Littérature scientifique sur le sujet « Sephin1, autophagy, integrated stress response »

Sephin1 is a derivative of guanabenz that inhibits the dephosphorylation of the eukaryotic initiation factor 2 alpha (eIF2α) and therefore may enhance the integrated stress response (ISR), an adaptive mechanism against different cellular stresses, such as accumulation of misfolded proteins. Unlike guanabenz, Sephin1 provides neuroprotection without adverse effects on the α2-adrenergic system and therefore it is considered a promising pharmacological therapeutic tool. Here, we have studied the effects of Sephin1 on N-methyl-D-aspartic acid (NMDA) receptor signaling which may modulate the ISR and contribute to excitotoxic neuronal loss in several neurodegenerative conditions. Time-course analysis of peIF2α levels after NMDA receptor overactivation showed a delayed dephosphorylation that occurred in the absence of activating transcription factor 4 (ATF4) and therefore independently of the ISR, in contrast to that observed during endoplasmic reticulum (ER) stress induced by tunicamycin and thapsigargin. Similar to guanabenz, Sephin1 completely blocked NMDA-induced neuronal death and was ineffective against AMPA-induced excitotoxicity, whereas it did not protect from experimental ER stress. Interestingly, both guanabenz and Sephin1 partially but significantly reduced NMDA-induced cytosolic Ca2+ increase, leading to a complete inhibition of subsequent calpain activation. We conclude that Sephin1 and guanabenz share common strong anti-excitotoxic properties with therapeutic potential unrelated to the ISR.

Eukaryotic cells are exposed to many internal and external stimuli that affect their fate. In particular, the exposure to some of these stimuli induces stress triggering a variety of stress responses aimed to re-establish cellular homeostasis. It is now established that the deregulation of stress response pathways plays a central role in cancer initiation and progression, allowing the adaptation of cells to an altered state in the new environment. Autophagy is a tightly regulated pathway which exerts “housekeeping” role in physiological processes. Recently, a growing amount of evidence highlighted the crucial role of autophagy in the regulation of integrated stress responses, including nucleolar and endoplasmic reticulum. In this review, we attempt to afford an overview of the complex role of nucleolar and endoplasmic reticulum stress-response mechanisms in the regulation of autophagy in cancer and cancer treatment.

Nanoparticle drug carriers trigger a variety of cellular stress responses, including ER stress and antioxidant responses, but may also affect the intracellular degradative pathway autophagy. This can impose profound effects on drug delivery, cellular treatment responses, and nanoparticle cytotoxicity. We recently demonstrated that even small variations in the alkyl side chains of poly(alkylcyanoacrylate) (PACA) drug carrier nanoparticles, namely butyl (PBCA), ethylbutyl (PEBCA), or octyl (POCA), differentially induce ER stress and redox imbalance in human cell lines. Here, we systematically investigate how these PACA variants affect autophagy. Interestingly, treatment with PEBCA or POCA particles led to intracellular accumulation of the autophagosome marker LC3-II, but via different mechanisms. PEBCA induced an integrated stress response-and ATF4-mediated increase in LC3B mRNA, whereas POCA blocked autophagic degradation of LC3-II and long-lived proteins in bulk. PBCA also increased LC3B mRNA via the integrated stress response and ATF4, but unlike PEBCA, it inhibited LC3 lipidation and autophagic cargo degradation. Our data demonstrate that even subtle variations in NP structure can have profoundly different impacts on autophagy, and that careful monitoring of autophagic flux and cargo degradation is critical for drawing accurate conclusions. Our findings have important implications for the choice of PACA monomer in different therapeutic settings.

Cells respond to starvation by shutting down protein synthesis and by activating catabolic processes, including autophagy, to recycle nutrients. This two-pronged response is mediated by the integrated stress response (ISR) through phosphorylation of eIF2α, which represses protein translation, and by inhibition of mTORC1 signaling, which promotes autophagy also through a stress-responsive transcriptional program. Implementation of such a program, however, requires protein synthesis, thus conflicting with general repression of translation. How is this mismatch resolved? We found that the main regulator of the starvation-induced transcriptional program, TFEB, counteracts protein synthesis inhibition by directly activating expression of GADD34, a component of the protein phosphatase 1 complex that dephosphorylates eIF2α. We discovered that GADD34 plays an essential role in autophagy by tuning translation during starvation, thus enabling lysosomal biogenesis and a sustained autophagic flux. Hence, the TFEB-GADD34 axis integrates the mTORC1 and ISR pathways in response to starvation.

