Kinase Glycogen Synthase Kinase-3 Research Articles

Aucubin provides protection against cerebral ischaemia-reperfusion injury by suppressing neuronal apoptosis, oxidative stress, and inflammation through the modulation of the AKT-GSK-3β-Nrf2 signal cascade

Aucubin (AU) is a naturally occurring iridoid glycoside known to possess a wide range of pharmacological properties and exhibit a notable protective effect against various pathological conditions. Studies have shown that AU has neuroprotective properties in different neurological diseases. However, its potential protective effects against cerebral ischemia-reperfusion (CIR) injury have not been thoroughly investigated. This study aimed to investigate the impact of AU on CIR injury and explore the underlying mechanism. Cultured neurons treated with AU showed a significant reduction in apoptosis, oxidative stress, and inflammation caused by oxygen-glucose deprivation and reoxygenation (OGD/R). In a rat model of CIR, treatment with AU resulted in a significant decrease in cerebral infarct size and neurological deficits. AU treatment also reversed the increased apoptosis, oxidative stress, and inflammation in the brains of CIR rats. Furthermore, AU was found to enhance the activation of nuclear factor-erythroid 2-related factor 2 (Nrf2), accompanied by increased phosphorylation of serine/threonine-protein kinase AKT and glycogen synthase kinase-3 beta (GSK-3β). The activation of Nrf2 induced by AU was reversed when the AKT-GSK-3β cascade was blocked. Additionally, the neuroprotective effect of AU was significantly reduced when Nrf2 was pharmacologically suppressed. In conclusion, these findings suggest that AU exerts a neuroprotective effect on CIR injury, and this effect is mediated by the activation of Nrf2 through the AKT-GSK-3β axis. This work highlights the potential of AU as a drug candidate for the treatment of CIR injury.

GSK3 and lamellipodin balance lamellipodial protrusions and focal adhesion maturation in mouse neural crest migration

Neural crest cells are multipotent cells that delaminate from the neuroepithelium, migrating throughout the embryo. Aberrant migration causes developmental defects. Animal models are improving our understanding of neural crest anomalies, but invivo migration behaviors are poorly understood. Here, we demonstrate that murine neural crest cells display actin-based lamellipodia and filopodia invivo. Using neural crest-specific knockouts or inhibitors, we show that the serine-threonine kinase glycogen synthase kinase-3 (GSK3) and the cytoskeletal regulator lamellipodin (Lpd) are required for lamellipodia formation while preventing focal adhesion maturation. Lpd is a substrate of GSK3, and phosphorylation of Lpd favors interactions with the Scar/WAVE complex (lamellipodia formation) at the expense of VASP and Mena interactions (adhesion maturation and filopodia formation). This improved understanding of cytoskeletal regulation in mammalian neural crest migration has general implications for neural crest anomalies and cancer.

Mapping the signaling network of BIN2 kinase using TurboID-mediated biotin labeling and phosphoproteomics.

Elucidating enzyme-substrate relationships in posttranslational modification (PTM) networks is crucial for understanding signal transduction pathways but is technically difficult because enzyme-substrate interactions tend to be transient. Here, we demonstrate that TurboID-based proximity labeling (TbPL) effectively and specifically captures the substrates of kinases and phosphatases. TbPL-mass spectrometry (TbPL-MS) identified over 400 proximal proteins of Arabidopsis thaliana BRASSINOSTEROID-INSENSITIVE2 (BIN2), a member of the GLYCOGEN SYNTHASE KINASE 3 (GSK3) family that integrates signaling pathways controlling diverse developmental and acclimation processes. A large portion of the BIN2-proximal proteins showed BIN2-dependent phosphorylation in vivo or in vitro, suggesting that these are BIN2 substrates. Protein-protein interaction network analysis showed that the BIN2-proximal proteins include interactors of BIN2 substrates, revealing a high level of interactions among the BIN2-proximal proteins. Our proteomic analysis establishes the BIN2 signaling network and uncovers BIN2 functions in regulating key cellular processes such as transcription, RNA processing, translation initiation, vesicle trafficking, and cytoskeleton organization. We further discovered significant overlap between the GSK3 phosphorylome and the O-GlcNAcylome, suggesting an evolutionarily ancient relationship between GSK3 and the nutrient-sensing O-glycosylation pathway. Our work presents a powerful method for mapping PTM networks, a large dataset of GSK3 kinase substrates, and important insights into the signaling network that controls key cellular functions underlying plant growth and acclimation.

