Glycogen Synthase Kinase-3 Inhibitors Research Articles

Wnt/β-Catenin Signaling Activation Induces Differentiation in Human Limbal Epithelial Stem Cells Cultured Ex Vivo.

Human limbal epithelial stem cells (hLESCs) continuously replenish lost or damaged human corneal epithelial cells. The percentage of stem/progenitor cells in autologous ex vivo expanded tissue is essential for the long-term success of transplantation in patients with limbal epithelial stem cell deficiency. However, the molecular processes governing the stemness and differentiation state of hLESCs remain uncertain. Therefore, we sought to explore the impact of canonical Wnt/β-catenin signaling activation on hLESCs by treating ex vivo expanded hLESC cultures with GSK-3 inhibitor LY2090314. Real-time qRT-PCR and microarray data reveal the downregulation of stemness (TP63), progenitor (SOX9), quiescence (CEBPD), and proliferation (MKI67, PCNA) genes and the upregulation of genes for differentiation (CX43, KRT3) in treated- compared to non-treated samples. The pathway activation was shown by AXIN2 upregulation and enhanced levels of accumulated β-catenin. Immunocytochemistry and Western blot confirmed the findings for most of the above-mentioned markers. The Wnt/β-catenin signaling profile demonstrated an upregulation of WNT1, WNT3, WNT5A, WNT6, and WNT11 gene expression and a downregulation for WNT7A and DKK1 in the treated samples. No significant differences were found for WNT2, WNT16B, WIF1, and DKK2 gene expression. Overall, our results demonstrate that activation of Wnt/β-catenin signaling in ex vivo expanded hLESCs governs the cells towards differentiation and reduces proliferation and stem cell maintenance capability.

Synthesis, Characterization and Biological Evaluation of Glycogen Synthase Kinase-3β Inhibitors as Antidiabetic Agents

Diabetes is a substantially growing unhealthiest in the globe that causes severe morbidity and mortality. Currently a day’s there’s an imperative for the invention, development and improvement of novel antidiabetic drug molecules. A chain of compound (5-imidazol-2-yl-4-phenylpyrimidin-2-yl) [2-(2- pyridyl amino) ethyl] amine were developed and synthesized by using suitable chemical synthetic techniques. These recently synthesized derivatives were characterized with the help of modern analytical techniques. This compound was evaluated for glycogen synthase kinase-3 enzyme inhibitor activities for managing of polygenic disease. GSK-3 inhibitors may advance and emerge as a novel strategy for the management of diabetes.

Elraglusib (formerly 9-ING-41) possesses potent anti-lymphoma properties which cannot be attributed to GSK3 inhibition

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.7CsbmemxsJeEQuU9mL3kzMVideo

Toward countering muscle and bone loss with spaceflight: GSK3 as a potential target

We examined the effects of ∼30days of spaceflight on glycogen synthase kinase 3 (GSK3) content and inhibitory serine phosphorylation in murine muscle and bone samples from four separate missions (BION-M1, rodent research [RR]1, RR9, and RR18). Spaceflight reduced GSK3β content across all missions, whereas its serine phosphorylation was elevated with RR18 and BION-M1. The reduction in GSK3β was linked to the reduction in type IIA fibers commonly observed with spaceflight as these fibers are particularly enriched with GSK3. We then tested the effects of inhibiting GSK3 before this fiber type shift, and we demonstrate that muscle-specific Gsk3 knockdown increased muscle mass, preserved muscle strength, and promoted the oxidative fiber type with Earth-based hindlimb unloading. In bone, GSK3 activation was enhanced after spaceflight; and strikingly, muscle-specific Gsk3 deletion increased bone mineral density in response to hindlimb unloading. Thus, future studies should test the effects of GSK3 inhibition during spaceflight.

GSK-3 inhibitor elraglusib (9-ING-41) to enhance tumor-infiltrating immune cell activation in tumor biopsies and synergize with anti-PD-L1 in a murine model of colorectal cancer.

