Integrated Stress Response Signaling Research Articles

Design, synthesis and anticancer/antiestrogenic activities of novel indole-benzimidazoles.

Indole-benzimidazoles have recently gained attention due to their antiproliferative and antiestrogenic effects. However, their structural similarities and molecular mechanisms shared with selective estrogen receptor modulators (SERMs) have not yet been investigated. In this study, we synthesized novel ethylsulfonyl indole-benzimidazole derivatives by substituting the first (R1) and fifth (R2) positions of benzimidazole and indole groups, respectively. Subsequently, we performed 1H NMR, 13C NMR, and Mass spectral and in silico docking analyses, and anticancer activity screening studies of these novel indole-benzimidazoles. The antiproliferative effects of indole-benzimidazoles were found to be more similar between the estrogen (E2) responsive cell lines MCF-7 and HEPG2 in comparison to the Estrogen Receptor negative (ER-) cell line MDA-MB-231. R1:p-fluorobenzyl group members were selected as lead compounds for their potent anticancer effects and moderate structural affinity to ER. Microarray expression profiling and gene enrichment analyses (GSEA) of the selected compounds (R1:p-fluorobenzyl: 48, 49, 50, 51; R1:3,4-difluorobenzyl: 53) helped determine the similarly modulated cellular signaling pathways among derivatives. Moreover, we identified known compounds that have significantly similar gene signatures to that of 51 via queries performed in LINCS database; and further transcriptomics comparisons were made using public GEO datasets (GSE35428, GSE7765, GSE62673). Our results strongly demonstrate that these novel indole-benzimidazoles can modulate ER target gene expression as well as dioxin-mediated aryl hydrocarbon receptor and amino acid deprivation-mediated integrated stress response signaling in a dose-dependent manner.

The Integrated Stress Response and Phosphorylated Eukaryotic Initiation Factor 2α in Neurodegeneration.

The proposed molecular mechanisms underlying neurodegenerative pathogenesis are varied, precluding the development of effective therapies for these increasingly prevalent disorders. One of the most consistent observations across neurodegenerative diseases is the phosphorylation of eukaryotic initiation factor 2α (eIF2α). eIF2α is a translation initiation factor, involved in cap-dependent protein translation, which when phosphorylated causes global translation attenuation. eIF2α phosphorylation is mediated by 4 kinases, which, together with their downstream signaling cascades, constitute the integrated stress response (ISR). While the ISR is activated by stresses commonly observed in neurodegeneration, such as oxidative stress, endoplasmic reticulum stress, and inflammation, it is a canonically adaptive signaling cascade. However, chronic activation of the ISR can contribute to neurodegenerative phenotypes such as neuronal death, memory impairments, and protein aggregation via apoptotic induction and other maladaptive outcomes downstream of phospho-eIF2α-mediated translation inhibition, including neuroinflammation and altered amyloidogenic processing, plausibly in a feed-forward manner. This review examines evidence that dysregulated eIF2a phosphorylation acts as a driver of neurodegeneration, including a survey of observations of ISR signaling in human disease, inspection of the overlap between ISR signaling and neurodegenerative phenomenon, and assessment of recent encouraging findings ameliorating neurodegeneration using developing pharmacological agents which target the ISR. In doing so, gaps in the field, including crosstalk of the ISR kinases and consideration of ISR signaling in nonneuronal central nervous system cell types, are highlighted.

EXTH-57. PRECLINICAL COMBINATION OF ONC201 WITH RADIOTHERAPY OR TEMOZOLOMIDE IN GBM, DIPG AND ATRT CELL LINES RESULTS IN DOPAMINE RECEPTOR ANTAGONISM, ATF4 INDUCTION AND CELL DEATH