The budding yeast GSK-3 homologue Mck1 is an essential component of the spindle position checkpoint.

The spindle position checkpoint (SPOC) is a mitotic surveillance mechanism in Saccharomyces cerevisiae that prevents cells from completing mitosis in response to spindle misalignment, thereby contributing to genomic integrity. The kinase Kin4, one of the most downstream SPOC components, is essential to stop the mitotic exit network (MEN), a signalling pathway that promotes the exit from mitosis and cell division. Previous work, however, suggested that a Kin4-independent pathway contributes to SPOC, yet the underlying mechanisms remain elusive. Here, we established the glycogen-synthase-kinase-3 (GSK-3) homologue Mck1, as a novel component that works independently of Kin4 to engage SPOC. Our data indicate that both Kin4 and Mck1 work in parallel to counteract MEN activation by the Cdc14 early anaphase release (FEAR) network. We show that Mck1's function in SPOC is mediated by the pre-replication complex protein and mitotic cyclin-dependent kinase (M-Cdk) inhibitor, Cdc6, which is degraded in a Mck1-dependent manner prior to mitosis. Moderate overproduction of Cdc6 phenocopies MCK1 deletion and causes SPOC deficiency via its N-terminal, M-Cdk inhibitory domain. Our data uncover an unprecedented role of GSK-3 kinases in coordinating spindle orientation with cell cycle progression.

Design, synthesis, in silico and antibacterial evaluation of curcumin derivatives loaded nanofiber as potential wound healing agents

ObjectiveDrug resistance can result in mortality and morbidity due to treatment failures. Therefore, researchers are actively involved in the urgent development of new antibacterial agents which are more effective against drug-resistant bacteria. This study is aimed to prepare curcumin derivatives (CD) nanofiber and test its antibacterial activity by disk diffusion test and evaluate their role in wound healing when potentially binds with transforming growth factor-beta type 1 kinase domain (TGF-β) and Glycogen synthase kinase-3 beta (GSK3-β). MethodMolecular docking and molecular dynamic have been used to select the most potent dual-action molecules on both TGF-β and GSK3-β, most active 2 compounds have been synthesized and characterized by 1H and 13C (NMR), IR spectroscopy, Electrospinning of CD in ethanol were studied by adding Poly vinyl alcohol (PVA) as a polymer. The properties of the CD\\PVA solutions measured, morphology of electrospun CD\\PVA was tested by Scanning Electrode Microscope (SEM), antibacterial activity of electrospun nanofiber against Staphylococcus aureus (S. aureus) was tested by disk diffusion method.Results and Conclusions: CD nanofiber were synthesized successfully. The results showed that CD were successfully synthesized with good yield (72% and 78%) for compound1 and 2 respectively, Disc diffusion antibacterial test demonstrated an increased inhibition zone from 0.5 to 5.0 mg loaded CD with the largest inhibition zone of 8.5 ± 0.71 and 9.67 ± 0.29 mm for compound1 and 2 respectively. Hence, it can be concluded that these CD\\PVA could be a new source in designing new antibacterial wound dressing for diabetic patients.

