PKR)-like Endoplasmic Reticulum Kinase Inhibitor Research Articles

PERK specific inhibitor AMG-44 protects against Trimethylamine N-oxide (TMAO)-induced Endoplasmic Reticular Stress and Endothelial Dysfunction

The gut microbiota is known to release metabolites detrimental to the endothelial cell lining of the vascular wall leading to endothelial dysfunction. We have earlier reported that Trimethylamine N-oxide (TMAO), an intestinal microbe derived metabolite, caused endothelial dysfunction. In the present study we hypothesized that protein kinase R-like endoplasmic reticulum kinase (PERK)/eIF2α/ATF4 signaling is involved in TMAO-induced endoplasmic reticular (ER) stress and that treatment with PERK inhibitor AMG-44 protects against TMAO-induced ER stress. The endothelial cells were treated with a range of increasing doses of TMAO (0-300 μM) to induce ER stress. PERK inhibitor AMG-44 (20 – 200nM) was used to treat the TMAO treated and control cells. TAMO showed a dose-dependent upregulation of ER stress marker proteins involved in the PERK/eIF2α/ATF4 ER-stress signaling pathway. Western blot analysis showed significant increase in p-PERK/PERK, p-eIF2α/eIF2α, and IRE1α, AFT-4 protein expression which was further confirmed by immunofluorescent analysis in endothelial cells. Treatment with AMG-44 significantly attenuated the PERK/eIF2α/ATF4 axis also protected against increased endothelial cell permeability induced by TMAO (n=6/group; p<0.05). Our results demonstrate that TMAO-induced endothelial dysfunction via the ER stress-mediated PERK- eIF2 α -ATF4 signaling pathway and that treatment with PERK specific inhibitor AMG-44 could be a novel therapeutic strategy in protection against TMAO-induced ER stress and endothelial dysfunction. NIH R01HL148711 to Dr. Sai Sudha Koka. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Selenium nanoparticles alleviate deoxynivalenol-induced intestinal epithelial barrier dysfunction by regulating endoplasmic reticulum stress in IPEC-J2 cells

The intestinal epithelial barrier plays a crucial role in maintaining human and animal health. Deoxynivalenol (DON) is a mycotoxin that contaminates cereal-based foods worldwide, which is a serious threat to human and animal health. This study was aimed to investigate the protective effect of selenium nanoparticles (SeNPs) synthesized by Lactobacillus casei ATCC 393 against DON-induced intestinal epithelial barrier dysfunction and its relationship with PERK-mediated signaling pathway. IPEC-J2 cells were randomly assigned to four groups: Con (vehicle), DON (0.6 μg DON/mL, 48 h), SeNPs+DON (8 μg Se/mL, 24 h; 0.6 μg DON/mL, 48 h) and SeNPs (8 μg Se/mL, 24 h). Compared with Con group, the transepithelial electrical resistance (TEER) and the tight junction proteins expression of IPEC-J2 cells exposed to DON was increased and decreased, respectively. In addition, DON exposure led to increased ROS content, decreased antioxidant capacity, structural damage of endoplasmic reticulum (ER), and activation of endoplasmic reticulum stress (ERS)-related protein kinase R-like endoplasmic reticulum kinase (PERK) pathway in IPEC-J2. Compared with SeNPs+DON group, SeNPs alleviated oxidative stress, ER structure damage and PERK pathway activation and the increase of intestinal epithelial permeability of IPEC-J2 cells exposed to DON. PERK agonist (CCT020312) and inhibitor (GSK2656157) treatments were performed to identify the role of PERK signaling pathway in the regulatory effects of SeNPs on DON-induced intestinal epithelial barrier dysfunction. Compared with SeNPs+DON group, PERK agonist increased the expression levels of p-PERK. PERK inhibitor exerted a similar inhibitory effect to SeNPs on the p-PERK expression. In conclusion, SeNPs effectively alleviate DON-induced intestinal epithelial barrier dysfunction in IPEC-J2 cells, which are closely associated with ERS-related PERK signaling pathway. This will provide a potential solution for prevention and control of DON in the aquaculture industry.