e15138 Background: Glycogen synthase kinase 3 (GSK-3) is a serine/threonine kinase with key roles in myriad biological processes such as tumor progression, and inhibition of GSK-3 using a novel small-molecule elraglusib has shown promising preclinical antitumor activity in multiple tumor types. Methods: We characterize the effects of elraglusib in vitro on tumor and immune cells including cell killing and cytokine profiling , in vivo in combination with checkpoint inhibitors in a syngeneic murine colon carcinoma BALB/c model using MSS cell line CT-26, and in human tumor biopsies and plasma samples from patients with refractory solid tumors of multiple tissue origins enrolled in a Phase 1 clinical trial investigating elraglusib ( NCT03678883 ).We used human plasma samples from patients treated with elraglusib, paired pre- and post-treatment tumor biopsies and performed digital spatial proteomics. Results: Elraglusib promoted immune cell-mediated tumor cell killing, enhanced tumor cell pyroptosis, decreased tumor cell NF-κB-regulated survival protein expression, and increased immune cell effector molecule secretion. Synergy was observed between elraglusib and anti-PD-L1 in an immunocompetent murine model of colorectal cancer. Murine responders had more tumor-infiltrating T-cells, fewer tumor-infiltrating Tregs, lower tumorigenic circulating cytokine concentrations, and higher immunostimulatory circulating cytokine concentrations. Murine responders had lower serum concentrations of BAFF, CCL7, CCL12, VEGF, VEGFR2, and CCL21, and higher serum concentrations of CCL4, TWEAK, GM-CSF, CCL22, and IL-12p70 as compared to non-responders. We utilized human plasma samples from patients treated with elraglusib and correlated cytokine profiles with survival. Elevated baseline plasma levels of proteins such as IL-1 β and reduced levels of proteins such as VEGF correlated with improved PFS and OS. PFS was also found to be positively correlated with elevated plasma levels of immunostimulatory analytes such as Granzyme B, IFN-γ, and IL-2 at 24 hours post-treatment with elraglusib. Several of these secreted proteins correlated with results from the in vivo study where expression of proteins such as IL-1 β, CCL22, CCL4, and TWEAK was positively correlated with improved response to therapy while expression of proteins such as BAFF and VEGF negatively correlated with response to therapy. Using paired tumor biopsies, we found that CD45+ tumor-infiltrating immune cells had lower expression of inhibitory immune checkpoints and higher expression of T-cell activation markers in post-elraglusib patient biopsies. Conclusions: These results introduce several immunomodulatory mechanisms of GSK-3 inhibition using elraglusib, providing a rationale for the clinical evaluation of elraglusib in combination with immunotherapy.

Discovery of 2-(Anilino)pyrimidine-4-carboxamides as Highly Potent, Selective, and Orally Active Glycogen Synthase Kinase-3 (GSK-3) Inhibitors.

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase that serves as an important regulator of a broad range of cellular functions. It has been linked to Alzheimer's disease as well as various other diseases, including mood disorders, type 2 diabetes, and cancer. There is considerable evidence indicating that GSK-3β in the central nervous system plays a role in the production of abnormal, hyperphosphorylated, microtubule-associated tau protein found in neurofibrillary tangles associated with Alzheimer's disease. A series of analogues containing a pyrimidine-based hinge-binding heterocycle was synthesized and evaluated, leading to the identification of highly potent GSK-3 inhibitors with excellent kinase selectivity. Further evaluation of 34 and 40 in vivo demonstrated that these compounds are orally bioavailable, brain-penetrant GSK-3 inhibitors that lowered levels of phosphorylated tau in a triple-transgenic mouse Alzheimer's disease model.

AKT inhibition sensitizes acute leukemia cells to S63845-induced apoptosis

ABSTRACTThe MCL1 inhibitors are undergoing clinical testing for multiple leukemia. However, because that MCL1 inhibition has on-target hematopoietic, hepatic and cardiac toxicities, there is substantial interest in finding agents can sensitize leukemia cells to the MCL1 inhibitors. Here we describe that the AKT inhibitors MK-2206 and Gsk690693 sensitize multiple leukemia cells to the MCL1 inhibitor S63845. Further experiments demonstrate that MK-2206 and Gsk690693 sensitize S63845 through the mitochondrial apoptosis pathway. Moreover, MK-2206 downregulates the anti-apoptotic protein BCLXL and induces the BH3-only pro-apoptotic protein BAD dephosphorylation and mitochondrial translocation. Knockdown of BAD significantly inhibits MK-2206-induced sensitization to S63845. Thus, our results suggest that MK-2206 sensitizes multiple leukemia cells to S63845-induced apoptosis, with the mechanisms involving BAD dephosphorylation and BCLXL downregulation.

P38MAPK- and GSK3-Mediated Phosphorylation of Snakehead Vesiculovirus Phosphoprotein at Threonine 160 Facilitates Viral Replication.

