An increased level of phosphorylation of eukaryotic translation initiation factor 2 subunit-α (eIF2α, encoded by EIF2S1; eIF2α-p) coupled with decreased guanine nucleotide exchange activity of eIF2B is a hallmark of the 'canonical' integrated stress response (c-ISR)1. It is unclear whether impaired eIF2B activity in human diseases including leukodystrophies2, which occurs in the absence of eIF2α-p induction, is synonymous with the c-ISR. Here we describe a mechanism triggered by decreased eIF2B activity, distinct from the c-ISR, which we term the split ISR (s-ISR). The s-ISR is characterized by translational and transcriptional programs that are different from those observed in the c-ISR. Opposite to the c-ISR, the s-ISR requires eIF4E-dependent translation of the upstream open reading frame 1 and subsequent stabilization of ATF4 mRNA. This is followed by altered expression of a subset of metabolic genes (for example, PCK2), resulting in metabolic rewiring required to maintain cellular bioenergetics when eIF2B activity is attenuated. Overall, these data demonstrate a plasticity of the mammalian ISR, whereby the loss of eIF2B activity in the absence of eIF2α-p induction activates the eIF4E-ATF4-PCK2 axis to maintain energy homeostasis.

• MeSH
• Energy Metabolism genetics   MeSH
• Eukaryotic Initiation Factors * metabolism   MeSH
• Eukaryotic Initiation Factor-2 metabolism   MeSH
• Eukaryotic Initiation Factor-2B metabolism   deficiency   MeSH
• Eukaryotic Initiation Factor-4E metabolism   MeSH
• Phosphorylation MeSH
• Stress, Physiological * genetics   MeSH
• Humans MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Mice MeSH
• Protein Biosynthesis MeSH
• RNA Stability MeSH
• Activating Transcription Factor 4 genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• ATF4 protein, human MeSH Browser
• Eukaryotic Initiation Factors * MeSH
• Eukaryotic Initiation Factor-2 MeSH
• Eukaryotic Initiation Factor-2B MeSH
• Eukaryotic Initiation Factor-4E MeSH
• RNA, Messenger MeSH
• Activating Transcription Factor 4 MeSH

Protein synthesis plays a major role in homeostasis and when dysregulated leads to various pathologies including cancer. To this end, imbalanced expression of eukaryotic translation initiation factors (eIFs) is not only a consequence but also a driver of neoplastic growth. eIF3 is the largest, multi-subunit translation initiation complex with a modular assembly, where aberrant expression of one subunit generates only partially functional subcomplexes. To comprehensively study the effects of eIF3 remodeling, we contrasted the impact of eIF3d, eIF3e or eIF3h depletion on the translatome of HeLa cells using Ribo-seq. Depletion of eIF3d or eIF3e, but not eIF3h reduced the levels of multiple components of the MAPK signaling pathways. Surprisingly, however, depletion of all three eIF3 subunits increased MAPK/ERK pathway activity. Depletion of eIF3e and partially eIF3d also increased translation of TOP mRNAs that encode mainly ribosomal proteins and other components of the translational machinery. Moreover, alterations in eIF3 subunit stoichiometry were often associated with changes in translation of mRNAs containing short uORFs, as in the case of the proto-oncogene MDM2 and the transcription factor ATF4. Collectively, perturbations in eIF3 subunit stoichiometry exert specific effect on the translatome comprising signaling and stress-related transcripts with complex 5' UTRs that are implicated in homeostatic adaptation to stress and cancer.

• Keywords
• MAPK pathway, eIF3, genetics, genomics, human, ribosomal proteins, ribosome, translation, translational control,
• MeSH
• Eukaryotic Initiation Factor-3 * metabolism   genetics   MeSH
• HeLa Cells MeSH
• Humans MeSH
• MAP Kinase Signaling System * MeSH
• Protein Biosynthesis MeSH
• Proto-Oncogene Mas * MeSH
• Ribosomal Proteins * metabolism   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Eukaryotic Initiation Factor-3 * MeSH
• MAS1 protein, human MeSH Browser
• Proto-Oncogene Mas * MeSH
• Ribosomal Proteins * MeSH

Activating transcription factor 4 (ATF4) is a master transcriptional regulator of the integrated stress response, leading cells toward adaptation or death. ATF4's induction under stress was thought to be due to delayed translation reinitiation, where the reinitiation-permissive upstream open reading frame 1 (uORF1) plays a key role. Accumulating evidence challenging this mechanism as the sole source of ATF4 translation control prompted us to investigate additional regulatory routes. We identified a highly conserved stem-loop in the uORF2/ATF4 overlap, immediately preceded by a near-cognate CUG, which introduces another layer of regulation in the form of ribosome queuing. These elements explain how the inhibitory uORF2 can be translated under stress, confirming prior observations but contradicting the original regulatory model. We also identified two highly conserved, potentially modified adenines performing antagonistic roles. Finally, we demonstrated that the canonical ATF4 translation start site is substantially leaky scanned. Thus, ATF4's translational control is more complex than originally described, underpinning its key role in diverse biological processes.

• Keywords
• ATF4, CP: Molecular biology, integrated stress response, ribosome, ribosome queuing, translation reinitiation, translational control, unfolded protein response,
• MeSH
• Stress, Physiological MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Open Reading Frames * genetics   MeSH
• Protein Biosynthesis * MeSH
• Ribosomes * metabolism   MeSH
• Base Sequence MeSH
• Activating Transcription Factor 4 * metabolism   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• ATF4 protein, human MeSH Browser
• Activating Transcription Factor 4 * MeSH

Drug repurposing is a versatile strategy to improve current therapies. Disulfiram has long been used in the treatment of alcohol dependency and multiple clinical trials to evaluate its clinical value in oncology are ongoing. We have recently reported that the disulfiram metabolite diethyldithiocarbamate, when combined with copper (CuET), targets the NPL4 adapter of the p97VCP segregase to suppress the growth of a spectrum of cancer cell lines and xenograft models in vivo. CuET induces proteotoxic stress and genotoxic effects, however important issues concerning the full range of the CuET-evoked tumor cell phenotypes, their temporal order, and mechanistic basis have remained largely unexplored. Here, we have addressed these outstanding questions and show that in diverse human cancer cell models, CuET causes a very early translational arrest through the integrated stress response (ISR), later followed by features of nucleolar stress. Furthermore, we report that CuET entraps p53 in NPL4-rich aggregates leading to elevated p53 protein and its functional inhibition, consistent with the possibility of CuET-triggered cell death being p53-independent. Our transcriptomics profiling revealed activation of pro-survival adaptive pathways of ribosomal biogenesis (RiBi) and autophagy upon prolonged exposure to CuET, indicating potential feedback responses to CuET treatment. The latter concept was validated here by simultaneous pharmacological inhibition of RiBi and/or autophagy that further enhanced CuET's tumor cytotoxicity, using both cell culture and zebrafish in vivo preclinical models. Overall, these findings expand the mechanistic repertoire of CuET's anti-cancer activity, inform about the temporal order of responses and identify an unorthodox new mechanism of targeting p53. Our results are discussed in light of cancer-associated endogenous stresses as exploitable tumor vulnerabilities and may inspire future clinical applications of CuET in oncology, including combinatorial treatments and focus on potential advantages of using certain validated drug metabolites, rather than old, approved drugs with their, often complex, metabolic profiles.

• MeSH
• Zebrafish metabolism   MeSH
• Disulfiram * metabolism   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• Neoplasms * metabolism   MeSH
• Ribosomes metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Disulfiram * MeSH
• Tumor Suppressor Protein p53 MeSH