Inhibition Of GSK-3beta Research Articles

Synaptophysin and GSK-3beta activity in the prefrontal cortex may underlie the effects of REM sleep deprivation and lithium on behavioral functions and memory performance in male rats

Rapid-eye movement (REM) stage of sleep serves a critical role in processing cognitive and behavioral functions. Evidence shows that REM sleep deprivation (REM SD) strongly affects the mood state and cognitive abilities. However, there are many inconsistent reports. Although the exact molecular mechanisms underlying REM SD effects have not well been discovered, however, molecular factors including those affected synaptic plasticity and mood state may be involved. There are two important molecular factors that have not been well studied: synaptophysin and glycogen synthase kinase-3 beta (GSK-3beta). The present study aimed to investigate the role of synaptophysin and GSK-3beta in the modulation of memory and behavioral changes induced by REM SD and lithium (as a potent GSK-3beta inhibitor and mood stabilizer). Multiple platform apparatus was used to induce REM SD for 48 h. Lithium was injected at the dose of 50 mg/kg, intraperitoneal (i.p.). Locomotor activity, anxiety-like behavior, pain threshold, novel object recognition memory, and synaptophysin and GSK-3beta level in the prefrontal cortex were evaluated. Results showed REM SD increased locomotor activity, decreased pain threshold, impaired novel object recognition memory, decreased synaptophysin and increased GSK-3beta levels. Lithium reversed these effects. Anxiety-like behavior was unaffected. For the first time, the present study showed that GSK-3beta and synaptophysin may be involved in the modulation of behavior and cognition induced by REM SD and lithium. In conclusion, we suggested that GSK-3beta upregulation and synaptophysin downregulation may underlie the deleterious effects of REM SD, while lithium may counteract REM SD effects via restoring the level of both.

Abstract C073: FOLFIRINOX with Glycogen Synthase Kinase-3 Beta (GSK-3β) Inhibitor Elraglusib and Transforming Growth Factor- β (TGFβ) Inhibitor Losartan in Untreated Metastatic Pancreatic Ductal Adenocarcinoma(PDAC): Interim analysis of safety cohort

Abstract Introduction: Epithelial-mesenchymal transition (EMT) is one of the key drivers of aggressive biology and development of chemoresistance in PDAC. Inhibition of glycogen synthase kinase 3β (GSK3β) may offer a promising novel approach for modulating dynamic EMT plasticity to overcome resistance to FOLFIRINOX (5-Fluororuacil, Irinotecan, Oxaliplatin, FFX). Methods: Cell line panels comprising both epithelial (E) and quasi-mesenchymal (QM) subtypes were used and treated with IC50 (0.3125 mM) and IC100 (1.25 mM) doses of elraglusib (ELR). Gene expression patters in response to treatment were analyzed using bulk RNA sequencing. 6 patients in the safety run-in cohort of NCT05077800 treated with standard doses of FFX, ELR and losartan were included in this interim analysis. Biopsy specimens were processed for RNA-in situ hybridization (RNA-ISH), a clinically validated assay, and Nanostring (GeoMx) spatial transcriptomics. Results: In preclinical models, exposure to the higher IC100 (1.25 mM) doses of ELR in the QM cell lines caused a shift in transcriptional expression patterns similar to the E cell line, with strong downregulation of EMT. FFX exposure alone in QM cell lines was associated with significant resistance, but addition of ELR (even at IC50 dose) caused a synergistic effect with decreased cell viability. However, in the E cell lines, there was no benefit from addition of ELR. In the safety cohort, clinical results are encouraging with a long-term (>6 month) response in 3/6 patients. 2/6 achieved partial response, 1/6 had stable disease. Nanostring (GeoMx) spatial transcriptomic profiling on available biopsy samples showed significant global gene expression changes before and after treatment. There were concordant changes in 97 differentially expressed genes (DEGs) within the epithelial compartment with treatment, similar to ELR induced changes in PDAC cell lines, with a shift in transcriptional expression patterns from QM to E subtype. Gene set enrichment analysis demonstrated downregulation of the EMT pathway, as well as decreased TGF-β signaling. RNA-ISH analysis on baseline biopsy samples showed that the two partial (best) responders had higher degrees of QM marker expression (82.58% and 63.76%). The regimen was well tolerated with no dose limiting toxicity noted in the safety cohort. Conclusion: Our experiments demonstrate that the E subtype PDAC cell lines are inherently sensitive to FFX chemotherapy with no added advantage with GSK3β inhibition. Meanwhile, GSK3β inhibition in QM cell lines induces mesenchymal to epithelial transition, thereby rendering it sensitive to FFX. The data from our safety cohort provide a clinical proof of concept that GSK-3β inhibition with ELR, in combination with FFX, may be an effective therapeutic strategy in metastatic PDAC. NCT05077800 is currently enrolling. Citation Format: Priyadarshini Pathak, Eric Lin, Ildiko Phillips, David Ting, Colin Weekes. FOLFIRINOX with Glycogen Synthase Kinase-3 Beta (GSK-3β) Inhibitor Elraglusib and Transforming Growth Factor- β (TGFβ) Inhibitor Losartan in Untreated Metastatic Pancreatic Ductal Adenocarcinoma(PDAC): Interim analysis of safety cohort [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C073.

