General Control Nonderepressible-2 Kinase Research Articles

Discovery of HC-7366: An Orally Bioavailable and Efficacious GCN2 Kinase Activator.

A series of activators of GCN2 (general control nonderepressible 2) kinase have been developed, leading to HC-7366, which has entered the clinic as an antitumor therapy. Optimization resulted in improved permeability compared to that of the original indazole hinge binding scaffold, while maintaining potency at GCN2 and selectivity over PERK (protein kinase RNA-like endoplasmic reticulum kinase). The improved ADME properties of this series led to robust in vivo compound exposure in both rats and mice, allowing HC-7366 to be dosed in xenograft models, demonstrating that activation of the GCN2 pathway by this compound leads to tumor growth inhibition.

Abstract A012: Exploring the role of adaptive amino acid responses in Ras-driven leukemia progression and therapy

Abstract Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, affecting the lymphoid cells of both B and T lineages. Most ALL cells are auxotrophic for asparagine, a nonessential amino acid for protein synthesis, due to the low expression of asparagine synthetase (ASNS), a rate limiting enzyme for de novo biosynthesis of asparagine. As a result, standard ALL treatment takes advantage of this vulnerability by giving patients L-asparaginase, a bacterial enzyme that depletes the circulating asparagine. However, previous work from our lab and others have shown that some ALL cells become resistance to L-asparaginase treatment through the induction of ASNS expression. Mechanistically, amino acid starvation actives the general control nonderepressible 2 (GCN2) kinase, leading to the accumulation of ATF4 transcriptional factor. ATF4, in turn, is recruited to the promoter of the ASNS gene to activate its transcription. However, the role of GCN2 kinase in the process of leukemogenesis under nutrient limiting environment has not been established. In this study, our goal is to use a mouse model of T-ALL driven by mutant KRas to determine the role of GCN2 in leukemogenesis and the therapeutic response to L-asparaginase treatment. Citation Format: Ji Zhang, Rodney Claude, Sankalp Srivastava, Sandeep Batra, Minghua Zhong, Utpal Dave, Ronald C. Wek. Exploring the role of adaptive amino acid responses in Ras-driven leukemia progression and therapy [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A012.

Multiple Roles of the Stress Sensor GCN2 in Immune Cells.

The serine/threonine-protein kinase general control nonderepressible 2 (GCN2) is a well-known stress sensor that responds to amino acid starvation and other stresses, making it critical to the maintenance of cellular and organismal homeostasis. More than 20 years of research has revealed the molecular structure/complex, inducers/regulators, intracellular signaling pathways and bio-functions of GCN2 in various biological processes, across an organism's lifespan, and in many diseases. Accumulated studies have demonstrated that the GCN2 kinase is also closely involved in the immune system and in various immune-related diseases, such as GCN2 acts as an important regulatory molecule to control macrophage functional polarization and CD4+ T cell subset differentiation. Herein, we comprehensively summarize the biological functions of GCN2 and discuss its roles in the immune system, including innate and adaptive immune cells. We also discuss the antagonism of GCN2 and mTOR pathways in immune cells. A better understanding of GCN2's functions and signaling pathways in the immune system under physiological, stressful, and pathological situations will be beneficial to the development of potential therapies for many immune-relevant diseases.

Limiting glutamine utilization activates a GCN2/TRAIL-R2/Caspase-8 apoptotic pathway in glutamine-addicted tumor cells

Oncogenic transformation leads to changes in glutamine metabolism that make transformed cells highly dependent on glutamine for anabolic growth and survival. Herein, we investigated the cell death mechanism activated in glutamine-addicted tumor cells in response to the limitation of glutamine metabolism. We show that glutamine starvation triggers a FADD and caspase-8-dependent and mitochondria-operated apoptotic program in tumor cells that involves the pro-apoptotic TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2), but is independent of its cognate ligand TRAIL. In glutamine-depleted tumor cells, activation of the amino acid-sensing general control nonderepressible-2 kinase (GCN2) is responsible for TRAIL-R2 upregulation, caspase-8 activation, and apoptotic cell death. Interestingly, GCN2-dependent ISR signaling induced by methionine starvation also leads to TRAIL-R2 upregulation and apoptosis. Moreover, pharmacological inhibition of transaminases activates a GCN2 and TRAIL-R2-dependent apoptotic mechanism that is inhibited by non-essential amino acids (NEAA). In addition, metabolic stress upon glutamine deprivation also results in GCN2-independent FLICE-inhibitory protein (FLIP) downregulation facilitating caspase-8 activation and apoptosis. Importantly, downregulation of the long FLIP splice form (FLIPL) and apoptosis upon glutamine deprivation are inhibited in the presence of a membrane-permeable α-ketoglutarate. Collectively, our data support a model in which limiting glutamine utilization in glutamine-addicted tumor cells triggers a previously unknown cell death mechanism regulated by GCN2 that involves the TRAIL-R2-mediated activation of the extrinsic apoptotic pathway.

