Glycogen Synthase Kinase-3alpha Research Articles

GSK-3α-BNIP3 axis promotes mitophagy in human cardiomyocytes under hypoxia

Dysregulated autophagy/mitophagy is one of the major causes of cardiac injury in ischemic conditions. Glycogen synthase kinase-3alpha (GSK-3α) has been shown to play a crucial role in the pathophysiology of cardiac diseases. However, the precise role of GSK-3α in cardiac mitophagy remains unknown. Herein, we investigated the role of GSK-3α in cardiac mitophagy by employing AC16 human cardiomyocytes under the condition of acute hypoxia. We observed that the gain-of-GSK-3α function profoundly induced mitophagy in the AC16 cardiomyocytes post-hypoxia. Moreover, GSK-3α overexpression led to increased ROS generation and mitochondrial dysfunction in cardiomyocytes, accompanied by enhanced mitophagy displayed by increased mt-mKeima intensity under hypoxia. Mechanistically, we identified that GSK-3α promotes mitophagy through upregulation of BNIP3, caused by GSK-3α-mediated increase in expression of HIF-1α and FOXO3a in cardiomyocytes post-hypoxia. Moreover, GSK-3α displayed a physical interaction with BNIP3 and, inhibited PINK1 and Parkin recruitment to mitochondria was observed specifically under hypoxia. Taken together, we identified a novel mechanism of mitophagy in human cardiomyocytes. GSK-3α promotes mitochondrial dysfunction and regulates FOXO3a -mediated BNIP3 overexpression in cardiomyocytes to facilitate mitophagy following hypoxia. An interaction between GSK-3α and BNIP3 suggests a role of GSK-3α in BNIP3 recruitment to the mitochondrial membrane where it enhances mitophagy in stressed cardiomyocytes independent of the PINK1/Parkin.

Differential role of bovine serum albumin and HCO3- in the regulation of GSK3 alpha during mouse sperm capacitation.

To become fertile, mammalian sperm are required to undergo capacitation in the female tract or in vitro in defined media containing ions (e.g. HCO3 -, Ca2+, Na+, and Cl-), energy sources (e.g. glucose, pyruvate) and serum albumin (e.g. bovine serum albumin (BSA)). These different molecules initiate sequential and concomitant signaling pathways, leading to capacitation. Physiologically, capacitation induces changes in the sperm motility pattern (e.g. hyperactivation) and prepares sperm for the acrosomal reaction (AR), two events required for fertilization. Molecularly, HCO3 - activates the atypical adenylyl cyclase Adcy10 (aka sAC), increasing cAMP and downstream cAMP-dependent pathways. BSA, on the other hand, induces sperm cholesterol release as well as other signaling pathways. How these signaling events, occurring in different sperm compartments and with different kinetics, coordinate among themselves is not well established. Regarding the AR, recent work has proposed a role for glycogen synthase kinases (GSK3α and GSK3β). GSK3α and GSK3β are inactivated by phosphorylation of residues Ser21 and Ser9, respectively, in their N-terminal domain. Here, we present evidence that GSK3α (but not GSK3β) is present in the anterior head and that it is regulated during capacitation. Interestingly, BSA and HCO3 - regulate GSK3α in opposite directions. While BSA induces a fast GSK3α Ser21 phosphorylation, HCO3 - and cAMP-dependent pathways dephosphorylate this residue. We also show that the HCO3--induced Ser21 dephosphorylation is mediated by hyperpolarization of the sperm plasma membrane potential (Em) and by intracellular pH alkalinization. Previous reports indicate that GSK3 kinases mediate the progesterone-induced AR. Here, we show that GSK3 inhibition also blocks the Ca2+ ionophore ionomycin-induced AR, suggesting a role for GSK3 kinases downstream of the increase in intracellular Ca2+ needed for this exocytotic event. Altogether, our data indicate a temporal and biphasic GSK3α regulation with opposite actions of BSA and HCO3 -. Our results also suggest that this regulation is needed to orchestrate the AR during sperm capacitation.

Active Ingredient Paeonol of Jijiu Huiyang Decoction Alleviates Isoproterenol-Induced Chronic Heart Failure via the GSK3A/PPARα Pathway.

