Activation Of GCN2 Research Articles

Abstract LB234: Activation of the integrated stress response by the developmental HSF1 pathway inhibitor NXP800

Abstract HSF1 is a stress-inducible transcription factor that regulates the eukaryotic heat shock response (HSR). HSF1 activation induces the expression of multiple proteins needed for cellular recovery from stress. HSF1 also plays a key role in tumorigenesis and regulates the expression of a cancer-specific gene signature which is unique to malignant cells and distinct from that activated during the HSR. We discovered NXP800, the first-in-class orally active HSF1 pathway inhibitor which is now undergoing Phase 1 clinical trial, based on a phenotypic pathway screen. Here we employed various techniques to investigate the mechanism of action of NXP800. Using RNAseq in a panel of human carcinoma cell lines, we identified overlapping gene expression changes in response to NXP800. These included genes regulated by HSF1 and interestingly also genes associated with activation of the integrated stress response (ISR). In contrast, we found no evidence for activation of the unfolded protein response. Consistent with the observed NXP800-induced phosphorylation of eIF2α which is a critical regulator of the ISR, NXP800 increased the protein expression of downstream ISR markers ATF4, CHOP and CHAC1, both in human tumor cells in vitro and in human tumor xenograft models in vivo. Induction of the ISR is controlled by four stress-activated protein kinases (PKs) that phosphorylate eIF2α. To further explore these kinases in the mechanism of action of NXP800 we used genetic knockdown by siRNA and inhibition by small-molecule tool compounds. Silencing each of the ISR-regulatory PKs revealed that GCN2 was required for ISR activation by NXP800. This was confirmed using two GCN2 inhibitors from different chemical series. Global phospho-proteome analysis showed that altered protein phosphorylation following NXP800 exposure was reversed upon co-treatment with a GCN2 inhibitor. We also demonstrated that activation of the ISR caused inhibition of HSF1 activation when stimulated with an HSF1 activator, thus confirming the link between ISR induction and inhibition of HSF1 activation. Activation of GCN2 and the ISR can occur in response to a variety of stimuli including amino acid deprivation. However, we did not detect a difference in the uptake of amino acids following exposure to NXP800, indicating that NXP800 does not directly impair amino acid uptake. Using an siRNA approach to determine if activation of the ISR components was contributing to growth inhibition following NXP800 exposure, we found that blocking the induction of ATF4 reduced the response of NXP800-sensitive SK-OV-3 human ovarian carcinoma cells to NXP800 treatment. In summary, NXP800 acts on cancer cells to induce activation of the ISR pathway via GCN2, which then leads to inhibition of HSF1 activation. Further studies are underway to determine the precise molecular target of NXP800 and the mechanism of HSF1 pathway inhibition. Citation Format: Marissa V. Powers, Swee Y. Sharp, Eirini-Maria Lampraki, Toby Roe, Loredana Pellegrino, Maria Taskinen, Robert te Poele, Florence Raynaud, Suzanne Eccles, Matthew Cheeseman, Keith Jones, Paul A. Clarke, Paul Workman. Activation of the integrated stress response by the developmental HSF1 pathway inhibitor NXP800 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB234.

Differential requirements for P stalk components in activating yeast protein kinase Gcn2 by stalled ribosomes during stress

The General Amino Acid Control is a conserved response to amino acid starvation involving activation of protein kinase Gcn2, which phosphorylates eukaryotic initiation factor 2 (eIF2α) with attendant inhibition of global protein synthesis and increased translation of yeast transcriptional activator GCN4. Gcn2 can be activated by either amino acid starvation or conditions that stall elongating ribosomes without reducing aminoacylation of tRNA, but it is unclear whether distinct molecular mechanisms operate in these two circumstances. We identified three regimes that activate Gcn2 in yeast cells by starvation-independent (SI) ribosome-stalling: treatment with tigecycline, eliminating the sole gene encoding tRNAArgUCC, and depletion of translation termination factor eRF1. We further demonstrated requirements for the tRNA- and ribosome-binding domains of Gcn2, the positive effector proteins Gcn1/Gcn20, and the tethering of at least one of two distinct P1/P2 heterodimers to the uL10 subunit of the ribosomal P stalk, for detectable activation by SI-ribosome stalling. Remarkably, no tethered P1/P2 proteins were required for strong Gcn2 activation elicited by starvation for histidine or branched-chain amino acids isoleucine/valine. These results indicate that Gcn2 activation has different requirements for the P stalk depending on how ribosomes are stalled. We propose that accumulation of deacylated tRNAs in amino acid-starved cells can functionally substitute for the P stalk in binding to the histidyl-tRNA synthetase-like domain of Gcn2 for eIF2α kinase activation by ribosomes stalled with A sites devoid of the eEF1A∙GTP∙aminoacyl-tRNA ternary complex.

Abstract 6231: Activation of GCN2 by HC-7366 results in significant antitumor efficacy as monotherapy and in combination with multiple standard of care agents in various solid cancer models

