Colorectal cancer (CRC) is a highly complex disease with multiple risk factors. The orphan nuclear receptor 4A1 (NR4A1) is overexpressed in several cancers and is a negative prognostic factor for cancer patient survival. Previous reports indicate a potential role for overexpression of NR4A1 in T-cell exhaustion and in this study, we aim to investigate the antitumorigenic activity of two bis-indole derived ligands (DIMs) that act as receptor antagonists. Immune competent C57BL/6 mice and mouse MC-38 colon cancer cells were used and tumor Infiltrating Lymphocytes (TILs) were isolated from mice either untreated or treated with CDIM/NR4A1 antagonists. FACS analysis and Real -Time PCR were performed to determine expression of exhaustion markers in these tumor T-cell population. 1,1-Bis(3-indolyl)-1-(3-bromo-5-trifluoromethoxyphenyl)methane (DIM-3-Br-5-OCF3) and the 3,5-dichlorophenyl analog (DIM-3,5-Cl2) at doses of 2.5 and 7.5 mg/kg/d inhibited tumor growth and downregulated expression of PD-L1, an NR4A1-regulated gene in MC-38 - derived tumors and cells. The mouse MC-38 colon cancer cell line was used in a syngeneic mouse model of colon cancer and tumor infiltrating lymphocytes (TILs) from MC-38 cell-derived colon tumors exhibited multiple markers of T-cell exhaustion in both CD8+ and CD4+ T-cells. Lymphocytes from an enlarged spleen in these tumor-bearing animals also exhibited markers of CD8+ and CD4+ T-cell exhaustion. Analysis of CD8+ cells from TILs showed that treatment with the NR4A1 antagonists modulated expression of several genes associated with T-cell exhaustion namely a decrease in TOX, TOX2 and NFAT mRNAs, activation of T-Bet. The percentage of CD8+ and CD4+ T-cells from tumors and spleen expressing PD-1, 2B4 and TIGIT was decreased in the treated vs control mice and TIM3 expression was also decreased in CD8+ (tumors and spleen) and CD4+ (tumors) T-cells. These findings suggest that NR4A1 antagonists are highly effective as anticancer agents in this mouse syngeneic colon tumor model by inactivating the pro-oncogenic activities of NR4A1 in tumors and by remediating NR4A1-regulated T-cell exhaustion in tumor and splenic lymphocytes.
Citation Format: Kumaravel Mohankumar, Gus Wright, Subhashree Kumaravel, Rupesh Shrestha, Lei Zhang, Maen Abdelrahim, Robert S. Chapkin, Stephen Safe. Nuclear receptor 4A1 ligands target T-cell exhaustion in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 236.