Determining the role of nuclear envelope reformation proteins in regulating the cGAS/STING innate immune response in cancer

确定核膜重组蛋白在调节癌症 cGAS/STING 先天免疫反应中的作用

• 批准号: 10750669
• 负责人: Anthony Wayne Isenhour
• 金额: $ 4.77万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10750669
• 关键词: Auxins; Biochemical; CRISPR/Cas technology; Cancer Biology; Cancer cell line; Cell Line; Cellular biology; Chromatin; Chromosomes; Clupeidae; Complex; Cyclic GMP; Cytosol; DNA; DNA Binding; DNA Repair Pathway; DNA-Binding Proteins; Data; Data Analyses; Dedications; Defect; Electron Microscopy; Endosomes; Enzyme-Linked Immunosorbent Assay; Exposure to; Fluorescence Microscopy; Gene Expression; Genes; Genetic; Genomic DNA; Genomic Instability; Goals; Home; Immune signaling; Immunofluorescence Immunologic; Immunotherapeutic agent; Individual; Innate Immune Response; Interferons; Interphase; Intervention; Learning; Malignant Neoplasms; Measures; Membrane Proteins; Mitosis; Modeling; Molecular; Nuclear; Nuclear Envelope; Nuclear Inner Membrane; Outcome; Pathway interactions; Phosphorylation; Production; Productivity; Proteins; Publishing; Regulation; Role; Rupture; Scientist; Signal Transduction; Site; Sorting; Source; Stimulator of Interferon Genes; System; Testis; Training; Transcript; Transfection; Tumor Promotion; Western Blotting; Work; cancer cell; career; chromosome missegregation; design; env Gene Products; experimental study; innate immune pathways; insight; light microscopy; live cell imaging; micronucleus; novel; recruit; response; seal; sensor; spatiotemporal; synergism; tumor; tumor growth

PROJECT SUMMARY/ABSTRACT: In cancer cells, exposure of self DNA to the cytosol is driven by a variety of genomic instabilities such as micronuclei, chromatin bridges, and nuclear ruptures. This cytosolic DNA can be recognized by cytosolic DNA sensors such as cGAS (cyclic GMP-AMP synthase), which triggers a downstream innate immune response. Interestingly and confoundingly, the activation of the cGAS/STING innate immune pathway can protect or sensitize tumors to immunotherapeutic interventions depending on the specific context. Therefore, insight into the ways in which cGAS/STING signaling is regulated in cancer can inform targeted intervention. Sources of cytosolic DNA in cancer cells arise primarily from defects in mitosis that lead to the enclosure of chromosomes in micronuclei that are prone to rupture. These ruptured micronuclei recruit cGAS and nuclear envelope reformation (NER) factors—such as LEM2, CHMP7, and BAF—but it remains unknown how, or if, these NER factors impact cGAS/STING signaling but there is emerging evidence in published and in our preliminary data that there is potential crosstalk between cGAS/STING signaling and NER proteins. The goal of this proposal is to provide key insights into the regulation of the innate immune response to cytosolic DNA in cancer cells by nuclear envelope reformation factors. In order to achieve this goal, I will use transfected herring testes (HT) DNA and transfected DNA-coated beads as models for cytosolic DNA as this can be more readily controlled compared to the stochastic formation of micronuclei, only some of which are unstable and prone to rupture. With this model, I will use CRISPR/Cas9 gene-editing and the auxin-inducible-degron (AID) conditional degradation system to probe the roles of NER factors in cGAS/STING signaling in response to transfected HT DNA and DNA beads. This proposal will address fundamental aspects of cell biology and innate immune signaling that will shed light on immunotherapeutic targets for cancer.

项目摘要/摘要： 在癌细胞中，自我DNA暴露于胞质溶胶是由多种基因组不稳定性驱动的 微核，染色质桥和核破裂。该胞质DNA可以通过胞质DNA识别 CGA（环状GMP-AMP合酶）等传感器，它会触发下游的先天免疫反应。 有趣而令人困惑的是，CGA/STING先天免疫途径的激活可以保护或 根据特定情况，对免疫治疗干预的敏感肿瘤。因此，深入了解 CGA/STING信号在癌症中调节的方式可以为有针对性的干预提供信息。来源 癌细胞中的胞质DNA来自有丝分裂的缺陷，导致染色体的围栏 在容易破裂的微核中。这些破裂的微核募集了CGA和核包膜 改革（NER）因素（例如LEM2，CHMP7和BAF），但仍然未知，或者是否ner 因素会影响CGA/STING信号传导，但已发布和初步数据中有新的证据 CGA/STING信号传导和NER蛋白之间存在潜在的串扰。该提议的目标 是为了调节癌胞质DNA的先天免疫激素的调节，提供关键的见解 细胞通过核包膜报告因素。为了实现这一目标，我将使用翻译的鲱鱼 睾丸（HT）DNA和翻译DNA涂层珠作为胞质DNA的模型，因为这很容易 与微核的随机形成相比，受控的只有一些 破裂。使用此模型，我将使用CRISPR/CAS9基因编辑和生长素 - 诱导型 - 脱糖（AID）条件 降解系统以探测NER因子在CGA/STING信号传导中响应翻译HT的作用 DNA和DNA珠。该建议将介绍细胞生物学和先天免疫的基本方面 信号传达将阐明癌症的免疫治疗靶标。