Cuproptosis

Cuproptosis is a genetically and biochemically distinct modality of metal-dependent regulated cell death in cell biology, biochemistry, and oncology driven by the excessive accumulation of intracellular copper ions (Cu⁺) inside the mitochondria. Discovered and named by Peter Tsvetkov, Todd R. Golub, and colleagues at the Broad Institute of MIT and Harvard in a landmark March 2022 study in Science ("Copper induces cell death by targeting lipoylated TCA cycle proteins"), cuproptosis is mechanistically independent of all previously characterized regulated cell death pathways—including caspase-driven apoptosis, RIPK3/MLKL-mediated necroptosis, gasdermin-mediated pyroptosis, and iron-dependent lipid-peroxidation-driven ferroptosis. Instead of cleaving cellular substrates or peroxidizing membrane phospholipids, cuproptosis operates through an ancient mitochondrial metabolic vulnerability: the mitochondrial matrix reductase ferredoxin 1 (FDX1) reduces divalent cupric ions (Cu²⁺) to more reactive monovalent cuprous ions (Cu⁺) while simultaneously regulating the protein lipoylation of four mitochondrial multienzyme complexes—most notably dihydrolipoamide S-acetyltransferase (DLAT), the E2 subunit of the pyruvate dehydrogenase complex. Excess Cu⁺ binds directly to the sulfur-containing lipoyl moieties of lipoylated DLAT, driving insoluble disulfide-bonded DLAT oligomerization and the concurrent destabilization of mitochondrial iron–sulfur (Fe–S) cluster proteins, which culminates in acute proteotoxic stress and cell death selectively in cells reliant on mitochondrial oxidative phosphorylation (Tsvetkov et al., 2019, 2022; Tang et al., 2022; Cobine & Brady, 2022).

For decades, investigators knew that while copper is an indispensable redox cofactor for cytochrome c oxidase (Complex IV), copper/zinc superoxide dismutase (SOD1), lysyl oxidase, and dopamine β-hydroxylase, dysregulated intracellular copper overload—whether induced genetically in Wilson disease (ATP7B loss-of-function) or pharmacologically by copper ionophores such as elesclomol, disulfiram, and NSC-319726 (elesclomol/pyrithione)—is rapidly lethal to mammalian cells. However, the exact death mechanism remained controversial until Tsvetkov et al. (2022) demonstrated that copper-ionophore lethality cannot be rescued by any canonical cell-death inhibitor:

• Pan-Caspase Inhibitors (Z-VAD-FMK, Boc-D-FMK) & BAX/BAK1 Double Knockout: Genetic ablation of the intrinsic apoptotic pore proteins BAX and BAK1 or chemical inhibition of caspases-3/7/8/9 completely blocks paclitaxel- or bortezomib-induced apoptosis, yet has zero protective effect against elesclomol–Cu²⁺ lethality (and dying cuproptotic cells show no caspase-3 cleavage).
• Ferroptosis Inhibitors (Ferrostatin-1, Liproxstatin-1, α-Tocopherol): Radical-trapping antioxidants that completely block GPX4-inhibitor-induced ferroptosis fail to prevent copper-induced death.
• Necroptosis & ROS Inhibitors (Necrostatin-1, N-Acetylcysteine [NAC], Ebselen): Neither RIPK1 inhibition nor general cytosolic reactive oxygen species scavengers block cuproptosis.
• Selective Rescue by Copper Chelators and Mitochondrial Respiration Blockade: Only hydrophilic copper chelators (tetrathiomolybdate [TTM] and bathocuproinedisulfonic acid [BCS]), endogenous reduced glutathione (GSH) (which chelates intracellular free Cu⁺; conversely, depleting GSH with buthionine sulfoximine [BSO] sensitizes cells 100-fold to cuproptosis), and inhibitors of mitochondrial electron transport chain Complex I and Complex III (rotenone, piericidin A, antimycin A) or the mitochondrial pyruvate carrier (UK-5099) prevent cell death.

Crucially, Tsvetkov et al. observed a ~1,000-fold metabolic selectivity: cells forced to rely on mitochondrial respiration (by replacing glucose in the culture medium with galactose, which forces ATP production through the tricarboxylic acid (TCA) cycle and oxidative phosphorylation) are nearly 1,000 times more sensitive to cuproptosis than isogenic cells growing primarily via anaerobic glycolysis ( the Warburg effect) or under hypoxia (1% O₂).

To map the genetic pathway of cuproptosis, Tsvetkov et al. (2022) conducted whole-genome positive-selection CRISPR-Cas9 knockout screens across human cancer cell lines treated with structurally distinct copper ionophores (elesclomol and disulfiram). Remarkably, out of ~19,000 human genes, loss of only seven positively acting genes rescued cells from copper-induced death, while loss of three negatively acting genes sensitized them:

