General Control Nonderepressible 2 Activity Research Articles

GCN2 drives diurnal patterns in the hepatic integrated stress response and maintains circadian rhythms in whole body metabolism during amino acid insufficiency.

Disruptions in circadian rhythms are associated with an increased risk of developing metabolic diseases. General control nonderepressible 2 (GCN2), a primary sensor of amino acid insufficiency and activator of the integrated stress response (ISR), has emerged as a conserved regulator of the circadian clock in multiple organisms. The objective of this study was to examine diurnal patterns in hepatic ISR activation in the liver and whole body rhythms in metabolism. We hypothesized that GCN2 activation cues hepatic ISR signaling over a natural 24-h feeding-fasting cycle. To address our objective, wild-type (WT) and whole body Gcn2 knockout (GCN2 KO) mice were housed in metabolic cages and provided free access to either a control or leucine-devoid diet (LeuD) for 8 days in total darkness. On the last day, blood and livers were collected at CT3 (CT = circadian time) and CT15. In livers of WT mice, GCN2 phosphorylation followed a diurnal pattern that was guided by intracellular branched-chain amino acid concentrations (r2 = 0.93). Feeding LeuD to WT mice increased hepatic ISR activation at CT15 only. Diurnal oscillations in hepatic ISR signaling, the hepatic transcriptome including lipid metabolic genes, and triglyceride concentrations were substantially reduced or absent in GCN2 KO mice. Furthermore, mice lacking GCN2 were unable to maintain circadian rhythms in whole body energy expenditure, respiratory exchange ratio, and physical activity when fed LeuD. In conclusion, GCN2 activation functions to maintain diurnal ISR activation in the liver and has a vital role in the mechanisms by which nutrient stress affects whole body metabolism.NEW & NOTEWORTHY This work reveals that the eIF2 kinase GCN2 functions to support diurnal patterns in the hepatic integrated stress response during natural feeding and is necessary to maintain circadian rhythms in energy expenditure, respiratory exchange ratio, and physical activity during amino acid stress.

5 A Phase 1b, Open-Label, Safety, Tolerability, and Efficacy Study of HC-7366 in Combination with Belzutifan (WELIREGTM) in Patients with Advanced or Metastatic Renal Cell Carcinoma, NCT06234605

Abstract Background HC-7366 is a novel, orally administered, highly selective and potent activator of general control nonderepressible 2 (GCN2) kinase, a core regulator of metabolic stress through activation of the integrated stress response (ISR). Activation of GCN2 promotes cell survival, whereas prolonged activation induces apoptosis. GCN2 activation also suppresses general protein synthesis and induces cell cycle arrest, thereby preventing cell growth during nutrient scarcity. Additionally, HC-7366 decreases HIF expression and inhibits glycolysis, oxidative phosphorylation, and TCA cycle function. HC-7366 also inhibits HIF expression in immunosuppressive myeloid cells, including macrophages. These effects of HC-7366 on metabolism, HIF signaling, and immune suppression suggest therapeutic benefit in ccRCC with clear rationale for combinations with HIF2a antagonists and immune checkpoint inhibitors. HC-7366 (0.5-1 mg/kg), combined with belzutifan (1 mg/kg), exhibited combination benefit in HIF-2 dependent A-498 and 786-O RCC xenografts, yielding 90% tumor growth inhibition and a three-fold increase in complete responses, respectively. Additionally, HC-7366 drives significant monotherapy antitumor activity in PDX models that demonstrated belzutifan resistance. Mechanism of action studies have identified several pathway engagement and potential efficacy biomarkers (Stokes, et al., 2024). Trial design/schema This multicenter, open-label phase 1b study will identify the Maximum Tolerated Dose (MTD) and/or the Recommended Phase 2 Dose (RP2D) of HC-7366 in combination with fixed-dose belzutifan, 120 mg po qd, in patients with advanced or metastatic RCC with renal cell histology irrespective of VHL gene mutation. Monotherapy HC-7366 will be evaluated in parallel. The monotherapy arm, HC-7366, 60 mg p.o. qd, includes patients who have relapsed after 1 to 4 prior lines of standard of care that may include belzutifan or another HIF-2α inhibitor. The combination arm includes patients who have received 1 to 3 prior lines of standard of care and are belzutifan or HIF-2α naïve. HC-7366 dose escalation arm evaluates combination fixed dose belzutifan, 120 mg po qd plus HC-7366 at 20, 40, or 60 mg po qd. Enrollment will start with the HC-7366 monotherapy arm. Subsequently, patients will begin to enroll in the combination therapy HC-7366 dose escalation arm, while the monotherapy arm will continue to enroll. Expansion will evaluate the combination of fixed dose belzutifan with two doses of HC-7366 selected from escalation. Assessments include safety, PK, and anti-tumor activity. The study will enroll up to 80 patients at US study sites. Significance and vision Combination HC-7366 plus belzutifan, supported by preclinical evidence, is being studied to assess antitumor activity in the RCC relapsed setting. Determining safety and evaluation of dose are foundational to this study. Reference Stokes M, Tameire F, Wojnarowicz P, et al. HC-7366, a potent GCN2 activator, complements belzutifan, a HIF-2⍺ antagonist, by providing combination benefit in belzutifan-sensitive models and monotherapy activity in belzutifan-resistant models. Meeting of the American Association for Cancer Research; 2024 Apr 5-10; San Diego (CA); AACR; 2024. Abstract 4615. This study is in collaboration with Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA

