Super‐enhancer Activates Master Transcription Factor NR3C1 Expression and Promotes 5‐FU Resistance in Gastric Cancer

Abstract Poor response to 5‐fluorouracil (5‐FU) remains an obstacle in the treatment of gastric cancer (GC). Super enhancers (SEs) are crucial for determining tumor cell survival under drug pressure. SE landscapes related to 5‐FU‐resistance are mapped to GC using chromatin immunoprecipitation‐sequencing (ChIP‐Seq). SiRNA transcription factors (TFs) screen determines master TF Nuclear Receptor Subfamily 3 Group C Member 1 (NR3C1) activated by SE. High NR3C1 expression driven by SE correlated with 5‐FU resistance in patient‐derived organoids (PDOs). Phase separation formed by NR3C1 is observed using fluorescence recovery after photobleaching (FRAP). NR3C1 protein and Mediator promoted SE‐related gene transcription via phase separation. SEs and NR3C1 co‐binding patterns are explored using Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing. 5‐FU‐related genes driven by NR3C1 are identified using epigenetic reader inhibitor JQ1 and NR3C1 specific inhibitor Cort108297. NR3C1 knockdown increases 5‐FU sensitivity and alters the SE landscape through enhancer reprogramming, reducing downstream 5‐FU‐related target genes. JQ1 and Cort108297 both improve 5‐FU efficacy in PDOs and patient‐derived xenografts (PDXs) by destroying SEs or inhibiting NR3C1. In conclusion, SE‐driven NR3C1 promotes 5‐FU resistance in GC. SE destruction and NR3C1 inhibition lead to enhancer reconstruction and reduce 5‐FU‐related gene transcription, providing alternative therapeutic strategies for improving 5‐FU sensitivity.