Elraglusib causes a mitotic arrest. A and B, Example flow cytometry plots (A) and quantifications (B) showing the effect of 24-hour elraglusib (Elr) treatment on the percentage of 2N, 4N, and 4N-mitotic K299 WT cells. C and D, Example flow cytometry plots (C) and quantifications (D) following 24-hour treatments with two structurally distinct GSK3 inhibitors, CT99021 and LY2090314 in K299 WT cells. E and F, Example flow cytometry plots (E) and quantifications (F) showing the effect of 24-hour elraglusib (Elr) treatment on the percentage of 2N, 4N, and 4N-mitotic K299 GSK3A/B dual paralog shRNA knockdown cells (DKD). G and H, Total GSK3 immunoblot (G) with quantification confirming knockdown efficiency in K299 DKD compared with K299 WT cells (H). Cells were treated for 24 hours prior to cell-cycle analysis by flow cytometry using propidium iodide and anti–phospho-histone-H3 (Ser10) staining. Ten thousand events were analyzed per sample. Cell-cycle histograms are representative of three independent experiments, and quantifications combine three independent experiments. Immunoblot representative of three independent experiments; quantification was performed by normalizing to actin loading control and combines three independent experiments. Fold change calculated vs. control, error bars represent SEM, and statistical significance was determined using one-way ANOVA, with P ≤ 0.05. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Bifunctional targeted protein degraders, also known as Proteolysis Targeting Chimeras (PROTACs), are an emerging drug modality that may offer a new approach for treating neurodegenerative diseases. Identifying chemical starting points for PROTACs remains a largely empirical process and the design rules for identifying Central Nervous System (CNS) active PROTACs have yet to be established. Here we demonstrate a concept of using orthogonally reactive linker reagents, that allow the construction of screening libraries whereby the E3 ligase binder, the target protein binder and the linker can be simultaneously varied and tested directly in cellular assays. This approach enabled the discovery of Glycogen Synthase Kinase 3 (GSK3) PROTACs which are CNS in vivo active in female mice. Our findings provide opportunities to investigate the role of GSK3 paralogs in cellular and in vivo disease models and for the rapid discovery of in vivo quality bifunctional chemical probes for CNS disease concepts.

Mutations in glycogen synthase kinase-3 (GSK3) are associated with oncogenesis, but the opposing roles of GSK3 in tumour suppression and promotion, inadequate methods for assessing GSK3 activity and off-target effects of commonly used inhibitors on cell proliferation have stalled progress to clinical benefit.This review evaluates evidence linking GSK3 dysfunction to specific cancer types and explores how this knowledge could help develop clinical applications. Very few, if any, of the published studies on GSK3 and oncogenesis demonstrate significant alteration in GSK3 activity associated with malignancy. Unfortunately, identifying GSK3 mutations or quantifying expression levels offers limited insights into functional activity and this underpins the lack of progress towards clinical use.Developing precise, user-friendly biomarkers of GSK3 activity would allow researchers and clinicians to accurately assess its role in cancer progression and drug resistance, helping to overcome barriers to effective GSK3-targeted therapies.

