Simultaneous Targeting of NQO1 and SOD1 Eradicates Breast Cancer Stem Cells via Mitochondrial Futile Redox Cycling

Triple-negative breast cancer (TNBC) contains the highest proportion of cancer stem-like cells (CSC), which display intrinsic resistance to currently available cancer therapies. This therapeutic resistance is partially mediated by an antioxidant defense coordinated by the transcription factor NRF2 and its downstream targets that include NAD(P)H quinone oxidoreductase 1 (NQO1). In this study, we identified the antioxidant enzymes NQO1 and superoxide dismutase 1 (SOD1) as therapeutic vulnerabilities of ALDH⁺ epithelial-like CSCs and CD24^-/loCD44^+/hi mesenchymal-like CSCs in TNBC. Effective targeting of these CSC states was achieved by using isobutyl-deoxynyboquinone (IB-DNQ), a potent and specific NQO1-bioactivatable futile redox cycling molecule, which generated large amounts of reactive oxygen species including superoxide and hydrogen peroxide. Furthermore, the CSC killing effect was specifically enhanced by genetic or pharmacologic inhibition of SOD1, a copper-containing superoxide dismutase highly expressed in TNBC. Mechanistically, a significant portion of NQO1 resides in the mitochondrial intermembrane space, catalyzing futile redox cycling from IB-DNQ to generate high levels of mitochondrial superoxide, and SOD1 inhibition markedly potentiated this effect, resulting in mitochondrial oxidative injury, cytochrome c release, and activation of the caspase-3–mediated apoptotic pathway. Treatment with IB-DNQ alone or together with SOD1 inhibition effectively suppressed tumor growth, metastasis, and tumor-initiating potential in xenograft models of TNBC expressing different levels of NQO1. This futile oxidant-generating strategy, which targets CSCs across the epithelial–mesenchymal continuum, could be a promising therapeutic approach for treating patients with TNBC.