Dietary leucine supplementation improves growth performance, metabolic responses of liver via GCN2/ATF4, and insulin signaling pathways in largemouth bass (Micropterus salmoides).

An 8-week growth experiment was conducted to investigate the effects of dietary leucine on growth performance, body composition, and gene expression of hepatic nutrient metabolism in the largemouth bass (Micropterus salmoides). Six isonitrogenous (49.87%) diets with graded leucine levels (2.62, 3.07, 3.60, 3.87, 4.20, 4.71% of dry diet) were fed to triplicate groups with 20 juvenile fish (20.00 ± 0.13g). The results revealed that the specific growth rate (SGR) and weight gain (WG) increased significantly with increasing dietary leucine levels, reached their maximal value in the Leu-4.20% groups, and then decreased slightly. Although the feed conversion ratio (FCR) showed decreasing trends, no significant difference was detected. Leucine supplementation significantly improved the content of body protein and total plasma protein (TP). Additionally, a higher expression level of target of rapamycin (TOR) and ribosomal protein S6 (S6) mRNA was observed in the Leu-3.87% and Leu-4.20% diets, whereas the GCN2 (general control nonderepressible2 kinase) and AFT4 (activating transcription factor 4) mRNA expression levels were suppressed. The lipid content of the body was not influenced by leucine levels, whereas the content of total triglyceride (TG) first decreased significantly with increasing dietary leucine levels from 2.62 to 3.87% and then increased with increasing leucine levels (4.20% to 4.71%). The total cholesterol (TC) and low-density lipoproteins (LDL) trended in a similar direction but did not achieve statistical significance (P > 0.05). The expression of insulin receptor substrate 1 (IRS-1) was significantly elevated by dietary leucine levels, while protein kinase B (AKT) and phosphatidylinositol 3-kinase (PI3K) expression was inconsistently upregulated. Furthermore, leucine supplementation decreased plasma glucose and hepatic glycogen contents, and the expression levels of glucokinase (GK), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6pase) were significantly inhibited at 4.20% and 4.71% leucine diets. Analyses of the change in SGR and FCR using the quadratic regression model estimated that the optimum dietary leucine requirement of juvenile largemouth bass was 4.42% and 4.63% of the dry diet (8.86% and 9.28% of dietary protein), respectively.

Modulation of GCN2 activity under excess light stress by osmoprotectants and amino acids

ABSTRACT The protein kinase GCN2 (General Control Nonderepressible2) and its phosphorylation target, the eukaryotic translation initiation factor (eIF)2α represent the core module of the plant’s integrated stress response, a signaling pathway widely conserved in eukaryotes that can rapidly regulate translation in response to stressful conditions. Recent findings indicate that the Arabidopsis thaliana GCN2 protein operates under the command of reactive oxygen species (ROS) emanating from the chloroplast under a variety of abiotic stresses such as excess light. To get deeper insights into the mechanism of GCN2 activation under excess light, we assessed the role of amino acids in view of the classic function of GCN2 as a sensor of amino acid status. Additionally, given that osmoprotectants can counteract ROS-related stresses, we tested their ability to mitigate GCN2 activity. Our results demonstrate that certain amino acids and osmoprotectants attenuate eIF2α-phosphorylation under excess light stress to some degree. Future investigations into the biochemical mechanisms of these natural compounds on GCN2 signaling activity will provide better insights into the GCN2-eIF2α regulation.

The Role of GCN2 Kinase in Mediating the Effects of Amino Acids on Longevity and Feeding Behaviour in Drosophila.

