EZH2和MLL2基因突变在滤泡性淋巴瘤发生中的作用及分子机制

Follicular lymphoma is a common subtype of B cell lymphoma derived from germinal center B cell, of which tumorigenesis is unclear at present. EZH2 and MLL2 belong to the histone 3(H3) methyltransferase, which catalyze the methylation of the lysine 27(K27）and 4(K4) residue of H3 respectively. Methylation of H3K27 is a repressive mark of gene expression, and methylation of H3K4 is an active mark on the contrary, which is the important epigenetic control playing important role in normal embryonic development and tissue differentiation. According to references and our study, EZH2 Y641 and MLL2 mutated in 7.2%-30.7% and 89% follicular lymphoma respectively,and 30.7% follicular lymphoma harboring EZH2 and MLL2 mutation simultaneously. It has been demonstrated that EZH2 Y641 mutation is gain-of-function, and MLL2 mutation is loss-of-function.We detected the frequency of EZH2 mutation in follicular lymphoma and analyzed the gene expression profiling of follicular lymphoma with or without EZH2 mutation.A group of significantly differential expression genes was found between the two cohorts.The function and significance of certain differentially expressed gene was studied, however,it is unclear about the biology significance of the histone methyltransferase EZH2 and MLL2 mutants in normal germinal center B cell development and malignant transformation.Simultaneous mutation of EZH2 and MLL2 probably result in the imbalance of the epegenetic control of gene expression in germinal center B cell development and enhance the tumorigenesis of follicular lymphoma.On the basis of our previous study, we are planning to deeply investigate the role of EZH2 and MLL2 mutation in the tumorigenesis of follicular lymphoma, the role of both mutants in controlling proliferation and differentiation of germinal center B lymphocytes. The project is expected to expand the study of epigenetic control of proliferation and differentiation of germinal center B lymphocytes, to explore the new mechanism of tumorigenesis of follicular lymphoma, and to provide the basis for specific rational combinatorial targeted therapy for follicular lymphoma.

滤泡性淋巴瘤是生发中心B淋巴细胞来源的一种常见恶性肿瘤，EZH2和MLL2分别催化组蛋白H3K27和H3K4甲基化，是一对调控生发中心B细胞正常发育的表观遗传学开关。文献和我们的研究表明，滤泡性淋巴瘤中存在EZH2功能获得性突变（7.2-30.7%）和MLL2功能缺失性突变（89%），30.7%滤泡性淋巴瘤同时有二者的突变，但EZH2和MLL2同时突变的生物学意义及其在滤泡性淋巴瘤发生中的作用及分子机制尚不清楚。我们对EZH2突变和野生型的滤泡性淋巴瘤基因表达谱分析，发现EZH2突变的滤泡性淋巴瘤，B淋巴细胞增殖、分化和凋亡基因异常表达。本课题拟在此基础上，深入研究EZH2和MLL2突变对生发中心B淋巴细胞发育的影响及分子机制；揭示EZH2和MLL2突变在滤泡性淋巴瘤发生中的作用和分子机制；阐明其在滤泡性淋巴瘤发生中的意义。本研究可发现滤泡性淋巴瘤发生的新机制，为分子靶向治疗提供理论依据。

滤泡性淋巴瘤和弥漫大B细胞淋巴瘤（Diffuse large B cell lymphoma, DLBCL）是两种最常见的恶性淋巴瘤，组蛋白甲基转移酶EZH2和MLL2分别催化组蛋白H3K27和H3K4甲基化而调控靶基因的转录，本课题研究了滤泡性淋巴瘤和DLBCL中EZH2和MLL2基因突变及意义。首先研究EZH2和MLL2蛋白在正常淋巴结的表达，结果发现EZH2和MLL2仅在淋巴滤泡生发中心B细胞强表达，具有时间和空间的有序性表达的特点。而在滤泡性淋巴瘤和DLBCL中EZH2蛋白的表达丧失时了正常淋巴结的时空有序性表达特征，60% 滤泡性淋巴瘤和75.4% DLBCL均有EZH2蛋白过表达，而且与肿瘤细胞H3K27me3高水平、高侵袭性和预后差有显著相关，35.85%和52.84% DLBCL有MLL2蛋白过表达和H3K4me3高水平，但未发现MLL2蛋白过表达与滤泡性淋巴瘤和DLBCL临床病理学特征的显著相关性。进一步研究EZH2和MLL2基因突变，结果发现EZH2 的SET结构域第641位酪氨酸（Y641）编码基因是突变热点，且仅发生于滤泡性淋巴瘤和生发中心型DLBCL，突变率分别为16.9%和19.5%，共有5种突变类型，包括Y641F、Y641S、Y641N、Y641H和Y641C。EZH2基因Y641点突变为功能获得性突变，导致H3K27 me3水平增高，使参与生发中心B细胞分化的基因如TCF4等表达降低。同时，EZH2基因Y641点突变与凋亡抑制基因BCL2异常密切相关。同时，EZH2基因突变的滤泡性淋巴瘤凋亡促进基因BIK的表达也显著降低。此外，对特殊类型DLBCL包括胃肠道原发性DLBCL和中枢神经系统原发性DLBCL的研究显示EZH2蛋白过表达提示预后差。但未发现MLL2异常与滤泡性淋巴瘤和DLBCL临床病理学特征之间的显著相关性。总之，EZH2在滤泡性淋巴瘤和DLBCL中发生突变和蛋白异常表达，而且提示肿瘤预后差。EZH2基因突变和蛋白质异常表达使B淋巴细胞锁定在淋巴滤泡生发中心而不能正常分化，并与BCL2基因协同作用促进滤泡性淋巴瘤和DLBCL的发生。