Xanthatin Targets CISD1 to Drive Ferroptosis and Mitophagy as a Dual Anticancer Strategy in Triple‐Negative Breast Cancer

ABSTRACT Triple‐negative breast cancer (TNBC) is an aggressive subtype with poor prognosis. Here, we identify xanthatin, a sesquiterpene lactone from Xanthium species, as a potent inhibitor of TNBC cell growth with minimal toxicity to normal cells. Transcriptomic analyses revealed that xanthatin activates ferroptosis, evidenced by elevated ROS, lipid peroxidation, and Fe 2 + accumulation, together with GSH depletion and downregulation of SLC7A11 and GPX4. Target identification by drug affinity responsive target stability and mass spectrometry uncovered CDGSH iron sulfur domain 1 (CISD1) as the direct binding partner of xanthatin. Cellular thermal shift assay, surface plasmon resonance, and dynamics simulations consistently demonstrated that tryptophan‐75 is the critical residue mediating this interaction. Functionally, xanthatin promotes CISD1 ubiquitination and proteasomal degradation, thereby disrupting mitochondrial iron homeostasis and inducing ferroptosis. CISD1 destabilization further impaired mitochondrial integrity and activated PINK1/Parkin‐dependent mitophagy, establishing a dual ferroptosis–mitophagy mechanism. Importantly, genetic knockdown of CISD1 markedly attenuated the anticancer activity of xanthatin, confirming its essential role. In an orthotopic TNBC mouse model, xanthatin significantly suppressed tumor growth without causing systemic toxicity. Collectively, our findings provide the first demonstration that xanthatin directly targets CISD1 at the Trp‐75 site to trigger ferroptosis and mitophagy, highlighting its promise as a therapeutic candidate for TNBC.