Glycogen synthase kinase-3 beta inhibitors as novel cancer treatments and modulators of antitumor immune responses

ABSTRACT

As a kinase at the crossroads of numerous metabolic and cell growth signaling pathways, glycogen synthase kinase-3 beta (GSK-3β) is a highly desirable therapeutic target in cancer. Despite its involvement in pathways associated with the pathogenesis of several malignancies, no selective GSK-3β inhibitor has been approved for the treatment of cancer. The regulatory role of GSK-3β in apoptosis, cell cycle, DNA repair, tumor growth, invasion, and metastasis reflects the therapeutic relevance of this target and provides the rationale for drug combinations. Emerging data on GSK-3β as a mediator of anticancer immune response also highlight the potential clinical applications of novel selective GSK-3β inhibitors that are entering clinical studies. This manuscript reviews the preclinical and early clinical results with GSK-3β inhibitors and delineates the developmental therapeutics landscape for this potentially important target in cancer therapy.

KEYWORDS:

• GSK-3β
• cancer
• immunotherapy
• clinical trial

Introduction

Glycogen synthase kinase-3 (GSK-3), a serine/threonine protein kinase with two functionally distinct isoforms, α and β, was discovered in the context of glycogen metabolism and emerged as a ubiquitous regulator of multiple signaling pathways (Figure 1).¹ Tyrosine 216 and serine 9 phosphorylation prime the GSK-3 beta (GSK-3β) enzyme to either activation or inhibition, of its kinase properties, respectively.² Approximately, 100 proteins have been reported as phosphorylation targets of GSK-3β.³ GSK-3β can serve as either a tumor suppressor by priming oncogene products for proteasomal destruction or as a pro-oncogene, mainly through proliferative pathways such as Wnt/β-catenin.⁴ GSK-3 has recently been linked to increased programed cell death-1 (PD-1) expression and its inhibition enhanced T-cell response.^5,6 The majority of inhibitors developed compete with ATP for GSK-3’s ATP-binding site with low selectivity. No selective GSK-3β inhibitor has been FDA approved for treatment of cancer or other diseases as of yet. This manuscript summarizes the evidence supporting GSK‐3β as a promising cancer therapeutic target and reviews the clinical development of GSK-3β inhibitors.

Figure 1. GSK-3β-mediated signaling pathways. When the Wnt ligand is present, it inhibits GSK-3β on the target cell, which suppresses β-catenin phosphorylation and makes it stable in cytoplasm. Stable β-catenin translocates into the nucleus promoting transcription of target genes such as c-Myc and cyclin-D. GSK-3β prevents epithelial–mesenchymal transition (EMT) by inhibiting Snail, a repressor of E-cadherin gene. GSK-3β phosphorylates several upstream and downstream components of the PI3K/AKT/mTOR pathway, and AKT can phosphorylate GSK-3β and inhibit its activity.

Rationale for therapeutic targeting of GSK-3β in cancer

GSK-3 has been described as a tumor suppressor in malignancies such as skin,^7,8 breast,^9–11 oral cavity,^12,13 and lung cancers,^14,15 and as a tumor promoter in other malignancies such as pancreas,^16–19 colorectal,^20,21 hepatocellular carcinoma,^22,23 kidney,²⁴ leukemia,^25,26 and glioblastoma multiforme (GBM).^27,28

The role of GSK-3 in signaling pathways was initially described in the Wnt/β-catenin pathway. GSK-3-mediated β-catenin phosphorylation has a critical role in Wnt–β-catenin signaling. Wnt signaling inhibits GSK-3β, thus preventing β-catenin degradation and stabilizes β-catenin, which in turn increases transcriptional activity of c-Myc and cyclin-D.²⁹ GSK-3β modulates the double strand homologous repair pathway by phosphorylating FAAP2 (Fanconi Anemia-Associated Protein, 20 kDa), an important component of the Fanconi Anemia complex involved in the repair of DNA interstrand cross-links.³⁰ In GBM, GSK-3β mediates repair of DNA double strand-break through phosphorylation of 53BP1 (p53 binding protein 1).³¹ GSK-3β can also phosphorylate several upstream and downstream components of the PI3K/AKT/mTOR pathway including AKT, RICTOR, and PTEN.³² Moreover, the regulatory roles of GSK-3β in cell cycle,³³ apoptosis,³⁴ tumor invasion, and metastasis have been well described.^35,36 GSK-3β also affects cellular responses to kinases.^37,38