Identification of a novel GSK3β inhibitor involved in abrogating KRas dependent pancreatic tumors in Wnt/beta-catenin and NF-kB dependent manner

Pancreatic cancer is an aggressive malignancy with a poor survival rate because it is difficult to diagnose the disease during its early stages. The currently available treatments, which include surgery, chemotherapy and radiation therapy, offer only limited survival benefit. Pharmacological interventions to inhibit Glycogen Synthase Kinase-3beta (GSK3β) activity is an important therapeutic strategy for the treatment of pancreatic cancer because GSK3β is one of the key factors involved in the onset, progression as well as in the acquisition of chemoresistance in pancreatic cancer. Here, we report the identification of MJ34 as a potent GSK3β inhibitor that significantly reduced growth and survival of human mutant KRas dependent pancreatic tumors. MJ34 mediated GSK3β inhibition was seen to induce apoptosis in a β-catenin dependent manner and downregulate NF-kB activity in MiaPaCa-2 cells thereby impeding cell survival and anti-apoptotic processes in these cells as well as in the xenograft model of pancreatic cancer. In vivo acute toxicity and in vitro cardiotoxicity studies indicate that MJ34 is well tolerated without any adverse effects. Taken together, we report the discovery of MJ34 as a potential drug candidate for the therapeutic treatment of mutant KRas-dependent human cancers through pharmacological inhibition of GSK3β.

β-Sitosterol alleviates the malignant phenotype of hepatocellular carcinoma cells via inhibiting GSK3B expression

To explore the effects of β-Sitosterol upon hepatocellular carcinoma cell proliferation, apoptosis, migration, invasion, and epithelial–mesenchymal transition (EMT), and to investigate the underlying mechanism using network pharmacology. Human hepatocellular carcinoma cell lines (Huh-7 and HCCLM3) were expose to gradient concentrations of β-Sitosterol (5 μg/mL, 10 μg/mL, and 20 μg/mL). Cell viability and proliferation were assessed using MTT, CCK-8, colony formation, and EdU assays.Flow cytometry was employed to evaluate cell cycle and apoptosis. Scratch and Transwell assays were performed, respectively, to detect cell migration and invasion. The levels of apoptosis-associated proteins (BAX, BCL2, and cleaved caspase3) as well as EMT-associated proteins (E-cadherin, N-cadherin, Snail, and Vimentin) were detected in Huh-7 and HCCLM3 cell lines using Western blot analysis. The drug target gene for β-Sitosterol was screened via PubChem and subsequently evaluated for expression in the GSE112790 dataset. In addition, the expression level of glycogen synthase kinase 3 beta (GSK3B) within the Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) database was analyzed, along with its correlation to the survival outcomes of patients with hepatocellular carcinoma. The diagnostic efficiency of GSK3B was assessed by analyzing the ROC curve. Subsequently, Huh-7 and HCCLM3 cell lines were transfected with the overexpression vector of GSK3B and then treated with β-Sitosterol to further validate the association between GSK3B and β-Sitosterol. GSK3B demonstrated a significantly elevated expression in patients with hepatocellular carcinoma, which could predict hepatocellular carcinoma patients’ impaired prognosis based on GEO dataset and TCGA database. GSK3B inhibitor (CHIR-98014) notably inhibited cell proliferation and invasion, promoted cell apoptosis and cell cycle arrest at G0/G1 phase in hepatocellular carcinoma cells. β-Sitosterol treatment further promoted the efffects of GSK3B inhibitor on hepatocellular carcinoma cells. GSK3B overexpression has been found to enhance the proliferative and invasive capabilities of hepatocellular carcinoma cells. Furthermore it has been observed that GSK3B overexpression, it has been obsear can partially reverse the inhibitory effect of β-Sitosterol upon hepatocellular. β-Sitosterol suppressed hepatocellular carcinoma cell proliferation and invasion, and enhanced apoptosis via inhibiting GSK3B expression.

Multitarget Ensemble Docking of Potent Anticancer and Antioxidant Active Compounds from the Acacia auriculiformis and Acacia crassicarpa

Bioactive chemicals derived from Acacia auriculiformis and A. crassicarpa have the potential to be developed as sources of anti-cancer raw materials and antioxidants, given that these plants are fast-growing species with medicinal capability. The in silico method was successful in identifying these bioactive chemicals for the preliminary study. Using an in silico approach, this work aimed to identify the most potent compounds as inhibitors of six cancer and stress oxidative therapy-targeted proteins from these two distinct Acacia species. Seventeen out of the 37 compounds examined exhibited low affinity and satisfied the drug-likeness criterion. Five active chemicals were identified by redocking analysis: auriculoside, 3-(3,4-dihydroxybenzyl)-7-hydroxychroman-4-one, kaempferol 7-O-glucoside, quercetin 7-O-glucoside, and keto-teracacidin. According to simulations of molecular dynamics, molecular motion occurs with a root mean square deviation of less than four and generates at least eleven receptor conformations for 0 to 100 ns. Auriculoside showed the lowest average binding energy against four receptors in colorectal and breast cancer, as determined by ensemble docking, followed by 3-(3,4-dihydroxybenzyl)-7-hydroxychroman-4-one, quercetin 7-O-glucoside, and kaempferol 7-O-glucoside. Auriculoside shown multitarget inhibitory effect against colorectal cancer by inhibiting cyclin dependent kinase-6 and breast cancer by inhibiting epidermal growth factor receptor and mammalian target of rapamycin. Auriculoside has the powerful ability to inhibit glycogen synthase kinase-3 beta, hence regulating oxidative stress. Kaempferol 7-O-glucoside and quercetin 7-O-glucoside also exhibited a possible single protein targeting method against breast cancer. These findings are essential for future research targeted at developing these plants as potent natural therapeutic raw materials and for isolating or synthesizing compounds with anticancer and oxidative stress-regulating antioxidant properties.

