Mechanistic and proteomic analyses of NF-kappaB-driven lymphomas

NF-қB家族转录因子c-Rel基因位点在霍奇金淋巴瘤、弥漫性大B细胞淋巴瘤（DLBCL）和原发性纵隔B细胞淋巴瘤（PMBCL）中经常扩增。此外，在DLBCL中检测到缺乏外显子10的过度活跃的c-Rel剪接变体。我们最近发现外显子10编码一个功能性的聚合位点。因此，我们的目的是阐明c-Rel过表达和选择性剪接在淋巴瘤发生过程中的作用。为此，我们在细菌人工染色体（BAC）上生成了转基因小鼠c-Rel基因组位点。这些修饰包括将人类第10号外显子的小鼠同源物第1位加在第9号外显子的侧翼，将c-Rel与GFP的n端融合，引入c-末端3x FLAG标签及其组合。BACs受内源性启动子或CAG启动子的条件控制而过表达。我们用BACs重组了c-Rel敲除mef，并表明c-Rel及其剪接变体及其融合蛋白可以以条件方式显着过表达。我们现在正在培育bac转基因小鼠。我们将评估c-Rel过表达和选择性剪接在小鼠B细胞发育和功能中的影响。它们在淋巴瘤形成过程中的作用将与我们团队和合作伙伴提供的其他致癌修饰物结合进行评估。这些研究将通过shrna介导的c-Rel在相关人类淋巴瘤系中的敲除来补充。通过细胞系和患者样本的研究，人们对各种人类淋巴瘤实体的差异基因表达谱了解甚多。然而，目前尚不清楚mRNA水平的差异在多大程度上与全球范围内蛋白质含量的差异相关。为了解决这个问题，我们希望与Matthias Mann合作，采用定量质谱法的最新进展。我们生成了一种淋巴瘤super-SILAC混合物，并将其用于尖入方法，以确定和量化5个活化B细胞（ABC）样DLBCL和5个生发中心B细胞（GCB）样DLBCL细胞系和4个B细胞慢性淋巴细胞白血病（BCLL）患者样本的蛋白质组。在最初的无监督分层分析中，样本根据其分类（ABC-, GCB-DLBCL， BCLL）聚类。我们现在希望将我们的范围扩展到合作者提供的大量患者样本。蛋白质组数据将与可用的基因和miRNA表达数据相关联，以产生独特完整的分子表征。

The gene locus of the NF-қB family transcription factor c-Rel is frequently amplified in Hodgkin, diffuse large B cell (DLBCL) and primary mediastinal B cell (PMBCL) lymphoma. In addition, a hyperactive c-Rel splice variant lacking exon 10 has been detected in DLBCL. We recently discovered that exon 10 encodes a functional sumoylation site. We therefore aim to elucidate the role of c-Rel overexpression and alternative splicing during lymphomagenesis. To this end we have generated genetically modified mouse c-Rel genomic loci on bacterial artificial chromosomes (BAC). The modifications include flanking exon 9, the mouse homologue of human exon 10, with Frt sites, N-terminal fusion of c-Rel with GFP, introduction of a C-terminal 3x FLAG tag and combinations thereof. The BACs are under control of the endogenous promoter or under conditional control of the CAG promoter for overexpression. We have reconstituted c-Rel knockout MEFs with the BACs and shown that c-Rel, its splice-variant and its fusion proteins can be dramatically overexpressed in a conditional fashion. We are now in the process of generating BAC-transgenic mice. We will evaluate the impact of c-Rel overexpression and alternative splicing during B cell development and function in the mouse. Their role during lymphomagenesis will be evaluated in combination with other oncogenic modifications available in our group and from our collaboration partners. These studies will be complemented by shRNA-mediated knock-down of c-Rel in relevant human lymphoma lines. Much is known about the differential gene expression profiles of various human lymphoma entities through work with cell lines and patient samples. However, it is not clear to what extent differences in mRNA levels correlate with differences in protein content on a global scale. To address this point we want to employ recent advances in quantitative mass spectrometry in collaboration with Matthias Mann. We have generated a lymphoma super-SILAC mix and used it in a spike-in approach to determine and quantify the proteomes of 5 activated B cell(ABC)-like DLBCL and 5 germinal center B cell(GCB)-like DLBCL cell lines and 4 B cell chronic lymphocytic leukemia (BCLL) patient samples. In an initial unsupervised hierarchical analysis the samples clustered according to their classification (ABC-, GCB-DLBCL, BCLL). We now want to extend our scope to a large collection of patient samples available from our collaborators. The proteome data will be correlated with available gene and miRNA expression data to yield a uniquely complete molecular characterization.