Abstract 152: Discovery and characterization of GSK3 allosteric activators

Abstract The intestinal epithelium undergoes constant regeneration and is tightly regulated. Disruption of its homeostasis can result in the development of colorectal cancer (CRC). Glycogen synthase kinase 3 (GSK3) is a kinase that plays a central role in multiple signaling pathways, including cellular proliferation, where it inhibits the activity of its substrates. GSK3 dynamics can be described as a multiple input multiple output system. Numerous upstream growth factors can regulate and thus inhibit GSK3 from acting on its many substrates. Mutations in these growth signaling pathways (e.g., Wnt and KRAS) have been well established to have a role in CRC progression. Therefore, aberrantly activated signaling due to these proteins are presumed to result in the constitutive inhibition of GSK3 activity in CRC, thus making it an ideal therapeutic target. The aim of this study is to discover an allosteric GSK3 small molecule activator that can restore GSK3 kinase activity within CRC. Method: We screened 50,000 small molecule compounds using a luciferase-based in-vitro kinase assay. We are further confirming our top hits using both chemical and cell-based assays. For the cell-based assays, we test the compounds in human colonic epithelial cells (HCEC) and its three isogenic cell lines, which mimic CRC progression, and monitor changes in GSK3 kinase activity indirectly, via changes in its substrate protein levels, and directly via a live-cell GSK3 specific reporter. In addition, the top hits are also tested in both normal colon and CRC organoids. Results: Prior to running our large screen, we performed various optimization steps to determine the ideal condition for our screening assay. Our screening assay scored a Z-Score of 0.5 and higher, which indicated that it was a high-quality screening assay. Additionally, we have developed two cell-based orthogonal HTS assays. One using immunofluorescence and single-cell quantification of 24 GSK3 substrates from various pathways in our cell lines. Second, we engineered a GSK3 live-cell reporter and expressed it in the HCEC cells and three isogenic cell lines and organoid models. Impact: There is a need for advancements in preventative or therapeutic targets against CRC. Research indicates that suppression of GSK3 enzymatic activity decreases sensitivity to certain chemotherapeutic and targeted therapies. Therefore, restoring GSK3 enzymatic activity in CRC may re-sensitize tumors to other targeted therapies and establish GSK3 as a potential therapeutic target. Citation Format: Reeba P. Varghese, Curtis A. Thorne. Discovery and characterization of GSK3 allosteric activators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 152.

Pharmacological suppression of glycogen synthase kinase-3 reactivates HIV-1 from latency via activating Wnt/β-catenin/TCF1 axis in CD4+ T cells

ABSTRACT HIV-1 latency posts a major obstacle for HIV-1 eradication. Currently, no desirable latency reversing agents (LRAs) have been implicated in the “Shock and Kill” strategy to mobilize the latently infected cells to be susceptible for clearance by immune responses. Identification of key cellular pathways that modulate HIV-1 latency helps to develop efficient LRAs. In this study, we demonstrate that the Wnt downstream β-catenin/TCF1 pathway is a crucial modulator for HIV-1 latency. The pharmacological activation of the β-catenin/TCF1 pathway with glycogen synthase kinase-3 (GSK3) inhibitors promoted transcription of HIV-1 proviral DNA and reactivated latency in CD4+ T cells; the GSK3 kinase inhibitor 6-bromoindirubin-3′-oxime (6-BIO)-induced HIV-1 reactivation was subsequently confirmed in resting CD4+ T cells from cART-suppressed patients and SIV-infected rhesus macaques. These findings advance our understanding of the mechanisms responsible for viral latency, and provide the potent LRA that can be further used in conjunction of immunotherapies to eradicate viral reservoirs.

The GSK3 kinase and LZTR1 protein regulate the stability of Ras family proteins and the proliferation of pancreatic cancer cells.

Ras family proteins are membrane-bound GTPases that control proliferation, survival, and motility. Many forms of cancers are driven by the acquisition of somatic mutations in a RAS gene. In pancreatic cancer (PC), more than 90% of tumors carry an activating mutation in KRAS. Mutations in components of the Ras signaling pathway can also be the cause of RASopathies, a group of developmental disorders. In a subset of RASopathies, the causal mutations are in the LZTR1 protein, a substrate adaptor for E3 ubiquitin ligases that promote the degradation of Ras proteins. Here, we show that the function of LZTR1 is regulated by the glycogen synthase kinase 3 (GSK3). In PC cells, inhibiting or silencing GSK3 led to a decline in the level of Ras proteins, including both wild type Ras proteins and the oncogenic Kras protein. This decline was accompanied by a 3-fold decrease in the half-life of Ras proteins and was blocked by the inhibition of the proteasome or the knockdown of LZTR1. Irrespective of the mutational status of KRAS, the decline in Ras proteins was observed and accompanied by a loss of cell proliferation. This loss of proliferation was blocked by the knockdown of LZTR1 and could be recapitulated by the silencing of either KRAS or GSK3. These results reveal a novel GSK3-regulated LZTR1-dependent mechanism that controls the stability of Ras proteins and proliferation of PC cells. The significance of this novel pathway to Ras signaling and its contribution to the therapeutic properties of GSK3 inhibitors are both discussed.

