Major efforts are under way to identify agents that can potentiate effects of immune checkpoint inhibition. Ascorbic acid (AA) has been previously shown to cause DNA demethylation and corresponding increase in hydroxymethylation in multiple malignancies by enhancing the activity of the Ten-Eleven Translocation (TET) enzymes (Shenoy et al., BCJ 2017; Shenoy et al., JCI 2019; Cimmino et al., Cancer Cell 2017; Agathocleous et al., Nature 2017). Furthermore, AA has re-emerged as a promising anticancer agent based on recent knowledge of pharmacokinetics, discovery of unexpected mechanisms of action, and early phase trials with IV AA (Shenoy et al., Cancer Cell 2018). Here, we report that AA treatment caused genome-wide demethylation and enhanced expression of endogenous retroviral elements in lymphoma cells. AA also increased 5-hydroxymethylcytosine (5hmC) levels of CD8+ T cells and enhanced their cytotoxic activity in a lymphoma coculture system. High-dose AA treatment synergized with anti-PD1 therapy in a syngeneic lymphoma mouse model, resulting in marked inhibition of tumor growth compared with either agent alone. Analysis of the intratumoral epigenome revealed increased 5hmC with AA treatment, consistent with in vitro findings. Analysis of the tumor immune microenvironment revealed that AA strikingly increased intratumoral infiltration of CD8+ T cells and macrophages, suggesting enhanced tumor immune recognition. The combination treatment markedly enhanced intratumoral infiltration of macrophages and CD8+ T lymphocytes, granzyme B production by cytotoxic cells (cytotoxic T cells and natural killer cells), and IL-12 production by antigen-presenting cells compared with single-agent anti-PD1. These data strongly indicate that AA potentiates anti-PD1 checkpoint inhibition through synergistic mechanisms. The study provides a compelling rationale for testing combinations of AA and anti-PD1 agents in lymphoma patients as well as in preclinical models of other malignancies.
Citation Format: Rebecca Luchtel, Tushar Bhagat, Kith Pradhan, William Jacobs, Mark Levine, Amit Verma, Niraj Shenoy. High-dose ascorbic acid synergizes with anti-PD1 in a lymphoma mouse model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B4.