DNA去甲基化药物地西他滨抑制人T细胞中IκBα蛋白甲基化的功能和机制研究

The effects and mechanism of non-histone protein methylation attracts attention nowadays. Recent studies indicated that protein lysine methylation regulates the activation of the intracellular molecules. The DNA demethylating agents such as decitabine have displayed significant clinical responses in cancer treatment. However, the previous studies mainly focused on the regulation of tumor cells while the direct function of these drugs on the host immunocytes was still unclear. Based on the observations from our clinical trials of “low dose decitabine based therapy in patients with refractory and/or chemotherapy resistant solid tumors”, we demonstrated that low-dose decitabine promoted the activation, proliferation and cytolytic capacity of CTL cells probably by inducing NF-κB activation through the downregulation of IκBα. Moreover, we observed the IκBα methylation level was decreased using the pan-lysine methylation antibody. Thus, we supposed that decitabine could be the novel “immunosensitizer” and possess the capacity to “regulate the non-histone protein methylation”. In this project, we intend to investigate the different effects of low-dose decitabine in controlling the proliferation between distinct T lymphocyte subsets and tumor cells; analyze the regulation mechanism of IκBα protein methylation and NF-κB activation following low dose decitabine treatment; clarify the novel role of decitabine in non-histone protein methylation regulation and the detailed anti-tumor T cell immunity of low-dose decitabine in solid tumor treatment; and explore the anti-tumor immunity-related biomarkers of clinical reactivity in response to this epigenetic therapy, to eventually realize the precision medicine.

目前非组蛋白甲基化修饰调控效应及其机制受广泛关注，胞内信号分子赖氨酸甲基化在其活化和调控中发挥重要作用。DNA去甲基化药物如地西他滨等已证实具有抗肿瘤临床疗效，但以往多关注药物对肿瘤细胞的调节而对机体免疫细胞的直接作用尚不清楚。在本团队低剂量去甲基化药物治疗实体瘤的临床试验中，我们发现低剂量地西他滨能够增强T细胞活化、增殖及抗肿瘤杀伤活性，可能通过促进T细胞IκBα降解而激活NF-κB信号通路，且观察到IκBα蛋白甲基化程度降低。据此，我们开创性提出地西他滨享有“免疫增效”效应，且具有“调控非组蛋白甲基化”全新作用机理。本课题拟研究地西他滨对不同T细胞亚群及肿瘤细胞差异调控效应，进而解析地西他滨对IκBα蛋白甲基化及NF-κB活化的调控作用机制，阐明低剂量地西他滨抗肿瘤免疫效应及其对胞内信号分子甲基化调控新机制，并从机体免疫功能角度挖掘地西他滨表观治疗临床反应性预测指标，实现肿瘤精准治疗。

肿瘤发生发展过程中，除了遗传学突变，表观遗传调控异常同样作为重要致癌因素，导致肿瘤治疗抵抗并逃逸机体免疫监视。DNA去甲基化表观遗传修饰药物地西他滨已用于血液系统肿瘤治疗，但在实体瘤中单药作用有限，表观药物联合治疗的最佳治疗策略及其全面系统抗瘤作用机制有待深入探讨。生物治疗科团队国际注册开展了应用低剂量去甲基化药物地西他滨联合治疗复发难治性实体瘤的临床试验，临床研究发现，在复发难治性卵巢癌及消化道肿瘤中，低剂量地西他滨具有化疗增敏作用，且联合CIK免疫细胞治疗能够进一步显著提高疾病反应率及持久性。在复发难治性霍奇金淋巴瘤中，低剂量地西他滨与PD-1抗体联合治疗，肿瘤完全缓解率显著提高。体内外实验证明低剂量地西他滨能够直接促进T淋巴细胞增殖、活化及其杀伤活性。对于CD4+ T细胞，低剂量地西他滨促进IFN-γ+ Th1细胞增殖及活性，而NF-κB通路抑制剂能够显著减弱此效应。具体机制，地西他滨上调泛素连接酶β-TrCP 表达水平，促进IκBα泛素化及降解，进而导致NF-κB激活和IFN-γ的合成。因此，我们提出地西他滨调控新模式，通过促进IκBα降解及NF-κB活化而增强IFN-γ+CD4+ T细胞激活与抗瘤功能。