Molecular mechanisms of B cell malignant transformation

描述（由申请人提供）：TRAF3是一种新的肿瘤抑制基因，在多种人类B淋巴瘤中发现，包括脾边缘区淋巴瘤、B细胞慢性淋巴细胞白血病和套细胞淋巴瘤以及多发性骨髓瘤（MM）。为了探索TRAF3的作用，我们最近建立了一种新的转基因小鼠模型，该模型在B细胞中特异性地删除了TRAF3基因（B-TRAF3-/-小鼠）。我们发现TRAF3缺失导致成熟B细胞存活时间延长，最终导致小鼠B淋巴瘤的自发发展。这项提议旨在了解TRAF3失活如何促进致癌B细胞存活。我们首先发现TRAF3失活介导的异常B细胞存活机制与生理刺激诱导的正常B细胞存活有着本质的不同。为了确定TRAF3失活的新靶点，我们进行了微阵列分析，比较了从年轻、无肿瘤的B-TRAF3-/-小鼠和同窝对照小鼠纯化的脾脏B细胞的整体基因表达谱。引人注目的是，在我们的微阵列分析中发现的一些基因与亚细胞细胞器或囊泡运输有关，包括Rhbdf1、Rasgrp3、Ehd1和Kif11等。目前的证据表明，许多存活蛋白在两个重要的细胞器——内质网（ER）和线粒体之间运输，线粒体促存活蛋白和抗存活蛋白水平之间的平衡控制着细胞死亡的阈值。有趣的是，初步结果表明，在癌前TRAF3-/- B细胞中，线粒体抗生存蛋白Bak水平降低，线粒体促生存蛋白Mcl-1水平升高。然而，在这些细胞中，Bak和Mcl-1的总细胞水平没有改变。基于我们的新发现，我们将验证TRAF3失活调节Bak和Mcl-1在内质网和线粒体之间的运输以增加B细胞死亡阈值的中心假设。为了解决这个问题，我们建议使用TRAF3缺失或突变的B-TRAF3-/-小鼠和人类患者来源的多发性骨髓瘤细胞系作为模型系统进行补充研究。在Aim 1中，使用微阵列分析和严格的优先级方案，我们的初步结果使我们将重点放在TRAF3的新靶标Rhbdf1上，该靶标与内质网蛋白运输有关。为了描述Rhbdf1在致癌B细胞存活中的因果作用，我们将采用慢病毒shRNA载体介导的Rhbdf1的敲低、异位过表达和突变。在Aim 2中，我们将进行广泛的生化分离、共聚焦显微成像和共免疫沉淀研究，以了解TRAF3失活和Rhbdf1如何调节Bak和Mcl-1在内质网和线粒体之间的运输。此外，我们将探讨TRAF3信号如何与内质网应激反应相互作用和交叉对话。综上所述，本研究将为TRAF3失活介导的癌性B细胞存活的确切机制提供新的见解，并将为B淋巴瘤和MM的治疗开辟新的途径。

DESCRIPTION (provided by applicant): TRAF3 is a novel tumor suppressor gene identified in a variety of human B lymphoma, including splenic marginal zone lymphoma, B cell chronic lymphocytic leukemia and mantle cell lymphoma, as well as multiple myeloma (MM). To explore the role of TRAF3, we recently generated a new genetically modified mouse model that has the TRAF3 gene specifically deleted in B cells (B-TRAF3-/- mice). We found that TRAF3 deletion results in prolonged survival of mature B cells, which eventually leads to spontaneous development of B lymphomas in mice. This proposal aims to understand how TRAF3 inactivation promotes oncogenic B cell survival. We first found that the mechanism of aberrant B cell survival mediated by TRAF3 inactivation is fundamentally different from that of normal B cell survival induced by physiological stimuli. To identify novel targets of TRAF3 inactivation, we performed microarray analysis to compare the global gene expression profiles of splenic B cells purified from young, tumor-free B-TRAF3-/- mice and littermate control mice. Strikingly, a number of genes identified in our microarray analysis are implicated in subcellular organelle or vesicle trafficking, including Rhbdf1, Rasgrp3, Ehd1, and Kif11, etc. Current evidence indicates that many survival proteins are trafficking between two important organelles, endoplasmic reticulum (ER) and mitochondria, and that the balance between mitochondrial levels of pro-survival proteins and anti-survival proteins controls the threshold of cell death. Interestingly, or preliminary results demonstrated that mitochondrial levels of the anti-survival protein Bak were decreased, and mitochondrial levels of the pro- survival protein Mcl-1 were increased in premalignant TRAF3-/- B cells. However, total cellular levels of Bak and Mcl-1 were not altered in these cells. Based on our new findings, we will test the central hypothesis that TRAF3 inactivation modulates the trafficking of Bak and Mcl-1 between ER and mitochondria to increase the threshold of cell death in B cells. To address this, we propose complementary studies using B-TRAF3-/- mice and human patient-derived multiple myeloma cell lines with TRAF3 deletions or mutations as model systems. In Aim 1, using microarray analysis and a stringent prioritization scheme, our preliminary results led us to focus on a novel target of TRAF3, Rhbdf1, which is implicated in ER protein trafficking. To delineate the causal role of Rhbdf1 in oncogenic B cell survival, we will employ lentiviral shRNA vector-mediated knockdown, ectopic overexpression, and mutagenesis of Rhbdf1. In Aim 2, we will perform extensive biochemical fractionation, confocal microscopic imaging, and co-immunoprecipitation studies to understand how TRAF3 inactivation and Rhbdf1 modulate the trafficking of Bak and Mcl-1 between ER and mitochondria. Furthermore, we will interrogate how TRAF3 signaling interplays and cross-talks with ER stress responses. In summary, this study will provide new insights into the exact mechanisms of TRAF3 inactivation-mediated oncogenic B cell survival, and will open up new avenues for the treatment of B lymphoma and MM.