Angiopoietin-like protein 8 deficiency attenuates thoracic aortic aneurysm/dissection development in β-aminopropionitrile monofumarate-induced model mice

Thoracic aortic aneurysm/dissection (TAAD) is a life-threatening cardiovascular disorder. Endoplasmic reticulum stress (ERS) and vascular smooth muscle cell (VSMC) apoptosis are involved in TAAD progression. The Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) pathway is associated with VSMC apoptosis. Serum Angiopoietin-Like Protein 8 (ANGPTL8) levels are associated with aortic diameter and rupture rate of TAAD. However, a direct role of ANGPTL8 in TAAD has not been determined. β-Aminopropionitrile monofumarate (BAPN) was used to induce TAAD in C57BL/6 mice. ANGPTL8 knockout mice were used to detect the effects of ANGPTL8 on TAAD development. ANGPTL8knockdown in vitro was used to analyze the role of ANGPTL8 in VSMCs and ERS. In addition, over-expression of ANGPTL8 in VSMCs and a PERK inhibitor were used to assess the effect of ANGPTL8 on the PERK pathway. ANGPTL8 levels were increased in the aortic wall and VSMCs of BAPN-induced TAAD mice. Compared with BAPN-treated wild-type mice, ANGPTL8 knockout significantly reduced the rupture rate of TAAD to 0 %. In addition, the protein levels of proinflammatory cytokines and matrix metalloproteinase 9 (MMP9) and ERS proteins were decreased in the aorta wall. Angptl8 shRNA decreased MMP9 and ERS protein levels in VSMCs in vitro. Overexpression of ANGPTL8 significantly increased the levels of ERS proteins and MMPs, while a PERK inhibitor significantly decreased the effects of ANGPTL8 in VSMCs. ANGPTL8 contributed to TAAD development by inducing ERS activation and degradation of extracellular matrix in the aorta wall. Inhibition of ANGPTL8 may therefore represent a new strategy for TAAD therapy.

Optimization of a Novel Mandelamide-Derived Pyrrolopyrimidine Series of PERK Inhibitors.

The protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is one of three endoplasmic reticulum (ER) transmembrane sensors of the unfolded protein response (UPR) responsible for regulating protein synthesis and alleviating ER stress. PERK has been implicated in tumorigenesis, cancer cell survival as well metabolic diseases such as diabetes. The structure-based design and optimization of a novel mandelamide-derived pyrrolopyrimidine series of PERK inhibitors as described herein, resulted in the identification of compound 26, a potent, selective, and orally bioavailable compound suitable for interrogating PERK pathway biology in vitro and in vivo, with pharmacokinetics suitable for once-a-day oral dosing in mice.

Pharmacological inhibition of UPR sensor PERK attenuates HIV Tat-induced inflammatory M1 phenotype in microglial cells.

HIV-1-associated neurocognitive disorders (HAND) are a major concern in HIV-infected individuals despite the currently available antiretroviral therapy regime. Impaired M1 pro-inflammatory microglial activation is considered one of the hallmark features of HAND neuropathogenesis, and it has been suggested that circulant HIV-1 transactivator protein (Tat) can play a critical role in this process. At the same time, endoplasmatic reticulum (ER) stress has also been implicated in neurodegenerative conditions resulting from the accumulation of misfolded proteins and subsequent unfolded protein response (UPR) deflagration. Here, we demonstrate that pharmacological inhibition of UPR-related protein kinase R-like endoplasmic reticulum kinase (PERK) can attenuate HIV-1 Tat-induced M1 inflammatory state in microglia in vitro. Our initial experiments demonstrate that the bystander stimulus of recombinant Tat on BV-2 microglial cells result in the coupled overexpression of central UPR markers and pro-inflammatory mediators such as iNOS, surface CD16/32 and secreted tumour necrosis factor-α (TNF-α), IL-6, monocyte chemoattractant protein (MCP)-1 and NO. We show that blocking PERK-eIF2-α-ATF4 signalling using the PERK inhibitor GSK2606414 leads to reduced inflammatory response in M1-like BV-2 cells activated by recombinant Tat. Taken together, these findings suggest that PERK targeting may provide a therapeutic intervention to mitigate against lasting neuroinflammation and neuronal loss in of HAND.

