Defining deregulated ubiquitylation events in B-NHL

B细胞非霍奇金淋巴瘤(B-NHL)的特点是基因组高度不稳定和累积水平。最近，人们认识到泛素蛋白酶体系统(UPS)在细胞DNA损伤反应机制中的核心作用，从而表明UPS在B-NHL的发生和作为靶结构中的作用。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去调节泛素化事件。然而，他们在B-NHL中的身份在很大程度上仍然不为人知。从全基因组aCGH的系统分析和表达研究出发，我们确定了先前孤立的SCF型E3泛素Fbxo25和USP型脱泛素酶(DUB)USP9X分别是预测在B-NHL中缺失和过表达的候选基因。使用无偏倚的质谱学筛选，我们随后发现Fbxo25针对促生存蛋白HAX-1，以降解蛋白酶体，以响应凋亡刺激。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稳定促进了淋巴肿大的发生。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，尤其是在套细胞淋巴瘤(MCL)样本中。这些发现表明Fbxo25可能是B-NHL中一种新的单倍体不足的肿瘤抑制因子。关于USP9X，我们发现有丝分裂磷酸酶CDC14B是USP9X的上游调节因子，XIAP(X连锁的细胞凋亡抑制因子)是一种新的泛素化底物。我们的数据表明，CDC14B-USP9X-XIAP轴参与了有丝分裂细胞死亡的调节。对患者样本的初步IHC研究显示，USP9X的过度表达丰富，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的解除调控如何促进B-NHL的发展和进展。这些方法将包括对顺从性细胞培养模型的研究，以在生化和细胞生物学水平上对相关的连接酶/双链底物对进行功能表征。随后，这些机制的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，并提供可用的临床随访数据。此外，作为一种补充方法，我们建议建立一个有条件的Fbxo25基因敲除小鼠，以进一步研究Fbxo25作为一种新的肿瘤抑制因子的作用。综上所述，我们的跨学科方法包括功能蛋白质组学、细胞生物学、小鼠模型和对确定的患者队列的分析，有望进一步从机制上深入了解B-NHL的病理生理学，并确定相关的泛素蛋白酶体系统的可药物靶向结构，用于这些疾病的特定治疗。

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.