Glycogen Synthase Kinase 3 Inactivation Drives T-bet-Mediated Downregulation of Co-receptor PD-1 to Enhance CD8(+) Cytolytic T Cell Responses.

Despite the importance of the co-receptor PD-1 in T cell immunity, the upstream signaling pathway that regulates PD-1 expression has not been defined. Glycogen synthase kinase 3 (GSK-3, isoforms α and ß) is a serine-threonine kinase implicated in cellular processes. Here, we identified GSK-3 as a key upstream kinase that regulated PD-1 expression in CD8(+) T cells. GSK-3 siRNA downregulation, or inhibition by small molecules, blocked PD-1 expression, resulting in increased CD8(+) cytotoxic T lymphocyte (CTL) function. Mechanistically, GSK-3 inactivation increased Tbx21 transcription, promoting enhanced T-bet expression and subsequent suppression of Pdcd1 (encodes PD-1) transcription in CD8(+) CTLs. Injection of GSK-3 inhibitors in mice increased in vivo CD8(+) OT-I CTL function and the clearance of murine gamma-herpesvirus 68 and lymphocytic choriomeningitis clone 13 and reversed T cell exhaustion. Our findings identify GSK-3 as a regulator of PD-1 expression and demonstrate the applicability of GSK-3 inhibitors in the modulation of PD-1 in immunotherapy.

Bisindolylmaleimide IX: A novel anti-SARS-CoV2 agent targeting viral main protease 3CLpro demonstrated by virtual screening pipeline and in-vitro validation assays.

SARS-CoV-2, the virus that causes COVID-19 consists of several enzymes with essential functions within its proteome. Here, we focused on repurposing approved and investigational drugs/compounds. We targeted seven proteins with enzymatic activities known to be essential at different stages of the viral cycle including PLpro, 3CLpro, RdRP, Helicase, ExoN, NendoU, and 2'-O-MT. For virtual screening, energy minimization of a crystal structure of the modeled protein was carried out using the Protein Preparation Wizard (Schrodinger LLC 2020-1). Following active site selection based on data mining and COACH predictions, we performed a high-throughput virtual screen of drugs and investigational molecules (n = 5903). The screening was performed against viral targets using three sequential docking modes (i.e., HTVS, SP, and XP). Virtual screening identified ∼290 potential inhibitors based on the criteria of energy, docking parameters, ligand, and binding site strain and score. Drugs specific to each target protein were further analyzed for binding free energy perturbation by molecular mechanics (prime MM-GBSA) and pruning the hits to the top 32 candidates. The top lead from each target pool was further subjected to molecular dynamics simulation using the Desmond module. The resulting top eight hits were tested for their SARS-CoV-2 anti-viral activity in-vitro. Among these, a known inhibitor of protein kinase C isoforms, Bisindolylmaleimide IX (BIM IX), was found to be a potent inhibitor of SARS-CoV-2. Further, target validation through enzymatic assays confirmed 3CLpro to be the target. This is the first study that has showcased BIM IX as a COVID-19 inhibitor thereby validating our pipeline.

Glycogen synthase kinase 3ß as a potential therapeutic target in synovial sarcoma and fibrosarcoma.

Soft tissue sarcomas (STSs) are a rare cancer type. Almost half are unresponsive to multi-pronged treatment and might therefore benefit from biologically targeted therapy. An emerging target is glycogen synthase kinase (GSK)3ß, which is implicated in various diseases including cancer. Here, we investigated the expression, activity and putative pathological role of GSK3ß in synovial sarcoma and fibrosarcoma, comprising the majority of STS that are encountered in orthopedics. Expression of the active form of GSK3ß (tyrosine 216-phosphorylated) was higher in synovial sarcoma (SYO-1, HS-SY-II, SW982) and in fibrosarcoma (HT1080) tumor cell lines than in untransformed fibroblast (NHDF) cells that are assumed to be the normal mesenchymal counterpart cells. Inhibition of GSK3ß activity by pharmacological agents (AR-A014418, SB-216763) or of its expression by RNA interference suppressed the proliferation of sarcoma cells and their invasion of collagen gel, as well as inducing their apoptosis. These effects were associated with G0/G1-phase cell cycle arrest and decreased expression of cyclin D1, cyclin-dependent kinase (CDK)4 and matrix metalloproteinase 2. Intraperitoneal injection of the GSK3ß inhibitors attenuated the growth of SYO-1 and HT1080 xenografts in athymic mice without obvious detrimental effects. It also mitigated cell proliferation and induced apoptosis in the tumors of mice. This study indicates that increased activity of GSK3ß in synovial sarcoma and fibrosarcoma sustains tumor proliferation and invasion through the cyclin D1/CDK4-mediated pathway and enhanced extracellular matrix degradation. Our results provide a biological basis for GSK3ß as a new and promising therapeutic target for these STS types.

