Fraile-Bethencourt Eugenia, Foss Marie H, Nelson Dylan, Malhotra Sanjay V, Anand Sudarshan
Department of Cell, Developmental and Cancer Biology, Oregon Health and Science University, Portland, OR, United States.
Center for Experimental Therapeutics, Knight Cancer Institute, Oregon Health and Science University, Portland, OR, United States.
Front Mol Biosci. 2022 Feb 14;9:837610. doi: 10.3389/fmolb.2022.837610. eCollection 2022.
Enhancing the immune microenvironment in cancer by targeting the nucleic acid sensors is becoming a potent therapeutic strategy. Among the nucleic acid sensors, activation of the RNA sensor Retinoic Acid-inducible Gene (RIG-I) using small hairpin RNAs has been shown to elicit powerful innate and adaptive immune responses. Given the challenges inherent in pharmacokinetics and delivery of RNA based agonists, we set out to discover small molecule agonists of RIG-I using a cell-based assay. To this end, we established and validated a robust high throughput screening assay based on a commercially available HEK293 reporter cell line with a luciferase reporter downstream of tandem interferon stimulated gene 54 (ISG54) promoter elements. We first confirmed that the luminescence in this cell line is dependent on RIG-I and the interferon receptor using a hairpin RNA RIG-I agonist. We established a 96-well and a 384-well format HTS based on this cell line and performed a proof-of-concept screen using an FDA approved drug library of 1,200 compounds. Surprisingly, we found two HDAC inhibitors Entinostat, Mocetinostat and the PLK1 inhibitor Volasertib significantly enhanced ISG-luciferase activity. This luminescence was substantially diminished in the null reporter cell line indicating the increase in signaling was dependent on RIG-I expression. Combination treatment of tumor cell lines with Entinostat increased RIG-I induced cell death in a mammary carcinoma cell line that is resistant to either Entinostat or RIG-I agonist alone. Taken together, our data indicates an unexpected role for HDAC1,-3 inhibitors in enhancing RIG-I signaling and highlight potential opportunities for therapeutic combinations.
通过靶向核酸传感器来增强癌症中的免疫微环境正成为一种有效的治疗策略。在核酸传感器中，使用小发夹RNA激活RNA传感器视黄酸诱导基因（RIG-I）已被证明能引发强大的先天性和适应性免疫反应。鉴于基于RNA的激动剂在药代动力学和递送方面存在固有的挑战，我们着手使用基于细胞的检测方法来发现RIG-I的小分子激动剂。为此，我们基于一种市售的HEK293报告细胞系建立并验证了一种强大的高通量筛选检测方法，该细胞系在串联干扰素刺激基因54（ISG54）启动子元件下游带有荧光素酶报告基因。我们首先使用发夹RNA RIG-I激动剂证实了该细胞系中的发光依赖于RIG-I和干扰素受体。我们基于该细胞系建立了96孔和384孔格式的高通量筛选方法，并使用一个包含1200种化合物的FDA批准药物库进行了概念验证筛选。令人惊讶的是，我们发现两种组蛋白去乙酰化酶抑制剂恩替诺特、莫西诺特以及PLK1抑制剂沃拉替尼能显著增强ISG-荧光素酶活性。在无报告基因的细胞系中，这种发光显著减弱，表明信号增强依赖于RIG-I的表达。恩替诺特与肿瘤细胞系联合处理可增加RIG-I诱导的细胞死亡，在单独对恩替诺特或RIG-I激动剂耐药的乳腺癌细胞系中也是如此。综上所述，我们的数据表明组蛋白去乙酰化酶1、3抑制剂在增强RIG-I信号传导中具有意想不到的作用，并突出了治疗联合的潜在机会。
