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

项目摘要/摘要 高剂量的电离辐射会导致大量的细胞死亡和严重的胃肠道损伤，这是 被称为胃肠道综合征。胃肠道综合征是癌症患者在接受手术时常见和严重的并发症。 放射治疗或化疗，但其细胞和分子机制尚不清楚，也没有有效的治疗方法。 黑色素瘤缺失2(AIM2)与DNA和连接蛋白ASC一起形成一个炎症体，启动caspase-1 炎症细胞因子的激活、分泌和一种称为上睑下垂的细胞死亡。然而，功能角色 AIM2在GI综合征中的作用尚未得到重视。 我们最近利用次全身照射(SBI)诱导的GI综合征小鼠模型的研究表明：(1)AIM2-/-、ASC-/- CASP-1-/-和小鼠减少了胃肠道综合征和死亡率。(Ii)值得注意的是，核AIM2结合在DNA的位点 具有-组蛋白2AX焦点的复合体中的双链断裂(DSB)启动炎症小体激活 辐射，提示AIM2可能通过感应DSB来监测基因组的不稳定性。我们方法的基本原理是基于 在支持AIM2作为DNA传感器检测DSB启动信号以促进GI细胞死亡的科学前提下 综合症。因此，我们假设AIM2感应DSBs信号是新发现的基因组感应的核心特征 完整性和炎症性小体协调了炎症性IEC的死亡。因此，我们将测试以下三个具体目标：目标1： 确定AIM2炎性小体介导的DNA损伤导致细胞死亡的机制：1)测定 不同形式的细胞死亡在胃肠道综合征中的作用。2)。鉴定AIM2的动态亚细胞定位 稳定状态和辐射激活时的炎性小体成分。3)。测试临床相关药物的疗效 Caspase-1抑制剂VX-765减轻APCmin小鼠模型GI综合征的研究AIM2：确定 AIM2炎症体具有典型的DSB反应；1)研究AIM2、ASC和Gsdmd缺陷的DSB反应 老鼠。2)。确定AIM2炎症体在典型DNA损伤反应中的作用。3)。描述动态 AIM2与ATM/DNA-PK/P53通路中DSB修复蛋白的相互作用Aim3：识别新的AIM2 伙伴(S)作为GI综合征放射/化学增敏的潜在靶点。1)。确定AIM2的分子组成 对辐射有反应的互动体。2)。确定新的基因AIM2互作组在控制黄瓜枯萎病中的作用 通过全基因组CRISPR/Cas9文库筛选体外辐射诱导的DSB反应和细胞死亡。3)。验证 并对新的AIM2蛋白(S)在胃肠道综合征中的作用进行了体内外研究。 总而言之，我们拟议的研究将阐明GI综合征背后的新调控机制，并为 通过操纵DSB感应AIM2的激活来开发治疗GI综合征的新疗法的基础。

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.