IU Indianapolis ScholarWorks :: Browsing by Author "Staschke, Kirk A."

Browsing by Author "Staschke, Kirk A."

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A PERK-Specific Inhibitor Blocks Metastatic Progression by Limiting Integrated Stress Response-Dependent Survival of Quiescent Cancer Cells
(American Association for Cancer Research, 2023) Calvo, Veronica; Zheng, Wei; Adam-Artigues, Anna; Staschke, Kirk A.; Huang, Xin; Cheung, Julie F.; Nobre, Ana Rita; Fujisawa, Sho; Liu, David; Fumagalli, Maria; Surguladze, David; Stokes, Michael E.; Nowacek, Ari; Mulvihill, Mark; Farias, Eduardo F.; Aguirre-Ghiso, Julio A.; Biochemistry and Molecular Biology, School of Medicine
Purpose: The integrated stress response (ISR) kinase PERK serves as a survival factor for both proliferative and dormant cancer cells. We aim to validate PERK inhibition as a new strategy to specifically eliminate solitary disseminated cancer cells (DCC) in secondary sites that eventually reawake and originate metastasis. Experimental design: A novel clinical-grade PERK inhibitor (HC4) was tested in mouse syngeneic and PDX models that present quiescent/dormant DCCs or growth-arrested cancer cells in micro-metastatic lesions that upregulate ISR. Results: HC4 significantly blocks metastasis, by killing quiescent/slow-cycling ISRhigh, but not proliferative ISRlow DCCs. HC4 blocked expansion of established micro-metastasis that contained ISRhigh slow-cycling cells. Single-cell gene expression profiling and imaging revealed that a significant proportion of solitary DCCs in lungs were indeed dormant and displayed an unresolved ER stress as revealed by high expression of a PERK-regulated signature. In human breast cancer metastasis biopsies, GADD34 expression (PERK-regulated gene) and quiescence were positively correlated. HC4 effectively eradicated dormant bone marrow DCCs, which usually persist after rounds of therapies. Importantly, treatment with CDK4/6 inhibitors (to force a quiescent state) followed by HC4 further reduced metastatic burden. In HNSCC and HER2+ cancers HC4 caused cell death in dormant DCCs. In HER2+ tumors, PERK inhibition caused killing by reducing HER2 activity because of sub-optimal HER2 trafficking and phosphorylation in response to EGF. Conclusions: Our data identify PERK as a unique vulnerability in quiescent or slow-cycling ISRhigh DCCs. The use of PERK inhibitors may allow targeting of pre-existing or therapy-induced growth arrested "persister" cells that escape anti-proliferative therapies.
Activation of Gcn2 by Pharmacological Agents Designed to be Inhibitors
(2023-01) Carlson, Kenneth Reed; Wek, Ronald C.; Georgiadis, Millie M.; Liu, Yunlong; Staschke, Kirk A.; Turchi, John J.
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 stressadaptive 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. This thesis reports that one such Gcn2 inhibitor, Gcn2iB, can activate Gcn2, probes the mechanism by which this activation occurs, and compares the mechanism of Gcn2 activation by Gcn2iB to that of uncharged tRNA. In this study, Gcn2 activation was measured in cultured human cells by immunoblot and luciferase reporter assays making use of a genetic complementation assay to assess the contribution of various Gcn2 residues to its activation. 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 fromGcn2-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. However, 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.
Activation of Gcn2 by small molecules designed to be inhibitors
(Elsevier, 2023) Carlson, Kenneth R.; Georgiadis, Millie M.; Tameire, Feven; Staschke, Kirk A.; Wek, Ronald C.; Biochemistry and Molecular Biology, School of Medicine
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.