Pim Kinase Inhibitor Enhances FLT3 Inhibitor Efficacy through GSK-3β Activation and GSK-3β-Mediated Proteasomal Degradation of c-Myc

BACKGROUNDInternal tandem duplication in the fms-like tyrosine kinase 3 receptor tyrosine kinase (FLT3-ITD) is present in acute myeloid leukemia (AML) in 30% of patients, associated with poor treatment outcomes due to rapid relapse. FLT3 inhibitors are used in the clinic, but with incomplete efficacy and development of resistance. Further treatment options are needed. The serine/threonine kinase proviral integration site for Moloney murine leukemia virus (Pim-1) is upregulated downstream of FLT3-ITD; it directly stimulates cell growth and inhibits apoptosis, and also phosphorylates and stabilizes FLT3 in a positive feedback loop in cells with FLT3-ITD. Dual targeting of Pim-1 and FLT3 is a promising treatment strategy. The c-Myc transcription factor contributes to dysregulation of cell growth and apoptosis in cancers, including AML. In addition to transcriptional regulation, c-Myc is regulated post-translationally by T58 phosphorylation by the serine/threonine kinase glycogen synthase kinase-3- β (GSK-3β). Here we show that concurrent treatment of cells with FLT3-ITD with Pim and FLT3 inhibitors activates GSK-3β, which phosphorylates and post-translationally downregulates c-Myc.METHODSBa/F3-ITD and MV4-11 cells, with FLT3-ITD, and FLT3-ITD AML patient blasts were cultured with the pan-Pim inhibitor AZD1208 (1 μM) and/or the FLT3 inhibitors gilteritinib or quizartinib (15 nM, 1 nM), with and without the GSK-3β inhibitor TCG-24 (20 μM). c-Myc, p-GSK3-α/β (S21/9) and GSK3-α/β protein expression was measured by immunoblotting. c-Myc mRNA was measured by qPCR. Cells were also cultured with cycloheximide (100 μg/mL) with and without the proteasome inhibitor MG-132 (20 μM) to measure protein half-life and proteasomal degradation. To study the role of c-Myc overexpression and activation, Ba/F3-ITD cells were infected with retroviral estrogen receptor (ER)-Myc plasmid, causing c-Myc nuclear translocation when activated by 4-hydroxytamoxifen (4-OHT; 300 nM). To study the role of c-Myc phosphorylation at T58, Ba/F3-ITD cells were infected with MycT58A plasmid, preventing c-Myc phosphorylation at T58. Apoptosis was detected by Annexin V and propidium iodide staining, measured by flow cytometry.RESULTSTreatment with Pim inhibitor AZD1208 and FLT3 inhibitor gilteritinib or quizartinib combination rapidly downregulated c-Myc protein expression in Ba/F3-ITD and MV4-11 cells, with FLT3-ITD, and in primary FLT3-ITD AML patient blasts, compared to quizartinib or gilteritinib alone. Pim inhibitor and FLT3 inhibitor combination treatment did not decrease c-Myc mRNA levels, but markedly decreased c-Myc protein half-life, from 36 mins without drugs and 24 mins with gilteritinib to 18 mins with combination. Half-life did not decrease when cells were pre-treated with the proteasome inhibitor MG-132, consistent with post-translational downregulation through proteasomal degradation. Apoptosis induction by Pim inhibitor and FLT3 inhibitor combination decreased by more than 50% in Ba/F3-ITD cells infected with ER-Myc plasmid and treated with 4-OHT, demonstrating the major role of c-Myc downregulation in apoptosis induction by combination treatment. GSK-3b is inactivated by phosphorylation, and combination treatment rapidly decreased p-GSK-3b levels, while total GSK-3b levels were unchanged, indicating activation of GSK-3b. Treatment of cells with FLT3-ITD with the GSK-3b inhibitor TCG-24 in addition to Pim and FLT3 inhibitors abrogated c-Myc protein downregulation, demonstrating that Pim and FLT3 inhibitor combination downregulates c-Myc through activation of GSK-3b. Finally, Pim and FLT3 inhibitor combination treatment did not downregulate c-Myc in Ba/F3-ITD cells transfected with c-Myc T58A, preventing c-Myc phosphorylation at T58, showing that c-Myc phosphorylation at T58 is necessary for its downregulation by combination treatment.CONCLUSIONSConcurrent treatment of cells with FLT3-ITD with Pim kinase inhibitor enhances the efficacy of FLT3 inhibitors through activation of GSK-3β and GSK-3β-mediated phosphorylation of c-Myc at T58, with resulting c-Myc downregulation through increased proteasomal degradation. This work and previous work in our laboratory on PP2A activating drugs and FLT3 inhibitor combination (Mol Cancer Ther 20:676, 2021) support GSK-3β activation as a mechanism for enhancing efficacy of FLT3 inhibitors in AML with FLT3-ITD. DisclosuresNo relevant conflicts of interest to declare.

