ER stress and dsRNA sensing with Rt3D and palbociclib - Whole Proteome

Cytoplasmic pattern recognition receptors (PRRs) for double-stranded RNA (RIG-I/MDA5) are key mediators of anti-viral responses. PRR agonists, such as dsRNA oncolytic Reovirus type 3 Dearing (Rt3D), potently activate RNA sensors. We used an unbiased cytotoxicity screen to reveal synergistic drug-virotherapy combinations and found potent effects of Rt3D combined with the CDK4/6 inhibitor, palbociclib. The combination augmented oncolytic virus-induced endoplasmic reticulum (ER) stress/unfolded protein response (UPR) and the expression and activation/signaling of RNA sensors. Combined Rt3D-palbociclib treatment potently increased interferon production and signaling, and knockdown studies implicated key UPR proteins and the RNA sensor, RIG-I, as essential to the phenotype observed. Further experiments, using canonical RIG-I agonists and an ER stress inducer, thapsigargin, confirmed cross-talk between RNA sensing and ER stress pathways that augmented cancer cell death and interferon production. Combined Rt3D-palbociclib also increased innate immune activation within tumour cells and IFN-induced HLA expression. Analysis of the immunopeptidome revealed changes to HLA-captured peptides with Rt3D-palbociclib, including altered expression of peptides from cancer/testis antigens (CTA) and endogenous retroviral elements (ERVs). Our findings highlight cross-talk between UPR signaling and RNA-mediated PRR activation as a means of enhancing anti-cancer efficacy with potential pro-immunogenic consequences. This has implications for future clinical development of PRR agonists and oncolytic viruses, and broadens the therapeutic remit of CDK4/6 inhibitors to include roles as both ER stress and dsRNA PRR sensitizers.

识别双链RNA的胞质模式识别受体（PRRs，RIG-I/MDA5）是抗病毒免疫应答的关键介导因子。模式识别受体激动剂，如双链RNA溶瘤呼肠孤病毒3型迪林株（Rt3D），可强效激活RNA感受器。本研究通过无偏倚细胞毒性筛选发掘协同性药物-病毒治疗联合方案，发现Rt3D与CDK4/6抑制剂帕博西尼（palbociclib）联用可产生显著抗肿瘤效应。该联合方案可增强溶瘤病毒诱导的内质网（ER）应激与未折叠蛋白反应（UPR），并上调RNA感受器的表达、激活及其信号通路活性。Rt3D-帕博西尼联合治疗可强效促进干扰素（IFN）产生与信号通路激活；基因敲低实验证实，关键UPR蛋白与RNA感受器RIG-I是观测到的表型产生的必需调控因子。后续研究采用经典RIG-I激动剂与内质网应激诱导剂毒胡萝卜素（thapsigargin）进行验证，证实RNA感知通路与内质网应激通路间存在串扰，该串扰可增强癌细胞死亡与干扰素产生。Rt3D-帕博西尼联合治疗还可增强肿瘤细胞内的先天免疫激活，以及干扰素（IFN）诱导的人类白细胞抗原（HLA）表达。对免疫肽组的分析显示，Rt3D-帕博西尼处理可改变HLA捕获的肽段谱，包括癌-睾丸抗原（CTA）与内源性逆转录病毒元件（ERVs）来源肽段的表达异常。本研究揭示，未折叠蛋白反应信号通路与RNA介导的模式识别受体激活之间的串扰，可作为提升抗癌疗效的策略，同时具备潜在的促免疫原性效应。该成果对模式识别受体激动剂与溶瘤病毒的未来临床开发具有重要指导价值，同时拓展了CDK4/6抑制剂的治疗适应证范围，使其可同时作为内质网应激与双链RNA模式识别受体增敏剂。