The Inflammasome as a novel mediator and therapeutic target of GI syndrome

炎症小体作为胃肠道综合征的新型介质和治疗靶点

• 批准号: 10320010
• 负责人: Richard A. Flavell
• 金额: $ 37.55万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10320010
• 关键词: AIM2 gene; Adaptor Signaling Protein; Address; Apoptosis; CASP1 gene; CASP3 gene; CRISPR library; CRISPR screen; CRISPR/Cas technology; Cancer Patient; Cell Cycle Progression; Cell Death; Cells; Cessation of life; Clustered Regularly Interspaced Short Palindromic Repeats; Co-Immunoprecipitations; Complex; Complication; Coupled; DNA; DNA Binding; DNA Damage; DNA Double Strand Break; DNA Repair; DNA-dependent protein kinase; Development; Dose; Double Strand Break Repair; Ecology; Epithelial Cells; Epitopes; Foundations; Gastrointestinal tract structure; Genetic; Genetic Engineering; Genomic Instability; Histones; Immunoprecipitation; In Vitro; Individual; Inflammasome; Inflammation; Inflammatory; Intestines; Ionizing radiation; Knock-in Mouse; Knock-out; Knockout Mice; Maps; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Mitotic; Molecular; Molecular Target; Monitor; Multiprotein Complexes; Mus; Nuclear; Pathway interactions; Patients; Pattern recognition receptor; Pharmacology; Phosphotransferases; Process; Proteins; Radiation; Radiation Injuries; Radiation Toxicity; Radiation induced double strand break; Radiation therapy; Radiosensitization; Research; Resistance; Role; Signal Transduction; Site; Syndrome; TP53 gene; Technology; Testing; Western Blotting; ataxia telangiectasia mutated protein; base; chemotherapy; clinically relevant; confocal imaging; cytokine; ds-DNA; effective therapy; efficacy testing; gastrointestinal; genome integrity; genome-wide; in vivo; interleukin-1beta-converting enzyme inhibitor; intestinal crypt; intestinal epithelium; intestinal homeostasis; irradiation; microbial; mortality; mouse model; novel; novel therapeutics; protein complex; radiation response; response; screening; sensor; therapeutic target; tissue repair

Project Summary/Abstract High doses of ionizing radiation causes massive cell death and severe damage to the gastrointestinal (GI) tract which is referred to as GI syndrome. The GI syndrome is a common and severe complication in cancer patients undergoing radiotherapy or chemotherapy, but the cellular and molecular mechanisms are unclear and there is no effective therapy. Absent in melanoma 2 (AIM2) forms an inflammasome with DNA and the adapter protein ASC to initiate caspase-1 activation, secretion of inflammatory cytokines and a form of cell death known as pyroptosis. However, the functional role of AIM2 in the GI syndrome was not appreciated. Our recent studies using mouse models of subtotal body irradiation (SBI) induced GI syndrome showed (i) Aim2-/-, Asc-/- Casp-1-/- and mice had reduced GI syndrome and mortality. (ii) Remarkably, nuclear AIM2 bound DNA at sites of DNA double-strand breaks (DSBs) in a complex with -Histone 2AX foci to initiate inflammasome activation in response to radiation, suggesting AIM2 may monitor genomic instability through sensing DSBs. The rationale to our approach is based on the scientific premise supporting AIM2 as a DNA sensor in detecting DSB initiated signals to facilitate cell death in GI syndrome. Thus, we hypothesize that AIM2 sensing DSBs signal is a newly identified core feature of the sensing of genomic integrity and inflammasome coordinated pyroptotic IEC death. We will therefore test following three specific aims: Aim1: Determine the mechanism of cell death mediated by the AIM2 inflammasome in response to DNA damage; 1). Determine the contributions of different forms of cell death to GI syndrome. 2). Identify the dynamic subcellular localization of AIM2 inflammasome components during both steady state and activation by radiation. 3). Test the efficacy of a clinically relevant Caspase-1 inhibitor VX-765 to mitigate GI syndrome in APCmin mouse model. Aim2: Determine the intersection of the AIM2 inflammasome with canonical DSB response; 1) Investigate the DSB response in Aim2-, Asc- and Gsdmd-deficient mice. 2). Identify the role of the AIM2 inflammasome in the canonical DNA damage response. 3). Characterize the dynamic interaction of AIM2 with the DSB repair proteins of the ATM/DNA-PK/p53 pathway. Aim3: Identify novel AIM2 partner(s) as a potential radio-/chemo-sensitizing target in GI syndrome. 1). Define the molecular composition of the AIM2 interactome that responds to radiation. 2). Determine the novel genetic AIM2 interactome in the control of radiation-induced DSB response and cell death in vitro through genome-wide CRISPR/cas9 library screening. 3). Validate and determine the functions of the novel AIM2 partner(s) in GI syndrome both in vitro and in vivo. Collectively, our proposed research will elucidate the novel regulatory mechanisms underlying the GI syndrome, and lay the foundation for developing novel therapeutics for GI syndrome by manipulation of DSBs sensing AIM2 activation.