Glycogen Synthase Kinase-3: A Focal Point for Advancing Pathogenic Inflammation in Depression.

Increasing evidence indicates that the host immune response has a monumental role in the etiology of major depressive disorder (MDD), motivating the development of the inflammatory hypothesis of depression. Central to the involvement of chronic inflammation in MDD is a wide range of signaling deficits induced by the excessive secretion of pro-inflammatory cytokines and imbalanced T cell differentiation. Such signaling deficits include the glutamatergic, cholinergic, insulin, and neurotrophin systems, which work in concert to initiate and advance the neuropathology. Fundamental to the communication between such systems is the protein kinase glycogen synthase kinase-3 (GSK-3), a multifaceted protein critically linked to the etiology of MDD and an emerging target to treat pathogenic inflammation. Here, a consolidated overview of the widespread multi-system involvement of GSK-3 in contributing to the neuropathology of MDD will be discussed, with the feed-forward mechanistic links between all major neuronal signaling pathways highlighted.

Lithium and Therapeutic Targeting of GSK-3.

Lithium salts have been in the therapeutic toolbox for better or worse since the 19th century, with purported benefit in gout, hangover, insomnia, and early suggestions that lithium improved psychiatric disorders. However, the remarkable effects of lithium reported by John Cade and subsequently by Mogens Schou revolutionized the treatment of bipolar disorder. The known molecular targets of lithium are surprisingly few and include the signaling kinase glycogen synthase kinase-3 (GSK-3), a group of structurally related phosphomonoesterases that includes inositol monophosphatases, and phosphoglucomutase. Here we present a brief history of the therapeutic uses of lithium and then focus on GSK-3 as a therapeutic target in diverse diseases, including bipolar disorder, cancer, and coronavirus infections.

Insulin directly stimulates mitochondrial glucose oxidation in the heart

BackgroundGlucose oxidation is a major contributor to myocardial energy production and its contribution is orchestrated by insulin. While insulin can increase glucose oxidation indirectly by enhancing glucose uptake and glycolysis, it also directly stimulates mitochondrial glucose oxidation, independent of increasing glucose uptake or glycolysis, through activating mitochondrial pyruvate dehydrogenase (PDH), the rate-limiting enzyme of glucose oxidation. However, how insulin directly stimulates PDH is not known. To determine this, we characterized the impacts of modifying mitochondrial insulin signaling kinases, namely protein kinase B (Akt), protein kinase C-delta (PKC-δ) and glycogen synthase kinase-3 beta (GSK-3β), on the direct insulin stimulation of glucose oxidation.MethodsWe employed an isolated working mouse heart model to measure the effect of insulin on cardiac glycolysis, glucose oxidation and fatty acid oxidation and how that could be affected when mitochondrial Akt, PKC-δ or GSK-3β is disturbed using pharmacological modulators. We also used differential centrifugation to isolate mitochondrial and cytosol fraction to examine the activity of Akt, PKC-δ and GSK-3β between these fractions. Data were analyzed using unpaired t-test and two-way ANOVA.ResultsHere we show that insulin-stimulated phosphorylation of mitochondrial Akt is a prerequisite for transducing insulin’s direct stimulation of glucose oxidation. Inhibition of mitochondrial Akt completely abolishes insulin-stimulated glucose oxidation, independent of glucose uptake or glycolysis. We also show a novel role of mitochondrial PKC-δ in modulating mitochondrial glucose oxidation. Inhibition of mitochondrial PKC-δ mimics insulin stimulation of glucose oxidation and mitochondrial Akt. We also demonstrate that inhibition of mitochondrial GSK3β phosphorylation does not influence insulin-stimulated glucose oxidation.ConclusionWe identify, for the first time, insulin-stimulated mitochondrial Akt as a prerequisite transmitter of the insulin signal that directly stimulates cardiac glucose oxidation. These novel findings suggest that targeting mitochondrial Akt is a potential therapeutic approach to enhance cardiac insulin sensitivity in condition such as heart failure, diabetes and obesity.

