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

描述(由申请人提供)：通过拟议的工作，我将描述Tribble同源基因1(Trib1)在NF-κB信号转导中的作用，并确定这一作用在急性髓系白血病发病机制中的意义。我们在筛选中发现MALT1是一种Trib1相互作用蛋白，以确定新的Trib结合伙伴。在Jurkat T细胞系中过表达Trib1可在核因子-κB反应报告实验中增强核因子-κB活性。鉴于MALT1在介导NF-κB信号转导中的作用，我假设Trib1通过调节MALT1的活性来增强NF-κB的活性。我的实验室发表的研究发现，Trib1是急性髓系白血病的癌基因。逆转录病毒转导Trib1基因可诱导重组小鼠急性髓系白血病此外，Trib1在多个人类AML患者亚群中高度表达。我推测TRIB1通过增强促炎因子-κB信号的激活而促进急性髓系白血病的发病。在目标1中，我将评估TRIB1：MALT1相互作用在TRIB1调节NF-κB中的作用。我将首先通过检测TRIB1在MALT1被敲除或MALT1缺乏的情况下增强信号的能力，将TRIB1与MALT1介导的NF-κB途径联系起来。此外，我将评估TRIB1在不依赖刺激的情况下增强MALT1诱导的NF-κB激活的能力。确认Trib1：Malt1相互作用在 Trib1介导的NF-κB调控，我将产生Trib1的结合突变体，该突变体保持结构完整性，但取消结合MALT1.MALT1支架和蛋白酶的功能都受泛素化的调节。鉴于Trib1与E3泛素连接酶的关联，我假设Trib1通过调节MALT1的泛素化状态来增强MALT1的活性。我将使用标记的野生型和突变型泛素结构来鉴定Trib1对MALT1泛素化的影响。在目标2中，我将确定核因子-κB I Trib1介导的急性髓细胞白血病的作用。我将首先评估在TRIB1介导的小鼠白血病细胞中的NF-κB活性，以及在被选为TRIB1高表达的人急性髓细胞白血病样本中。为了确定这些白血病细胞对 核因子-κB，我将用核因子-κB抑制剂处理细胞，并检测细胞存活、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抑制NF-κB在诱导和维持TRIB1介导的急性髓细胞白血病中的作用。我将通过在MALT1缺乏/抑制的背景下分析AML诱导或通过AIM 1中开发的Trib1结合突变体来确定Trib1：Malt1相互作用在AML中的作用。这项工作将为AML的发病机制提供关键的见解，并可能为治疗干预寻找新的靶点。

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.