9-ING-41, a small-molecule glycogen synthase kinase-3 inhibitor, is active in neuroblastoma.

Advanced stage neuroblastoma is a very aggressive pediatric cancer with limited treatment options and a high mortality rate. Glycogen synthase kinase-3ß (GSK-3ß) is a potential therapeutic target in neuroblastoma. Using immunohistochemical staining, we observed positive GSK-3ß expression in 67% of human neuroblastomas (34 of 51 cases). Chemically distinct GSK-3 inhibitors (AR-A014418, TDZD-8, and 9-ING-41) suppressed the growth of neuroblastoma cells, whereas 9-ING-41, a clinically relevant small-molecule GSK-3ß inhibitor with broad-spectrum preclinical antitumor activity, being the most potent. Inhibition of GSK-3 resulted in a decreased expression of the antiapoptotic molecule XIAP and an increase in neuroblastoma cell apoptosis. Mouse xenograft studies showed that the combination of clinically relevant doses of CPT-11 and 9-ING-41 led to greater antitumor effect than was observed with either agent alone. These data support the inclusion of patients with advanced neuroblastoma in clinical studies of 9-ING-41, especially in combination with CPT-11.

Involvement of Spinal CCR5/PKCγ Signaling Pathway in the Maintenance of Cancer-Induced Bone Pain.

Cancer-induced bone pain (CIBP) is a challenging medical problem that considerably influences cancer patients' quality of life. Currently, few treatments have been developed to conquer CIBP because of a poor understanding of the potential mechanisms. Our previous work has proved that spinal RANTES (a major ligand for CCR5) was involved in the maintenance of CIBP. In this study, we attempted to investigate whether spinal CCR5 and its downstream PKCγ pathway is involved in the maintenance of CIBP. Inoculation of Walker 256 cells into the tibia could induce a marked mechanical allodynia with concomitant upregulation of spinal CCR5 and p-PKCγ expression from day 6 to day 15 after inoculation. Spinal CCR5 was prominently expressed in microglia, and mechanical allodynia was attenuated by intrathecal injection of DAPTA (a specific antagonist of CCR5) with downregulation of spinal CCR5 and p-PKCγ expression levels at day 15 in inoculated rats. Pre-intrathecal injection of RANTES could reverse the anti-allodynia effects of DAPTA. Intrathecal administration of GF109203X (an inhibitor of PKC) could alleviate mechanical allodynia as well as decrease of spinal p-PKCγ expression level, but no influence on spinal CCR5 level. Our findings suggest that CCR5/PKCγ signaling pathway in microglia may contribute to the maintenance of CIBP in rats.

Inhibition of glycogen synthase kinase-3 by SB 216763 affects acquisition at lower doses than expression of amphetamine-conditioned place preference in rats.

