The Role of RNA Editing in the DNMTi-induced interferon response in ovarian cancer

RNA 编辑在 DNMTi 诱导的卵巢癌干扰素反应中的作用

基本信息

批准号：
10203828
负责人：
Stephanie Gomez
金额：
$ 3.44万
依托单位：
GEORGE WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10203828
关键词：
ADAR1 Antibiotics Antiviral Agents Apoptosis Ascites Attenuated Bioinformatics CD8-Positive T-Lymphocytes Cancer Patient Cancer Prognosis Cancer cell line Cell Culture Techniques Cell Line Cell model Cells Cessation of life Chemoresistance Coupled Cytoplasm DNA DNA Methyltransferase Inhibitor Data Data Set Diagnosis Disease Double-Stranded RNA Drug Targeting Endogenous Retroviruses Enzyme-Linked Immunosorbent Assay Enzymes Epigenetic Process Flow Cytometry Genes Genetic Transcription Goals Growth Human Human Cell Line Immune Immune response Immune system Immunocompetent Immunologic Tests Immunophenotyping Immunosuppression Immunotherapy In Vitro Innate Immune Response Interferon Type I Interferon-beta Interferons Knock-out Knowledge Lead Learning Luciferases Malignant Neoplasms Malignant neoplasm of ovary Measures Mediating Melanoma Cell Modeling Mus Natural Immunity Nucleotides Operative Surgical Procedures Ovarian Patients Pharmaceutical Preparations Platinum Play Protein Isoforms Proteins RNA Editing RNA analysis Regulation Repetitive Sequence Reporter Research Resistance development Role Sampling Serous Signal Pathway Signal Transduction Survival Rate TP53 gene Testing Therapeutic Training Tumor Burden Tumor-infiltrating immune cells Up-Regulation Western Blotting Woman adenosine deaminase advanced disease anti-CTLA4 anti-PD-1 base cancer cell cancer immunotherapy cancer therapy cancer type career career development chemotherapy cytokine design drug development epigenetic therapy fighting immunoregulation improved in vitro Model insight mouse model novel novel strategies pre-clinical preclinical study recruit response sensor stable cell line tissue culture transcriptome sequencing tumor tumor microenvironment tumor-immune system interactions viral RNA

项目摘要

High-grade serous ovarian cancer (OC) is a deadly disease that is often diagnosed at a late stage. Despite significant research, the five-year survival rate for OC has remained unchanged for decades. Many OC tumors develop chemoresistance, and only about 10% of OC patients respond to immunotherapy. Better OC prognosis is associated with tumor-infiltrating CD8 T cells, so many research efforts focus on strategies to activate the immune system against OC. DNA hypomethylating drugs have been shown to increase the transcription of repetitive elements to induce an anti-viral innate immune response in OC cancer cell lines and to reduce tumor burden and increase survival in mouse models through the same mechanism. RNA editing by ADAR enzymes inhibits this anti-viral innate immune response. In preclinical mouse models, knockout of ADAR1 in tumors increases the type I interferon response and reverses the immunosuppressive tumor microenvironment (TME) to sensitize melanoma cells to immune therapy. We hypothesize that ADAR1 dampens the DNA methyltransferase inhibitor (DNMTi) immune response. Little is known about RNA editing of different repetitive element species in cancers, and the role of RNA editing in the DNMTi-induced immune response has not yet been explored. Studies in this application will test approaches to reduce tumor immunosuppression and activate the immune system in OC, coupled with promising epigenetic therapies. In Aim 1, RNA editing of repetitive elements in human OC cell lines following DNMTi treatment will be assessed through RNA editing analysis of RNA-sequencing data sets. In a second approach, RNA editing reporter constructs will be stably transduced into OC cell lines and ADAR1 knockout cell lines will be generated. Resulting RNA-editing levels, quantified by the luciferase reporter, will be measured in mock or DNMTi-treated WT or reporter cell lines. Together, these studies will provide novel information about ADAR- mediated regulation of RNA editing after DNMTi treatment and interferon signaling. Our preliminary data in mouse OC cells suggest that ADAR loss and type I interferon treatment induces growth arrest and death. Adar1 knockout cells will be generated in the ID8 p53-/- syngeneic mouse OC cell line. C57BL/6J mice will be injected with these syngeneic Adar1 KO cells and mice will be treated with DNMTi and anti-PD1 or anti-CTLA-4. Tumor burden and survival will be assessed and infiltrating immune cells from the tumor microenvironment (ascites) will be immunophenotyped to determine effects of epigenetic and immune therapy. These studies may lead to development of drugs targeting ADAR1 or other proteins for cancer immunotherapy. Knowledge gained from these studies is not only pertinent to the improvement of OC treatment but may also be broadly applied to the improvement of treatment for other cancer types as well.

高级别浆液性卵巢癌（OC）是一种致命的疾病，通常在晚期才被诊断出来。尽管一项重要的研究表明，OC 的五年生存率几十年来一直保持不变。许多 OC 肿瘤产生化疗耐药性，只有约 10% 的 OC 患者对免疫治疗有反应。更好的超频预后与肿瘤浸润性 CD8 T 细胞有关，因此许多研究工作都集中在以下策略上：激活免疫系统对抗 OC。 DNA 低甲基化药物已被证明可以增加重复元件的转录可在 OC 癌细胞系中诱导抗病毒先天免疫反应，通过相同的机制减少小鼠模型的肿瘤负荷并提高生存率。 RNA 编辑 ADAR 酶抑制这种抗病毒先天免疫反应。在临床前小鼠模型中，敲除肿瘤中的 ADAR1 增强 I 型干扰素反应并逆转肿瘤的免疫抑制微环境（TME）使黑色素瘤细胞对免疫治疗敏感。我们假设 ADAR1 抑制 DNA 甲基转移酶抑制剂 (DNMTi) 的免疫反应。人们对RNA编辑知之甚少癌症中不同的重复元件种类，以及 RNA 编辑在 DNMTi 诱导的免疫中的作用尚未探索回应。该应用的研究将测试减少肿瘤的方法免疫抑制并激活 OC 中的免疫系统，再加上有前途的表观遗传疗法。在目标 1 中，在 DNMTi 处理后对人类 OC 细胞系中的重复元件进行 RNA 编辑通过 RNA 测序数据集的 RNA 编辑分析进行评估。第二种方法是RNA编辑报告构建体将被稳定转导至 OC 细胞系中，ADAR1 敲除细胞系将被生成的。由荧光素酶报告基因量化的 RNA 编辑水平将在模拟或 DNMTi 处理的 WT 或报告细胞系。这些研究将共同提供有关 ADAR 的新信息 DNMTi 治疗和干扰素信号传导后介导的 RNA 编辑调节。我们在小鼠 OC 细胞中的初步数据表明，ADAR 缺失和 I 型干扰素治疗会诱导生长停滞和死亡。 Adar1 敲除细胞将在 ID8 p53-/- 同基因小鼠 OC 细胞系中产生。 C57BL/6J 小鼠将被注射这些同基因 Adar1 KO 细胞，小鼠将接受 DNMTi 和抗 PD1 或抗 CTLA-4。将评估肿瘤负荷和存活率，并从肿瘤细胞中浸润免疫细胞肿瘤微环境（腹水）将进行免疫表型分析，以确定表观遗传和免疫的影响治疗。这些研究可能会导致针对 ADAR1 或其他癌症蛋白的药物的开发免疫疗法。从这些研究中获得的知识不仅与 OC 的改进相关治疗，但也可以广泛应用于改善其他癌症类型的治疗。