Mechanistic and proteomic analyses of NF-kappaB-driven lymphomas

点击翻译按钮获取中文摘要

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.