Abstract CNS tumors are one of the most lethal cancers in children and young adults. We previously reported that first-in-class small-molecule imipridone ONC201 can antagonize dopamine receptor D2 (DRD2), inactivate ERK/AKT, induce the integrated stress response (ISR), upregulate pro-apoptotic TRAIL receptor DR5, deplete cancer stem cells, and induce growth arrest or cell death in tumor cells. ONC201 crosses the blood-brain barrier and has induced durable tumor regressions in adult and pediatric H3K27M-mutant glioma patients. We hypothesized that ONC201 may synergize with radiotherapy or temozolomide in brain tumors. Glioblastoma (GBM: SNB19, T98G and U251), diffuse intrinsic pontine glioma (DIPG: SF8628) and atypical teratoid rhabdoid tumor (ATRT: BT-12, BT-16) cell lines were tested using cell viability or colony formation assays with ONC201 up to 20 μM alone or in combination with radiotherapy up to 8 Gy or temozolomide up to 100 μM. We observed synergy between ONC201 and radiation or temozolomide by multiple assays. We observed induction of PKA substrate phosphorylation as a marker of DRD2 antagonism, induction of ATF4 as a marker of ISR activation, and multiple markers of cell death in treated brain tumor cells. We are evaluating the relevance of mitochondrial protease ClpP, a recently described binding target of ONC201, in ONC201-induced glioma cell death. We have observed that knockdown of ClpP strongly protects multiple human cancer cell lines from ONC201-mediated cytotoxicity. Thus, we are exploring the potential interplay between ClpP, dopamine receptors, the ISR and TRAIL signaling pathways after single agent or combinatorial treatments. Our data suggests that ONC201 may be combined synergistically with temozolomide or radiation to address gliomas, along with potential pharmacodynamic biomarkers.

Small Molecule Modulation of the Integrated Stress Response Governs the Keratoconic Phenotype In Vitro.

PurposeThe degenerative corneal disease keratoconus is a leading indicator for corneal transplant with an unknown etiology. We recently identified the activation of the integrated stress response (ISR) in ex vivo human corneas and in vitro cell culture. Utilizing small molecules to modulate the ISR we sought to investigate the effects of stimulating the ISR in healthy cells to recapitulate aspects of the in vitro keratoconic phenotype and whether relieving the ISR signaling would recover the disease phenotype.MethodsCorneal fibroblasts were extracted from patients undergoing corneal transplant or unaffected cadaverous donor limbal rings. Cells were exposed to the DNA damage-inducible protein (GADD34) inhibitor SAL003 to stimulate the ISR, or Trans-ISRIB to relieve ISR signaling pathway. Collagen production was assessed through hydroxyproline production, Sirius Red incorporation, or quantitative (q)PCR. Western blotting, hydroxyproline, and qPCR were used to assess components of the ISR pathway and collagen production.ResultsISR stimulation through SAL003 resulted in significant decrease of hydroxyproline and COL1A1 transcription and eventual apoptosis in normal fibroblasts. Patient (KC) fibroblast production of hydroxyproline was increased in response to ISRIB, while matrix metalloproteinase (MMP)9 production was lowered. The prospective biomarker of keratoconus prolactin-inducible factor was also upregulated in KC fibroblast cultures in response to ISRIB. Inflammatory markers TNFα and IL-1β were unaffected.ConclusionsActivation of the ISR is sufficient to recapitulate many key aspects of the KC phenotype in unaffected cells in vitro. Inhibition of the ISR also relieves many of the hallmarks of KC in affected cells. Therefore, targeting of the ISR through small molecules is a potential therapeutic path for small molecule treatment of keratoconus.

Quercetin Regulates the Integrated Stress Response to Improve Memory.

The initiation of protein synthesis is suppressed under several stress conditions, inducing phosphorylation of the α-subunit of the eukaryotic initiation factor 2 (eIF2α), thereby inactivating the GTP-GDP recycling protein eIF2B. By contrast, the mammalian activating transcription factor 4 (ATF4, also known as cAMP response element binding protein 2 (CREB2)) is still translated under stress conditions. Four protein kinases (general control nonderepressible-2 (GCN2) kinase, double-stranded RNA-activated protein kinase (PKR), PKR-endoplasmic reticulum (ER)-related kinase (PERK), and heme-regulated inhibitor kinase (HRI)) phosphorylate eIF2α in the presence of stressors such as amino acid starvation, viral infection, ER stress, and heme deficiency. This signaling reaction is known as the integrated stress response (ISR). Here, we review ISR signaling in the brain in a mouse model of Alzheimer’s disease (AD). We propose that targeting ISR signaling with quercetin has therapeutic potential, because it suppresses amyloid-β (Aβ) production in vitro and prevents cognitive impairments in a mouse model of AD.

