Translation Initiation Factor 2α Research Articles

Amidino-rocaglates (ADRs), a class of synthetic rocaglates, are potent inhibitors of SARS-CoV-2 replication through inhibition of viral protein synthesis

Coronaviruses are highly transmissible respiratory viruses that cause symptoms ranging from mild congestion to severe respiratory distress. The recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has underscored the need for new antivirals with broad-acting mechanisms to combat increasing emergence of new variants. Currently, there are only a few antivirals approved for treatment of SARS-CoV-2. Previously, the rocaglate natural product silvestrol and synthetic rocaglates such as CR-1-31b were shown to have antiviral effects by inhibiting eukaryotic translation initiation factor 4A1 (eIF4A) function and virus protein synthesis. In this study, we evaluated amidino-rocaglates (ADRs), a class of synthetic rocaglates with the most potent eIF4A-inhibitory activity to-date, for inhibition of SARS-CoV-2 infection. This class of compounds showed low nanomolar potency against multiple SARS-CoV-2 variants and in multiple cell types, including human lung-derived cells, with strong inhibition of virus over host protein synthesis and low cytotoxicity. The most potent ADRs were also shown to be active against two highly pathogenic and distantly related coronaviruses, SARS-CoV and MERS-CoV. Mechanistically, cells with mutations of eIF4A1, which are known to reduce rocaglate interaction displayed reduced ADR-associated loss of cellular function, consistent with targeting of protein synthesis. Overall, ADRs and derivatives may offer new potential treatments for SARS-CoV-2 with the goal of developing a broad-acting anti-coronavirus agent.

Acute exposure to polystyrene nanoplastics induces unfolded protein response and global protein ubiquitination in lungs of mice

Inhaling microplastics (MPs) and nanoplastics (NPs) in the air can damage lung function. Xenobiotics in the body can cause endoplasmic reticulum (ER) stress, and the unfolded protein response (UPR) activation alleviates ER stress. Degradation of unfolded or misfolded proteins is an important pathway for recovering cellular homeostasis. The UPR and protein degradation induced by MPs/NPs in lung tissues are not well understood. Here, we investigated the UPR and protein ubiquitination in the lungs of mice exposed to polystyrene (PS)-NPs and their possible molecular mechanisms leading to protein ubiquitination. Mice were intratracheally administered with 5.6, 17, and 51 mg/kg PS-NPs once for 24 h. Exposure to PS-NPs elevated protein ubiquitination in the lungs of mice in a dose-dependent manner. PS-NPs activated three branches of UPR including inositol-requiring protein 1α (IRE1α), eukaryotic translation initiator factor 2α (eIF2α), and activating transcription factor 6α (ATF6α) in the lungs of mice. However, activated IRE1α did not trigger X-box binding protein 1 (XBP1) mRNA splicing. Exposure to PS-NPs induced an increase in the levels of E3 ubiquitin ligase hydroxymethyl glutaryl-coenzyme A reductase degradation protein 1 (HRD1) and carboxy terminus of Hsc70 interacting protein (CHIP) in the lungs of mice and BEAS-2B cells. ATF6α siRNA inhibited the levels of HRD1 and CHIP proteins induced by PS-NPs in BEAS-2B cells. These results suggest that ATF6α plays a critical role in increasing ubiquitination of unfolded or misfolded proteins by alleviating PS-NPs induced ER stress through UPR to achieve ER homeostasis in the lungs of mice.

Abstract B007: Unexpected synthetic lethality mechanisms in eIF4A-targeted therapy

Abstract BACKGROUND Dysregulation of mRNA translation, which is prevalent in cancer, presents an underexplored therapeutic vulnerability. The eukaryotic translation initiation factor 4A (eIF4A) promotes protein synthesis by unwinding secondary structures in the 5’-untranslated region of mRNAs to facilitate translation initiation. While perturbation of eIF4A minimally affects the health normal cells, it is crucial for the translation of many oncogenes and is considered essential in most cancer cell lines. Zotatifin, a first-in-class eIF4A inhibitor, is currently under evaluation in a Phase I/II clinical trial for KRAS-mutant tumors and ER+ breast cancers. In addition to the tumor-intrinsic effect, our recent publication in the Journal of Clinical Investigation revealed that pharmacological targeting of eIF4A not only affects tumor cells but also improves the tumor immune microenvironment. This results in tumor inhibition, heightened sensitivity to immune checkpoint blockade, and remarkable synergism with platinum in various preclinical triple-negative breast cancer mouse models. OBJECTIVES To investigate the mechanism underlying the synergy between eIF4A-targeted therapy and platinum. UNPUBLISHED RESULT Mass spectrometry proteomic analysis coupled with ELISA and qPCR analysis unveiled that eIF4A-targeted therapy promotes type I interferon (IFN) secretion and induces a robust type I IFN response both in vivo and in vitro. This suggests a tumor cell-autonomous effect of IFN induction by eIF4A inhibition. Combining platinum with eIF4A-targeted therapy further heightens the type I IFN response in vivo, leading to T cell-and macrophage-dependent synergistic tumor regression. Interestingly, this synergy was not observed in vitro, indicating the involvement of the host immune system in the efficacy of combination therapy. It is possible that platinum may stimulate certain immune populations to secrete IFN, which acts on tumor cells to upregulate their IFN response. CONCLUSION Activation of the type I IFN response by eIF4A inhibition combined with platinum in vivo can lead to synthetic lethality of tumor cells. These studies not only offer novel insights into eIF4A biology but also provide valuable guidance for ongoing and future clinical trials. Citation Format: Na Zhao, Elena Kabotyanski, Alexander Saltzman, Jeffrey Rosen. Unexpected synthetic lethality mechanisms in eIF4A-targeted therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B007.

