c-FLIPL调控PD-L1表达对黑色素瘤PD-1抗体疗效的影响及机制研究

The immune checkpoint inhibitor PD-1 blockade is an important method for the treatment of metastatic melanoma, but the complete remission rate was limited. Studies showed that non-cytokine induced PD-L1 expression was up regulated by oncogene, which could induce T cell exhaustion and increase apoptosis rate and result in tumor immune escape and primary drug resistance. Therefore, down regulation of tumor specific PD-L1 expression by blocking the oncogene signal is very important to improve the curative effect. Our previous studies found that the expression of c-FLIPL was associated with the efficacy of PD-1 blockade, c-FLIPL could up-regulate the expression of PD-L1 and increase the apoptosis of T cells. Moreover, c-FLIPL could interact with AKT and affect its phosphorylation. Therefore, we hypothesize that c-FLIPL can regulate the expression of PD-L1 through the AKT pathway in melanoma and affect the efficacy of PD-1 antibody. In this study, the correlation between c-FLIPL and AKT in melanoma will be analyzed by immune precipitation and GST pull-down, and the effect of c-FLIPL on the stability of PD-L1 protein by regulating the activity of AKT kinase was analyzed by western bolt and QPCR. Furthermore, the effect of inhibiting c-FLIPL on the efficacy of PD-1 antibody will be verified by in vivo experiments. This study is expected to reveal the new mechanism of c-FLIPL regulating immunotherapy in melanoma and provide new combined therapeutic targets for further improving the efficacy of PD-1 blockade.

PD-1抗体作为晚期黑色素瘤的重要疗法目前疗效有限。研究表明非细胞因子诱导的PD-L1表达上调多由癌基因驱动所致，可引起T细胞耗竭并导致肿瘤免疫逃逸及原发耐药，通过阻断癌基因信号而实现肿瘤特异性的PD-L1表达下调对提高疗效至关重要。我们研究发现c-FLIPL高表达的黑色素瘤患者PD-1抗体疗效不佳，c-FLIPL能够上调PD-L1表达并增加T细胞凋亡，且c-FLIPL可与AKT结合并影响其磷酸化。据此我们提出假设：黑色素瘤中c-FLIPL通过AKT调控PD-L1的表达而影响PD-1抗体疗效。本项目拟通过免疫共沉淀、融合蛋白沉降等技术证实c-FLIPL与AKT的相互作用，分析c-FLIPL通过调控AKT激酶活性对PD-L1蛋白稳定性的影响，并在体内实验评价抑制c-FLIPL联合PD-1抗体的疗效。本项目有望揭示c-FLIPL调节免疫治疗的新机制，为提高PD-1抗体疗效提供新的联合治疗靶点。

c-FLIP是外源凋亡通路中的负调控因子,c-FLIPL的表达与黑色素瘤的组织学类型，Clark分级，Ki67指数和患者预后密切相关。我们的前期研究发现c-FLIPL高表达的黑色素瘤患者PD-1抗体疗效不佳，c-FLIPL能够上调PD-L1表达并增加T细胞凋亡。本项目通过动物实验发现黑色素瘤中c-FLIPL表达下调能够提高PD-1抗体的疗效，使肿瘤微环境中招募更多的CD3+T细胞并引起CD8+T细胞绝对数的增加；在肿瘤细胞和淋巴细胞共培养体系当中，c-FLIPL低表达的细胞可通过PD-L1/PD-1通路降低T淋巴细胞的凋亡并促进IFN-γ的释放。我们的研究表明c-FLIPL的表达水平有可能成为预测抗PD-1抗体疗效的标志物，也为开发新的联合免疫治疗靶点提供理论依据。