Abstract 14071: Gut Microbial Metabolite Trimethylamine N-oxide Enhances Endoplasmic Reticular Stress and Promotes Endothelial Dysfunction

Background: The intestinal microbe-derived metabolite trimethylamine N-oxide (TMAO) is known to the increase risk of cardiovascular diseases (CVDs). Endothelial dysfunction is associated with most forms of CVDs and is characterized by damage to the endoplasmic reticulum (ER). The current study was aimed to investigate the involvement of endoplasmic reticulum stress (ERS)-mediated protein kinase R-like endoplasmic reticulum kinase (PERK) signaling in TMAO induced endothelial dysfunction. Methods and Results: Biochemical analysis showed that PERK, CHOP, IRE-1α, AFT-4, eIF2 α which are indicative of ER stress, increased significantly in a dose dependent manner in response to TMAO treatment in endothelial cells. Western blot and immunofluorescent analysis showed significant increase in the phosphorylation of PERK and eIF2α along with upregulation of CHOP, IRE-1α and AFT-4 in TMAO group (30μM) compared to the control group (n=4/group; p<0.05). TMAO (30μM) treatment for 24h induced mitochondrial dysfunction as evidenced by decreased mitochondrial membrane potential, increased mitochondrial ROS levels, and also enhanced endothelial cell permeability compared to the control (n=6/group; p<0.05) . Furthermore, treatment with a PERK inhibitor (GSK2606414, 50nM, 24h) inhibited TMAO-induced phosphorylation of ER stress-related proteins, PERK and eIF2α and downregulated IRE1α, AFT-4 and CHOP and protected against increased endothelial cell permeability and mitochondrial ROS production. Conclusion: Our findings suggest that TMAO induced endothelial dysfunction through the ER stress-mediated PERK- eIF2 α -ATF4signaling pathway. Treatment with PERK inhibitors like (GSK2606414) could be a novel therapeutic strategy in TMAO-induced endothelial dysfunction.

PERK-Dependent Activation of the JAK2/STAT3 Pathway Contributes to High Glucose-Induced Extracellular Matrix Deposition in Renal Tubular Epithelial Cells.

Background Although the deposition of extracellular matrix (ECM) is critical leading to tubular damage in diabetic kidney disease (DKD), the mechanism still remains unclear. The purpose of this study was to demonstrate a role for protein kinase R-like endoplasmic reticulum kinase (PERK) (a protein located in the endoplasmic reticulum membrane) in this pathologic process. Methods NRK-52E cells were grown in the media containing different concentrations of glucose or thapsigargin for different durations. Cells were subsequently incubated with or without AG490, a selective inhibitor of Janus kinase 2 (JAK2) or GSK2606414 (a selective PERK inhibitor). We evaluated the production of TGF-β1, fibronectin, and collagen I proteins by ELISA. The levels of 78 kD-glucose-regulated protein (GRP78) and PERK, as well as the phosphorylation statues of PERK and JAK2/signal transducer and activator of transcription (STAT3), were determined by western blotting analysis. Results We showed that the increased phosphorylation of JAK2 and STAT3 was accompanied by overexpression of TGF-β1 and ECM deposition in high glucose medium. Disruption of the JAK2/STAT3 pathway with AG490 significantly prevents the high glucose-induced increase in TGF-β1, fibronectin, and collagen I. High glucose induced the overproduction of GRP78 and phosphorylation of PERK, which indicated that endoplasmic reticulum stress (ERS) was triggered in NRK-52E cells cultured under high glucose condition. Inhibition of PERK phosphorylation with GSK2606414, however, blocked the effect of JAK2/STAT3 on the production of TGF-β1 and ECM components in NRK-52E cells. Conclusion Our data indicated that the ECM accumulation induced by high glucose arouse via the PERK-dependent JAK2/STAT3-signaling pathway in renal tubular epithelial cells.

Activation of the Protein Kinase R-Like Endoplasmic Reticulum Kinase (PERK) Pathway of the Unfolded Protein Response after Experimental Traumatic Brain Injury and Treatment with a PERK Inhibitor.

Neurodegeneration after traumatic brain injury (TBI) is increasingly recognized as a key factor contributing to poor chronic outcomes. Activation (i.e., phosphorylation) of the protein kinase R-like endoplasmic reticulum kinase (PERK) pathway has been implicated in neurodegenerative conditions with pathological similarities to TBI and may be a potential target to improve TBI outcomes. Here, we aimed to determine whether a moderate TBI would induce activation of the PERK pathway and whether treatment with the PERK inhibitor, GSK2606414, would improve TBI recovery. Male mice were administered a lateral fluid percussion injury (FPI) or sham injury and were euthanized at either 2 h, 24 h, or 1 week post-injury (n = 5 per injury group and time point) to assess changes in the PERK pathway. In the injured cortex, there was increased phosphorylated-PERK at 2 h post-FPI and increased phosphorylation of eukaryotic translation initiation factor α at 24 h post-FPI. We next examined the effect of acute treatment with GSK2606414 on pathological and behavioral outcomes at 4 weeks post-injury. Thus, there were a total of four groups: sham + VEH (n = 9); sham + GSK4606414 (n = 10); FPI + VEH (n = 9); and FPI + GSK2606414 (n = 9). GSK2606414 (50 mg/kg) or vehicle treatment was delivered by oral gavage beginning at 30 min post-injury, followed by two further treatments at 12-h increments. There were no significant effects of GSK2606414 on any of the outcomes assessed, which could be attributable to several reasons. For example, activation of PERK may not be a significant contributor to the neurological consequences 4 weeks post-FPI in mice. Further research is required to elucidate the role of the PERK pathway in TBI and whether interventions that target this pathway are beneficial.