Knockdown of EIF2AK2-OAS1 axis reduces ATP production inducing AMPK phosphorylation to inhibit the malignant behavior of gastric cancer cells.

Energy metabolism has always been a hot topic in cancer progression and targeted therapy, and exploring the role of genes in energy metabolic pathways in cancer cells has become key to address this issue. Eukaryotic translation initiation factor 2α kinase 2 (EIF2AK2) plays regulatory roles in cancer and disorders of energy metabolism. Indeed, the role of EIF2AK2 in energy metabolism has been underestimated. The aim of this study is to reveal the expression specificity of EIF2AK2 in gastric cancer (GC) progression and metastasis, and to demonstrate the role of EIF2AK2 in energy metabolism, cytoskeleton, proliferation, death and metastasis pathways in GC cells. Mechanistically, EIF2AK2 overexpression promoted cytoskeleton remodeling and ATP production, mediated cell proliferation and metastasis, upregulated OAS1 expression, decreases p-AMPK expression and inhibited apoptosis in GC cells. Conversely, knockdown of EIF2AK2 resulted in the opposite effect. However, overexpression of OAS1 mediated the upregulation of mitochondrial membrane potential and promoted ATP production and NAD+/NADH ratio, but knockdown of OAS1 inhibited the above effects. In addition, knockdown of OAS1 had no effect on EIF2AK2 expression, but inhibited AMPK and upregulated p-AMPK expression. In conclusion, our study identified EIF2AK2 and OAS1 as previously undescribed regulators of energy metabolism in GC cells. We hypothesized that EIF2AK2-OAS1 axis may regulate energy metabolism and inhibit cellular malignant behavior in cancer cells by affecting ATP production to induce AMPK phosphorylation, suggesting EIF2AK2 as a potential therapeutic target for cancer cell progression.

Nuclear eNOS interacts with and S-nitrosates ADAR1 to modulate endothelial gene expression

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Collaborative Research Centre (CRC 1366); Chinese Scholarship Council (CSC) Introduction and Aim Nitric oxide (NO) generated by the endothelial NO synthase (eNOS) regulates vascular tone and endothelial homeostasis to counteract vascular inflammation. The effects of NO are attributable to its interaction with heme-containing proteins e.g., soluble guanylyl cyclase, or to the S-nitrosation of proteins. Most eNOS is localized to the cell membrane or the Golgi apparatus, however, it has also been detected in the nucleus. In this study, we assessed the role of nuclear eNOS in endothelial cells. Methods and Results Confocal microscopy studies confirmed the presence of eNOS in the nucleus of unstimulated human and murine endothelial cells (ex vivo and in vitro) and stimulation with vascular endothelial growth factor (VEGF) increased eNOS nuclear translocation. Co-immunoprecipitation studies coupled with proteomics revealed the VEGF-dependent association of nuclear eNOS with 81 proteins involved in RNA binding and processing. Among these, nuclear eNOS bound double-stranded RNA-specific adenosine deaminase 1 (ADAR1), an enzyme involved in RNA editing via the deamination of adenosine (A) to inosine (I) in double-stranded RNA (dsRNA). ADAR1 was S-nitrosated in human endothelial cells and the knockdown of eNOS was associated with an increase in dsRNA (immunofluorescence) and altered ADAR1-mediated A-I editing (RNA seq). The accumulation of dsRNA in human endothelial cells lacking eNOS led to the activation of the interferon α/β signalling pathway (e.g., MX2, OASL, OAS1/2/3, RNASEL) and the downregulation of cell cycle-related genes. As a result, growth factor-stimulated cell proliferation was abrogated and basal as well as tumour necrosis factor- or H2O2-induced cell death were increased. Furthermore, the increased dsRNA in endothelial cells lacking eNOS elicited the activation of interferon-induced, dsRNA-activated protein kinase, which in turn phosphorylated the eukaryotic translation initiation factor 2A. Accordingly, new protein synthesis was inhibited following eNOS knockdown (immunofluorescence and mass spectrometry). Conclusion These results demonstrated that eNOS/NO signalling regulates endothelial type I interferon signalling and affects translation, cell cycle and cell death through S-nitrosation of ADAR1.