Dopamine (DA) drives incentive learning, whereby neutral stimuli acquire the ability to elicit responses. DA influences the signaling molecule glycogen synthase kinase-3 (GSK3). Inhibition of GSK3 attenuates the development of behavioral sensitization to stimulant drugs and conditioned place preference (CPP), a measure of incentive learning. We examined the role of GSK3 in the nucleus accumbens (NAc) of rats in CPP produced by amphetamine (1.5 mg/kg, i.p. or 20.0 µg/0.5 µl/side intra-NAc) by administering the inhibitor SB 216763 (1.0, 2.0, and 2.5 mg/kg, i.p. or 0.03, 0.30, 3.00, and 5.00 µg/0.5 µl/side intra-NAc) during acquisition or expression. We hypothesized a dose-dependent effect of SB 216763 and that acquisition would be affected by smaller doses than expression. For the systemic groups, 1.0 mg/kg of SB 216763 did not block CPP; 2.0 mg/kg administered in acquisition but not expression blocked CPP; and 2.5 mg/kg administered in either phase blocked CPP. For the central groups, 0.03 µg/0.5 µl/side of SB 216763 prevented acquisition but not expression, whereas larger doses administered in either phase blocked CPP. Thus, systemic or NAc inhibition of GSK3 by SB 216763 during acquisition or expression blocks amphetamine-produced CPP and acquisition is sensitive to lower doses than expression.

Co-suppression of vitamin C composite nano-drug carrier and its drug delivery to nidus in tumor cells.

This study aimed to discuss the co-suppression of vitamin C-contained composite nano-drug carrier and its drug delivery to nidus in tumor cells. Amphiphilic polymers PLA-block-PAAA and block polymer PLA-PEG4000-Maleimide, PLA-block-PAAA and PLA-PEG4000-Maleimide composite nano-micelles were prepared, and, PLA-block-PAAA polymer-coated Nile red nano-micelle, PLA-block-PAA and PLA-PEG4000-Maleimide composite nano-micelles as well as paclitaxel-carrying composite nano-micelle in different molar ratios were given stability tests. Lastly, PLA-block-PAAA and PLA-PEG4000-Maleimide composite nano-micelle cancer cells and paclitaxel-carrying composite nano-micelle cancer cells were given toxicity tests. Stability tests showed that self stability of PLA-block-PAAA (63/8) nano-micelle was not sufficient; the stability was good when the molar ratio of PLA-block-PAAA and PLA-PEG4000-Maleimide composite nano-micelle was 3:1; paclitaxel-carrying composite nano-micelle had good stability within 48 hours; PAAA segment had an inhibiting effect on C6 cancer cells and paclitaxel-carrying composite nano-micelle had a strong inhibiting effect also on tumors. After 24 hours, with the continuous release of paclitaxel, the tumor inhibiting effect of paclitaxel-carrying composite nano-micelle enhanced gradually, and the controlled-release of drugs had continuous inhibiting effect on tumor cells. Therefore, PAAA segment and paclitaxel had time-postponed synergistic effect. In conclusion, vitamin C-contained composite nanometer drug carrier materials can deliver anti-cancer drugs to nidus and thus inhibit tumor cells.

Combination of long-acting HIV fusion inhibitor albuvirtide and LPV/r showed potent efficacy in HIV-1 patients.

BACKGROUND: Long acting antiretroviral drugs represent a promising approach for chronic treatment of HIV infection. Here, we study the efficacy and safety of albuvirtide (ABT), an HIV-1 fusion inhibitor with a half life of 11-12 days in human. METHODS: ABT was evaluated in a 7-week, open-label and randomized trial, combining with LPV/r. Twenty HIV-1-infected adults were assigned to two dose groups, receiving ABT (160 or 320 mg) given weekly and LPV/r given twice daily. RESULTS: At week 7, the decline of HIV-1 RNA from baseline was 1.9 (1.3-2.3) log10 and 2.2 (1.6-2.7) log10 copies/ml, and suppression of HIV-1 RNA to below 50 copies/ml was achieved in 11.1 % (1/9) and 55.6 % (5/9) patients, for the 160 and 320 mg dose group respectively. CONCLUSION: A clear dose-efficacy correlation of ABT was demonstrated. ABT combining with LPV/r is a promising two-drug regimen to be tested in larger patient population.

Hyperosmolarity-Induced Down-Regulation of Claudin-2 Mediated by Decrease in PKCß-Dependent GATA-2 in MDCK Cells.