Phosphoprotein (P), co-factor of the polymerase (large protein, L) of single-stranded negative-sense RNA viruses, is phosphorylated during viral infection and its phosphorylation has been reported to play important roles in viral replication. However, the function of P phosphorylation in viral replication is still far from clear. Snakehead vesiculovirus (SHVV) is a kind of fish rhabdovirus that has caused serious economic losses in snakehead fish culture in China without any effective preventive or therapeutical measures currently. In this study, 4D label-free phosphoproteomics sequencing of SHVV-infected cells identified five phosphorylated sites on SHVV P, among which threonine 160 (T160) was proved to be phosphorylated. Overexpression of wild-type P, but not P-T160A or P-T160E mutant, promoted SHVV replication, suggesting that the T160 phosphorylation on the P protein is critical for SHVV replication. Moreover, we found that T160A or T160E mutation on SHVV P had no effect on the interactions of P-nucleoprotein (N), P-P, or P-L. Further study revealed that p38 mitogen-activated protein kinase (p38MAPK) and glycogen synthase kinase 3 (GSK3) interacted with SHVV P and mediated the T160 phosphorylation. Besides, overexpression of p38MAPK or GSK3 facilitated, while knockdown or activity inhibition of p38MAPK or GSK3 suppressed, SHVV replication. Overall, p38MAPK- and GSK3-mediated phosphorylation of the P protein at T160 is required for SHVV replication, which provided targets for designing anti-SHVV drugs and developing live-attenuated SHVV vaccines. Our study helps understand the role of P phosphorylation in the replication of single-stranded negative-sense RNA viruses. IMPORTANCE Phosphorylation of viral proteins plays important roles in viral replication. Currently, the role of phosphorylation of phosphoprotein (P) in the replication of single-stranded negative-sense RNA viruses is far from clear. Identification of the phosphorylated sites on viral P protein and the related host kinases is helpful for developing live-attenuated vaccines and designing antiviral drugs. This study focused on identifying the phosphorylated sites on P protein of a fish rhabdovirus SHVV, determining the related host kinases, and revealing the effects of the phosphorylated sites and kinases on SHVV replication. We found that SHVV P was phosphorylated at T160, which was mediated by the kinases p38MAPK and GSK3 to promote SHVV replication. This study is the first time to study the role of P phosphorylation in fish rhabdovirus replication.

Wip1 phosphatase inhibition enhances etoposide mediated apoptosis by increasing p53 phosphorylation in Rhabdoid tumor cells

Protein phosphatase magnesium-dependent 1 delta (PPM1D/Wip1) is a genotoxic stress-activated serine/threonine phosphatase playing a central role in the timely termination of DNA damage response (DDR). Wip1 is frequently mutated, amplified, and overexpressed in common human cancers and is recognized as an oncogene. Rhabdoid tumors (RT) are a type of soft tissue sarcoma being quite aggressive and resistant to chemotherapy. In this study, the role of Wip1 in the regulation of genotoxic stress-induced apoptosis was studied in RT cell line A204. First PPM1D mRNA levels were analyzed in RT compared to normal tissues by in silico analysis utilizing RNA-seq data generated by TARGET from TCGA Pan Cancer. Second, MCF-7, BJ, and A204 cells were used to test the gene copy number and mRNA levels of Wip1 by qRT-PCR. Small molecule inhibitor GSK2830371 and etoposide were used to target Wip1 and to trigger genotoxic stress, respectively. MTT and AnexinV/7AAD analyzes were used to determine the viability and apoptotic response of A204 cells. Inhibition of Wip1 and total and phosphorylated levels of p53 was demonstrated by WB analysis. Thus, here we showed that Wip1 is overexpressed and amplified in the A204 RT cell line. Wip1 overexpressing A204 cells display resistance to etoposide-induced apoptosis due to decreased phosphorylation of p53. Targeting of Wip1 by GSK2830371 increased the phosphorylation of p53 in response to etoposide treatment. Additionally combined use of GSK2830371 and etoposide enhances p53-mediated apoptosis in A204 cells.

A quantitative analysis method for a new glycogen synthase kinase-3 inhibitor and its pharmacokinetics in rats.

Alzheimer's disease (AD), as a chronic and frequent neurodegenerative disease in the elderly population, has caused a huge economic burden to society, family, and other aspects. (E)-N-(4-(((2-amino-5-phenylpyridin-3-yl) imino) methyl) pyridine-2-yl) cyclopropanecarboxamide (PIMPC), a new glycogen synthase kinase-3 (GSK-3) inhibitor, has been designed and synthesized as a potential anti-AD compound with antioxidant and metal chelating properties. In this study, we established an HPLC method for the determination of PIMPC, which has high accuracy, good sensitivity, and repeatability. This method determined the PIMPC content in rat plasma at different time points after intragastric administration to understand the pharmacokinetics (PK) process of PIMPC in rats. In addition, we preliminarily evaluated the effect of PIMPC on the liver and kidney in rats at pharmacodynamic doses. In conclusion, we have established a quantitative analysis method for PIMPC with excellent performance. And the PK process of PIMPC in rats, which was characterized by fast absorption, rapid distribution, and rapid elimination, conformed to the characteristics of the two-compartment model. In addition, long-term administration of PIMPC at therapeutic doses would not affect liver and kidney function. These studies have a certain reference for the development and research of PIMPC as a potential anti-AD drug.