The pharmacological mechanism of the traditional Chinese medicine formula-Jijiu Huiyang decoction (JJHYD), which contains several herbal medicines for the treatment of chronic heart failure (CHF), is yet unknown. Method and Materials. The main active components of JJHYD were analyzed by ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS/MS). The target genes of JJHYD and CHF were retrieved through multiple databases, a drug-ingredient-target-disease network was created, and KEGG enrichment and GO analyses were carried out. The binding ability of paeonol and Glycogen Synthase Kinase-3 alpha (GSK3A) was confirmed by molecular docking. CHF animal model and cell model were constructed. The effects of paeonol on cardiac dysfunction, myocardial hypertrophy, cardiac lipid accumulation, and myocardial apoptosis were detected by echocardiography, histopathology, and flow cytometry, respectively. The effects of paeonol on the expression of myocardial hypertrophy index, GSK3A, and genes or proteins related to the PPARα pathway were determined by qRT-PCR or western blot. UHPLC-MS/MS analysis combined with database verification showed a total of 227 chemical components in JJHYD, among which paeonol was the one with heart-protective roles and had the highest content. Paeonol alleviated isoproterenol-induced cardiac lipid accumulation, cardiac hypertrophy, and myocardial dysfunction and inhibited the activation of the PPARα pathway, while overexpression of GSK3A reversed these effects of paeonol. However, the reversal effects of GSK3A overexpression could be offset by siPPARα. As the main active substance of JJHYD, paeonol participates in the protection of CHF by targeting the GSK3A/PPARα signaling pathway to reduce lipid toxicity.

Design, synthesis and molecular docking of new fused 1H-pyrroles, pyrrolo[3,2-d]pyrimidines and pyrrolo[3,2-e][1, 4]diazepine derivatives as potent EGFR/CDK2 inhibitors

A new series of 1H-pyrrole (6a–c, 8a–c), pyrrolo[3,2-d]pyrimidines (9a–c) and pyrrolo[3,2-e][1, 4]diazepines (11a–c) were designed and synthesised. These compounds were designed to have the essential pharmacophoric features of EGFR Inhibitors, they have shown anticancer activities against HCT116, MCF-7 and Hep3B cancer cells with IC50 values ranging from 0.009 to 2.195 µM. IC50 value of doxorubicin is 0.008 µM, compounds 9a and 9c showed IC50 values of 0.011 and 0.009 µM respectively against HCT-116 cells. Compound 8b exerted broad-spectrum activity against all tested cell lines with an IC50 value less than 0.05 µM. Compound 8b was evaluated against a panel of kinases. This compound potently inhibited CDK2/Cyclin A1, DYRK3 and GSK3 alpha kinases with 10–23% compared to imatinib (1–10%). It has also arrested the cell cycle of MCF-7 cells at the S phase. Its antiproliferative activity was further augmented by molecular docking into the active sites of EGFR and CDK2 cyclin A1.

Modeling Sporadic Alzheimer's Disease in Human Brain Organoids under Serum Exposure.

Alzheimer's disease (AD) is a progressive neurodegenerative disease with no cure. Huge efforts have been made to develop anti‐AD drugs in the past decades. However, all drug development programs for disease‐modifying therapies have failed. Possible reasons for the high failure rate include incomplete understanding of complex pathophysiology of AD, especially sporadic AD (sAD), and species difference between humans and animal models used in preclinical studies. In this study, sAD is modeled using human induced pluripotent stem cell (hiPSC)‐derived 3D brain organoids. Because the blood–brain barrier (BBB) leakage is a well‐known risk factor for AD, brain organoids are exposed to human serum to mimic the serum exposure consequence of BBB breakdown in AD patient brains. The serum‐exposed brain organoids are able to recapitulate AD‐like pathologies, including increased amyloid beta (Aβ) aggregates and phosphorylated microtubule‐associated tau protein (p‐Tau) level, synaptic loss, and impaired neural network. Serum exposure increases Aβ and p‐Tau levels through inducing beta‐secretase 1 (BACE) and glycogen synthase kinase‐3 alpha / beta (GSK3α/β) levels, respectively. In addition, single‐cell transcriptomic analysis of brain organoids reveals that serum exposure reduced synaptic function in both neurons and astrocytes and induced immune response in astrocytes. The human brain organoid‐based sAD model established in this study can provide a powerful platform for both mechanistic study and therapeutic development in the future.

Phosphoproteomic analysis of lung tissue from patients with pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rare disorder associated with high morbidity and mortality despite currently available treatments. We compared the phosphoproteome of lung tissue from subjects with idiopathic PAH (iPAH) obtained at the time of lung transplant with control lung tissue. The mass spectrometry-based analysis found 60,428 phosphopeptide features from which 6622 proteins were identified. Within the subset of identified proteins there were 1234 phosphopeptides with q < 0.05, many of which are involved in immune regulation, angiogenesis, and cell proliferation. Most notably there was a marked relative increase in phosphorylated (S378) IKZF3 (Aiolos), a zinc finger transcription factor that plays a key role in lymphocyte regulation. In vitro phosphorylation assays indicated that GSK3 alpha and/or GSK3 beta could phosphorylate IKZF3 at S378. Western blot analysis demonstrated increased pIKZF3 in iPAH lungs compared to controls. Immunohistochemistry demonstrated phosphorylated IKZF3 in lymphocytes surrounding severely hypertrophied pulmonary arterioles. In situ hybrization showed gene expression in lymphocyte aggregates in PAH samples. A BCL2 reporter assay showed that IKZF3 increased BCL2 promoter activity and demonstrated the potential role of phosphorylation of IKZF3 in the regulation of BCL mediated transcription. Kinase network analysis demonstrated potentially important regulatory roles of casein kinase 2, cyclin-dependent kinase 1 (CDK1), mitogen-associated protein kinases (MAPKs), and protein kinases (PRKs) in iPAH. Bioinformatic analysis demonstrated enrichment of RhoGTPase signaling and the potential importance of cGMP-dependent protein kinase 1 (PRKG). In conclusion, this unbiased phosphoproteomic analysis demonstrated several novel targets regulated by kinase networks in iPAH, and reinforced the potential role of immune regulation in the pathogenesis of iPAH. The identified up- and down-regulated phosphoproteins have potential to serve as biomarkers for PAH and to provide new insights for therapeutic strategies.