PDCD4 restricts PRRSV replication in an eIF4A-dependent manner and is antagonized by the viral nonstructural protein 9.

As obligate parasites, viruses have evolved multiple strategies to evade the host immune defense. Manipulation of the host proteasome system to degrade specific detrimental factors is a common viral countermeasure. To identify host proteins targeted for proteasomal degradation by porcine reproductive and respiratory syndrome virus (PRRSV), we conducted a quantitative proteomics screen of PRRSV-infected Marc-145 cells under the treatment with proteasome inhibitor MG132. The data revealed that the expression levels of programmed cell death 4 (PDCD4) were strongly downregulated by PRRSV and significantly rescued by MG132. Further investigation confirmed that PRRSV infection induced the translocation of PDCD4 from the nucleus to the cytoplasm, and the viral nonstructural protein 9 (Nsp9) promoted PDCD4 proteasomal degradation in the cytoplasm by activating the Akt-mTOR-S6K1 pathway. The C-terminal domain of Nsp9 was responsible for PDCD4 degradation. As for the role of PDCD4 during PRRSV infection, we demonstrated that PDCD4 knockdown favored viral replication, while its overexpression significantly attenuated replication, suggesting that PDCD4 acts as a restriction factor for PRRSV. Mechanistically, we discovered eukaryotic translation initiation factor 4A (eIF4A) was required for PRRSV. PDCD4 interacted with eIF4A through four sites (E249, D253, D414, and D418) within its two MA3 domains, disrupting eIF4A-mediated translation initiation in the 5'-untranslated region of PRRSV, thereby inhibiting PRRSV infection. Together, our study reveals the antiviral function of PDCD4 and the viral strategy to antagonize PDCD4. These results will contribute to our understanding of the immune evasion strategies employed by PRRSV and offer valuable insights for developing new antiviral targets.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) infection results in major economic losses in the global swine industry and is difficult to control effectively. Here, using a quantitative proteomics screen, we identified programmed cell death 4 (PDCD4) as a host protein targeted for proteasomal degradation by PRRSV. We demonstrated that PDCD4 restricts PRRSV replication by interacting with eukaryotic translation initiation factor 4A, which is required for translation initiation in the viral 5'-untranslated region. Additionally, four sites within two MA3 domains of PDCD4 are identified to be responsible for its antiviral function. Conversely, PRRSV nonstructural protein 9 promotes PDCD4 proteasomal degradation in the cytoplasm by activating the Akt-mTOR-S6K1 pathway, thus weakening the anti-PRRSV function. Our work unveils PDCD4 as a previously unrecognized host restriction factor for PRRSV and reveals that PRRSV develops countermeasures to overcome PDCD4. This will provide new insights into virus-host interactions and the development of new antiviral targets.

Abstract 2109: Nox1-mediated Endothelial Ros Propagates Hypoxia-induced Pulmonary Hypertension Via The Unfolded Protein Response.

Pulmonary arterial hypertension (PAH) is a devastating disease in which reactive oxygen species (ROS) play an important role. The disease is characterized by lung arterial cell proliferation (inward remodeling) leading to right ventricle (RV) failure. We postulated that NADPH oxidase 1 (NOX1)-derived ROS mediate endothelial cell (EC) proliferation and hemodynamic changes in PAH. Indeed, we found increased NOX1 (1.6±0.10-fold; n=7-9, p<0.01) in human pulmonary artery ECs (HPAECs) exposed to 24 hr hypoxia (Hx) vs. normoxia (Nx) (1% v. 21% O 2 , in vitro PAH phenotype). To test an in vivo NOX1 role, C57BL/6J (WT), mice were implanted with pumps delivering a NOX1-selective peptidic inhibitor (NoxA1ds) or scrambled control (Scr) and exposed to Nx (21% O 2 ) or Hx (10% O 2 ) for 3 wks plus vehicle or VEGF receptor antagonist Sugen 5416. Sugen/Hx (Su/Hx) increased RV max pressure (RVMP) 2-fold ± 0.20-fold (n=7-9, p<0.001) along with elevated mean pulmonary artery pressure (mPAP, 2-fold 0.25-fold, n=7-9, p<0.001), pulmonary vascular resistance (PVR, 2-fold±0.2-fold, n=7-9, p<0.001), and right ventricular max dP/dt (RVM dP/dt, 2-fold±0.2-fold, n=7-9, p<0.001) vs. Nx. NoxA1ds infusion significantly attenuated these rises. In parallel, RNA-Seq of HPAECs treated with NOX1 siRNA and exposed to Nx or Hx revealed the protein kinase R-like ER kinase (PERK) branch of the unfolded protein response (UPR) as NOX1-mediated. UPR promotes tumor growth in cancer due to positive effects on cell survival and proliferation. Indeed, phospho-PERK expression measured via WB increased by 1.5-fold ± 0.3-fold (n=9, p<0.01) in HPAECs exposed to Hx, which was completely reversed by siNOX1. Further, downstream members of the PERK-UPR pathway followed similar trends including phospho-eukaryotic translation initiation factor 2A (eIF2α, 3-fold ± 0.6-fold, n=9, p<0.05), activating transcription factor 4 (ATF4, 2.1-fold ± 0.4-fold, n=9, p<0.01), and protein disulfide-isomerase (PDI, 2.3-fold ± 0.65-fold, n=9, p<0.01). We corroborated these findings in cross-sections of Su/Hx mouse lungs and in autopsied iPAH patient lungs. The findings are consistent with NOX1 mediating activation of the UPR through PERK, which, in turn, induces EC hyperproliferation in PAH.

