Molecular mechanisms of B cell malignant transformation

B细胞恶性转化的分子机制

• 批准号: 8686772
• 负责人: PING XIE
• 金额: $ 31.28万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-04 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8686772
• 关键词: Address; Affect; Apoptosis; Apoptotic; B-Cell Lymphomas; B-Cell Neoplasm; B-Lymphocytes; B-Lymphoma Development; BCL2 gene; Biochemical; Biological Assay; Biological Models; Cell Death; Cell Line; Cell Survival; Cells; Cessation of life; Chronic Lymphocytic Leukemia; Co-Immunoprecipitations; Communication; Deletion Mutation; Development; Endoplasmic Reticulum; Equilibrium; Family; Fractionation; Gene Expression; Genes; Grant; Human; Image; Knowledge; Luciferases; Lymphoma; MCL1 protein; Malignant - descriptor; Malignant Neoplasms; Mantle Cell Lymphoma; Mature B-Lymphocyte; Mediating; Mediator of activation protein; Microarray Analysis; Microscopic; Mitochondria; Modeling; Molecular; Molecular Profiling; Multiple Myeloma; Mus; Mutagenesis; Mutation; Non-Hodgkin' s Lymphoma; Oncogenic; Organelles; Pathway interactions; Patients; Physiological; Premalignant; Protein Family; Proteins; Reporter; Role; Scheme; Signal Transduction; Stimulus; Structure-Activity Relationship; T-Lymphocyte; TNF receptor-associated factor 3; TNFRSF5 gene; Testing; Therapeutic Intervention; Tumor Suppressor Genes; Tumor Suppressor Proteins; United States; Up-Regulation; Vesicle; bak protein; base; biological adaptation to stress; chromatin immunoprecipitation; combat; design; endoplasmic reticulum stress; human RTN4 protein; information gathering; insight; interest; macrophage; mitochondrial membrane; mouse model; mutant; novel; overexpression; protein transport; small hairpin RNA; therapeutic target; trafficking; transcription factor; treatment strategy; tumor; tumorigenesis; vector

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.

描述（申请人提供）：TRAF3是在多种人类B淋巴瘤中发现的新型抑癌基因，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，我们最近构建了一种新的转基因小鼠模型，该模型在 B 细胞中专门删除了 TRAF3 基因（B-TRAF3-/- 小鼠）。我们发现 TRAF3 缺失会导致成熟 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 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​​存活机制有根本不同。为了确定 TRAF3 失活的新靶点，我们进行了微阵列分析，比较从年轻无肿瘤 B-TRAF3-/- 小鼠和同窝对照小鼠中纯化的脾 B 细胞的整体基因表达谱。引人注目的是，我们的微阵列分析中发现的许多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，许多存活蛋白在内质网 (ER) 和线粒体这两个重要细胞器之间运输，并且线粒体水平之间的平衡 促存活蛋白和抗存活蛋白控制细胞死亡的阈值。有趣的是，初步结果表明，在癌前 TRAF3-/- B 细胞中，抗​​存活蛋白 Bak 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，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，我们将测试以下中心假设：TRAF3 失活调节 Bak 和 Mcl-1 在 ER 和线粒体之间的运输，从而提高 B 细胞的细胞死亡阈值。为了解决这个问题，我们建议使用具有 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重点关注 TRAF3 的新靶点 Rhbdf1，该靶点与 ER 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 敲低、异位过度表达和诱变。在目标 2 中，我们将进行广泛的生化分离、共焦显微成像和免疫共沉淀研究，以了解 TRAF3 失活和 Rhbdf1 如何调节 Bak 和 Mcl-1 在 ER 和线粒体之间的运输。此外，我们将探讨 TRAF3 信号如何与 ER 应激反应相互作用和串扰。总之，这项研究将为TRAF3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的治疗开辟新途径。