Voluntary wheel running and lithium supplementation improves muscle force production without altering SERCA function in the D2 mdx mouse

Background: Duchenne muscular dystrophy (DMD) is an X-linked recessive muscle wasting disease that, with no cure, leads to early mortality (30-40 years of age). Previous studies have shown that voluntary wheel running can mitigate disease pathology and improve muscle contractility in the preclinical mdx mouse model. We have shown that treating wild-type (WT) mice with a low dose of lithium, a natural inhibitor of glycogen synthase kinase 3, enhanced soleus (SOL) and extensor digitorum longus (EDL) muscle contractility. Our objectives were to test our hypothesis of whether combining VWR with low dose lithium supplementation could provide further benefits to muscle contractility in DBA/2J mdx mice – a model that displays far worse disease severity compared with the traditional C57BL/10 mdx mouse. We also examined if this combined intervention could improve muscle calcium regulation in muscle by assessing sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA) function as we have recently shown that SERCA function is drastically impaired in D2 mdx mice, which could also contribute to deficits in muscle force production. Methods: Young (5-6 week old) male D2 WT and mdx mice were ordered from Jackson Laboratory. D2 mdx mice were assigned into each of the following conditions: sedentary ( mdxSED), voluntary wheel running ( mdxVWR), or VWR+lithium chloride ( mdxVWR+Li). The VWR groups were given access to a cagewheel for a total of 6 weeks, and the mdxVWR+Li group were provided a low dose of LiCl (50 mg/kg/day) via drinking water for the entire duration of the study. After 6 weeks, all mice were euthanized and the SOL and EDL muscles were subjected to force-frequency curve analysis (1-160 Hz). SERCA function was assessed by performing SERCA activity assays and a Ca 2+ uptake assay. Results: In the SOL, the combined effect of lithium and VWR improved specific force production to near WT levels across submaximal and maximal frequencies. Specific force production in the mdxVWR+Li group was significantly greater compared to the mdxSED group at all stimulation frequencies. Rate of SOL force development was significantly lower in all mdx groups compared to WT. However, rate of relaxation was only significantly reduced (vs. WT) within the mdxSED group, suggesting that VWR and VWR+Li improved relaxation. This could not be explained by any changes in SERCA function. In the EDL, specific force was significantly (p<0.001) reduced across mdx groups compared to WT; however, of all mdx groups, the mdxVWR+Li produced the most force and was significantly different compared to mdxVWR at 80 and 160 Hz. Rates of force development and relaxation were all slower in all mdx groups compared to WT, and there were no differences in SERCA activity. Conclusions: Combining lithium supplementation with VWR improved SOL and EDL force production in the D2 mdx mouse without any changes to SERCA function. V.A.F is a Tier 2 Canada Research Chair in Muscle Plasticity and Tissue-Remodelling. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Voluntary wheel running and lithium supplementation promotes fatigue resistance, fat oxidation, and improves insulin tolerance in D2 mdx mice

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked disorder characterized by progressive muscle wasting and premature death. Many DMD patients will also exhibit signs of metabolic dysfunction such as insulin resistance that predisposes them to type 2 diabetes and worsened cardiovascular disease. Most treatments for DMD aim to lower muscle inflammation and improve strength, however, none have addressed the complication of insulin resistance. The current standard of treatment is corticosteroids, which prolongs ambulation, however, chronic use increases susceptibility to obesity, insulin resistance, and type 2 diabetes. It is well-established that regular aerobic exercise enhances insulin sensitivity. Further, we have recently found that inhibiting glycogen synthase kinase 3 with lithium (Li), a known insulin mimetic, improves force production and fatigue resistance in wildtype (WT) mice. Here, we tested whether voluntary wheel running (VWR) and Li supplementation, together, could improve whole-body fatigue, metabolism, and insulin sensitivity in the DBA/2J (D2) mdx mouse model of DMD. METHODS: 5-week old male D2 WT and mdx mice were separated into four groups: WT, mdx sedentary (SED), mdx VWR, and mdx Li+VWR (n=11/group). The mdx VWR and mdx Li+VWR mice had unlimited access to a cagewheel, and mdx Li+VWR mice were given a low dose of Li (50 mg/kg/day) via their drinking water throughout the study. A treadmill time-to-fatigue test was performed to assess whole-body fatigue. Mice were housed for 48-hours in metabolic cages to measure energy expenditure. Glucose handling was measured using glucose and insulin tolerance tests. RESULTS: As expected, WT mice had ~2-fold greater total cage activity compared to all mdx groups. When examining cagewheel distance, we found that mdx Li+VWR mice ran half the total distance in kilometres than mdx VWR mice (39.1 ± 9.6 km vs. 74.8 ± 13.6 km). Despite this, VWR with and without Li improved whole-body fatigue as only mdx SED mice (22.8 ± 0.9 min) had a shorter time to exhaustion than WT mice (32.6 ± 0.9 min). All mdx groups had higher energy expenditure than WT mice, suggestive of a hypermetabolic phenotype. When examining the respiratory exchange ratio (RER) among mdx mice, mdxLi+VWR (0.87) mice had a lower RER than mdx SED mice (0.91), indicative of enhanced fat oxidation. Glucose tolerance was equally impaired among mdx groups compared to WT mice. However, insulin tolerance was notably improved in mdx VWR and mdx Li+VWR mice, with a greater improvement observed in the mdx Li+VWR mice. CONCLUSION: Despite running less than mdx VWR mice, mdx Li+VWR mice had enhanced fatigue resistance, fat utilization, and insulin sensitivity, suggesting Li may be a viable treatment to improve muscle and metabolic function in mdx mice. CIHR CRC (Tier II) to VAF This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

