Mechanistic and proteomic analyses of NF-kappaB-driven lymphomas

NF-κB 驱动的淋巴瘤的机制和蛋白质组学分析

• 批准号: 224805578
• 负责人: Professor Dr. Marc Schmidt-Supprian
• 金额: --
• 依托单位: Klinik und Poliklinik für Innere Medizin III: Hämatologie und Onkologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/224805578?language=en
• 关键词: Mechanistic; proteomic; analyses; NF; kappaB

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.

NF-κ B B家族转录因子c-Rel的基因座在霍奇金、弥漫性大B细胞（DLBCL）和原发性纵隔B细胞（PMBCL）淋巴瘤中频繁扩增。此外，在DLBCL中已检测到缺乏外显子10的过度活跃的c-Rel剪接变体。我们最近发现外显子10编码一个功能性小泛素化位点。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，我们已经在细菌人工染色体（BAC）上产生了遗传修饰的小鼠c-Rel基因组位点。修饰包括侧翼外显子9，人外显子10的小鼠同源物，具有Frt位点，c-Rel与GFP的N-末端融合，C-末端3x FLAG标签的引入及其组合。BAC在内源启动子的控制下或在CAG启动子的条件控制下过表达。我们已经用BAC重建了c-Rel敲除MEFs，并表明c-Rel、其剪接变体及其融合蛋白可以以条件方式显著过表达。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细胞发育和功能过程中的影响。它们在淋巴瘤发生过程中的作用将与我们小组和我们合作伙伴提供的其他致癌修饰相结合进行评估。这些研究将通过shRNA介导的c-Rel在相关人淋巴瘤细胞系中的敲低来补充。通过对细胞系和患者样本的研究，我们对各种人类淋巴瘤实体的差异基因表达谱有了很大的了解。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。为了解决这一点，我们希望与Matthias Mann合作，采用定量质谱法的最新进展。我们已经产生了淋巴瘤超级SILAC混合物，并将其用于加标方法，以确定和定量5种活化的B细胞（ABC）样DLBCL和5种生发中心B细胞（GCB）样DLBCL细胞系和4种B细胞慢性淋巴细胞白血病（BCLL）患者样品的蛋白质组。在最初的无监督分层分析中，样本根据其分类（ABC-，GCB-DLBCL，BCLL）进行聚类。现在，我们希望将我们的范围扩展到从我们的合作者那里获得的大量患者样本。蛋白质组数据将与可用的基因和miRNA表达数据相关联，以产生独特的完整分子表征。

项目成果

Canonical NF-κB signaling is uniquely required for the long-term persistence of functional mature B cells

• DOI: 10.1073/pnas.1604529113
• 发表时间: 2016-05-03
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Derudder, Emmanuel;Herzog, Sebastian;Rajewsky, Klaus
• 通讯作者: Rajewsky, Klaus

An oncogenic role for alternative NF-κB signaling in DLBCL revealed upon deregulated BCL6 expression.

• DOI: 10.1016/j.celrep.2015.03.059
• 发表时间: 2015-05-05
• 期刊: Cell reports
• 影响因子: 8.8
• 作者: Zhang B;Calado DP;Wang Z;Fröhler S;Köchert K;Qian Y;Koralov SB;Schmidt-Supprian M;Sasaki Y;Unitt C;Rodig S;Chen W;Dalla-Favera R;Alt FW;Pasqualucci L;Rajewsky K
• 通讯作者: Rajewsky K