m6A去甲基化酶ALKBH5促进肝内胆管癌PD-L1表达参与免疫逃逸的机制研究

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary hepatic malignancy. The prognosis of ICC after curative resection remains extremely dismal because of high recurrence rates and lack of effective therapies. Blocking PD-1/PD-L1 signal is the curent emphasis of cancer therapy, and the associated clinical trails is ongoing in ICC. Immune cell infiltration is the basis of cancer immune therapy and is partly controlled by PD-1/PD-L1 immune check-point axis signal. N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic mRNAs. RNA m6A modification has been reported to play important roles in physiological processes, but its role in cancer is poorly studied. Especially, there is no report about the role of RNA m6A modification in ICC or in tumor immune microenvironment. Our pre-experiments found that the ALKBH5 expression level positively correlates with the recurrence-free survival of ICC patient. Further, the protein level of ALKBH5 significantly correlates with that of PD-L1 in human ICC tumor tissue. Moreover, knockdown of ALKBH5 in ICC cell lines significantly decreases the protein level of PD-L1, which can be reversed upon global methylation inhibitor treatment. Therefore, we hypothesize that ALKBH5 regulates PD-L1 expresison via RNA m6A modification, thereby promoting ICC immune escape and impacting anti-PD-L1 immunotherapy. This project is planned to: 1) confirm the clinical significance of ALKBH5/PD-L1 in ICC patients; 2) clarify the “ALKBH5-m6A-PD-L1-tumor immune escape” axis; 3) explore the impact of ALKBH5 on anti-PD-L1 immunotherapy in hCD34+ humanized mice. The results will broaden our knowledge on RNA m6A methylation modification, and provide theoretical basis or new ideas for ICC immunotherapy.

肝内胆管癌（ICC）恶性程度极高，目前尚无有效抗复发转移手段。阻断PD-1/PD-L1信号是当前肿瘤免疫治疗的热点。RNA m6A甲基化修饰是mRNA上最常见的表观修饰，已报道参与很多生理过程，然而其是否参与肿瘤免疫调节尚未见报道。前期我们发现ICC患者癌组织m6A去甲基化酶ALKBH5和PD-L1表达水平显著正相关；多种ICC细胞敲降ALKBH5显著下调PD-L1水平，使用甲基化抑制剂可逆转该现象，因此我们推测ALKBH5通过m6A修饰调控PD-L1表达从而促进肿瘤免疫逃逸。本项目拟：1）临床病理和随访资料明确ALKBH5和PD-L1表达的临床意义；2）体外实验明确ALKBH5调控PD-L1水平促进肿瘤细胞免疫逃逸的分子机理；3）人源嵌合小鼠探究ALKBH5对PD-L1/PD-1抗体治疗反应性的影响。研究结果将加深我们对m6A甲基化修饰的认识，为ICC免疫治疗提供理论依据或新思路。

N6-甲基腺苷 (m6A) 修饰是一种重要的转录后调控机制。程序性死亡配体1 (PD-L1) 是一种主要的免疫抑制分子，在肿瘤细胞上表达以促进免疫逃避。在本课题中，我们证实了ALKBH5通过其m6A去甲基化酶的功能调控肝内胆管癌 (ICC) 肿瘤细胞PD-L1的表达。首先，细胞实验证实ALKBH5调控ICC细胞PD-L1表达。m6A-seq发现PD-L1 mRNA为m6A修饰的直接靶标，其水平受ALKBH5调节。此外，我们通过RIP实验了验证ALKBH5和PD-L1 mRNA之间的相互作用。在分子机制上，敲降ALKBH5增加PD-L1 mRNA 3'UTR区域的m6A修饰，并以YTHDF2依赖性方式促进PD-L1 mRNA降解。此外，我们证实了肿瘤内源性ALKBH5通过在体外和体内维持肿瘤细胞PD-L1表达来抑制T细胞扩增和细胞毒性作用。ALKBH5-PD-L1调节轴在人ICC标本中得到进一步证实。单细胞质谱流式检测人源小鼠皮下瘤免疫细胞结果进一步揭示了ALKBH5可以促进PD-L1 在单核细胞/巨噬细胞上的表达和减少MDSC细胞的浸润。最后，对接受anti-PD1免疫治疗的患者标本的研究表明，肿瘤细胞高表达ALKBH5的患者对anti-PD1免疫治疗更敏感。综上所述，本课题研究了ALKBH5对PD-L1 mRNA表观遗传水平上新调控机制以及ALKBH5在免疫治疗中的潜在作用，这可能为癌症免疫治疗策略提供新思路。