Regulation of GSK-3β by microRNAs (miRNAs), a class of noncoding small RNAs, has been implicated in cancer progression and treatment resistance. This interaction has been shown with multiple miRNAs in various malignancies including gastric cancer (miR-96, miR-182, and miR-183), colorectal cancer (miR-224), lung cancer (miR-26a), breast cancer (miR-1229), melanoma (miR-769), pancreatic cancer (miR-940), bladder cancer (miR-433), endometrial cancer (miR-129), ovarian cancer (let-7), and nasopharyngeal carcinoma (miR-15a).^{8,10,18,21,39–43}

The cBio Cancer Genomics Portal dataset analysis reveals the landscape of GSK-3β genomic alterations among various cancer types (Figure 2).^44,45 These results highlight the rationale to target certain cancers such as myeloid, penile and colon neoplasms carrying GSK-3β gene mutation in about 15% of cases. Based on the search on Catalogue of Somatic Mutations in Cancer dataset, there are no available data on targeted drugs that have been used to treat GSK-3β mutant tumors (cancer.sanger.ac.uk).⁴⁶

Figure 2. Genomic alterations in GSK-3β. The landscape of GSK-3β genomic alterations among cancer types from the cBio Cancer Genomics Portal dataset.

Mutations at GSK-3β phosphorylation sites prevent its ability to phosphorylate its substrates.⁴⁷ For instance, mutation in GSK-3β arginine 96 to alanine abrogates the ability of GSK-3 to phosphorylate pre-primed substrates.⁴⁸ Mutations in GSK-3 targets also limit the ability of GSK-3β to phosphorylate its substrates. Mutation of c-Myc Thr58 phosphorylation site blocks GSK-3-induced phosphorylation of c-Myc preventing its proteasomal degradation.⁴⁹ Similarly, genetic mutations changing the GSK-3 phosphorylation sites S33, S37, S45, or T41 present in β-catenin inhibited the GSK-3β-mediated phosphorylation of β-catenin.^47,50 Thus, mutations at GSK-3 or its substrates can impact its kinase activity.⁴⁷

Cellular localization of GSK-3β also seems important to its signaling properties. Increased nuclear accumulation of GSK-3β has been demonstrated in a number of malignancies including breast, head and neck, pancreatic, renal, and bladder cancers.^51–53 Nuclear GSK-3β accumulation is believed to enhance NF-κB binding to its target gene promoters increasing transcriptional activity of antiapoptotic molecules expression in cancer cells.^4,54

GSK-3β role in specific cancers

Prostate cancer

GSK-3β is a positive regulator of androgen receptor (AR) transactivation and tumor growth independent of the Wnt/β-catenin pathway.⁵⁵ Compared to normal prostate tissue, GSK‐3β was upregulated in prostate cancer specimens, and cytoplasmic accumulation was associated with more aggressive clinicopathological features.^56,57 TDZD-8, first non-ATP competitive inhibitor of GSK-3β, and L-803-mts, a substrate-competitive inhibitor, inhibited prostate cancer growth in preclinical models.⁵⁸ Recently, the kinase inhibitor 6-bromoindirubine-3′-monoxime decreased AR expression in prostate cancer cells via inhibition of both GSK-3α and GSK-3β.⁵⁹ The recruitment in a phase 1 study evaluating lithium, a known inhibitor of GSK-3 and its effect on localized prostate cancer, was completed with no published data as yet⁶⁰ (NCT02198859).

