Mechanism of mitochondria-independent cuproptosis

Copper is an essential trace element that functions as a cofactor for numerous metabolic and detoxification enzymes. In excess, however, copper exerts marked cytotoxicity in cancer cells and can induce a recently characterized form of regulated cell death termed cuproptosis . Currently, cuproptosis is believed to depend on mitochondrial activity, involving the reduction of bivalent copper ions to monovalent, leading to subsequent toxic effects via the oligomerization of lipoylated DLAT (dihydrolipoyl transacetylase) and the loss of iron-sulfur cluster proteins. Here, we evaluated several copper ionophores in their copper-complexed forms, including bis(diethyldithiocarbamate) (CuET), pyrithione, NSC319726, and 8-hydroxyquinoline, to assess their ability to induce cuproptosis. Surprisingly, their cytotoxic effects were comparable in oxidative-phosphorylation-dependent cancer cells and in glycolysis-driven counterparts. Consistently, neither chemical inhibition of individual mitochondrial complexes nor the use of mitochondrial DNA-deficient Rho0 cells revealed differential sensitivity. Instead, all tested ionophores induced aggregation and immobilization of the essential p97 cofactor NPL4, mirroring the mechanism previously reported for CuET. NPL4 dysfunction disrupts the p97/proteasome axis, and its aggregation strongly correlates with cytotoxic outcomes. Ionophore treatment also triggered canonical proteotoxic stress pathways, including the unfolded protein response, the heat-shock response, and accumulation of polyubiquitinated proteins. Importantly, additional treatment with the non-toxic, more potent divalent copper chelator, dibenzyldithiocarbamate, reversed NPL4 aggregation and ionophore-induced cytotoxicity. Together, these findings refine the mechanistic framework of copper-dependent cell killing by revealing a prominent proteotoxic component of bivalent copper associated with targeting the NPL4. This insight strengthens the rationale for targeting copper-regulated proteostasis pathways as a potential anticancer strategy.