Direct-Acting Small Molecule Activators of PP2A (SMAPs) in Myeloid Malignancies: Understanding Mechanisms of Cytotoxicity to Inform Rational Combinatorial Therapeutic Strategies

The protein phosphatase 2A (PP2A) functions as a tumor suppressor by counteracting oncogenic kinase signaling. Suppression of PP2A activity represents a critical process in the development and maintenance of many neoplasms, including myeloid malignancies; PP2A inhibition de-represses numerous proliferative and survival signaling pathways. However, complete loss of PP2A is lethal to cells. As such, indirect reactivation of PP2A has emerged as a promising therapeutic strategy for myeloid leukemias. Yet the broad applicability of such a strategy remains uncertain, as it relies on targeting endogenous PP2A inhibitors. In the present work we investigated the effects of direct acting small molecule activators of PP2A (SMAPs) (Sandokar et al. JCI 2017) on myeloid leukemia cells and uncovered a novel link between PP2A reactivation and NOXA-mediated apoptosis. This mechanism explains our observation of cooperativity between SMAPs and the BH3 mimetic ABT-199 (venetoclax). Additionally, we found that SMAPs elicit a protective stress response that can be overcome by inhibition ofprotein kinase R (PKR)-like endoplasmic reticulum kinase (PERK).Given the known role of PP2A inactivation in both acute and chronic myeloid leukemia (AML and CML), we first tested the activity of SMAPs against cell lines derived from both diseases. Across all cell lines tested (CML- K562, Meg-01; AML- MOLM-14, HL-60, OCI-AML3, THP-1) SMAPs caused a dose-dependent decrease in cell viability. It is known that reactivation of PP2A induces apoptosis in myeloid leukemia cells, though this area has yet to be thoroughly investigated. Therefore, we first sought to clarify the mode of cell death in response to PP2A reactivation. Both by biochemical and flow cytometric analyses, SMAPs showed activation of the intrinsic apoptotic cascade. Furthermore, SMAP-mediated cell death was blocked by co-treatment with the caspase inhibitor z-VAD-FMK (50 μM). We previously demonstrated the target specificity of SMAPs for PP2A with methods including expression of SV40 small T antigen and PP2A alpha subunit drug-binding mutants (Sandokar et al. JCI 2017). Here, we confirmed that pre-treatment with the PP2A inhibitor okadaic acid at a dose that retains selectivity for PP2A (10 nM) attenuated SMAP-induced cell death and cleavage of PARP and caspase-3.Next, we investigated the effectors of SMAP-mediated apoptosis. To this end, we performed Western blots to detect changes in abundance and/or phosphorylation of a panel of Bcl-2 family member proteins. Interestingly, a strong upregulation of the pro-apoptotic protein NOXA occurred consistently across AML and CML cell lines. Further RT-PCR studies showed this increase to occur at the transcript level. Knockdown studies confirmed the importance of NOXA for SMAP-mediated apoptosis. These data represent the first reported link between PP2A and NOXA regulation. Since SMAPs target the NOXA-MCL1 axis of the Bcl-2 family, we next tested their efficacy in combination with the Bcl-2-selective BH3 mimetic ABT-199. We found co-treatment with SMAPs and ABT-199 to exhibit enhanced cell death compared to either agent alone.Concurrent with NOXA elevation, SMAP treatment modulated protein expression and phosphorylation levels for several known pathways downstream of PP2A. Interestingly, we additionally observed increased phosphorylation of eukaryotic translation initiation factor 2 subunit alpha (eIF2α) in SMAP-treated cells which correlated with upregulation of mRNA and protein for the downstream transcription factor ATF4. Blockade of eIF2α phosphorylation with PERK inhibitor GSK2656157 (350 nM) prevented ATF4 elevation and enhanced SMAP-induced apoptosis. This result supports the idea that the integrated stress response (ISR) via PERK-eIF2α-ATF4 acts to counter apoptosis in the context of PP2A reactivation in myeloid leukemia cells. Thus, the efficacy of SMAPs and other PP2A-activating therapeutics may be enhanced by combination with both BH3 mimetics and inhibitors of the ISR. DisclosuresNarla:Mount Sinai School of Medicine: Patents & Royalties: patents on the small molecule PP2A activators (GN).