Mechanism of the combined action of green tea polyphenols and concurrent radiochemotherapy in regulating GSK-3β to treat non-small cell lung cancer through the Wnt∕β-catenin pathway.

Green tea, derived from Camellia sinensis, contains polyphenolic active compounds that exhibit diverse pharmacological effects including anticancer, anti-inflammatory, antioxidant, and immunomodulatory properties. Employing various concentrations of green tea polyphenols (GTPs; 0, 100, 200, 300, 400, 500 μg∕mL), human normal lung epithelial cells (BEAS-2B) and non-small cell lung cancer (NSCLC) cells (A549) underwent treatment. The cell viability was assessed using the cell counting kit-8 (CCK-8) assay, proliferation was examined through the colony formation assay, apoptosis was monitored via flow cytometry, cell migration, and epithelial-mesenchymal transition (EMT)-associated proteins (E-cadherin, N-cadherin) were determined by Western blot. A549 cells were subjected to Cisplatin (0, 0.5, 1, 1.5 μM) and X-ray irradiation (0, 2, 4, 6 Gy) for treatment to probe the influence of GTPs on A549 cells in response to chemoradiotherapy. The functioning mechanism of GTPs in the context of NSCLC was validated using lithium chloride (LiCl) [a glycogen synthase kinase-3 beta (GSK-3β) inhibitor], which activates the Wnt∕β-catenin pathway. GTPs suppressed NSCLC cell viability in a concentration-dependent pattern, with a half-maximal inhibitory concentration (IC50) of 362.5 μg∕mL, while showing little impact on BEAS-2B cells' viability (at concentrations not exceeding 500 μg∕mL). Treatment with GTPs dampened colony formation of NSCLC cells, while promoting apoptosis. LiCl treatment vigorously attenuated the inhibitory impact of GTPs on the malignant phenotype of NSCLC cells. Mechanistic studies suggested that GTPs strengthened GSK-3β stability, thereby impeding the Wnt∕β-catenin pathway. Tea polyphenols (TPs) in conjunction with concurrent radiochemotherapy (CRCT) enhance the stability of GSK-3β and dampen the Wnt∕β-catenin pathway, hence exerting anticancer effects in NSCLC.

Hyperinsulinemic milieu elicits glomerular podocyte impairment and dysfunction via inducing GSK3beta hyperactivity

Background: Epidemiological evidence suggests that hyperinsulinemia or insulin resistance is a significant risk factor for the development of diabetic complications such as DKD. However, whether hyperinsulinemia per se plays a causative role in the development of diabetic kidney injury is unknown and was explored here.
 Methods: Pre-diabetic db/db mice were examined for serum insulin levels, urinary albumin to creatinine ratios and renal histology. Conditionally immortalized mouse podocytes were cultured under non-permissive conditions and exposed to high ambient insulin conditions, following GSK3beta silencing, ectopic expression of a constitutively active GSK3beta mutant (S9A), or treatment with a small molecule GSK3beta inhibitor tideglusib. Podocyte injury was assessed and signaling pathways examined. 
 Results: In pre-diabetic db/db mice, hyperinsulinemia was evident and associated with microalbuminuria and early signs of podocyte impairment, including diminished expression of homeostatic marker proteins like synaptopodin, as compared with db/m littermates. In vitro, prolonged exposure of differentiated podocytes to high ambient insulin induced podocytopathic changes, including cellular hypertrophy, loss of synaptopodin, and disruption of actin cytoskeleton integrity. This was associated with a desensitized insulin signaling and diminished inhibitory phosphorylation of GSK3beta, denoting GSK3beta hyperactivity. In pre-diabetic db/db mice, GSK3beta hyperactivity was confirmed in glomerular podocytes, correlating with the level of hyperinsulinemia or microalbuminuria. In cultured podocytes, ectopic expression of S9A caused podocyte hypertrophy and podocytopathic changes, reminiscent of the harmful effect of the hyperinsulinemic milieu. Conversely, GSK3beta knockdown mitigates podocyte injury elicited by hyperinsulinemic milieu. This protective effect was mimicked by the small molecule inhibitor tideglusib.
 Conclusions: GSK3beta hyperactivity is required and sufficient for Hyperinsulinemic milieu-elicited glomerular podocyte impairment and dysfunction.

