A new tool in the cancer immunotherapy toolbox?

Programmed cell death-1 (PD-1), a member of the CD28 superfamily, is primarily expressed on the surface of T cells. The normal function of PD-1 is to dampen the immune activity and promote self-tolerance of activated CD8⁺ cytotoxic T cells by suppressing T cell inflammatory reactions. Binding of PD-1 with its ligand PD-L1 (or B7-H1) or PD-L2 (or B7-DC) excessively leads to the exhaustion of CD8⁺ cytotoxic T cells (1). In most of patients with advanced cancers, CD8⁺ cytotoxic T cells are exhausted owing to the engagement of PD-1 by the aberrantly expressed PD-L1 on tumor cells and/or nurse cells in the tumor microenvironment. Cancer immunotherapy aims to rejuvenate the exhausted CD8⁺ cytotoxic T cells by targeting the PD-1 immune checkpoint through blocking of PD-1/PD-L1 binding with antibodies specific to either PD-1 (Nivolumab or Pembrolizumab) or PD-L1 (Atezolizumab, Avelumab, or Durvalumab) (1). Such antibody mediated-blocking overcomes the exhaustion of these CD8⁺ cytotoxic T cells. Recently, Rudd’s group identified that glycogen synthase kinase 3 (GSK3) is an upstream kinase that regulates the expression of PD-1 in CD8⁺ cytotoxic T cells. By use of siRNA and small molecule inhibitors they demonstrated that inactivation of GSK3 could down-regulate PD-1 transcription resulting in enhanced CD8⁺ cytolytic T cell response measured by viral clearance activity (2). This study suggests the tantalizing possibility that inactivation of GSK3 may constitute a new way of reversing the exhausted CD8⁺ cytotoxic T cells to become functional cancer killing cytotoxic T cells.

In a recently published paper in Cancer Research (3), the same group tested this very idea and demonstrated the potential utility of GSK3 inhibitors in cancer immunotherapy. Using different mouse model systems, they showed that SB415286, a small molecule inhibitor to GSK3, provided impressive tumor suppression benefits similar to that of anti-PD-1 blocking antibody. Mechanistically, GSK3 inhibition resulted in induced expression of T-bet, a repressor of PD-1 gene transcription, which leads to the reduction of PD-1 expression in CD8⁺ cytotoxic T cells. Specifically, the authors first demonstrated that SB415286 significantly reduced the number of tumor metastasis nodules in lungs harvested from C57/B6 mice injected with mouse B16 tumor cells IV 14 days prior. This anti-tumor effect of SB415286 was comparable to that of using the anti-PD-1 blocking antibody. Furthermore, the combination of the anti-PD-1 blocking antibody and SB415286 did not show much additional effect suggesting the two agents are pharmacologically similar. Using conditional knockout mouse models in which GSK3 genes were exclusively deleted from T cells, they further showed that mice with GSK3 deficient T cells had a significant reduction in tumor growth (or engraftment), highlighting the direct role of GSK3 in modulating the antitumor function of cytotoxic T cells. Finally, the authors addressed the antigen specificity aspect of the tumor suppression using an Ova specific OT-1 transgenic mouse model engrafted with EL4 lymphoma tumor. They observed that Ova-antigen-bearing tumor xenografts grow slower than non-Ova-antigen-bearing ones; however, dramatic tumor clearance occurred only when the mice were treated with SB415286. This provides further evidence that the effect of GSK3 is to modulate antigen-specific tumor killing by CD8⁺ T cells. These results are consistent with an earlier report that in graft-versus-host disease hyperactive and hyperproliferative T cells from the transplant possess much lower GSK3 activity (4).

Aside from its role in the function of CD8⁺ cytotoxic T cells, GSK3 activity is also important to the function of NK cells. Parameswaran and colleagues (5) reported that NK cells in acute myelogenous leukemia patients have heightened expression of GSK3; inhibition of GSK3 activity significantly improved NK cell-mediated anti-tumor activity through the increased production of TNF-α and increased NK-tumor cell conjugation. Finally, Cichocki and coauthors (6) found in addition to enhanced tumor-killing activity, GSK3 inhibition could also drive the maturation of NK cells both in vitro and in mouse xenograft models.