HnRNPA1 ISOFORM BRING DIVERSITY IN GLIOMA CELL SURVIVAL

Hypoxia response plays a critical impact on tumor growth and drug resistance development. Under hypoxic and non-hypoxic growth environment U87MG and A172 glioma cells behaved differently. The presence of hnRNPA1 isoforms variant showed critical regulatory step in directing glioma cell adaptability. Knocking hnRNPA1, distinctly affecting the hnRNPA1 isoforms in two glioma cell lines. U87 MG cells, in response two different siRNA against hnRNPA1, suppresses higher isoform of hnRNPA1 compare to lower isoforms, on contrary A172 responses predominantly with up regulation of hnRNPA1vII (higher isoform) only. Leading expression of HnRNPA1vII isoform down regulates phospho-ERK½ activation. Additionally, knock down of GSK3 alpha also enhances the generation of higher isoform of hnRNPA1 in non-hypoxic cells. Remarkably, cells grown under hypoxic condition, phospho-ERK½ activation was observed only in presence of lower abundant isoform of hnRNPA1. Furthermore, immunohistochemistry was conducted to recapitulate the moderate to weak expression of GSK3 alpha correlated with strong expression of hnRNPA1 in glioma hypoxic tumor. Nonetheless, selective knock down of hnRNPA1vII (higher form) and vI (lower isoform) in U87MG were examined, evidently the phospho-ERK½ expression was downregulated in hnRNPA1vI knock down, compared with higher phospho-ERK½ in hnRNPA1 higher isoform (vII) knock down was observed. Interestingly, knock down of hnRNPA1 vII (higher isoform) also reduces the expression of hnRNPA2/B1 and SF2/ASF1 (splice factor RNA binding protein), in U87 MG cells. In conclusion, selective targeting hnRNPA1 isoforms attenuate glioma cell metabolism, and promote therapeutic sensitization.

2092-P: Invalidation of GSK3B in Pancreatic Beta Cell as a Therapeutic Target for T2D

Insufficient pancreatic β-cell mass or function is one of the main causes underlying diabetes mellitus. Glycogen synthase kinase 3 (GSK3) is a ubiquitously expressed serine/threonine kinase. GSK3 is encoded by two known genes, GSK3 alpha (GSK3A) and GSK3 beta (GSK3B), and small molecule inhibitors targeting both are known to induce micronucleation. Isoform specific small molecule inhibitors targeting GSK3B have demonstrated increased cell proliferation and expansion of islets mass in human and rodents. However, many of the inhibitors target dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) in addition to GSK3B making the data difficult to interpret (Shen et al, Nature comms 2015). Mice with β-cell overexpression of GSK3B have reduced β-cell mass and proliferation through modulation of PDX-1 stability. Recently, Ämmälä et al. demonstrated conjugation of ASOs to glucagon-like-peptide-1 receptor (GLP-1R) ligand can productively deliver ASO cargo to the insulin secreting β-cells. The doses needed for GLP-1-conjugated ASOs to silence target genes in the β-cells are below what is needed to affect target gene expression elsewhere allowing for evaluation of specific inhibition in the β-cells. In lean C57B6 mice a single administration of GLP-1-conjugated GSK3B ASO achieved an ED50=0.016µmol/kg with no change to DYRK1A RNA expression. Here we demonstrate robust, specific GSK3B reduction in the β-cells after GLP-1-conjugated GSK3B ASO treatment in db/db mice. In contrast to the SMI data, specific inhibition of GSK3B in β-cells worsened the health based on reductions in β-cell specific genes, PDX1 and MAFA, and decreased the β-cell mass (2.9 ± 1.3mg GLP-1-Con ASO vs. 1.4 ± 0.6mg GLP-1-GSK3B ASO). In addition, no effect on glucose or insulin was observed suggesting specific inhibition of GSK3B in the β-cell is not enough to preserve the function and increase β-cell survival. In summary, we have invalidated GSK3B as a therapeutic target for protecting the β-cell against glucotoxicity and T2D. Disclosure S. Murray: Employee; Self; Ionis Pharmaceuticals. J. Zhang: None. A. Ferng: None. T. Prakash: None. R. Lee: Employee; Self; Ionis Pharmaceuticals, Inc. E. Andersson: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. D. Janzén: None. L. Knerr: None. C. Ammala: None. S. Guo: Employee; Self; Ionis Pharmaceuticals, Inc.