Elraglusib acts via direct MT destabilization (A–J). Example images (A) of MCF7 cells treated with nocodazole (Noco), paclitaxel (Taxol), elraglusib (Elr), and CT99021 (CT). MCF7 cells were treated for 24 hours before fixation and IF microscopy. Ten mitotic cells were imaged per coverslip; images are representative of three independent experiments. Tubulin fractionation experiments comparing soluble (S) and insoluble (I) tubulin by immunoblot in MCF7 (B and C), K299 (D and E) and HH (F and G) cells. Cells were treated for 5 hours and lysed in MT stabilization buffer before fractionation into soluble (S) and insoluble (I) tubulin and analysis by immunoblot. Quantification was performed by normalizing to actin loading control and then to the experimental control. Blots are representative of three independent experiments, and quantification combines three independent experiments. Fold change calculated vs. control, error bars represent SEM, and statistical significance was determined using one-way ANOVA, with P ≤ 0.05. Scale bars, 10 μm. H, Kymographs illustrating dynamics of MT growth in vitro in the absence of drug (control) or the presence of 3.3 μmol/L nocodazole, 1, 2, 3, or 10 μmol/L elraglusib. Rhodamine-labeled short stable MTs were seeded on the coverslip, and 12 μmol/L tubulins (containing 4% fluorescent HiLyte 488 tubulins) were added to the reaction to generate and visualize dynamic MTs. Scale bars, 2 μm. I and J, Quantification of MT growth rate (I) and catastrophe frequency (J) with no drug treatment (control) or with 3.3 μmol/L nocodazole, 1, 2, 3 or 10 μmol/L elraglusib. The number of analyzed MT growth events: n = 34 for control; n = 42 for 3.3 μmol/L nocodazole; and n = 43, 24, and 21 for 1, 2, and 3 μmol/L elraglusib, respectively. MT catastrophe frequency was analyzed for dynamic MT on individual MT seeds–the number of analyzed MT seeds: n = 13 for control; n = 20 for 3.3 μmol/L nocodazole; and n = 13, 18, and 15 for 1, 2, and 3 μmol/L elraglusib, respectively. Error bars represent SD. Statistical significance was determined using one-way ANOVA, with P ≤ 0.05. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Accumulation of reactive oxygen species (i.e., oxidative stress) is a leading cause of beta cell dysfunction and apoptosis in diabetes. NRF2 (NF-E2 p45-related factor-2) regulates the adaptation to oxidative stress, and its activity is negatively regulated by the redox-sensitive CUL3 (cullin-3) ubiquitin ligase substrate adaptor KEAP1 (Kelch-like ECH-associated protein-1). Additionally, NRF2 is repressed by the insulin-regulated Glycogen Synthase Kinase-3 (GSK3). We have demonstrated that phosphorylation of NRF2 by GSK3 enhances β-TrCP (beta-transducin repeat-containing protein) binding and ubiquitylation by CUL1 (cullin-1), resulting in increased proteasomal degradation of NRF2. Thus, we hypothesise that inhibition of GSK3 activity or β-TrCP binding upregulates NRF2 and so protects beta cells against oxidative stress. We have found that treating the pancreatic beta cell line INS-1 832/13 with the KEAP1 inhibitor TBE31 significantly enhanced NRF2 protein levels. The presence of the GSK3 inhibitor CT99021 or the β-TrCP-NRF2 protein-protein interaction inhibitor PHAR, along with TBE31, resulted in prolonged NRF2 stability and enhanced nuclear localisation (P < 0.05). TBE31-mediated induction of NRF2-target genes encoding NAD(P)H quinone oxidoreductase 1 (Nqo1), glutamate-cysteine ligase modifier (Gclm) subunit and heme oxygenase (Hmox1) was significantly enhanced by the presence of CT99021 or PHAR (P < 0.05) in both INS-1 832/13 and in isolated mouse islets. Identical results were obtained using structurally distinct GSK3 inhibitors and inhibition of KEAP1 with sulforaphane. In summary, we demonstrate that GSK3 and β-TrCP/CUL1 regulate the proteasomal degradation of NRF2, enhancing the impact of KEAP1 regulation, and so contributes to the redox status of pancreatic beta cells. Inhibition of GSK3, or β-TrCP/CUL1 binding to NRF2 may represent a strategy to protect beta cells from oxidative stress.

Elraglusib (9-ING-41) is an ATP-competitive inhibitor of glycogen synthase kinase-3 (GSK3) with preclinical studies demonstrating broad activity against many tumor types. Promising early-phase clinical trial data led to FDA orphan drug status, and a randomized phase II study in combination with cytotoxic chemotherapy in pancreatic cancer has recently completed its recruitment. Similarly, single-agent responses in adult T-cell leukemia/lymphoma and melanoma and combination treatment data in several other tumor types have been encouraging. The elraglusib mechanism of action is unknown, but it is unlikely to act through GSK3 inhibition because cytotoxicity is observed below the IC50 for GSK3, and other small molecule GSK3 inhibitors do not produce cytotoxic effects, at least in lymphoma cells. We show here that elraglusib perturbs chromosomal alignment to cause a mitotic arrest in multiple tumor lines. This arrest is caused by direct microtubule (MT) depolymerization, which prevents the attachment of kinetochores to MTs. At clinically relevant doses, these mitotically arrested cells eventually undergo mitotic slippage, leading to gross chromosome missegregation, DNA damage, and apoptosis. These effects explain the cytotoxicity of elraglusib because temporarily pausing cell-cycle progression with the CDK4/6 inhibitor palbociclib abolishes any drug-induced genotoxicity and apoptosis. In summary, elraglusib acts as a direct MT destabilizer both in vitro and across multiple cancer types, resulting in mitotic arrest, DNA damage, and apoptosis. These effects likely account for its broad pan-cancer activity, which does not rely upon GSK3 inhibition as they are not replicated by other GSK3 inhibitors.Significance: Elraglusib was designed as a GSK3 inhibitor and is currently in clinical trials for several cancers. We show conclusively that the target of elraglusib that leads to cytotoxicity is MTs and not GSK3. This has significant implications for ongoing clinical trials of the compound and will help in understanding off-target side effects, inform future clinical trial design, and facilitate the development of biomarkers to predict response.

