Background: The Klf6 gene, belonging to Krüppel-like family of C2H2 zinc finger transcription
factors, is strongly associated with tumor formation through high somatic mutations in
carcinomas of the prostate, liver, colon, stomach, lung, neck, pituitary gland and nervous system.
Recently, Klf6 super-enhancer which strongly regulates Klf6 gene expression has been identified,
and the function of Klf6 super-enhancer which regulates cell growth was studied.
Objective: The development of inhibitors targeting BRD4-binding super-enhancers is a potential
target therapeutic strategy for tumor therapy. However, the suppression of Klf6 super-enhancer
function by BRD4 inhibitors is not known.
Methods: CRISPR-Cas9 editing technique was used for the Klf6 super-enhancer deletion experiment,
and the expression levels of several genes for cell clones were detected by qRT-PCR analysis
and Western blotting. Cell proliferation assay was applied to evaluate the functional role of
Klf6 super-enhancer using several BRD4 inhibitors. The interaction of several BRD4 inhibitors
against the target protein was analyzed by molecular docking simulation.
Results: JQ-1, a human BRD4 inhibitor, inhibited Klf6 gene expression and its activity in HepG2
cells in a time and dose-dependent manner while simultaneously inhibiting cell growth. Besides,
BETd-246, a human BRD4 inhibitor, strongly inhibited Klf6 gene expression, significantly inhibited
cell growth, and exhibited higher efficacy than JQ-1. Molecular docking studies revealed that
some key residues were critical for ligand-receptor interactions by forming hydrogen bonds with ligands
(JQ-1: ASN140, BETd-246: ASN140, TYR106, LYS65, GLN58, MET105, and MET53).
Conclusion: Our findings suggest that KLF6 is regulated by Klf6 super-enhancer and the targeting
of Klf6 super-enhancer by BRD4 inhibitors may be an effective therapeutic strategy for liver cancer