Dietary Leucine Supplementation Restores Serum Glucose Levels, and Modifying Hepatic Gene Expression Related to the Insulin Signal Pathway in IUGR Piglets.

Simple SummaryIntrauterine malnutrition may compromise the size and structure of fetal organs and tissues, which leads to lower birth weight and a slower rate of growth after weaning. Intrauterine growth restriction/retardation (IUGR) impairs pancreas function, resulting in the decreased glucose levels in serum. Leucine, one of branched chain amino acids, is an essential amino acid and the substrate of protein synthesis. Leucine also acts as a major regulator of hormone signal transduction, like insulin. Dietary branched chain amino acids or leucine have beneficial effects on the glucose metabolism and glycogen synthesis of muscle. Leucine supplementation improves the insulin sensitivity in liver and muscle and then influences the systemic glucose homeostasis. However, it is still unclear whether leucine supplementation would alter insulin sensitivity in IUGR neonatal piglets. Our results showed that dietary leucine supplementation restored serum glucose concentrations, increased insulin and creatinine concentrations, and enhanced protein kinase adenosine monophosphate-activated γ 3-subunit and glucose transporter type 2 expression. These findings suggest that leucine might play a positive role in hepatic lipid metabolism and glucose metabolism in IUGR.This study aimed to investigate the effects of leucine with different levels on the insulin resistance in intrauterine growth restriction/retardation (IUGR) piglets. Thirty-two weaned piglets were arranged in a 2 × 2 factorial design and four treatments (n = 8) were as follow: (1) normal weaned piglets fed a basal diet (CONT), (2) IUGR weaned piglets fed a basal diet (IUGR), (3) normal weaned piglets fed a basal diet with the addition of 0.35% l-leucine (C-LEU), and (4) IUGR fed a basal diet with the addition of 0.35% l-leucine (I-LEU) for a 21-days trial. The results showed that compared to the IUGR group, the I-LEU group had higher final body weight and body weight gain, higher serum glucose concentrations, and higher serum insulin concentrations (p < 0.05). The gene expression of phosphatidylinositol 3-kinase p110 gamma, protein kinase adenosine monophosphate-activated γ 3-subunit, glycogen synthase kinase-3 alpha, and glucose transporter type 2 were increased in the I-LEU group as compared to the IUGR group (p < 0.05). It was concluded that dietary leucine supplementation restored serum glucose concentrations, increased insulin and creatinine concentrations, and enhanced protein kinase adenosine monophosphate-activated γ 3-subunit and glucose transporter type 2 expression, suggesting that leucine might play a positive role in hepatic lipid metabolism and glucose metabolism in IUGR.

Cross-talk between ER stress and mitochondrial pathway mediated adriamycin-induced testicular toxicity and DA-9401 modulate adriamycin-induced apoptosis in Sprague\u2013Dawley rats

BackgroundDA-9401 was prepared as a mixture of Chinese medicinal herb extracts from roots of Morinda officinalis How (Rubiaceae), outer scales of Allium cepa L. (Liliceae) and seeds of Cuscuta chinensis Lamark (Convolvulaceae). The present study was designed to investigate the possible protective role of DA-9401 in adriamycin (ADR)-induced testicular toxicity associated with oxidative stress, endoplasmic reticulum (ER) stress, and apoptosis.MethodsFifty healthy 8-week-old male Sprague–Dawley rats were equally divided into five groups. The first CTR group was treated with normal saline 2 ml/day by gavage. The second was treated with DA-100 (DA-9401 100 mg/kg/day). The third (ADR) group received ADR (2 mg/kg/once a week) intraperitoneally, while the combination of ADR and DA-9401 was given to the fourth ADR + DA-100 (100 mg/kg/day p.o) group and fifth ADR + DA-200 (200 mg/kg/day p.o) group. At the end of the 8-week treatment period, body weight, reproductive organ weights, fertility rate, pups per female were recorded, and serum were assayed for hormone concentrations. Tissues were subjected to semen analysis, histopathological changes, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), oxidative stress markers and expression levels of endoplasmic reticulum (ER) stress markers, apoptosis markers, tight junction protein markers, steroidogenic acute regulatory protein (StAR), cation channel of sperm (CatSper) and glycogen synthase kinase-3 (GSK-3) by western blot.ResultsDA-9401 administration to ADR-treated rats significantly decreased serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels, interleukin-6, TNF-α, MDA level, ROS/RNS level, ER stress response protein levels, tunnel positive cells, cleaved caspase-3, and Bax/Bcl2 ratio. Moreover, pretreatment with DA-9401 significantly increased body weight, reproductive organ weights, fertility rate, pups per female, Johnsen’s score, spermatogenic cell density, sperm count and sperm motility, serum testosterone concentration, testicular superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), tight junction protein markers, star protein level, CatSper, and GSK-3 level.ConclusionsADR treatment can markedly impair testicular function and induce testicular cell death presumably by causing significant changes in oxidative stress, ER stress, and mitochondrial pathway. DA-9401 exerts beneficial effects against oxidative stress, ER stress, and mitochondria-mediated cell death pathway in testis tissue by up-regulating expression levels of tight junction protein markers, steroidogenic acute regulatory protein, GSK-3 alpha, and cation channels of sperm.