In Silico-Motivated Discovery of Novel Potent Glycogen Synthase-3 Inhibitors: 1-(Alkyl/arylamino)-3H-naphtho[1,2,3-de]quinoline-2,7-dione Identified as a Scaffold for Kinase Inhibitor Development.

Glycogen synthase kinase-3 (GSK-3) isoforms α and β have diverse roles within cell biology, and have been linked with multiple diseases that include prominent CNS conditions such as Alzheimer's disease and several psychiatric disorders. In this study, motivated by computation, we aimed to identify novel ATP-binding site inhibitors of GSK-3 with CNS-active potential. A ligand screening (docking) protocol against GSK-3β was first optimized, employing an active/decoy benchmarking set, with the final protocol selected based on statistical performance analysis. The optimized protocol involved pre-filtering of ligands using a three-point 3D-pharmacophore, followed by Glide-SP docking applying hinge region hydrogen bonding constraints. Using this approach, the Biogenic subset of the ZINC15 compound database was screened, focused on compounds with potential for CNS-activity. Twelve compounds (generation I) were selected for experimental validation using in vitro GSK-3β binding assays. Two hit compounds, 1 and 2, with 6-amino-7H-benzo[e]perimidin-7-one and 1-(phenylamino)-3H-naphtho[1,2,3-de]quinoline-2,7-dione type scaffolds were identified with IC50 values of 1.63 µM and 20.55 µM, respectively. Ten analogues of 2 (generation II) were selected for structure activity relationship (SAR) analysis and revealed four low micromolar inhibitors (<10 µM), with 19 (IC50 = 4.1 µM)~five times more potent than initial hit compound 2. Selectivity screening of low micromolar inhibitors 14 and 19 (comparing aryl- and alkyl-substituents) against 10 homologous kinases revealed unique selectivity profiles, with both compounds more potent against the GSK-3α isoform (IC50s~2 µM) and, additionally, inhibitors of PKBβ (IC50s < 25 µM). Compound 14 also inhibited ERK2 and 19, PKCγ, but generally good selectivity for GSK-3 isoforms over the other kinases was observed. The compounds had excellent predicted oral bioavailability and CNS-activity profiles, presenting promising candidates for future testing in cellular models of disease.

The Role of Glycogen Synthase Kinase-3β in the Zinc-Mediated Neuroprotective Effect of Metformin in Rats with Glutamate Neurotoxicity.

Metformin has been suggested to have protective effects on the central nervous system, but the mechanism is unknown. The similarity between the effects of metformin and the inhibition of glycogen synthase kinase (GSK)-3β suggests that metformin may inhibit GSK-3β. In addition, zinc is an important element that inhibits GSK-3β by phosphorylation. In this study, we investigated whether the effects of metformin on neuroprotection and neuronal survival were mediated by zinc-dependent inhibition of GSK-3β in rats with glutamate-induced neurotoxicity. Forty adult male rats were divided into 5 groups: control, glutamate, metformin + glutamate, zinc deficiency + glutamate, and zinc deficiency + metformin + glutamate. Zinc deficiency was induced with a zinc-poor pellet. Metformin was orally administered for 35days. D-glutamic acid was intraperitoneally administered on the 35th day. On the 38th day, neurodegeneration was examined histopathologically, and the effects on neuronal protection and survival were evaluated via intracellular S-100β immunohistochemical staining. The findings were examined in relation to nonphosphorylated (active) GSK-3β levels and oxidative stress parameters in brain tissue and blood. Neurodegeneration was increased (p < 0.05) in rats fed a zinc-deficient diet. Active GSK-3β levels were increased in groups with neurodegeneration (p < 0.01). Decreased neurodegeneration, increased neuronal survival (p < 0.01), decreased active GSK-3β (p < 0.01) levels and oxidative stress parameters, and increased antioxidant parameters were observed in groups treated with metformin (p < 0.01). Metformin had fewer protective effects on rats fed a zinc-deficient diet. Metformin may exert neuroprotective effects and increase S-100β-mediated neuronal survival by zinc-dependent inhibition of GSK-3β during glutamate neurotoxicity.