Elraglusib-induced genotoxicity and cytotoxicity are abolished by cell-cycle arrest (A–D). K299 (A and B), and HH cells (C and D) were arrested in palbociclib (Palbo; or treated with DMSO control) for 24 hours before the addition of elraglusib (Elr), doxorubicin (Dox), or DMSO for a further 24 hours. Cells were then lysed and analyzed by immunoblot. PARP cleavage was used for apoptosis assessment and γH2AX for DNA damage. Quantification was performed by normalizing to actin and the experimental control. Blots are representative of three independent experiments, and quantification combines three independent experiments. Fold change calculated vs. control, error bars represent SEM, and statistical significance was determined using one-way ANOVA, with P ≤ 0.05. ns, not significant; *, P < 0.05; ****, P < 0.0001.

Elraglusib impairs kinetochore–MT attachment to cause mitotic arrest and mitotic slippage (A–C). Mitotic duration (A) with CT99021 (CT) and elraglusib (Elr), example images (B), and mitotic fate quantifications (C). U2OS H2B-GFP/mCherry-α-tubulin cells were treated and imaged every 5 minutes for 24 hours. Ten cells were analyzed per field of view, with two fields per condition for each experiment. Mitotic duration (A) and fate plots (C) combine two independent experiments. For graph A, red data points indicate the movie ended with the cell remaining in mitosis, and the thick vertical lines represent a 95% confidence interval around the median, which can be used for statistical comparison of treatments by eye (see “Materials and Methods”). For graph C, error bars represent SD. D and E, Example images (D), number of MAD1-positive kinetochores with nocodazole (Noco), elraglusib (Elr), CT99021 (CT), and LY2090314 (LY); (E). MCF7 cells were synchronized in G1 with palbociclib for 24 hours and then released for 16 hours in CM before a 2-hour RO3306 block, wash off and MG132 treatment for 30 minutes. Cells were treated for 10 minutes with nocodazole (Noco), elraglusib (Elr), CT99021 (CT), or LY209014 (LY) before fixing and IF microscopy. CENPC was used to identify kinetochores, and MAD1 positivity was used to assess kinetochore attachment. Five mitotic cells were imaged per coverslip. Images are representative of three independent experiments. Quantification represents MAD1-positive kinetochores from five mitotic cells per condition and combines three independent experiments. For graph E, the thick vertical lines represent a 95% confidence interval around the median, which can be used for statistical comparison of treatments by eye (see “Materials and Methods”). Scale bars, 10 μm.

Calorie-restricted diets cause weight loss and can drive type 2 diabetes remission. However, many patients struggle to achieve clinically relevant weight loss, and the reasons are not well understood. Chemical exposure is associated with obesity and type 2 diabetes development, and some evidence from preclinical experiments suggests it can limit the clinical benefits of calorie restriction. We systematically reviewed the evidence for the effects of environmental chemical exposure on mass loss and glycemic control during diet-induced weight management in humans (PROSPERO: CRD42022339993). Of 222 unique citations, only six papers directly examined this question. Only one targeted people with type 2 diabetes. One linked phthalates and parabens, but not bisphenols, with slower fat loss. Two showed per- and polyfluoroalkyl substances were not associated with mass loss, but with faster subsequent mass regain. One linked impaired adiposity improvements with air pollutants. Two papers reported weight loss-induced elevation in plasma organochlorines associated with altered glycemic control. The risk of bias largely arose from the potential for deviation from the intended diet, and statistics and reporting. The role of chemical exposure in impeding the effectiveness of weight management programs needs to be better understood to provide suitable support to people living with obesity and type 2 diabetes.