ER stress and its PERK branch enhance TCR-induced activation in regulatory T cells

Although accumulating evidence indicates participation of endoplasmic reticulum (ER) stress pathway or the unfolded protein response (UPR) in immunity, there still exists little information linking ER stress to regulatory T cells (Tregs). To evaluate the potential contribution of the UPR, we tested the effects of thapsigargin (TG), an ER stress inducer, on the function of Tregs. Here we reported that TG stimulation induced the up-regulation of the endoplasmic reticulum (ER)-stress chaperon Glucose-Regulated Protein 78 (GRP78), which is a master regulator of the UPR, the phosphorylation of eukaryotic initiation factor 2 alpha (elF2α) and the induction of activating transcription factor 4 (ATF4), which are both protein kinase R (PKR)-like ER kinase (PERK) downstream targets in Tregs. Simultaneously, we demonstrated that, under ER stress conditions, Tregs presented enhanced functional activity upon TCR stimulation, as illustrated with forkhead box transcription factor (Foxp3) expression, interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) production and suppressive functional analysis. Notably, pretreatment with GSK2656157, a potent and selective PERK inhibitor, markedly diminished the TG-induced hyperresponsiveness of Tregs upon T cell antigen receptor (TCR) stimulation. Therefore, our findings illustrated the inter-connection and coordination of the evolutionarily conserved ER stress response and TCR signaling in Tregs and uncover a critical new role of the PERK branch of UPR in the regulation of Tregs function.

Discovery of 2-amino-3-amido-5-aryl-pyridines as highly potent, orally bioavailable, and efficacious PERK kinase inhibitors

The protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is one of the three endoplasmic reticulum (ER) transmembrane sensors of the unfolded protein response (UPR) that regulates protein synthesis, alleviates cellular ER stress and has been implicated in tumorigenesis and prolonged cancer cell survival. In this study, we report a series of 2-amino-3-amido-5-aryl-pyridines that we have identified as potent, selective, and orally bioavailable PERK inhibitors. Amongst the series studied herein, compound (28) a (R)-2-Amino-5-(4-(2-(3,5-difluorophenyl)-2-hydroxyacetamido)-2-ethylphenyl)-N-isopropylnicotinamide has demonstrated potent biochemical and cellular activity, robust pharmacokinetics and 70% oral bioavailability in mice. Given these data, this compound (28) was studied in the 786-O renal cell carcinoma xenograft model. We observed dose-dependent, statistically significant tumor growth inhibition, supporting the use of this tool compound in additional mechanistic studies.

Characterization of a PERK Kinase Inhibitor with Anti-Myeloma Activity.

Simple SummaryMultiple myeloma is a bone marrow cancer that represents a severe health threat. The drugs used nowadays in chemotherapy often encounter resistance leading to a dramatic loss of their efficacy, which consequently affects patients’ survival. Previous studies have shown that the protein kinase R (PKR)-like ER kinase (PERK) pathway, which is one of the three branches of the unfolded protein response, is highly activated in multiple myeloma, possibly contributing to the chemotherapy resistance that these patients develop. In this study, we have used the compound GSK2606414, which is a PERK inhibitor, and found that myeloma cells are highly sensitive to this molecule. These effects were more pronounced when the inhibitor was used in combination with an anti-myeloma drug such as the proteasome inhibitor bortezomib, suggesting that the PERK pathway could be a potential therapeutic target for the treatment of multiple myeloma patients.Due to increased immunoglobulin production and uncontrolled proliferation, multiple myeloma (MM) plasma cells develop a phenotype of deregulated unfolded protein response (UPR). The eIF2-alpha kinase 3 [EIF2αK3, protein kinase R (PKR)-like ER kinase (PERK)], the third known sensor of endoplasmic reticulum (ER) stress, is a serine-threonine kinase and, like the other two UPR-related proteins, i.e., IRE1 and ATF6, it is bound to the ER membrane. MM, like other tumors showing uncontrolled protein secretion, is highly dependent to UPR for survival; thus, inhibition of PERK can be an effective strategy to suppress growth of malignant plasma cells. Here, we have used GSK2606414, an ATP-competitive potent PERK inhibitor, and found significant anti-proliferative and apoptotic effects in a panel of MM cell lines. These effects were accompanied by the downregulation of key components of the PERK pathway as well as of other UPR elements. Consistently, PERK gene expression silencing significantly increased cell death in MM cells, highlighting the importance of PERK signaling in MM biology. Moreover, GSK2606414, in combination with the proteasome inhibitor bortezomib, exerted an additive toxic effect in MM cells. Overall, our data suggest that PERK inhibition could represent a novel combinatorial therapeutic approach in MM.

