1944P - Super-enhancer driven NR3C1 expression promotes 5-FU resistance in gastric cancer

Background

This study aims to describe the Super enhancers (SEs) landscape in 5-FU resistant GC, and to illustrate the biological mechanism of SE-driven NR3C1 mediating 5-FU resistance.

Methods

The ChIP-Seq and ROSE algorithms were applied to calculate SE in induced 5-FU resistanceGC cells. The correlation between NR3C1 expression and the sensitivity to 5-FU was verified by drug sensitivity curves. Confocal microscopy was used to observe the purified NR3C1 fluorescent protein droplets in vitro, and to observe dynamic droplets in the nucleus. FRAP verified the droplet fluidity. CUT&Tag of NR3C1 was applied to analyze the co-binding pattern between NR3C1 and SE on the genome. 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.

Conclusions

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. 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.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

Shanghai Key Laboratory of Gastric Neoplasms, Shanghai Institute of Digestive Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine.

Funding

National Natural Science Foundation of China.

Disclosure

All authors have declared no conflicts of interest.