The PERK Inhibitor GSK2606414 Enhances Reovirus Infection in Head and Neck Squamous Cell Carcinoma via an ATF4-Dependent Mechanism

Martin Mclaughlin,Malin Pedersen,Victoria Roulstone,Katharina F Bergerhoff,Henry G Smith,Harriet Whittock,Joan N Kyula,Magnus T Dillon,Hardev S Pandha,Richard Vile,Alan A Melcher,Kevin J Harrington

doi:10.1016/j.omto.2020.01.001

Abstract

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.

Highlights

Reovirus type 3 Dearing is an immuno-oncolytic virus under active clinical development
Studies indicated reovirus tumor selectivity was linked to inactivation of the protein kinase R (PKR)-mediated anti-viral response driven by oncogenic transformation events.[3,4]
Inhibition of PKR-like endoplasmic reticulum kinase (PERK) Sensitizes HNSCC to Reovirus The HPV-negative head and neck squamous cell carcinoma (HNSCC) cell lines HN5 and FaDu were used; both are characterized by TP53 mutations and high EGFR and HER2 levels.[20]

Summary

Introduction

Reovirus type 3 Dearing (abbreviated hereafter as reovirus) is an immuno-oncolytic virus under active clinical development. It has been granted orphan drug status by the FDA for malignant glioma. It is a segmented, double-stranded RNA virus with 10 genome segments. Viral proteins provide self-sufficiency in relation to cellular entry via JAM1 and b1-integrins, endosomal escape allowing entry to the cytoplasm, and viral genomic replication.[1,2] Reovirus is dependent on the translational machinery of infected cells for protein synthesis following release of viral RNA into the cytoplasm.

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Conclusion