Restriction of amino acids in the diet can extend lifespan in diverse species ranging from flies to mammals. However, the role of individual amino acids and the underlying molecular mechanisms are only partially understood. The evolutionarily conserved serine/threonine kinase General Control Nonderepressible 2 (GCN2) is a key sensor of amino acid deficiency and has been implicated in the response of lifespan to dietary restriction (DR). Here, we generated a novel Drosophila GCN2 null mutant and analyzed its response to individual amino acid deficiency. We show that GCN2 function is essential for fly development, longevity and feeding behaviour under long-term, but not short-term, deprivation of all individual essential amino acids (EAAs) except for methionine. GCN2 mutants were longer-lived than control flies and showed normal feeding behaviour under methionine restriction. Thus, in flies at least two systems regulate these responses to amino acid deprivation. Methionine deprivation acts via a GCN2-independent mechanism, while all other EAA are sensed by GCN2. Combined deficiency of methionine and a second EAA blocked the response of GCN2 mutants to methionine, suggesting that these two pathways are interconnected. Wild type flies showed a short-term rejection of food lacking individual EAA, followed by a long-term compensatory increase in food uptake. GCN2 mutants also showed a short-term rejection of food deprived of individual EAA, but were unable to mount the compensatory long-term increase in food uptake. Over-expression of the downstream transcription factor ATF4 partially rescued the response of feeding behaviour in GCN2 mutants to amino acid deficiency. Phenotypes of GCN2 mutants induced by leucine and tryptophan, but not isoleucine, deficiency were partially rescued by ATF4 over-expression. The exact function of GCN2 as an amino acid sensor in vivo and the downstream action of its transcription factor effector ATF4 are thus context-specific with respect to the EAA involved.

A multicenter, open-label, phase 1a/b study of HC-7366, a modulator of integrated stress response (ISR) kinase GCN2 in subjects with advanced solid tumors.

TPS3179 Background: To survive harsh tumor microenvironments, cancer cells actively utilize ISR as an adaptive stress response and survival mechanism. General control nonderepressible 2 (GCN2), a serine-threonine kinase is essential for maintaining cellular homeostasis in amino acid stress conditions. HC-7366 is a novel, highly selective, and potent GCN2 kinase modulator. Single agent HC-7366 has demonstrated potent anti-tumor activity resulting in regression and complete responses in several pre-clinical tumor models. Methods: This is a first in human, multicenter, open label, Phase 1a/b dose escalation and expansion study to establish the maximum tolerated dose, evaluate safety and tolerability, and determine the recommended Phase 2 dose of daily oral dosing of HC-7366 in patients (pts) with advanced solid tumors. Up to 36 pts with squamous cell carcinoma of the head and neck (SCCHN), colorectal cancer (CRC), non-small cell lung cancer (NSCLC), or transitional cell carcinoma of the bladder (TCC) will be enrolled into Phase 1a; other solid tumors are eligible if selection criteria are met (capped at 50%). A 3+3 design will be employed and dose escalation determined by occurrence of dose limiting toxicities (DLT) within Cycle 1 (21 days). A Safety Monitoring Committee will review each cohort when the planned number of pts complete the DLT period. Six dose levels (10, 20, 40, 75, 125, 150 mg) of HC-7366 will be evaluated. Phase 1b will be an expansion of up to two Phase 1a dose levels and enroll 15 pts per cohort. Secondary endpoints include ORR, DOR, TTF, PFS, and OS. Pharmacokinetic data will be profiled. Exploratory objectives include evaluation of pharmacodynamic markers in tumor biopsies and immunophenotyping in blood samples. Main inclusion criteria include: SCCHN, CRC, NSCLC, TCC or other solid tumors; >1 radiologically measurable lesion per RECISTv1.1; >1 biopsiable lesion at baseline; no immune checkpoint inhibitor within 4 weeks (wks) of 1st dose; ECOG 0 or 1; <10% body weight loss in 4 wks before 1st dose & serum albumin >3 g/dL; and normal/adequately controlled pan-endocrine function. Main exclusion criteria include: autoimmune disease, organ transplant, retinitis or photosensitive skin disorders; history of interstitial lung disease or pneumonitis within 1yr; and overt or latent disorders of the exocrine pancreas. Formal hypothesis testing will not be performed. Descriptive statistics of parameters of interest will be presented by dose level and safety parameters will be summarized. The trial is sponsored by HiberCell, Inc. Approximately 9 US sites will participate. Clinical trial information: NCT05121948.

GCN2 kinase activation by ATP-competitive kinase inhibitors.

Small-molecule kinase inhibitors represent a major group of cancer therapeutics, but tumor responses are often incomplete. To identify pathways that modulate kinase inhibitor response, we conducted a genome-wide knockout (KO) screen in glioblastoma cells treated with the pan-ErbB inhibitor neratinib. Loss of general control nonderepressible 2 (GCN2) kinase rendered cells resistant to neratinib, whereas depletion of the GADD34 phosphatase increased neratinib sensitivity. Loss of GCN2 conferred neratinib resistance by preventing binding and activation of GCN2 by neratinib. Several other Food and Drug Administration (FDA)-approved inhibitors, such erlotinib and sunitinib, also bound and activated GCN2. Our results highlight the utility of genome-wide functional screens to uncover novel mechanisms of drug action and document the role of the integrated stress response (ISR) in modulating the response to inhibitors of oncogenic kinases.