The MYCN 5′ UTR as a therapeutic target in neuroblastoma

Tumor MYCN amplification is seen in high-risk neuroblastoma, yet direct targeting of this oncogenic transcription factor has been challenging. Here, we take advantage of the dependence of MYCN-amplified neuroblastoma cells on increased protein synthesis to inhibit the activity of eukaryotic translation initiation factor 4A1 (eIF4A1) using an amidino-rocaglate, CMLD012824. Consistent with the role of this RNA helicase in resolving structural barriers in 5' untranslated regions (UTRs), CMLD012824 increased eIF4A1 affinity for polypurine-rich 5' UTRs, including that of the MYCN and associated transcripts with critical roles in cell proliferation. CMLD012824-mediated clamping of eIF4A1 spanned the full lengths of mRNAs, while translational inhibition was mediated through 5' UTR binding in a cap-dependent and -independent manner. Finally, CMLD012824 led to growth inhibition in MYCN-amplified neuroblastoma models without generalized toxicity. Our studies highlight the key role of eIF4A1 in MYCN-amplified neuroblastoma and demonstrate the therapeutic potential of disrupting its function.

Social defeat stress induces liver injury by modulating endoplasmic reticulum stress in C57BL/6J mice

Social defeat stress is associated with endoplasmic reticulum (ER) stress, inflammation and apoptosis. ER stress is thought to contribute to many lifestyle diseases such as liver injury, cardiovascular dysfunction and depression. We investigated the expression of the ER stress markers RNA-dependent protein kinase-like ER kinase (PERK), eukaryotic translation initiation factor 2α (eIF2α) and C/EBP homologous protein (CHOP), as well as inflammatory and apoptotic factors, to assess how social defeat stress induces liver injury. Furthermore, we evaluated the effects of the ER stress inhibitor phenylbutyric acid (PBA) and ER stress inducer thapsigargin (TG) on liver injury. Adult mice were divided into the control, social defeat, social defeat + PBA, TG, PBA and TG + PBA groups. The social defeat and social defeat + PBA groups were simultaneously exposed to social defeat stress for 10 days. The social defeat + PBA, TG, PBA and TG + PBA groups were treated with PBA or TG via intraperitoneal injections. PBA was injected 1 h before the TG injection into the TG + PBA group. Liver samples from six groups of mice were analyzed by histological analysis and western blotting. Social defeat stress promoted ER stress, increased the expression of inflammatory factors and induced apoptosis in the liver of socially defeated mice, which was reversed by PBA. Moreover, ER stress induces TG-induced liver injury by initiating ER stress. Social defeat stress initiates ER stress, promotes the expression of inflammatory and apoptotic factors, and induces liver injury. PBA suppresses liver injury caused by social defeat stress and TG treatment.

Abstract 4284: Macrophage reprogramming and immune suppression through extracellular vesicles loaded eIF4A1in ovarian cancer