Massive expansion of functional human iPSC-derived cardiomyocytes by concomitant glycogen synthase kinase-3 beta inhibition and removal of cell-cell contact

Abstract Background Adult mammalian heart has limited capacity for regeneration. Cardiac injuries such as myocardial infarction lead to significant cardiomyocyte (CM) loss and subsequent heart failure with significant morbidity and mortality. Cell-based therapeutic approaches such as injection of stem cell-derived cells or transplantation of engineered cardiac tissue patches have shown promise to re-muscularize the damaged myocardium and enhance cardiac functions. Rationale: Modulating signaling pathways including Wnt and Hippo can induce cardiomyocyte proliferation in vivo. Applying these signaling modulators to human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro can expand CMs modestly (< 5-fold). Results Here, we demonstrate massive expansion of hiPSC-CMs in vitro (i.e. 100 to 250-fold) by glycogen synthase kinase-3β (GSK-3β) inhibition using CHIR99021 and concurrent removal of cell-cell contact. We show GSK-3β inhibition suppresses CM maturation while contact removal prevents CMs from cell cycle exit. Remarkably, contact removal enabled 10- to 25-times greater expansion beyond GSK-3β inhibition alone. Mechanistically, persistent CM proliferation required both LEF/TCF activity and AKT phosphorylation but was independent from Yes associated protein (YAP) signaling. Engineered heart tissues from expanded hiPSC-CMs showed comparable contractility to those from unexpanded hiPSC-CMs, demonstrating uncompromised cellular functionality after expansion. Conclusion In summary, we uncovered a molecular interplay that enables massive hiPSC-CMs expansion for large-scale drug screening and tissue engineering applications.

Bone-Targeted Nanoparticle Drug Delivery System-Mediated Macrophage Modulation for Enhanced Fracture Healing.

Despite decades of progress, developing minimally invasive bone-specific drug delivery systems (DDS) to improve fracture healing remains a significant clinical challenge. To address this critical therapeutic need, nanoparticle (NP) DDS comprised of poly(styrene-alt-maleic anhydride)-b-poly(styrene) (PSMA-b-PS) functionalized with a peptide that targets tartrate-resistant acid phosphatase (TRAP) and achieves preferential fracture accumulation has been developed. The delivery of AR28, a glycogen synthase kinase-3 beta (GSK3β) inhibitor, via the TRAP binding peptide-NP (TBP-NP) expedites fracture healing. Interestingly, however, NPs are predominantly taken up by fracture-associated macrophages rather than cells typically associated with fracture healing. Therefore, the underlying mechanism of healing via TBP-NP is comprehensively investigated herein. TBP-NPAR28 promotes M2 macrophage polarization and enhances osteogenesis in preosteoblast-macrophage co-cultures in vitro. Longitudinal analysis of TBP-NPAR28 -mediated fracture healing reveals distinct spatial distributions of M2 macrophages, an increased M2/M1 ratio, and upregulation of anti-inflammatory and downregulated pro-inflammatory genes compared to controls. This work demonstrates the underlying therapeutic mechanism of bone-targeted NP DDS, which leverages macrophages as druggable targets and modulates M2 macrophage polarization to enhance fracture healing, highlighting the therapeutic benefit of this approach for fractures and bone-associated diseases.

Recent Progress in the Development of New Antiepileptic Drugs with Novel Targets.

Epilepsy is a chronic neurological disorder that affects approximately 50-70 million people worldwide. Epilepsy has a significant economic and social burden on patients as well as on the country. The recurrent, spontaneous seizure activity caused by abnormal neuronal firing in the brain is a hallmark of epilepsy. The current antiepileptic drugs provide symptomatic relief by restoring the balance of excitatory and inhibitory neurotransmitters. Besides, about 30% of epileptic patients do not achieve seizure control. The prevalence of adverse drug reactions, including aggression, agitation, irritability, and associated comorbidities, is also prevalent. Therefore, researchers should focus on developing more effective, safe, and disease-modifying agents based on new molecular targets and signaling cascades. This review overviews several clinical trials that help identify promising new targets like lactate dehydrogenase inhibitors, c-jun n-terminal kinases, high mobility group box-1 antibodies, astrocyte reactivity inhibitors, cholesterol 24-hydroxylase inhibitors, glycogen synthase kinase-3 beta inhibitors, and glycolytic inhibitors to develop a new antiepileptic drug. Approximately 30% of epileptic patients do not achieve seizure control. The current anti-seizure drugs are not disease modifying, cure or prevent epilepsy. Lactate dehydrogenase inhibitor, cholesterol 24-hydroxylase inhibitor, glycogen synthase kinase-3 beta inhibitors, and mTOR inhibitors have a promising antiepileptogenic effect.

