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Defining deregulated ubiquitylation events in B-NHL

定义 B-NHL 中解除管制的泛素化事件

基本信息

批准号：
258522452
负责人：
Professor Dr. Florian Bassermann
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：
2014
资助国家：
德国
起止时间：
2013-12-31 至 2017-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/258522452?language=en
关键词：
Defining deregulated ubiquitylation events NHL

项目摘要

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.

B细胞非霍奇金斯淋巴瘤（B-NHL）的特征在于高水平和累积水平的基因组不稳定性。最近，泛素蛋白酶体系统（UPS）在细胞DNA损伤反应机制中的中心作用已被认识到，从而表明在B-NHL发展中的作用和作为靶结构。事实上，蛋白酶体抑制已成功地引入到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失调的泛素化事件。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，我们确定了以前的孤儿SCF型E3泛素Fbxo 25和USP型去泛素化酶（DUB）USP 9 X作为预测在B-NHL中分别缺失和过表达的有希望的候选物。使用无偏质谱筛选，我们随后发现Fbxo 25靶向促生存蛋白Hax-1，用于响应凋亡刺激的蛋白酶体降解。在B-NHL细胞系和鼠B-NHL体内模型中的进一步研究表明，Fbxo 25的缺失通过Hax-1稳定化促进淋巴瘤发生。事实上，我们在不同的B-NHL患者样本中发现Fbxo 25的单等位基因缺失和低表达，在套细胞淋巴瘤（MCL）样本中特别富集。这些发现将Fbxo 25区分为B-NHL中潜在的新型单倍不足肿瘤抑制因子。关于USP 9 X，我们确定了有丝分裂磷酸酶Cdc 14 B作为USP 9 X的上游调节因子，XIAP（X连锁凋亡抑制剂）作为新的泛素化底物。我们的数据表明，Cdc 14 B-USP 9 X-XIAP轴有助于有丝分裂细胞死亡的调节。患者样本中的初始IHC研究揭示了USP 9 X过表达的富集，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Fbxo 25和USP 9 X的失调如何有助于B-NHL的发展和进展。这些方法将包括在适合的细胞培养模型中进行研究，以在生物化学和细胞生物学水平上对相关连接酶/DUB-底物对进行功能表征。随后，将在不同的人B-NHL组织培养模型、B-NHL小鼠模型和具有可用临床随访数据的B-NHL患者队列中进一步研究这些机制发现。此外，作为一种互补的方法，我们建议产生一个条件Fbxo 25敲除小鼠，以进一步研究Fbxo 25作为一种新的肿瘤抑制因子的作用。总之，我们的跨学科的方法，包括功能蛋白质组学，细胞生物学，小鼠模型，并确定患者队列的分析，预计将获得进一步的机制洞察B-NHL的病理生理学，并确定相关的药物靶结构的泛素蛋白酶体系统的具体治疗这些疾病。