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

描述（由申请人提供）：Ezh 2是Polycomb抑制复合物2的酶组分，催化组蛋白H3赖氨酸27（K27）的甲基化，这是一种与抑制基因相关的组蛋白翻译后修饰。许多癌症类型的特征在于基因失活。Ezh 2 mRNA水平在多种肿瘤中增加，与抑制关键基因的作用一致。然而，最近，在一种特定的癌症亚型中高频率地检测到Ezh 2（Y 641）的催化位点中的特异性突变，其过度激活其H3 K27三甲基转移酶活性，同时消除其单甲基化H3 K27的能力：弥漫性大B细胞淋巴瘤（GCB-DLBCL）的生发中心（GC）B细胞亚型。这种突变仅发生在肿瘤细胞中，而不是在生殖系DNA中，因此是体细胞获得的正选择突变。如此独特的功能突变增益是前所未有的。Ezh 2在正常GC B细胞中高表达，但在幼稚B细胞中低表达。我们假设Ezh 2在GC B细胞中的表达增加是为了抑制特定的基因亚群，这些基因必须被下调以允许幼稚B细胞分化为GC B细胞，和/或GC B细胞分化为GC后B细胞。我们进一步假设， Y 461 F突变将导致过度抑制通常被激活或再激活以使B细胞离开GC区室的基因子集。如果这个假设是正确的，那么我们预测这种过度抑制可能使GC B细胞在GC的致突变环境中保持的时间比通常更长。这种Ezh 2突变对B细胞的直接影响不能在患者的淋巴瘤样品中直接解决，因为每个个体的基因组是独特的，并且因为淋巴瘤可能具有易位或各种其他遗传或表观遗传变化。在这里，我们将直接测试这种过度活跃的Ezh 2在近交系小鼠中的作用，这样我们就可以明确地确定这种淋巴瘤相关突变的下游后果。我们将制备具有靶向ROSA 26基因座的Ezh 2 Y 641 F的小鼠。靶向构建体在启动子和Ezh 2 Y 641 F之间具有floxed转录终止。当与C1-Cre小鼠杂交时，突变体Ezh 2将在GC B细胞中表达。我们将确定Ezh 2 Y 641 F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。我们将确定是否会导致 延迟从GC中退出，这可能会在GC的诱变环境中提供额外的时间。我们将对Ezh 2 Y 641 F和WT小鼠的GC B细胞进行基因表达谱分析，并对H3 K27 me 3进行ChIP-seq。我们将通过ChIP-seq确定Ezh 2 Y 641的酶活性改变对PRC 1组分和泛素化H2 AK 119的下游影响，后者由PRC 1催化。该研究将深入了解该突变对基因表达、表观遗传学特征、抗原刺激后B细胞分化和淋巴瘤发生的下游影响。如果Ezh 2 Y 641本身足以导致淋巴瘤发生，那么突变体Ezh 2的特异性抑制剂可能被设计为治疗剂。) 公共卫生相关性：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%。大部分DLBCL的GCB亚群在酶Ezh 2的催化结构域中具有独特的突变，其标记基因用于抑制。在这里，我们将测试这种过度活跃的Ezh 2本身是否足以在小鼠模型中引起淋巴瘤;如果是这样，那么突变体Ezh 2的等位基因特异性抑制剂很可能是具有这种突变的GCB-DLBCL患者的未来治疗靶标。

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.)