Different Effects and Mechanisms of Selenium Compounds in Improving Pathology in Alzheimer’s Disease

Owing to the strong antioxidant capacity of selenium (Se) in vivo, a variety of Se compounds have been shown to have great potential for improving the main pathologies and cognitive impairment in Alzheimer's disease (AD) models. However, the differences in the anti-AD effects and mechanisms of different Se compounds are still unclear. Theoretically, the absorption and metabolism of different forms of Se in the body vary, which directly determines the diversification of downstream regulatory pathways. In this study, low doses of Se-methylselenocysteine (SMC), selenomethionine (SeM), or sodium selenate (SeNa) were administered to triple transgenic AD (3× Tg-AD) mice for short time periods. AD pathology, activities of selenoenzymes, and metabolic profiles in the brain were studied to explore the similarities and differences in the anti-AD effects and mechanisms of the three Se compounds. We found that all of these Se compounds significantly increased Se levels and antioxidant capacity, regulated amino acid metabolism, and ameliorated synaptic deficits, thus improving the cognitive capacity of AD mice. Importantly, SMC preferentially increased the expression and activity of thioredoxin reductase and reduced tau phosphorylation by inhibiting glycogen synthase kinase-3 beta (GSK-3β) activity. Glutathione peroxidase 1 (GPx1), the selenoenzyme most affected by SeM, decreased amyloid beta production and improved mitochondrial function. SeNa improved methionine sulfoxide reductase B1 (MsrB1) expression, reflected in AD pathology as promoting the expression of synaptic proteins and restoring synaptic deficits. Herein, we reveal the differences and mechanisms by which different Se compounds improve multiple pathologies of AD and provide novel insights into the targeted administration of Se-containing drugs in the treatment of AD.

PS-B09-1: ENOS/NO SIGNALING REGULATES RENAL INTERSTITIAL FIBROSIS BY INCREASING CYTOPLASMIC BETA-CATENIN DEGRADATION

Renal interstitial fibrosis is a final common pathway in progressive renal disease. Recently, activation of the wnt/beta-catenin signaling pathway has been demonstrated to play an important role in the development of renal fibrosis. It is also well known that endothelial dysfunction is associated with chronic kidney disease. However, the role of endothelial dysfunction in the progression of renal fibrosis remains largely elusive. We hypothesized that protein kinase G (PKG) activation by nitric oxide (NO) could phosphorylate beta-catenin and downregulate beta-catenin signaling in the process of renal fibrosis. We showed that beta-catenin levels in the cytoplasm were decreased by activation of the NO-PKG pathway. Renal fibrotic changes were not observed in wild type (WT)-lithium (Li; glycogen synthase kinase-3beta inhibitor) mice. However, renal interstitial fibrosis developed in endothelial nitric oxide synthase knockout (eNOSKO)-Li mice compared with WT-Li mice. beta-catenin protein expression was remarkably increased in eNOSKO-Li mice. These changes were suppressed in eNOSKO-Li/Bay (soluble guanylate cyclase [sGC] stimulator) mice. Moreover, increased interstitial renal fibrosis was observed in eNOSKO mice subjected to unilateral ureteral obstruction (eNOSKO-UUO) compared with WT-UUO mice. beta-catenin protein expression was significantly increased in eNOSKO-UUO mice. These phenotypic changes in eNOSKO-UUO mice were suppressed by Bay treatment. Activation of eNOS signaling could be protective against renal interstitial fibrosis. These findings indicate the potential therapeutic utility of eNOS-NO-PKG stimulation for the amelioration of renal fibrosis.

Clinical activity of 9-ING-41, a small molecule selective glycogen synthase kinase-3 beta (GSK-3β) inhibitor, in refractory adult T-Cell leukemia/lymphoma

ABSTRACT GSK-3β is a serine/threonine kinase implicated in tumorigenesis and chemotherapy resistance. GSK-3β blockade downregulates the NF-κB pathway, modulates immune cell PD-1 and tumor cell PD-L1 expression, and increases CD8 + T cell and NK cell function. We report a case of adult T-cell leukemia/lymphoma (ATLL) treated with 9-ING-41, a selective GSK-3β inhibitor in clinical development, who achieved a durable response. A 43-year-old male developed diffuse lymphadenopathy, and biopsy of axillary lymph node showed acute-type ATLL. Peripheral blood flow cytometry revealed a circulating clonal T cell population, and CSF was positive for ATLL involvement. After disease progression on the 3rd line of treatment, he started treatment with 9-ING-41 monotherapy in a clinical trial (NCT03678883). CT imaging after seven months showed a partial response. Sustained reduction of peripheral blood ATLL cells lasted 15 months. Treatment of patient-derived CD8 + T cells with 9-ING-41 increased the secretion of IFN-γ, granzyme B, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In conclusion, treatment of a patient with refractory ATLL with the GSK-3β inhibitor 9-ING-41 resulted in a prolonged response. Ongoing experiments are investigating the hypothesis that 9-ING-41-induced T cell activation and immunomodulation contributes to its clinical activity. Further clinical investigation of 9-ING-41 for treatment of ATLL is warranted.