Abstract The integrated stress response (ISR) is an adaptive signaling pathway that cells utilize to respond to a wide range of extrinsic and intrinsic stresses, which are important for tumorigenesis. Activation of ISR is suggested to play a dual role in cell fate decisions. While the ISR promotes survival, prolonged activation of ISR induces apoptosis. We are developing HC-7366, a first-in-class, first-in-human GCN2 activator, and are currently evaluating it in a phase 1 clinical trial in solid tumors (NCT05121948). In this study, we present the characterization of the antitumor effects of HC-7366 in solid tumors. In vivo efficacy studies using HC-7366 montherapy showed significant tumor growth inhibition (TGI%) in preclinical cancer models of colorectal (78-95%), head and neck (33% regression), sarcoma (80%) and prostate (65%). HC-7366 activated the ISR in tumors from treated mice as evidenced by induction of the ATF4 target genes ASNS and PSAT1. Additionally, HC-7366 induced the proapoptotic protein PUMA and reduced HIF1⍺ and HIF2⍺ levels. Furthermore, HC-7366 showed significant benefit in colorectal models when combined with DC101 (anti-VEGFR2 antibody), 5-fluorouracil (chemotherapy), alpelisib (PI3Kα inhibitor), or trametinib (MEK1/2 inhibitor). Using GCN2 CRISPR-knockout cells, we confirmed that the HC-7366 mediated reduction of cell growth and induction of ISR markers was dependent on GCN2. We performed multi-omics analyses to further understand the mechanism of action. Metabolomics analysis of tumors treated with HC-7366 revealed that HC-7366 altered several metabolites involved in amino acid metabolism, oxidative stress, the urea cycle, and pyrimidine biosynthesis. Additionally, proteomics analysis showed that HC-7366 significantly reduced proteins involved in oxidative phosphorylation. Analysis of the transcriptome in tumors from treated mice demonstrated that HC-7366 reduced the activity of HIF and E2F1-driven transcription, including expression of metaphase-anaphase transition genes, consistent with decreased Ki67 staining in tumors. ATF4 and JUN transcriptional activity was enhanced with HC-7366 treatment consistent with activation of ISR. Collectively, our in vitro and in vivo results demonstrate that HC-7366 is a potent GCN2 activator with strong antitumor activity across multiple solid tumor models as a monotherapy or in combination with standard of care agents. Citation Format: Feven Tameire, Paulina Wojnarowicz, Crissy Dudgeon, Sho Fujisawa, Sharon Huang, Owen B. Reilly, Nicholas Collette, Jeremy Drees, Kathryn Bieging-Rolett, Takashi O. Kangas, Weiyu Zhang, Maria Fumagalli, Iman Dewji, Yunfang Li, Anissa SH Chan, Xiaohong Qiu, Ben Harrison, Ashley LaCayo, Ricardo A. Cordova, Kirk A. Staschke, Alan C. Rigby, Savithri Ramurthy, Eric S. Lightcap, David Surguladze, Nandita Bose. Activation of GCN2 by HC-7366 results in significant antitumor efficacy as monotherapy and in combination with multiple standard of care agents in various solid cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6231.

Abstract 1639: DP-9149, an investigational small molecule modulator of the Integrated Stress Response kinase GCN2, pre-clinically causes solid tumor growth inhibition as a single agent and regression in combination with standard of care agents

Abstract Background: The Integrated Stress Response (ISR) is one of the major adaptive stress response pathways in cancer and plays an important role in cell fate determination. Oncogene addicted solid tumors are under high stress levels, both extrinsic as well as intrinsic, and are dependent on a well-balanced ISR pathway activity to cope with the high demand for accelerated growth. The ISR is well known to be a double edge sword of survival and cell death and depending on context, the activation of the ISR kinase, GCN2, and downstream pathway can have either cytoprotective or cytotoxic effects. Given the context-dependent nature of the ISR pathway, the inhibition or stimulation of GCN2 in solid tumors can be pharmacologically leveraged to induce anti-tumoral effects. Methods: Modulation of ISR kinases was characterized using enzymatic assays. Kinome selectivity profiling was determined using enzymatic and cellular assays. Cellular modulation of the ISR pathway (phospho-GCN2, ATF4, CHOP) or the apoptosis pathway (PARP and Caspase3/7) was assessed via Western blot or ELISA. In vivo upregulation of tumoral ATF4 was determined in a fibrosarcoma PK/PD xenograft model. In vivo inhibition of tumor growth was determined in solid tumor xenografts. Results: Selective and potent modulators of GCN2 kinase with favorable drug-like properties were designed. These compounds were found to upregulate components of the ISR pathway (phospho-GCN2, ATF4, CHOP). The mechanism by which GCN2 modulator DP-9149 treatment induced the ISR pathway was found to be through the direct binding and activation of GCN2. Upregulation of the ISR pathway downstream of GCN2 led to induction of a programmed cell death pathway in oncogene-driven solid tumor cell lines in vitro. DP-9149-mediated activation of the ISR pathway led to cell growth arrest both as a single agent and in combination with standard-of-care (SOC) agents. Furthermore, oral dosing of DP-9149 in RAS mutant and other oncogene-driven xenograft models in vivo induced ATF4, and significantly inhibited tumor growth as a single agent and in combination with SOC agents. Additionally, therapeutic agents targeting the tumor microenvironment, including anti-angiogenic agents, synergized with DP-9149 to induce tumor regressions in vivo. Conclusions: The ISR is a targetable vulnerability in oncogene addicted solid tumors. Upregulating the ISR by paradoxical activation of the ISR family member kinase, GCN2, by DP-9149 can be leveraged as a novel mechanism to cause anti-tumoral effects in solid tumors in vitro and in vivo, likely through the induction of an unresolved stress response. In particular, DP-9149 exhibited robust activity in RAS mutant cancers and in VHL-mutant renal cancers as a single agent and in combination with SOC agents in vivo. Citation Format: Gada Al-Ani, Qi Groer, Kristin M. Elliott, Aaron J. Rudeen, Patrick C. Kearney, Jeffery D. Zwicker, Yu Mi Ahn, Stacie L. Bulfer, Cale L. Heiniger, Molly M. Hood, Salim Javid, Joshua W. Large, Max D. Petty, Kristen L. Stoltz, Bertrand Le Bourdonnec, Bryan D. Smith, Daniel L. Flynn. DP-9149, an investigational small molecule modulator of the Integrated Stress Response kinase GCN2, pre-clinically causes solid tumor growth inhibition as a single agent and regression in combination with standard of care agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1639.

