FDX1-independent non-canonical cuproptosis establishes a parallel mitochondria-centric death program

Objectives Copper is an essential redox cofactor required for mitochondrial respiration and antioxidant defense, yet its dysregulation creates a therapeutic vulnerability that can be exploited by copper active agents. Elesclomol and neocuproine are two potent copper active compounds, but whether they induce the same form of cuproptosis , a copper-dependent cell death mechanism, has remained unclear. We aimed to elucidate the cytotoxic programs triggered by these two agents and to determine whether distinct mechanistic forms of cuproptosis exist. Methods Our study integrates limited-proteolysis proteomics to identify compound-responsive changes in protein structural accessibility, whole-genome CRISPRa (activation) screening, transcriptomic analysis of 436 CCLE cancer cell lines, mass spectrometry-based analysis of cellular and tumor copper abundance, genetic perturbation studies, mitochondrial functional assays, and mitophagy analyses. Pathway enrichment and functional validation were used to identify adaptive survival programs and compound-specific copper-handling mechanisms. Results Building on the previously established FDX1-dependent cuproptosis mechanism induced by elesclomol, we designate this pathway as canonical cuproptosis (c-CPX) and identify neocuproine as inducing a mechanistically distinct, FDX1-independent form of cuproptosis, which we term non-canonical cuproptosis (nc-CPX). In c-CPX, FDX1 mediates elesclomol delivered Cu(II) to Cu(I) reduction, leading to rapid oligomerization of lipoylated mitochondrial proteins and subsequent proteotoxic stress. In contrast, neocuproine induces n-CPX, a FDX1-independent copper-dependent death program that preferentially engages neocuproine stabilized Cu(I)-handling and stress-adaptation networks. Despite its FDX1 independence, neocuproine rapidly induces oligomerization of lipoylated DLAT, identifying an early point of convergence with canonical cuproptosis. Large-scale analysis of CCLE cancer cell lines further revealed that sensitivity to both neocuproine and elesclomol is associated with enrichment of oxidative phosphorylation (OXPHOS) transcriptional programs. Notably, neocuproine-sensitive cancer cells additionally displayed significantly higher mesenchymal/EMT scores across multiple independent gene-expression signatures, linking an OXPHOS-enriched, mesenchymal cell state to increased susceptibility to neocuproine-induced copper-dependent cell death. Limited-proteolysis profiling identified ATOX1 among the prominent neocuproine-responsive proteins. Functional validation demonstrated that ATOX1 depletion partially attenuates neocuproine-induced cytotoxicity, but not elesclomol toxicity, while ATOX1 loss itself promotes DLAT oligomerization, supporting a specific contribution of ATOX1-dependent Cu(I) handling to nc-CPX. CRISPRa screening further revealed that adaptive programs enriched during neocuproine exposure predominantly involve proteasomal and lysosomal pathways; whereas elesclomol is associated with distinct signaling and bioenergetic programs, including PI3K, JAK–STAT, and MAPK pathways, as well as oxidative phosphorylation and glutathione metabolism. The functional relevance of the neocuproine-associated pathways was supported by enhanced cytotoxicity upon pharmacological inhibition of proteasomal or lysosomal function. Despite their divergent initiation mechanisms, both canonical and non-canonical cuproptosis converge on early oligomerization of lipoylated mitochondrial proteins followed by Fe–S-protein loss, respiratory-chain dysfunction, mitochondrial remodeling, impaired mitochondrial quality control, and respiratory failure. Both compounds also induce cytoplasmic and mitochondrial ROS and late apoptotic features. Continuous N-acetylcysteine (NAC) exposure reduced the labile intracellular Cu(I) signal and rescued cytotoxicity; whereas NAC pretreatment followed by washout did not protect against subsequent neocuproine or elesclomol exposure. Together with the lack of rescue by caspase inhibition and the delayed appearance of apoptotic features, these findings support ROS accumulation and apoptosis as predominantly downstream consequences rather than defining initiating mechanisms. Conclusion Our findings establish a mechanistic framework distinguishing canonical FDX1 dependent cuproptosis from non-canonical FDX1 independent cuproptosis. Rather than representing separate downstream death programs, c-CPX and nc-CPX constitute distinct upstream copper-handling routes that converge on a shared mitochondria-centered proteotoxic program. These results provide insight into copper-driven cytotoxicity and suggest FDX1 dependency, ATOX1-dependent copper-handling, EMT/OXPHOS state, and proteostasis pathways as candidate biomarkers and therapeutic vulnerabilities for further investigation.