PD-L1 N-糖基化提高MSI结直肠癌免疫治疗疗效的分子机制研究

Clinical studies have confirmed that microsatellite instable colorectal cancer patients (MSI) are more likely to benefit from PD-L1 antagonist therapy than microsatellite stable patients (MSS), but the reasons remain unknown. In our previous studies, we found that compared with that of MSS, the molecular weight of PD-L1 in clinical specimens and cells of MSI ususlly increased 12KD, which has been proved to be caused by the N-glycosylation of PD-L1 through mass spectrometric analysis. N-glycosylated PD-L1 would be much more stable and therefore less susceptible to degradation. However, phosphorylated PD-L1 by GSK3β would be easier to degrade (the specific phosphorylation sites were unknown). We further identified N192/219 as the critical site for the stability PD-L1 among four potential glycosylation sites. Taken together, we assume that the reason why MSI patients are sensitive to PD-L1 antagonistic therapy is that PD-L1 is less likely be degraded after being N-glycosylated, while MSS patients with deficient expressions of PD-L1 are insensitive to the therapy due to low level of N-glycosylation but high phosphorylation. This project intends to use gene knockout, PDX model combined with T cell killing test and other methods, combined with clinical verification, to clarify the above molecular mechanisms, providing a theoretical basis for immunotherapy.

临床研究证实，较之微卫星稳定结直肠癌（MSS）患者，微卫星不稳定（MSI）患者易从PD-L1单抗治疗中获益，机制不明。我们的研究发现：较之MSS，MSI组织及细胞株中PD-L1分子量发生改变：升高12KD；质谱分析证实分子量升高源于PD-L1被N-糖基化，且糖基化修饰后更稳定不易降解，反之GSK3β通过磷酸化PD-L1（位点未知）促进其降解；在PD-L1四个潜在N-糖基化位点中鉴定出N192/219为稳定PD-L1的关键位点。据此，我们提出MSI者对PD-L1单抗治疗反应良好的分子机制：PD-L1的N192/219被N-糖基化后不易被降解，表达量高，故对PD-L1单抗治疗有效；MSS患者低N-糖基化的PD-L1被GSK3β磷酸化降解，表达量低，故对PD-L1单抗治疗无效。本项目拟采用基因敲除、PDX模型结合T细胞杀伤实验等方法，结合临床验证，阐明以上分子机制，为基于此的免疫治疗提供理论依据

PD-L1单抗联合新辅助放化疗已经成为了肿瘤内科治疗的“新宠”，越来越多的患者通过抗PD-L1的免疫治疗延长了生存期。我们发现，较之微卫星稳定的结直肠癌（MSS）患者，微卫星不稳定（MSI）患者更易从PD-L1单抗治疗中获益。那么，其机制是什么呢？本研究发现：较之MSS，MSI患者肿瘤组织或MSI直肠癌细胞株中PD-L1分子量明显升高；质谱分析证实分子量升高源于PD-L1被N-糖基化，且糖基化修饰后PD-L1蛋白更稳定，半衰期增加。通过生物学预测、点突变等技术我们发现PD-L1蛋白的糖基化位点为N35、N192、N200、N219，其中N192/219为抑制PD-L1泛素化降解的关键位点。在对PD-L1糖基化修饰上游基因的探索中我们发现，p53通路下游的糖基转移酶Mgat4a、Mgat5、Pomgnt1能够糖基化修饰PD-L1,同时PD-L1的糖基化响应于放射产生的肿瘤细胞损伤。本项研究阐明了部分直肠癌患者放疗抵抗或者PD-L1靶向治疗失败的原因，为结直肠癌的PD-L1/PD-1 免疫治疗和新辅助治疗的联合治疗提供了理论基础。