Mitochondrial pyruvate import is a metabolic vulnerability in androgen receptor-driven prostate cancer

Specific metabolic underpinnings of androgen receptor (AR)-driven growth in prostate adenocarcinoma (PCa) are largely undefined, hindering the development of strategies to leverage the metabolic dependencies of this disease when hormonal manipulations fail. Here we show that the mitochondrial pyruvate carrier (MPC), a critical metabolic conduit linking cytosolic and mitochondrial metabolism, is transcriptionally regulated by AR. Experimental MPC inhibition restricts proliferation and metabolic outputs of the citric acid cycle (TCA) including lipogenesis and oxidative phosphorylation in AR-driven PCa models. Mechanistically, metabolic disruption resulting from MPC inhibition activates the eIF2α/ATF4 integrated stress response (ISR). ISR signaling prevents cell cycle progression while coordinating salvage efforts, chiefly enhanced glutamine assimilation into the TCA, to regain metabolic homeostasis. We confirm that MPC function is operant in PCa tumors in-vivo using isotopomeric metabolic flux analysis. In turn, we apply a clinically viable small molecule targeting the MPC, MSDC0160, to pre-clinical PCa models and find that MPC inhibition suppresses tumor growth in hormone-responsive and castrate-resistant conditions. Collectively, our findings characterize the MPC as a tractable therapeutic target in AR-driven prostate tumors.

AP-SWATH Reveals Direct Involvement of VCP/p97 in Integrated Stress Response Signaling Through Facilitating CReP/PPP1R15B Degradation

The ubiquitin-directed AAA-ATPase VCP/p97 facilitates degradation of damaged or misfolded proteins in diverse cellular stress response pathways. Resolving the complexity of its interactions with partner and substrate proteins and understanding its links to stress signaling is therefore a major challenge. Here, we used affinity-purification SWATH mass spectrometry (AP-SWATH) to identify proteins that specifically interact with the substrate-trapping mutant, p97-E578Q. AP-SWATH identified differential interactions over a large detection range from abundant p97 cofactors to pathway-specific partners and individual ligases such as RNF185 and MUL1 that were trapped in p97-E578Q complexes. In addition, we identified various substrate proteins and candidates including the PP1 regulator CReP/PPP1R15B that dephosphorylates eIF2α and thus counteracts attenuation of translation by stress-kinases. We provide evidence that p97 with its Ufd1-Npl4 adapter ensures rapid constitutive turnover and balanced levels of CReP in unperturbed cells. Moreover, we show that p97-mediated degradation, together with a reduction in CReP synthesis, is essential for timely stress-induced reduction of CReP levels and, consequently, for robust eIF2α phosphorylation to enforce the stress response. Thus, our results demonstrate that p97 not only facilitates bulk degradation of misfolded proteins upon stress, but also directly modulates the integrated stress response at the level of signaling.

Chronic oxycodone induces axonal degeneration in rat brain

BackgroundChronic opioid therapy for non-malignant pain conditions has significantly increased over the last 15 years. Recently, the correlation between opioid analgesics and alternations in brain structure, such as leukoencephalopathy, axon demyelination, and white matter lesions, has been demonstrated in patients with a history of long-term use of prescription opioids. The exact mechanisms underlying the neurotoxic effect of opioids on the central nervous system are still not fully understood. We investigated the effect of chronic opioids using an animal model in which female rats were orally gavaged with 15 mg/kg of oxycodone every 24 h for 30 days. In addition we tested oxycodone, morphine and DAMGO in breast adenocarcinoma MCF7 cells, which are known to express the μ-opioid receptor.ResultsWe observed several changes in the white matter of animals treated with oxycodone: deformation of axonal tracks, reduction in size of axonal fascicles, loss of myelin basic protein and accumulation of amyloid precursor protein beta (β-APP), suggesting axonal damages by chronic oxycodone. Moreover, we demonstrated activation of pro-apoptotic machinery amid suppression of anti-apoptotic signaling in axonal tracks that correlated with activation of biomarkers of the integrated stress response (ISR) in these structures after oxycodone exposure. Using MCF7 cells, we observed induction of the ISR and pro-apoptotic signaling after opioid treatment. We showed that the ISR inhibitor, ISRIB, suppresses opioid-induced Bax and CHOP expression in MCF7 cells.ConclusionsAltogether, our data suggest that chronic opioid administration may cause neuronal degeneration by activation of the integrated stress response leading to induction of apoptotic signaling in neurons and also by promoting demyelination in CNS.