Hyperosmolarity decreases claudin-2 expression in renal tubular epithelial cells, but the molecular mechanism remains undefined. Here, we found that the hyperosmolarity-induced decrease in claudin-2 expression is inhibited by Go6983, a non-selective protein kinase C (PKC) inhibitor, and PKCß specific inhibitor in Madin-Darby canine kidney II cells. Hyperosmolarity increased intracellular free Ca(2+) concentration and phosphorylated PKCß level, which were inhibited by RN-1734, an antagonist of transient receptor potential vanilloid 4 channel. Phorbol 12-myristate 13-acetate, a PKC activator, decreased claudin-2 expression. These results indicate hyperosmolarity decreases claudin-2 expression mediated by the activation of RN-1734-sensitive channel and PKCß. Hyperosmolarity decreased promoter activity of claudin-2, which was inhibited by Go6983 and PKCß inhibitor similar to those in real-time PCR and Western blotting. The effect of hyperosmolarity on promoter activity was not observed in the construct of -469/-6, a deletion mutant. Claudin-2 has hyperosmolarity-sensitive region in its promoter, which includes GATA binding site. Hyperosmolarity decreased the nuclear level of GATA-2, which was inhibited by Go6983 and PKCß inhibitor. Mutation of GATA binding site decreased the basal promoter activity and inhibited the effect of hyperosmolarity. In contrast, the hyperosmolarity-induced decrease in reporter activity and claudin-2 expression were rescued by over-expression of wild type GATA-2. Chromatin immunoprecipitation assay showed that GATA-2 bound to promoter region of claudin-2. These results suggest that hyperosmolarity decreases the expression level of claudin-2 via a decrease in PKCß-dependent GATA-2 transcriptional activity in renal tubular epithelial cells.

Administration of sulfosuccinimidyl-4-[N-maleimidomethyl] cyclohexane-1-carboxylate conjugated GP100(25-33) peptide-coupled spleen cells effectively mounts antigen-specific immune response against mouse melanoma.

It remains a top research priority to develop immunotherapeutic approaches to induce potent antigen-specific immune responses against tumors. However, in spite of some promising results, most strategies are ineffective because they generate low numbers of tumor-reactive cytotoxic T lymphocytes (CTLs). Here we designed a strategy to enhance antigen-specific immune response via administering sulfosuccinimidyl-4-[N-maleimidomethyl] cyclohexane-1-carboxylate (sulfo-SMCC)-conjugated melanoma tumor antigen GP10025-33 peptide-coupled syngeneic spleen cells in a mouse model of melanoma. We found that infusion of GP10025-33 peptide-coupled spleen cells significantly attenuated the growth of melanoma in prophylactic and therapeutic immunizations. Consistent with these findings, the adoptive transfer of spleen cells from immunized mice to naïve syngeneic mice was able to transfer anti-tumor effect, suggesting that GP10025-33 peptide-specific immune response was induced. Further studies showed that, CD8+ T cell proliferation and the frequency of interferon (IFN)-γ-producing CD8+ T cells upon ex vivo stimulation by GP10025-33 were significantly increased compared to control groups. Tumor antigen, GP10025-23 specific immune response was also confirmed by ELISpot and GP100-tetramer assays. This approach is simple, easy-handled, and efficiently delivering antigens to lymphoid tissues. Our study offers an opportunity for clinically translating this approach into tumor immunotherapy.

A first-in-human phase I dose-escalation, pharmacokinetic, and pharmacodynamic evaluation of intravenous LY2090314, a glycogen synthase kinase 3 inhibitor, administered in combination with pemetrexed and carboplatin.