Abstract 4818: GCN2 eIF2 kinase and p53 coordinate amino acid homeostasis and metabolism in prostate cancer

Abstract Activation of the integrated stress response (ISR) contributes to the progression of many cancers, including prostate cancer (PCa). The ISR features a family of protein kinases that phosphorylate the eukaryotic translation initiation factor 2 (eIF2) during different stress conditions, resulting in repression of global protein synthesis. In parallel, eIF2 phosphorylation also enhances the translation of select gene transcripts, such as ATF4, which directs the transcription of ISR-target genes that are critical for cancer stress adaptation. We recently reported (Cordova et al., 2022 eLife) that the eIF2 kinase GCN2 is a driver of the ISR in PCa and is critical for the maintenance of essential amino acid (EAA) homeostasis. GCN2 is activated in PCa due to EAA limitations, resulting in increased expression of key amino acid transporters which provide for nutrient import to facilitate protein synthesis and metabolism that drive PCa proliferation. Genetic loss or pharmacological inhibition of GCN2 results in lowered expression of amino acid transporters, leading to severe depletion of intracellular essential amino acids and reduced proliferation in PCa cell lines and xenograft models. These results support the therapeutic potential of targeting GCN2 in PCa.We recently determined that loss of GCN2 in PCa triggers a G1 nutrient-sensitive cell cycle checkpoint that is dependent on p53 and its target gene CDKN1A, encoding p21 inhibitor of cell cycle. Induced G1 arrest and p53/p21 signaling by GCN2 inhibition is reversed by supplementation with EAAs, suggesting amino acid limitation is critical for activation of p53. Metabolic stresses, such as amino acid starvation, have been suggested to activate p53 in different cancers and p53 regulates metabolic pathways that are critical for cancer cells adaptation to stresses. We also showed that depletion of specific amino acids activates GCN2 and p53 in cultured PCa cells. Using transcriptomic and metabolomic analyses, we determined that loss of GCN2 or p53 in PCa cells impacted multiple metabolic pathways, specifically those involved in amino acid and nucleotide metabolism, supporting the importance of GCN2 and p53 in metabolic homeostasis. In addition, deletion of p53 in PCa cells exacerbates the activation of GCN2 and the ISR, suggesting that loss of p53 results in amino acid imbalances that further activate GCN2. Importantly, inhibition of GCN2 in combination with loss of p53 resulted in increased cell death and apoptosis in PCa. Our study suggests that GCN2 and p53 can be activated in parallel in PCa and these stress response pathways are crucial for PCa cells to maintain homeostasis and adapt to metabolic stress. We propose that GCN2 and p53 function to coordinate PCa growth and progression by regulating metabolism and cell cycle control. Targeting these stress pathways in combination may provide enhanced efficacy for the treatment of PCa. Citation Format: Ricardo A. Cordova, Noah R. Sommers, Angela J. Klunk, Haroon M. Mohiuddin, Roberto Pili, Ronald C. Wek, Kirk A. Staschke. GCN2 eIF2 kinase and p53 coordinate amino acid homeostasis and metabolism in prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4818.

Abstract 1615: NMS-812, a novel potent PERK inhibitor that also inhibits GCN2, exhibits strong anti-tumor activity as single agent and in combination in preclinical models

Abstract PERK and GCN2 are eIF-2α serine-threonine kinases activated by endoplasmic reticulum (ER) stress and amino acid deprivation respectively. These kinases are key effectors of the unfolded protein response (UPR) and amino acid Response (AAR) branches of the integrated stress response (ISR). ISR downregulates protein synthesis while upregulating specific genes in response to various cellular stresses, thus allowing adaptation and survival. PERK and GCN2 cooperate to modulate survival under stress conditions in cancer cells, with GCN2 activation compensating when PERK is inhibited, therefore suggesting a certain degree of redundancy between the two. Multiple myeloma (MM) and other protein-secreting tumors are highly dependent on UPR for survival, due to their high protein load. Therefore, PERK inhibition represents a promising anticancer approach in these tumor types. Notably, in these contexts, dual PERK/GCN2 inhibition may be more effective than inhibiting the single kinase.NMS-03597812 is a novel, very potent, ATP-competitive PERK/GCN2 inhibitor, NMS-812 is preferentially active in vitro on multiple myeloma cell lines; activity has however been observed also on lymphoma and selected solid tumor cell lines, suggesting a broader but still specific role of PERK and GCN2 inhibition depending on cell context. In assays revealing the mechanism of action, as expected, a transient dose-dependent eIF2α pathway activation is observed, suggesting the attempt of GCN2 to compensate for PERK inhibition. However, at higher doses, NMS-812 strongly induces pathway downmodulation and apoptosis compared to prototype PERK selective inhibitors. NMS-812 treatment of a MM xenograft model significantly prolongs the median survival time as single agent and demonstrates synergistic activity with MM standard of care (SOC) agents. In lymphoma, NMS-812 shows activity both as single agent and in combination with SOC.NMS-812 shows a favorable in vitro ADME profile and good PK properties with oral bioavailability in preclinical species and an acceptable toxicity profile. NMS-812 is a novel dual, PERK/GCN2 inhibitor with potential for first-in-class, that validates dual PERK and GCN2 inhibition as a therapeutic approach for MM and other PERK/GCN2-dependent settings. Based on these promising results, a Ph1 study has been started in Multiple Myeloma with plans to expand to other malignancies in the near future. Citation Format: Claudia Perrera, Maurizio Pulici, Patrizia Banfi, Nilla Avanzi, Davide Carenzi, Daniele Casero, Antonella Ciavolella, Laura Gianellini, Fabio Gasparri, Grazia Saturno, Barbara Valsasina, Elena Ardini, Alessia Montagnoli, Arturo P. Galvani, Antonella Isacchi. NMS-812, a novel potent PERK inhibitor that also inhibits GCN2, exhibits strong anti-tumor activity as single agent and in combination in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1615.