The ATPase activity of Asna1/TRC40 is required for pancreatic progenitor cell survival.

Asna1, also known as TRC40, is implicated in the delivery of tail-anchored (TA) proteins into the endoplasmic reticulum (ER), in vesicle-mediated transport, and in chaperoning unfolded proteins during oxidative stress/ATP depletion. Here, we show that Asna1 inactivation in pancreatic progenitor cells leads to redistribution of the Golgi TA SNARE proteins syntaxin 5 and syntaxin 6, Golgi fragmentation, and accumulation of cytosolic p62+ puncta. Asna1−/− multipotent progenitor cells (MPCs) selectively activate integrated stress response signaling and undergo apoptosis, thereby disrupting endocrine and acinar cell differentiation, resulting in pancreatic agenesis. Rescue experiments implicate the Asna1 ATPase activity and a CXXC di-cysteine motif in ensuring Golgi integrity, syntaxin 5 localization and MPC survival. Ex vivo inhibition of retrograde transport reproduces the perturbed Golgi morphology, and syntaxin 5 and syntaxin 6 expression, whereas modulation of p53 activity, using PFT-α and Nutlin-3, prevents or reproduces apoptosis in Asna1-deficient and wild-type MPCs, respectively. These findings support a role for the Asna1 ATPase activity in ensuring the survival of pancreatic MPCs, possibly by counteracting p53-mediated apoptosis.

Integrated Stress Response Mediates Epithelial Injury in Mechanical Ventilation.

Ventilator-induced lung injury (VILI) is a severe complication of mechanical ventilation that can lead to acute respiratory distress syndrome. VILI is characterized by damage to the epithelial barrier with subsequent pulmonary edema and profound hypoxia. Available lung-protective ventilator strategies offer only a modest benefit in preventing VILI because they cannot impede alveolar overdistension and concomitant epithelial barrier dysfunction in the inflamed lung regions. There are currently no effective biochemical therapies to mitigate injury to the alveolar epithelium. We hypothesize that alveolar stretch activates the integrated stress response (ISR) pathway and that the chemical inhibition of this pathway mitigates alveolar barrier disruption during stretch and mechanical ventilation. Using our established rat primary type I-like alveolar epithelial cell monolayer stretch model and in vivo rat mechanical ventilation that mimics the alveolar overdistension seen in acute respiratory distress syndrome, we studied epithelial responses to mechanical stress. Our studies revealed that the ISR signaling pathway is a key modulator of epithelial permeability. We show that prolonged epithelial stretch and injurious mechanical ventilation activate the ISR, leading to increased alveolar permeability, cell death, and proinflammatory signaling. Chemical inhibition of protein kinase RNA-like endoplasmic reticulum kinase, an upstream regulator of the pathway, resulted in decreased injury signaling and improved barrier function after prolonged cyclic stretch and injurious mechanical ventilation. Our results provide new evidence that therapeutic targeting of the ISR can mitigate VILI.

The integrated stress response.

In response to diverse stress stimuli, eukaryotic cells activate a common adaptive pathway, termed the integrated stress response (ISR), to restore cellular homeostasis. The core event in this pathway is the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) by one of four members of the eIF2α kinase family, which leads to a decrease in global protein synthesis and the induction of selected genes, including the transcription factor ATF4, that together promote cellular recovery. The gene expression program activated by the ISR optimizes the cellular response to stress and is dependent on the cellular context, as well as on the nature and intensity of the stress stimuli. Although the ISR is primarily a pro-survival, homeostatic program, exposure to severe stress can drive signaling toward cell death. Here, we review current understanding of the ISR signaling and how it regulates cell fate under diverse types of stress.