PURPOSE: LY2090314 (LY) is a glycogen synthase kinase 3 inhibitor with preclinical efficacy in xenograft models when combined with platinum regimens. A first-in-human phase 1 dose-escalation study evaluated the combination of LY with pemetrexed/carboplatin. PATIENTS AND METHODS: Forty-one patients with advanced solid tumors received single-dose LY monotherapy lead-in and 37 patients received LY (10-120 mg) plus pemetrexed/carboplatin (500 mg/m(2) and 5-6 AUC, respectively) across 8 dose levels every 21 days. Primary objective was maximum tolerated dose (MTD) determination; secondary endpoints included safety, antitumor activity, pharmacokinetics, and beta-catenin pharmacodynamics. RESULTS: MTD of LY with pemetrexed/carboplatin was 40 mg. Eleven dose-limiting toxicities (DLTs) occurred in ten patients. DLTs during LY monotherapy occurred at ≥ 40 mg: grade 2 visual disturbance (n = 1) and grade 3/4 peri-infusional thoracic pain during or shortly post infusion (n = 4; chest, upper abdominal, and back pain). Ranitidine was added after de-escalation to 80 mg LY to minimize peri-infusional thoracic pain. Following LY with pemetrexed/carboplatin therapy, DLTs included grade 3/4 thrombocytopenia (n = 4) and grade 4 neutropenia (n = 1). Best overall response by RECIST included 5 confirmed partial responses (non-small cell lung cancer [n = 3], mesothelioma, and breast cancer) and 19 patients having stable disease. Systemic LY exposure was approximately linear over dose range studied. Transient upregulation of beta-catenin measured in peripheral blood mononuclear cells (PBMCs) occurred at 40 mg LY. CONCLUSIONS: The initial safety profile of LY2090314 was established. MTD LY dose with pemetrexed/carboplatin is 40 mg IV every 3 weeks plus ranitidine. Efficacy of LY plus pemetrexed/carboplatin requires confirmation in randomized trials.

Targeting glycogen synthase kinase-3ß to prevent hyperoxia-induced lung injury in neonatal rats.

The pathological hallmarks of bronchopulmonary dysplasia (BPD), a chronic lung disease of premature infants, include inflammation, arrested alveolarization, and dysregulated angiogenesis. Severe BPD is often complicated by pulmonary hypertension (PH) that significantly increases morbidity and mortality. Glycogen synthase kinase (GSK)-3ß plays a pivotal role in embryonic development, cell proliferation and survival, and inflammation by modulating multiple signaling pathways, particularly the nuclear transcription factor, NF-κB, and Wnt/ß-catenin pathways. Aberrant GSK-3ß signaling is linked to BPD. We tested the hypothesis that inhibition of GSK-3ß is beneficial in preventing hyperoxia-induced neonatal lung injury, an experimental model of BPD. Newborn rats were exposed to normoxia or hyperoxia (90% oxygen), and received daily intraperitoneal injections of placebo (DMSO) or SB216763, a specific pharmacological inhibitor of GSK-3ß, for 14 days. Hyperoxia exposure in the presence of the placebo increased GSK-3ß phosphorylation, which was correlated with increased inflammation, decreased alveolarization and angiogenesis, and increased pulmonary vascular remodeling and PH. However, treatment with SB216763 decreased phosphorylation of NF-κB p65, expression of monocyte chemotactic protein-1, and lung inflammation during hyperoxia. Furthermore, treatment with the GSK-3ß inhibitor also improved alveolarization and angiogenesis, and decreased pulmonary vascular remodeling and PH. These data indicate that GSK-3ß signaling plays an important role in the pathogenesis of hyperoxia-induced neonatal lung injury, and that inhibition of GSK-3ß is beneficial in preventing inflammation and protecting alveolar and vascular structures during hyperoxia. Thus, targeting GSK-3ß signaling may offer a novel strategy to prevent and treat preterm infants with BPD.

Inhibition of GSK3α/ß promotes increased pulmonary endothelial permeability to albumin by reactive oxygen/nitrogen species.

Glycogen synthase kinase 3α/ß (GSK3α/ß) is a serine/threonine kinase that participates in numerous processes in many cell types. Importantly, the role of GSK3α/ß in homeostatic maintenance of the pulmonary endothelial cell barrier to protein is not known. We tested the hypothesis that GSK3α/ß regulates endothelial barrier function by measuring the permeability to albumin of a rat pulmonary microvessel endothelial cell monolayer (PMECM) treated with and without the selective GSK3α/ß inhibitor SB 216763 (1.0, 5.0 and 10 uM) for 1 h. The treatment with the inhibitor SB 216763 caused a dose dependent decrease in phospho-ß-catenin-Ser(33/37) levels indicating effective suppression of GSK3α/ß. SB216763 caused an increase in both permeability to albumin and DCFDA (6-Carboxy-2',7'-Dichlorodihydrofluorescein Diacetate, Di(Acetoxymethyl Ester)) oxidation that were prevented by co-treatment with the anti-oxidant tiron or the nitric oxide synthase inhibitor L-NAME (Nω-nitro-l-arginine-methyl ester). In separate studies PMECMs were treated with the Akt inhibitor triciribine (12.5 uM) for 1 h to unmask Akt dependent constitutive suppression of GSK3α/ß. Triciribine decreased phospho-GSK3α/ß-Ser(21)/9 (i.e., the product of Akt) which was associated with an increase in phospho-ß-catenin-Ser(33/37) (i.e., the product of GSK3α/ß) indicating constitutive activity of Akt for GSK3α/ß-Ser(21/9). The data indicates GSK3α/ß inhibition causes increased endothelial monolayer protein permeability which is mediated by reactive oxygen/nitrogen species.

