Defining deregulated ubiquitylation events in B-NHL

b细胞非霍奇金淋巴瘤（B-NHL）的特点是高度累积的基因组不稳定性。最近，泛素蛋白酶体系统（UPS）在细胞DNA损伤反应机制中的核心作用已被认识到，因此提示在B-NHL的发展和作为靶结构中都有作用。事实上，蛋白酶体抑制已经成功地引入到B-NHL的治疗中，但不同实体之间存在相当大的差异，表明存在疾病特异性的无调节泛素化事件。然而，他们在B-NHL中的身份在很大程度上仍然未知。从全基因组aCGH和表达研究的系统分析开始，我们确定了先前孤儿的scf型E3泛素Fbxo25和usp型去泛素酶（DUB） USP9X分别是预计在B-NHL中被删除和过表达的有希望的候选基因。使用无偏质谱筛选，我们随后发现Fbxo25靶向促生存蛋白Hax-1，以响应凋亡刺激降解蛋白酶体。对B-NHL细胞系和小鼠B-NHL体内模型的进一步研究表明，Fbxo25的缺失通过Hax-1稳定促进了淋巴瘤的发生。事实上，我们在不同的B-NHL患者样本中发现了Fbxo25的单等位基因缺失和低表达，并在套细胞淋巴瘤（MCL）样本中特别富集。这些发现将Fbxo25区分为B-NHL中潜在的新型单倍不足肿瘤抑制因子。关于USP9X，我们发现有丝分裂磷酸酶Cdc14B是USP9X的上游调节因子，而XIAP （X-linked inhibitor of apoptosis）是一种新的泛素化底物。我们的数据表明，Cdc14B-USP9X-XIAP轴参与有丝分裂细胞死亡的调控。患者样本的初步免疫组化研究显示USP9X过表达富集，特别是在DLBCL中。基于这些结果，我们建议进一步从功能上研究Fbxo25和USP9X的失调如何促进B-NHL的发展和进展。这些方法将包括在可调节的细胞培养模型中进行研究，以在生化和细胞生物学水平上表征相关连接酶/ dub -底物对的功能。随后，这些机制发现将在不同的人类B-NHL组织培养模型、B-NHL小鼠模型和B-NHL患者队列中进一步研究，并获得临床随访数据。此外，作为补充方法，我们建议生成条件Fbxo25敲除小鼠，以进一步研究Fbxo25作为新型肿瘤抑制因子的作用。总之，我们的跨学科方法包括功能蛋白质组学、细胞生物学、小鼠模型和确定的患者队列分析，预计将进一步深入了解B-NHL的病理生理学机制，并确定泛素蛋白酶体系统的相关可药物靶标结构，以用于这些疾病的特异性治疗。

B-Cell Non Hodgkins Lymphomas (B-NHL) are characterized by high and cumulative levels of genomic instability. Recently, the central role of the ubiquitin proteasome system (UPS) in the cellular DNA damage response machinery has been appreciated, thus suggesting roles in both B-NHL development and as a target structures. Indeed, proteasomal inhibition has been successfully introduced into the therapy of B-NHL, yet considerable variations exist between different entities, indicating the presence of disease-specific deregulated ubiquitylation events. Their identity in B-NHL has however remained largely unknown. Starting from systematic analyses of genomewide aCGH and expression studies, we identified the previously orphan SCF-type E3 ubiquitin Fbxo25 and the USP-type deubiquitylase (DUB) USP9X as promising candidates predicted to be deleted and overexpressed in B-NHL, respectively. Using unbiased mass-spectrometric screens, we subsequently found that Fbxo25 targets the pro-survival protein Hax-1 for proteasomal degradation in response to apoptotic stimuli. Further studies in B-NHL cell lines and a murine B-NHL in vivo model suggest that deletions of Fbxo25 contribute to lymphomagenesis through Hax-1 stabilization. Indeed, we find monoallelic deletion and low expression of Fbxo25 in different B-NHL patient samples with a particular enrichment in mantle cell lymphoma (MCL) samples. These findings distinguish Fbxo25 as a potential novel haploinsufficient tumorsuppressor in B-NHL. With regard to USP9X, we identified the mitotic phosphatase Cdc14B as an upstream regulator of USP9X, and XIAP (X-linked inhibitor of apoptosis) as a novel ubiquitylation substrate. Our data suggest that the Cdc14B-USP9X-XIAP axis contributes to the regulation of mitotic cell death. Initial IHC studies in patient samples revealed an enrichment of USP9X overexpression particularly in DLBCL. Based on these results, we propose to further functionally investigate how deregulation of Fbxo25 and USP9X contributes to B-NHL development and progression. These approaches will include studies in amenable cell culture models to functionally characterize the relevant ligase/DUB-substrate pairs on the biochemical and cell biological level. Subsequently, these mechanistic findings will be further investigated in different human B-NHL tissue culture models, B-NHL mouse models and B-NHL patient cohorts with available clinical follow up data. Moreover, as a complimentary approach, we propose to generate a conditional Fbxo25 knock-out mouse to further investigate the role of Fbxo25 as a novel tumorsuppressor. In summary, our interdisciplinary approach comprising functional proteomics, cell biology, mouse models, and analyses of defined patient cohorts is anticipated to gain further mechanistic insight into the pathophysiology of B-NHL, and identify relevant drugable target structures of the ubiquitin proteasome system for the specific treatment of these diseases.