DataSheet_1_Decreased Expression of Programmed Death Ligand-L1 by Seven in Absentia Homolog 2 in Cholangiocarcinoma Enhances T-Cell–Mediated Antitumor Activity.docx

N6-methyladenosine (m6A) has been reported as an important mechanism of post-transcriptional regulation. Programmed death ligand 1 (PD-L1) is a primary immune inhibitory molecule expressed on tumor cells that promotes immune evasion. In addition, seven in absentia homolog 2 (Siah2), a RING E3 ubiquitin ligase, has been involved in tumorigenesis and cancer progression. However, the role of m6A-METTL14-Siah2-PD-L1 axis in immunotherapy remains to be elucidated. In this study, we showed that METTL14, a component of the m6A methyltransferase complex, induced Siah2 expression in cholangiocarcinoma (CCA). METTL14 was shown to enrich m6A modifications in the 3’UTR region of the Siah2 mRNA, thereby promoting its degradation in an YTHDF2-dependent manner. Furthermore, co-immunoprecipitation experiments demonstrated that Siah2 interacted with PD-L1 by promoting its K63-linked ubiquitination. We also observed that in vitro and in vivo Siah2 knockdown inhibited T cells expansion and cytotoxicity by sustaining tumor cell PD-L1 expression. The METTL14-Siah2-PD-L1–regulating axis was further confirmed in human CCA specimens. Analysis of specimens from patients receiving anti-PD1 immunotherapy suggested that tumors with low Siah2 levels were more sensitive to anti-PD1 immunotherapy. Taken together, our results evidenced a new regulatory mechanism of Siah2 by METTL14-induced mRNA epigenetic modification and the potential role of Siah2 in cancer immunotherapy.

N6-甲基腺苷酸（N6-methyladenosine, m6A）已被证实为一类重要的转录后调控机制。程序性死亡配体1（Programmed death ligand 1, PD-L1）是肿瘤细胞表面表达的核心免疫抑制分子，可介导肿瘤免疫逃逸。此外，七缺失同源物2（seven in absentia homolog 2, Siah2）作为一种环指E3泛素连接酶（RING E3 ubiquitin ligase），已被发现参与肿瘤发生与癌症进展过程。然而，m6A-METTL14-Siah2-PD-L1轴在肿瘤免疫治疗中的作用仍有待阐明。本研究发现，作为m6A甲基转移酶复合物组分之一的METTL14，可在胆管癌（cholangiocarcinoma, CCA）细胞中诱导Siah2的表达。实验结果显示，METTL14能够在Siah2 mRNA的3'非翻译区（3’UTR）富集m6A修饰，进而以YTH N6-甲基腺苷RNA结合蛋白2（YTHDF2）依赖的方式促进该mRNA的降解。此外，免疫共沉淀实验证实，Siah2可通过促进PD-L1的K63连接泛素化，与其发生相互作用。研究还观察到，无论在体外还是体内实验中，敲低Siah2均可通过维持肿瘤细胞PD-L1的表达水平，抑制T细胞的增殖与细胞毒性功能。该METTL14-Siah2-PD-L1调控轴在人类胆管癌组织样本中得到了进一步验证。对接受抗PD-1免疫治疗的患者队列样本进行分析后发现，Siah2低表达的肿瘤组织对抗PD-1免疫治疗的响应更为敏感。综上，本研究结果揭示了METTL14诱导的mRNA表观遗传修饰对Siah2的新型调控机制，同时证实了Siah2在癌症免疫治疗中的潜在应用价值。