Colorectal cancer (CRC) is a highly complex disease with multiple risk factors and both genetic and environmental components contribute to disease incidence.1 2 Cancer immunotherapy using immune-checkpoint blockades represents a major advance in treatment strategy.3 4 The orphan nuclear receptor 4A1 (NR4A1) is overexpressed in lung, colon, liver and breast cancers and in Rhabdomyosarcoma and is a negative prognostic factor for cancer patient survival.58 Previous studies in breast cancer cells showed that PD-L1 was regulated by NR4A1 which activates transcription factor Sp1 bound to the PD-L1 gene promoter. Genome-wide studies have identified NR4A1 as a key mediator of T-cell dysfunction and NR4A1 also plays an important role in regulating genes which are involved in tumor-induced T-cell exhaustion.9 Bis-indole derived NR4A1 ligand that act as receptor antagonists have been developed in this laboratory and these compounds block pro-oncogenic NR4A1-regulated genes/pathways.
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.
Results Two compounds:
1,1-bis(3-indolyl)-1-(3-bromo-5-trifluoromethoxyphenyl)methane (DIM-3-Br-5-OCF3) and 3,5-dichlorophenyl analog (DIM-3,5-Cl2) inhibited tumor growth and weight at doses of 2.5 and 7.5mg/kg/day (figure 1). Tumor CD8+ T-cells isolated from mice treated with DIM-3,5-Cl2 and DIM-3-Br-5-OCF3 exhibited decreased mRNA expression of NR4A1 and high mobility group box transcription factors NFAT, TOX and TOX2 and increased mRNA levels of Interferon- (IFN), granzyme (GzB) and perforin compared to control animals (figure 2). As TOX and TOX2 cooperate with NR4A1 to modulate CD8+ T-cell exhaustion, we investigated the expression of several inhibitory receptors of T-cell exhaustion in CD8+ TILs, including PD-1, 2B4, TIGIT and TIM3. Following treatment with DIM-3,5-Cl2 or DIM-3-Br-5-OCF3, there was a significant decrease in the percentage of PD1 and 2B4 cells and a decrease in TIGIT and TIM3 (figure 3). These results indicate that NR4A1 antagonists reverses T-cell exhaustion.
NR4A1 plays a critical role in T-cell dysfunction, and this includes T-cell exhaustion.10 11 Our results demonstrate that the NR4A1 antagonists reverse many markers of T-cell exhaustion including activation of cytokines. The combined effects of NR4A1 antagonists in both tumors and T-cells result in inhibition of colon tumorigenesis by targeting pathways/genes in tumor cells and by enhancing immune surveillance via reversal of T-cell exhaustion.
Ahmed M. Colon cancer: a Clinician's perspective in 2019.
Gastroenterology Res2020; 13(1):110. Epub 2020/02/26. doi: 10.14740/gr1239.
Keum N, Giovannucci E. Global burden of colorectal cancer: emerging trends, risk factors and prevention strategies.
Nat Rev Gastroenterol Hepatol2019; 16(12):71332. Epub 2019/08/29. doi: 10.1038/s41575-019-0189-8.
Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy.
Nat Rev Cancer2012; 12(4):25264. Epub 2012/03/23. doi: 10.1038/nrc3239.
Ribas A, Wolchok JD. Cancer immunotherapy using checkpoint blockade.
Yang et al. Distinct epigenetic features of tumor-reactive CD8+ T cells in colorectal cancer patients revealed by genome-wide DNA methylation analysis.
Genome Biol2019; 21(1):2. doi: 10.1186/s13059-019-1921-y.
Safe S, Karki K. The paradoxical roles of orphan nuclear receptor 4A (NR4A) in cancer.
Mol Can Res2020. Epub 2020/10/28. doi: 10.1158/1541-7786.MCR-20-0707.
Lee SO, et al. Diindolylmethane analogs bind NR4A1 and are NR4A1 antagonists in colon cancer cells.
Molecular Endocrinology. 2014; 28(10):172939. doi: 10.1210/me.2014-1102.
Hedrick E, et al. The nuclear orphan receptor NR4A1 regulates 1-integrin expression in pancreatic and colon cancer cells and can be targeted by NR4A1 antagonists.
Mol Carcinog2017; 56(9):20662075.
Liu X, et al. Genome-wide analysis identifies NR4A1 as a key mediator of T cell dysfunction.
Nature2019; 567(7749):5259. Epub 2019/03/01. doi: 10.1038/s41586-019-0979-8.
Chen J et al. NR4A transcription factors limit CAR T cell function in solid tumours.
Nature. 2019; 567(7749):5304. Epub 2019/03/01. doi: 10.1038/s41586-019-0985-x. PubMed PMID: 30814732; PMCID: PMC6546093.
Seo H, et al. TOX and TOX2 transcription fa