(E,E)-bisantrene silences c-MYC expression by stabilizing its promotor region G-quadruplex

Background: G-quadruplex (G4) DNA and RNA are important non-canonical nucleic acid secondary structures that play key roles in many cellular processes. They regulate the expression and translation of several oncogenes, including the master cell growth regulator, MYC. Bisantrene is a small-molecule anticancer agent that has been shown to be safe and effective in >1500 clinical trial patients. This study characterized the binding and stabilization of a G4 region in the c-MYC promoter by the (E,E)-bisantrene isomer and silencing of c-MYC expression in cancer cells. Methods: Circular dichroism spectroscopy established if (E,E)-bisantrene stabilizes the c-MYC promotor G4 structure, with surface plasmon resonance used to measure the binding affinity. Nuclear magnetic resonance spectroscopy provided structural insights into the binding interactions within the complex. Molecular dynamics simulations modelled the 3-dimensional structure of the complex. Changes in c-MYC gene expression were assessed across a range of cancer cell lines after treatment with (E,E)-bisantrene and RNA-seq with pathway analysis was performed. Results: (E,E)-bisantrene was found to stabilize the c-MYC promoter G4 region, producing similar increases in melting temperature to other G4 ligands (i.e., pidnarulex and pyridostatin). NMR spectroscopy and molecular modelling suggests (E,E)-bisantrene binds with a 2:1 stoichiometry to the planar surfaces of the top and bottom G-tetrads. (E,E)-bisantrene potently inhibited c-MYC expression in multiple cancer cell lines. RNA-seq analysis showed (E,E)-bisantrene also decreased expression of other oncogenes containing G4 regions in their promoters, including MET, TERT, VEGFA, ATF4 and MDM2 . Pathway analysis of the RNA-Seq data demonstrated a transcriptomic profile similar to pidnarulex, a known G4-binding drug in early-stage clinical development. Conclusion: (E,E)-bisantrene binds to and stabilizes the G4 structure contained within the c-MYC promotor region, leading to silencing of c-MYC gene expression. These studies support clinical evaluation of (E,E)-bisantrene as a new G4-targeting drug in MYC-driven tumors.