BACKGROUND:Hepatic expression of several gene products involved in glucose metabolism, including phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase) and insulin-like growth factor binding protein-1 (IGFBP-1), is rapidly and completely inhibited by insulin. This inhibition is mediated through the regulation of a DNA element present in each of these gene promoters, that we call the Thymine-rich Insulin Response Element (TIRE). The insulin signalling pathway that results in the inhibition of these gene promoters requires the activation of phosphatidylinositol 3-kinase (PI 3-kinase). However, the molecules that connect PI 3-kinase to these gene promoters are not yet fully defined. Glycogen Synthase Kinase 3 (GSK-3) is inhibited following activation of PI 3-kinase. We have shown previously that inhibitors of GSK-3 reduce the activity of two TIRE-containing gene promoters (PEPCK and G6Pase), whose products are required for gluconeogenesis. RESULTS:In this report we demonstrate that in H4IIE-C3 cells, four distinct classes of GSK-3 inhibitor mimic the effect of insulin on a third TIRE-containing gene, IGFBP-1. We identify the TIRE as the minimum requirement for inhibition by these agents, and demonstrate that the target of GSK-3 is unlikely to be the postulated TIRE-binding protein FOXO-1. Importantly, overexpression of GSK-3 in cells reduces the insulin regulation of TIRE activity as well as endogenous IGFBP-1 expression. CONCLUSIONS:These results implicate GSK-3 as an intermediate in the pathway from the insulin receptor to the TIRE. Indeed, this is the first demonstration of an absolute requirement for GSK-3 inhibition in insulin regulation of gene transcription. These data support the potential use of GSK-3 inhibitors in the treatment of insulin resistant states such as Type 2 diabetes mellitus, but suggest that it will be important to identify all TIRE-containing genes to assess potential side effects of these agents.

Elraglusib impairs proliferation and reduces cell viability in stimulated primary human T cells but does not affect unstimulated cells (A and B). Primary human T cells were isolated from PBMCs from healthy volunteers. Unstimulated controls (A) remained in CM, and stimulated cells (B) were treated with a CD3/CD28-co-stimulation kit. Cells were treated with DMSO control or elraglusib (Elr). Cell viability was assayed using Promega RealTime-Glo MT assay and the plate read daily for 120 hours. Data combine three independent experiments with T cells from three individual volunteers. Data normalized to the experimental control peak luminescence. Elraglusib increases the soluble tubulin fraction and decreases the insoluble tubulin fraction in stimulated primary human T cells (C and D). Proliferating stimulated primary human T cells were treated for 5 hours and assayed for soluble and insoluble tubulin as described above. Blots are representative of three independent experiments, and quantification combines three independent experiments. Fold change calculated vs. control, error bars represent SEM, and statistical significance was determined using one-way ANOVA, with P ≤ 0.05. ns, not significant; *, P < 0.05; ****, P < 0.0001.

Background: Point-of-care (POC) analysers in community settings can provide opportunistic and regular HbA1c monitoring. Community pharmacies in NHS Scotland are utilised by populations at greatest risk of type two diabetes (T2D). This study describes initial development of an HbA1c pathway using a POC analyser in community pharmacies. Methods: The Abbott Afinion analyser was compared in (i) NHS Tayside's Blood Sciences Service and (ii) community pharmacies from four Scottish Health Boards. A side by side comparison with standard operating procedures for HbA1c quantification using 80 T2D patient venous samples. The machine was implemented into 11 community pharmacies and 144 samples obtained from patients for comparison to their recent laboratory HbA1c. Four focus groups examined themes around the intervention and an exit questionnaire was administered. Results: Laboratory assessment verified the efficacy of the POC test machine. The value for level 1 quality control was 44 mmol/mol and the mean during testing 42.7 mmol/mol. The greatest percent coefficient of variation (cv) was within-run for both levels of quality control material, at a value of 1.63% and 1.62%, respectively. The analyser performed robustly within the pharmacy assessment, with a mean difference of 1.68 and a standard deviation of 0.71 (CV 0.423). Patients with T2D reported positive experiences of using a pharmacy. The focus groups identified an appreciation of the convenience of pharmacies and of the longitudinal relationships with pharmacy staff. Conclusion: POC HbA1c analysers can be successfully established in community pharmacies. The target patient group responded positively to the opportunity to use a pharmacy service.