Kidney cancer

Several lines of evidence highlight the involvement of GSK-3β in the pathogenesis of renal cell carcinoma (RCC). Increased nuclear expression of GSK-3β was described in RCC and GSK-3β inhibition decreased the expression of NF-κB target genes Bcl-2 and XIAP leading to increased apoptosis in RCC models.^51,61 GSK-3β also phosphorylated 4EBP1 leading to activation of mTORC1 downstream signaling cascades promoting cell proliferation in RCC and the effect was reversed by GSK-3 inhibition (AR-A014418).⁶² Inhibition of GSK-3 also potentiated antitumor activity of gemcitabine and tyrosine kinase inhibitors sorafenib and gemcitabine in preclinical models of RCC.^63,64 These results align with significant antitumor activity of 9-ING-41, a maleimide-based small molecule-specific GSK-3β inhibitor, in two xenograft models of RCC.⁶⁴

Pancreas cancer

Pancreatic cancers express aberrant nuclear accumulation of GSK-3β particularly in advanced disease state.^52,65 Nuclear accumulation of GSK-3β regulates NF-κB transcriptional activity but exact mechanism is unknown. Possible mechanisms include direct interaction with NF-κB leading to effects on DNA binding for antiapoptotic genes or through increasing accessibility of NF-κβ through chromatin structure.⁶⁵ Ding et al.⁶⁶ demonstrated that GSK‐3β promotes TGF‐α‐induced acinar to ductal metaplasia and GSK‐3β deletion reduced pancreatic intraepithelial neoplasia progression in KrasG12D transgenic mice. Another possible mechanism inducing pancreatic cancer cell proliferation is GSK-3β-dependent SP2 phosphorylation which helps NFATc2 (nuclear factor of activated T cells), oncogenic transcription factor, and protein stability in the nucleus and maintains NFATc2 activation via stabilization of NFATc2–STAT3 complexes.⁶⁷ In a recent study, LY2090314, a selective ATP-competitive GSK-3 inhibitor, was evaluated for possible additive effect with nab-paclitaxel, gemcitabine, oxaliplatin, and SN-38.⁶⁸ Inhibition of GSK-3 enhanced the effect of chemotherapy in pancreatic cancer cells via modulation of stability of oncogenic effectors, yes-associated protein, and transcriptional coactivator with PDZ-binding motif (TAZ).⁶⁸ A phase 1/2 study of LY2090314 in combination with chemotherapy in advanced pancreatic cancer was conducted but was terminated due to slow enrollment (NCT01632306).

GBM

Higher expression and activity of GSK-3β have been documented in GBM cell lines compared to normal brain tissue.⁶⁹ Tumor expression of the active form of GSK-3β (pGSK3β^y216) among 57 human tumor specimens was associated with worse progression-free survival and overall survival (OS) and multivariate analysis showed that activation of GSK-3β was an independent predictor of poor outcome.⁷⁰ Multiple mechanisms involving GSK-3β in GBM pathophysiology have been described.^69,71 GSK-3β inhibition can cause c-Myc-dependent glioma cell cytotoxicity.⁷² A study by Koru et al.⁷³ suggested that GSK-3 inhibition might specifically target a subpopulation of GBM with stem cell-like characteristics. To evaluate the potential role of GSK-3β in glioblastoma stem-like cells, protein profiles from patients and normal brain samples were analyzed in another study and results suggested that GSK-3β displays distinct roles based on EGFR expression/regulation and protein phosphatase 2A (PP2A) expression.⁷⁴

Interestingly, GSK-3β had a tumor suppressor role in GBM cells with EGFR overexpression, while constitutively active GSK-3β in PP2A overexpression led to an oncogenic role. Several studies showed that GSK-3β inhibition attenuates the proliferation and invasion of GBM cancer cells and sensitizes them to chemotherapeutic agents and radiation.^69,72,75,76 A recent study demonstrated that transcriptional targets of GSK-3β regulate GBM invasion and suggested potential therapeutic targets.⁷⁷ Ugolkov et al. showed that GSK-3β inhibitor 9-ING-41 in combination with lomustine, one of the standard therapies for recurrent GBM, enhanced antitumor activity in PDX models of human GBM.⁷⁸ The effect of 9-ING-41 was shown in staged orthotopic GBM12 and GBM6 PDX models, which had minimal response to single agent lomustine. Importantly, combination treatment resulted in histologically confirmed cures with significant increase in OS.