The Role of Indoleamine 2,3-Dioxygenase in Renal Tubular Epithelial Cells Senescence under Anoxia or Reoxygenation.

Ischemia-reperfusion injury is the commonest form of acute kidney injury (AKI). Tubular epithelial cell senescence contributes to incomplete recovery from AKI and predisposes to subsequent chronic kidney disease. In cultures of primary proximal renal tubular epithelial cells (RPTECs) subjected to anoxia or reoxygenation, we evaluated the role of indoleamine 2,3-dioxygenase 1 (IDO) in cellular senescence. Proteins of interest were assessed with Western blotting or enzyme-linked immunosorbent assay or histochemically. Under anoxia or reoxygenation, IDO expression and activity were increased. Moreover, the two IDO-derived pathways, the general control nonderepressible 2 kinase (GCN2K) pathway and the aryl-hydrocarbon receptor (AhR) pathway, were also activated. A DNA damage response (DDR) took place and led to increased levels of the cell-cycle inhibitors p21 and p16, and senescence-associated β-galactosidase (SA-β-Gal) activity. Cell proliferation was inhibited, and more IL-6 was produced. The IDO inhibitor 1-DL-methyl-tryptophan ameliorated the DDR; decreased p21, p16, and SA-β-Gal activity; restored cell proliferation; and decreased IL-6 production. The AhR inhibitor CH223191 did not affect the above parameters. In conclusion, anoxia and the subsequent reoxygenation upregulate IDO. IDO depletes tryptophan and activates GCN2K. The latter enhances the anoxia- or reoxygenation-induced DDR, resulting in increased p21 and p16 expression and eventually leading to RPTEC senescence. Since cellular senescence affects AKI outcome, the role of IDO in cellular senescence and the possible therapeutic role of IDO inhibitors deserve further investigation.

Abstract LB136: Targeting GCN2 kinase-driven stress response inactivation by orally available small molecules to restore immune tumor microenvironment in prostate cancers

Abstract In patients with metastatic castration-resistant prostate cancers (mCRPC) who have previously been treated with abiraterone or enzalutamide, the median overall survival is only 14 months. Unlike many other cancers, immunotherapies have had limited successes, due to the fact that there are very few T cells in the tumor microenvironment of prostate cancer (PC) patients. Identifying ways to boost immunotherapy responses could change the paradigm of mCRPC, a disease still difficult to treat. The highly proliferative nature of tumor cells, along with infiltration of myeloid cells into the tumors, leads to depletion of nutrients such as functional/natural amino acids. This metabolically stressful milieu causes activation of nutrient stress pathways, autophagy, and repressed immune responses. A key meditator of this nutrient stress pathway is a cytoplasmic Ser/Thr protein kinase called General Control Nonderepressible 2 (GCN2), also called EIF2AK4. GCN2 switches on by a reduction of amino acids, and its activity results in T cell inactivation, T cell death, regulatory T cell expansion, and the potentiation of myeloid-derived suppressor cells (MDSCs). We have developed and synthesized a series of novel small molecule immunotherapeutic agents that reversibly bind to GCN2 kinase, competitively block the ATP site, and elicit pharmacological responses in immune cells. GCN2 cell-free kinase binding, kinome selectivity, pGCN2, pEIF2α, ATF-4 phosphorylation inhibition assays were performed and confirmed it's on-target efficacy and potency of lead GCN2 inhibitor HCI-1046. In these studies, our lead GCN2 kinase inhibitor HCI-1046 demonstrated potent activity with an IC50 of 36 nM in inhibiting GCN2. GCN2 expression has been detected in PC-3, DU-145, and LNCap cell lines, and HCI-1046 exhibited cellular efficacy with an IC50 of 0.9 to 8 μM range, reduced phosphorylation of GCN2, ATF-4 significantly and its downstream target eIF2α. HCI-1046 inhibited pGCN2 and pEIF2α in human prostate cancer clinical patient samples derived MDSC cultured in amino-acid starved conditions. HCI-1046, in a dose-dependent fashion, restored CD8+ T cell proliferation and function in clinical samples of PC patients. Our preliminary results support the hypothesis that inhibition of GCN2 reinstates anti-tumor immunity and blocks tumor progression in PC cellular models. In vivo PK studies of HCI-1046 in rodent species showed excellent PK properties; 55% oral bioavailability, low clearance, and >5 hours half-life. HCI-1046 is nominated as a pre-clinical agent. Additional 3D cellular efficacy studies, FACS for cellular apoptosis, cell migration, live PC cells including immuno-ELISA, ELISpot experimental results for MDSCs suppressive function of T cells, and restoration by HCI-1046 will be presented at the conference. PC mouse model efficacy studies will also be discussed. Citation Format: Kyle Medley, Zhaoliang Li, Dongqing Yan, Umang Swami, Neeraj Agarwal, Hariprasad Vankayalapati. Targeting GCN2 kinase-driven stress response inactivation by orally available small molecules to restore immune tumor microenvironment in prostate cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB136.

