肿瘤细胞内在PD-L1直接激活AKT/mTOR信号通路对弥漫大B细胞淋巴瘤代谢与生长的影响及调控机制研究

The interaction between PD-L1 (Programmed death ligand 1), which is reported to overexpress in different tumors, and PD-1 (Programmed death 1), which is reported to express on the surface of T cells, leads to the suppression of T-cell function through PD-1 and then further facilitates the immune escape of tumor. However, the report that PD-L1 is involved in this signal transduction is rare. Our group has previously demonstrated that PD-L1 overexpressed in diffuse large B-cell lymphoma (DLBCL) through several genetic alterations including gene translocation. After stimulating the PD-L+ DLBCL cells using recombinant human PD1/Fc, the AKT/mTOR pathway in DLBCL was directly activated, which was validated in primary DLBCL lymphoma. Second-generation sequencing analysis found that the metabolism and growth-related genes were regulated by this signal transduction. In this project, we will utilize the cellular and molecular biology techniques and mice transplanted tumor model to systematically issue the effect and mechanism of tumor cell-intrinsic PD-L1-mediated metabolism and growth of DLBCL through directly activating AKT/mTOR signaling. And we will also explore the theoretical basis for the clinical application of PD-1/PD-L1 immunotherapy combined with mTOR targeted drug therapy as the followings: (1) To investigate that tumor cell-intrinsic PD-L1 regulates the metabolism and growth in DLBCL. (2) To explore the relationship between PD-L1 expression and the activation of metabolic and growth signaling in primary DLBCL. (3) To evaluate the value of PD-L1 blockage combined with mTOR targeted drug therapy.

肿瘤细胞过表达PD-L1与T细胞表达PD-1结合通过PD-1介导T细胞功能缺失，促进肿瘤免疫逃逸；然而PD-L1参与信号传导的研究甚少。我们前期研究发现DLBCL淋巴瘤通过基因易位等遗传学改变介导PD-L1过表达；重组人PD-1刺激PD-L1阳性DLBCL细胞可直接激活AKT/mTOR信号通路，并在原发肿瘤中得到验证；测序分析发现代谢与生长相关基因被该信号传导所调控。本项目我们将利用细胞分子生物学技术和小鼠移植瘤模型，从三方面系统深入阐明肿瘤细胞内在PD-L1直接激活AKT/mTOR信号通路对DLBCL代谢与生长的影响及调控机制，并探索PD-1/PD-L1免疫治疗联合mTOR靶向药物治疗临床应用的理论基础：（1）肿瘤细胞内在PD-L1对DLBCL代谢与生长的调控；（2）原发DLBCL病例PD-L1表达与代谢和生长途径活化的关联；（3）阻断PD-L1联合mTOR靶向药物治疗DLBCL的价值。

弥漫大B细胞淋巴瘤(Diffuse large B-cell lymphoma, DLBCL)是最常见恶性淋巴瘤，免疫检查点抗体在部分DLBCL如原发中枢、原发睾丸、EBV阳性等患者中展现可佳疗效，然而PD-L1基因遗传机制及癌基因活化有待进一步研究。我们在该研究中发现PD-L1新伙伴基因SP140，断裂位于PD-L1的3’-UTR段；还发现PD-L1与PD-L2倒位，这些基因遗传学改变可致PD-L1 mRNA和蛋白水平上调，且偏向于发生在Non-GCB。同时揭示了DLBCL中PD-L1和PDL-2新突变位点，这些突变致其蛋白表达下调。该研究丰富了PD-L1的遗传学内容。阐明了美罗华时代，利妥昔单抗不能改善PD-L1阳性DLBCL患者的生存。揭露了DLBCL两种新免疫检查点即Tim-3/galectin-9和CD73/A2aR的遗传特征，发现galection-9涉及3种突变即c.13G>A、c.905G>A和c.716A>G，分别影响着galection-9蛋白第5、第302和第239位氨基酸，其中c.905G>A为有害突变，伴Tim-3+TIL的患者分期晚，IPI评分高，并且预后差，这部分患者肿瘤微环境T细胞处于更严重的免疫耗竭状态。发现CD73 A62V和M379T两种新突变形式，这两种突变使CD73表达下调，进而改变其功能，减弱免疫抑制效应；经公共数据库数据证实了发生CD73改变的患者具有更长的长期生存。揭示了A2aR伴有R205和N359S突变。根据PD-1和A2aR，定义了不同耗竭程度的T细胞，这些患者具有极差预后。揭露了DLBCL中PD-L1可直接激活胞内AKT/mTOR通路，影响细胞代谢和生长基因eIF4E、PIKFYVE、TBC1D4、BCL2、MDM2、BCL2L11、Bax、TP53。然而在T细胞淋巴瘤中则直接激活ERK通路，体现PD-L1功能异质性。