Abstract 1461: Super-enhancer driven NR3C1 expression promotes 5-FU resistance in gastric cancer

Abstract Background: This study aims to describe the Super-enhancer (SE) landscape in 5-FU resistant gastric cancer (GC), and to illustrate the biological mechanism of SE-driven NR3C1 mediating 5-FU resistance. The study might provide new possibilities of improving chemotherapy efficacy for GC. Methods: The induced drug-resistant gastric cancer cells were constructed with long-term 5-FU medication. The cChIP-Seq and rank ordering of super-enhancers (ROSE) algorithms were applied to calculate SE in GC cells. The core regulating transcription factor (TF) NR3C1 was determined by siRNA libraries. The correlation between NR3C1 expression and the sensitivity to 5-FU was verified by drug sensitivity curves. In induced resistant cells, NR3C1 expression was detected and was knocked down as a rescue experiment. Confocal microscopy was used to observe the purified NR3C1 fluorescent protein droplets in vitro, and to observe dynamic droplets in the nucleus. Fluorescence recovery after photobleaching (FRAP) verified the droplet fluidity. The CUT&Tag of NR3C1 was applied to analyze the co-binding pattern between NR3C1 and SE on the genome. The SE changes of NR3C1 knockdown cells were detected and were analyzed for downstream target genes. PDX models and GC cells were used to evaluate the inhibitory effect of epigenetic inhibitors JQ1 on NR3C1 and its target genes, and to improve 5-FU responsivity in GC cells. Results: The SE profiles of 5-FU-sensitive, spontaneously resistant and induced resistant cells were described. SiRNA libraries were used to identify the master TF NR3C1. The 5-FU drug sensitivity curve verified that GC cells with high NR3C1 expression were resistant to 5-FU. Moreover, NR3C1 expression was increased in 5-FU-resistant cells, and knocking down NR3C1 in resistant cells could restore the 5-FU sensitivity. NR3C1 inhibition also improved the sensitivity of GC organoids to 5-FU. NR3C1 protein formed phase separation in vitro, and also formed droplets in the nucleus, together with transcription cofactors. With NR3C1 knockdown, SE profile changed through enhancer reprogramming, and the SE-associated genes transcription was inhibited. Then, NR3C1 downstream target genes, which were associated with 5-FU resistance, were significantly reduced. The use of JQ1 effectively inhibited the expression of downstream target genes, and efficiently improve the tumor inhibition effect of 5-FU in PDX models. Conclusion: The high expression of NR3C1 driven by SE is positively correlated with 5-FU resistance in GC patients. SE-driven NR3C1 formed phase separation at the SE region in the nucleus, activating the positive feedback loop of TF. NR3C1 inhibition led to remarkable enhancer reconstruction of GC cells. SE-driven NR3C1 strongly promotes the transcription of downstream target genes and mediates the resistance of GC to 5-FU. JQ1 can effectively improve the 5-FU sensitivity of GC Citation Format: Bingya Liu, Junxian Yu, Haorang Feng, Jianfang Li. Super-enhancer driven NR3C1 expression promotes 5-FU resistance in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1461.