Background Diacylglycerol kinases (DGK) convert diacylglycerol (DAG) into phosphatidic acid which limits DAG-induced propagation of T cell activation.¹ T cells predominantly express DGK alpha and DGK zeta isoforms (DGKalpha/zeta),² and deletion of either enhances T cell activation and function.^3,4 DGKalpha/zeta are overexpressed in tumor infiltrating T cells and may limit the anti-tumor activity of these cells.⁵ Thus, inhibition of DGKalpha/zeta is a potential therapeutic avenue to enhance T cell anti-tumor responses. We demonstrate that inhibition of DGKalpha/zeta with novel dual DGKalpha/zeta inhibitors (DGKi) enhances anti-tumor T cell activity.

Methods The role of DGKalpha/zeta in T cell activation was studied using wildtype and CRISPR-knockout T cells. In vitro studies assessing the effect of DGKi were conducted using various mouse and human T cell functional assays. T cell exhaustion studies were performed in T cells from lymphocytic choriomeningitis virus (LCMV) clone 13-infected mice. In vivo activity of DGKi was assessed by measuring T cell activation in OT-I mice challenged with OVA-peptides. Efficacy studies and memory rechallenge experiments were performed in the MC38 syngeneic tumor model.

Results CRISPR knockout studies characterizing the role of DGKalpha/zeta on T cells demonstrated that both isoforms regulate T cell activation and function. DGKi enhanced IFN gamma production by human melanoma-specific T cells stimulated with the weak affinity MART1 antigen. Similarly, treatment of mouse OT-I cells with DGKi enhanced responses to low-affinity OVA-peptides and OT-I-mediated cytotoxicity of MC38-OVA tumor cells. Treatment of exhausted CD8 T cells with DGKi reverted exhaustion caused by chronic LCMV infection, and enhanced T cell function with PDL1 blockade. DGKi rescued cytokine release from T cells suppressed by adenosine, TGF beta or PGE₂ treatment. Oral administration of DGKi resulted in dose-dependent increase in T cell activation in mice. Furthermore, DGKi in combination with anti-PD-1 therapy was efficacious in the MC38 model with complete tumor regressions in treated animals. These mice eradicated tumors upon rechallenge with MC38 cells two months later, demonstrating durable anti-tumor memory responses.

Conclusions We demonstrate that DGKalpha/zeta negatively regulates T cell activity. Dual DGKalpha/zeta inhibition restores T cell responses to weak tumor antigens, overcomes cell exhaustion, and enhances CD8 T cell cytotoxic activity. In vivo efficacy data demonstrates that DGKi in combination with anti-PD-1 antibody therapy promotes robust anti-tumor responses and generates durable T cell memory. These data support dual DGKalpha/zeta inhibition in combination with anti-PD-1 antibodies as a therapeutic approach for cancer treatment.

References

1. Mérida I, Avila-Flores A, Merino E. Diacylglycerol kinases: at the hub of cell signalling. Biochem J. 2008; 409(1):1–18.

2. Krishna S, Zhong X. Role of diacylglycerol kinases in T cell development and function. Crit Rev Immunol. 2013; 33(2):97–118.

3. Guo R, Wan C-K, Carpenter JH, et al. Synergistic control of T cell development and tumor suppression by diacylglycerol kinase alpha and zeta. Proc Natl Acad Sci U S A. 2008; 105(33):11909–14.

4. Riese MJ, Grewal J, Das J, et al. Decreased diacylglycerol metabolism enhances ERK activation and augments CD8+ T cell functional responses. J Biol Chem. 2011; 286(7):5254–65.

5. Prinz PU, Mendler AN, Masouris I, et al. High DGK-α and disabled MAPK pathways cause dysfunction of human tumor-infiltrating CD8+ T cells that is reversible by pharmacologic intervention. J Immunol. 2012; 188(12):5990–6000.

Ethics Approval All animal studies were approved and conducted in accordance with the Explora IACUC Program of Veterinary Care.