Downregulation of nutrition sensor GCN2 in macrophages contributes to poor wound healing in diabetes

Poor skin wound healing, which is common in patients with diabetes, is related to imbalanced macrophage polarization. Here, we find that nutrition sensor GCN2 (general control nonderepressible 2) and its downstream are significantly upregulated in human skin wound tissue and mouse skin wound macrophages, but skin wound-related GCN2 expression and activity are significantly downregulated by diabetes and hyperglycemia. Using wound healing models of GCN2-deleted mice, bone marrow chimeric mice, and monocyte-transferred mice, we show that GCN2 deletion in macrophages significantly delays skin wound healing compared with wild-type mice by altering M1 and M2a/M2c polarization. Mechanistically, GCN2 inhibits M1 macrophages via OXPHOS-ROS-NF-κB pathway and promotes tissue-repairing M2a/M2c macrophages through eukaryotic translation initiation factor 2 (eIF2α)-hypoxia-inducible factor 1α (HIF1α)-glycolysis pathway. Importantly, local supplementation of GCN2 activator halofuginone efficiently restores wound healing in diabetic mice with re-balancing M1 and M2a/2c polarization. Thus, the decreased macrophage GCN2 expression and activity contribute to poor wound healing in diabetes and targeting GCN2 improves wound healing in diabetes.

Activation of GCN2 By HC-7366 Results in Significant Anti-Tumor Efficacy As Monotherapy and Overcomes Resistance Mechanisms When Combined with Venetoclax in AML