Cells remove unstable polypeptides through protein quality-control (PQC) pathways such as ubiquitin-mediated proteolysis and autophagy. In the present study, we investigated how these pathways are used in β-thalassemia, a common hemoglobinopathy in which β-globin gene mutations cause the accumulation and precipitation of cytotoxic α-globin subunits. In β-thalassemic erythrocyte precursors, free α-globin was polyubiquitinated and degraded by the proteasome. These cells exhibited enhanced proteasome activity, and transcriptional profiling revealed coordinated induction of most proteasome subunits that was mediated by the stress-response transcription factor Nrf1. In isolated thalassemic cells, short-term proteasome inhibition blocked the degradation of free α-globin. In contrast, prolonged in vivo treatment of β-thalassemic mice with the proteasome inhibitor bortezomib did not enhance the accumulation of free α-globin. Rather, systemic proteasome inhibition activated compensatory proteotoxic stress-response mechanisms, including autophagy, which cooperated with ubiquitin-mediated proteolysis to degrade free α-globin in erythroid cells. Our findings show that multiple interregulated PQC responses degrade excess α-globin. Therefore, β-thalassemia fits into the broader framework of protein-aggregation disorders that use PQC pathways as cell-protective mechanisms.

Hypoxia in the microenvironment of many solid tumours is an important determinant of malignant progression. The ISR (integrated stress response) protects cells from the ER (endoplasmic reticulum) stress caused by severe hypoxia. Likewise, autophagy is a mechanism by which cancer cells can evade hypoxic cell death. In the present paper we report that the autophagy-initiating kinase ULK1 (UNC51-like kinase 1) is a direct transcriptional target of ATF4 (activating transcription factor 4), which drives the expression of ULK1 mRNA and protein in severe hypoxia and ER stress. We demonstrate that ULK1 is required for autophagy in severe hypoxia and that ablation of ULK1 causes caspase-3/7-independent cell death. Furthermore, we report that ULK1 expression is associated with a poor prognosis in breast cancer. Collectively, the findings of the present study identify transcriptional up-regulation of ULK1 as a novel arm of the ISR, and suggest ULK1 as a potentially effective target for cancer therapy.

