Biological consequences of a lymphoma-associated mutation in Ezh2 in mice

小鼠 Ezh2 淋巴瘤相关突变的生物学后果

• 批准号: 8309668
• 负责人: ANN J FEENEY
• 金额: $ 23.69万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8309668
• 关键词: Accounting; Address; Affect; Alleles; Antigens; B cell differentiation; B-Lymphocytes; Biological; CD2 gene; Catalytic Domain; Cells; ChIP-seq; Complementary DNA; Complex; DNA; Disease; Environment; Enzymes; Epigenetic Process; Frequencies; Future; Gene Expression; Gene Expression Profiling; Gene Silencing; Gene Targeting; Generations; Genes; Genetic Polymorphism; Genome; Histology; Histone H3; Histones; Human; Hypermethylation; Immunization; Inbred Strains Mice; Individual; Lead; Lymphoma; Lymphomagenesis; Lysine; Malignant Neoplasms; Memory B-Lymphocyte; Messenger RNA; Methylation; Methyltransferase; Monitor; Mono-S; Mus; Mutant Strains Mice; Mutation; Non-Hodgkin' s Lymphoma; Other Genetics; PRC1 Protein; Patients; Pattern; Plasma Cells; Polycomb; Population; Post-Translational Protein Processing; RNA; Repression; Role; Sampling; Staging; Structure of germinal center of lymph node; Testing; Therapeutic; Time; Trisomy; Tumor Suppressor Genes; Wild Type Mouse; cancer type; design; gain of function mutation; gene repression; genome-wide; inhibitor/antagonist; insight; large cell Diffuse non-Hodgkin' s lymphoma; mouse model; mutant; neoplastic cell; prevent; promoter; protein complex; therapeutic target; tumor; tumorigenesis; uH2A

DESCRIPTION (provided by applicant): Ezh2 is the enzymatic component of Polycomb Repressive Complex 2, and it catalyzes the methylation of lysine 27 (K27) of histone H3, a histone post-translational modification associated with repressed genes. Many cancer types are characterized by gene inactivation. Ezh2 mRNA levels are increased in a variety of tumors, consistent with a role in repressing critical genes. Recently, however, a specific mutation in the catalytic site of Ezh2 (Y641) that hyperactivates its H3K27 tri-methyltransferase activity while eliminating its ability to mono- methylate H3K27 been detected with high frequency in one specific subtype of cancer: the germinal center (GC) B cell subtype of diffuse large B cell lymphoma (GCB-DLBCL). This mutation occurs only in the tumor cell, not in the germline DNA, and therefore is a somatically acquired and positively selected mutation. Such a unique gain of function mutation is unprecedented. Ezh2 is highly expressed in normal GC B cells, but is low in na¿ve B cells. We hypothesize that Ezh2 expression is increased in GC B cells to repress a specific subset of genes that must be downregulated to allow the differentiation of a na¿ve B cell into a GC B cell, and/or of a GC B cell into a post-GC B cell. We further hypothesize that the Y461F mutation will result in over-repression of a subset of genes that normally are activated or re-activated in order for a B cell to exit the GC compartment. If this hypothesis is correct, then we predict that this over-repression might retain the GC B cells in the mutagenic environment of the GC longer than usual. The direct effect to B cells of this Ezh2 mutation cannot be directly addressed in patients' lymphoma samples, since the genome of each individual is unique, and because the lymphomas may have translocations or a variety of other genetic or epigenetic changes. Here we will directly test the effect of this hyperactive Ezh2 in an inbred strain of mouse, so that we can unambiguously determine the downstream consequences of this lymphoma-associated mutation. We will make a mouse with the Ezh2 Y641F targeted into the ROSA26 locus. The targeting construct has a floxed transcriptional stop between the promoter and Ezh2 Y641F. When crossed to C¿1-Cre mice, the mutant Ezh2 will be expressed in GC B cells. We will determine if Ezh2 Y641F, with all of its downstream epigenetic, transcriptional and biological consequences, will be sufficient to lead to lymphoma. We will determine if it results in delayed exit from the GC, which may provide additional time in the mutagenic environment of the GC. We will perform gene expression profiling of GC B cells from Ezh2 Y641F and WT mice, and also ChIP-seq for H3K27me3. We will determine the downstream consequences of the altered enzymatic activity of Ezh2 Y641 by ChIP-seq for PRC1 components and ubiquitinated H2AK119, which is catalyzed by PRC1. The proposed studies will provide insight into the downstream effects of this mutation on gene expression, epigenetic profile, B cell differentiation after antigen stimulation, and lymphomagenesis. If this Ezh2 Y641 is sufficient in itself to result in lymphomagenesis, then specific inhibitors of the mutant Ezh2 could potentially be designed as a therapeutic. ) PUBLIC HEALTH RELEVANCE: Diffuse large B cell lymphoma (DLBCL) is an aggressive disease that accounts for 40% of all non- Hodgkin's lymphomas. A substantial fraction of the GCB subset of DLBCL have a unique mutation in the catalytic domain of the enzyme Ezh2 which marks genes for repression. Here we will test whether this hyperactive Ezh2 by itself is sufficient to cause lymphoma in a mouse model; if so, then allele- specific inhibitors of the mutant Ezh2 could well be a future therapeutic target for GCB-DLBCL patients with this mutation.)

