利用GSK-3β异常剪接对慢粒急变进行早期预警及靶向消除

Treatment may effectively arrest the progress of the chronic-phase chronic myeloid leukemia (CML-CP), but usually does not to help control the rapid deterioration of the blastic crisis (CML-BC). So it is urgent to identify new molecular targets for early diagnosis and targeted therapy of CML-BC. Previous studies from our laboratory and others have revealed that the splicing pattern of glycogen synthase kinase 3β (GSK3β) is not affected by imatinib which is now recognized as the gold standard for CML treatment, but its aberrant splicing (generates misspliced GSK3β transcript) is an early molecular events which leads to the formation of leukemia stem cell and CML-BC. These findings suggest that m-GSK3β could be a special molecular for the diagnosis and therapy of CML-BC. Based on this, we propose to establish a real time quantitative PCR (qRT-PCR) method to detect the expressions of GSK3β and m-GSK3β, and to assess the predictive diagnostic value of m-GSK3β/GSK3β for CML-BC by combining with cross-sectional study and prospective cohort study. In addition, the trans-splicing ribozyme which especially againsts m-GSK3β will be designed and used to selectively induce apoptosis of CML-BC cells. Luciferase reporter assay, cytotoxicity testing, flow cytometry, RT-PCR and western blot will also be used to investigate the molecular mechanism. In short, the main objective of this project is to carry out an exceptionally useful exploration work for early diagnosis and targeted therapy of CML-BC.

慢性粒细胞白血病（CML）慢性期症状可控，一旦急变预后极差，针对急变的早期诊疗亟待寻求突破。我们前期研究发现，糖原合酶激酶3β（GSK3β）剪接模式不受BCR/ABL抑制剂imatinib调控，而文献报道其异常剪接体m-GSK3β导致了CML干细胞形成及病程急性化，提示其有可能作为CML急变早期诊疗的靶点。因此，本研究拟建立荧光定量PCR方法检测GSK3β剪接体，开展临床随访，以横断面研究和前瞻性队列研究相结合，统计分析m-GSK3β/GSK3β比值从而确定早期预警CML急变的cut-off值，评价其诊断效率；探讨应用特殊设计的反式剪接核酶，靶向m-GSK3β并激活自杀基因，诱导体外培养和动物模型体内急变CML细胞凋亡，综合运用荧光素酶实验、细胞毒性检测、流式细胞术、RT-PCR、蛋白印迹等方法，阐明其分子机制。对CML急变诊疗过程中早期预警和靶向治疗这两个紧密相连的重要环节作有益的探索。

慢性粒细胞白血病（chronic myeloid leukemia, CML）慢性期症状可控，一旦急变预后极差，针对急变的早期诊疗亟待寻求突破。糖原合成酶激酶3β（glycogen synthase kinase 3β, GSK3β）是一种丝氨酸/苏氨酸蛋白激酶，其功能失调与多种疾病的发病机制密切相关。近期研究发现，GSK3β基因的异常选择性剪接在CML的病程进展中起着关键作用。本项目中我们首先针对GSK3β及其剪接异构体m-GSK3β建立荧光定量PCR方法检测方法并进行方法学评价，并应用于CML急变的早期预警；随后收集临床代表性病例1）慢性CML患者、2）急变CML患者和3）正常人，针对CML疾病进展过程中的选择性剪接事件进行了高通量转录组测序分析，分析发现随着CML疾病病程的进展，差异选择性剪接事件逐渐增加，异常剪接事件主要聚焦于Wnt信号通路，提示Wnt/GSK-3β/β-catenin通路中基因的选择性剪接水平变化与CML急变相关。同时我们还针对CML一线治疗药物伊马替尼的作用机制进行了探讨，研究了伊马替尼对细胞周期相关基因剪接的影响，发现调控细胞周期相关基因剪接是伊马替尼发挥治疗作用的基础之一。通过本项目研究，课题组共发表中英文期刊论文6篇，参编大型专业书、医学本科教材3部，国际会议论文1篇，培养研究骨干2名、培养研究生5名。