Abstract 4658: Activation mechanisms of Integrated Stress Response by inhibition of oncogenic BRAF in melanoma cell lines

Abstract The eIF2α-ATF4 signaling pathway termed “Integrated stress response (ISR)” plays an important role in protecting cells against various cellular stresses such as deprivation of glucose and amino acids, virus infection, and deficiency of heme. Under such stress conditions, eIF2α kinases, including PERK, GCN2, PKR and HRI, phosphorylate eIF2α, resulting in reduction of general protein synthesis while facilitating selective translation of ATF4, a central transcription factor that upregulates expression of stress-induced genes for cellular adaptation. Recently, it was reported that BRAF inhibitor vemurafenib triggers activation of the eIF2α-ATF4 signaling in BRAF-mutant melanoma cell lines; however, activation mechanisms of vemurafenib-induced ISR signaling not fully understood. In this study, we first compared vemurafenib-induced ISR activation in BRAF-mutant and -wild type melanoma cell lines. Consistent with previous studies, vemurafenib activated ISR signaling preferentially in BRAF-mutant cell lines, as determined by phosphorylation of eIF2α and ATF4 expression. We also found that the ISR activation by vemurafenib was impaired by knockdown of BRAF in BRAF-mutant cell lines. These results indicated that existence of mutated BRAF is important for vemurafenib to trigger activation of ISR signaling pathway. We next examined which eIF2α kinase is involved in the ISR signaling activation by vemurafenib. We found that knockdown of GCN2, an eIF2α kinase sensing depletion of amino acids, impaired phosphorylation of eIF2α and ATF4 induction by vemurafenib. In addition, we confirmed that vemurafenib induced phosphorylation of GCN2 on Thr899 that is known to occur in response to amino acids starvation. In agreement with these results, gene expression profiling with ontology analysis revealed that vemurafenib upregulated genes that are involved in amino acids biosynthesis via ISR signaling. Collectively, our results suggested that vemurafenib activated ISR signaling pathway possibly through mimicking amino acid deprivation in BRAF-mutant melanoma cell lines. Citation Format: Ikuko Nagasawa, Satomi Tsukahara, Kazuhiro Kunimasa, Akihiro Tomida. Activation mechanisms of Integrated Stress Response by inhibition of oncogenic BRAF in melanoma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4658.

Abstract 230: The role of integrated stress response (ISR) transcription factor ATF4 in metabolic adaptation of tumors to c-Myc activation

Abstract The ability of cancer cells to adapt to non-cell autonomous (extrinsic) and cell autonomous (intrinsic) stresses is critical for maintaining cell viability and growth. The Integrated Stress Response (ISR) pathway is essential in cancer cell adaptation to extrinsic stresses such as hypoxia and nutrient deprivation both in vitro and in vivo. The ISR kinases, PERK and GCN2, phosphorylate eIF2α, transiently inhibiting protein translation during times of stress. Phosphorylation of eIF2α promotes the preferential translation of the transcription factor ATF4, which activates a gene expression program that enhances uptake and synthesis of amino acids, antioxidant defense and chaperone expression to promote recovery from nutrient deprivation and ER stress. Here, we demonstrate the importance of ISR signaling in MYC oncogene induced intrinsic stress both in vitro and in vivo. We previously reported that activation of PERK, during c-Myc induction is vital for activating cytoprotective autophagy and suppressing c-Myc induced apoptosis. Here, we demonstrate GCN2 is also robustly activated during c-Myc induction. GCN2 activation after c-Myc induction regulates ATF4 transcription and translation. ATF4 was necessary for survival and suppression of oxidative stress during c-Myc activation. We have identified ATF4 targets that regulate amino acid uptake and involved in amelioration of oxidative stress. Furthermore, siRNA mediated knockdown or genetic knockout of GCN2 was synthetic lethal with c-Myc activation. Interestingly, PERK is still able to phosphorylate eIF2α in the absence of GCN2. However, eIF2α phosphorylation was completely inhibited in the absence of both kinases suggesting that these kinases compensate for each other and both are required for optimal phosphorylation of eIF2α after c-Myc activation. Consistent with our in vitro data, we observe higher levels of p-eIF2α and ATF4 in lymphoma cells compared to WT B cells in vivo in a mouse model of Myc driven lymphoma. These findings highlight a novel mechanism of stress response pathway activation to support c-Myc driven tumorigenesis involving an amino acid sensor kinase GCN2. Citation Format: Feven Tameire, Maria A. Monroy, Constantinos Koumenis. The role of integrated stress response (ISR) transcription factor ATF4 in metabolic adaptation of tumors to c-Myc activation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 230.