Activation of Gcn2 by small molecules designed to be inhibitors

The integrated stress response (ISR) is an important mechanism by which cells confer protection against environmental stresses. Central to the ISR is a collection of related protein kinases that monitor stress conditions, such as Gcn2 (EIF2AK4) that recognizes nutrient limitations, inducing phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Gcn2 phosphorylation of eIF2 lowers bulk protein synthesis, conserving energy and nutrients, coincident with preferential translation of stress-adaptive gene transcripts, such as that encoding the Atf4 transcriptional regulator. While Gcn2 is central for cell protection to nutrient stress and its depletion in humans leads to pulmonary disorders, Gcn2 can also contribute to the progression of cancers and facilitate neurological disorders during chronic stress. Consequently, specific ATP-competitive inhibitors of Gcn2 protein kinase have been developed. In this study, we report that one such Gcn2 inhibitor, Gcn2iB, can activate Gcn2, and we probe the mechanism by which this activation occurs. Low concentrations of Gcn2iB increase Gcn2 phosphorylation of eIF2 and enhance Atf4 expression and activity. Of importance, Gcn2iB can activate Gcn2 mutants devoid of functional regulatory domains or with certain kinase domain substitutions derived from Gcn2-deficient human patients. Other ATP-competitive inhibitors can also activate Gcn2, although there are differences in their mechanisms of activation. These results provide a cautionary note about the pharmacodynamics of eIF2 kinase inhibitors in therapeutic applications. Compounds designed to be kinase inhibitors that instead directly activate Gcn2, even loss of function variants, may provide tools to alleviate deficiencies in Gcn2 and other regulators of the ISR.

A genetic approach to identify amino acids in Gcn1 required for Gcn2 activation.

The protein kinase Gcn2 is present in virtually all eukaryotic cells. It is best known for its role in helping cells cope with amino acid starvation. Under starvation, Gcn2 phosphorylates the α subunit of the eukaryotic translation initiation factor 2 (eIF2α), to stimulate a signal transduction pathway that allows cells to cope and overcome starvation. Gcn2 has been implicated in many additional biological functions. It appears that for all functions, Gcn2 must directly bind to its effector protein Gcn1, mediated via a region in Gcn1 called the RWD binding domain (RWDBD). Arg-2259 in this region is important for Gcn2 binding. Overexpression of a Gcn1 fragment only encompassing the RWDBD binds Gcn2, thereby disrupting endogenous Gcn1-Gcn2 interaction which dampens Gcn2 activation. Consequently, cells are unable to increase eIF2α phosphorylation under starvation conditions, visible by impaired growth. This dominant negative phenotype is reverted by the R2259A substitution, again allowing Gcn1-Gcn2 interaction and enhanced eIF2α phosphorylation. We have found that the amino acid substitutions, R2289A, R2297A, and K2301A, also reverted the dominant negative phenotype as well as allowed enhanced eIF2α phosphorylation, as found previously for the R2259A substitution. This suggests that the respective amino acids are relevant for the overexpressed RWDBD to disrupt Gcn1-Gcn2 interaction and impair Gcn2 activation, supporting the idea that in Gcn1 these amino acids mediate Gcn2-binding. Our findings suggest that two helices in Gcn1 constitute a Gcn2 binding site. We serendipitously found amino acid substitutions that enhanced the dominant negative phenotype that correlated with a further reduction in eIF2α-P levels, suggesting that the respective RWDBD variants are more potent in disrupting Gcn1-Gcn2 interaction.

Differential integrated stress response and asparagine production drive symbiosis and therapy resistance of pancreatic adenocarcinoma cells

The pancreatic tumor microenvironment drives deregulated nutrient availability. Accordingly, pancreatic cancer cells require metabolic adaptations to survive and proliferate. Pancreatic cancer subtypes have been characterized by transcriptional and functional differences, with subtypes reported to exist within the same tumor. However, it remains unclear if this diversity extends to metabolic programming. Here, using metabolomic profiling and functional interrogation of metabolic dependencies, we identify two distinct metabolic subclasses among neoplastic populations within individual human and mouse tumors. Furthermore, these populations are poised for metabolic cross-talk, and in examining this, we find an unexpected role for asparagine supporting proliferation during limited respiration. Constitutive GCN2 activation permits ATF4 signaling in one subtype, driving excess asparagine production. Asparagine release provides resistance during impaired respiration, enabling symbiosis. Functionally, availability of exogenous asparagine during limited respiration indirectly supports maintenance of aspartate pools, a rate-limiting biosynthetic precursor. Conversely, depletion of extracellular asparagine with PEG–asparaginase sensitizes tumors to mitochondrial targeting with phenformin.

A cell-based chemical-genetic screen for amino acid stress response inhibitors reveals torins reverse stress kinase GCN2 signaling

mTORC1 and GCN2 are serine/threonine kinases that control how cells adapt to amino acid availability. mTORC1 responds to amino acids to promote translation and cell growth while GCN2 senses limiting amino acids to hinder translation via eIF2α phosphorylation. GCN2 is an appealing target for cancer therapies because malignant cells can harness the GCN2 pathway to temper the rate of translation during rapid amino acid consumption. To isolate new GCN2 inhibitors, we created cell-based, amino acid limitation reporters via genetic manipulation of Ddit3 (encoding the transcription factor CHOP). CHOP is strongly induced by limiting amino acids and in this context, GCN2-dependent. Using leucine starvation as a model for essential amino acid sensing, we unexpectedly discovered ATP-competitive PI3 kinase-related kinase inhibitors, including ATR and mTOR inhibitors like torins, completely reversed GCN2 activation in a time-dependent way. Mechanistically, via inhibiting mTORC1-dependent translation, torins increased intracellular leucine, which was sufficient to reverse GCN2 activation and the downstream integrated stress response including stress-induced transcriptional factor ATF4 expression. Strikingly, we found that general translation inhibitors mirrored the effects of torins. Therefore, we propose that mTOR kinase inhibitors concurrently inhibit different branches of amino acid sensing by a dual mechanism involving direct inhibition of mTOR and indirect suppression of GCN2 that are connected by effects on the translation machinery. Collectively, our results highlight distinct ways of regulating GCN2 activity.