Venetoclax (anti-BCL2) is approved for elderly patients with acute myeloid leukemia (AML) in combination with hypomethylating agents such as azacitidine or decitabine. However, several resistance mechanisms arise, impeding responses in patients. These resistance mechanisms include TP53 or FLT3-ITD mutations, suppression of pro-apoptotic proteins NOXA or PUMA, increases in oxidative phosphorylation and energy metabolism, or elevated expression of S100A8/A9. Recent reports have highlighted that hyperactivation of the integrated stress response (ISR) pathway overcomes venetoclax resistance through NOXA activation and glycolytic activity inhibition. However, these studies used drugs that indirectly activate the ISR with other off-target effects. General Control Nonderepressible 2 (GCN2) is an ISR kinase that senses and responds to nutrient stress conditions, and activation of GCN2 results in antitumor activity. We are developing HC-7366, a first-in-class, first-in-human direct GCN2 activator, and are currently evaluating this agent in a phase 1 clinical trial in solid tumors (NCT05121948). Here we present a series of studies characterizing the antitumor effects of HC-7366 in AML, and how it impacts venetoclax resistance. AML patients with TP53 mutations are known to have lower response rates to venetoclax.The in vivo efficacy of HC-7366 in both CDX and PDX models was independent of TP53 mutation, resulting in 100% complete response and 100% tumor growth inhibition in TP53-mutated MOLM-16 and KG-1 tumor models, respectively. Analysis of tumors from treated mice by IHC demonstrated activation of the ISR as evidenced by increased expression of the ATF4 targets ASNS and PSAT1. A genome-wide CRISPR screen in KG-1 cells revealed that GCN2 sgRNAs were significantly enriched in HC-7366-treated cells, highlighting the importance of GCN2 activation in mediating the mechanism of action of HC-7366. Interestingly, BCL2 sgRNAs were significantly depleted in HC-7366 treated cells, suggesting a synthetic lethal relationship with BCL2 inhibitors, such as venetoclax, when combined with HC-7366. Indeed, in the MV4-11 FLT3-ITD mutant model (a differentiated subtype of AML that shows limited response to venetoclax), the combination of HC-7366 and venetoclax produced substantial benefit resulting in 26% tumor regression and enhanced activation of the ISR pathway. HC-7366 treatment also increased mRNA and protein levels of NOXA and PUMA while reducing mitochondrial respiration and glycolysis in a GCN2-dependent manner, leading to a low energetic state. Consistent with its impact on cellular energetics, global metabolomic analyses of AML xenograft tumors showed that HC-7366 significantly altered metabolites associated with glycolysis and the TCA cycle. Additionally, HC-7366 reduced protein levels of S100A8/A9, also known to correlate with venetoclax resistance. Our in vitro and in vivo results demonstrate that HC-7366 is a potent GCN2 activator with strong antitumor activity in AML as a single agent and in combination with venetoclax. The potential of HC-7366 to counteract multiple known resistance mechanisms to venetoclax could provide a viable treatment option for patients with relapsed/refractory AML when used in combination with venetoclax.

GCN2 is required to maintain core body temperature in mice during acute cold.

Nonshivering thermogenesis in rodents requires macronutrients to fuel the generation of heat during hypothermic conditions. In this study, we examined the role of the nutrient sensing kinase, general control nonderepressible 2 (GCN2) in directing adaptive thermogenesis during acute cold exposure in mice. We hypothesized that GCN2 is required for adaptation to acute cold stress via activation of the integrated stress response (ISR) resulting in liver production of FGF21 and increased amino acid transport to support nonshivering thermogenesis. In alignment with our hypothesis, female and male mice lacking GCN2 failed to adequately increase energy expenditure and veered into torpor. Mice administered a small molecule inhibitor of GCN2 were also profoundly intolerant to acute cold stress. Gcn2 deletion also impeded liver-derived FGF21 but in males only. Within the brown adipose tissue (BAT), acute cold exposure increased ISR activation and its transcriptional execution in males and females. RNA sequencing in BAT identified transcripts that encode actomyosin mechanics and transmembrane transport as requiring GCN2 during cold exposure. These transcripts included class II myosin heavy chain and amino acid transporters, critical for maximal thermogenesis during cold stress. Importantly, Gcn2 deletion corresponded with higher circulating amino acids and lower intracellular amino acids in the BAT during cold stress. In conclusion, we identify a sex-independent role for GCN2 activation to support adaptive thermogenesis via uptake of amino acids into brown adipose.NEW & NOTEWORTHY This paper details the discovery that GCN2 activation is required in both male and female mice to maintain core body temperature during acute cold exposure. The results point to a novel role for GCN2 in supporting adaptive thermogenesis via amino acid transport and actomyosin mechanics in brown adipose tissue.

Activating mutations in EGFR and PI3K promote ATF4 induction for NSCLC cell survival during amino acid deprivation

Some oncoproteins along with stress kinase general control non-derepressible 2 (GCN2) can ensure the induction of activating transcription factor 4 (ATF4) to counteract amino acid deprivation; however, little is known regarding the role of the oncogenic EGFR-PI3K pathway. In this study, we demonstrate that both mutated EGFR and PIK3CA contribute to ATF4 induction following GCN2 activation in NSCLC cells. The inhibition of EGFR or PI3K mutant proteins, pharmacologically or through genetic knockdown, inhibited ATF4 induction without affecting GCN2 activation. A downstream analysis revealed that the oncogenic EGFR-PI3K pathway may utilize mTOR-mediated translation control mechanisms for ATF4 induction. Furthermore, in NSCLC cells harboring co-mutations in EGFR and PIK3CA, the combined inhibition of these oncoproteins markedly suppressed ATF4 induction and the subsequent gene expression program as well as cell viability during amino acid deprivation. Our findings establish a role for the oncogenic EGFR-PI3K pathway in the adaptive stress response and provide a strategy to improve EGFR-targeted NSCLC therapy.