Antagonistic role of GSK3 isoforms in glioma survival.

GSK3 (Glycogen Synthase Kinase-3) function in brain is contributed by two distinct gene GSK3 alpha and GSK3 beta. Present findings indicate that imbalance in between GSK3 alpha and beta isoform contributes oncogenesis. In gliomas, GSK3 isoform specific functions are different then as reported for melanoma, prostate cancer, lung cancer etc. Both the isoforms of GSK3 are inversely regulating hnRNPA1 (RNA binding protein) expression, subsequently affecting RNA alternative splicing (BIN1, RON, Mcl1, PKM) in gliomas. Elevated expression of c-Myc, hnRNPA1, Phospo-ERK1/2 and Cyclin D1 in GSK3 alpha knock down cells, resembles GSK3 beta isoform overexpressing glioma cells, promotes cell survival. HnRNPA1 dependent survival signaling pathway were elaborated using si RNA approach or by over expressing cloned hnRNPA1 gene in U87 glioma cells. Therefore, performed study empirically support GSK3β inhibition along with restoration of GSK3α would be a good strategy to target gliomas.

Identification of a First in Class GSK3-Alpha Selective Inhibitor As a New Differentiation Therapy for AML

Isoform-selective enzyme targeting poses a potential clinical challenge when there is significant homology in the protein activity domains. Glycogen synthase kinase 3-alpha (GSK3α) has been identified previously as a therapeutic target to promote differentiation in acute myeloid leukemia (AML) (Banerji et al, JCI, 2012). The GSK3 family consists of two highly homologous, but functionally non-redundant, isoforms (α and β). These proteins are serine-threonine kinases implicated in a diversity of cellular processes. Concurrent inhibition of both isoforms is known to induce β-catenin stabilization and can therefore increase self-renewal of leukemia initiating cells. Isoform-selective GSK3 suppression does not increase β-catenin levels (Doble et al, Dev Cell, 2007) and thus may offer a new therapeutic approach. GSK3α selective inhibitors, with cell-based activity, have not been reported. Our collaborative study aimed to identify first-in-class GSK3α selective inhibitors and evaluate their activity in AML, a disease in great need for new therapeutic approaches.A series of potent and selective inhibitors for GSK3α/β were discovered through a screening campaign supported by the NIH's Molecular Libraries Probe Centers Network (MLPCN) program. While these compounds are equipotent for GSK3α/β, they demonstrate remarkable selectivity versus the larger kinome (> 100x selective vs 311 kinases). Analysis of the high resolution x-ray crystal structure obtained with BRD9421, one of the lead compounds, and GSK3β revealed a tridentate hydrogen bond binding mode within the ATP catalytic domain. One of these interactions, is to aspartic 133 in GSK3β. This same residue in GSK3α is a glutamic acid and represents one of the two amino acid differences within the ATP binding domain between these two enzymes. GSK3α and β share 98% homology in their catalytic domains, but the exploitation of this hydrogen bond interaction through rational drug design offered an excellent opportunity to produce selective inhibitors for GSK3α and GSK3β. BRD0705 was thus identified as an 8-fold GSK3α isoform-selective inhibitor over GSK3β (IC50 GSK3α: 0.045 vs. GSK3β: 0.350 μM).We first validated BRD0705 isoform-selectivity in a panel of AML cell lines and primary patients (n=3 cell lines and n= 2 patient samples). Treatment with BRD0705 resulted in a dose- and time-dependent decrease in tyr279 GSK3α phosphorylation without any effect on tyr216 GSK3β phosphorylation. No increase in β-catenin protein levels by western blotting and no nuclear localization by immunofluorescence were observed. GSK3α inhibition with BRD0705 led to morphological and surface marker changes (CD11b, CD11c, CD14, CD117) consistent with AML differentiation in a panel of AML cell lines (n=4) and primary AML samples (n=5). BRD0705 also induced a G2/M arrest and impaired methylcellulose colony formation in AML cell lines and primary patient samples. BRD0705 possesses favorable pharmacokinetic properties and was well tolerated without any observed adverse events in C57BL/6 mice with repeated dosing. In vivo efficacy studies in AML models are ongoing.In conclusion, we identified a first-in-class GSK3α isoform-selective inhibitor and conducted preclinical studies validating BRD0705 as a promising new differentiation therapeutic candidate in AML. Small-molecule inhibition of GSK3α induced myeloid differentiation, cell cycle arrest and impaired colony formation without β-catenin stabilization in AML. DisclosuresDeAngelo:Incyte: Consultancy; Novartis: Consultancy; Amgen: Consultancy; Pfizer: Consultancy; Ariad: Consultancy; Agios: Consultancy; Bristol Myers Squibb: Consultancy; Celgene: Consultancy. Stone:Sunesis: Consultancy, Other: DSMB for clinical trial; Roche/Genetech: Consultancy; Agios: Consultancy; Karyopharm: Consultancy; Merck: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Novartis: Research Funding; Celator: Consultancy; Amgen: Consultancy; Pfizer: Consultancy; AROG: Consultancy; Juno: Consultancy. Stegmaier:Novartis Pharmaceuticals: Consultancy.