Ganoderic Acid D Protects Human Amniotic Mesenchymal Stem Cells against Oxidative Stress-Induced Senescence through the PERK/NRF2 Signaling Pathway.

Aging is an important risk factor in the occurrence of many chronic diseases. Senescence and exhaustion of adult stem cells are considered as a hallmark of aging in organisms. In this study, a senescent human amniotic mesenchymal stem cell (hAMSC) model subjected to oxidative stress was established in vitro using hydrogen peroxide. We investigated the effects of ganoderic acid D (GA-D), a natural triterpenoid compound produced from Ganoderma lucidum, on hAMSC senescence. GA-D significantly inhibited β-galactosidase (a senescence-associated marker) formation, in a dose-dependent manner, with doses ranging from 0.1 μM to 10 μM, without inducing cytotoxic side-effects. Furthermore, GA-D markedly inhibited the generation of reactive oxygen species (ROS) and the expression of p21 and p16 proteins, relieved the cell cycle arrest, and enhanced telomerase activity in senescent hAMSCs. Furthermore, GA-D upregulated the expression of phosphorylated protein kinase R- (PKR-) like endoplasmic reticulum kinase (PERK), peroxidase III (PRDX3), and nuclear factor-erythroid 2-related factor (NRF2) and promoted intranuclear transfer of NRF2 in senescent cells. The PERK inhibitor GSK2656157 and/or the NRF2 inhibitor ML385 suppressed the PERK/NRF2 signaling, which was activated by GA-D. They induced a rebound for the generation of ROS and β-galactosidase-positive cells and attenuated the differentiation capacity. These findings suggest that GA-D retards hAMSC senescence through activation of the PERK/NRF2 signaling pathway and may be a promising candidate for the discovery of antiaging agents.

ROS-mediated PERK-eIF2α-ATF4 pathway plays an important role in arsenite-induced L-02 cells apoptosis via regulating CHOP-DR5 signaling.

Chronic exposure to arsenic remains a worldwide environmental health issue, affecting hundreds of millions of people. Although, arsenic-induced oxidative stress and apoptosis have been determined, the underlying apoptosis mechanism has not been fully elucidated yet. Oxidative stress integrated-ER stress plays an important role in Life-and-Death decision of cells. The current study was to investigate whether NaAsO2 utilizes oxidative stress integrated-ER stress signaling to exert pro-apoptotic activity in L-02 cells. Results showed that death receptor 5 (DR5) was a mediator of NaAsO2 -induced apoptosis by enhancing construction of the death-inducing signaling complex (DISC). NaAsO2 -sensitized DR5 elevation required maintainable transcription and its transcription factor C/EBP homologous protein (CHOP). Further results showed that NaAsO2 increased expression in biomarker of endoplasmic reticulum (ER) stress and activated the protein kinase R-like ER kinase (PERK)-eukaryotic translation initiation 2α (eIF2α)-activating transcription factor 4 (ATF4) pathway. PERK inhibitor and ATF4 siRNA significantly attenuated NaAsO2 -induced CHOP and DR5 expressions. In addition, the antioxidant N-acetyl-l-cysteine (NAC) treatment led to amelioration of NaAsO2 -induced production of reactive oxygen species (ROS) and some ER stress- and apoptosis- related protein levels and cell viability. Taken together, the results indicate that ROS-mediated PERK-eIF2α-ATF4 pathway activated by NaAsO2 is the critical upstream event for subsequent apoptosis induction via regulating CHOP-DR5 signaling in L-02 cells when chronic exposure to arsenic, and support that antioxidants might be potential therapeutic agents for preventing or delaying the onset and progress of arsenic-induced hepatotoxicity.

The PKR-Like Endoplasmic Reticulum Kinase Promotes the Dissemination of Myc-Induced Leukemic Cells.

