Abstract

Previous work has shown that innate immune sensing of tumors involves the host STING pathway, which leads to IFN-{beta} production, dendritic cell (DC) activation, and T cell priming against tumor antigens. This observation has led to the development of STING agonists as a potential cancer therapeutic. However, despite promising results in mouse studies using transplantable tumor models, clinical testing of STING agonists has shown activity in only a minority of patients. Thus, further study of innate immune pathways in anti-tumor immunity is paramount. Innate immune activation in response to a pathogen rarely occurs through stimulation of only one signaling pathway, and activating multiple innate immune pathways similar to a natural infection is one possible strategy to improve the efficacy of STING agonists. To test this, we performed experiments with the STING agonist DMXAA alone or in combination with several TLR agonists. We found that LPS + DMXAA induced significantly greater IFN-{beta} transcription than the sum of either agonist alone. To explain this synergy, we assayed each step of STING pathway signaling. LPS did not increase STING protein aggregation, IRF3 phosphorylation, or IRF3 nuclear translocation beyond what occurred with DMXAA alone. However, since the IFN-{beta} promoter also includes NF-{kappa}B binding sites, we additionally examined the NF-{kappa}B pathway. In fact, LPS increased the phosphorylation and nuclear translocation of the NF-{kappa}B subunit p65, and NF-{kappa}B signaling was required for the observed synergy. Intratumoral injection of suboptimal doses of LPS + DMXAA resulted in significantly improved tumor control of B16 melanoma in vivo compared to either agonist alone. Our results suggest that combinatorial signaling through TLR4 and STING results in optimal innate signaling via co-involvement of NF-{kappa}B and IRF3, and that combined engagement of these two pathways has therapeutic potential.