Integrated stress response is involved in the 24(S)-hydroxycholesterol-induced unconventional cell death mechanism

Perturbation of proteostasis triggers the adaptive responses that contribute to the homeostatic pro-survival response, whereas disruption of proteostasis can ultimately lead to cell death. Brain-specific oxysterol—i.e., 24(S)-hydroxycholesterol (24S-OHC)—has been shown to cause cytotoxicity when esterified by acyl-CoA:cholesterol acyltransferase 1 (ACAT1) in the endoplasmic reticulum (ER). Here, we show that the accumulation of 24S-OHC esters caused phosphorylation of eukaryotic translation initiator factor 2α (eIF2α), dissociation of polysomes, and formation of stress granules (SG), resulting in robust downregulation of global protein de novo synthesis in human neuroblastoma SH-SY5Y cells. We also found that integrated stress response (ISR) activation through PERK and GCN2 activation induced by 24S-OHC treatment caused eIF2α phosphorylation. 24S-OHC-inducible SG formation and cell death were suppressed by inhibition of ISR. These results show that ACAT1-mediated 24S-OHC esterification induced ISR and formation of SG, which play crucial roles in 24S-OHC-inducible protein synthesis inhibition and unconventional cell death.

Mitochondrial ROS-mediated ribosome stalling and GCN2 activation are partially involved in 1-nitropyrene-induced steroidogenic inhibition in testes

In the past 50years, testosterone (T) level in men has declined gradually. In this research, we discovered that acute exposure to 1-nitropyrene (1-NP), an environmental stressor from polluted atmosphere, reduced T contents by downregulating steroidogenic proteins in mouse testes and Leydig cells. Acute 1-NP exposure caused GCN2 activation and eIF2α phosphorylation, a marker of integrated stress, in mouse testes and Leydig cells. GCN2iB, a selective GCN2 kinase inhibitor, and siGCN2, the GCN2-targeted short interfering RNA, attenuated 1-NP-induced reduction of steroidogenic proteins in Leydig cells. Mechanistically, mitochondrial membrane potential was reduced and ATP5A, UQCRC2, SDHB and NDUFB8, four OXPHOS subunits, were reduced in 1-NP-exposed Leydig cells. Cellular mitochondrial respiration was inhibited and ATP production was reduced. Moreover, mitochondrial reactive oxygen species (ROS) were elevated in 1-NP-exposed Leydig cells. The interaction between GCN2 and uL10, a marker of ribosome stalling, was observed in 1-NP-exposed Leydig cells. MitoQ, a mitochondria-targeted antioxidant, attenuated1-NP-evoked ATP depletion and ribosome stalling in Leydig cells. Moreover, MitoQ suppressed 1-NP-caused GCN2 activation and eIF2α phosphorylation in Leydig cells. In addition, MitoQ alleviated 1-NP-induced steroidogenic inhibition in mouse testes. In conclusion, mitochondrial ROS-mediated ribosome stalling and GCN2 activation are partially involved in environmental stress-induced steroidogenic inhibition in testes.

Activation of the eIF2α-ATF4 Pathway by Chronic Paracetamol Treatment Is Prevented by Dietary Supplementation with Cysteine.

Chronic treatment with acetaminophen (APAP) induces cysteine (Cys) and glutathione (GSH) deficiency which leads to adverse metabolic effects including muscle atrophy. Mammalian cells respond to essential amino acid deprivation through the phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α). Phosphorylated eIF2α leads to the recruitment of activating transcription factor 4 (ATF4) to specific CCAAT/enhancer-binding protein-ATF response element (CARE) located in the promoters of target genes. Our purpose was to study the activation of the eIF2α-ATF4 pathway in response to APAP-induced Cys deficiency, as well as the potential contribution of the eIF2α kinase GCN2 and the effect of dietary supplementation with Cys. Our results showed that chronic treatment with APAP activated both GCN2 and PERK eIF2α kinases and downstream target genes in the liver. Activation of the eIF2α-ATF4 pathway in skeletal muscle was accompanied by muscle atrophy even in the absence of GCN2. The dietary supplementation with cysteine reversed APAP-induced decreases in plasma-free Cys, liver GSH, muscle mass, and muscle GSH. Our new findings demonstrate that dietary Cys supplementation also reversed the APAP-induced activation of GCN2 and PERK and downstream ATF4-target genes in the liver.