Ribosome Elongation Stall Directs Gene-specific Translation in the Integrated Stress Response

Upon exposure to environmental stress, phosphorylation of the α subunit of eIF2 (eIF2α-P) represses global protein synthesis, coincident with preferential translation of gene transcripts that mitigate stress damage or alternatively trigger apoptosis. Because there are multiple mammalian eIF2 kinases, each responding to different stress arrangements, this translational control scheme is referred to as the integrated stress response (ISR). Included among the preferentially translated mRNAs induced by eIF2α-P is that encoding the transcription factor CHOP (DDIT3/GADD153). Enhanced levels of CHOP promote cell death when ISR signaling is insufficient to restore cell homeostasis. Preferential translation of CHOP mRNA occurs by a mechanism involving ribosome bypass of an inhibitory upstream ORF (uORF) situated in the 5'-leader of the CHOP mRNA. In this study, we used biochemical and genetic approaches to define the inhibitory features of the CHOP uORF and the biological consequences of loss of the CHOP uORF on CHOP expression during stress. We discovered that specific sequences within the CHOP uORF serve to stall elongating ribosomes and prevent ribosome reinitiation at the downstream CHOP coding sequence. As a consequence, deletion of the CHOP uORF substantially increases the levels and modifies the pattern of induction of CHOP expression in the ISR. Enhanced CHOP expression leads to increased expression of key CHOP target genes, culminating in increased cell death in response to stress.

Integrated Stress Response Signaling Pathways Induced by Supraphysiological Concentrations of Thyroid Hormone Inhibit Viral Replication

Supraphysiological concentrations (SPCs) of triiodo-L-thyronine (T3) have been used in the treatment of a number of nonviral diseases. However, the signaling mechanisms that regulate the function of T3 at these concentrations and their role in modulating cellular stress pathways and antiviral responses are unknown. Here, we have investigated the effects of SPCs of T3 on integrated stress response (ISR) signaling pathways and the replication of vesicular stomatitis virus (VSV). T3 amplified Poly IC-induced activation of RNA-dependent protein kinase, induced phosphorylation of eIF2α, stress granule (SG) formation, IRE1α phosphorylation, XBP1 splicing, and the expression of stress markers. T3 inhibited VSV replication by modulating SG formation and the expression of stress response markers. ISR activator guanabenz also inhibited VSV replication and amplified T3-induced anti-VSV response. To summarize, we have uncovered novel functions of T3 at SPCs as an activator of ISR signaling pathways and an inhibitor of VSV replication. This study offers a proof of principle of the concept that ISR activating agents like SPC of T3 and guanabenz can be potential antiviral agents.

Signaling through alternative Integrated Stress Response pathways compensates for GCN2 loss in a mouse model of soft tissue sarcoma.

The tumor microenvironment is characterized by deficiencies in oxygen and nutrients, such as glucose and amino acids. Activation of the GCN2 arm of the Integrated Stress Response (ISR) in response to amino acid deprivation is one mechanism by which tumor cells cope with nutrient stress. GCN2 phosphorylates the alpha subunit of the eukaryotic translation initiation factor eIF2, leading to global downregulation of translation to conserve amino acids and initiation of a transcriptional program through ATF4 to promote recovery from nutrient deprivation. Loss of GCN2 results in decreased tumor cell survival in vitro under amino acid deprivation and attenuated tumor growth in xenograft tumor models. However, it is not known what effects GCN2 loss has on the growth of autochthonous tumors that arise in their native microenvironment. Here, we demonstrate in a genetically engineered mouse model of soft tissue sarcoma that loss of GCN2 has no effect on tumor growth or animal survival. The sarcomas displayed compensatory activation of PERK or phospho-eIF2α independent upregulation of ATF4 in order to maintain ISR signaling, indicating that this pathway is critical for tumorigenesis. These results have important implications for the development and testing of small molecule inhibitors of ISR kinases as cancer therapeutics.

