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

高级别浆液性卵巢癌(OC)是一种致命的疾病，通常在晚期被诊断出来。尽管 重大研究表明，OC的五年存活率几十年来一直保持不变。许多OC肿瘤 出现化疗耐药，只有大约10%的OC患者对免疫治疗有反应。更好的OC 预后与肿瘤浸润性CD8 T细胞有关，因此许多研究努力集中在策略上 激活免疫系统对抗OC。DNA去甲基化药物已被证明可以增加 转录重复元件在OC癌细胞系中诱导抗病毒先天免疫反应 通过同样的机制减轻肿瘤负担，提高小鼠存活率。RNA编辑者 ADAR酶抑制这种抗病毒的先天免疫反应。在临床前小鼠模型中，基因敲除 肿瘤中的ADAR1增加I型干扰素反应并逆转免疫抑制肿瘤 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甲基转移酶抑制物(DNMTi)的免疫反应。对RNA的编辑知之甚少 肿瘤中不同重复元件的种类以及RNA编辑在DNMTi诱导免疫中的作用 目前还没有人对此做出回应。这项应用的研究将测试减少肿瘤的方法 免疫抑制和激活OC的免疫系统，结合有前景的表观遗传疗法。 在目标1中，DNMTi处理后人OC细胞系中重复元件的RNA编辑将是 通过对RNA测序数据集的RNA编辑分析进行评估。在第二种方法中，RNA编辑 报告基因的构建将稳定地转导入OC细胞系和ADAR1基因敲除细胞系 已生成。由此产生的RNA编辑水平，由荧光素酶报告者量化，将以模拟或 DNMTi处理的WT或报告细胞系。总之，这些研究将提供有关ADAR的新信息- DNMTi治疗和干扰素信号转导后RNA编辑的中介调节。 我们在小鼠OC细胞中的初步数据表明，ADAR丢失和I型干扰素治疗可诱导 生长停滞和死亡。在ID8-p53-/-同基因小鼠OC细胞系中将产生ADAR1基因敲除细胞。 C57BL/6J小鼠将被注射这些同基因ADAR1 KO细胞，小鼠将被DNMTi和 抗PD1或抗CTLA-4。将评估肿瘤负担和生存期，并从 肿瘤微环境(腹水)将进行免疫表型，以确定表观遗传和免疫的影响 心理治疗。这些研究可能导致针对ADAR1或其他癌症蛋白质的药物的开发 免疫疗法。从这些研究中获得的知识不仅与提高组织能力有关 但也可广泛应用于其他癌症类型的治疗方法的改进。

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.