Cellular response to stress is diverse, ranging from adaptation through activation of survival pathways to induction of cell death. We designed a multicolor flow cytometry panel to gain insight into multifaceted stress response and to assess multiple cell death modes at the single cell level. The panel included antibodies against RIP3, active caspase 3, cleaved PARP, ATF4, H2AX, p21, Ki-67 and a dead cell discriminating dye. This enabled simultaneous interrogation of a multitude of cell death modes including necrosis, necroptosis, apoptosis and parthanatos as well as proliferation, autophagy and endoplasmic reticulum (ER) stress. Notably, we utilized high dimensional analytic approaches to better elucidate stress response and cell death modes. We leveraged t-SNE for dimension reduction, PhenoGraph to identify distinct phenotypes and diffusion map to map cell trajectories. First, we aimed at delineating response patterns and cell death modes associated with targeted therapies currently being investigated for the treatment of acute myeloid leukemias (AML), including inhibitors targeting anti-apoptotic molecules (Bcl-2i and Mcl-1i), propagating p53-mediated apoptosis (MDM-2i and exportin 1i [XPO1i]), abrogating adaptive circuits through blocking autophagic degradation (SBI-0206965) and depleting anti-oxidant pool (Buthionine sulfoximine [BSO]), and mitochondrial proteasome ClpP activator (ClpPa) (ONC201). Initially, we generated two-dimensional t-SNE plots to interrogate agent-specific response landscapes. Unsupervised high-dimensional mapping demonstrated that Bcl-2 or Mcl-1 inhibition alone did not alter cellular landscape. However, treatment with MDM2i or XPO1i and ClpPa elicited divergent stress responses and cell death modes. Two-dimensional plots showed differential induction of autophagy and ER stress following treatment with MDM2i, XPO1i or ClpPa. Remarkably, we observed that MDM2i and XPO1i were associated with emergence of quiescent cells, based on high expression of p21, and higher levels of ER stress and autophagy while ClpPa induced DNA damage, and was associated with persistent Ki-67 expression and lower levels of p21. This approach enabled us to dissect single agent specific stress signatures. Next, we assessed the response landscapes of prior knowledge-based, data-driven synergistic dual drug combinations. Mapping of response landscapes of multiple dual drug combinations at single-cell resolution revealed distinct associations among integrated stress responses, divergent cellular progression trajectories and previously unidentified response patterns. We observed that autophagic cells were associated with high levels of ER stress and cell kinetic quiescence, suggesting that perturbation-specific stress responses are integrated at the cellular level and are triggered concomitantly. Unsupervised clustering and partitioning of response landscapes to identify major phenotypes revealed two distinct autophagic cell phenotypes: 1) Quiescent autophagic cells without DNA damage and 2) proliferating autophagic cells with DNA damage. Strikingly, combinatorial use of MDM2i and XPO1i almost completely eliminated all AML cells. The surviving cells were quiescent, had high levels of autophagy and ER stress, and were spared of DNA damage. On the other hand, addition of either Bcl-2i or Mcl-1i to MDM2i markedly reduced p21, ER stress and autophagy, indicating that these anti-apoptotic molecules may play a role in cellular adaptation. Addition of Bcl-2i or Mcl-1i inhibitors may specifically deplete autophagic cells with high p21 and ER stress (as we have reported, Pan et al. Cancer Cell 2017). To map cellular trajectories and identify the sequence of events we leveraged diffusion map algorithm and aligned the clusters along pseudo-time. This approach enabled us to identify the earliest stage of cell death, characterized by expression of LC3B, H2AX and cleaved PARP while dead cell dyes marked the latest stage. These findings provide proof of concept for the utility of single cell mapping of cellular stress in delineating stressor-specific response patterns and identifying potential resistance mechanisms. Single cell mapping of cell stress and cell death can inform the development of more effective combinatorial drug regimens. Disclosures Carter: Syndax: Research Funding; Ascentage: Research Funding; Amgen: Research Funding; AstraZeneca: Research Funding. Andreeff:Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.

The maintenance of protein homeostasis is vital for all cells, but it is of utmost importance in post-mitotic cells, such as neurons that cannot dilute aggregates by cell division. Dysregulation of the proteostasis network can lead to neurodegenerative disorders such as Parkinson’s disease (PD), Alzheimer’s disease, Huntington’s disease, Amyotrophic Lateral Sclerosis (ALS), and prion diseases. The small molecule Sephin1 is a promising lead against proteostasis disruption, but its mechanism of action is uncertain. We assessed the therapeutic efficacy of Sephin1 in an established PD mouse model. Our laboratory has recently characterized a mouse expressing via bacterial artificial chromosome (BAC) the human LRRK2 G2019S protein, a variant linked to PD. Our data show that Sephin1 treatment rescues the motor deficit observed in BAC human-G2019S mice. Our experimental evidence shows that Sephin1 binds the monomeric globular actin (G-actin) in cell-free assays. By combining PAL chemistry to MS/MS analysis we identified the putative Sephin1 binding site on actin. In vitro, Sephin1 drives actin misfolding, and eventually, its precipitation. Upon Sephin1 treatment in HeLa cells, we visualized actin clusters localized to the lysosomes. This event at the lysosome impairs the normal autophagic flux. At the same time, Sephin1 induces the inactivation of the mammalian target of rapamycin (mTORC1), thus allowing the nuclear translocation of the transcription Factor EB (TFEB) and the expression of TFEB-direct target genes, on the longer term. In parallel, Sephin1 elicits the phosphorylation of the α subunit of the Eukaryotic Initiation Factor 2 (eIF2) and the ER-stress independent expression of the C/EBP homologous protein (CHOP). CHOP is a transcription factor that contributes to the integrated stress response as well as to autophagy. As such, Sephin1 triggers the activation of two main players in the autophagic response, TFEB and CHOP. Accordingly, we reported that, after the initial impairment, Sephin1 stimulates autophagy. Taken together, our results reveal a novel Sephin1 molecular mechanism in which lysosomal stress may regulate autophagy via mTORC1-TFEB complemented with the eIF2α signalling pathway. Although several questions remain to be answered, Sephin1, which successfully completed the phase I clinical trial for ALS and Charcot–Marie–Tooth disease, represents a promising therapeutic strategy that targets autophagy to regulate the homeostatic balance of proteins in neurodegenerative diseases.

