Mitoxantrone inhibits and downregulates ERα through binding at the DBD-LBD interface

Targeting the estrogen receptor (ER or ER) through competitive antagonists, receptor downregulators, or estrogen synthesis inhibition remains the primary therapeutic strategy for luminal breast cancer. We have identified a novel mechanism of ER inhibition by targeting the critical interface between its DNA-binding domain (DBD) and ligand-binding domain (LBD). We demonstrate that mitoxantrone (MTO), a topoisomerase II inhibitor, binds at this previously unexplored DBD-LBD interface. Using comprehensive computational, biophysical, biochemical, and cellular analyses, we show that independent of its DNA damage response activity, MTO binding induces distinct conformational changes in ER, leading to its cytoplasmic redistribution and subsequent proteasomal degradation. Notably, MTO effectively inhibits clinically relevant ER mutations (Y537S and D538G) that confer resistance to current endocrine therapies, outperforming fulvestrant in both in vitro and in vivo assays. Our findings establish domain-domain interaction targeting as a viable therapeutic strategy for ER, with translational implications for other nuclear receptors.