描述（由申请人提供）：Ezh2 是 Polycomb 抑制复合物 2 的酶成分，它催化组蛋白 H3 的赖氨酸 27 (K27) 甲基化，这是一种与抑制基因相关的组蛋白翻译后修饰。然而，Ezh2 mRNA 水平在多种肿瘤中增加，这与抑制关键基因的作用一致。 Ezh2 (Y641) 催化位点的特定突变会过度激活其 H3K27 三甲基转移酶活性，同时消除其单甲基化 H3K27 的能力，这种突变在一种特定癌症亚型：生发中心 (GC) B 细胞亚型中被高频率检测到弥漫性大 B 细胞淋巴瘤 (GCB-DLBCL) 的突变仅发生在肿瘤细胞中，而不发生在种系 DNA 中，因此是体细胞获得性突变。这种独特的功能增益突变在正常 GC B 细胞中高表达，但 na¿我们发现GC B细胞中Ezh2表达增加，以抑制必须下调的特定基因子集，以允许na的分化。将B细胞转化为GC B细胞，和/或将GC B细胞转化为GC后B细胞。 Y461F 突变将导致 B 细胞正常情况下被激活或重新激活的基因子集被过度抑制，如果这个假设是正确的，那么我们预测这种过度抑制可能会保留 B 细胞。 GC B 细胞在 GC 诱变环境中的存活时间比平常长，这种 Ezh2 突变对 B 细胞的直接影响无法在患者的淋巴瘤样本中直接确定，因为每个个体的基因组都是独特的，而且淋巴瘤可能具有相同的特征。在这里，我们将直接测试这种高度活跃的 Ezh2 在近交系小鼠中的影响，以便我们能够明确地确定这种淋巴瘤相关突变的下游后果。当 Ezh2 Y641F 靶向 ROSA26 基因座时，靶向构建体在启动子和 Ezh2 Y641F 之间有一个 floxed 转录终止点。在 1-Cre 小鼠中，突变型 Ezh2 将在 GC B 细胞中表达，我们将确定 Ezh2 Y641F 及其所有下游表观遗传、转录和生物学后果是否足以导致淋巴瘤。 延迟退出 GC，这可能会在 GC 的诱变环境中提供额外的时间。我们将对 Ezh2 Y641F 和 WT 小鼠的 GC B 细胞进行基因表达谱分析，并对 H3K27me3 进行 ChIP-seq。通过 ChIP-seq 检测 PRC1 成分和泛素化 H2AK119 的 Ezh2 Y641 酶活性改变，即拟议的研究将深入了解该突变对基因表达、表观遗传谱、抗原刺激后 B 细胞分化和淋巴瘤发生的下游影响。如果 Ezh2 Y641 本身足以导致淋巴瘤发生，则可以使用特异性抑制剂。突变体 Ezh2 可能被设计为一种治疗药物。 公共健康相关性：弥漫性大 B 细胞淋巴瘤 (DLBCL) 是一种侵袭性疾病，占所有非霍奇金淋巴瘤的 40%。DLBCL 的 GCB 亚型的很大一部分在酶 Ezh2 的催化结构域中具有独特的突变。在这里，我们将测试这种过度活跃的 Ezh2 本身是否足以在小鼠模型中引起淋巴瘤；如果是的话，那么突变 Ezh2 的等位基因特异性抑制剂很可能成为具有该突变的 GCB-DLBCL 患者未来的治疗靶点。）