Abstract Background- In ovarian carcinoma (OvCa), macrophages are reprogrammed toward pro-tumorigenic phenotypes, including releasing anti-inflammatory cytokines and expressing immunosuppressive molecules. However, how these macrophages acquire their phenotype remains largely unexplored. To understand the mechanism driving pro-tumorigenic phenotype in macrophages, we studied the role of tumor cell-derived extracellular vesicles (EVs) in the cross-talk with macrophages in OvCa. We found that EVs from ovarian cancer cells contain eukaryotic translation initiation factor 4A1 (eIF4A1). eIF4A1 containing EVs from ovarian cells enhanced macrophage translational activity and PD-L1 expression. However, the detailed mechanism that explains how eIF4A1-packaged EVs induce immunosuppressive phenotype in macrophages is necessary to develop novel immunotherapy strategies for advanced OvCa treatment. Methods- We performed SUnSET assay (surface sensing of translation) to determine whether eIF4E-EVs affect global translation in macrophages. SILAC (Stable isotope labeling by amino acids) based mass spectrometry was used to identify the downstream targets of eIF4A1 in macrophages. We isolated murine peritoneal macrophages from naïve C57BL/6 female mice and co-cultured the macrophages with EVs derived from ovarian cancer cell lines. The expression of PD-L1, CD206, interleukin-6 (IL-6), and other inflammatory cytokines on EVs treated macrophages was evaluated using flow cytometry and a cytokine array kit. Further, we created a Rab27aKO ovarian cancer in vivo model to study the effect of eIF4A1 packaged EVs on the immune suppressive ability of macrophages and tumor progression. Results- Our study shows that tumor cells employ EVs to deliver eIF4A1 to the tumor microenvironment, facilitating the release of cytokines such as IL-6 and the expression of PD-L1 on macrophages and supporting tumor growth. In addition, our data shows that eIF4A1-EVs enhance protein synthesis in macrophages. Our data show that in the Rab27aKO ovarian cancer in vivo mouse model, intravenous injection of eIF4A1-packaged EVs increased the tumor burden, metastasis, and ascites accumulation as compared to PBS control. eIF4A1-EVs treatment increased the infiltration of tumor-associated macrophages (TAM) in ascites. We also found increased expression of PD-L1 and CD206 on TAM isolated from ascites Citation Format: Sonam Mittal, Ishaque Pulikkal Kadamberi, Sudhir Kumar, Jasmine George, Mona Singh, Anjali Geethadevi, Pradeep Chaluvally-Raghavan, Sunila Pradeep. Macrophage reprogramming and immune suppression through extracellular vesicles loaded eIF4A1in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4284.

Hypoxia leads to gill endoplasmic reticulum stress and disruption of mitochondrial homeostasis in grass carp (Ctenopharyngodon idella): Mitigation effect of thiamine

Hypoxia in water environment is one of the important problems faced by intensive aquaculture. Under hypoxia stress, the effects of dietary thiamine were investigated on grass carp gill tissue damage and their mechanisms. Six thiamine diets with different thiamine levels (0.22, 0.43, 0.73, 1.03, 1.33 and 1.63 mg/kg) were fed grass carp (Ctenopharyngodon idella) for 63 days. Then, 96-hour hypoxia stress test was conducted. This study described that thiamine enhanced the growth performance of adult grass carp and ameliorated nutritional status of thiamine (pyruvic acid, glucose, lactic acid and transketolase). Additionally, thiamine alleviated the deterioration of blood parameters [glutamic oxalacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), glucose, cortisol, lactic dehydrogenase (LDH), erythrocyte fragility, and red blood cell count (RBC count)] caused by hypoxia stress, and reduced reactive oxygen species (ROS) content and oxidative damage to the gills. In addition, thiamine alleviated endoplasmic reticulum stress in the gills, which may be related to its inhibition of RNA-dependent protein kinase-like ER kinase (PERK)/eukaryotic translation initiation factor-2α (eIF2α)/activating transcription factor4 (ATF4), inositol-requiring enzyme 1 (IRE1)/X-Box binding protein 1 (XBP1) and activating transcription factor 6 (ATF6) pathways. Furthermore, thiamine maintaining mitochondrial dynamics balance was probably related to promoting mitochondrial fusion and inhibiting mitochondrial fission, and inhibiting mitophagy may involve PTEN induced putative kinase 1 (PINK1)/Parkin-dependent pathway and hypoxia-inducible factor (HIF)-Bcl-2 adenovirus E1B 19 kDa interacting protein 3 (BNIP3) pathway. In summary, thiamine alleviated hypoxia stress in fish gills, which may be related to reducing endoplasmic reticulum stress, regulating mitochondrial dynamics balance and reducing mitophagy. The thiamine requirement for optimum growth [percent weight gain (PWG)] of adult grass carp was estimated to be 0.81 mg/kg diet. Based on the index of anti-hypoxia stress (ROS content in gill), the thiamine requirement for adult grass carp was estimated to be 1.32 mg/kg diet.

Abstract P29: Zotatifin, the eukaryotic translation initiation factor 4A (eIF4A) inhibitor, synergizes with Venetoclax to inhibit acute myeloid leukemia cell growth

