYC/Trp53-/-肝癌小鼠模型中，抑制PUS1可使肿瘤对抗PD-1治疗增敏。类似地，我们发现5-氟尿嘧啶（5-fluorouracil）可抑制假尿苷合成，并显著增强PD-1抑制剂的抗肿瘤疗效。临床数据显示，PUS低表达的患者对免疫检查点阻断治疗具有更高的响应率与更佳的临床结局。综上，本研究结果表明PUS1是免疫逃逸的关键调控因子，靶向假尿苷合成或可通过激活双链RNA感知通路，成为增强免疫治疗疗效的全新策略。

鉴于PUS酶类在肝细胞癌（hepatocellular carcinoma, HCC）发生发展中的临床相关性，我们进一步探究了其在肝癌进展中的促肿瘤生成作用。我们采用化学诱导肝癌模型，将肝脏特异性Pus1敲除小鼠（Alb-cre; Pus1f/f，下称Pus1-cKO）与对照小鼠（Pus1f/f，野生型WT）进行对比分析。为明确PUS1在肿瘤进展与免疫逃逸中的作用是否依赖于其假尿苷合酶活性，我们通过将天冬氨酸146突变为丙氨酸（D146A），构建了催化失活突变体（PUS1-Mut）。随后，我们在MYC/Trp53-/-新发肝癌小鼠模型中评估了野生型PUS1（PUS1-WT）与PUS1-Mut的功能影响。我们对两种小鼠模型均开展了高通量测序（high-throughput sequencing）。