Activation of the integrated stress response (ISR) pathways in response to Ref-1 inhibition in human pancreatic cancer and its tumor microenvironment
(Frontiers Media, 2023-04-27) Mijit, Mahmut; Boner, Megan; Cordova, Ricardo A.; Gampala, Silpa; Kpenu, Eyram; Klunk, Angela J.; Zhang, Chi; Kelley, Mark R.; Staschke, Kirk A.; Fishel, Melissa L.; Pediatrics, School of Medicine
Pancreatic cancer or pancreatic ductal adenocarcinoma (PDAC) is characterized by a profound inflammatory tumor microenvironment (TME) with high heterogeneity, metastatic propensity, and extreme hypoxia. The integrated stress response (ISR) pathway features a family of protein kinases that phosphorylate eukaryotic initiation factor 2 (eIF2) and regulate translation in response to diverse stress conditions, including hypoxia. We previously demonstrated that eIF2 signaling pathways were profoundly affected in response to Redox factor-1 (Ref-1) knockdown in human PDAC cells. Ref-1 is a dual function enzyme with activities of DNA repair and redox signaling, responds to cellular stress, and regulates survival pathways. The redox function of Ref-1 directly regulates multiple transcription factors including HIF-1α, STAT3, and NF-κB, which are highly active in the PDAC TME. However, the mechanistic details of the crosstalk between Ref-1 redox signaling and activation of ISR pathways are unclear. Following Ref-1 knockdown, induction of ISR was observed under normoxic conditions, while hypoxic conditions were sufficient to activate ISR irrespective of Ref-1 levels. Inhibition of Ref-1 redox activity increased expression of p-eIF2 and ATF4 transcriptional activity in a concentration-dependent manner in multiple human PDAC cell lines, and the effect on eIF2 phosphorylation was PERK-dependent. Treatment with PERK inhibitor, AMG-44 at high concentrations resulted in activation of the alternative ISR kinase, GCN2 and induced levels of p-eIF2 and ATF4 in both tumor cells and cancer-associated fibroblasts (CAFs). Combination treatment with inhibitors of Ref-1 and PERK enhanced cell killing effects in both human pancreatic cancer lines and CAFs in 3D co-culture, but only at high doses of PERK inhibitors. This effect was completely abrogated when Ref-1 inhibitors were used in combination with GCN2 inhibitor, GCN2iB. We demonstrate that targeting of Ref-1 redox signaling activates the ISR in multiple PDAC lines and that this activation of ISR is critical for inhibition of the growth of co-culture spheroids. Combination effects were only observed in physiologically relevant 3D co-cultures, suggesting that the model system utilized can greatly affect the outcome of these targeted agents. Inhibition of Ref-1 signaling induces cell death through ISR signaling pathways, and combination of Ref-1 redox signaling blockade with ISR activation could be a novel therapeutic strategy for PDAC treatment.
Asparagine bioavailability regulates the translation of MYC oncogene
(Springer Nature, 2022) Srivastava, Sankalp; Jiang, Jie; Misra, Jagannath; Seim, Gretchen; Staschke, Kirk A.; Zhong, Minghua; Zhou, Leonardo; Liu, Yu; Chen, Chong; Davé, Utpal; Kapur, Reuben; Batra, Sandeep; Zhang, Chi; Zhou, Jiehao; Fan, Jing; Wek, Ronald C.; Zhang, Ji; Pediatrics, School of Medicine
Amino acid restriction has recently emerged as a compelling strategy to inhibit tumor growth. Recent work suggests that amino acids can regulate cellular signaling in addition to their role as biosynthetic substrates. Using lymphoid cancer cells as a model, we found that asparagine depletion acutely reduces the expression of c-MYC protein without changing its mRNA expression. Furthermore, asparagine depletion inhibits the translation of MYC mRNA without altering the rate of MYC protein degradation. Of interest, the inhibitory effect on MYC mRNA translation during asparagine depletion is not due to the activation of the general controlled nonderepressible 2 (GCN2) pathway and is not a consequence of the inhibition of global protein synthesis. In addition, both the 5' and 3' untranslated regions (UTRs) of MYC mRNA are not required for this inhibitory effect. Finally, using a MYC-driven mouse B cell lymphoma model, we found that shRNA inhibition of asparagine synthetase (ASNS) or pharmacological inhibition of asparagine production can significantly reduce the MYC protein expression and tumor growth when environmental asparagine becomes limiting. Since MYC is a critical oncogene, our results uncover a molecular connection between MYC mRNA translation and asparagine bioavailability and shed light on a potential to target MYC oncogene post-transcriptionally through asparagine restriction.