Abstract New targeted therapies such as the selective and potent BCL-2 inhibitor Venetoclax have revolutionized the treatment of acute myeloid leukemia (AML). However, due to the cellular heterogeneity of the disease, drug-resistant cell types often emerge and drive relapse. We found that the eukaryotic translation initiation factor 4A (EIF4A) is highly expressed in multiple primitive AML cell types compared to their healthy counterparts, suggesting its potential as a therapeutic target. By unwinding complex structures in the 5’ UTR of transcripts, eIF4A promotes the translation of oncogene transcripts. A first-in-class drug, Zotatifin, inhibits the helicase activity of eIF4A and curtails tumor growth in receptor tyrosine kinase-driven tumors. Zotatifin is currently being evaluated in phase 1/2 trials across multiple solid tumors (NCT04092673). However, its effectiveness in blood cancers such as AML remains unexplored, and it is unknown whether Zotatifin may work synergistically with Venetoclax. Here, we treated AML cell lines in vitro and primary healthy or AML bone marrow ex vivo with Zotatifin or Venetoclax alone and in combination, and demonstrate synergistic killing of AML cells by the combination therapy (ZIP synergy score = 57.0 in MOLM-13, 19.3 in MV4-11, P < 0.0001). Mechanistically, we show that Zotatifin decreases eIF4A and AKT expression, and its combination with Venetoclax drives increased BAK and cleaved BAK expression, as well as increased cleavage of MCL-1 and BCL-2, consistent with drug synergy by convergence on the apoptotic pathway. In pilot colony-forming unit (CFU) assays, we found the combination to be tolerable in healthy CD34+ human hematopoietic stem and progenitor cells (HSPCs). To test drug efficacy in vivo, we generated a patient-derived xenograft (PDX) AML model from a patient who experienced relapse with complex cytogenetics, FLT3 and NPM1 mutations. Only the combination of Zotatifin and Venetoclax but not monotherapies effectively suppressed tumor burden as assessed by human CD45 engraftment in the peripheral blood. Further, the combination of Zotatifin and Venetoclax significantly prolonged survival of xenografted mice (median survival after treatment: vehicle 11 days, Venetoclax 13 days, Zotatifin 15 days, combination 42 days, P = 0.0002). Together, our data indicates that Zotatifin is synergistic with Venetoclax and may represent an effective treatment option for patients who are either primary refractory or have developed resistance to existing Venetoclax-containing regimens. Citation Format: Yoke Seng Lee, Jonathan Good, Noha Awais, Benjamin Eschle, Jenny Noel, Jean Acosta, Erica DePasquale, David Cucchi, Prafulla Gokhale, Fadi Najm, Peter van Galen. Zotatifin, the eukaryotic translation initiation factor 4A (eIF4A) inhibitor, synergizes with Venetoclax to inhibit acute myeloid leukemia cell growth [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P29.

Translation initiation factor eIF1A rescues hygromycin B sensitivity caused by deleting the carboxy-terminal tail in the GPN-loop GTPase Npa3.

The essential yeast protein GPN-loop GTPase 1 (Npa3) plays a critical role in RNA polymerase II (RNAPII) assembly and subsequent nuclear import. We previously identified a synthetic lethal interaction between a mutant lacking the carboxy-terminal 106-amino acid tail of Npa3 (npa3ΔC) and a bud27Δ mutant. As the prefoldin-like Bud27 protein participates in ribosome biogenesis and translation, we hypothesized that Npa3 may also regulate these biological processes. We investigated this proposal by using Saccharomyces cerevisiae strains episomally expressing either wild-type Npa3 or hypomorphic mutants (Npa3ΔC, Npa3K16R, and Npa3G70A). The Npa3ΔC mutant fully supports RNAPII nuclear localization and activity. However, the Npa3K16R and Npa3G70A mutants only partially mediate RNAPII nuclear targeting and exhibit a higher reduction in Npa3 function. Cell proliferation in these strains displayed an increased sensitivity to protein synthesis inhibitors hygromycin B and geneticin/G418 (npa3G70A > npa3K16R > npa3ΔC > NPA3 cells) but not to transcriptional elongation inhibitors 6-azauracil, mycophenolic acid or 1,10-phenanthroline. In all three mutant strains, the increase in sensitivity to both aminoglycoside antibiotics was totally rescued by expressing NPA3. Protein synthesis, visualized by quantifying puromycin incorporation into nascent-polypeptide chains, was markedly more sensitive to hygromycin B inhibition in npa3ΔC, npa3K16R, and npa3G70A than NPA3 cells. Notably, high-copy expression of the TIF11 gene, that encodes the eukaryotic translation initiation factor 1A (eIF1A) protein, completely suppressed both phenotypes (of reduced basal cell growth and increased sensitivity to hygromycin B) in npa3ΔC cells but not npa3K16R or npa3G70A cells. We conclude that Npa3 plays a critical RNAPII-independent and previously unrecognized role in translation initiation.

Glucose Induces ER Stress Response-Mediated Peritoneal Mesothelial Cell Death.

Peritoneal dialysis (PD) fluid, which contains a high concentration of glucose, is involved in peritoneal damage after long-term use. The mechanisms through which glucose induces damage to the mesothelium have not been clearly elucidated. Although, endoplasmic reticulum (ER) stress response is associated with several diseases, the involvement of ER stress in peritoneal damage has not yet been demonstrated. Primary-cultured rat peritoneal mesothelial cells (RPMCs) and rat PD model were used to investigate the influence of glucose on the peritoneum. Cells treated with glucose were examined for cytotoxicity, induction of apoptosis, and activation of the ER stress pathway. Glucose treatment of RPMCs induced cell death at concentrations higher than 3%. Annexin V positive, that is a feature of apoptosis, occurred in dead cells. Treatment with glucose led to the activation of protein kinase R-like ER kinase (PERK) and eukaryotic translation initiation factor-2α (eIF-2α). Glucose also induced the expression and nuclear translocation of homologous protein C/EBP. Cell death was rescued by the integrated stress response inhibitor, ISRIB, which suppresses the integrated stress response pathway, including ER stress. Glucose in PD fluid induces PERK/eIF-2α-mediated ER stress in RPMCs, resulting in apoptosis. This cellular stress may cause peritoneal damage in patients receiving PD.