Pharmacological inhibition of GSK-3ß produces late phase of cardioprotection in hyperlipidemic rat: possible involvement of HSP 72.

The acute, as well as late, phase of cardioprotection induced by ischemic preconditioning is abolished in hyperlipidemic (HL) rat heart. The pharmacological inhibition of glycogen synthase kinase-3ß (GSK-3ß), has earlier been reported to restore this attenuated acute cardioprotective effect. However, it not known whether GSK-3ß inhibitors administered 24 h before the ischemic injury would restore the late cardioprotective in HL rat and, if yes, the role of heat shock protein 72 (HSP 72) in its modulation. Hyperlipidemia was produced in rat by feeding high-fat diet for 6 weeks. Isolated perfused rat heart was subjected to 30 min of ischemia followed by 120 min of reperfusion (I/R). Myocardial infarct size was estimated by triphenyltetrazolium chloride staining, while lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) levels were analyzed from coronary effluent. GSK-3ß inhibitors, SB 216763 (SB, 0.6 mg/kg, i.p.), and indirubin-3 monoxime (IND, 0.4 mg/kg, i.p.), administered 24 h before the isolation of heart, significantly decreased the I/R-induced myocardial infarct size and the release of LDH and CK-MB. The cardioprotective effect of GSK-3ß inhibitors was significantly attenuated by quercetin (4 mg/kg, i.p.), a HSP 72 inhibitor, administered 1 h before the administration of SB or IND. That the late phase of cardioprotection induced by pretreatment with GSK-3ß inhibitors is not attenuated/lost in HL rat heart is a new finding in our study. Our results indicate that HSP 72 acts on pathway of GSK-3ß and plays a significant role in cardioprotection.

A cell-based drug delivery platform for treating central nervous system inflammation.

Mesenchymal stem cells (MSCs) are promising candidates for the development of cell-based drug delivery systems for autoimmune inflammatory diseases, such as multiple sclerosis (MS). Here, we investigated the effect of Ro-31-8425, an ATP-competitive kinase inhibitor, on the therapeutic properties of MSCs. Upon a simple pretreatment procedure, MSCs spontaneously took up and then gradually released significant amounts of Ro-31-8425. Ro-31-8425 (free or released by MSCs) suppressed the proliferation of CD4+ T cells in vitro following polyclonal and antigen-specific stimulation. Systemic administration of Ro-31-8425-loaded MSCs ameliorated the clinical course of experimental autoimmune encephalomyelitis (EAE), a murine model of MS, displaying a stronger suppressive effect on EAE than control MSCs or free Ro-31-8425. Ro-31-8425-MSC administration resulted in sustained levels of Ro-31-8425 in the serum of EAE mice, modulating immune cell trafficking and the autoimmune response during EAE. Collectively, these results identify MSC-based drug delivery as a potential therapeutic strategy for the treatment of autoimmune diseases. KEY MESSAGES: MSCs can spontaneously take up the ATP-competitive kinase inhibitor Ro-31-8425. Ro-31-8425-loaded MSCs gradually release Ro-31-8425 and exhibit sustained suppression of T cells. Ro-31-8425-loaded MSCs have more sustained serum levels of Ro-31-8425 than free Ro-31-8425. Ro-31-8425-loaded MSCs are more effective than MSCs and free Ro-31-8425 for EAE therapy.

Sulfhydryl-based tumor antigen-carrier protein conjugates stimulate superior antitumor immunity against B cell lymphomas.