GSK3 includes functionally related GSK3α and GSK3β each encoded by two distinct genes. Both GSK3α and GSK3β are multifunctional serine/threonine protein kinases involved in many key signaling pathways. Aside from its roles in CD8⁺ cytotoxic T cells and NK cells, GSK3 has long been suspected to play direct roles in the biology of cancer. However, Taylor et al. showed GSK3 inhibitor SB415286 did not have any direct effect on B16 or EL4 derived tumors engrafted in T cell deficient RAG2 knockout mice (3). The status of the expression and function of GSK3 proteins in these two cell lines used in their study is not known.

The latest work by Taylor et al. nicely validated their earlier prediction that GSK3 inhibition may be a viable alternative to PD-1/PD-L1 checkpoint blocking antibodies in cancer immunotherapy. However, this was done only in mouse systems; whether it also holds true in human patients remains to be demonstrated by clinical trials. Given that GSK3 is one of the earliest discovered protein kinases, it is not surprising that there have been over 20 different GSK3 inhibitors with varying specificity, potency and adverse effects developed to date. However, none are approved for cancer therapy. Only lithium is FDA approved and on available for the treatment of bipolar disorder. Lithium compound is unlikely to be useful in cancer given that it is neither a specific nor potent GSK3 inhibitor. Many of the other agents are primarily useful as tool compounds and are not undergoing clinical development. However, it is likely, given these reports, that pharmaceutical companies will be developing new GSK inhibitors. It is important that these new GSK inhibitors be evaluated in vivo since their primary anti-tumor effects may be mediated through immune stimulatory activity rather than a direct effect on the tumor cells. These trials will need to carefully evaluate not only tumor response but also effects on T cells and NK cells as described above. Indeed, it will be fascinating to see if GSK3 inhibitors alone, and in combination with immune checkpoint inhibitors, can induce similar PD-1/PD-L1 checkpoint blocking function in human CD8⁺ T cells, as well as NK cell mediated anti-tumor activities in patients. If so, GSK3 inhibitors could become a new tool in the ever-expanding immunotherapy armamentarium available to the oncologist.

Acknowledgments

Funding: This work was supported in part by CA97274-16.

Footnote

Provenance and Peer Review: This article was commissioned and reviewed by the Section Editor Dr. Yi Miao, MD, PhD (Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA).

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/aol.2018.03.01). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Chen L, Han X. Anti-PD-1/PD-L1 therapy of human cancer: past, present, and future. J Clin Invest 2015;125:3384-91. [Crossref] [PubMed]
2. Taylor A, Harker JA, Chanthong K, et al. Glycogen Synthase Kinase 3 Inactivation Drives T-bet-Mediated Downregulation of Co-receptor PD-1 to Enhance CD8(+) Cytolytic T Cell Responses. Immunity 2016;44:274-86. [Crossref] [PubMed]
3. Taylor A, Rothstein D, Rudd CE. Small-Molecule Inhibition of PD-1 Transcription Is an Effective Alternative to Antibody Blockade in Cancer Therapy. Cancer Res 2018;78:706-17. [Crossref] [PubMed]
4. Orbach A, Bassan-Levin T, Dan P, et al. Utilizing glycogen synthase kinase-3beta as a marker for the diagnosis of graft-versus-host disease. Transplant Proc 2013;45:2051-5. [Crossref] [PubMed]
5. Parameswaran R, Ramakrishnan P, Moreton SA, et al. Repression of GSK3 restores NK cell cytotoxicity in AML patients. Nat Commun 2016;7:11154. [Crossref] [PubMed]
6. Cichocki F, Valamehr B, Bjordahl R, et al. GSK3 Inhibition Drives Maturation of NK Cells and Enhances Their Antitumor Activity. Cancer Res 2017;77:5664-75. [Crossref] [PubMed]