An upstream open reading frame (5′-uORF) links oxidative stress to translational control of ALCAT1 through phosphorylation of eIF2α

Acyl-CoA:lysocardiolipin acyltransferase 1 (ALCAT1) is an enzyme that promotes mitochondrial dysfunction by catalyzing pathological remodeling of cardiolipin. Upregulation of ALCAT1 protein expression by oxidative stress is implicated in the pathogenesis of age-related metabolic diseases, but the underlying molecular mechanisms remain elusive. In this study, we identified a highly conserved upstream open reading frame (uORF) at the 5′-untranslated region (5′-UTR) of ALCAT1 mRNA as a key regulator of ALCAT1 expression in response to oxidative stress. We show that the uORF serves as a decoy that prevents translation initiation of ALCAT1 under homeostatic condition. The inhibitory activity of the uORF on ALCAT1 mRNA translation is mitigated by oxidative stress but not ER stress, which requires the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α). Consequently, ablation of uORF or eIF2α phosphorylation at Ser51 renders ALCAT1 protein expression unresponsive to induction by oxidative stress. Taken together, our data show that the uORF links oxidative stress to translation control of ALCAT1 mRNAs through phosphorylation of eIF2α at Ser51.

RNA Activators of Stress Kinase PKR within Human Genes That Control Splicing or Translation Create Novel Targets for Hereditary Diseases.

Specific sequences within RNA encoded by human genes essential for survival possess the ability to activate the RNA-dependent stress kinase PKR, resulting in phosphorylation of its substrate, eukaryotic translation initiation factor-2α (eIF2α), either to curb their mRNA translation or to enhance mRNA splicing. Thus, interferon-γ (IFNG) mRNA activates PKR through a 5'-terminal 203-nucleotide pseudoknot structure, thereby strongly downregulating its own translation and preventing a harmful hyper-inflammatory response. Tumor necrosis factor-α (TNF) pre-mRNA encodes within the 3'-untranslated region (3'-UTR) a 104-nucleotide RNA pseudoknot that activates PKR to enhance its splicing by an order of magnitude while leaving mRNA translation intact, thereby promoting effective TNF protein expression. Adult and fetal globin genes encode pre-mRNA structures that strongly activate PKR, leading to eIF2α phosphorylation that greatly enhances spliceosome assembly and splicing, yet also structures that silence PKR activation upon splicing to allow for unabated globin mRNA translation essential for life. Regulatory circuits resulting in each case from PKR activation were reviewed previously. Here, we analyze mutations within these genes created to delineate the RNA structures that activate PKR and to deconvolute their folding. Given the critical role of intragenic RNA activators of PKR in gene regulation, such mutations reveal novel potential RNA targets for human disease.

Hugan Qingzhi tablets attenuates endoplasmic reticulum stress in nonalcoholic fatty liver disease rats by regulating PERK and ATF6 pathways

BackgroundEndoplasmic reticulum (ER) stress, promoting lipid metabolism disorders and steatohepatitis, contributes significantly to the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Hugan Qingzhi tablets (HQT) has a definite effect in the clinical treatment of NAFLD patients, but its mechanism is still unclear. This study aims to investigate the effects of HQT on ER stress in the liver tissues of NAFLD rats and explore the underlying mechanism.MethodsThe NAFLD rat model was managed with high-fat diet (HFD) for 12weeks. HQT was administrated in a daily basis to the HFD groups. Biochemical markers, pro-inflammatory cytokines, liver histology were assayed to evaluate HQT effects in HFD-induced NAFLD rats. Furthermore, the expression of ER stress-related signal molecules including glucose regulating protein 78 (GRP78), protein kinase RNA-like endoplasmic reticulum kinase (PERK), p-PERK, eukaryotic translation initiation factor 2α (EIF2α), p-EIF2α, activating transcription factor 4 (ATF4), acetyl-coenzyme A-carboxylase (ACC), activating transcription factor (ATF6), and nuclear factor-kappa B-p65 (NF-κB-p65) were detected by western blot and/or qRT-PCR.ResultsThe histopathological characteristics and biochemical data indicated that HQT exhibited protective effects on HFD-induced NAFLD rats. Furthermore, it caused significant reduction in the expression of ERS markers, such as GRP78, PERK, p-PERK, and ATF6, and subsequently downregulated the expression of EIF2α, p-EIF2α ATF4, ACC, and NF-κB-p65.ConclusionsThe results suggested that HQT has protective effect against hepatic steatosis and inflammation in NAFLD rats by attenuating ER stress, and the potential mechanism is through inhibition of PERK and ATF6 pathways.