Role of GADD34 in age‐related differences in hyperoxiainduced stress signaling

Hyperoxic exposure activates an adaptive signal transduction pathway in the lung known as the integrated stress response (ISR), which promotes the expression of factors that modulate redox balance, the immune response, and DNA repair. Given that newborns (NB) are more tolerant to the toxic effects of O2 than adults, we conducted the presence study to explore age‐related differences in ISR signaling within the lung. In adult (AD) mice exposed to 95% O2 (Ox), phosphorylated eIF2α, the initiating event of the ISR, increased within 24 hrs and declined by 72 hrs coincident with enhanced GADD34, ATF3, and CHOP expression. In NB, increased p‐2α was delayed until 72 hrs and coincided with lesser increases in GADD34 and ATF3. In MEF and mouse lung epithelial cells (MLE15), Ox increased p‐2α in MLE15 but decreased p‐2α in MEF. Assessment of GADD34/PP1 phosphatase complexes revealed that the amount of PP1 precipitating with GADD34 was 3‐fold greater in MEF. Ox also decreased the association of the PP1 inhibitor I‐1 with GADD34 in both cell types, while the inhibitor BiP was present only in MLE complexes. In animals, 72 hrs of Ox increased GADD34/PP1 association by 70% and decreased I‐1/GADD34 association by 87% in AD relative to NB. These findings show that age‐related and cell specific differences in extent and duration of Ox‐induced ISR activation are likely to be mediated by the composition of GADD34/PP1 phosphatase complexes.

Salubrinal Increases Human Fetal Hemoglobin Production Through a Post-Transcriptional Mechanism

Abstract 250Sickle cell disease and β-thalassemia continue to cause significant morbidity and mortality. Strategies to increase fetal hemoglobin (HbF) levels can ameliorate symptoms and improve the lives of patients with these diseases. While most previous studies have focused on induction of γ-globin gene expression as an approach to induce HbF, there is evidence that HbF may be post-transcriptionally regulated. For example, butyrate was shown to increase the translational efficiency of γ-globin mRNA and 5-azacytidine (5-Aza) induces HbF to a much greater degree than γ-globin mRNA steady state levels. These findings suggest that translational regulation may play an underappreciated yet important role in controlling HbF levels and that investigating the molecular mechanisms involved in this control may provide new therapeutic targets for HbF induction. We hypothesized that the Integrated Stress Response (ISR) pathway is involved in differentially regulating fetal and adult hemoglobin production. The ISR pathway has been shown to modulate globin protein synthesis in response to heme availability and other stresses. In the absence of heme, the heme-regulated inhibitor kinase phosphorylates eIF2α, downregulates general protein synthesis, but enables translation of a limited number of transcripts that are critical for coordinating the stress response. To test our hypothesis, we first evaluated the effects of salubrinal (Sal), a small molecule that activates ISR signaling by selectively inhibiting p-eIF2α dephosphorylation, in K562 cells. 3μM and 6μM Sal increased p-eIF2α and activated ISR signaling as evidenced by increased ATF4 and GADD34 protein levels and increased gene expression of ATF3 and CHOP, two transcriptional targets of ATF4. Once we verified that Sal increased p-eIF2α and ISR signaling, we extended testing to primary human erythroid cells to evaluate its effect on hemoglobin production. We first determined a dose range of Sal that increased p-eIF2α in primary cells without reducing cell viability. Both 3μM and 6μM Sal increased p-eIF2α and only reduced cell number by 15% when applied on days 15 and 18 of differentiation, the period of maximal hemoglobin synthesis. Next, we determined that 3μM and 6μM Sal slightly reduced γ-globin and β-globin steady state mRNA levels but did not change the γ/(γ+β) ratio relative to control. In contrast, Sal significantly induced HbF when evaluated by HPLC at the end of differentiation on day 20. Compared to untreated cells, 3μM Sal increased the percent HbF from 2.7% to 5.0% (1.8 fold) and 6μM Sal resulted in 12.9% HbF (4.7 fold) (n=4, p<0.05). The enhanced %HbF was due to increased HbF but also reduced HbA, providing evidence that HbF and HbA may be differentially or reciprocally regulated at the translational level. Importantly, Sal treatment did not significantly reduce the total hemoglobin content relative to the untreated control and did not alter cellular differentiation when assessed by flow cytometry for CD71 and CD235a. These results suggest that Sal increases HbF by a post-transcriptional mechanism potentially through ISR activation. Sal treatments earlier in the differentiation process (days 9 and 12) before considerable amounts of hemoglobin are synthesized failed to significantly increase HbF, further supporting this conclusion. We then evaluated whether Sal treatment could enhance HbF induction by known activators of γ-globin transcription, such as 5-Aza and hydroxyurea (HU). 200nM 5-Aza alone increased %HbF from 2.7% to 12.4%. When 200nM 5-Aza was combined with 3μM and 6μM Sal, the %HbF increased to 18.0% and 22.8%, respectively. Similarly, 10μM HU alone increased HbF from 2.9% to 4.9%, but co-treatment with 3μM and 6μM Sal increased HbF to 7.7% and 15.0%, respectively. For both HU and 5-Aza, combined treatment with Sal did not alter the γ/(γ+β) ratio from what was seen with HU or 5-Aza alone. Taken together, these results indicate that the novel method of HbF induction by Sal enhances the effect of transcriptional activators of γ-globin. In the future, utilization of transcriptional and translational mechanisms of HbF induction may provide an opportunity for combination therapy to achieve therapeutic HbF levels at reduced doses, thereby reducing toxicity. Disclosures:No relevant conflicts of interest to declare.