AKT signaling restrains tumor suppressive functions of FOXO transcription factors and GSK3 kinase in multiple myeloma

AbstractThe phosphatidylinositide-3 kinases and the downstream mediator AKT drive survival and proliferation of multiple myeloma (MM) cells. AKT signaling is active in MM and has pleiotropic effects; however, the key molecular aspects of AKT dependency in MM are not fully clear. Among the various downstream AKT targets are the Forkhead box O (FOXO) transcription factors (TFs) and glycogen synthase kinase 3 (GSK3), which are negatively regulated by AKT signaling. Here we show that abrogation of AKT signaling in MM cells provokes cell death and cell cycle arrest, which crucially depends on both FOXO TFs and GSK3. Based on gene expression profiling, we defined a FOXO-repressed gene set that has prognostic significance in a large cohort of patients with MM, indicating that AKT-mediated gene activation is associated with inferior overall survival. We further show that AKT signaling stabilizes the antiapoptotic myeloid cell leukemia 1 (MCL1) protein by inhibiting FOXO- and GSK3-mediated MCL1 turnover. In concordance, abrogation of AKT signaling greatly sensitized MM cells for an MCL1-targeting BH3-mimetic, which is currently in clinical development. Taken together, our results indicate that AKT activity is required to restrain the tumor-suppressive functions of FOXO and GSK3, thereby stabilizing the antiapoptotic protein MCL1 in MM. These novel insights into the role of AKT in MM pathogenesis and MCL1 regulation provide opportunities to improve targeted therapy for patients with MM.

Modulated Expression of the Protein Kinase GSK3 in Motor and Dopaminergic Neurons Increases Female Lifespan in Drosophila melanogaster.

Most eukaryotic genes express multiple transcripts and proteins, and a sophisticated gene expression strategy plays a crucial role in ensuring the cell-specificity of genetic information and the correctness of phenotypes. The Drosophila melanogaster gene shaggy encodes several isoforms of the conserved glycogen synthase kinase 3 (GSK3), which is vitally important for multiple biological processes. To characterize the phenotypic effects of differential shaggy expression, we explored how the multidirectional modulation of the expression of the main GSK3 isoform, Shaggy-PB, in different tissues and cells affects lifespan. To this end, we used lines with transgenic constructs that encode mutant variants of the protein. The effect of shaggy misexpression on lifespan depended on the direction of the presumed change in GSK3 activity and the type of tissue/cell. The modulation of GSK3 activity in motor and dopaminergic neurons improved female lifespan but caused seemingly negative changes in the structural (mitochondrial depletion; neuronal loss) and functional (perturbed locomotion) properties of the nervous system, indicating the importance of analyzing the relationship between lifespan and healthspan in invertebrate models. Our findings provide new insights into the molecular and cellular bases of lifespan extension, demonstrating that the fine-tuning of transcript-specific shaggy expression in individual groups of neurons is sufficient to provide a sex-specific increase in survival and slow aging.

The Role of GSK-3 in Cancer Immunotherapy: GSK-3 Inhibitors as a New Frontier in Cancer Treatment.

The serine/threonine kinase glycogen synthase kinase-3 (GSK-3) was initially identified because of its key role in the regulation of glycogen synthesis. However, it is now well-established that GSK-3 performs critical functions in many cellular processes, such as apoptosis, tumor growth, cell invasion, and metastasis. Aberrant GSK-3 activity has been associated with many human diseases, including cancer, highlighting its potential therapeutic relevance as a target for anticancer therapy. Recently, newly emerging data have demonstrated the pivotal role of GSK-3 in the anticancer immune response. In the last few years, many GSK-3 inhibitors have been developed, and some are currently being tested in clinical trials. This review will discuss preclinical and initial clinical results with GSK-3β inhibitors, highlighting the potential importance of this target in cancer immunotherapy. As described in this review, GSK-3 inhibitors have been shown to have antitumor activity in a wide range of human cancer cells, and they may also contribute to promoting a more efficacious immune response against tumor target cells, thus showing a double therapeutic advantage.

