7-Dehydrocholesterol is an endogenous suppressor of ferroptosis.

Ferroptosis is a form of cell death that has received considerable attention not only as a means to eradicate defined tumour entities but also because it provides unforeseen insights into the metabolic adaptation that tumours exploit to counteract phospholipid oxidation1,2. Here, we identify proferroptotic activity of 7-dehydrocholesterol reductase (DHCR7) and an unexpected prosurvival function of its substrate, 7-dehydrocholesterol (7-DHC). Although previous studies suggested that high concentrations of 7-DHC are cytotoxic to developing neurons by favouring lipid peroxidation3, we now show that 7-DHC accumulation confers a robust prosurvival function in cancer cells. Because of its far superior reactivity towards peroxyl radicals, 7-DHC effectively shields (phospho)lipids from autoxidation and subsequent fragmentation. We provide validation in neuroblastoma and Burkitt's lymphoma xenografts where we demonstrate that the accumulation of 7-DHC is capable of inducing a shift towards a ferroptosis-resistant state in these tumours ultimately resulting in a more aggressive phenotype. Conclusively, our findings provide compelling evidence of a yet-unrecognized antiferroptotic activity of 7-DHC as a cell-intrinsic mechanism that could be exploited by cancer cells to escape ferroptosis.

A non-canonical vitamin K cycle is a potent ferroptosis suppressor.

Ferroptosis, a non-apoptotic form of cell death marked by iron-dependent lipid peroxidation1, has a key role in organ injury, degenerative disease and vulnerability of therapy-resistant cancers2. Although substantial progress has been made in understanding the molecular processes relevant to ferroptosis, additional cell-extrinsic and cell-intrinsic processes that determine cell sensitivity toward ferroptosis remain unknown. Here we show that the fully reduced forms of vitamin K-a group of naphthoquinones that includes menaquinone and phylloquinone3-confer a strong anti-ferroptotic function, in addition to the conventional function linked to blood clotting by acting as a cofactor for γ-glutamyl carboxylase. Ferroptosis suppressor protein 1 (FSP1), a NAD(P)H-ubiquinone reductase and the second mainstay of ferroptosis control after glutathione peroxidase-44,5, was found to efficiently reduce vitamin K to its hydroquinone, a potent radical-trapping antioxidant and inhibitor of (phospho)lipid peroxidation. The FSP1-mediated reduction of vitamin K was also responsible for the antidotal effect of vitamin K against warfarin poisoning. It follows that FSP1 is the enzyme mediating warfarin-resistant vitamin K reduction in the canonical vitamin K cycle6. The FSP1-dependent non-canonical vitamin K cycle can act to protect cells against detrimental lipid peroxidation and ferroptosis.

Radical-Trapping Antioxidant Activity of Copper and Nickel Bis(Thiosemicarbazone) Complexes Underlies Their Potency as Inhibitors of Ferroptotic Cell Death.

Herein we demonstrate that copper(II)-diacetyl-bis(N4-methylthiosemicarbazone)(CuATSM), clinical candidate for the treatment of ALS and Parkinson's disease, is a highly potent radical-trapping antioxidant (RTA) and inhibitor of (phospho)lipid peroxidation. In THF autoxidations, CuATSM reacts with THF-derived peroxyl radicals with kinh = 2.2 × 106 M-1 s-1─roughly 10-fold greater than α-tocopherol (α-TOH), Nature's best RTA. Mechanistic studies reveal no H/D kinetic isotope effects and a lack of rate-suppressing effects from H-bonding interactions, implying a different mechanism from α-TOH and other canonical RTAs, which react by H-atom transfer (HAT). Similar reactivity was observed for the corresponding Ni2+ complex and complexes of both Cu2+ and Ni2+ with other bis(thiosemicarbazone) ligands. Computations corroborate the experimental finding that rate-limiting HAT cannot account for the observed RTA activity and instead suggest that the reversible addition of a peroxyl radical to the bis(thiosemicarbazone) ligand is responsible. Subsequent HAT or combination with another peroxyl radical drives the reaction forward, such that a maximum of four radicals are trapped per molecule of CuATSM. This sequence is supported by spectroscopic and mass spectrometric experiments on isolated intermediates. Importantly, the RTA activity of CuATSM (and its analogues) translates from organic solution to phospholipid bilayers, thereby accounting for its (their) ability to inhibit ferroptosis. Experiments in mouse embryonic fibroblasts and hippocampal cells reveal that lipophilicity as well as inherent RTA activity contribute to the potency of ferroptosis rescue, and that one compound (CuATSP) is almost 20-fold more potent than CuATSM and among the most potent ferroptosis inhibitors reported to date.

Metabolic determinants of cancer cell sensitivity to canonical ferroptosis inducers.

Cancer cells rewire their metabolism and rely on endogenous antioxidants to mitigate lethal oxidative damage to lipids. However, the metabolic processes that modulate the response to lipid peroxidation are poorly defined. Using genetic screens, we compared metabolic genes essential for proliferation upon inhibition of cystine uptake or glutathione peroxidase-4 (GPX4). Interestingly, very few genes were commonly required under both conditions, suggesting that cystine limitation and GPX4 inhibition may impair proliferation via distinct mechanisms. Our screens also identify tetrahydrobiopterin (BH4) biosynthesis as an essential metabolic pathway upon GPX4 inhibition. Mechanistically, BH4 is a potent radical-trapping antioxidant that protects lipid membranes from autoxidation, alone and in synergy with vitamin E. Dihydrofolate reductase catalyzes the regeneration of BH4, and its inhibition by methotrexate synergizes with GPX4 inhibition. Altogether, our work identifies the mechanism by which BH4 acts as an endogenous antioxidant and provides a compendium of metabolic modifiers of lipid peroxidation.