A phase 1/2 study evaluated a combination of drugs with GSK-3β-inhibitory activity (i.e., cimetidine, lithium, olanzapine, and valproate—CLOVA cocktail) plus temozolomide among 20 patients with recurrent GBM.⁷⁰ Preclinical studies had demonstrated that CLOVA cocktail inhibited GSK-3β activity in GBM cells. CLOVA cocktail in combination with temozolomide showed increased survival in recurrent GBM patients compared to the control group treated with temozolomide alone (median OS 11.2 vs. 4.3 months; p = 0.004).⁷⁰ While limited by the lack of specificity of multiple inhibitors and small number of patients, this phase 1/2 study supports the therapeutic potential of GSK-3β inhibitors in GBM in combination with temozolomide. In fact, preclinical studies had shown that GSK-3β inhibition increased sensitivity of temozolomide in GBM by silencing methylguanine DNA methyltransferase expression via c-Myc-mediated promoter methylation.⁷⁶

Acute myeloid leukemia

GSK-3β has been involved in the signaling pathways of hematologic malignancies particularly acute myeloid leukemia (AML) given its regulation of Wnt/β-catenin pathway which is associated with drug resistance in AML.⁷⁹ Recent data suggested that GSK-3β localization has clinical significance with worse patient survival and mediates drug resistance in both in vitro and in vivo.⁸⁰ GSK-3β is also upregulated in natural killer (NK) cells from AML patients compared to normal donors.⁸¹ Same study showed that GSK-3β overexpression in NK cells impairs their ability to kill AML cells, while genetic or pharmacological GSK-3β inhibition enhances NK cell cytotoxic activity in human AML mouse models. Gupta et al.⁸² showed that all-trans retinoic acid (ATRA) receptor is a target of GSK-3β and its inhibition with combination of ATRA significantly enhances ATRA-mediated AML differentiation and growth inhibition. Other studies also suggested GSK-3β as promising target in AML cells since inhibition of this kinase leads to the growth inhibition and differentiation of leukemic cells.^33,81 These results provided the rationale for a phase 2 study evaluating the GSK-3 inhibitor LY2090314 as monotherapy among 20 patients with AML that showed an acceptable safety profile but no clinical responses.⁸³ A transient increase in the levels of β-catenin following treatment was observed in peripheral blood mononuclear cell (PBMC)s and blast cells suggesting target inhibition, but levels returned to baseline by 24 h suggesting limited exposure to the drug. Most frequent drug-related adverse events included decreased appetite, nausea, dry mouth, dyspepsia, fatigue, and prolonged QT interval (8/19 AEs were Grade 3/4). Possibly drug-related visual disturbances were reported in two patients (blurred vision, flashing lights). No further studies are being pursuing with LY2090314 in AML.

GSK-3β inhibitors

Several GSK-3β inhibitors have been developed and advanced to early stage clinical trials including multiple cancer histologies.⁸⁴

Tideglusib, an ATP noncompetitive GSK-3β inhibitor, originally developed to decrease tau phosphorylation in Alzheimer disease, increases proapoptotic proteins in murine models of human neuroblastoma.⁸⁵ Tideglusib also decreased colony formation and increased G0/G1 population in tumor cells. One study showed that tideglusib sensitizes intracranial glioblastoma xenografts to temozolomide with improvement in mouse survival.⁸⁶ Tideglusib also increased NK cell cytotoxic activity in human AML mouse models.⁸¹ The drug was tested in phase 2 studies for Alzheimer’s disease and progressive supranuclear palsy and was found to be well tolerated.^87,88 To our knowledge, there is no active cancer clinical trials with tideglusib.

