Abstract：
Novel therapies are urgently needed for ovarian cancer (OC), the fifth deadliest cancer in women. Preclinical work has shown that DNA methyltransferase inhibitors (DNMTis) can reverse the immunosuppressive tumor microenvironment in OC. Inhibiting DNA methyltransferases activate transcription of double-stranded (ds)RNA, including transposable elements. These dsRNAs activate sensors in the cytoplasm and trigger type I interferon (IFN) signaling, recruiting host immune cells to kill the tumor cells. Adenosine deaminase 1 (ADAR1) is induced by IFN signaling and edits mammalian dsRNA with an A-to-I nucleotide change, which is read as an A-to-G change in sequencing data. These edited dsRNAs cannot be sensed by dsRNA sensors, and thus ADAR1 inhibits the type I IFN response in a negative feedback loop. We hypothesized that decreasing ADAR1 editing would enhance the DNMTi-induced immune response.
Human OC cell lines were treated in vitro with DNMTi and then RNA-sequenced to measure RNA editing. Adar1 was stably knocked down in ID8 Trp53-/- mouse OC cells. Control cells (shGFP) or shAdar1 cells were tested with mock or DNMTi treatment. Tumor-infiltrating immune cells were immunophenotyped using flow cytometry and cell culture supernatants were analyzed for secreted chemokines/cytokines. Mice were injected with syngeneic shAdar1 ID8 Trp53-/- cells and treated with tetrahydrouridine/DNMTi while given anti-interferon alpha and beta receptor 1, anti-CD8, or anti-NK1.1 antibodies every 3 days.
We show that ADAR1 edits transposable elements in human OC cell lines after DNMTi treatment in vitro. Combining ADAR1 knockdown with DNMTi significantly increases pro-inflammatory cytokine/chemokine production and sensitivity to IFN-β compared with either perturbation alone. Furthermore, DNMTi treatment and Adar1 loss reduces tumor burden and prolongs survival in an immunocompetent mouse model of OC. Combining Adar1 loss and DNMTi elicited the most robust antitumor response and transformed the immune microenvironment with increased recruitment and activation of CD8+ T cells.
In summary, we showed that the survival benefit from DNMTi plus ADAR1 inhibition is dependent on type I IFN signaling. Thus, epigenetically inducing transposable element transcription combined with inhibition of RNA editing is a novel therapeutic strategy to reverse immune evasion in OC, a disease that does not respond to current immunotherapies.
Human OC cell lines were treated in vitro with DNMTi and then RNA-sequenced to measure RNA editing. Adar1 was stably knocked down in ID8 Trp53-/- mouse OC cells. Control cells (shGFP) or shAdar1 cells were tested with mock or DNMTi treatment. Tumor-infiltrating immune cells were immunophenotyped using flow cytometry and cell culture supernatants were analyzed for secreted chemokines/cytokines. Mice were injected with syngeneic shAdar1 ID8 Trp53-/- cells and treated with tetrahydrouridine/DNMTi while given anti-interferon alpha and beta receptor 1, anti-CD8, or anti-NK1.1 antibodies every 3 days.
We show that ADAR1 edits transposable elements in human OC cell lines after DNMTi treatment in vitro. Combining ADAR1 knockdown with DNMTi significantly increases pro-inflammatory cytokine/chemokine production and sensitivity to IFN-β compared with either perturbation alone. Furthermore, DNMTi treatment and Adar1 loss reduces tumor burden and prolongs survival in an immunocompetent mouse model of OC. Combining Adar1 loss and DNMTi elicited the most robust antitumor response and transformed the immune microenvironment with increased recruitment and activation of CD8+ T cells.
In summary, we showed that the survival benefit from DNMTi plus ADAR1 inhibition is dependent on type I IFN signaling. Thus, epigenetically inducing transposable element transcription combined with inhibition of RNA editing is a novel therapeutic strategy to reverse immune evasion in OC, a disease that does not respond to current immunotherapies.
摘要：
卵巢癌（OC）迫切需要新的治疗方法。女性第五大致命癌症。临床前工作表明，DNA甲基转移酶抑制剂（DNMTis）可以逆转OC中的免疫抑制肿瘤微环境。抑制DNA甲基转移酶激活双链（ds）RNA的转录，包括转座元素。这些dsRNA激活细胞质中的传感器并触发I型干扰素（IFN）信号，招募宿主免疫细胞杀死肿瘤细胞。腺苷脱氨酶1（ADAR1）由IFN信号诱导，并编辑哺乳动物dsRNA，其被读取为测序数据中的A到G变化。这些编辑的dsRNA不能被dsRNA传感器感知，因此ADAR1在负反馈回路中抑制I型IFN应答。我们假设减少ADAR1编辑将增强DNMTi诱导的免疫应答。
人OC细胞系在体外用DNMTi处理,然后RNA测序以测量RNA编辑。Adar1在ID8Trp53-/-小鼠OC细胞中稳定敲低。用模拟或DNMTi处理测试对照细胞(shGFP)或shAdar1细胞。使用流式细胞术对肿瘤浸润性免疫细胞进行免疫表型分析,并分析细胞培养上清液的分泌趋化因子/细胞因子。将小鼠注射同基因shAdar1ID8Trp53-/-细胞并用四氢尿苷/DNMTi处理,同时每3天给予抗-干扰素α和β受体1、抗-CD8或抗-NK1.1抗体。
我们显示,在体外DNMTi处理后,ADAR1在人OC细胞系中编辑转座因子。与单独的任一种扰动相比,将ADAR1敲低与DNMTi组合显著增加促炎细胞因子/趋化因子的产生和对IFN-β的敏感性。此外，在OC的免疫活性小鼠模型中,DNMTi治疗和Adar1损失减少肿瘤负荷并延长存活。Adar1损失和DNMTi的组合引发了最强大的抗肿瘤反应，并通过增加CD8T细胞的募集和激活来转化免疫微环境。
总之，我们表明,DNMTi+ADAR1抑制的生存获益依赖于I型IFN信号传导.因此，表观遗传诱导转座因子转录结合抑制RNA编辑是逆转OC免疫逃避的一种新的治疗策略，一种对目前的免疫疗法没有反应的疾病。
人OC细胞系在体外用DNMTi处理,然后RNA测序以测量RNA编辑。Adar1在ID8Trp53-/-小鼠OC细胞中稳定敲低。用模拟或DNMTi处理测试对照细胞(shGFP)或shAdar1细胞。使用流式细胞术对肿瘤浸润性免疫细胞进行免疫表型分析,并分析细胞培养上清液的分泌趋化因子/细胞因子。将小鼠注射同基因shAdar1ID8Trp53-/-细胞并用四氢尿苷/DNMTi处理,同时每3天给予抗-干扰素α和β受体1、抗-CD8或抗-NK1.1抗体。
我们显示,在体外DNMTi处理后,ADAR1在人OC细胞系中编辑转座因子。与单独的任一种扰动相比,将ADAR1敲低与DNMTi组合显著增加促炎细胞因子/趋化因子的产生和对IFN-β的敏感性。此外，在OC的免疫活性小鼠模型中,DNMTi治疗和Adar1损失减少肿瘤负荷并延长存活。Adar1损失和DNMTi的组合引发了最强大的抗肿瘤反应，并通过增加CD8T细胞的募集和激活来转化免疫微环境。
总之，我们表明,DNMTi+ADAR1抑制的生存获益依赖于I型IFN信号传导.因此，表观遗传诱导转座因子转录结合抑制RNA编辑是逆转OC免疫逃避的一种新的治疗策略，一种对目前的免疫疗法没有反应的疾病。