Phase 1/2 study of elraglusib (9-ING-41), a small molecule selective glycogen synthase kinase-3 beta (GSK-3β) inhibitor, alone or with irinotecan, temozolomide/irinotecan or cyclophosphamide/topotecan in pediatric patients with refractory malignancies: Interim results.

e22015 Background: GSK-3β overexpression is associated with aggressive malignancies, treatment resistance and poor prognosis. The GSK-3β inhibitor Elraglusib induces apoptosis via NFΚB and p53 pathways and has potent anti-fibrotic and immunomodulatory activity. Adult studies of elraglusib demonstrate clinical activity in pancreatic cancer, melanoma, lymphoma and sarcoma as a single agent or in combination with cytotoxic chemotherapy. Elraglusib is active in in vivo models of neuroblastoma (NBL) and malignant glioma. This first-in-pediatrics study (NCT04239092) is evaluating the safety, pharmacokinetics (PK), and efficacy of elraglusib monotherapy and in combination with chemotherapy in patients with refractory malignancies. Methods: Elraglusib is given intravenously (IV) twice-weekly at 3 dose levels (DL) (9.3, 12.4 and 15 mg/kg) as a single agent or in combination with irinotecan, cyclophosphamide/topotecan or temozolomide/irinotecan in 21-day cycles. A cohort of pts with refractory NBL will be treated at the recommended phase 2 dose (RP2D) of elraglusib with temozolomide/irinotecan. Results: As of January 2022, 23 pts (n = 7 female, median age 14.2 years) have received at least one dose of elraglusib. Tumor types: 5 NBL, 3 diffuse midline glioma (DMG), 3 osteosarcoma (OS), 3 ependymoma (EP), 2 alveolar rhabdomyosarcoma (aRMS), 1 angiosarcoma (AS), 1 Ewing sarcoma (ES), 1 glioblastoma (GBM), 1 hepatoblastoma (HB), 1 embryonal CNS tumor NOS, 1 NUT midline carcinoma, 1 pineoblastoma (PB). Median time from diagnosis is 26 months (range: 6.7 – 156.3) and median number of lines of prior systemic therapy is 2 (range 0-14). Two DLs of single agent (6 pts) have been completed (9.3 and 12.4 mg/kg) without elraglusib-attributable severe adverse events (SAEs). Of the 15 patients on the combination arm with irinotecan or cyclophosphamide/topotecan, a single adverse event (Grade 4 hypotension/infusion reaction) was reported. Grade 1/2 elraglusib attributable-AEs include: transient visual change (n = 10), nausea (n = 7), vomiting (n = 6), fatigue (n = 2), hypotension (n = 2) and infusion reaction (n = 1). One pt with recurrent ES had a radiographic and pathologic CR after 3 cycles of elraglusib/cyclophosphamide/topotecan. 6 pts (26.1%) had SD (2 NBL, 1 aRMS, 1 EP, 1 OS, 1 GBM). 8 pts (35%) remained on study treatment ≥ 3 months (2 NBL, 2 EP, 1 OS, 1 aRMS, 1 ES, 1 PB). Median treatment duration was 40 days (range 1 - 126). 4 pts remain on therapy. Conclusions: Elraglusib is well tolerated as a single agent and with several chemotherapy regimens in this heavily pretreated pediatric population with refractory cancers. It has encouraging antitumor activity, with 1 CR in a patient with recurrent ES. Enrollment is ongoing; a RPD2 has not been reached. Clinical trial information: NCT04239092.

Dexmedetomidine inhibits oxidative stress in sepsis-induced acute kidney injury in rats by regulating GSK-3β/Nrf2/ARE axis

Purpose: To determine the effect of dexmedetomidine on sepsis-mediated acute kidney injury (SAKI) in rats.
 Methods: Twenty-four SD rats were randomly divided into blank untreated control group, model group given lipopolysaccharide at a dose of 10 mg/kg, dexmedetomidine (Dex) group, and glycogen synthase kinase-3 beta (GSK-3β) inhibitor group given sb216763. In addition, Dex and inhibitor groups received the same dose of lipopolysaccharide as model group. There were 10 rats in each group. Pathological changes in kidney tissue, and the levels of related oxidative stress indices were determined.
 Results: Compared with the model group, granular degeneration of renal tubular epithelial cells was significantly reduced in Dex group, and the population of exfoliated cells decreased (p < 0.05). The levels of malondialdehyde (MDA) and reactive oxygen species (ROS) in dexmedetomidine and GSK-3β inhibitor groups were significantly reduced, while superoxide dismutase (SOD), glutathione (GSH) and catalase (CAT) activities were markedly raised, relative to model group (p < 0.05). Protein levels of p- GSK-3β and Nrf2 markedly were raised in dexmedetomidine and GSK-3β inhibitor groups (p < 0.01). After intervention with Dex and GSK-3β inhibitor, there were marked upregulations in mRNA and protein expression levels of heme oxygenase-1 (HO-1), when compared with model group (p < 0.05).
 Conclusion: Dexmedetomidine ameliorates SAKI injury in rats via suppression of oxidative stress. Its mechanism of action may be related to the regulation of GSK-3β/Nrf2/ARE signaling pathway. This finding may be a novel approach to the clinical management of SAKI.

Phase 2 study of 9-ING-41, a small molecule selective glycogen synthase kinase-3 beta (GSK-3β) inhibitor, with gemcitabine/nab-paclitaxel (GnP) in first-line advanced pancreatic ductal adenocarcinoma (PDAC).

