Journal of Mucosal Immunology Research
Open Access

Immune Checkpoint Blockade (ICB) is at the forefront of immunotherapy regimes in the treatment of cancer [1-3]. Antibody blockade of programmed cell death-1 (PD-1) or its ligand, PD-L1 has played a prominent role in this immunotherapeutic approach with Nivolumab, Pembrolizumab and Atezolizumab [4-6] being the first FDA approved anti-PD1/PD-L1 antibodies alongside more recently approved Cemiplimab, Avelumab and Durvalumab [7,8]. Over 1300 studies involving combinations of PD-1 or PD-L1 antibodies are listed on the Clinicaltrials.gov registry.

The use of PD-1 mAb prevents T cells from recognising the PD-1 ligand (PDL-1) on tumour cells. As part of the body’s natural defence, T cells patrol the body for foreign cells and mount an immune response against them in order to destroy them. This recognition is carried out through PD-1-PDL-1/2 interaction; cells expressing PD1- L1/2 are recognised by the T cell and inhibitory signals are sent preventing effector cytotoxic responses. However, cancer cells may also express PDL-1/2 and this can lead to evasion of the immune response and formation of a tumour [9].

PD-1 mAbs have been used alone [10] or in combination with mAbs against other checkpoint molecules, such as cytotoxic Tlymphocyte- associated protein 4 (CTLA-4) [11]. Both mono and combination therapies have shown much success, however not all patients are cured, resistance may develop and there is a correlation with increased immune-related adverse events (irAEs) which include colitis, hepatitis, pneumonitis, cardiotoxicity, nephritis and vitiligo [12-16]. Around 10% of patients receiving anti-PD-1/PD-L1 antibodies suffer from grade 3-4 irAEs. These are serious side effects which need to be addressed in the development of new/improved treatments as well as improving efficacy.

A major advance would be to develop small molecules that modulate co-receptors or their signalling pathways for enhanced antitumour activity. The use of Small Molecule Inhibitors (SMIs) would provide several advantages over antibodies including, a short pharmacokinetic profile allowing flexible dosing and rapid withdrawal should signs of irAEs develop, the ability to cross membranes leading to better distribution and tumour penetration, and oral bioavailability, which will have a positive impact on the patient`s quality of life.

One approach for enhanced anti-tumour immunity is to inhibit pathways that control the expression of inhibitory co-receptors such as PD-1. SMIs have been used to impair PD-1/PD-L1 interaction by recognizing the PDL-1 binding pockets at the interface of PD-1 and blocking PD-1/PDL-1 binding directly and/or by inducing dimerization of PDL-1 (i.e. BMS-1001 and BMS-1166 Bristols-Myers- Squibb) [17]. Other SMIs can act simultaneously against two checkpoint inhibitor pathways due to their recognition of binding pockets with high sequence similarity [18,19]. Our recent work has highlighted the serine/threonine kinase glycogen synthase kinase-3 (GSK-3) as an alternative target. There are two ubiquitously expressed and highly conserved isoforms of GSK-3, GSK-3α and GSK-3β, which have shared and distinct substrates as well as functional effects. Both forms have been implicated in processes ranging from glycogen metabolism to gene transcription, apoptosis and microtubule stability.

GSK-3 is constitutively active in resting T cells [20,21] and is inhibited by receptor induced activation signals [22]. During T cell activation, the co-receptor CD28 binds to phosphoinositide 3-kinase, activating Akt, which phosphorylates Ser-21 and Ser-9 on GSK-3α and GSK-3, respectively [23], inhibiting GSK-3 activity. Inactivation of GSK-3 occurs by serine phosphorylation (Ser9:, Ser21: α) which allows its own phospho-serine tail to bind and block the active site [24,25]. This is a highly dynamic event whereby the serine tail switches rapidly between phosphorylated and dephosphorylated states causing a fluctuation of binding and release from the active site. This allows “primed” substrates that have accumulated in high levels to compete for the active site and become phosphorylated by GSK-3.

