Molecular mechanisms of B cell malignant transformation

描述（由申请人提供）：TRAF 3是在多种人类B淋巴瘤（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））中鉴定的新型肿瘤抑制基因。为了探索TRAF 3的作用，我们最近产生了一种新的转基因小鼠模型，该模型在B细胞中特异性缺失TRAF 3基因（B-TRAF 3-/-小鼠）。我们发现TRAF 3缺失导致成熟B细胞存活时间延长，最终导致小鼠自发发展为B淋巴瘤。该提案旨在了解TRAF 3失活如何促进致癌B细胞存活。我们首先发现TRAF 3失活介导的异常B细胞存活的机制与生理刺激诱导的正常B细胞存活的机制根本不同。为了鉴定TRAF 3失活的新靶点，我们进行了微阵列分析，以比较从年轻的无肿瘤B-TRAF 3-/-小鼠和同窝对照小鼠纯化的脾B细胞的整体基因表达谱。令人惊讶的是，我们的微阵列分析中发现的许多基因涉及亚细胞器或囊泡运输，包括Rhbdf 1，Rasgrp 3，Ehd 1和Kif 11等。目前的证据表明，许多生存蛋白在两个重要的细胞器，内质网（ER）和线粒体之间运输，并且促存活蛋白和抗存活蛋白的线粒体水平之间的平衡控制细胞死亡的阈值。有趣的是，初步结果表明，在癌前TRAF 3-/- B细胞中，抗存活蛋白巴克的线粒体水平降低，而促存活蛋白Mcl-1的线粒体水平升高。然而，在这些细胞中，巴克和Mcl-1的总细胞水平没有改变。基于我们的新发现，我们将测试中心假设，即TRAF 3失活调节ER和线粒体之间的巴克和Mcl-1的运输，以增加B细胞的细胞死亡阈值。为了解决这个问题，我们提出了补充研究使用B-TRAF 3-/-小鼠和人类患者来源的多发性骨髓瘤细胞系与TRAF 3缺失或突变作为模型系统。在目标1中，使用微阵列分析和严格的优先级排序方案，我们的初步结果使我们专注于TRAF 3的新靶点Rhbdf 1，它与ER蛋白运输有关。为了阐明Rhbdf 1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Rhbdf 1敲除、异位过表达和诱变。在目标2中，我们将进行广泛的生化分离，共聚焦显微镜成像和免疫共沉淀研究，以了解TRAF 3失活和Rhbdf 1如何调节ER和线粒体之间的巴克和Mcl-1的运输。此外，我们将询问TRAF 3信号如何与ER应激反应相互作用和交叉对话。总之，本研究将为TRAF 3失活介导的致癌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.