Antiproliferative effects of LY2090314, ATP-competitive GSK-3 inhibitor, were observed in preclinical studies for melanoma and neuroblastoma.^89,90 A phase 1 study found that LY2090314 could be dosed at a safe and tolerable level in patients with cancer.⁹¹ Signal of antitumor activity was observed in combination with pemetrexed and carboplatin in patients with mesothelioma, non-small cell lung, and breast cancers. An open-label phase 2 study showed that the drug was well tolerated with minimal non-hematologic side effects (i.e., nausea and decreased appetite) found in 11 out of 20 patients.⁸³ Hematologic effects were minimal but included anemia, thrombocytopenia, and neutropenia. None of the patients in the study had complete or partial remission making LY2090314 likely a poor candidate as single agent but combination treatments might be of interest in future studies.

Solasodine, a naturally occurring aglycone of glycoalkaloid, displayed antitumor activity in different cancers by inhibiting the GSK-3β pathway and inducing apoptosis.^92,93 AR-A014418, a highly specific GSK-3β inhibitor, enhanced the cytotoxic effects of temozolomide and gemcitabine in models of GBM and pancreatic cancer, respectively.⁷⁵ A preclinical study using AR-A014418 demonstrated that inhibition of GSK-3 decreased gastric cancer cell survival and proliferation through decreasing telomerase reverse transcriptase expression and telomerase activity.⁹⁴ Clinical studies are warranted to investigate safety and efficacy of AR-A014418.

9-ING-41 is a small ATP-competitive GSK-3 inhibitor molecule which inhibits both α and β isoforms but more selective for GSK-3β than for 320 other related kinases.²⁴ In renal cancer, GSK-3β inhibition by 9-ING-41 decreased proliferation via G0–G1 and G2-M phase arrest and induced autophagy by altering glucose metabolism.²⁴ The study also showed the antitumor activity of 9-ING-41 in two xenograft models of RCC. In addition to its antiproliferative effects, 9-ING-41 decreased tumorigenicity of cancer cells by changing their phenotype from mesenchymal state to epithelial direction via increased Ksp-cadherin level and decrease Id-1 level. Similarly, another study demonstrated that 9-ING-41 significantly enhances the cytotoxicity of gemcitabine and cabozantinib in RCC cell lines.⁶⁴

GSK-3β inhibition in breast cancer cells in vitro decreased cancer cell survival by suppressing the expression of antiapoptotic proteins Bcl-2 and XIAP and enhanced tumor response to irinotecan in vivo.⁹⁵ The study also demonstrated that 9-ING-41 provided more potent inhibition of tumor growth than other tested GSK-3 inhibitors including AR-A014418, SB-216763, and LY2090314.

Constitutively active NF-κβ has been a hallmark feature of chemotherapy-resistant glioblastoma, making GSK-3β inhibition a particularly promising target. In GBM models, single agent 9-ING-41 showed modest antitumor activity but combination with lomustine led to significant tumor regression and survival benefit.⁷⁸ Similar to breast cancer models, results from GBM models suggested that potential additive effect form the combination of 9-ING-41 and chemotherapy.

Antitumor effect of 9-ING-41 was also observed in animal models of neuroblastoma.⁹⁶ GSK-3 inhibition decreased expression of the NF-κB target XIAP leading to apoptosis. Mouse models showed that combination of 9-ING-41 with irinotecan provided a significant antitumor effect. Compared to other GSK-3 inhibitors AR-A014418 and TDZD-8, 9-ING-41 was found to be the most potent inhibitor. These results provided the support for FDA granting Rare Pediatric Disease Designation to 9-ING-41, critical to advance the clinical development in the setting of this rare and devastating malignancy.