CD28 and glycogen synthase kinase-3 (GSK-3) inhibitor combination therapy generates tumor infiltrating T-cells (TILs) with a reversal of markers for T-cell exhaustion

Abstract Immune checkpoint blockade (ICB) of inhibitory receptors such as PD-1 has revolutionized the treatment of cancer. ICB is dependent on CD28 co-receptor expression introducing the question of whether the positive co-receptor could be targeted for the reversal of T-cell exhaustion in immunotherapy. We previously showed that the serine/threonine kinase glycogen synthase kinase-3 (GSK-3) is a central regulator of programmed cell death-1 (PD-1) transcription (Taylor et al., 2016, Immunity) and that small molecule inhibitors of GSK-3 (GSK-3 SMI) are as effective as anti-PD-1 blocking antibody in the control of tumor growth (Taylor et al., 2018, Cancer Research). In this study, we now show that anti-CD28 can synergise with GSK-3α/β inhibitors to regress tumors that are resistant to anti-PD-1 immunotherapy. Combination therapy showed responses in >55% mice with an overall reduction in tumor volume of 70% in responders compared to control mice (182 mm3 vs. 611 mm3). Flow cytometry analysis of tumor infiltrating lymphocytes (TILs) shows that, while non-treated mice were enriched with TOX+TIM3+PD1+CD8+, combination therapy increased tumor infiltration of TOX-negative CD8 T cells with TIM3-PD1int CD8 phenotype. Remarkably, this reversal involved only a single injection of anti-CD28 (PV-1) and GSK-3 SMI (SB415286). These results showed that CD28/GSK-3 combination therapy greatly reduced TOX, TIM-3 and PD1markers indicative of exhaustion. Our findings offer a small molecule approach for synergy with anti-CD28 that leads to an increase tumor infiltration with TOX-negative CD8 T cells that have decrease expression of inhibitory receptors.

Inhibiting Glycogen Synthase Kinase-3 Mitigates the Hematopoietic Acute Radiation Syndrome in a Sex- and Strain-dependent Manner in Mice.

The Radiation and Nuclear Countermeasures Program at the National Institute of Allergy and Infectious Diseases (NIAID) mandated that medical countermeasures for treating Acute Radiation Syndrome (ARS) must have efficacy when administered at least 24 h after radiation exposure. At this time point, many cells within key target tissues, such as the hematopoietic system and the gastrointestinal (GI) tract, will already be dead. Therefore, drugs that promote the regeneration of surviving cells may improve outcomes. The serine/threonine kinase glycogen synthase kinase-3 (GSK-3) regulates stem and progenitor cell self-renewal and regeneration in the hematopoietic and GI compartments. We tested inhibition of GSK-3β by SB216763 24 h after total body irradiation (TBI) and sub-total body irradiation (SBI). Here, we show that subcutaneous administration of SB216763 promotes the regeneration of surviving hematopoietic stem/progenitor cells (HSPCs), including myeloid progenitor cells, and improves survival of C57Bl/6 male mice when administered 24 h after TBI. However, these results were not recapitulated in female C57Bl/6 animals, suggesting a sex difference in GSK-3β signaling in HSPCs. Subcutaneous administration of SB216763 in male mice stimulated activation of Sox2 transcription but failed to induce Sox2 transcription in female C57Bl/6 mice. Using TCF/lef-GFP reporter mice, we examined Wnt signaling in HSPCs of irradiated male and female mice treated with SB216763. GSK-3 inhibition elevated Wnt reporter activity in HSPCs isolated from male but not female mice. SB216763 did not mitigate hematopoietic ARS in males or females of a second strain of wild-type mice, C3H. In addition, administration of SB216763 did not mitigate hematopoietic ARS beyond the currently available standard approved therapy of ciprofloxacin and granulocyte-colony stimulating factor (G-CSF) in male C57Bl/6 mice. Further, SB216763 did not mitigate GI-ARS after SBI in C57Bl/6 male mice. The lack of efficacy in both sexes and multiple strains of mice indicate that SB216763 is not suitable for further drug development as a mitigator of ARS. Our studies demonstrate that activation of Wnt signaling in HSPCs promotes hematopoietic regeneration following radiation exposure, and targeting this pathway downstream of GSK-3β may mitigate ARS in a sex- and strain-independent manner.

A Single Amino Acid Substitution in STKc_GSK3 Kinase Conferring Semispherical Grains and Its Implications for the Origin of Triticum sphaerococcum.

