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，通常在晚期诊断。尽管 根据一项重要的研究，OC的五年生存率几十年来一直保持不变。许多OC肿瘤 OC患者中只有约10%对免疫疗法有反应。更好的OC 预后与肿瘤浸润性CD 8 T细胞相关，因此许多研究工作集中在 激活免疫系统对抗OC DNA低甲基化药物已被证明可以增加 转录重复元件以在OC癌细胞系中诱导抗病毒先天免疫应答， 通过相同的机制降低肿瘤负荷并增加小鼠模型的存活率。RNA编辑 阿达尔酶抑制这种抗病毒先天免疫应答。在临床前小鼠模型中， 肿瘤中的ADAR 1增加I型干扰素应答并逆转免疫抑制性肿瘤 微环境（TME）以使黑素瘤细胞对免疫疗法敏感。我们假设ADAR 1 抑制DNA甲基转移酶抑制剂（DNMTi）免疫应答。关于RNA编辑的知之甚少。 癌症中的不同重复元件种类，以及RNA编辑在DNMTi诱导的免疫应答中的作用。 尚未探讨对策。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，加上有前途的表观遗传疗法。 在目标1中，将在DNMTi处理后对人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。在第二种方法中， 报告基因构建体将被稳定地转导到OC细胞系中，并且ADAR 1敲除细胞系将被 生成的.通过荧光素酶报告基因定量的所得RNA编辑水平将在模拟或非模拟环境中测量。 DNMTi处理的WT或报告细胞系。总之，这些研究将提供有关阿达尔的新信息- DNMTi处理后的RNA编辑和干扰素信号传导介导的调节。 我们在小鼠OC细胞中的初步数据表明，阿达尔缺失和I型干扰素治疗诱导了 生长停滞和死亡。Adar 1敲除细胞将在ID 8 p53-/-同基因小鼠OC细胞系中产生。 C57 BL/6 J小鼠将注射这些同基因Adar 1 KO细胞，小鼠将用DNMTi和 抗PD 1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。 将对肿瘤微环境（腹水）进行免疫表型分析，以确定表观遗传和免疫治疗的效果。 疗法这些研究可能会导致开发针对ADAR 1或其他癌症蛋白质的药物 免疫疗法从这些研究中获得的知识不仅与OC的改善有关， 本发明不仅适用于治疗，而且还可以广泛地应用于改善其他癌症类型的治疗。

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.