一个染色体脆性位点基因调控抗肿瘤免疫的作用及细胞和分子机制研究

In recent years, investigations on the tumor immunotherapy, including immune checkpoint therapy and CAR-T therapy, undergo rapid progress and reveal a very remarkable effect. However, CAR-T therapy could hardly penetrate the solid tumors and the effective rate of immune checkpoints therapy are limited to 20%. Therefore, there is an urgent need to develop new immune target and immune treatment strategies. Chromosomal common fragile sites (CFS) are conservative regions in human genome that to some extent play a protective role against cancer owing to their tendency towards genomic instability under conditions of multiple factors such as microbial toxins, radiation, associated with abnormal DNA replication and tumorigenesis. Bioinformatics analysis showed that CFSs were enriched for genes associated to the immune response and to mechanisms involved in tumor progression. CFS may perform immunoprotective functions and further experimental evidence are warranted. We identify a novel Fragile-site Associated Immune Regulator (termed FAIR). FAIR-/- mice exhibit the slow tumor growth rate, enhanced T cell activation, macrophage toward to M1 type polarization and reversed the immune suppressive tumor microenvironment to anti-tumor immune microenvironment. This project is aimed at disclosing the role and molecular mechanism of FAIR regulating immunosuppressive tumor immune microenvironment as well as the interactions between key immune cells (M1 type macrophage) and CD4, CD8+ T cells. Investigate the role of FAIR-/- (or FAIR knockdown) macrophages and T cells in the tumor treatment and the regulation of tumor immune microenvironment. Uncover the immune regulating function of chromosomal fragile sites gene, develop a novel immune therapeutic target and immune treatment strategies for tumor immunotherapy.

近年来免疫检查点抗体和CAR-T细胞等肿瘤免疫治疗进展迅速、疗效显著。但是CAR-T细胞很难攻破实体瘤，免疫检查点抗体有效率约20%。因此亟待开发新的免疫靶点和治疗策略。染色体脆性位点进化保守，在微生物毒素及辐射等因素诱导下容易断裂重排，导致DNA非正常复制和肿瘤发生，具有一定生物学功能。生物信息学分析显示脆性位点可能具有免疫保护功能，但仍缺少实验证据。我们发现一个位于脆性位点的新的免疫调节因子（命名为FAIR）, FAIR敲除鼠中肿瘤生长极慢，T细胞激活，巨噬细胞向M1型极化，免疫抑制性肿瘤微环境全面逆转。该项目拟在此基础上深入研究FAIR调控免疫抑制性肿瘤免疫微环境中巨噬细胞M1极化及其协同CD4和CD8阳性T细胞调节抗肿瘤免疫的作用和分子机制；研究敲除和敲降FAIR的巨噬细胞和T细胞的肿瘤免疫治疗作用及其对肿瘤免疫微环境的调节作用，揭示脆性位点基因的新功能，开辟肿瘤免疫治疗新途径。

染色体常见脆性位点(CFSs)是特定的易碎基因组区域，常出现在癌症细胞中。E3泛素结合酶FATS（脆性位点相关肿瘤抑制基因）在多种癌症细胞中缺失或突变，具有抑制肿瘤活性，但FATS在免疫细胞中的功能尚不清楚。该项目通过体内及体外实验研究发现FATS调节肿瘤免疫的作用和分子机制。应用小鼠黑色素瘤和胰腺肿瘤移植实验研究发现，与野生型对照组小鼠相比，Fats-/-小鼠皮下移植的黑色素瘤和胰腺肿瘤生长显著减慢。进一步对脾脏和肿瘤内浸润的CD4+、CD8+、Mφ、M1、M2等各种免疫细胞变化分析，以及应用氯膦酸脂质体清除巨噬细胞研究发现Fats-/-小鼠中肿瘤生长减慢是巨噬细胞依赖性的，与肿瘤内巨噬细胞从促肿瘤的M2样巨噬细胞向抗肿瘤的M1样巨噬细胞的表型转变有关。进一步在巨噬细胞中测序研究发现FATS与抗原提呈、T细胞激活、先天性免疫应答、免疫抑制以及趋化因子等分子网络相关。深入研究发现FATS通过抑制上游激酶对IκBα的磷酸化作用抑制IκBα泛素化，进而促进IκBα的细胞质积累和抑制NF-κB的转录活性。即FATS缺乏通过破坏NF-κB/IκBα负反馈回路刺激和延长NF-κB活化，进而促进M1极化。FATS可以调控巨噬细胞的重编程并间接增强CD4+T辅助细胞1型（Th1）和细胞毒性T淋巴细胞（CTL）适应性免疫应答反应，以促进肿瘤消退。过继细胞治疗研究发现，Fats-/-及siRNA敲降的巨噬细胞以及T细胞移植可治疗小鼠黑色素瘤。.总之，本研究发现了一个染色体脆性位点相关的新的肿瘤免疫开关基因，敲除巨噬细胞中的FATS后可全面逆转肿瘤免疫抑制微环境，首次证明染色体脆性位点区基因的肿瘤免疫监视功能，革新了以前一直认为染色体脆性位点断裂导致的基因缺失或者重排诱导肿瘤发生的认识，为将染色体脆性位点区基因作为临床诊断及治疗新靶点提供科学依据。.此项目研究主要内容已发表在Nature Communications杂志，项目共支持发表论文8篇，其中SCI论文7篇，国内领军期刊论文1篇，中文期刊论文1篇。并获得国家发明专利1项及国际PCT专利1项，已申请美国专利。