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.

项目总结/摘要 高剂量的电离辐射会导致大量细胞死亡和严重损害胃肠道（GI）， 称为GI综合征。胃肠道综合征是癌症患者接受化疗时常见的严重并发症。 放疗或化疗，但细胞和分子机制尚不清楚，没有有效的治疗方法。 在黑色素瘤2（AIM 2）中缺失，与DNA和衔接蛋白ASC形成炎性小体以启动caspase-1 活化、分泌炎性细胞因子和一种称为细胞凋亡的细胞死亡形式。然而，职能作用 AIM 2在GI综合征中的作用未得到重视。 我们最近的研究使用次全身照射（SBI）诱导的GI综合征（GI syndrome）的小鼠模型显示：（i）Aim 2-/-，Asc-/-， Casp-1-/-和小鼠的GI综合征和死亡率降低。(ii)值得注意的是，核AIM 2在DNA的位点结合DNA， 在具有α-组蛋白2AX焦点的复合物中的双链断裂（DSB），以响应于 这表明AIM 2可以通过检测DSB来监测基因组的不稳定性。我们的方法的基本原理是基于 在支持AIM 2作为DNA传感器检测DSB引发的信号以促进GI中细胞死亡的科学前提下， 综合征因此，我们假设AIM 2感知DSB信号是一个新发现的基因组DNA传感的核心特征。 完整性和炎性小体协调的炎性IEC死亡。因此，我们将测试以下三个具体目标：目标1： 确定由AIM 2炎性体介导的响应于DNA损伤的细胞死亡的机制; 1）.确定 不同形式的细胞死亡对胃肠道综合征的影响。2）。确定AIM 2的动态亚细胞定位 在稳态和辐射激活期间，炎性体组分的浓度增加。3）。测试临床相关的 Caspase-1抑制剂VX-765减轻APCmin小鼠模型中的GI综合征。目标2：确定 具有典型DSB应答的AIM 2炎性小体; 1）研究Aim 2缺陷型、Asc缺陷型和Gsdmd缺陷型中的DSB应答。 小鼠2）。确定AIM 2炎性小体在典型DNA损伤反应中的作用。3）。描述动态 AIM 2与ATM/DNA-PK/p53途径的DSB修复蛋白的相互作用。目标3：确定新的AIM 2 伴侣作为GI综合征的潜在放射/化学增敏靶点。1）。定义AIM的分子组成2 对辐射有反应的相互作用体。2）。确定新的遗传AIM 2相互作用组在控制 通过全基因组CRISPR/cas9文库筛选，在体外检测辐射诱导的DSB应答和细胞死亡。3）。验证 并在体外和体内确定新的AIM 2配偶体在GI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的新的调控机制，并奠定了基础。 通过操纵DSB传感AIM 2激活来开发GI综合征的新疗法的基础。