Stress- and metabolic responses of Candida albicans require Tor1 kinase N-terminal HEAT repeats

Whether to commit limited cellular resources toward growth and proliferation, or toward survival and stress responses, is an essential determination made by Target of Rapamycin Complex 1 (TORC1) for a eukaryotic cell in response to favorable or adverse conditions. Loss of TORC1 function is lethal. The TORC1 inhibitor rapamycin that targets the highly conserved Tor kinase domain kills fungal pathogens like Candida albicans, but is also severely toxic to human cells. The least conserved region of fungal and human Tor kinases are the N-terminal HEAT domains. We examined the role of the 8 most N-terminal HEAT repeats of C. albicans Tor1. We compared nutritional- and stress responses of cells that express a message for N-terminally truncated Tor1 from repressible tetO, with cells expressing wild type TOR1 from tetO or from the native promoter. Some but not all stress responses were significantly impaired by loss of Tor1 N-terminal HEAT repeats, including those to oxidative-, cell wall-, and heat stress; in contrast, plasma membrane stress and antifungal agents that disrupt plasma membrane function were tolerated by cells lacking this Tor1 region. Translation was inappropriately upregulated during oxidative stress in cells lacking N-terminal Tor1 HEAT repeats despite simultaneously elevated Gcn2 activity, while activation of the oxidative stress response MAP kinase Hog1 was weak. Conversely, these cells were unable to take advantage of favorable nutritional conditions by accelerating their growth. Consuming oxygen more slowly than cells containing wild type TOR1 alleles during growth in glucose, cells lacking N-terminal Tor1 HEAT repeats additionally were incapable of utilizing non-fermentable carbon sources. They were also hypersensitive to inhibitors of specific complexes within the respiratory electron transport chain, suggesting that inefficient ATP generation and a resulting dearth of nucleotide sugar building blocks for cell wall polysaccharides causes cell wall integrity defects in these mutants. Genome-wide expression analysis of cells lacking N-terminal HEAT repeats showed dysregulation of carbon metabolism, cell wall biosynthetic enzymes, translational machinery biosynthesis, oxidative stress responses, and hyphal- as well as white-opaque cell type-associated genes. Targeting fungal-specific Tor1 N-terminal HEAT repeats with small molecules might selectively abrogate fungal viability, especially when during infection multiple stresses are imposed by the host immune system.

Cytosolic aspartate aminotransferase moonlights as a ribosome-binding modulator of Gcn2 activity during oxidative stress.

Regulation of translation is a fundamental facet of the cellular response to rapidly changing external conditions. Specific RNA-binding proteins (RBPs) co-ordinate the translational regulation of distinct mRNA cohorts during stress. To identify RBPs with previously under-appreciated roles in translational control, we used polysome profiling and mass spectrometry to identify and quantify proteins associated with translating ribosomes in unstressed yeast cells and during oxidative stress and amino acid starvation, which both induce the integrated stress response (ISR). Over 800 proteins were identified across polysome gradient fractions, including ribosomal proteins, translation factors, and many others without previously described translation-related roles, including numerous metabolic enzymes. We identified variations in patterns of PE in both unstressed and stressed cells and identified proteins enriched in heavy polysomes during stress. Genetic screening of polysome-enriched RBPs identified the cytosolic aspartate aminotransferase, Aat2, as a ribosome-associated protein whose deletion conferred growth sensitivity to oxidative stress. Loss of Aat2 caused aberrantly high activation of the ISR via enhanced eIF2α phosphorylation and GCN4 activation. Importantly, non-catalytic AAT2 mutants retained polysome association and did not show heightened stress sensitivity. Aat2 therefore has a separate ribosome-associated translational regulatory or 'moonlighting' function that modulates the ISR independent of its aspartate aminotransferase activity.

Targeting GCN2 Regulation of Amino Acid Homeostasis in Prostate Cancer

The Integrated Stress Response (ISR) plays a critical role in the adaptation and survival of tumor cells to exogenous and endogenous stresses. The ISR features four protein kinases (PERK, GCN2, PKR, and HRI), each activated by different stresses, that phosphorylate the eukaryotic translation initiation factor eIF2, resulting in repression of global protein synthesis. Paradoxically, eIF2 phosphorylation also enhances translation of select gene transcripts, including the transcription factor ATF4, which is central for ISR‐directed gene transcription. Therefore, the ISR directs translational and transcriptional control that is critical for cancer stress adaptation. Moreover, eIF2 phosphorylation and ATF4 have recently been suggested to play a role in prostate cancer (PCa) growth and survival; however, the specific function of ISR kinases, their mode of activation, and the mechanisms by which the ISR facilitate PCa progression are unknown.We discovered that GCN2 is activated in a range of PCa cell lines, contributing to enhanced eIF2 phosphorylation and ATF4 expression. Genetic or pharmacological inhibition of GCN2 reduces growth in androgen‐sensitive and castration‐resistant PCa cell lines in culture and cell line‐derived and patient‐derived xenograft mouse models in vivo. Induction of GCN2 is accompanied by limitations of select amino acids and accumulation of cognate tRNAs that are reported to be activators of GCN2.A transcriptome analysis of PCa cells treated with a specific GCN2 small molecular inhibitor indicates that GCN2 is critical for expression of SLCgenes involved in metabolite transport. We found that GCN2 inhibition decreases intracellular amino acid levels accounting for reduced growth in PCa cells. Using CRISPR‐based phenotypic screens and genome‐wide gene expression analyses of wild‐type and GCN2‐depleted PCa cells, we confirmed the importance of the transporter genes in PCa fitness. One transporter, SLC3A2 (4F2), is induced by GCN2 and is essential for PCa proliferation. SLC3A2 engages with many nutrient transporters, allowing for their localization to the plasma membrane. Importantly, expression of SLC3A2 reduced GCN2 activation and rescued decreased amino acid levels and growth inhibition due to loss of GCN2.Our results indicate that select amino acid limitations activate GCN2 in PCa, resulting in the upregulation of key amino acid transporters, including 4F2 (SLC3A2), which provide for nutrient import to facilitate protein synthesis and metabolism required for PCa progression. We conclude that GCN2 and the ISR are promising therapeutic targets for both androgen‐sensitive and castration‐resistant prostate cancer.