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.

Environmental cadmium exposure during gestation impairs fetal brain and cognitive function of adult offspring via reducing placenta-derived E2 level

Early-life exposure to environmental cadmium (Cd) is known to cause developmental disorders, yet the effect and mechanism of gestational exposure to Cd on the offspring's cognitive function remains unclear. Placenta as a well-established target organ for Cd-impaired fetal development, its role in estrogen regulation and offspring cognitive function is unknown. Our in vivo experiments found that gestational Cd exposure impaired cognitive function in adult male offspring, accompanied with lowered 17β-estradiol (E2) level in the male fetal brain upon Cd exposure. Correspondingly, the expression of synapse-associated proteins including brain-derived neurotrophic factor (BDNF), post-synaptic density protein 95 (PSD95) and synapsin-1 were downregulated, which were reversed when supplemented with E2 hormone during gestation. Further observation showed placental estrogen synthesis inhibition and general control non-derepressible 2 (GCN2) signaling activation upon Cd exposure, whereas placental estrogen synthesis could be restored through inhibiting GCN2 activity. Based on ovariectomy (OVX) of pregnant mice, we confirmed that Cd exposure reduced E2 level in fetal brain via inhibiting placenta-derived estrogen synthesis. The aforementioned Cd-induced fetal brain injury and cognitive impairment in adult offspring were significantly alleviated when pregnant dams were supplemented with anti-stress agent N-Acetyl-l-cysteine. In summary, Cd disrupted placenta-derived estrogen synthesis via activating GCN2 signaling, and thereby caused cognitive impairment in adult offspring mice. Our findings suggest that placenta-derived estrogen may be an effect marker of environmental toxicants-evoked cognitive dysfunction in adult offspring and suggest that environmental toxicants may affect the fetal brain development via placenta-fetal-brain axis.

Ethylene modulates translation dynamics in Arabidopsis under submergence via GCN2 and EIN2.

General translational repression is a key process that reduces energy consumption under hypoxia. Here, we show that plant stress-activated general control nonderepressible 2 (GCN2) was activated to regulate the reduction in polysome loading during submergence in Arabidopsis. GCN2 signaling was activated by ethylene under submergence. GCN2 activity was reduced in etr1-1, but not in ein2-5 or eil1ein3, under submergence, suggesting that GCN2 activity is regulated by a noncanonical ethylene signaling pathway. Polysome loading was not reduced in ein2-5 under submergence, implying that ethylene modulates translation via both EIN2 and GCN2. Transcriptomic analysis demonstrated that EIN2 and GCN2 regulate not only general translational repression but also translational enhancement of specific mRNAs under submergence. Together, these results demonstrate that during submergence, entrapped ethylene triggers GCN2 and EIN2 to regulate translation dynamics and ensure the translation of stress response proteins.

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.

Disrupting ATF4 Expression Mechanisms Provides an Effective Strategy for BRAF-Targeted Melanoma Therapy.

SummaryBRAF V600 mutation influences cellular signaling pathways for melanoma development. However, the role of oncogenic BRAF in adaptive stress response pathways is not fully understood. Here, we show that oncogenic BRAF plays an essential role in the induction of ATF4 following the activation of general control non-derepressible 2 (GCN2) kinase during nutrient stress and BRAF-targeted, therapeutic stress. Under GCN2 activation, BRAF ensures ATF4 induction by utilizing mTOR and eIF4B as downstream regulators. In contrast to the MEK-ERK pathway, this signaling pathway remains temporarily active even during treatment with BRAF inhibitors, thereby enabling the transient induction of ATF4. We also identify a chemical compound that prevents BRAF inhibitor-induced activation of the GCN2-ATF4 pathway and produces synergistic cell killing with BRAF inhibitors. Our findings establish a collaborative relationship between oncogenic BRAF and the GCN2-ATF4 signaling pathway, which may provide a novel therapeutic approach to target the adaptive stress response.

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.

