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

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

Mechanism-based screen for G1/S checkpoint activators identifies a selective activator of EIF2AK3/PERK signalling.

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

Establishment of a system for monitoring endoplasmic reticulum redox state in mammalian cells

The endoplasmic reticulum (ER) performs a critical role in the oxidative folding of nascent proteins such that perturbations to ER homeostasis may lead to protein misfolding and subsequent pathological processes. Among the mechanisms for maintaining ER homeostasis is a redox regulation, which is a critical determinant of the fate of ER stressed cells. Here we report the establishment of a system for monitoring ER redox state in mammalian cells. The new ER redox sensing system was developed based on the previously described monitoring system in yeast. Our system could successfully monitor the dynamic ER redox state in mammalian cells. Using this system, we find that manipulation of ER oxidases changes ER redox state. The mammalian ER redox sensing system could be used to study the mechanisms of ER redox regulation and provide a foundation for an approach to develop novel therapeutic modalities for human diseases related to dysregulated ER homeostasis including diabetes, neurodegeneration and Wolfram syndrome

Delayed Ras/PKA signaling augments the unfolded protein response

During environmental, developmental, or genetic stress, the cell’s folding capacity can become overwhelmed, and misfolded proteins can accumulate in all cell compartments. Eukaryotes evolved the unfolded protein response (UPR) to counteract proteotoxic stress in the endoplasmic reticulum (ER). Although the UPR is vital to restoring homeostasis to protein folding in the ER, it has become evident that the response to ER stress is not limited to the UPR. Here, we used engineered orthogonal UPR induction, deep mRNA sequencing, and dynamic flow cytometry to dissect the cell’s response to ER stress comprehensively. We show that budding yeast augments the UPR with time-delayed Ras/PKA signaling. This second wave of transcriptional dynamics is independent of the UPR and is necessary for fitness in the presence of ER stress, partially due to a reduction in general protein synthesis. This Ras/PKA-mediated effect functionally mimics other mechanisms, such as translational control by PKR-like ER kinase (PERK) and regulated inositol-requiring enzyme 1 (IRE1)-dependent mRNA decay (RIDD), which reduce the load of proteins entering the ER in response to ER stress in metazoan cells