项目概要/摘要 高剂量的电离辐射会导致大量细胞死亡并对胃肠道（GI）造成严重损害 称为胃肠道综合症。胃肠道综合征是癌症患者常见且严重的并发症 放疗或化疗，但其细胞和分子机制尚不清楚，尚无有效治疗方法。 黑色素瘤 2 中不存在的 (AIM2) 与 DNA 和接头蛋白 ASC 形成炎症小体，以启动 caspase-1 炎症细胞因子的激活、分泌和一种称为细胞焦亡的细胞死亡形式。然而，功能性角色 AIM2 在胃肠道综合征中的作用尚未得到重视。 我们最近使用次全体照射 (SBI) 诱发胃肠道综合征的小鼠模型进行的研究表明 (i) Aim2-/-、Asc-/- Casp-1-/- 和小鼠降低了胃肠道综合征和死亡率。 (ii) 值得注意的是，核 AIM2 在 DNA 位点结合 DNA 具有 -组蛋白 2AX 焦点的复合物中的双链断裂 (DSB) 可启动炎症小体激活以响应 辐射，表明 AIM2 可以通过感知 DSB 来监测基因组不稳定性。我们的方法的基本原理是基于 基于支持 AIM2 作为 DNA 传感器检测 DSB 启动信号以促进胃肠道细胞死亡的科学前提 综合症。因此，我们假设 AIM2 传感 DSB 信号是基因组传感的一个新发现的核心特征。 完整性和炎性体协调焦亡 IEC 死亡。因此，我们将测试以下三个具体目标： 目标 1： 确定 AIM2 炎症小体响应 DNA 损伤介导的细胞死亡机制； 1）。决定 不同形式的细胞死亡对胃肠道综合征的影响。 2）。识别 AIM2 的动态亚细胞定位 稳态和辐射激活期间的炎性体成分。 3）。测试临床相关的功效 Caspase-1 抑制剂 VX-765 可减轻 APCmin 小鼠模型中的胃肠道综合征。目标2：确定交集 具有典型 DSB 反应的 AIM2 炎性体； 1) 研究 Aim2、Asc 和 Gsdmd 缺陷中的 DSB 响应 老鼠。 2）。确定 AIM2 炎性体在典型 DNA 损伤反应中的作用。 3）。表征动态 AIM2 与 ATM/DNA-PK/p53 途径的 DSB 修复蛋白的相互作用。目标 3：识别新颖的 AIM2 伴侣作为胃肠道综合征的潜在放射/化学敏化目标。 1）。定义 AIM2 的分子组成 对辐射作出反应的相互作用组。 2）。确定新型遗传 AIM2 相互作用组的控制 通过全基因组 CRISPR/cas9 文库筛选，体外辐射诱导 DSB 反应和细胞死亡。 3）。证实 并确定新型 AIM2 伴侣在体外和体内胃肠道综合征中的功能。 总的来说，我们提出的研究将阐明胃肠道综合征背后的新调节机制，并奠定基础 为通过操纵 DSB 感应 AIM2 激活来开发胃肠道综合征新疗法奠定了基础。