Abstract 3153: Targeting the stress response kinase GCN2 to restore immunity in the tumor microenvironment

Abstract Recent advances in cancer metabolism suggest that targeting amino acid metabolism represents a promising strategy for the development of novel therapeutic agents. Tumor, stromal and myeloid-derived suppressor cells (MDSC) within the tumor microenvironment (TME) create a nutrient-poor environment that inhibit immune function and support tumor growth. GCN2 (general control nonderepressible 2), a stress response kinase, plays a key role in maintaining cellular homeostasis under a wide range of stressors. Phosphorylation of GCN2 (pGCN2) in response to stress leads to inhibition of global protein synthesis and subsequently leads to 1) T cell anergy and apoptosis, 2) enhanced MDSC-dependent immune suppression and 3) tumor cell survival. Collectively, these roles suggest that GCN2 inhibition could have both a direct anticancer and an immune-activating effect. Treating nutrient-deprived T cells in vitro with a RAPT GCN2 inhibitor (RPT-GCN2i) rescued CD4+ and CD8+ T cell proliferation and effector functions. The RPT-GCN2i also reversed T cell suppression mediated by MDSCs derived from healthy donors or cancer patients. Using syngeneic mouse tumor models, we demonstrated that translational induction of activating transcription factor 4 (ATF4) is a strong marker of GCN2 pathway activation in vivo. Oral administration of an RPT-GCN2i exhibited notable drug-target occupancy and potently inhibited GCN2 kinase and ATF4 in the TME. RPT-GCN2i as a single agent and in combination with checkpoint blockade or angiogenesis inhibitor (anti-VEGFR) led to delays in tumor growth rate in various syngeneic tumor models. In addition, GCN2 inhibition redirected MDSC within the TME from a suppressive to inflammatory phenotype through downregulation of Arg1 and iNOS. Our results show that inhibition of GCN2 is an attractive approach for enhancing antitumor immune response and therefore GCN2 is a promising therapeutic target for the treatment of cancer. Citation Format: Lisa Marshall, Buvana Ravishankar, Urvi Kolhatkar, Mengshu Xu, Lavanya Adusumilli, Deepa Pookot, Thant Zaw, Chandru Ramana, Raashi Sreenivasan, Mikhail Zibinsky, Jeffrey Jackson, Grant Shibuya, Paul Leger, Omar Robles, Anqi Ma, Andrew Ng, Anton Shakhmin, Justy Guagua, Scott Jacobson, Steve Wong, Delia Bradford, Tracy L. McGaha, M Teresa Ciudad, James E. Talmadge, Holly C. Britton, George Katibah, Gene Cutler, David Wustrow, Paul D. Kassner, Dirk G. Brockstedt. Targeting the stress response kinase GCN2 to restore immunity in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3153.

Discordant regulation of eIF2 kinase GCN2 and mTORC1 during nutrient stress.

Appropriate regulation of the Integrated stress response (ISR) and mTORC1 signaling are central for cell adaptation to starvation for amino acids. Halofuginone (HF) is a potent inhibitor of aminoacylation of tRNAPro with broad biomedical applications. Here, we show that in addition to translational control directed by activation of the ISR by general control nonderepressible 2 (GCN2), HF increased free amino acids and directed translation of genes involved in protein biogenesis via sustained mTORC1 signaling. Deletion of GCN2 reduced cell survival to HF whereas pharmacological inhibition of mTORC1 afforded protection. HF treatment of mice synchronously activated the GCN2-mediated ISR and mTORC1 in liver whereas Gcn2-null mice allowed greater mTORC1 activation to HF, resulting in liver steatosis and cell death. We conclude that HF causes an amino acid imbalance that uniquely activates both GCN2 and mTORC1. Loss of GCN2 during HF creates a disconnect between metabolic state and need, triggering proteostasis collapse.