Hyperactive oncogenic Myc stimulates protein synthesis that induces the unfolded protein response, which requires the function of the eukaryotic translation initiation factor 2-alpha kinase 3, also known as protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK). Activated PERK acts to limit mRNA translation, enable proper protein folding, and restore the homeostasis in the endoplasmic reticulum. Given that Myc activation contributes to many types of lymphoid and myeloid human leukemias, we used a mouse model to examine the importance of PERK in development and progression of Myc-induced leukemias. We found that genetic ablation of Perk does not suppress the generation of the leukemic cells in the bone marrow. However, the cell-autonomous Perk deficiency restricts the dissemination of leukemic cells into peripheral blood, lymph nodes, and vital peripheral organs. Whereas the loss of the IFNAR1 chain of type I IFN receptor stimulated leukemia, Perk ablation did not stabilize IFNAR1, suggesting that PERK stimulates the leukemic cells' dissemination in an IFNAR1-independent manner. We discuss the rationale for using PERK inhibitors against Myc-driven leukemias. IMPLICATIONS: The role of PERK in dissemination of Myc-induced leukemic cells demonstrated in this study argues for the use of PERK inhibitors against leukemia progression.

Breaking the DNA Damage Response via Serine/Threonine Kinase Inhibitors to Improve Cancer Treatment.

Multiple, both endogenous and exogenous, sources may induce DNA damage and DNA replication stress. Cells have developed DNA damage response (DDR) signaling pathways to maintain genomic stability and effectively detect and repair DNA lesions. Serine/ threonine kinases such as Ataxia-telangiectasia mutated (ATM) and Ataxia-telangiectasia and Rad3-Related (ATR) are the major regulators of DDR, since after sensing stalled DNA replication forks, DNA double- or single-strand breaks, may directly phosphorylate and activate their downstream targets, that play a key role in DNA repair, cell cycle arrest and apoptotic cell death. Interestingly, key components of DDR signaling networks may constitute an attractive target for anti-cancer therapy through two distinct potential approaches: as chemoand radiosensitizers to enhance the effectiveness of currently used genotoxic treatment or as single agents to exploit defects in DDR in cancer cells via synthetic lethal approach. Moreover, the newest data reported that serine/threonine protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is also closely associated with cancer development and progression. Thereby, utilization of small-molecule, serine/threonine kinase inhibitors may provide a novel, groundbreaking, anti-cancer treatment strategy. Currently, a range of potent, highlyselective toward ATM, ATR and PERK inhibitors has been discovered, but after foregoing study, additional investigations are necessary for their future clinical use.

Endoplasmic Reticulum Stress Contributes to Mitochondrial Exhaustion of CD8+ T Cells.

Tumor antigen-specific T cells rapidly lose energy and effector function in tumors. The cellular mechanisms by which energy loss and inhibition of effector function occur in tumor-infiltrating lymphocytes (TILs) are ill-defined, and methods to identify tumor antigen-specific TILs that experience such stress are unknown. Processes upstream of the mitochondria guide cell-intrinsic energy depletion. We hypothesized that a mechanism of T-cell-intrinsic energy consumption was the process of oxidative protein folding and disulfide bond formation that takes place in the endoplasmic reticulum (ER) guided by protein kinase R-like endoplasmic reticulum kinase (PERK) and downstream PERK axis target ER oxidoreductase 1 (ERO1α). To test this hypothesis, we created TCR transgenic mice with a T-cell-specific PERK gene deletion (OT1 + Lckcre+ PERK f/f , PERK KO). We found that PERK KO and T cells that were pharmacologically inhibited by PERK or ERO1α maintained reserve energy and exhibited a protein profile consistent with reduced oxidative stress. These T-cell groups displayed superior tumor control compared with T effectors. We identified a biomarker of ER-induced mitochondrial exhaustion in T cells as mitochondrial reactive oxygen species (mtROS), and found that PD-1+ tumor antigen-specific CD8+ TILs express mtROS. In vivo treatment with a PERK inhibitor abrogated mtROS in PD-1+ CD8+ TILs and bolstered CD8+ TIL viability. Combination therapy enabled 100% survival and 71% tumor clearance in a sarcoma mouse model. Our data identify the ER as a regulator of T-cell energetics and indicate that ER elements are effective targets to improve cancer immunotherapy.

Hypoxia-Activated Prodrugs of PERK Inhibitors.