Abstract 675: Off-target based drug repurposing opportunities for tivantinib in acute myeloid leukemia

Abstract GSK3 alpha has been shown to be a new target in the treatment of acute myeloid leukemia (AML); however current GSK3 inhibitors are unselective and target both GSK3 alpha and GSK3 beta. Interestingly, a known pan-GSK3 inhibitor LiCl has been previously investigated for the treatment of AML yet has met limited clinical success. This could be partly due to the fact that pan-GSK3 inhibition results in beta-catenin stabilization, which has been shown to mediate hematopoietic self-renewal and leukemogenesis. As beta-catenin stabilization requires inhibition of both kinases and most GSK3 inhibitors target GSK3 alpha and GSK3 beta with equal potency, these compounds may possess some significant limitations. We have previously shown GSK3 alpha to be a prominent target of the intended MET inhibitor tivantinib (ARQ197) and that tivantinib shows some specificity for GSK3 alpha over GSK3 beta. We thus hypothesized that tivantinib would be an effective therapy for the treatment of AML. Consistently, tivantinib potently inhibited cellular viability across several AML cell lines. Using an unbiased, mass-spectrometry based chemical proteomics approach; we confirmed GSK3 alpha (and to a slightly lesser extent GSK3 beta) to be targeted by tivantinib in AML. Tivantinib strongly induced apoptosis as compared to the pan-GSK3 inhibitor LiCl in these cells while LiCl showed larger effects on cell differentiation. Interestingly tivantinib caused less stabilization of beta-catenin as compared to LiCl. Subsequent drug combination studies identified the Bcl-2 inhibitor ABT-199 to synergize with tivantinib and to amplify apoptosis as seen by PARP1 cleavage. Furthermore, the combination of tivantinib with ABT-199 completely abrogated beta-catenin stabilization. Tivantinib was able to significantly inhibit colony formation of primary AML patient bone marrow mononuclear cells (BMNCs) and ABT-199 combination showed significant benefit over tivantinib or ABT-199 alone. In summary, tivantinib has potent anticancer activity in AML based on targeting GSK3 alpha; and tivantinib single agent or combination with ABT-199 may represent a novel and exciting opportunity for the treatment of AML. Citation Format: Brent M. Kuenzi, Lily L. Remsing Rix, Sateesh S. Kunigal, Fumi Kinose, Claire E. Knezevic, Gabriela Wright, Jodi L. Kroeger, Jeffrey E. Lancet, Eric Padron, Uwe Rix. Off-target based drug repurposing opportunities for tivantinib in acute myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 675. doi:10.1158/1538-7445.AM2015-675

Loss of GSK-3 Causes Abnormal Astrogenesis and Behavior in Mice.

Altered activity of glycogen synthase kinase-3 (GSK-3) is associated with psychiatric diseases and neurodegenerative diseases. GSK-3 is a key regulator in multiple aspects of neuronal differentiation in the brain. However, little is known about the role of GSK-3 in astrocyte development. To examine the role of GSK-3 in astrocytes, we generated a conditional knockout mouse using a glial fibrillary acidic protein (GFAP)-cre driver, in which the GSK-3 alpha and beta genes are deleted in astrocytes. We found that GFAP-cre-mediated GSK-3 deletion led to a larger brain. The number and size of astrocytes were increased in GSK-3 mutant brains. The levels of GFAP and phospho-STAT3, indicators of astrogenesis, were elevated in GSK-3 mutants. Furthermore, we found upregulation of astrocyte regulatory molecules such as phospho-AKT, phospho-S6, and cyclin D in GSK-3 mutant brains. Finally, GSK-3 mutant mice exhibited aberrant anxiety and social behavior. Our results suggest that GSK-3 plays a significant role in astrocyte development and behavioral control in mice.

In vitro streptozotocin model for modeling Alzheimer-like changes: effect on amyloid precursor protein secretases and glycogen synthase kinase-3.