We have previously shown that inhibition of GSK-3 resulted in a down-regulation of Pdcd1 (PD-1) transcription via upregulation of the transcription factor Tbet [26]. This led to enhanced cytotoxic functionality of CD8+ T cells and increased levels of IFN-γ and Granzyme B expression, promoting viral clearance [26]. Further to this our current work shows that inhibition of GSK-3 can control B16 and EL4 tumour growth and is as effective as PD-1 blockade [27].

We have shown in vitro inhibition of GSK-3 by SMIs or siRNA to act primarily in CD8+ T cells reducing PD-1 expression. This inhibition has been shown further using SMIs in vivo in comparison to anti-PD-1 mAb treatment. T cells from GSK-3-/- mice also showed a reduction in PD-1 expression and B16 pulmonary metastasis was reduced to a similar extent in both Pdcd-/- and GSK-3-/- mice. Both models revealed a decrease in Pdcd1 transcription, with an increase in Tbx21 (Tbet) transcription and elevated numbers of CD8+ TILs expressing CD107a+ (LAMP1) and granzyme B (GZMB). Downregulation of Tbet with siRNA resulted in increased PD-1 expression indicating that Tbet inhibits PD-1 transcription, a finding consistent with that of another lab [28]. Inhibition of GSK-3 in T cells with downregulated Tbet had no effect on PD-1 expression indicating GSK-3 to operate upstream of and dependent on Tbet which in turn inhibits PD-1 expression.

Despite this, it is important to note that GSK-3 is likely to affect other aspects of T cell function in a PD-1 independent fashion. GSK-3 SMIs may eventually be found to alter the expression of other receptors and mediators and provide a potential advantage over anti-PD-1 blockade. However, in the context of the models examined to date, the down-regulatory effect on PD-1 plays a central role in generating antitumour immunity.

Overall, there are potential advantages and disadvantages to the use of GSK-3 SMIs versus anti-PD-1 antibody therapies.

Anti-PD-1 immunotherapy is associated with irAEs such as fatigue, rash and possible autoimmune complications such as colitis and although we cannot exclude these effects with GSK-3 SMIs, to date, we have seen no evidence of autoimmunity in the GSK-3-/- mice. However, there is the potential for GSK-3 inactivation to effect the function of other host cells or the tumour itself. We have not seen any direct effect of GSK-3 SMI on the growth of B16 melanoma cells, but GSK-3 inhibition has been reported to directly inhibit the growth of multiple myeloma, neuroblastoma, hepatoma and prostate tumours [29-33]. This may however be of added benefit whereby GSK-3 inhibitors can directly inhibit the growth of some tumours in addition to an enhancing effect on the immune system. However, in our studies, the major effect of GSK-3 SMIs was the amplification of the immune system. This was shown by the effects on ex vivo T cells, adoptive transfer experiments and by the elimination of tumours in mice with GSK-3 specifically deleted in their T cells.

With regard to patient benefit, several inhibitors are now moving forward into clinical trials. Lithium chloride is a classical inhibitor of GSK-3 which has been used for decades for the treatment of bipolar disease. Tideglusib has been investigated in a phase 2 oral study to treat progressive supranuclear palsy [34] and will be used in a new clinical trial in congenital Myotonic Dystrophy (ClinicalTrials.gov Identifier: NCT03692312). More recently, 9-ING-41, a potent GSK-3β inhibitor is being used in a phase 1/2 study to evaluate its safety and efficacy, as a single agent and in combination with cytotoxic agents, in patients with refractory cancers (ClinicalTrials.gov Identifier: NCT03678883).

Overall this shows numerous possibilities for GSK-3 SMIs in clinical applications and as research progresses, it is likely that developments in immunotherapy will move beyond the targeting of immune checkpoint blockade pathways such as CTLA-4 and PD-1 and focus will move to other approaches such as SMIs. Further work is needed to uncover the full range of down-stream effects that may be regulated by GSK-3 regulation in anti-tumour immunity, but overall these findings identify a potential alternate approach in the treatment of cancer.