578 Background: GSK-3β overexpression is associated with worse prognosis and chemotherapy resistance in PDAC. GSK-3β inhibition reverses chemotherapy-resistance by inhibiting chemotherapy-induced, ATR-mediated, DNA damage response. 9-ING-41 has significant anti-tumor activity through apoptosis induction, anti-fibrotic activity and NK/T-cell effector stimulation. We hypothesized that 9-ING-41 in combination with GnP chemotherapy would lead to anti-tumor activity, with improved tumor responses in patients with advanced PDAC in the first line setting. Methods: Primary endpoint is disease control rate (DCR). DCR = confirmed complete response (CR), partial response (PR), or stable disease (SD) ≥ 16 weeks (wks). Secondary endpoints are safety and ORR (overall response rates). Eligibility: Advanced PDAC, ECOG PS 0-2, no prior therapy in the metastatic setting and no systemic therapy in prior 6 months. Pts received 9-ING-41 15mg/kg IV twice-weekly with G 1,000 mg/m2 and nP 125 mg/m2 on days 1,8,15 of a 28-day cycle. Simon 2-Stage Design for DCR of 65% and null hypothesis of 50% (historical control), 80% power and 2 sided-significance level of.05. Up to 23 fully evaluable pts planned for stage 1 and if ≥ 12 evaluable patients achieve a DCR 37 additional pts will be enrolled on a second stage or a randomized study commenced. Results: As of Sept 27, 2021, 42 pts enrolled. Median age: 67. 24 females, 18 males. 38 pts with metastatic and 4 with locally advanced disease. Prior adjuvant therapy: 4 pts each FOLFIRINOX and gemcitabine-based. No 9-ING-41-attributable SAEs to date. 9-ING-41 attributed AEs: visual disturbance: 24 (75%) G1/2, 1 (3%) G3; infusion reactions 9 (28%) G1/2. Chemotherapy-related AEs: anemia 13 (40%) G1/2, 1 (3%) G3; neutropenia 2 (6%) G1/2, 13 (40%) G3/4; thrombocytopenia 9 (28%) G1/2, 2 (6%) G3/4; diarrhea 8 (25%) G1/2, 4 (13%) G3; fatigue 9 (28%) G1/2, 3 (9%) G3; nausea/vomiting 24 (75%) G1/2, 1 (3%) G3; constipation 9 (28%) G1/2; febrile neutropenia 5 (16%) G3/4. In 21 pts currently evaluable for response, DCR was 62% and ORR 43%. There were 2 confirmed CRs, 6 confirmed and 1 unconfirmed PRs, 4 SD and 8 disease progressions were observed. Amongst responder’s median duration of response has not yet been reached. Conclusions: 9-ING-41 plus GnP demonstrated encouraging clinical activity but chemotherapy-related AEs were significant. Based on efficacy data to date, including confirmed CRs, we have commenced a randomized phase 2 study, evaluating 9-ING-41 plus GnP vs Gn P alone. Enrollment to the randomized study is now open (NCT03678883). Clinical trial information: NCT03678883.

Lithium modulates multiple tau kinases with distinct effects in cortical and hippocampal neurons according to concentration ranges.

The hyperphosphorylation of tau is a central mechanism in the pathogenesis of Alzheimer's disease (AD). Lithium is a potent inhibitor of glycogen synthase kinase-3beta (GSK3β), the most important tau kinase in neurons, and may also affect tau phosphorylation by modifying the expression and/or activity of other kinases, such as protein kinase A (PKA), Akt (PKB), and calcium calmodulin kinase-II (CaMKII). The aim of the present study is to determine the effect of chronic lithium treatment on the protein expression of tau and its major kinases in cortical and hippocampal neurons, at distinct working concentrations. Primary cultures of cortical and hippocampal neurons were treated with sub-therapeutic (0.02mM and 0.2mM) and therapeutic (2mM) concentrations of lithium for 7days. Protein expression of tau and tau-kinases was determined by immunoblotting. An indirect estimate of GSK3β activity was determined by the GSK3β ratio (rGSKβ). Statistically significant increments in the protein expression of tau and CaMKII were observed both in cortical and hippocampal neurons treated with subtherapeutic doses of lithium. GSK3β activity was increased in cortical, but decreased in hippocampal neurons. Distinct patterns of changes in the expression of the remaining tau tau-kinases were observed: in cortical neurons, lithium treatment was associated with consistent decrements in Akt and PKA, whereas hippocampal neurons displayed increased protein expression of Akt and decreased PKA. Our results suggest that chronic lithium treatment may yield distinct biological effects depending on the concentration range, with regional specificity. We further suggest that hippocampal neurons may be more sensitive to the effect of lithium, presenting with changes in the expression of tau-related proteins at subtherapeutic doses, which may not be mirrored by the effects observed in cortical neurons.

GSK-3β in Pancreatic Cancer: Spotlight on 9-ING-41, Its Therapeutic Potential and Immune Modulatory Properties.