9-ING-41 also showed antiproliferative activity in B-cell lymphoma models where it reduced cell viability of up to 70%.⁹⁷ GSK-3β inhibition enhanced apoptosis in aggressive B-cell lymphoma lines through downregulation of pro-survival molecules. Combinations with BCL-2 inhibitor venetoclax and a CDK-9 inhibitor (BAY-1143572) enhanced the antitumor activity of 9-ING-41 and reduced IC₅₀ values significantly. These results provide foundational rationale to further investigate this agent in advanced non-Hodgkin’s lymphoma.

A phase I study designed to evaluate the safety and clinical efficacy of 9-ING-41 as a single agent and in combination with cytotoxic agents in patients with advanced malignancies is underway (NCT03678883).

GSK-3β regulation of immune checkpoints

Recent publications highlight the role of GSK-3β in regulating the immune response.

Gattinoni et al. described how effector T CD8+ cells are able to dedifferentiate into stem cells through GSK-3β inhibition.⁹⁸ GSK-3β was shown to regulate the Wnt/β-catenin pathway, which coordinates stemness through its TCF/LEF family of transcription factors by a gene expression program that suppresses differentiation in a subset of T cells. By blocking T-cell differentiation via GSK-3β inhibition, Wnt signaling was able to generate multipotent CD8+ memory stem cells with antitumor capacities. Moreover, GSK-3β inhibition enhanced the antitumoral capacity of CD8+ memory stem T cells both in vitro and in vivo via FAS ligand neutralization in a gastric cancer study.⁹⁹ Similarly, inhibition of GSK-3β in a model of GBM-specific CAR-T cells increased survival and memory phenotype generation with enhanced tumor-killing ability in GSK-3β-inhibited IL13CAR-T cells.^100,101 These results suggest that GSK-3 inhibitor could enhance antitumor response of T cells and improve CAR-T-cell treatments.

PD-L1 and PD-L2 expressed by tumors bind to PD-1 in the T CD8+ cells leading to immune system evasion. Taylor et al. identified GSK-3β as a key upstream kinase regulating PD-1 expression in CD8+ T cells and its inhibition blocked PD-1 expression, resulting in increased CTL function.⁵ In the presence of anti-PD-1, SHP-1/2 phosphorylates the CD28 YMNM motif which leads the recruitment of PI3K. PI3K phosphorylates and inhibits GSK-3α (Ser21) and GSK-3β (Ser9) by AKT. Ultimately, inhibition of GSK-3 upregulates the transcription of the transcription factor Tbx21 (Tbet) which inhibits PD-1 expression (Figure 3). Thus, the study suggested the following mechanistic model: GSK-3 inhibition increases Tbx21 transcription leading to enhanced T-bet expression which represses PD-1 expression and increases T-cell killing. Moreover, it was recently discovered that the rescue of exhausted CD8+ T cells by anti-PD-1 blockade requires CD28 expression¹⁰² and inactivation of GSK-3β was shown to substitute CD28 co-stimulation in priming of cytotoxic CD8+ T cells.^81,103 GSK-3 inhibition reversed the effects of CD28 blockade with CTLA-4-IgG in the cytotoxic response of CTLs. These results suggest a potential role of GSK-3 inhibition as a strategy to help restore exhausted CD8+ T cells.

Figure 3. Proposed model for regulation of PD-1 by GSK-3β signaling. In the presence of anti-PD-1, activation of Src homology region domain-containing phosphatase (SHP) is inhibited, thus allowing for the phosphorylation of the CD28 phosphoinositide 3-kinase (PI3K)-binding site which leads GSK-3β inhibition via AKT activation. GSK-3β induces the transcription of the transcription factor Tbx21 (Tbet) which in turn inhibits transcription and expression of PD-L1.