Selected Articles from This Issue

Screening and biomarker identification have improved prostate cancer outcomes, but prostate cancer progression remains a critical challenge. Mitochondria have garnered increasing attention in prostate and other cancers, and metabolic and non-metabolic mitochondrial contributions to cancer merit further investigation. In their study, Furnish and colleagues found that mitochondrial Rho GTPase 2 (MIRO2) expression is upregulated in recurrent and progressive prostate cancer, and that MIRO2 depletion via shRNA abrogates prostate cancer cell growth in vitro and in vivo. The authors identified MIRO2 protein interactors using MIRO2 immunoprecipitation and mass spectrometry and revealed general control nonderepressible (GCN1) is a MIRO2 binding partner. Mechanistically, the authors showed that MIRO2 facilitates GCN1-mediated activation of GCN2, which results in eukaryotic translation initiation factor 2 alpha (eIF2α) phosphorylation and transcriptional upregulation of transcriptional activator 4 (ATF4). Immunohistochemistry data from prostate cancer xenografts implicated HIF1α as an oncogenic MIRO2 regulator. Overall, this work presents a previously unknown mechanism by which mitochondria promote prostate cancer progression and suggests that MIRO2 could be a useful therapeutic target for recurrent and progressive prostate cancer.Few therapeutic options exist for patients with metastatic prostate cancer. Furthermore, longitudinal study challenges and therapeutic effects on tumor biology render elucidating mechanisms that promote prostate cancer metastasis difficult. To address these issues and interrogate molecular drivers of prostate cancer metastasis, Van den Broeck and colleagues performed genomic analysis using samples from patients with localized or locally advanced prostate cancer who participated in longitudinal studies. The authors demonstrated that copy numbers of the gene encoding antizyme inhibitor 1 (AZIN1) are often amplified in samples that progressed to metastasis. Furthermore, abrogating AZIN1 expression using siRNA in prostate cancer cells decreased cell growth and migration in vitro and lung metastasis in vivo. By surveying transcriptomic changes induced by AZIN1 depletion, the authors noted that AZIN1 expression negatively correlates with expression of several collagens that can be processed into matrikines. Accordingly, culture media from AZIN1-deficient and collagen-overexpressing cells both decreased invasion and migration of prostate cancer cells in vitro. Altogether, this study presents a novel molecular facet of prostate cancer metastasis that could inform future therapeutic strategies.The MUC1 C-terminal subunit (MUC1-C) is known to promote cancer stem cell (CSC)-associated dedifferentiation, but whether MUC1-C contributes to dedifferentiation-associated transcription factor transactivation and chromatin remodeling is not clear. In their study, Bhattacharya and colleagues abrogate MUC1-C expression in prostate and breast cancer cells using shRNA and utilize Assay for Transposase-Accessible Chromatin using sequencing (ATAC-Seq) to demonstrate that MUC1-C influences chromatin remodeling in cancer cells. Integrative analysis using ATAC-Seq, RNA-Seq, and ChIP-Seq revealed that genes whose chromatin accessibility is modified by MUC1-C and that are differentially expressed upon MUC1-C depletion are enriched with FOS, JUN, and NEF2 transcription factor binding motifs. The authors went on to demonstrate that MUC1-C interacts with JUN, and that a MUC1-C/JUN/ARID1A complex increases chromatin accessibility and expression of critical CSC genes, including NOTCH1, EGR1, and LY6E, in both prostate and breast cancer cells. Overall, in addition to documenting a previously unrecognized interaction between MUC1-C and JUN, this study furthers the understanding of how MUC1-C contributes to transcription factor transactivation, chromatin remodeling, and CSC biology.Inhibiting DNA double-strand break (DSB) repair augments DNA damage-inducing therapeutic efficacy, but mechanisms underlying this therapeutic potentiation have not been fully elucidated. In their study, Carr and colleagues used DNA-PK inhibitor peposertib and DNA damage-inducing radiation to demonstrate that DNA DSB repair inhibition enhances both radiation-mediated cell death and inflammation. The authors found that peposertib augments radiation-induced chromosomal abnormalities, causing distorted chromosomal segregation and cell death in cancer cells in vitro. Immunofluorescent imaging revealed that cancer cells treated with radiation and peposertib harbor numerous micronuclei that associate with inflammatory cytosolic DNA sensor cGAS. Accordingly, treatment with radiation and peposertib induced expression of inflammatory signaling mediators and cytokines, as well as immunoregulatory programmed cell death ligand 1 (PD-L1). The authors used bintrafusp alfa in vivo to demonstrate that PD-L1 blockade enhances CD8+ T cell recruitment to tumors and therapeutic efficacy of combination radiation–peposertib treatment. Taken together, this study reveals a previously unappreciated mechanism by which DNA DSB repair inhibition augments DNA damage-inducing therapy and demonstrates how that mechanism can be leveraged in therapeutic strategies.

Heme-Regulated eIF2α Kinase (HRI) Inhibition Decreases PKR Activation in HUDEP2 Cells