1. Copper Entry Across the Plasma and Mitochondrial Membranes: Under physiological homeostasis, the high-affinity plasma-membrane copper importer SLC31A1 (CTR1) imports monovalent Cu⁺, which is tightly buffered at sub-femtomolar free concentrations (<10⁻¹⁸ M) by cytosolic glutathione (GSH) and metallochaperones (ATOX1, CCS, COX17) while excess copper is exported across the trans-Golgi and plasma membrane by the P-type ATPases ATP7A and ATP7B (Lutsenko, 2010). Copper overload occurs either when copper ionophores (such as elesclomol, which binds extracellular Cu²⁺ in a 1:1 lipophilic chelate and shuttles it directly across the outer and inner mitochondrial membranes before releasing Cu⁺ and recycling out of the cell) bypass CTR1, or when SLC31A1 overexpression or ATP7A/ATP7B loss floods the cell with copper.
2. FDX1-Mediated Cu²⁺ Reduction and Fe–S Cluster Destabilization: Inside the mitochondrial matrix, FDX1 transfers electrons from NADPH/ferredoxin reductase (FDXR) to reduce Cu²⁺ to Cu⁺. Because Cu⁺ sits at the top of the Irving–Williams stability series (Mn²⁺ < Fe²⁺ < Co²⁺ < Ni²⁺ < Cu²⁺/Cu⁺ > Zn²⁺), excess Cu⁺ competitively displaces iron from solvent-exposed [4Fe–4S] iron–sulfur clusters in mitochondrial enzymes (such as LIAS, ACO2 [aconitase], NDUFS1, and SDHB), triggering rapid proteolytic degradation of the entire Fe–S cluster proteome.
3. Direct Cu⁺ Binding and Toxic Aggregation of Lipoylated DLAT and DLST: Simultaneously, Cu⁺ binds directly to the sulfur atoms of the covalently attached lipoyl moiety on DLAT (E2 of pyruvate dehydrogenase) and DLST (E2 of α-ketoglutarate dehydrogenase). Cu⁺ bridges adjacent lipoylated DLAT monomers into high-molecular-weight, insoluble oligomeric aggregates that recruit HSP70 (HSPA1A) chaperones, collapsing TCA cycle flux and inducing irreversible mitochondrial proteotoxic stress (Tsvetkov et al., 2022).

• Wilson Disease (ATP7B Deficiency): In Atp7b^−/− mouse models of Wilson disease (an autosomal recessive hepatolenticular degeneration disorder in which biliary copper excretion fails and copper accumulates 10- to 50-fold inside hepatocytes and basal ganglia neurons), Tsvetkov et al. (2022) demonstrated that aged Atp7b^−/− livers exhibit the exact molecular hallmarks of in vivo cuproptosis: severe depletion of FDX1, LIAS, and Fe–S cluster proteins alongside depletion of monomeric lipoylated DLAT and induction of HSP70.
• Targeting High-OXPHOS and Therapy-Resistant Cancer Stem Cells: Although the copper ionophore elesclomol previously failed to improve overall survival in unselected Phase III melanoma trials (where patients were not stratified by mitochondrial metabolism), retrospective subgroup analysis revealed significant clinical benefit specifically in patients with low serum lactate dehydrogenase (LDH)—a clinical biomarker of low glycolysis and high mitochondrial oxidative phosphorylation (OXPHOS). Because proteasome-inhibitor-resistant multiple myeloma, acute myeloid leukemia (AML) stem cells, and KRAS/BRAF-mutant persister cells rely heavily on mitochondrial respiration and express elevated FDX1 and lipoylated TCA enzymes, nanoparticle-delivered copper ionophores and FDX1/LIAS biomarker stratification represent an active frontier in precision oncology (Tsvetkov et al., 2019, 2022; Tang et al., 2022; Xue et al., 2023).

1. Lutsenko, S. (2010). Human copper homeostasis: A network of interconnected pathways. Current Opinion in Chemical Biology, 14(2), 211–217. https://doi.org/10.1016/j.cbpa.2010.01.003
2. Tsvetkov, P., Detappe, A., Cai, K., Keys, H. R., Brune, Z., Ying, W., Thiru, P., Reidy, M., Kugener, G., Rossen, J., Kocak, M., Kory, N., Tsherniak, A., Santagata, S., Whitesell, L., Ghobrial, I. M., Markley, J. L., Lindquist, S., & Golub, T. R. (2019). Mitochondrial metabolism promotes adaptation to proteotoxic stress. Nature Chemical Biology, 15(7), 681–689. https://doi.org/10.1038/s41589-019-0291-9
3. Tsvetkov, P., Coy, S., Petrova, B., Dreishpoon, M., Verma, A., Abdusamad, M., Rossen, J., Joesch-Cohen, L., Humeidi, R., Spangler, R. D., Eaton, J. K., Frenkel, E., Kocak, M., Corsello, S. M., Lutsenko, S., Kanarek, N., Santagata, S., & Golub, T. R. (2022). Copper induces cell death by targeting lipoylated TCA cycle proteins. Science, 375(6586), 1254–1261. https://doi.org/10.1126/science.abf0529
4. Cobine, P. A., & Brady, D. C. (2022). Cuproptosis: Cellular and molecular mechanisms underlying copper-induced cell death. Molecular Cell, 82(10), 1786–1787. https://doi.org/10.1016/j.molcel.2022.05.001
5. Tang, D., Chen, X., & Kroemer, G. (2022). Cuproptosis: A copper-triggered modality of mitochondrial cell death. Cell Research, 32(5), 417–418. https://doi.org/10.1038/s41422-022-00653-7
6. Dreishpoon, M. B., Bick, N. R., Petrova, B., Warui, D. M., Cameron, A., Booker, S. J., Kanarek, N., Golub, T. R., & Tsvetkov, P. (2023). FDX1 regulates cellular protein lipoylation through direct binding to LIAS. Journal of Biological Chemistry, 299(9), 105046. https://doi.org/10.1016/j.jbc.2023.105046
7. Xue, Q., Kang, R., Klionsky, D. J., Tang, D., Liu, J., & Chen, X. (2023). Copper metabolism in cell death and autophagy. Autophagy, 19(8), 2175–2195. https://doi.org/10.1080/15548627.2023.2200554