Effects of PERK eIF2α Kinase Inhibitor against Toxoplasma gondii
(American Society for Microbiology, 2018-10-24) Augusto, Leonardo; Martynowicz, Jennifer; Staschke, Kirk A.; Wek, Ronald C.; Sullivan, William J., Jr.; Biochemistry and Molecular Biology, School of Medicine
Toxoplasma gondii is an obligate intracellular parasite that has infected one-third of the population. Upon infection of warm-blooded vertebrates, the replicating form of the parasite (tachyzoite) converts into a latent form (bradyzoite) present in tissue cysts. During immune deficiency, bradyzoites can reconvert into tachyzoites and cause life-threatening toxoplasmosis. We previously reported that translational control through phosphorylation of the α subunit of T. gondii eukaryotic initiation factor 2 (eIF2α) (TgIF2α) is a critical component of the parasite stress response. Diverse stresses can induce the conversion of tachyzoites to bradyzoites, including those disrupting the parasite's endoplasmic reticulum (ER) (ER stress). Toxoplasma possesses four eIF2α kinases, one of which (TgIF2K-A) localizes to the parasite ER analogously to protein kinase R-like endoplasmic reticulum kinase (PERK), the eIF2α kinase that responds to ER stress in mammalian cells. Here, we investigated the effects of a PERK inhibitor (PERKi) on Toxoplasma Our results show that the PERKi GSK2606414 blocks the enzymatic activity of TgIF2K-A and reduces TgIF2α phosphorylation specifically in response to ER stress. PERKi also significantly impeded multiple steps of the tachyzoite lytic cycle and sharply lowered the frequency of bradyzoite differentiation in vitro Pretreatment of host cells with PERKi prior to infection did not affect parasite infectivity, and PERKi still impaired parasite replication in host cells lacking PERK. In mice, PERKi conferred modest protection from a lethal dose of Toxoplasma Our findings represent the first pharmacological evidence supporting TgIF2K-A as an attractive new target for the treatment of toxoplasmosis.
GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis
(eLife Sciences, 2022-09-15) Cordova, Ricardo A.; Misra, Jagannath; Amin, Parth H.; Klunk, Anglea J.; Damayanti, Nur P.; Carlson, Kenneth R.; Elmendorf, Andrew J.; Kim, Hyeong-Geug; Mirek, Emily T.; Elzey, Bennet D.; Miller, Marcus J.; Dong, X. Charlie; Cheng, Liang; Anthony, Tracy G.; Pili, Roberto; Wek, Ronald C.; Staschke, Kirk A.; Biochemistry and Molecular Biology, School of Medicine
A stress adaptation pathway termed the integrated stress response has been suggested to be active in many cancers including prostate cancer (PCa). Here, we demonstrate that the eIF2 kinase GCN2 is required for sustained growth in androgen-sensitive and castration-resistant models of PCa both in vitro and in vivo, and is active in PCa patient samples. Using RNA-seq transcriptome analysis and a CRISPR-based phenotypic screen, GCN2 was shown to regulate expression of over 60 solute-carrier (SLC) genes, including those involved in amino acid transport and loss of GCN2 function reduces amino acid import and levels. Addition of essential amino acids or expression of 4F2 (SLC3A2) partially restored growth following loss of GCN2, suggesting that GCN2 targeting of SLC transporters is required for amino acid homeostasis needed to sustain tumor growth. A small molecule inhibitor of GCN2 showed robust in vivo efficacy in androgen-sensitive and castration-resistant mouse models of PCa, supporting its therapeutic potential for the treatment of PCa.