Sterols from Centaurea pumilio L. with cell proliferative activity: In vitro and in silico studies

Abstract Numerous studies highlighted the impact of natural products, particularly phytosterols, in wound healing while providing less expensive alternatives to chemically synthesized drugs, with less side effects. Centaurea pumilio L. (family Asteraceae) is a rare and endangered species of genus Centaurea with few reports concerning its chemistry. Our phytochemical investigation for the non-polar fraction of its aerial parts led to the isolation and identification of the new compound (6) identified as stigmast-1,5-dien-3-O-β-d-glucopyranoside along with five known sterols and triterpenes (1–5) identified as taraxasterol, β-sitosterol, stigmasterol, β-sitosterol glucoside, and stigmasterol-3-O-β-d-glucopyranoside. Structures of the isolated compounds have been characterized using 1D, 2D NMR, and mass spectral analyses. The cell viability and proliferative activity of the isolated compounds were evaluated using an MTT assay on cultured human primary umbilical vein endothelial cells (HUVEC). None of the compounds exhibited any sign of cytotoxicity. Nonetheless, compounds 5 and 6 moderately enhanced the HUVEC cell growth by 14 and 16%, respectively, at the maximal tested dose (50 µg/mL). As inhibition of glycogen synthase kinase 3-β (GSK3-β) enzyme is important to enhance the wound healing process; therefore, molecular docking was performed to understand the possible interactions between bioactive compounds 5 and 6 and GSK-3β binding pocket active amino acid residues. Both compounds were able to bind to the substrate‑binding site of GSK-3β and potentially interact with the key active site residues, forming strong π and hydrogen interactions with the catalytic site residues, revealing lower binding energy (−7.185 and −6.303 kcal/mol, respectively) than that of indirubin-3-monooxime (−5.303 kcal/mol); thereby representing strong natural replacements candidates for GSK-3β inhibitors.

Multimodal Wnt signalling in the mouse neocortex

The neocortex is the site of higher cognitive functions and its development is tightly regulated by cell signalling pathways. Wnt signalling is inexorably linked with neocortex development but its precise role remains unclear. Most studies demonstrate that Wnt/β-catenin regulates neural progenitor self-renewal but others suggest it can also promote differentiation. Wnt/STOP signalling is a novel branch of the Wnt pathway that stabilizes proteins during G2/M by inhibiting glycogen synthase kinase 3 (GSK3)-mediated protein degradation. Recent data from our work in Da Silva et al. (2021) demonstrate that Wnt/STOP is involved in neocortex development where, by stabilizing the neurogenic transcription factors Sox4 and Sox11, it promotes neural progenitor differentiation. We also show that Wnt/STOP regulates asymmetric cell division and cell cycle dynamics in apical and basal progenitors, respectively. Our study reveals a division of labour in the Wnt signalling pathway by suggesting that Wnt/STOP is the primary driver of cortical neurogenesis while Wnt/β-catenin is mainly responsible for self-renewal. These results resolve a decades-old question on the role of Wnt signalling in cortical neural progenitors.

Abstract CT222: Multimodal mechanism of action of the GSK-3 inhibitor 9-ING-41 (elraglusib) includes an immunomodulatory component: preliminary results from the 1801 phase 1/2 trial

