Mechanism-Based Screen for G1/S Checkpoint Activators Identifies a Selective Activator of EIF2AK3/PERK Signalling

Simon R Stockwell,Edward Mcdonald,Julian Blagg,Mathilde Venet,Laurent Rigoreau,S Elaine Barrie,Sibylle Mittnacht,Michelle D Garrett,Georgia Zoumpoulidou,Georgina Platt,Stuart C Wilson,Paul Workman,G Wynne Aherne,Frédéric Elustondo,Peter Sheldrake,Christelle Soudy,Robert H Te Poele,Eric J Bernhard

doi:10.1371/journal.pone.0028568

Abstract

Human cancers often contain genetic alterations that disable G1/S checkpoint control and loss of this checkpoint is thought to critically contribute to cancer generation by permitting inappropriate proliferation and distorting fate-driven cell cycle exit. The identification of cell permeable small molecules that activate the G1/S checkpoint may therefore represent a broadly applicable and clinically effective strategy for the treatment of cancer. Here we describe the identification of several novel small molecules that trigger G1/S checkpoint activation and characterise the mechanism of action for one, CCT020312, in detail. Transcriptional profiling by cDNA microarray combined with reverse genetics revealed phosphorylation of the eukaryotic initiation factor 2-alpha (EIF2A) through the eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3/PERK) as the mechanism of action of this compound. While EIF2AK3/PERK activation classically follows endoplasmic reticulum (ER) stress signalling that sets off a range of different cellular responses, CCT020312 does not trigger these other cellular responses but instead selectively elicits EIF2AK3/PERK signalling. Phosphorylation of EIF2A by EIF2A kinases is a known means to block protein translation and hence restriction point transit in G1, but further supports apoptosis in specific contexts. Significantly, EIF2AK3/PERK signalling has previously been linked to the resistance of cancer cells to multiple anticancer chemotherapeutic agents, including drugs that target the ubiquitin/proteasome pathway and taxanes. Consistent with such findings CCT020312 sensitizes cancer cells with defective taxane-induced EIF2A phosphorylation to paclitaxel treatment. Our work therefore identifies CCT020312 as a novel small molecule chemical tool for the selective activation of EIF2A-mediated translation control with utility for proof-of-concept applications in EIF2A-centered therapeutic approaches, and as a chemical starting point for pathway selective agent development. We demonstrate that consistent with its mode of action CCT020312 is capable of delivering potent, and EIF2AK3 selective, proliferation control and can act as a sensitizer to chemotherapy-associated stresses as elicited by taxanes.

Highlights

G1/S checkpoint activation is recognized to play an important role in tumour suppression [1]
Our analysis indicates that CCT020312 promotes eukaryotic initiation factor 2-alpha (EIF2A) phosphorylation with the known consequence of D-type cyclin depletion and a loss of ability to phosphorylate pRB
Our results identify loss of cyclin D expression as a major and early event in CCT020312-treated cells and show this, along with inhibition of cell cycle transit by CCT020312 is a consequence of EIF2AK3/PERK signalling

Summary

Introduction

G1/S checkpoint activation is recognized to play an important role in tumour suppression [1]. Phosphorylation of pRB by the cyclin dependent kinases (CDK) 4 or 6 and CDK2 inhibits these different activities of pRB, permitting transit of cells into S-phase and facilitating DNA replication [4,5]. Loss of control is caused by genetic alterations that affect the functioning or expression of proteins that regulate the action of pRB Such alterations comprise inactivating mutations or gene loss of the p16INK4A CDK inhibitor, which inhibits the kinase activity of CDK4 and 6 [8]; mutations in CDK4 or CDK6, rendering these kinase catalytic subunits resistant to the action of INK4 family CDK inhibitors [9]; and the deregulated expression of D cyclin genes, arising from either gene translocation [10,11] or, more frequently, gene transcriptional activation as a consequence of oncogene activation. Signalling through the Ras, wingless (Wnt) and nuclear factor kappa B (NFkB) pathways all result in the transcriptional activation of D cyclin genes and mutational activation of these pathways in cancers is thought to contribute to unlicensed G1/S checkpoint transit [12,13]

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Conclusion