GCN2 eIF2 Kinase Promotes Prostate Cancer by Maintaining Amino Acid Homeostasis
(2024-04) Cordova E., Ricardo A.; Wek, Ronald C.; Staschke, Kirk A.; Pili, Roberto; Mosley, Amber L.; Elmendorf, Jeffrey S.; Zhang, Ji
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, repressing 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 critical for stress adaptation. We reported that the eIF2 kinase GCN2 is a critical driver of the ISR in PCa and is crucial to maintaining amino acid (AA) homeostasis. GCN2 is activated in PCa due to AA limitation, resulting in increased expression of key AA transporters which providing nutrient import to fuel protein synthesis and metabolism that drive prostate tumor cell proliferation. Inhibition of GCN2 results in lowered expression of AA transporters, leading to severe depletion of intracellular AA and reduced proliferation in PCa. We identified purine biosynthesis as a key metabolic pathway dependent on GCN2. Inhibition of GCN2 and the accompanying depletion of AAs decreases purine levels in PCa cells, ultimately resulting in reduced ribosome biogenesis leading to the activation of a p53-dependent cell cycle checkpoint, termed the Impaired Ribosome Biogenesis Checkpoint (IRBC). Interestingly, induction of p53 promotes survival of PCa following GCN2 inhibition by halting cell cycle progression and reprogramming metabolism to restore metabolic homeostasis. We found that reductions in select AAs that impact nucleotide pools activate GCN2 and p53 in parallel, and that cooperation of these stress pathways is critical for maintaining AA and purine pools. Of importance, deletion of p53 sensitizes PCa cells to GCN2 inhibition suggesting that loss of p53 creates a dependency for GCN2. Of importance, we demonstrate that a small molecule inhibitor of GCN2 showed robust in vivo efficacy in androgen-sensitive and castrationresistant mouse models of PCa, supporting its therapeutic potential for the treatment of PCa.
GCN2 is required to maintain core body temperature in mice during acute cold
(American Physiological Society, 2023) Levy, Jordan L.; Mirek, Emily T.; Rodriguez, Esther M.; Zalma, Brian; Burns, Jeffrey; Jonsson, William O.; Sampath, Harini; Staschke, Kirk A.; Wek, Ronald C.; Anthony, Tracy G.; Biochemistry and Molecular Biology, School of Medicine
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.
Inhibition of the Eukaryotic Initiation Factor-2-α Kinase PERK Decreases Risk of Autoimmune Diabetes in Mice
(bioRxiv, 2024-06-03) Muralidharan, Charanya; Huang, Fei; Enriquez, Jacob R.; Wang, Jiayi E.; Nelson, Jennifer B.; Nargis, Titli; May, Sarah C.; Chakraborty, Advaita; Figatner, Kayla T.; Navitskaya, Svetlana; Anderson, Cara M.; Calvo, Veronica; Surguladze, David; Mulvihill, Mark J.; Yi, Xiaoyan; Sarkar, Soumyadeep; Oakes, Scott A.; Webb-Robertson, Bobbie-Jo M.; Sims, Emily K.; Staschke, Kirk A.; Eizirik, Decio L.; Nakayasu, Ernesto S.; Stokes, Michael E.; Tersey, Sarah A.; Mirmira, Raghavendra G.; Pediatrics, School of Medicine
Preventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress-responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eIF2α. In T1D, maladaptive unfolded protein response (UPR) in insulin-producing β cells renders these cells susceptible to autoimmunity. We show that inhibition of the eIF2α kinase PERK, a common component of the UPR and ISR, reverses the mRNA translation block in stressed human islets and delays the onset of diabetes, reduces islet inflammation, and preserves β cell mass in T1D-susceptible mice. Single-cell RNA sequencing of islets from PERK-inhibited mice shows reductions in the UPR and PERK signaling pathways and alterations in antigen processing and presentation pathways in β cells. Spatial proteomics of islets from these mice shows an increase in the immune checkpoint protein PD-L1 in β cells. Golgi membrane protein 1, whose levels increase following PERK inhibition in human islets and EndoC-βH1 human β cells, interacts with and stabilizes PD-L1. Collectively, our studies show that PERK activity enhances β cell immunogenicity, and inhibition of PERK may offer a strategy to prevent or delay the development of T1D.

Browsing by Author "Staschke, Kirk A."

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