The amino acid sensor general control nonderepressible 2 (GCN2) controls TH9 cells and allergic airway inflammation

TH9 cells have emerged as important mediators of allergic airway inflammation. There is evidence that general control nonderepressible 2 (GCN2) affects the immune response under some stress conditions. However, whether GCN2 regulates CD4+ T-cell differentiation during allergic inflammation remains unknown. We sought to clarify the regulatory roles of GCN2 in CD4+ T-cell subset differentiation and its significance in patients with allergic airway inflammation. The effects of GCN2 in differentiation of TH cell subsets were detected by using the invitro induction system. GCN2 knockout mice, ovalbumin-induced allergic airway inflammation, and adoptive transfer mouse models were used to determine the significance of GCN2 in TH9 differentiation and allergic airway inflammation invivo. RNA sequencing, real-time PCR, Western blotting, and other molecular approaches were used to identify the molecular mechanisms relevant to regulation of GCN2 in TH9 cell differentiation. GCN2 deficiency significantly inhibited differentiation of TH9 cells but not TH1, TH2, and regulatory T cells. GCN2 knockout mice and recombination-activating gene 2 knockout (Rag2KO) mice that received adoptively transferred GCN2-deficient CD4+ T cells exhibited reduced TH9 differentiation and less severe allergic airway inflammation. Furthermore, the isolated GCN2-deficient TH9 cells also mediated less severe allergic airway inflammation on adoptive transfer. Mechanistically, GCN2 deficiency inhibits TH9 cell differentiation through a hypoxia-inducible factor 1α-dependent glycolytic pathway. Our results reveal a novel role of GCN2 in TH9 cell differentiation. Our findings indicate that new strategies to inhibit GCN2 activity might provide novel approaches to attenuate allergic airway inflammation.

Activation of GCN2 by the ribosomal P-stalk

Cells dynamically adjust their protein translation profile to maintain homeostasis in changing environments. During nutrient stress, the kinase general control nonderepressible 2 (GCN2) phosphorylates translation initiation factor eIF2α, initiating the integrated stress response (ISR). To examine the mechanism of GCN2 activation, we have reconstituted this process in vitro, using purified components. We find that recombinant human GCN2 is potently stimulated by ribosomes and, to a lesser extent, by tRNA. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) mapped GCN2-ribosome interactions to domain II of the uL10 subunit of the ribosomal P-stalk. Using recombinant, purified P-stalk, we showed that this domain of uL10 is the principal component of binding to GCN2; however, the conserved 14-residue C-terminal tails (CTTs) in the P1 and P2 P-stalk proteins are also essential for GCN2 activation. The HisRS-like and kinase domains of GCN2 show conformational changes upon binding recombinant P-stalk complex. Given that the ribosomal P-stalk stimulates the GTPase activity of elongation factors during translation, we propose that the P-stalk could link GCN2 activation to translational stress, leading to initiation of ISR.

GCN2 deficiency ameliorates cardiac dysfunction in diabetic mice by reducing lipotoxicity and oxidative stress

Excessive myocardial lipid accumulation is a major feature of diabetic cardiomyopathy (DCM). Although general control nonderepressible 2 (GCN2) has been identified as a sensor of amino acid availability, it also functions as an important regulator of hepatic lipid metabolism. Our previous studies have reported that GCN2 promotes pressure overload or doxorubicin-induced cardiac dysfunction by increasing cardiomyocyte apoptosis and myocardial oxidative stress. However, the impact of GCN2 on the development of DCM remains unclear. In this study, we investigated the effect of GCN2 on DCM in type 1 and type 2 diabetes animal models. After streptozotocin (STZ) or high-fat diet (HFD) plus low-dose STZ treatments, GCN2-/- mice developed less cardiac dysfunction, hyperlipidemia, myocardial hypertrophy, fibrosis, lipid accumulation, oxidative stress, inflammation and apoptosis compared with wild-type (WT) mice. In diabetic hearts, GCN2 deficiency attenuated the upregulation of peroxisome proliferator-activated receptor alpha (PPARα) and gamma (PPARγ), the phosphorylation of eIF2α and the induction of activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), as well as the reduction of Bcl-2. Furthermore, we found that knockdown of GCN2 attenuated, whereas overexpression of GCN2 exacerbated, high glucose or palmitic acid-induced cell death, oxidative and endoplasmic reticulum stress and lipid accumulation in H9C2 cells. Collectively, our data provide evidence that GCN2 deficiency protects cardiac function by reducing lipid accumulation, oxidative stress and cell death. Our findings suggest that strategies to inhibit GCN2 activity in the heart may be novel approaches for DCM therapy.