BackgroundGlycated haemoglobin (HbA1c) measurement using Point of Care (POC) testing may be of huge utility, providing convenient testing for early diagnosis and regular monitoring of hard-to-reach patient groups. This systematic review aimed to identify evidence for the successful deployment of these devices to improve patient outcomes in diabetes.MethodsA systematic review and meta-analysis was undertaken in February 2023, to identify all relevant articles: (CINAHL, Cochrane, PubMed, Scopus, and Web of Science). Studies were included if they reported outcomes of community POC testing for HbA1c for people with diabetes or at risk of diabetes. The Prospero database and trial registers were searched. Only English language articles were included.Title, abstract screening and full text review was carried out by two reviewers (AG/AR). The Cochrane risk of bias tool for randomised studies and the NIH Quality Assessment tool for observational cohort and cross-sectional studies were used. Publication bias was assessed visually using funnel plot and statistical assessment. We performed a meta-analysis on appropriate studies, applying a fixed effect model. We investigated heterogeneity using visual inspection of forest plots along with evaluative approaches (χ2, I2). Strength of evidence was assessed using GRADE.Findings24 studies fulfilled the criteria to be included in the narrative synthesis and 5 could be included in quantitative analysis. 13 studies evaluated HbA1c POC testing in non-diabetic patients, 9 reported results for diabetic patients and 2 included both groups. The narrative synthesis was constructed around 6 key themes: increased test access, diagnosis of people who would otherwise go undiagnosed, intervention/lifestyle change, POC testing effect on HbA1c and glycaemic control, follow-up time and patient satisfaction.InterpretationThe available published data supports the proposed use of POC devices in a community setting, with positive effects on diabetic care with limited evidence that patients can achieve better glycaemic control.

The ability of insulin to stimulate glucose transport in muscle and fat cells is mediated by the regulated delivery of intracellular vesicles containing glucose transporter-4 (GLUT4) to the plasma membrane, a process known to be defective in disease such as Type 2 diabetes. In the absence of insulin, GLUT4 is sequestered in tubules and vesicles within the cytosol, collectively known as the GLUT4 storage compartment. A subset of these vesicles, known as the 'insulin responsive vesicles' are selectively delivered to the cell surface in response to insulin. We have previously identified Syntaxin16 (Sx16) and its cognate Sec1/Munc18 protein family member mVps45 as key regulatory proteins involved in the delivery of GLUT4 into insulin responsive vesicles. Here we show that mutation of a key residue within the Sx16 N-terminus involved in mVps45 binding, and the mutation of the Sx16 binding site in mVps45 both perturb GLUT4 sorting, consistent with an important role of the interaction of these two proteins in GLUT4 trafficking. We identify Threonine-7 (T7) as a site of phosphorylation of Sx16 in vitro. Mutation of T7 to D impairs Sx16 binding to mVps45 in vitro and overexpression of T7D significantly impaired insulin-stimulated glucose transport in adipocytes. We show that both AMP-activated protein kinase (AMPK) and its relative SIK2 phosphorylate this site. Our data suggest that Sx16 T7 is a potentially important regulatory site for GLUT4 trafficking in adipocytes.

Introduction Diabetes mellitus has increased in prevalence worldwide and is causing an increasing burden on health services. The best patient outcomes occur with early diagnosis to prevent health complications. Glycated haemoglobin (HbA1c) is used to assess glycaemic control over 3–6 months and inform clinical management. Point-of-care (POC) HbA1c devices can be used in community settings, independent of clinical laboratories. This review aims to evaluate how these devices have been implemented in community settings and what patient outcomes have been documented.Methods and analysis This protocol follows the Preferred Reporting Items for Systematic Review and Meta-Analysis guidance. A systematic search was undertaken in October 2022, using the defined PICOS (population, intervention, comparison, outcomes, study type) statement to identify all relevant articles: CINAHL, Cochrane, PubMed, Scopus and Web of Science were searched (updated February 2023). Studies will be included if they report outcomes of community POC testing for HbA1c for people with diabetes or at risk of diabetes. We will review the PROSPERO database and trial registers.Title, abstract screening and full-text review will be carried out by two reviewers. The Cochrane risk-of-bias tool will be used to assess randomised studies and the National Institutes of Health (NIH) Quality Assessment tool for observational cohort and cross-sectional studies. Publication bias will be assessed visually with a funnel plot and statistical approaches if necessary. If a group of sufficiently comparable studies are identified, we will perform a meta-analysis applying a fixed or random effects model as appropriate. We will investigate heterogeneity using visual inspection of forest plots along with review of evaluative approaches such as Χ2 and the I2 statistic. Strength of evidence will be assessed using Grading of Recommendations, Assessment, Development and Evaluation.Ethics and dissemination Ethics approval is not required for this literature review. The results will be disseminated through peer-reviewed publication and conference presentations. Furthermore, this systematic review will be used to inform the design of a community pharmacy-based prediabetes intervention.PROSPERO registration number CRD42023383784.