Abstract 9-ING-41 (elraglusib) is a potent and selective GSK-3 inhibitor that has shown anti-tumor activity in patient-derived xenograft models and phase1/2 clinical studies in patients (pts) with advanced solid tumors. Preclinical studies have demonstrated that elraglusib downregulates PD-1, TIGIT and LAG-3, upregulates expression of MHC class I proteins in tumor cells from “cold tumors”, and shows synergy when combined with PD-1 blockade in mouse xenografts. Clinical activity has been observed both as a single agent and in combination with standard of care chemotherapies in several advanced cancer histologies. Here we present initial ‘omics data from our phase 1/2 study (NCT03678883; ACT1801) spanning &amp;gt;100 pts with advanced cancer evaluating both single agent and chemotherapy combinations. The patients (pts) included in this study received elraglusib as a ≥3rd line therapy for advanced disease. Most pts with melanoma treated with elraglusib monotherapy (8/9; 89%) stayed on study for &amp;gt;2 cycles. One patient with melanoma refractory to checkpoint inhibitors achieved a confirmed and durable CR. Clinical benefit was also observed among pts with colorectal cancer with 4/12 (33.3%) pts treated with monotherapy and 12/15 (80%) that received elraglusib plus irinotecan rechallenge stayed on study &amp;gt;2 cycles reaching median overall survival of 106 and 211 days, respectively. Based on emerging in vivo and in vitro results demonstrating that elraglusib activates T and NK cells promoting anti-tumor immune responses, we hypothesize immunomodulation by elraglusib may be contributing to anti-tumor immune response in the 1801 trial. We have acquired TCRseq and RNAseq profiles of PBMC samples from seven patients in 1801 during the first two weeks of treatment with elraglusib monotherapy. In these pts, reduced TCR clonality was observed and specific TCR clonotypes expanded after treatment, indicating T cell activation and expansion. These pts also showed changes in PBMC populations during elraglusib therapy as measured by immune deconvolution of PBMC RNAseq. Taken together, these data support a novel, previously unrecognized immunomodulatory mechanism of action for elraglusib and could provide rationale for future clinical development of elraglusib in pts with advanced malignancies. Additional analysis from TCRseq, immune profiling and cytokine analysis from expanded cohort of pts is ongoing. Citation Format: Taylor Weiskittel, Joseph McDermott, Daniel Billadeau, Hu Li, Anwaar Saeed, Devalingam Mahalingam, Solmaz Sahebjam, Pamela Munster, Ludimila Cavalcante, Frank J. Giles, Andrew Mazar, Benedito Carneiro. Multimodal mechanism of action of the GSK-3 inhibitor 9-ING-41 (elraglusib) includes an immunomodulatory component: preliminary results from the 1801 phase 1/2 trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT222.

Interrogating bromodomain inhibitor resistance in KMT2A-rearranged leukemia through combinatorial CRISPR screens

Bromo- and extra-terminal domain inhibitors (BETi) have exhibited therapeutic activities in many cancers. However, the mechanisms controlling BETi response and resistance are not well understood. We conducted genome-wide loss-of-function CRISPR screens using BETi-treated KMT2A-rearranged (KMT2A-r) cell lines. We revealed that Speckle-type POZ protein (SPOP) gene (Speckle Type BTB/POZ Protein) deficiency caused significant BETi resistance, which was further validated in cell lines and xenograft models. Proteomics analysis and a kinase-vulnerability CRISPR screen indicated that cells treated with BETi are sensitive to GSK3 perturbation. Pharmaceutical inhibition of GSK3 reversed the BETi-resistance phenotype. Based on this observation, a combination therapy regimen inhibiting both BET and GSK3 was developed to impede KMT2A-r leukemia progression in patient-derived xenografts invivo. Our results revealed molecular mechanisms underlying BETi resistance and a promising combination treatment regimen of ABBV-744 and CHIR-98014 by utilizing unique exvivo and invivo KMT2A-r PDX models.

Abstract 5636: Multiplex digital spatial profiling (DSP) of proteins in the tumor microenvironment in response to GSK-3 inhibition by 9-ING-41 (elraglusib) correlates with novel immunostimulatory effects observed in vivo