O-GlcNAc regulates the mitochondrial integrated stress response by regulating ATF4.

Accumulation of mitochondrial dysfunctional is a hallmark of age-related neurodegeneration including Alzheimer's disease (AD). Impairment of mitochondrial quality control mechanisms leading to the accumulation of damaged mitochondria and increasing neuronal stress. Therefore, investigating the basic mechanisms of how mitochondrial homeostasis is regulated is essential. Herein, we investigate the role of O-GlcNAcylation, a single sugar post-translational modification, in controlling mitochondrial stress-induced transcription factor Activating Transcription Factor 4 (ATF4). Mitochondrial dysfunction triggers the integrated stress response (ISRmt), in which the phosphorylation of eukaryotic translation initiation factor 2α results in the translation of ATF4. We used patient-derived induced pluripotent stem cells, a transgenic mouse model of AD, SH-SY5Y neuroblastoma and HeLa cell-lines to examine the effect of sustained O-GlcNAcase inhibition by Thiamet-G (TMG) on ISRmt using biochemical analyses. We show that TMG elevates ATF4 protein levels upon mitochondrial stress in SH-SY5Y neuroblastoma and HeLa cell-lines. An indirect downstream target of ATF4 mitochondrial chaperone glucose-regulated protein 75 (GRP75) is significantly elevated. Interestingly, knock-down of O-GlcNAc transferase (OGT), the enzyme that adds O-GlcNAc, in SH-SY5Y increases ATF4 protein and mRNA expression. Additionally, ATF4 target gene Activating Transcription Factor 5 (ATF5) is significantly elevated at both the protein and mRNA level. Brains isolated from TMG treated mice show elevated levels of ATF4 and GRP75. Importantly, ATF4 occupancy increases at the ATF5 promoter site in brains isolated from TMG treated mice suggesting that O-GlcNAc is regulating ATF4 targeted gene expression. Interestingly, ATF4 and GRP75 are not induced in TMG treated familial Alzheimer's Disease mice model. The same results are seen in a human in vitro model of AD. Together, these results indicate that in healthy conditions, O-GlcNAc regulates the ISRmt through regulating ATF4, while manipulating O-GlcNAc in AD has no effect on ISRmt.

Undersized telomeres in regulatory T cells link to the pathogenesis of allergic rhinitis

Telomeres are an important biomarker in the cell destiny. The relationship between telomeres and regulatory Tcells (Tregs) has not yet been investigated. The objective of this study is to evaluate the link between Tregs' telomere length and allergic rhinitis (AR)'s pathogenesis. Here, we report that low telomerase activity and high endoplasmic reticulum stress status were observed in Tregs from AR patients, as shown in the results. Immune regulatory molecules levels were correlated with the length of Tregs' telomeres. The immune-suppressive functions of Tregs were associated with the telomere length/Telomerase reverse transcriptase/Telomerase protein component 1 status in Tregs. The levels of telomere length/telomerase in airway Tregs were reduced by sensitization. Endoplasmic reticulum stress signaling pathway of proline-rich receptor-like protein kinase-eukaryotic translation initiation factor 2A (eIF2a) was associated with the regulation of telomerase. Inhibiting eIF2a had an effect on upregulating telomerase activity in Tregs and mitigating experimental AR.

Capsaicin supplementation prevents western diet-induced hyperleptinemia by reducing endoplasmic reticulum stress in apolipoprotein E-deficient mice

Background: Endoplasmic reticulum (ER) stress implicated in leptin resistance in the diet-induced obesity, which can accelerate the development of atherosclerosis forms the background of this study.
 Objective: This study aimed to investigate the effect of capsaicin on hyperleptinema by inhibiting ER stress in apolipoprotein E-deficient (ApoE-/-) mice fed a western diet (WD).
 Design: ApoE -/- mice were assigned one of three experimental diets: WD (60% kcal from fat, n = 10), WD + 0.015% capsaicin (n = 10, w/w), and WD + 1% PBA (n = 10, w/w) for 12 weeks.
 Results: In metabolic parameters, supplementation of dietary capsaicin displayed marked reduction of body weight gain and adipose tissue weight, plasma leptin, total cholesterol, and hepatic triglyceride levels without change in the plasma insulin level compared with WD fed ApoE-/- mice after 12 weeks. Capsaicin supplementation also attenuated the protein expression of ER stress markers such as eukaryotic translational initiation factor 2α and C/EBP homology protein in the liver, as well as glucose-related protein 78 localization in the aorta, indicating that capsaicin inhibits diet-induced hyperleptinemia in part by regulating the protein expression involved in ER stress.
 Conclusion: Capsaicin, therefore, may have potential as a therapeutic agent for individuals with diet-induced hyperleptinemia