Six subspecies of hexaploid wheat (Triticum aestivum) have been identified, but the origin of Indian dwarf wheat (Triticum sphaerococcum), the only subspecies with round grains, is currently unknown. Here, we isolated the grain-shape gene Tasg-D1 in T sphaerococcum via positional cloning. Tasg-D1 encodes a Ser/Thr protein kinase glycogen synthase kinase3 (STKc_GSK3) that negatively regulates brassinosteroid signaling. Expression of TaSG-D1 and the mutant form Tasg-D1 in Arabidopsis (Arabidopsis thaliana) suggested that a single amino acid substitution in the Thr-283-Arg-284-Glu-285-Glu-286 domain of TaSG-D1 enhances protein stability in response to brassinosteroids, likely leading to formation of round grains in wheat. This gain-of-function mutation has pleiotropic effects on plant architecture and exhibits incomplete dominance. Haplotype analysis of 898 wheat accessions indicated that the origin of T sphaerococcum in ancient India involved at least two independent mutations of TaSG-D1 Our results demonstrate that modest genetic changes in a single gene can induce dramatic phenotypic changes.

Glycogen synthase kinase 3 (GSK-3) synergizes with immune-checkpoint blockade (ICB) to overcome tumor resistance due to unique tumor infiltrating T-cell subsets as revealed by mass cytometry (CyTOF)

Abstract We have shown that the serine/threonine kinase glycogen synthase kinase-3 (GSK-3) is a major regulator of programmed cell death-1 (PD-1) transcription and expression in T-cells (Taylor et al., 2016, Immunity). Further, small molecule inhibitors (SMIs) increased Tbx21 transcription for enhanced Tbet expression which suppressed Pdcd1 transcription in CD8+cytolytic T-cells. SMIs against GSK-3 are as effective as anti-PD-1 blocking antibody in the control of spontaneous pulmonary metastasis of B16 melanoma and EL-4 lymphoma cells (Taylor et al., 2018, Cancer Research). This introduced a new chemical approach to down-regulate PD-1 for the treatment of cancer. In addition, GSK-3 effects in Tbet expression can induce the expression of interferon-γ1 (IFNγ1) and granzyme B (GZMB) for enhanced cytolytic T-cell (CTL) killing of tumors suggesting that GSK-3 SMIs might synergize with check-point blockade. We now show that GSK-3α/β inhibition can cooperate with anti-PD-1 blockade to overcome tumor resistance. Using high dimensional profiling of tumor infiltrated lymphocytes (TILs) with mass cytometry (CyTOF), we show that the synergy of GSK-3 SMI and anti-PD-1 is accompanied by 2 specific findings: (i) an increase in the presence of a unique subset of CD8+ TIL effector-memory T-cells beyond that observed with anti-PD-1 alone, and (ii) the significant decrease of a subset of CD4+ suppressor regulatory T-cells (Tregs) not observed with anti-PD-1 alone. Our findings offer a small molecule approach for synergy with anti-PD-1 that leads to an increase in CD8+ T-cells and the marked decrease of Tregs in tumors.

Hepatic Expression of Phosphorylated Kinase Akt and Glycogen Synthase Kinase-3 Isoforms in Patients with Chronic Hepatitis C.

Some patients with chronic hepatitis C also demonstrate liver steatosis, but the mechanism remains elusive. To analyze the hepatic expression of phosphorylated kinase Akt at Thr 308 and phosphorylated GSK-3 (Glycogen synthase kinase-3) isoforms, GSK3α at Ser 21 and GSK3β at Ser 9, in chronic hepatitis C patients with normal body weight, glucose, and lipid profiles depending on homeostasis model assessment of insulin resistance (HOMA-IR) levels and histological parameters. The study group consisted of 31 patients with chronic hepatitis C. The hepatic expression of kinase Akt (Thr308), GSK3β (Ser9), and GSK3α (Ser21) was measured using Western blot assay. Liver steatosis was observed in 41.93% of patients with HCV infection, in those with increased HOMA-IR index (p = 0.02). However, the hepatic expression of Akt (Thr308), GSK3β (Ser9), and GSK3α (Ser21) was not related to progression of liver steatosis, inflammation, and fibrosis. There was no significant difference in the hepatic expression of kinase Akt (Thr308), GSK3β (Ser9), and GSK3α (Ser21) in relation to HOMA-IR. Liver steatosis was found to be positively associated with HOMA-IR levels in patients with chronic hepatitis C without metabolic disorders. However, the hepatic expression of Akt (Thr308), GSK3β (Ser9), and GSK3α (Ser21) did not correspond to progression of liver disease.