Gating strategy employed for flow cytometry. A, The intratumoral cells were gated by FSC and SSC, followed by live single cells, CD45+, NK, and CD3+ T cell gating. B, Gated CD3+ T cells were gated on CD8a+ or CD4+ T cells. C, Gated CD4+ T cells were gated on CD25+Foxp3+ Treg cells. D-G, TIL analysis of intratumoral NK cells as in Fig. 5 (n = 10). D, E, and G, Scatter plots indicate the individual % or absolute number of cells and mean ± SEM are expressed as short horizontal lines and error bars. F, Expression profiles of representative data are shown. ****: P < 0.0001, ***: P < 0.001, **: P < 0.01, ns: not significant, compared with the vehicle group (unpaired Student’s t test).
ARTICLE ABSTRACT
Studies of diacylglycerol kinase ζ (DGKζ) in DGKζ knockout mice have revealed its role as an intracellular immune checkpoint in T cells. Although enhancing antitumor immunity by pharmacologic inhibition of DGKζ is desirable, selective DGKζ inhibitors for clinical use remain largely unexplored. In this study, we report a novel, small-molecule DGKζ inhibitor, ASP1570, which is currently under phase 1 development (NCT05083481), and characterize its effect on potential resistance mechanisms against approved immune checkpoint inhibitors in multiple immunosuppressive conditions: not only TGF-β, prostaglandin E2, adenosine, and PD-1 but also cytotoxic T-lymphocyte antigen-4 and T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif. First, our findings indicated that ASP1570 exhibited an inhibitory effect on the kinase activity of DGKζ. Unexpectedly, we observed that DGKζ protein was degraded in cells treated with ASP1570 in a proteasome-dependent manner. ASP1570 enhanced T-cell activation with increased diacylglycerol downstream signaling and released anergic T cells from their hyporesponsive state. Furthermore, ASP1570 restored T-cell functions suppressed by multiple immunosuppressive signals (TGF-β, prostaglandin E2, adenosine, PD-1, cytotoxic T-lymphocyte antigen-4, and T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif) and induced tumor growth inhibition in two types of syngeneic mouse models: anti–PD-1 antibody–sensitive MC38 and anti–PD-1 antibody–insensitive B16F1/F10. The antitumor efficacy of ASP1570 was canceled by CD8+ T-cell depletion, indicating that its antitumor effect depends on CD8+ cytotoxic T-cell activation. Collectively, ASP1570 potentially improves antitumor efficacy in both anti–PD-1 therapy–resistant and anti–PD-1 therapy–responsive tumors by overcoming multiple immunosuppressive signals.