Simple SummaryGlycogen synthase kinase-3 beta is a protein kinase implicated in the promotion and development of various cancers, including pancreatic cancer. In cell culture and animal studies, drugs targeting the inhibition of this protein show treatment potential in pancreatic cancer. Studies show targeting this protein for treatment may overcome resistance to conventional chemotherapy in pancreatic tumors. Early-stage clinical trials are currently studying small molecule inhibitors targeting glycogen synthase kinase-3 beta and interim results show favorable results. Recent studies also suggest that targeting this protein will create synergy with immunotherapy, such as checkpoint inhibitors. Future studies should aim to study new combination treatments involving glycogen synthase kinase-3 beta targeting drugs with chemotherapy and immunotherapy in pancreatic cancer.Glycogen synthase kinase-3 beta is a ubiquitously and constitutively expressed molecule with pleiotropic function. It acts as a protooncogene in the development of several solid tumors including pancreatic cancer through its involvement in various cellular processes including cell proliferation, survival, invasion and metastasis, as well as autophagy. Furthermore, the level of aberrant glycogen synthase kinase-3 beta expression in the nucleus is inversely correlated with tumor differentiation and survival in both in vitro and in vivo models of pancreatic cancer. Small molecule inhibitors of glycogen synthase kinase-3 beta have demonstrated therapeutic potential in pre-clinical models and are currently being evaluated in early phase clinical trials involving pancreatic cancer patients with interim results showing favorable results. Moreover, recent studies support a rationale for the combination of glycogen synthase kinase-3 beta inhibitors with chemotherapy and immunotherapy, warranting the evaluation of novel combination regimens in the future.

LiCl induces apoptosis via CHOP/NOXA/Mcl-1 axis in human choroidal melanoma cells

BackgroundChoroidal melanoma is the most common primary intraocular malignancy that occurs in adults. Lithium Chloride Promotes Apoptosis in Human Leukemia NB4 Cells by Inhibiting Glycogen Synthase Kinase-3 Beta. In this study, we aimed to understand whether LiCl exerts anticancer effects on choroidal melanoma cells and elucidate the underlying molecular mechanisms.MethodsHuman choroidal melanoma cells were treated with LiCl, and cell survival was assessed with MTT assays. Cell reproductive viability was measured by plate colony formation assays. Cell apoptosis was evaluated using flow cytometry, and proteins were detected using western blotting. A human choroidal melanoma xenograft model was established to demonstrate the effect of LiCl on human choroidal melanoma in vivo.ResultsWe found that LiCl inhibited cell survival and clonogenic potential and induced apoptosis in human choroidal melanoma cells. LiCl also reduced the proliferation of choroidal melanoma cells in vivo. Moreover, the upregulation of NOXA and downregulation of Mcl-1 were responsible for LiCl-induced apoptosis. Mcl-1 overexpression obviously impaired LiCl-induced apoptosis and cleavage of caspase8, caspase9, caspase3 and PARP. Moreover, the protein expression of endoplasmic reticulum stress markers, including IRE1α, Bip, p-eIF2α, ATF4 and CHOP, were upregulated following treatment with LiCl. When CHOP expression was knocked down and cells were treated with LiCl, the protein level of NOXA was partially increased, and Mcl-1 expression was increased, while the cleavage of caspase8, caspase9, caspase3 and PARP that was induced by the LiCl was reduced compared with the vehicle treated group. Prolonged ER stress results in the activation of the apoptotic pathway.ConclusionsIn summary, LiCl induced an endoplasmic reticulum stress response while activating intrinsic apoptosis. Furthermore, the CHOP/NOXA/Mcl-1 axis contributed to LiCl-induced apoptosis both in vitro and in vivo. The present study provides important mechanistic insight into potential cancer treatments involving LiCl and enhances the understanding of human choroidal melanoma.

Inhibitors of Gq signalling down-regulate β-catenin expression & function in human colon cancer cells.

Background & objectives:β-catenin signalling plays a key role in maintaining normal cellular physiology, and therefore, its deregulation can lead to many human diseases including cancers. Previously, we have shown that the activation of Gq signalling positively regulates β-catenin by inhibiting glycogen synthase kinase-3 beta and increasing the stability of β-catenin protein, however, these results were mainly based on overexpression experiments in either Xenopus oocytes or HEK293T cells. The present study was undertaken to evaluate the modulation of Gq signalling in human colon cancer cells.Methods:Gq signalling in SW480 and HT-29 colon cancer cells was specifically blocked to investigate the interaction between β-catenin and the Gq signalling pathways. GP antagonist-2A (a commercially available peptide) and a minigene expression construct encoding a peptide corresponding to the C-terminal 11 amino acids of Gαq were used to block Gq signalling. β-catenin expression and function were examined by western blotting, immunofluorescence microscopy, and quantitative real-time PCR experiments.Results:Transfection of cells with either of the blockers significantly decreased both β-catenin protein levels and β-catenin-mediated transcriptional activities. In addition, the migration of SW480 cells was reduced in the presence of the Gq blockers.Interpretation & conclusions:The results of this study further support the positive role of Gq signalling in regulating β-catenin expression and function and may provide a new means of preventing β-catenin-mediated carcinogenesis by blocking heterotrimeric G proteins.