Les leucémies aiguës myéloïdes (LAM) représentent un groupe d’hémopathies malignes agressives, de pronostic sombre en dépit des traitements intensifs actuellement proposés. Malgré une grande hétérogénéité clinique et moléculaire, les cellules de LAM sont caractérisées par l’activation de voies de signalisation essentielles à leur prolifération et leur survie, comme par exemple celle du complexe mTORC1 (mammalian target of rapamycin complex 1). Cependant, l’utilisation clinique d’inhibiteurs tels que la rapamycine ou des inhibiteurs catalytiques s’est avérée décevante, ce qui suggère qu’il n’y a pas d’addiction oncogénique à mTORC1 dans les LAM. Au cours de ce travail, nous avons démontré que l’activation de mTORC1 est au contraire une condition nécessaire à l’induction de la mort cellulaire en réponse à l’activation d’AMPK (AMP-activated protein kinase), établissant une relation de létalité synthétique entre ces deux voies. Pour cela, nous avons utilisé un nouveau composé activateur spécifique d’AMPK, le GSK621. En invalidant la sous-unité catalytique AMPKα1 par ARN interférence ou par le système CRISPR/Cas9, nous avons démontré que les effets antileucémiques de ce composé sont bien dépendants de l’activation d’AMPK. Nous avons observé que ce composé favorise l’autophagie, et que ce processus est impliqué dans la mort des cellules leucémiques puisque l’inhibition des protéines ATG5 ou ATG7 a un effet protecteur sur les cellules leucémiques. Les effets antileucémiques du composé GSK621 ont été confirmés sur des cellules primaires, ainsi que sur un panel de vingt lignées de LAM, et dans un modèle murin de xénogreffe. De façon intéressante, l’activation d’AMPK pourrait également compromettre la survie des cellules souches leucémiques, comme en atteste l’atténuation du potentiel clonogénique en méthylcellulose de cellules murines transformées par MLL-ENL ou FLT3-ITD. Nous avons observé que le composé GSK 621 n’avait pas de toxicité envers les progéniteurs hématopoïétiques normaux, ouvrant ainsi une fenêtre thérapeutique intéressante. Comme l’activation d’AMPK conduit dans de nombreux modèles cellulaires à l’inhibition de mTORC1, et comme l’activation de mTORC1 est observée dans les cellules de LAM mais pas dans les progéniteurs hématopoïétiques normaux, nous avons proposé l’hypothèse que le niveau d’activation de mTORC1 déterminait les effets de l’activateur d’AMPK. Pour cela, nous avons inhibé mTORC1 dans les cellules leucémiques d’une part, et activé mTORC1 dans les progéniteurs normaux d’autre part. De façon inattendue, mTORC1 échappe au contrôle d’AMPK dans les LAM, et nous avons observé que l’activation de mTORC1 est une condition nécessaire et suffisante pour que le composé GSK621 entraîne la mort des cellules. Le substrat moléculaire de cette létalité synthétique est le facteur de transcription proapoptotique ATF4, dont la transcription est favorisée par mTORC1, et la traduction par AMPK via la phosphorylation d’eIF2A. Ces travaux proposent donc que malgré l’absence d’addiction oncogénique, l’activation de mTORC1 dans les LAM représente une opportunité thérapeutique originale via une relation de létalité synthétique avec l’activation d’AMPK. Ils constituent un rationnel au développement clinique d’activateurs d’AMPK dans les LAM, voire dans d’autres cancers ayant une activation constitutive de mTORC1
Acute myeloid leukemia (AML) is a heterogeneous disease with poor prognosis despite intensive treatments. Virtually all recurrent molecular alterations in AML functionally converge to cause signal transduction pathway dysregulation that drives cellular proliferation and survival. The mammalian target of rapamycin complex 1 (mTORC1) is a rapamycin-sensitive signaling node defined by the interaction between mTOR and raptor. Constitutive mTORC1 activity is nearly universal in AML. However, pharmacologic inhibition with rapamycin or second-generation mTOR kinase inhibitors has shown limited anti-leukemic activity in both preclinical models as well as in clinical trials, suggesting that addiction to this oncogene is not a recurrent event in AML. Here we report that sustained mTORC1 activity is nonetheless essential for the cytotoxicity induced by pharmacologic activation of AMP-activated protein kinase (AMPK) in AML. Our studies employed a novel AMPK activator called GSK621. Using CRISPR/Cas9 and shRNA-mediated silencing of the AMPKa1 catalytic subunit, we showed that AMPK activity was necessary for the anti-leukemic response induced by this agent. GSK621-induced AMPK activation precipitated autophagy, and blocking autophagy via shRNA-mediated knockdown of ATG5 or ATG7 protected AML cells from cytotoxicity resulting from treatment with GSK621, suggesting that autophagy promotes cell death in the context of active AMPK. GSK621 cytotoxicity was consistently observed across twenty different AML cell lines, primary AML patient samples and AML xenografts in vivo. GSK621-induced AMPK activation also impaired the self-renewal capacity of MLL-ENL- and FLT3-ITD-induced murine leukemias as measured by serial methylcellulose replating assays. Strikingly, GSK621 did not induce cytotoxicity in normal CD34+ hematopoietic progenitor cells. We hypothesized that the differential sensitivity to GSK621 could be due to the difference in amplitude of mTORC1 activation between AML and normal CD34+ cells. In contrast to most reported cellular models in which AMPK inhibits mTORC1, sustained mTORC1 activity was seen following GSK621-induced AMPK activation in AML. Inhibition of mTORC1 either pharmacologically (using rapamycin) or genetically (using shRNAs targeting raptor and mTOR) abrogated AMPK-induced cytotoxicity in AML cells, including primary AML patient samples. The same synthetic lethality could be recapitulated in normal CD34+ progenitors by constitutive activation of mTORC1 using a lentivirally-transduced myrAKT construct. We further observed that the level of ATF4 protein is under a transcriptionnal control by mTORC1 and a translational control by AMPK (through eIF2A), and explains the synthetic lethal relationship between AMPK and mTORC1. Taken together, these data show that the magnitude of mTORC1 activity determines the degree of cytotoxicity triggered by AMPK activation. Our results therefore support AMPK activation as a promising therapeutic strategy in AML and other mTORC1-active malignancies which warrants further investigations in clinical trials