Abstract Glycogen synthase kinase 3 (GSK-3) is a serine/threonine kinase with key roles in myriad biological processes such as tumor progression, and inhibition of GSK-3 using the small molecule elraglusib has shown promising preclinical antitumor activity in multiple tumor types. Our preclinical experiments showed that elraglusib treatment increased tumor cell PD-L1 expression, downregulated angiogenic and immunosuppressive signaling pathways, and increased anti-tumor immune responses in vitro and in vivo. Subsequent studies showed that several circulating factors were predictive of response to PD-1/PD-L1 blockade and GSK-3 inhibition in a murine model of colorectal cancer. To determine the translational relevance of our prior results we evaluated tumor biopsies and plasma samples from patients with refractory solid tumors of multiple tissue origins enrolled in a Phase 1 clinical trial investigating elraglusib (NCT03678883). Plasma samples were collected from patients at baseline and 24 hours post-IV administration of elraglusib and were analyzed using Luminex technology. Paired FFPE tumor biopsies from patients with colorectal or pancreatic cancer before and after treatment were selected to analyze the tumor microenvironment using NanoString GeoMx DSP technology. The region of interest (ROI) selection strategy focused on mixed tumor and immune cell segments and ROIs were segmented using panCK+ and CD45+ morphology stains. Cytokine analysis revealed that elevated baseline plasma levels of IL-1 beta and reduced levels of VEGF correlated with improved progression-free survival (PFS) and overall survival (OS). PFS was also found to be positively correlated with elevated plasma levels of immunostimulatory analytes such as Granzyme B, IFN-gamma, and IL-2 at 24 hours post-treatment with elraglusib. CD45+ tumor-infiltrating immune cells had lower expression of VISTA, PD-1, and IDO-1 inhibitory checkpoint proteins and higher expression of OX40L and B7-H3 stimulatory checkpoint proteins in post-treatment biopsies as compared to pre-treatment biopsies. Moreover, time-on-study length negatively correlated with CD39 expression in PanCK+ segments and positively correlated with CD163 expression in CD45+ segments. This ongoing study, to our knowledge, represents the first digital spatial analysis of tumor biopsies from patients treated with elraglusib. These novel circulating biomarkers of response to GSK-3 inhibition could provide significant clinical utility and the spatial proteomics data may give us insights into the immunomodulatory mechanisms of GSK-3 inhibition. Citation Format: Kelsey E. Huntington, Christoph Schorl, Shaolei Lu, Daniel Newhouse, Benedito A. Carneiro, Wafik S. El-Deiry. Multiplex digital spatial profiling (DSP) of proteins in the tumor microenvironment in response to GSK-3 inhibition by 9-ING-41 (elraglusib) correlates with novel immunostimulatory effects observed in vivo. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5636.

Abstract 3884: Targeting the ARID1A mutations overcomes primary resistance to ALK inhibitors in EML4-ALK positive NSCLC

Abstract Introduction: Anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitors (TKIs) have improved an initial clinical response of non-small-cell lung cancer (NSCLC) patients with ALK-rearrangements. However, a subset of patients shows a poor response to ALK-TKIs. Here, we aimed to identify novel mechanism of primary resistance in preclinical model from a patient who presented an impressive resistance to sequential treatment with ALK TKIs. Experimental design: The PDC YU-1076 cells were established from pleural effusion of the patient at the time he was receiving chemotherapy and radiotherapy after failure of ALK-TKI treatment. Sanger sequencing confirmed ML4-ALK variant 1 detected at the initial diagnosis. No resistance mutations in the ALK kinase domain were detected. To investigate co-occurring genetic alterations, we performed whole exome sequencing (WES) on YU-1076 cells and the matched blood sample. We further investigated the molecular profile of YU-1076 cells using RNA-sequencing analysis. Results: YU-1076 cells exhibited cross-resistance to clinically available ALK-TKIs including crizotinib, ceritinib, alectinib, and lorlatinib. However, immunoblot analysis of YU-1076 cells treated with incremental doses of TKIs unexpectedly revealed an effective reduction of ALK activity and downstream signals. We noticed that YU-1076 cells gradually changed from a small, round shape to a fibroblast-like shape following the treatment of ALK-TKIs. Transcriptome analysis confirmed enrichment for gene signatures related epithelial-to-mesenchymal transition (EMT) in ALK TKI-treated YU-1076 cells, suggesting that ALK-TKIs treatment promotes YU-1076 cells toward a more mesenchymal phenotype. WES analysis identified a major chromatin remodeling complex subunit, AT-rich interacting domain 1A (ARID1A), as well as MYC amplification, CDKN2A loss, and TP53 mutations. We focused on a synthetic lethal strategies using the SFK inhibitor dasatinib and the EZH2 inhibitor GSK126 in ARID1A mutant cancers. The combination of dasatinib with ALK-TKIs restored the sensitivity to the ALK TKIs in YU-1076 cells, accompanied by suppression of EMT marker genes VIM and CDH2 and increase of apoptotic markers BIM, PARP, and CAS3. Congruently, dasatinib significantly impaired tumor growth in YU-1076-xenografts. GSK126 also induced synergistic inhibition of cell growth with upregulation of apoptosis marker genes in YU-1076 cells, but did not affect the expression of EMT marker genes. Conclusion: Our data indicate that ARID1A could be potentially used as a predictive biomarker for unfavorable ALK-TKI response. In this context, a combination strategy of ALK TKI with dasatinib may be effective in overcoming primary resistance. Citation Format: Seung Yeon Oh, You Won Lee, Eun Ji Lee, Ju Young Kim, Sewon Park, Ju Yeon Park, Su-Jin Choi, Mi Ra Yu, Sowon Aum, Jiyun Lee, Chang Gon Kim, Jii Bum Lee, Sun Min Lim, Min Hee Hong, Mi Ran Yun, Byoung Chul Cho. Targeting the ARID1A mutations overcomes primary resistance to ALK inhibitors in EML4-ALK positive NSCLC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3884.