Role of indoleamine 2,3-dioxygenase in ischemia-reperfusion injury of renal tubular epithelial cells.

The present study evaluated indoleamine 2,3-dioxygenase 1 (IDO) kinetics and how it affects cell survival during the two distinct phases of ischemia-reperfusion (I-R) injury. Primary renal proximal tubular epithelial cells (RPTECs) were cultured under anoxia or reoxygenation with or without the IDO inhibitor 1-DL-methyltryptophan, the aryl-hydrocarbon receptor (AhR) inhibitor CH223191 or the ferroptosis inhibitor α-tocopherol. Using cell imaging, colorimetric assays, PCR and western blotting, it was demonstrated that IDO was upregulated and induced apoptosis during anoxia. The related molecular pathway entails tryptophan degradation, general control non-derepressible-2 kinase (GCN2K) activation, increased level of phosphorylated eukaryotic translation initiation factor 2α, activating transcription factor (ATF)4, ATF3, C/EBP homologous protein, phosphorylated p53, p53, Bax, death receptor-5 and eventually activated cleaved caspase-3. Reoxygenation also upregulated IDO, which, in this case, induced ferroptosis. The related molecular pathway encompasses kynurenine production, AhR activation, cytochrome p450 enzymes increase, reactive oxygen species generation and eventually ferroptosis. In conclusion, in RPTECs, both anoxia and reoxygenation upregulated IDO, which in turn induced GCN2K-mediated apoptosis and AhR-mediated ferroptosis. Since both phases of I-R injury share IDO upregulation as a common point, its inhibition may prove a useful therapeutic strategy for preventing or attenuating I-R injury.

The effect of anti‑HLA classI antibodies on the immunological properties of human glomerular endothelial cells and theirmodification by mTOR inhibition or GCN2 kinase activation.

In antibody-mediated rejection (ABMR), the graft endothelium is at the forefront of the kidney transplant against the assault from the recipient's humoral immune system, and is a target of the latter. The present study investigated the effect of antibodies against human leukocyte antigen (HLA) class I (anti-HLAI) on the immunological properties of human glomerular endothelial cells. Additionally, the effect of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) inhibitor (everolimus), or the general control nonderepressible 2 kinase (GCN2K) activator (halofuginone) on anti-HLAI antibody-mediated alterations was assessed. Cell integrity was examined, an lactate dehydrogenase (LDH) release assay was performed and cleaved caspase-3 levels were determined. Furthermore, cell proliferation was analyzed by performing a bromodeoxyuridine assay and the cellular proteins involved in signal transduction or immune effector mechanisms were assessed via western blotting. IL-8, monocyte chemoattractive protein-1 (MCP-1), von Willebrand factor (vWF) and transforming growth factor-beta 1 (TGF-β1) were assayed via ELISA. The results revealed that anti-HLAI triggered integrin signaling, activated mTOR and GCN2K, preserved cell integrity and promoted cell proliferation. Additionally, by increasing intercellular adhesion molecule 1 (ICAM-1), HLA-DR, IL-8 and MCP-1 levels, anti-HLAI enhanced the ability of immune cells to interact with endothelial cells thus facilitating graft rejection. Contrarily, by upregulating CD46 and CD59, anti-HLAI rendered the endothelium less vulnerable to complement-mediated injury. Finally, by enhancing vWF and TGF-β1, anti-HLAI may render the endothelium prothrombotic and facilitate fibrosis and graft failure, respectively. According to our results, mTORC1 inhibition and GCN2K activation may prove useful pharmaceutical targets, as they prevent cell proliferation and downregulate ICAM-1, IL-8, MCP-1 and TGF-β1. mTORC1 inhibition also decreases vWF.