Introduction: Sickle Cell Disease (SCD) is a group of inherited disorders caused by mutations in the β-globin gene which encodes the hemoglobin subunit β in erythrocytes [1]. Hemoglobin containing the mutant β-globin polymerizes and causes sickling of erythrocytes, which subsequently leads to vaso-occlusion, hemolysis, and activation of the immune system by release of free heme [2]. Heme-regulated eIF2α kinase, also known as heme-regulated inhibitor (HRI/EIF2AK1), the dsRNA-activated kinase Protein Kinase R (PKR/EIF2AK2), PKR-like endoplasmic reticulum kinase (PERK/EIF2AK3) and General Control Non-Depressible 2 (GCN2/EIF2AK4) are four kinases belonging to the eIF2α kinases family and play key functions in the Integrated Stress Response-ATF4 pathway, which is critical for translational control in response to various stress conditions [3]. These kinases are activated respectively by heme depletion, viral infection, endoplasmic reticulum stress, and amino acid starvation, and they phosphorylate eukaryotic initiation factor-2α (eIF2α). Recently, it has been shown that HRI inhibition induces fetal hemoglobin in HUDEP-2 cells and CD34+ hematopoietic progenitor stem cells and prevents sickling, suggesting HRI as a potential therapeutic target for SCD [4, 5]. Moreover, it has been well documented that kinase inhibition can activate compensatory loops (bypass signaling) to circumvent the inhibited target, in particular by overexpression and activation of kinases having the same substrate [6, 7]. Thus, in this study, we investigated if the inhibition of HRI in HUDEP-2 cells lead to compensatory mechanisms by modulation of the expression and activation of the other eIF2α kinases.Methods: To inhibit HRI, we generated HUDEP-2 [8] HRI Knock-Out cells (HRI-KO) and HUDEP-2 HRI Kinase Dead cells (K196R) (clones HRI-KD1 and HRI-KD2) by using CRISPR-Cas9 gene editing technology [9]. We confirmed the induction of fetal hemoglobin for each clone by flow cytometry. To evaluate a potential compensatory mechanism, we measured the effects of HRI inhibition on the expression and activation of the other eIF2α kinases by western blot (WB) and the regulation at the transcriptomic level by qPCR. Based on the results of preliminary studies, we generated HUDEP-2 PKR Knock-Out cells (PKR-KO) by CRISPR-Cas9. We differentiated them for 7 days and we quantified the level of fetal hemoglobin by flow cytometry, AlphaLISA® and WB.Results: HRI-KO, HRI-KD1 and HRI-KD2 clones expressed fetal hemoglobin after 7-day of differentiation consistent with published data [4]. HRI inhibition did not result in any modulation of PKR protein expression, but the activation of PKR, measured by phosphorylation at its residue Threonine 446, was decreased in HRI-KD1, HRI-KD2, and in HRI-KO cells at day 0 and day 7 of differentiation. As PKR and HRI have the same downstream target eIF2α and HRI inhibition induces fetal hemoglobin through eIF2α-ATF4-Bcl11a axis, we verified if fetal hemoglobin induction is due to HRI inhibition exclusively and is not a consequence of the decrease in PKR activation when HRI is inhibited. We measured the protein expression level of fetal hemoglobin in PKR-KO cells and results obtained by flow cytometry, western blot and AlphaLISA® did not show any regulation in fetal hemoglobin in PKR-KO cells after 7 days of differentiation. Finally, HRI inhibition did not result in any regulation of kinases PERK and GCN2 activation and expression, at the RNA and protein level. The expression of these two eIF2α kinases was low compared to HRI and PKR.Conclusion: HRI inhibition does not cause any modulation in the expression and activation of GCN2 and PERK in HUDEP-2 cells but results in a decrease in PKR activation. This outcome could be explained by a possible increase in the expression of proteins that inhibit PKR, such as TRBP or Hsp40, and are induced by HRI silencing (Hsp40) [10-12]. Nonetheless PKR inhibition does not induce fetal hemoglobin in HUDEP-2 cells after 7 days of differentiation. Overall, this study provides evidence that fetal hemoglobin induction by HRI inhibition in HUDEP-2 cells is independent from the other eIF2α kinases and supports HRI as a potential therapeutic target in SCD. However, the biological implications of a potential compensatory effect on PKR signaling in HRI-expressing tissues warrant further investigation. DisclosuresKrishnamoorthy: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company.

The eIF2 kinase GCN2 directs keratinocyte collective cell migration during wound healing via coordination of reactive oxygen species and amino acids

Healing of cutaneous wounds requires the collective migration of epithelial keratinocytes to seal the wound bed from the environment. However, the signaling events that coordinate this collective migration are unclear. In this report, we address the role of phosphorylation of eukaryotic initiation factor 2 (eIF2) and attendant gene expression during wound healing. Wounding of human keratinocyte monolayers in vitro led to the rapid activation of the eIF2 kinase GCN2. We determined that deletion or pharmacological inhibition of GCN2 significantly delayed collective cell migration and wound closure. Global transcriptomic, biochemical, and cellular analyses indicated that GCN2 is necessary for maintenance of intracellular free amino acids, particularly cysteine, as well as coordination of RAC1-GTP-driven reactive oxygen species (ROS) generation, lamellipodia formation, and focal adhesion dynamics following keratinocyte wounding. In vivo experiments using mice deficient for GCN2 validated the role of the eIF2 kinase during wound healing in intact skin. These results indicate that GCN2 is critical for appropriate induction of collective cell migration and plays a critical role in coordinating the re-epithelialization of cutaneous wounds.

Endosomal trafficking and DNA damage checkpoint kinases dictate survival to replication stress by regulating amino acid uptake and protein synthesis.

Atg6Beclin 1 mediates autophagy and endosomal trafficking. We investigated how Atg6 influences replication stress. Combining genetic, genomic, metabolomic, and proteomic approaches, we found that the Vps34-Vps15-Atg6Beclin 1-Vps38UVRAG-phosphatydilinositol-3 phosphate (PtdIns(3)P) axis sensitizes cells to replication stress by favoring the degradation of plasma membrane amino acid (AA) transporters via endosomal trafficking and ESCRT proteins, while the PtdIns(3)P phosphatases Ymr1 and Inp53 promote survival to replication stress by reversing this process. An impaired AA uptake triggers activation of Gcn2, which attenuates protein synthesis by phosphorylating eIF2α. Mec1Atr-Rad53Chk1/Chk2 activation during replication stress further hinders translation efficiency by counteracting eIF2α dephosphorylation through Glc7PP1. AA shortage-induced hyperphosphorylation of eIF2α inhibits the synthesis of 65 stress response proteins, thus resulting in cell sensitization to replication stress, while TORC1 promotes cell survival. Our findings reveal an integrated network mediated by endosomal trafficking, translational control pathways, and checkpoint kinases linking AA availability to the response to replication stress.