There is accumulating evidence for a pathogenetic link between sporadic Alzheimer's disease (AD) and diabetes mellitus (DM). At subdiabetogenic doses, the cerebral administration of the diabetogenic substance streptozotocin (STZ) induces an insulin-resistant brain state (IRBS). The aim of the present pilot study was to investigate the effect of STZ on Alzheimer-like characteristics such as amyloid precursor protein (APP) cleavage secretases, betaA4 fragment, and glycogen synthase kinase (GSK) in vitro. Different STZ concentrations (0-5 mM) and incubation intervals (0-48 h) were tested to find appropriate cell culture conditions for further biochemical analyses in human neuroblastoma cells (SK-N-MC). Lactate dehydrogenase (LDH) was measured spectrophotometrically. Intracellular ATP was determined using bioluminescent luciferase assay. Secretase activity (alpha, beta, and gamma) was measured by employing commercial fluorometric secretase activity assay kits, betaA4 fragment by immunoprecipitation. Glycogen synthase kinase-3alpha/beta (total and phospho-GSK) content was assayed by ELISA technique. In vitro STZ administration (1 mM) induced a significant reduction in intracellular ATP concentration without pronounced cell death after 24 and 48 h as measured by LDH. Under these experimental conditions, a significant increase in beta-secretase and a significant drop in alpha-secretase were obtained, whereas gamma-secretase was not changed significantly. Simultaneously, the betaA4 concentration was increased by about threefold. Furthermore, STZ significantly increased total GSK and markedly decreased phospho-GSK. A direct link between STZ, intracellular ATP deficit, and Alzheimer-related enzymes was shown in this in vitro pilot study. Thus, these results support the hypothesis that sporadic AD is being recognized as an IRBS, which can be modulated by in vitro STZ model. Continuing investigations relating pathogenetic mechanisms and AD-like hallmarks are necessary to modulate different cascades of the IRBS using in vitro models.

GSK3A is redundant with GSK3B in modulating drug resistance and chemotherapy-induced necroptosis.

Glycogen Synthase Kinase-3 alpha (GSK3A) and beta (GSK3B) isoforms are encoded by distinct genes, are 98% identical within their kinase domain and perform similar functions in several settings; however, they are not completely redundant and, depending on the cell type and differentiative status, they also play unique roles. We recently identified a role for GSK3B in drug resistance by demonstrating that its inhibition enables necroptosis in response to chemotherapy in p53-null drug-resistant colon carcinoma cells. We report here that, similarly to GSK3B, also GSK3A silencing/inhibition does not affect cell proliferation or cell cycle but only abolishes growth after treatment with DNA-damaging chemotherapy. In particular, blocking GSK3A impairs DNA repair upon exposure to DNA-damaging drugs. As a consequence, p53-null cells overcome their inability to undergo apoptosis and mount a necroptotic response, characterized by absence of caspase activation and RIP1-independent, PARP-dependent AIF nuclear re-localization. We therefore conclude that GSK3A is redundant with GSK3B in regulating drug-resistance and chemotherapy-induced necroptosis and suggest that inhibition of only one isoform, or rather partial inhibition of overall cellular GSK3 activity, is enough to re-sensitize drug-resistant cells to chemotherapy.

GSK3 Alpha and Beta Are New Functionally Relevant Targets of Tivantinib in Lung Cancer Cells

Tivantinib has been described as a potent and highly selective inhibitor of the receptor tyrosine kinase c-MET and is currently in advanced clinical development for several cancers including non-small cell lung cancer (NSCLC). However, recent studies suggest that tivantinib's anticancer properties are unrelated to c-MET inhibition. Consistently, in determining tivantinib's activity profile in a broad panel of NSCLC cell lines, we found that, in contrast to several more potent c-MET inhibitors, tivantinib reduces cell viability across most of these cell lines. Applying an unbiased, mass-spectrometry-based, chemical proteomics approach, we identified glycogen synthase kinase 3 (GSK3) alpha and beta as novel tivantinib targets. Subsequent validation showed that tivantinib displayed higher potency for GSK3α than for GSK3β and that pharmacological inhibition or simultaneous siRNA-mediated loss of GSK3α and GSK3β caused apoptosis. In summary, GSK3α and GSK3β are new kinase targets of tivantinib that play an important role in its cellular mechanism-of-action in NSCLC.