OBJECTIVES:Metformin is the first line therapy recommended for type 2 diabetes. However, the precise mechanism of action remains unclear and up to a quarter of patients show some degree of intolerance to the drug, with a similar number showing poor response to treatment, limiting its effectiveness. A better understanding of the mechanism of action of metformin may improve its clinical use. SLC2A2 (GLUT2) is a transmembrane facilitated glucose transporter, with important roles in the liver, gut and pancreas. Our group previously identified single nucleotide polymorphisms in the human SLC2A2 gene, which were associated with reduced transporter expression and an improved response to metformin treatment. The aims of this study were to model Slc2a2 deficiency and measure the impact on glucose homoeostasis and metformin response in mice. METHODS:We performed extensive metabolic phenotyping and 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG)-positron emission tomography (PET) analysis of gut glucose uptake in high-fat diet-fed (HFD) mice with whole-body reduced Slc2a2 (Slc2a2+/-) and intestinal Slc2a2 KO, to assess the impact of metformin treatment. RESULTS:Slc2a2 partial deficiency had no major impact on body weight and insulin sensitivity, however mice with whole-body reduced Slc2a2 expression (Slc2a2+/-) developed an age-related decline in glucose homoeostasis (as measured by glucose tolerance test) compared to wild-type (Slc2a2+/+) littermates. Glucose uptake into the gut from the circulation was enhanced by metformin exposure in Slc2a2+/+ animals fed HFD and this action of the drug was significantly higher in Slc2a2+/- animals. However, there was no effect of specifically knocking-out Slc2a2 in the mouse intestinal epithelial cells. CONCLUSIONS:Overall, this work identifies a differential metformin response, dependent on expression of the SLC2A2 glucose transporter, and also adds to the growing evidence that metformin efficacy includes modifying glucose transport in the gut. We also describe a novel and important role for this transporter in maintaining efficient glucose homoeostasis during ageing.

Introduction Glycogen synthase kinase 3 (GSK3), a ubiquitously expressed serine/threonine kinase, is considered a therapeutic target in non-Hodgkin lymphomas (NHL) due to the poorer survival of patients with higher tumor expression, and the previously observed lethality of GSK3 ‘knock-out’ lymphoma cell lines. Elraglusib (formerly 9-ING-41), a small molecule reported to inhibit the GSK3β paralog, reduces proliferation and induces apoptosis in NHL cell lines, and is undergoing early-phase clinical trials (Wu et al, Blood 2019). However, in recent cell culture studies, the anti-lymphoma effects of elraglusib could not be phenocopied by structurally unrelated, small-molecule GSK3 inhibitors, raising questions about the importance of GSK3-dependent pathways in lymphoma biology and treatment (Coats et al, Cell Commun. Signal 2023). To further validate the lack of impact of GSK3 reduction on lymphoma viability and improve our understanding of the pharmacological properties of elraglusib, we have investigated the effects of reducing GSK3α, GSK3β, or both paralogs concomitantly in lymphoma cells, using paralog-specific and dual GSK3A/B genetic ablation techniques. Additionally, we performed cell-free kinase assays to explore the relative sensitivity of GSK3α and GSK3β as targets of elraglusib. Methods In the lymphoma cell line Karpas-299 (K299), single paralog GSK3A and GSK3B knockdowns (KD) were generated using commercial lentiviral shRNA transfection with subsequent puromycin-selection. Dual paralog KD (both GSK3A and GSK3B)were generated by transfecting stable GSK3A KD cells with a second commercial GSK3B lentiviral shRNA and subsequent neomycin-selection. KD efficiency was quantified by total-GSK3 immunoblot and apoptosis by poly (ADP-ribose) polymerase (PARP) cleavage immunoblot. Cell viability and proliferation were assessed by MTS assay following 72-hour incubations in complete media (RPMI 1640, 10% FBS, 100units/ml penicillin/100µg/ml streptomycin). Paralog-specific cell-free kinase assays were performed using 32P-γ-ATP, phospho-glycogen synthase-peptide-2 substrate and purified recombinant GSK3α and GSK3β. Results Viable and stable K299 single-paralog ( GSK3A or GSK3B) and dual-paralog ( GSK3A and GSK3B) KD cell lines were obtained. KD efficiency was quantified by immunoblot and compared to the parental cell line control for GSK3α (≈30% of control), GSK3β (≈20% of control) and dual GSK3αand GSK3β KD (≈35% control and ≈15% control respectively). All KD cell lines proliferated similarly to parental cell line cells by MTS assay (control vs GSK3B KD normalized to control, mean difference -5.1% (95% CI -11.7 to +1.37 p=0.14), control vs GSK3A/B KD +3.67% (95% CI -2.87 to +10.2 p=0.348)). Elraglusib 5µM induced apoptosis with a significant increase in cleaved/intact PARP (2.35x, p<0.0001) and reduced cell viability in K299 parental cell line and dual GSK3A/B KD cells (IC 50 0.158µM parental control vs 0.124µM dual GSK3A/B KD). The equivalence of paralog inhibition (GSK3β IC 50 0.494µM and GSK3α IC 50 0.446µM) was confirmed in cell-free 32P-γ-ATP kinase assays. Conclusion K299 cells do not depend upon high levels of GSK3 for survival or proliferation. This data extends previous findings regarding the redundancy of GSK3-dependent signalling in lymphoma biology and further suggests that limited therapeutic benefit may be derived from drugs specifically targeting the activity or expression of this protein kinase. The IC 50 of elraglusib remains similar despite a significant reduction in levels of GSK3, again suggesting that GSK3 is not its cytotoxic target. Importantly, elraglusib retains therapeutic potential in lymphoma however GSK3 is not a surrogate marker for its efficacy and additional studies are required to elucidate the mechanisms through which it mediates its cytotoxic effects.