Therapeutic vaccination of B cell lymphoma patients with tumor-specific Ig (idiotype, or Id) chemically coupled to the immunogenic foreign carrier protein keyhole limpet hemocyanin (KLH) using glutaraldehyde has shown promising results in early clinical trials, and phase III trials are underway. However, glutaraldehyde Id-KLH vaccines fail to elicit anti-Id immune and clinical responses in many patients, possibly because glutaraldehyde reacts with lysine, cysteine, tyrosine, and histidine residues, damaging critical immunogenic epitopes. A sulfhydryl-based tumor Ag-carrier protein conjugation system using maleimide chemistry was used to enhance the efficacy of Id-KLH vaccines. Maleimide Id-KLH conjugates eradicated A20 lymphoma from most tumor-bearing mice, whereas glutaraldehyde Id-KLH had little efficacy. Maleimide Id-KLH elicited tumor-specific IgG Abs and T cells, with CD8(+) T cells being the major effectors of antilymphoma immunity. Maleimide Id-KLH vaccines also demonstrated superior efficacy in 38C13 and BCL-1 lymphoma models, where Abs were shown to be critical for protection. Importantly, standard glutaraldehyde Id-KLH conjugation procedures could result in "overconjugation" of the tumor Ag, leading to decreased efficacy, whereas the heterobifunctional maleimide-based conjugation yielded potent vaccine product regardless of conjugation duration. Under lysosomal processing conditions, the Id-carrier protein linkage was cleavable only after maleimide conjugation. Maleimide KLH conjugation was easily performed with human Igs analogous to those used in Id-KLH clinical trials. These data support the evaluation of sulfhydryl-based Id-KLH vaccines in lymphoma clinical trials and possibly the use of tumor Ag-carrier protein vaccines for other cancers.

PKCß/NF-κB pathway in diabetic atrial remodeling.

Atrial remodeling in diabetes is partially attributed to NF-κB/TGF-ß signal transduction pathway activation. We examined whether the hyperglycemia-induced increased expression of NF-κB/TGF-ß was dependent upon protein kinase C-ß (PKCß) and tested the hypothesis that selective inhibition of PKCß using ruboxistaurin (RBX) can reduce NF-κB/TGF-ß expression and inhibit abnormal atrial remodeling in streptozotocin (STZ)-induced diabetic rats. The effects of PKCß inhibition on NF-κB/TGF-ß signal transduction pathway-mediated atrial remodeling were investigated in STZ-induced diabetic rats. Mouse atrial cardiomyocytes (HL-1 cells) were cultured in low- or high-glucose or mannitol conditions in the presence or absence of small interference RNA that targeted PKCß. PKCß inhibition using ruboxistaurin (RBX, 1 mg/kg/day) decreased the expression of NF-κBp65, p-IκB, P38MARK, TNF-α, TGF-ß, Cav1.2, and NCX proteins and inducibility of atrial fibrillation (AF) in STZ-induced diabetic rats. Exposure of cardiomyocytes to high-glucose condition activated PKCß and increased NF-κB/TGF-ß expression. Suppression of PKCß expression by small interference RNA decreased high-glucose-induced NF-κB and extracellular signal-related kinase activation in HL-1 cells. Pharmacological inhibition of PKCß is an effective method to reduce AF incidence in diabetic rat models by preventing NF-κB/TGF-ß-mediated atrial remodeling.

Modulating TAK1 Expression Inhibits YAP and TAZ Oncogenic Functions in Pancreatic Cancer.