GCN2 deficiency protects against high fat diet induced hepatic steatosis and insulin resistance in mice

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid deposition and oxidative stress. It has been demonstrated that general control nonderepressible 2 (GCN2) is required to maintain hepatic fatty acid homeostasis under conditions of amino acid deprivation. However, the impact of GCN2 on the development of NAFLD has not been investigated. In this study, we used Gcn2−/− mice to investigate the effect of GCN2 on high fat diet (HFD)-induced hepatic steatosis. After HFD feeding for 12 weeks, Gcn2−/− mice were less obese than wild-type (WT) mice, and Gcn2−/− significantly attenuated HFD-induced liver dysfunction, hepatic steatosis and insulin resistance. In the livers of the HFD-fed mice, GCN2 deficiency resulted in higher levels of lipolysis genes, lower expression of genes related to FA synthesis, transport and lipogenesis, and less induction of oxidative stress. Furthermore, we found that knockdown of GCN2 attenuated, whereas overexpression of GCN2 exacerbated, palmitic acid-induced steatosis, oxidative & ER stress, and changes of peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FAS) and metallothionein (MT) expression in HepG2 cells. Collectively, our data provide evidences that GCN2 deficiency protects against HFD-induced hepatic steatosis by inhibiting lipogenesis and reducing oxidative stress. Our findings suggest that strategies to inhibit GCN2 activity in the liver may provide a novel approach to attenuate NAFLD development.

GCN2 deficiency ameliorates doxorubicin-induced cardiotoxicity by decreasing cardiomyocyte apoptosis and myocardial oxidative stress

The clinical use of doxorubicin for cancer therapy is limited by its cardiotoxicity, which involves cardiomyocyte apoptosis and oxidative stress. Previously, we showed that general control nonderepressible 2 (GCN2), an eukaryotic initiation factor 2α (eIF2α) kinase, impairs the ventricular adaptation to chronic pressure overload by affecting cardiomyocyte apoptosis. However, the impact of GCN2 on Dox-induced cardiotoxicity has not been investigated. In the present study, we treated wild type (WT) and Gcn2−/− mice with four intraperitoneal injections (5 mg/kg/week) to induce cardiomyopathy. After Dox treatment, Gcn2−/− mice developed less contractile dysfunction, myocardial fibrosis, apoptosis, and oxidative stress compared with WT mice. In the hearts of the Dox-treated mice, GCN2 deficiency attenuated eIF2α phosphorylation and induction of its downstream targets, activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), and preserved the expression of anti-apoptotic factor Bcl-2 and mitochondrial uncoupling protein-2(UCP2). Furthermore, we found that GCN2 knockdown attenuated, whereas GCN2 overexpression exacerbated, Dox-induced cell death, oxidative stress and reduction of Bcl-2 and UCP2 expression through the eIF2α-CHOP-dependent pathway in H9C2 cells. Collectively, our data provide solid evidence that GCN2 has a marked effect on Dox induced myocardial apoptosis and oxidative stress. Our findings suggest that strategies to inhibit GCN2 activity in cardiomyocyte may provide a novel approach to attenuate Dox-related cardiotoxicity.

Triazolo[4,5-d]pyrimidines as Validated General Control Nonderepressible 2 (GCN2) Protein Kinase Inhibitors Reduce Growth of Leukemia Cells