The ability of insulin to stimulate glucose transport in muscle and fat cells is mediated by the regulated delivery of intracellular vesicles containing glucose transporter-4 (GLUT4) to the plasma membrane, a process known to be defective in disease such as Type 2 diabetes.In the absence of insulin, GLUT4 is sequestered in tubules and vesicles within the cytosol, collectively known as the GLUT4 storage compartment.A subset of these vesicles, known as the 'insulin responsive vesicles are selectively delivered to the cell surface in response to insulin.We have previously identified Syntaxin16 (Sx16) and its cognate Sec1/Munc18 protein family member mVps45 as key regulatory proteins involved in the delivery of GLUT4 into insulin responsive vesicles.Here we show that mutation of a key residue within the Sx16 N-terminus involved in mVps45 binding, and the mutation of the Sx16 binding site in mVps45 both perturb GLUT4 sorting, consistent with an important role of the interaction of these two proteins in GLUT4 trafficking.We identify Threonine-7 (T7) as a site of phosphorylation of Sx16 in vitro.Mutation of T7 to D impairs Sx16 binding to mVps45 in vitro and overexpression of T7D significantly impaired insulin-stimulated glucose transport in adipocytes.We show that both AMPactivated protein kinase (AMPK) and its relative SIK2 phosphorylate this site.Our data

Elraglusib (formerly 9-ING-41) is an ATP-competitive inhibitor of glycogen synthase kinase-3β (GSK3β) undergoing clinical trials for the treatment of various cancers including non-Hodgkin lymphoma (NHL). The drug reduces proliferation of several NHL cell lines and has efficacy in xenograft models of the disease. To confirm the importance of its action on GSK3β, we treated 3 lymphoma cell lines with selective, structurally distinct GSK3 inhibitors: CT99021, SB216763, LY2090314, tideglusib, and elraglusib. Stabilization of β-catenin and reduced phosphorylation of CRMP2, two validated targets of GSK3, were used as functional read-outs for GSK3 inhibition. CT99021, SB216763, and LY2090314 failed to reduce proliferation or viability in any cell line at concentrations that stabilized β-catenin and reduced CRMP2 phosphorylation. There was partial reduction of CRMP2 phosphorylation but no significant effect on β-catenin at cytotoxic doses of elraglusib. There was no indication of GSK3 inhibition at doses of tideglusib that affected cell viability and apoptosis. Cell-free kinase screening confirmed several other targets of elraglusib, distinct from the GSK3 inhibitors with no anti-lymphoma actions, including PIM kinases and MST2. These data question GSK3 as the target of elraglusib in lymphoma, and hence the utility of GSK3 expression as a ‘stand-alone’, therapeutic biomarker in NHL.