Targeting the ERa DBD-LBD interface with mitoxantrone disrupts receptor function through proteasomal degradation

The estrogen receptor (ER or ERa) remains the primary therapeutic target for luminal breast cancer, with current treatments centered on competitive antagonists, receptor down-regulators, and aromatase inhibitors. This therapeutic landscape frames our discovery of an alternative mechanism of ER inhibition that targets the critical interface between the DNA-binding domain (DBD) and ligand-binding domain (LBD) of the receptor. We identified mitoxantrone (MTO), an FDA-approved topoisomerase II inhibitor, as a specific ligand for this previously unexplored DBD-LBD interface. Through integration of computational, biophysical, biochemical, and cellular analyses, we demonstrate that independent of its DNA damage response activity, MTO binding induces ER conformational changes, leading to cytoplasmic redistribution and proteasomal degradation. Notably, MTO also effectively inhibits constitutively active ER mutants (Y537S and D538G) commonly associated with endocrine therapy resistance, suppressing ER/ER-mutant-dependent gene expression and tumor growth more potently than fulvestrant. These findings establish the DBD-LBD interface as a crucial regulatory point for ER function, suggesting a new paradigm for targeting domain interfaces across the nuclear receptor family. Overall design: Three MCF-7 cell lines (wildtype, Y537S ER knock-in, and D538G ER knock-in) were treated with four different conditions: vehicle control, fulvestrant (1 µM), MTO (1 µM), or combination treatment (fulvestrant + MTO) for 48 hours. Gene expression profiling was performed using RNA sequencing on the Illumina NextSeq 550 platform with 75 bp single-end reads. Two biological replicates were included for each treatment condition across all three cell lines, resulting in a total of 24 samples (3 cell lines × 4 treatments × 2 replicates).