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Alcoholic and nonalcoholic fatty liver disease is projected to be the most common cause of liver disease in developing countries. The main significant risk factors are obesity, diabetes mellitus type 2, cardiovascular disease, and dyslipidemia. Louisiana is ranked seventh in liver cancer diagnoses and ranked sixth in the leading cause of death. Recent findings indicated that multifaceted stress response due to the accumulation of fatty acids from the diet is the driving force of disease progression. We sought to study multifaceted integrated stress response (ISR) in liver cells cultured with saturated fatty acids. Understanding the process that ISR takes to either induce or inhibit autophagy, self-eating machinery, in strongly permissive HUH 7.5 cells is vital when treating liver abnormalities. The major protein kinase, P-EIF2 alpha, was the targeted factor contributing the most to autophagy due to its functional link to the endoplasmic reticulum, mitochondria, and cellular membrane by further assessment using the inductive drug, Sephin 1. HUH, 7.5 liver cells are treated with increasing amounts of palmitic acid for 24 hours in DMEM with 10% FBS. ISR activated after substantial cellular damage leading to autophagy impairment. The cell culture was assessed for lipid accumulation, and the expression of PKR, IRE1 alpha, PERK, ATF6, P-EIF2 alpha, HRI, MTORC1, GCN2, P62, and LC3B was achieved by immunoblot analysis. Membrane fluidity PKR, lysosomal MTORC1, and protein synthesis GCN2 activated to elicit an integral response to the ISR pathway. Endoplasmic reticulum protein kinases induced in response to UPR activation lead to an integration of the P-EIF2 alpha pathway. Mitochondrial stress heme regulated inhibitor proliferated to provoke an activation in the significant protein kinase leading to autophagy impairment. The P-EIF2 alpha kinase invoked autophagic deficiency even when dephosphorylation was prevented by Sephin 1 drug treatment. ISR constrained autophagy in the liver-derived cell line due to the accumulation of the toxic saturated fatty acid. Keywords: palmitate, autophagy, fatty liver disease, integrated stress response, Sephin 1
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Glory Ogunyinka