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.

项目总结/摘要： 在癌细胞中，自身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。这种胞质DNA可以被胞质DNA识别 传感器，如cGAS（环GMP-AMP合酶），其触发下游先天免疫应答。 有趣且令人困惑的是，cGAS/STING先天性免疫途径的激活可以保护或抑制免疫应答。 根据具体情况，使肿瘤对免疫干预敏感。因此，洞察 cGAS/STING信号在癌症中的调节方式可以为靶向干预提供信息。来源 癌细胞中的胞质DNA主要来自有丝分裂的缺陷，导致染色体的封闭 在微核中很容易破裂。这些破裂的微核募集cGAS和核膜 重组（NER）因素-如LEM 2，CHMP 7和BAF-但仍不清楚这些NER如何或是否 影响cGAS/STING信号传导的因素，但在已发表的和我们的初步数据中有新的证据 cGAS/STING信号传导和NER蛋白之间存在潜在的串扰。这项提案的目的是 是提供关键的见解调节先天免疫反应的细胞质DNA在癌症中 细胞核被膜重组因子。为了达到这个目的，我将使用转染鲱鱼 睾丸（HT）DNA和转染的DNA包被的珠作为细胞溶质DNA的模型，因为这可以更容易地 与微核的随机形成相比， 破裂有了这个模型，我将使用CRISPR/Cas9基因编辑和生长素诱导降解决定子（AID）条件 降解系统，以探测NER因子在响应于转染的HT的cGAS/STING信号传导中的作用 DNA和DNA珠。该提案将解决细胞生物学和先天免疫的基本方面 这将有助于阐明癌症的免疫靶点。