YAP and TAZ are central determinants of malignancy; however, their functions remain still undruggable. We identified TGFß-activated kinase 1 (TAK1) as a central hub integrating the most relevant signals sustaining pancreatic cancer aggressiveness and chemoresistance. Glycogen synthase kinase (GSK)3 is known to stabilize TAK1, and its inhibition causes a reduction in TAK1 levels. Here, we hypothesized that TAK1 could sustain YAP/TAZ program, and thus, modulation of TAK1 expression through the inhibition of GSK3 could impair YAP/TAZ functions in pancreatic cancer.Differentially expressed transcripts between pancreatic cancer cells expressing scramble or TAK1-specific shRNA were annotated for functional interrelatedness by ingenuity pathway analysis. TAK1 expression was modulated by using different GSK3 inhibitors, including LY2090314. In vivo activity of LY2090314 alone or in combination with nab-paclitaxel was evaluated in an orthotopic nude mouse model.Differential gene expression profiling revealed significant association of TAK1 expression with HIPPO and ubiquitination pathways. We measured a significant downregulation of YAP/TAZ and their regulated genes in shTAK1 cells. TAK1 prevented YAP/TAZ proteasomal degradation in a kinase independent manner, through a complex with TRAF6, thereby fostering their K63-ubiquitination versus K48-ubiquitination. Pharmacologic modulation of TAK1 by using GSK3 inhibitors significantly decreased YAP/TAZ levels and suppressed their target genes and oncogenic functions. In vivo, LY2090314 plus nab-paclitaxel significantly prolonged mice survival duration.Our study demonstrates a unique role for TAK1 in controlling YAP/TAZ in pancreatic cancer. LY2090314 is a novel agent that warrants further clinical development in combination with nab-paclitaxel for the treatment of pancreatic cancer.

Elevated glycogen synthase kinase-3 activity in Fragile X mice: key metabolic regulator with evidence for treatment potential.

Significant advances have been made in understanding the underlying defects of and developing potential treatments for Fragile X syndrome (FXS), the most common heritable mental retardation. It has been shown that neuronal metabotropic glutamate receptor 5 (mGluR5)-mediated signaling is affected in FX animal models, with consequent alterations in activity-dependent protein translation and synaptic spine functionality. We demonstrate here that a central metabolic regulatory enzyme, glycogen synthase kinase-3 (GSK3) is present in a form indicating elevated activity in several regions of the FX mouse brain. Furthermore, we show that selective GSK3 inhibitors, as well as lithium, are able to revert mutant phenotypes of the FX mouse. Lithium, in particular, remained effective with chronic administration, although its effects were reversible even when given from birth. The combination of an mGluR5 antagonist and GSK3 inhibitors was not additive. Instead, it was discovered that mGluR5 signaling and GSK3 activation in the FX mouse are coordinately elevated, with inhibition of mGluR5 leading to inhibition of GSK3. These findings raise the possibility that GSK3 is a fundamental and central component of FXS pathology, with a substantial treatment potential.

Liposomal formulation of alpha-tocopheryl maleamide: in vitro and in vivo toxicological profile and anticancer effect against spontaneous breast carcinomas in mice.

The vitamin E analogue alpha-tocopheryl succinate (alpha-TOS) is an efficient anti-cancer drug. Improved efficacy was achieved through the synthesis of alpha-tocopheryl maleamide (alpha-TAM), an esterase-resistant analogue of alpha-tocopheryl maleate. In vitro tests demonstrated significantly higher cytotoxicity of alpha-TAM towards cancer cells (MCF-7, B16F10) compared to alpha-TOS and other analogues prone to esterase-catalyzed hydrolysis. However, in vitro models demonstrated that alpha-TAM was cytotoxic to non-malignant cells (e.g. lymphocytes and bone marrow progenitors). Thus we developed lyophilized liposomal formulations of both alpha-TOS and alpha-TAM to solve the problem with cytotoxicity of free alpha-TAM (neurotoxicity and anaphylaxis), as well as the low solubility of both drugs. Remarkably, neither acute toxicity nor immunotoxicity implicated by in vitro tests was detected in vivo after application of liposomal alpha-TAM, which significantly reduced the growth of cancer cells in hollow fiber implants. Moreover, liposomal formulation of alpha-TAM and alpha-TOS each prevented the growth of tumours in transgenic FVB/N c-neu mice bearing spontaneous breast carcinomas. Liposomal formulation of alpha-TAM demonstrated anti-cancer activity at levels 10-fold lower than those of alpha-TOS. Thus, the liposomal formulation of alpha-TAM preserved its strong anti-cancer efficacy while eliminating the in vivo toxicity found of the free drug applied in DMSO. Liposome-based targeted delivery systems for analogues of vitamin E are of interest for further development of efficient and safe drug formulations for clinical trials.