Trib1 in NF-kappaB Signaling: Insights into MALT1 Regulation and Leukemia

NF-kappaB 信号传导中的 Trib1：深入了解 MALT1 调节和白血病

• 批准号: 8897857
• 负责人: Kelly Rome
• 金额: $ 4.31万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8897857
• 关键词: A20 protein; Acute Myelocytic Leukemia; Apoptosis; Binding; Biological Assay; Bone Marrow; Cell Proliferation; Cell Survival; Cell physiology; Cells; Collaborations; Data; Dependence; Development; Developmental Process; Diagnosis; Disease; Drosophila genus; Genetic; Health; Hematologic Neoplasms; Hematopoietic; Homologous Gene; Human; Knowledge; Leukemic Cell; Link; Lymphocyte; Maintenance; Maps; Mediating; Mediator of activation protein; Monoubiquitination; Mus; Myelogenous; NF-kappa B; Oncogenes; Oncogenic; Pathogenesis; Pathway interactions; Patients; Peptide Hydrolases; Phosphorylation; Polyubiquitination; Proteins; Publishing; Regulation; Reporter; Role; Sampling; Signal Transduction; Small Interfering RNA; Specificity; Structural Biologist; Structure; Therapeutic; Therapeutic Intervention; Ubiquitin; Ubiquitination; Universities; Wild Type Mouse; Work; cell motility; improved; inhibitor/antagonist; insight; leukemia; leukemia treatment; mouse model; mutant; novel; outcome forecast; overexpression; progenitor; protein function; reconstitution; research study; retroviral transduction; scaffold; small molecule; targeted treatment; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): Through the proposed work, I will characterize the role of Tribbles homolog 1 (Trib1) in NF-κB signaling, and identify the implications of this role in the pathogenesis of Acute Myeloid Leukemia (AML). We identified MALT1 as a Trib1interacting protein in a screen to identify novel Trib binding partners. Trib1 overexpression in the Jurkat Tcell line enhanced NF-κB activity in a NF-κB-responsive reporter assay. Given the function of MALT1 in mediating NF-κB signaling, I hypothesize that Trib1 enhances NF-κB activation by regulating MALT1 activity. Published work from my lab identified Trib1 as an oncogene in AML. Retroviral transduction of Trib1 in bone marrow progenitors induces AML in reconstituted mice. Furthermore, Trib1 is highly expressed in multiple human AML patient subsets. I hypothesize that Trib1 promotes AML pathogenesis by enhancing activation of proinflammatory NF-κB signaling. In Aim 1, I will assess the role of the Trib1:MALT1 interaction in Trib1 regulation of NF-κB. I will first link Trib1 to the MALT1-mediated NF-κB pathway by examining the ability of Trib1 to enhance signaling in the context of MALT1 knockdown or MALT1 deficiency. Furthermore, I will assess the ability of Trib1 to potentiate MALT1induced activation of NF-κB independently of stimulation. To confirm the role of the Trib1:MALT1 interaction in Trib1-mediated NF-κB regulation, I will generate a binding mutant of Trib1 that maintains structural integrity but abrogates the ability to bind MALT1. Both MALT1 scaffolding and protease function are regulated by ubiquitination. Given the association of Trib1 with E3 ubiquitin ligases, I hypothesize that Trib1 enhances MALT1 activity by regulating the ubiquitination status of MALT1. I will use tagged wildtype and mutant ubiquitin constructs to identify the effect of Trib1 on MALT1 ubiquitination. In Aim 2, I will determine the role of NF-κB i Trib1-mediated AML. I will first assess NF-κB activity in Trib1-mediated mouse leukemic cells, as well as in human AML samples selected for high Trib1 expression. To determine the dependence of these leukemic cells on NF-κB, I will treat cells with NF-κB inhibitors and examine cell survival, selfrenewal, proliferation and apoptosis. I will further assess the effect of NF-κB inhibition on both induction and maintenance of Trib1-mediated AML in our mouse model. I will determine the role of the Trib1:MALT1 interaction in AML by analyzing AML induction in the context of MALT1 deficiency/inhibition or by the Trib1 binding mutant developed in Aim 1. This work will provide critical insight into the pathogenesis of AML and may identify novel targets for therapeutic intervention.

描述（由申请人提供）：通过拟议的工作，我将描述Tribbles同源物1（Tribb 1）在NF-κB信号传导中的作用，并确定该作用在急性髓系白血病（AML）发病机制中的意义。我们在筛选中鉴定了MALT 1作为Trib 1相互作用蛋白，以鉴定新的Trib结合伴侣。在NF-κ B反应性报告分析中，Jurkat T细胞系中Trib 1过表达增强了NF-κB活性。鉴于MALT 1在介导NF-κB信号传导中的功能，我假设Trib 1通过调节MALT 1活性来增强NF-κB活化。我的实验室发表的工作将Trib 1确定为AML中的致癌基因。骨髓祖细胞中Trib 1的逆转录病毒转导在重建小鼠中诱导AML。此外，Trib 1在多种人类AML患者亚群中高度表达。我推测Trib 1通过增强促炎性NF-κB信号的激活促进AML的发病。在目标1中，我将评估Trib 1：MALT 1相互作用在Trib 1调节NF-κB中的作用。我将首先通过检查Trib 1在MALT 1敲低或MALT 1缺陷的情况下增强信号传导的能力，将Trib 1与MALT 1介导的NF-κB通路联系起来。此外，我将评估Trib 1增强MALT 1诱导的NF-κB活化的能力，而不依赖于刺激。为了证实Trib 1：MALT 1相互作用在 Trib 1介导的NF-κB调节，我将产生一个Trib 1的结合突变体，该突变体保持结构完整性，但消除了结合MALT 1的能力。MALT 1支架和蛋白酶功能都受泛素化的调节。鉴于Trib 1与E3泛素连接酶的关联，我推测Trib 1通过调节MALT 1的泛素化状态来增强MALT 1的活性。我将使用标记的野生型和突变体泛素结构，以确定Trib 1对MALT 1泛素化的影响。在目标2中，我将确定NF-κB在Trib 1介导的AML中的作用。我将首先评估NF-κB在Trib 1介导的小鼠白血病细胞以及选择高Trib 1表达的人AML样本中的活性。为了确定这些白血病细胞对 NF-κB，我将用NF-κB抑制剂处理细胞，并检查细胞存活、自我更新、增殖和凋亡。我将在我们的小鼠模型中进一步评估NF-κB抑制对Trib 1介导的AML诱导和维持的影响。我将通过分析MALT 1缺陷/抑制背景下的AML诱导或Aim 1中开发的Trib 1结合突变体来确定Trib 1：MALT 1相互作用在AML中的作用。这项工作将为AML的发病机制提供重要的见解，并可能为治疗干预确定新的靶点。