Immunosuppressive Amino-Acid Catabolizing Enzymes in Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease that affects the central nervous system. Although the pathogenesis of MS is not yet fully elucidated, several evidences suggest that autoimmune processes mediated by Th1, Th17, and B cells play an important role in the development of the disease. Similar to other cells, immune cells need continuous access to amino acids (AA) in order to maintain basal metabolism and maintain vitality. When immune cells are activated by inflammation or antigenic signals, their demand for AA increases rapidly. Although AA deprivation itself may weaken the immune response under certain conditions, cells also have AA sensitive pathways that can activate intense alterations in cell metabolism based on changes in AA levels. Several data indicate that cells expressing enzymes that can degrade AA can regulate the functions of antigen-presenting cells and lymphocytes, revealing that the AA pathways are essential for controlling the function, and survival of immune cells, as well as immune cell gene expression. Basal AA catabolism may contribute to immune homeostasis and prevent autoimmunity, while increased AA catalytic activity may enhance immune suppression. In addition, there is increasing evidence that some downstream AA metabolites are important biological mediators of autoimmune response regulation. Two of the most important AA that modulate the immune response are L-Tryptophan (Trp) and L-Arginine (Arg). Tryptophan is catabolized through 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) 1 and IDO2 enzymes, while three other enzymes catabolize Arg: inducible nitric oxide synthetase (iNOS), and two arginase isoforms (ARG1, ARG2). Genes encoding IDO, iNOS and ARG are induced by inflammatory cues such as cytokines, a key feature that distinguishes them from enzymes that catabolize other AA. Evidence suggests that AA catabolism is decreased in MS patients and that this decrease has functional consequences, increasing pro-inflammatory cytokines and decreasing Treg cell numbers. These effects are mediated by at least two distinct pathways involving serine/threonine kinases: the general control nonderepressible 2 kinase (GCN2K) pathway; and the mammalian target of rapamycin (mTOR) pathway. Similarly, IDO1-deficient mice showed exacerbation of experimental autoimmune encephalomyelitis (EAE), increased Th1 and Th17 cells, and decreased Treg cells. On the contrary, the administration of downstream Trp metabolite 3-HAA, inhibits Th1/Th17 effector cells and promotes Treg response by up-regulating TGF-β production by dendritic cells, thereby improving EAE. Collectively, these observations stand out the significance of AA catabolism in the regulation of the immune responses in MS patients. The molecules related to these pathways deserve further exploration as potential new therapeutic targets in MS

GZD824 Inhibits GCN2 and Sensitizes Cancer Cells to Amino Acid Starvation Stress.

Eukaryotic initiation factor 2α (eIF2α) kinase general control nonderepressible 2 (GCN2) drives cellular adaptation to amino acid limitation by activating the integrated stress response that induces activating transcription factor 4 (ATF4). Here, we found that a multikinase inhibitor, GZD824, which we identified using a cell-based assay with ATF4 immunostaining, inhibited the GCN2 pathway in cancer cells. Indeed, GZD824 suppressed GCN2 activation, eIF2α phosphorylation, and ATF4 induction during amino acid starvation stress. However, at lower nonsuppressive concentrations, GZD824 paradoxically stimulated eIF2α phosphorylation and ATF4 expression in a GCN2-dependent manner under unstressed conditions. Such dual properties conceivably arose from a direct effect on GCN2, as also observed in a cell-free GCN2 kinase assay and shared by a selective GCN2 inhibitor. Consistent with the GCN2 pathway inhibition, GZD824 sensitized certain cancer cells to amino acid starvation stress similarly to ATF4 knockdown. These results establish GZD824 as a multikinase GCN2 inhibitor and may enhance its utility as a drug under development. SIGNIFICANCE STATEMENT: GZD824, as a direct general control nonderepressible 2 (GCN2) inhibitor, suppresses activation of the integrated stress response during amino acid limitation, whereas it paradoxically stimulates this stress-signaling pathway at lower nonsuppressive concentrations. The pharmacological activity we identify herein will provide the basis for the use of GZD824 to elucidate the regulatory mechanisms of GCN2 and to evaluate the potential of the GCN2-activating transcription factor 4 pathway as a target for cancer therapy.

Activation of General Control Nonderepressible-2 Kinase Ameliorates Glucotoxicity in Human Peritoneal Mesothelial Cells, Preserves Their Integrity, and Prevents Mesothelial to Mesenchymal Transition.