Abstract 2586: Pomegranate components modulate distinct pathways in prostate cancer cells

Abstract Pomegranate juice, when provided as whole juice, modulates specific pathways in prostate cancer cells that are not reflected when its major constituents were tested independently. In order to identify these bioactive components of pomegranate, the peel (P) and arils (J) were isolated, juiced separately and extracted with 80% methanol (PE and JE). The components were enriched using a SPE C18 cartridge and, eluted as fraction I (P-I and J-I - contains mostly polar compounds) and fraction II (P-II and J-II - contains mostly non-polar compounds). Analysis by HPLC-PDA and by direct infusion analysis of these fractions by FTICR-MS revealed the J-I consisted of mostly phenolics including the three anthocyanins in its mono- and di-glycoside forms. P-I consisted mostly of punicalin and, the punicalagins A and B, while P-II and J-II consisted mostly of ellagitannins and free ellagic acid. The ORAC values as Trolox equivalent for the various fractions at 10 µg/ml were: Total peel extract (PE) - 1.64 compared with 2.74 for P-I and 4.01 for P-II, while for the total juice extract (JE) it was 2.62 compared with 7.01 for J-I and 5.47 for J-II. To identify the molecular mechanisms, the individual fractions of peel and juice were tested in prostate cancer cell lines, LNCaP and PC3 and analyzed by Cancer/Apoptosis Phospho Antibody Array and western blot analysis. In LNCaP cell line, BCL-2, PTEN, Cdc25A, p53 and STAT6 were hyper-phosphorylated by J-II, while they were unaltered by J-I. Similarly, GSK3 alpha and beta were hyper-phosphorylated by J-I and unmodified by J-II. When peel fractions were tested, Cdc25C, BCL-2, CREB and JAK were hypo-phosphorylated by P-II, whereas they were unaltered by P-I. Other molecules, such as IRS-1, MAP Kinase, PTEN, STAT3 and JAK2 were hypo-phosphorylated by P-II, and were hyper-phosphorylated by P-I. In PC3 cell line, BCL-2, p53 and CREB were hypo-phosphorylated by J-II, while they were unaltered by J-I. However, PTEN and p70 were hypo-phosphorylated by J-II and hyper-phosphorylated by J-I, while, CDK2, STATs 1, 3 and 6 were hypo-phosphorylated by P-II, they were unaltered by P-I fraction. Thus the components of peel and juice fractions demonstrate unique properties and the ability to modulate distinct pathways in androgen-dependent and independent prostate cancer cells providing a rich set of potential therapy or adjuvant applications. This work is supported by NCI grant CA143676. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2586. doi:1538-7445.AM2012-2586

Renal Collecting Duct Specific GSK 3 alpha Regulates Cellular Distribution and Lithium‐Induced NDI

Lithium (Li) induced nephrogenic diabetes insipidus is associated with reduced Aquaporin 2 (AQP2) levels and increased proliferation of collecting duct cells (CD) in rodent models. Li is a potent inhibitor of glycogen synthase kinase 3 (GSK3). GSK3 inhibition is known to increase cell proliferation in non‐renal cells. In the current study we examined isoform specific roles of GSK3α and GSK3β in CD structure and function in normal and Li treated mice. We found that gene deletion of GSK3 alpha (3 alpha −/−) caused urinary concentrating defect and polyuria at basal conditions. In these mice, AQP2 protein levels were lower and H‐ATPase, a marker for type A intercalated cells were significantly higher, compared to WT or 3β‐CDnull mice. LiCl treatment (4mmol/Kg, daily IP injection) for 6 days increased urine output and reduced AQP2 levels in WT, but not in 3 alpha −/− mice. Immunostaining for PCNA, marker for dividing cells, AQP4 for principal cells and H‐ATPase demonstrated increase in proliferation of principal cells of cortical and medullary CD cells in WT and 3beta‐CDnull mice, but not in 3 alpha −/− mice. These studies showed that gene deletion of GSK3 alpha changed normal cell distribution in renal CD, reduced AQP2 expression and caused polyuria and that, GSK3 alpha, rather than GSK3beta, is a critical target of Li in renal CDs.

Valproic Acid, a Drug with Multiple Molecular Targets Related to Its Potential Neuroprotective Action

Valproic acid (VA) is used worldwide as an antiepileptic drug and a mood stabilizer. Recently, VA was shown to act on cell growth, differentiation and apoptosis, by regulating gene expression at the molecular level, through epigenetic mechanisms. Thus, VA was demonstrated to act on the chromatin remodeling what is a consequence of the drug inhibition of histone deacetylases (HDACs) activity. Other studies uncovered the potential of VA to interfere with multiple regulatory mechanisms besides HDACs, as the GSK3 alpha and beta, Akt, ERK and phosphoinositol pathways, tricarboxylic acid cycle, GABA and OXPHOS system. The review focuses on the mechanisms of action of VA, showing that HDAC inhibitors, as VA, can be successfully used in the treatment of neurodegenerative disorders. This molecule, whose biological activities range from interactions with receptors and ion channels to the regulation of many catalytic reactions, has a central role in cellular cascades that regulate gene expression. Thus, inhibitors of HDACs, by positively affecting both neuronal degeneration and cognitive deficits, appear as promising drugs against various pathological conditions and neurodegenerative diseases. VA is known to present anti-inflammatory and antioxidative properties. And, since inflammation and oxidative stress are common links in neurodegeneration, VA is a drug that, from a clinical point of view, shows a great potential as a candidate for the treatment of neurodegenerative diseases related to excitotoxic events.