Taylor et al. investigated further how GSK-3 inhibition could downregulate PD-1 on T cells. GSK-3 inhibition decreased tumor growth and metastasis by downregulating PD-1 on CD8+ T cells in model of melanoma, while having a minimal effect on NK cells without obvious effect on CD4+ T cells.⁶ The effect was similar to anti-PD-1 pretreatment. Importantly, GSK-3 inhibitors did not show further inhibition of tumor growth in Pdcd1^−/− mice (PD-1 deficient). Anti-PD-1 treatment did not inhibit tumor growth in GSK-3α/β^−/− mice supporting the hypothesis that antitumor activity from GSK-3 inhibition was mediated by downregulation of anti-PD-1. Similar results were observed in lymphoma model. Although combination therapy did not have an additional effect compared to monotherapy, this study raises an important question whether GSK inhibitors could have comparable effect to anti-PD1 inhibitors in some models or could be used in combination with other checkpoint inhibitors in the future.

In addition to its pivotal role in PD-1 pathway, the potential role GSK-3β in PD-L1 pathway was demonstrated in a breast cancer study.¹⁰⁴ The study showed that PARP inhibition increased PD-L1 expression primarily through GSK-3β inactivation. The study also demonstrated that PARP inhibition induced GSK-3β Ser9 phosphorylation which was associated with increased PD-L1 expression. Knocking out GSK-3β had similar observation. Interestingly, PARP inhibition did not increase PD-L1 expression level in GSK-3β-knockout cells which suggest that inactivation of GSK-3β is required for the PARP inhibitor-induced PD-L1 expression. More importantly, anti-PD-L1 treatment was able to sensitize PARP inhibitor-treated cancer cells to T-cell killing. Combination of PARP inhibitor with anti-PD-L1 treatment was shown to be more effective than each treatment alone suggesting the therapeutic potential of such combinations. It might be particularly relevant to tumors with mutations in DNA repair genes (i.e., breast, ovarian, and prostate cancers) known to be sensitive to PARP inhibitors but developing resistance to such treatment in relatively short period.

The role of GSK-3 in NK cells was also described. GSK-3 overexpression impaired NK cell ability to kill AML cells, while GSK-3 inhibition enhances NK cell cytotoxic activity in human AML mouse models.⁸¹ In support of this data, a recent study explored inhibition of GSK-3 kinase in peripheral blood NK cells and expanded these cells ex vivo. GSK-3 inhibition caused elevated expression of several transcription factors, which are associated with NK cell maturation. Transferring conditioned NK cells to an established mouse xenograft model of ovarian cancer provided more robust and durable tumor control.¹⁰⁵

Future directions

GSK-3β is evolutionary an ancient protein that serves different roles in the cell as it is centrally located at the watershed of several signaling and metabolic pathways.

The evolution of precision medicine will move in parallel with the finding of new inhibitors. While the efficacy of new agents is proven in vivo, biomarkers from refined cancer pathways will inform the best molecular scenario in a continuous bench-to-bedside feedback loop. As an example, the absence of Myc mutations as the major player in c-Myc overexpression led scientists to focus upstream, revealing GSK-3 as a potential target in Myc overexpressing tumors.

Based on its interactions with apoptosis and chromatin remodeling process, combination strategies of GSK-3β inhibitors with Bcl-2 inhibitors, HDAC, hypomethylating agents, and PARP inhibitors have therapeutic relevance. New agents may be found while targeting bromodomains, chromodomains, Tudor, and PWWP domains, and their relationship to NF-κB (renal, pancreas) and STAT3 (pancreatic adenocarcinoma) transcription machinery may further our understanding of GSK-3 signaling.

GSK-3β inhibitors may also have an important role in regulating epithelial–mesenchymal transition and therefore metastasis control, especially in prostate and bladder cancers engendered by the Snail/E-cadherin pathway, which warrants further studies.

Finally, the understanding of GSK-3β role in regulating anticancer immune response is evolving rapidly with promising preclinical results. Particularly, dissecting the mechanisms of GSK-3β in PD-1 regulation may lead to future clinical trials with evaluation of GSK-3β inhibitors with checkpoint inhibitors.

Conflict of interest

The authors declare no potential conflicts of interest except Francis J. Giles who is a consultant and shareholder in Actuate Therapeutics Inc.