Along with infections, ultrafiltration failure due to the toxicity of glucose-containing peritoneal dialysis (PD) solutions is the Achilles’ heel of PD method. Triggered by the protective effect of general control nonderepressible-2 (GCN-2) kinase activation against high-glucose conditions in other cell types, we evaluated whether the same occurs in human peritoneal mesothelial cells. We activated GCN-2 kinase with halofuginone or tryptophanol, and assessed the impact of this intervention on glucose transporter-1, glucose transporter-3, and sodium-glucose cotransporter-1, glucose influx, reactive oxygen species (ROS), and the events that result in glucotoxicity. These involve the inhibition of glyceraldehyde 3-phosphate dehydrogenase and the diversion of upstream glycolytic products to the aldose pathway (assessed by D-sorbitol), the lipid synthesis pathway (assessed by protein kinase C activity), the hexosamine pathway (determined by O-linked β-N-acetyl glucosamine-modified proteins), and the advanced glycation end products generation pathway (assessed by methylglyoxal). Then, we examined the production of the profibrotic transforming growth factor-β1 (TGF-β1), the pro-inflammatory interleukin-8 (IL-8). Cell apoptosis was assessed by cleaved caspase-3, and mesothelial to mesenchymal transition (MMT) was evaluated by α-smooth muscle actin protein. High-glucose conditions increased glucose transporters, glucose influx, ROS, all the high-glucose-induced harmful pathways, TGF-β1 and IL-8, cell apoptosis, and MMT. Halofuginone and tryptophanol inhibited all of the above high glucose-induced alterations, indicating that activation of GCN-2 kinase ameliorates glucotoxicity in human peritoneal mesothelial cells, preserves their integrity, and prevents MMT. Whether such a strategy could be applied in the clinic to avoid ultrafiltration failure in PD patients remains to be investigated.

Arabidopsis GCN2 kinase contributes to ABA homeostasis and stomatal immunity

General Control Non-derepressible 2 (GCN2) is an evolutionarily conserved serine/threonine kinase that modulates amino acid homeostasis in response to nutrient deprivation in yeast, human and other eukaryotes. However, the GCN2 signaling pathway in plants remains largely unknown. Here, we demonstrate that in Arabidopsis, bacterial infection activates AtGCN2-mediated phosphorylation of eIF2α and promotes TBF1 translational derepression. Consequently, TBF1 regulates a subset of abscisic acid signaling components to modulate pre-invasive immunity. We show that GCN2 fine-tunes abscisic acid accumulation and signaling during both pre-invasive and post-invasive stages of an infection event. Finally, we also demonstrate that AtGCN2 participates in signaling triggered by phytotoxin coronatine secreted by P. syringae. During the preinvasive phase, AtGCN2 regulates stomatal immunity by affecting pathogen-triggered stomatal closure and coronatine-mediated stomatal reopening. Our conclusions support a conserved role of GCN2 in various forms of immune responses across kingdoms, highlighting GCN2’s importance in studies on both plant and mammalian immunology.

Abstract 1104: Targeting the stress response kinase GCN2 to restore immunity in the tumor microenvironment

Abstract The tumor microenvironment (TME) is characterized by deficiencies in oxygen and key nutrients, such as glucose and amino acids, resulting in an overall immune-suppressive environment. Key suppressive cell types in the TME include tumor, stromal and myeloid-derived suppressor cells (MDSC) which create a nutrient-poor environment that supports tumor growth and limits immune surveillance. General control nonderepressible 2 (GCN2), a stress response kinase, plays a key role in sensing and modulating the cellular response to amino acid deprivation. GCN2 activation in T cells triggers the integrated stress response pathway and promotes T cell anergy and apoptosis. We have developed small molecule GCN2 inhibitors (GCN2i) that are highly potent and selective in vitro. Culturing primary mouse or human immune cells under low nutrient conditions activates the GCN2 pathway limiting T cell proliferation and function. Treatment of these nutrient-deprived T cells with GCN2i resulted in rescue of CD8+ T cell proliferation and effector functions. In addition, GCN2 inhibition in MDSC alone fully reversed CD33+MDSC-induced T cell suppression and effector functions. Our GCN2 inhibitors are orally bioavailable with drug like in vivo ADME properties. Our GCN2i is currently being evaluated in vivo, in murine syngeneic tumor models. Our results demonstrate that inhibition of GCN2 is an attractive approach for relieving T cell suppression and promoting effector function, demonstrating GCN2 as a promising therapeutic target for the treatment of cancer. Note: This abstract was not presented at the meeting. Citation Format: Buvana Ravishankar, Lavanya Adusumilli, Deepa Pookot Pookot, Emily Huang, Raashi Sreenivasan, Lisa Marshall, Deepika Kaveri, Oezcan Talay, Silpa Suthram, Svetlana Miakicheva, Abood Okal, Mikhail Zibinsky, Jeffrey Jackson, Grant Shibuya, Paul Leger, Parcharee Tivitmahaisoon, Scott Jacobson, Steve Wong, Angela Wadsworth, Jerick Sanchez, Martin Brovarney, David Chian, Sachie Marubayashi, Aparna Jorapur, Delia Bradford, Christophe Colas, Gene Cutler, Jacob Schwartz, David Wustrow, Paul Kassner, Dirk Brockstedt. Targeting the stress response kinase GCN2 to restore immunity in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1104.