Tumour hypoxia plays an important role in tumour progression and resistance to therapy. Under hypoxia unfolded proteins accumulate in the endoplasmic reticulum (ER) and this stress is relieved through the protein kinase R-like ER kinase (PERK) signalling arm of the unfolded protein response (UPR). Targeting the UPR through PERK kinase inhibitors provides tumour growth inhibition, but also elicits on-mechanism normal tissue toxicity. Hypoxia presents a target for tumour-selective drug delivery using hypoxia-activated prodrugs. We designed and prepared hypoxia-activated prodrugs of modified PERK inhibitors using a 2-nitroimidazole bioreductive trigger. The new inhibitors retained PERK kinase inhibitory activity and the corresponding prodrugs were strongly deactivated. The prodrugs were able to undergo fragmentation following radiolytic reduction, or bioreduction in HCT116 cells, to release their effectors, albeit inefficiently. We examined the effects of the prodrugs on PERK signalling in hypoxic HCT116 cells. This study has identified a 2-substituted nitroimidazole carbamate prodrug with potential to deliver PERK inhibitors in a hypoxia-selective manner.

Role of Endoplasmic Reticulum Stress in Proinflammatory Cytokine–Mediated Inhibition of Trophoblast Invasion in Placenta-Related Complications of Pregnancy

Shallow extravillous trophoblast (EVT) invasion is central to the pathophysiology of many pregnancy complications. Invasion is mediated partially by matrix metalloproteinases (MMPs). MMP-2 is highly expressed in early pregnancy. MMP activity can be regulated by proinflammatory cytokines, which also induce endoplasmic reticulum (ER) stress in other cells. We investigated whether proinflammatory cytokines regulate MMP-2 activity through ER stress response pathways in trophoblast before exploring potential regulatory mechanisms. There was increased immunoreactivity of heat shock 70-kDa protein 5, also known as 78-kDa glucose regulated protein, in cells of the placental bed, including EVTs, in cases of early-onset preeclampsia compared with normotensive controls. Treating EVT-like JEG-3 and HTR8/SVneo cells with ER stress inducers (tunicamycin and thapsigargin) suppressed MMP2 mRNA and protein expression, secretion, and activity and reduced their invasiveness. A cocktail of proinflammatory cytokines (IL-1β, tumor necrosis factor-α, and interferon-γ) suppressed MMP-2 activity in JEG-3 cells and was accompanied by activation of the PKR-like ER kinase (PERK)–eukaryotic translation initiation factor 2A (EIF2A) arm of the ER stress pathway. Knockdown of ATF4, a downstream transcriptional factor of the PERK-EIF2A pathway, by small interference RNA, restored MMP2 expression but not cellular proteins. However, suppression of EIF2A phosphorylation with a PERK inhibitor, GSK2606414, under ER stress, restored MMP-2 protein. ER stress regulates MMP-2 expression at both the transcriptional and translational levels. This study provides the first mechanistic linkage by which proinflammatory cytokines may modulate trophoblast invasion through ER stress pathways.

PERK Pathway Activation Promotes Intracerebral Hemorrhage Induced Secondary Brain Injury by Inducing Neuronal Apoptosis Both in Vivo and in Vitro.

The protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) signaling pathway was reported to exert an important role in neuronal apoptosis. The present study was designed to investigate the roles of the PERK signaling pathway in the secondary brain injury (SBI) induced by intracerebral hemorrhage (ICH) and its potential mechanisms. Sprague–Dawley rats were used to establish ICH models by injecting autologous blood (100 μl), and cultured primary rat cortical neurons were exposed to oxyhemoglobin (10 μM) to mimic ICH in vitro. The PERK antagonist, GSK2606414, and inhibitor of eukaryotic translation initiation factor 2 subunit α (eIF2α) dephosphorylation, salubrinal, were used to study the roles of PERK signaling pathway in ICH-induced SBI. Our results showed that the protein levels of p-eIF2α and ATF4 were upregulated following ICH, peaking at 48 h. Application of GSK2606414 reversed this increase in vivo and in vitro, thereby preventing ICH-induced neuronal apoptosis. On the contrary, salubrinal inhibited the dephosphorylation of eIF2α, resulting in the elevation of p-eIF2α, which could activate downstream of PERK signaling and induce neuronal apoptosis and necrosis following ICH in vitro and in vivo. Thus, PERK signaling pathway plays an important role in ICH-induced apoptosis and blocking its activation has neuroprotective effects that alleviates SBI, suggesting that targeting this pathway could be a promising therapeutic strategy for improving patient outcome after ICH.