Reovirus type 3 Dearing (reovirus) is a tumor-selective oncolytic virus currently under evaluation in clinical trials. Here, we report that the therapeutic efficacy of reovirus in head and neck squamous cell cancer can be enhanced by targeting the unfolded protein response (UPR) kinase, protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK). PERK inhibition by GSK2606414 increased reovirus efficacy in both 2D and 3D models in vitro, while perturbing the normal host cell response to reovirus-induced endoplasmic reticulum (ER) stress. UPR reporter constructs were used for live-cell 3D spheroid imaging. Profiling of eIF2a-ATF4, IRE1a-XBP1, and ATF6 pathway activity revealed a context-dependent increase in eIF2a-ATF4 signaling due to GSK2606414. GSK2606414 blocked eIF2a-ATF4 signaling because of the canonical ER stress agent thapsigargin. In the context of reovirus infection, GSK2606414 induced eIF2a-ATF4 signaling. Knockdown of eIF2a kinases PERK, GCN2, and PKR revealed eIF2a-ATF4 reporter activity was dependent on either PERK or GCN2. Knockdown of ATF4 abrogated the GSK2606414-induced increase in reovirus protein levels, confirming eIF2a-ATF signaling as key to the observed phenotype. Our work identifies a novel approach to enhance the efficacy and replication of reovirus in a therapeutic setting.

3 型呼肠孤病毒 Dearing（呼肠孤病毒）是一种肿瘤选择性溶瘤病毒，目前正在临床试验中进行评估。在这里，我们报告说，通过靶向未折叠蛋白反应（UPR）激酶、蛋白激酶R（PKR）样内质网激酶（PERK），可以增强呼肠孤病毒对头颈鳞状细胞癌的治疗效果。GSK2606414 抑制 PERK 增加了体外2D 和 3D 模型中呼肠孤病毒的功效，同时扰乱了正常宿主细胞对呼肠孤病毒诱导的内质网 (ER) 应激的反应。UPR 报告基因构建体用于活细胞 3D 球体成像。对 eIF2a-ATF4、IRE1a-XBP1 和 ATF6 通路活性的分析表明，GSK2606414 导致 eIF2a-ATF4 信号传导出现环境依赖性增加。由于经典的 ER 应激剂毒胡萝卜素，GSK2606414 阻断了 eIF2a-ATF4 信号传导。在呼肠孤病毒感染的情况下，GSK2606414 诱导 eIF2a-ATF4 信号传导。eIF2a 激酶 PERK、GCN2 和 PKR 的敲低显示 eIF2a-ATF4 报告基因活性依赖于 PERK 或 GCN2。ATF4 的敲低消除了 GSK2606414 诱导的呼肠孤病毒蛋白水平的增加，证实 eIF2a-ATF 信号传导是观察到的表型的关键。我们的工作确定了一种新方法来增强呼肠孤病毒在治疗环境中的功效和复制。