Dissecting the Role Ubiquitin E3 Ligase UBR5 in Lymphomagenesis

摘要 套细胞淋巴瘤(MCL)是一种罕见的侵袭性非霍奇金淋巴瘤。不幸的是，有限 目前已有治疗MCL的方法，提示有必要进一步揭示分子机制。 调节疾病的转化和发展。大多数MCL患者有t(11；14) 易位导致细胞周期蛋白D1的过度表达，导致细胞广泛增殖和阻断 分化起源于淋巴结的外套膜区，但需要额外的突变 转型。下一代测序在MCL患者中发现了一些新的突变 包括泛素E3连接酶UBR5。E3泛素连接酶作为底物识别成分 泛素蛋白酶体系统对蛋白质的降解。在196名MCL患者的队列中，UBR5排在第3位 最常见的突变基因和~60%的突变发生在UBR5的Hect结构域，该结构域 可以接受泛素分子并将其转移到底物上。为了了解UBR5Hect的作用 我们使用新的CRISPR/CAS9技术产生了一只条件性小鼠。 Hect结构域的缺失导致脾中前生发中心B细胞受阻，两者均减少 B1和边缘B细胞亚群。此外，脾中的滤泡B细胞表型异常并衰竭。 终末分化为分泌抗体的浆细胞。蛋白质组学研究显示蛋白质表达上调 在缺乏UBR5的Hect结构域的B细胞中，通过剪接体进行mRNA剪接相关。这些 研究表明：1)UBR5突变与细胞周期蛋白D1的表达协同作用可能导致疾病 套细胞淋巴瘤的进展(目标1)，2)了解UBR5突变的分子机制 可为MCL提供潜在的治疗靶点(AIM 2)，3)U5剪接体的异常表达 蛋白质阻止B细胞成熟并促进淋巴肿大(目标3)。在本申请中，我们提出了研究 了解UBR5及其相互作用蛋白在B细胞淋巴瘤发生中的作用并确定分子 B细胞中受UBR5调控的通路。我们提议的研究的目标是提供对外套细胞的见解 淋巴瘤的转化、进展和未来潜在的治疗目标。

ABSTRACT Mantle cell lymphoma (MCL) is a rare and aggressive non-Hodgkin’s lymphoma. Unfortunately limited therapies for MCL are currently available suggesting a need to further unravel molecular mechanisms regulating transformation and progression of the disease. The majority of MCL patients have a t(11;14) translocation leading to overexpression of CyclinD1 resulting in extensive proliferation and block in differentiation originating in the mantle zone of the lymph node, however additional mutations are necessary for transformation. Next generation sequencing has identified a number of novel mutations in MCL patients including the ubiquitin E3 ligase UBR5. E3 ubiquitin ligases serve as the substrate-recognizing component for protein degradation by the ubiquitin proteasome system. In a cohort of 196 MCL patients UBR5 was the 3rd most frequently mutated gene and ~60% of the mutations were found within the HECT domain of UBR5, which can accept and transfer ubiquitin molecules to the substrate. In order to understand the role of UBR5 HECT domain in B-lymphoid development we generated a conditional mouse using novel CRISPR/Cas 9 technology. Loss of the HECT domain leads to a block in pre-germinal center B cells in the spleen with a reduction of both B1 and marginal B cell subsets. In addition, follicular B cells in the spleen are phenotypically abnormal and fail to terminally differentiate to anti-body secreting plasma cells. Proteomic studies reveal up-regulation of proteins associated with mRNA splicing via the spliceosome in B cells lacking the HECT domain of UBR5. These studies suggest that 1) cooperation of UBR5 mutations along with expression of cyclinD1 may led to disease progression of mantle cell lymphoma (Aim 1), 2) understanding molecular mechanism of UBR5 mutations could provide potential therapeutic targets in MCL (Aim 2), and 3) aberrant expression of U5 spliceosome proteins block B cell maturation and promote lymphomagenesis (Aim 3). In this application we propose studies to understand the role of UBR5 and its interacting proteins in B-cell lymphomagenesis and define molecular pathways regulated by UBR5 in B-cells. Our goal of the proposed studies is to provide insights to mantle cell lymphoma transformation, progression and potential future therapeutics targets.