A deregulated integrated stress response promotes interferon-γ-induced medulloblastoma.

Endoplasmic reticulum (ER) stress activates pancreatic ER kinase (PERK), which coordinates an adaptive program known as the integrated stress response (ISR) by phosphorylating translation initiation factor 2α (eIF2α). There is evidence that the ISR is involved in tumor development. Recent studies also show that the ISR stimulates the expression of vascular endothelial growth factor A (VEGF-A), a master regulator of angiogenesis. Our previous studies have demonstrated that enforced expression of interferon-γ (IFNγ) in the central nervous system during development induces sonic hedgehog expression and leads to cerebellar dysplasia or medulloblastoma. Here we report that PERK was activated in cerebellar dysplasia and medulloblastoma in IFNγ-expressing mice. We found that inactivation of the growth arrest and DNA damage 34 (GADD34) gene, encoding the stress-inducible regulatory subunit of a phosphatase complex that dephosphorylates eIF2α, enhanced ISR signaling and facilitated medulloblastoma formation in IFNγ-expressing mice. Moreover, we found that the induction of VEGF-A and enhanced angiogenesis were associated with medulloblastoma formation in IFNγ-expressing mice on the GADD34 mutation background. Thus, our data provide genetic evidence that the ISR facilitates medulloblastoma development.

ATF4 and the integrated stress response are induced by ethanol and cytochrome P450 2E1 in human hepatocytes

Molecular mechanisms underlying alcoholic liver disease (ALD) are still not fully understood. Activating transcription factor-4 (ATF4) is the master coordinator of the integrated stress response (ISR), an adaptive pathway triggered by multiple stressors. which can promote cell death and induce metabolic dysregulation if the stress is intense or prolonged. The aim of this study was to assess the effect of alcohol on the ISR signaling pathway in human liver cells and to define the role of cytochrome P450 2E1 (CYP2E1) in this response. Primary cultured human hepatocytes and human HepG2 cells over-expressing CYP2E1 by adenoviral infection were exposed to ethanol (25-100mM) for 8-48h. Ethanol treatment of both liver cells up-regulated ATF4 as well as the pro-survival and the pro-apoptotic transcriptional program of the ISR. Indeed, in CYP2E1-expressing HepG2 cells exposed to ethanol, the expression of ISR target genes (HMOX-1, GCLC, AsnS, IGFBP-1, GADD34,CHOP, ATF3, CHAC1) was induced. Up-regulation of ATF4 and the ISR transcriptional program was decreased by addition of the anti-oxidant glutathione. Several mechanisms mediated ATF4 protein induction, including, at early times, the phosphorylation of eIF2α which controls ATF4 translation, and, at later times, increased mRNA level and increased stability of the protein. A decrease in cell survival was also observed. This study demonstrates that both CYP2E1 and ethanol induce ATF4 and the integrated stress response, a pathway which coordinates signals from multiple stresses, as well as established risk factors for ALD, and can display detrimental cellular effects upon prolonged activation.