Cellular stress signals activate adaptive signaling pathways of the mammalian integrated stress response (ISR), of which the unfolded protein response (UPR) is a subset. These pathways converge at the phosporylation of eIF2α. Drug-like, potent and selective chemical inhibitors (valid chemical probes) targeting major ISR kinases have been previously identified, with the exception of GCN2. We synthesized and evaluated a series of GCN2 inhibitors based on a triazolo[4,5-d]pyrimidine scaffold. Several compounds potently inhibited GCN2 in vitro and displayed good selectivity over the related kinases PERK, HRI, and IRE1. The compounds inhibited phosporylation of eIF2α in HEK293T cells with an IC50 < 150 nM, validating them as chemical probes for cellular studies. These probes were screened against the National Cancer Institute NCI-60 human cancer cell line panel. Uniform growth inhibition was observed in the leukemia group of cell lines. Growth inhibition in the most sensitive cell lines coincided with high GCN2 mRNA expression levels. Oncomine analysis revealed high GCN2 expression accompanied by lower asparagine synthetase (ASNS) expression in patient-derived acute lymphoblastic leukemias with B-Cell origins (B-ALL) as well. Notably, asparaginase, which depletes amino acids and triggers GCN2 activity, is a licensed, first-line B-ALL treatment. Thus, we hypothesize that leukemias exhibiting high GCN2 expression and low ASNS expression may be susceptible to pharmacologic GCN2 inhibition.

The stress kinase GCN2 does not mediate suppression of antitumor T cell responses by tryptophan catabolism in experimental melanomas

ABSTRACTTryptophan metabolism is a key process that shapes the immunosuppressive tumor microenvironment. The two rate-limiting enzymes that mediate tryptophan depletion, indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO), have moved into the focus of research and inhibitors targeting IDO and TDO have entered clinical trials. Local tryptophan depletion is generally viewed as the crucial immunosuppressive mechanism. In T cells, the kinase general control non-derepressible 2 (GCN2) has been identified as a molecular sensor of tryptophan deprivation. GCN2 activation by tryptophan depletion induces apoptosis and mitigates T cell proliferation. Here, we investigated whether GCN2 attenuates tumor rejection in experimental B16 melanoma using T cell-specific Gcn2 knockout mice. Our data demonstrate that GCN2 in T cells did not affect immunity to B16 tumors even when animals were treated with antibodies targeting cytotoxic T lymphocyte antigen-4 (CTLA4). GCN2-deficient gp100 TCR-transgenic T cells were equally effective as wild-type pmel T cells against gp100-expressing B16 melanomas after adoptive transfer and gp100 peptide vaccination. Even augmentation of tumoral tryptophan metabolism in B16 tumors by lentiviral overexpression of Tdo did not differentially affect GCN2-proficient vs. GCN2-deficient T cells in vivo. Importantly, GCN2 target genes were not upregulated in tumor-infiltrating T cells. MALDI-TOF MS imaging of B16 melanomas demonstrated maintenance of intratumoral tryptophan levels despite high tryptophan turnover, which prohibits a drop in tryptophan sufficient to activate GCN2 in tumor-infiltrating T cells. In conclusion, our results do not suggest that suppression of antitumor immune responses by tryptophan metabolism is driven by local tryptophan depletion and subsequent GCN2-mediated T cell anergy.

Role of GCN2-Independent Signaling Through a Noncanonical PERK/NRF2 Pathway in the Physiological Responses to Dietary Methionine Restriction.

Restricting availability of essential amino acids (EAAs) limits aminoacylation of tRNAs by their cognate EAAs and activates the nutrient-sensing kinase, general control nonderepressible 2 (GCN2). Activated GCN2 phosphorylates eukaryotic initiation factor 2 (eIF2), altering gene-specific translation and initiating a transcriptional program collectively described as the integrated stress response (ISR). Central GCN2 activation by EAA deprivation is also linked to an acute aversive feeding response. Dietary methionine restriction (MR) produces a well-documented series of physiological responses (increased energy intake and expenditure, decreased adiposity, and increased insulin sensitivity), but the role of GCN2 in mediating them is unknown. Using Gcn2−/− mice, we found that the absence of GCN2 had no effect on the ability of MR to reduce body weight or adiposity, increase energy intake and expenditure, increase hepatic transcription and release of fibroblast growth factor 21, or improve insulin sensitivity. Interestingly, hepatic eIF2 phosphorylation by MR was uncompromised in Gcn2−/− mice. Instead, protein kinase R–like endoplasmic reticulum (ER) kinase (PERK) was activated in both intact and Gcn2−/− mice. PERK activation corresponded with induction of the ISR and the nuclear respiratory factor 2 antioxidant program but not ER stress. These data uncover a novel glutathione-sensing mechanism that functions independently of GCN2 to link dietary MR to its metabolic phenotype.