Targeting Ferroptosis New Hope for As-Yet-Incu.pdf

OpinionTargeting Ferroptosis:New Hope forAs-Yet-Incurable DiseasesMarcus Conrad,1,2,*Svenja M.Lorenz,1and Bettina Proneth1Attaining control over life and death decisions facilitates the identification of newtherapeutic strategies for diseases affected by early cell loss or resistance to celldeath.In this context,ferroptosis,a prevailing form of non-apoptotic cell deathmarked by the iron-dependent oxidative destruction of lipid bilayers and meta-bolic aberrations,has attracted overwhelming interest among basic researchersand clinicians due to its relevance for a number of degenerative diseases,suchas neurodegeneration,ischemia/reperfusion injury(IRI),and organ failure,aswell as therapy-resistant tumors.As the ferroptotic death pathway offers variousdruggable nodes,it is anticipated that the preclinical and clinical development offerroptosis modulators will unleash unprecedented opportunities for the treat-ment of as-yet-incurable diseases.Molecular Principles of FerroptosisThe traditional perception that cell death can be classified only as apoptosis,the archetypal formof programmed cell death,or necrosis(see Glossary),which was long considered to be unreg-ulated with no prospects for pharmacological intervention,is now being challenged by the notionthat necrotic cell death can also proceed in a regulated fashion,as first described fornecroptosis 1.More than a dozen forms of regulated necrosis have been described,althoughit remains uncertain whether all of them:(i)are trulyindependent;(ii)overlap;or(iii)are constrainedto certain cell types 2.Nonetheless,among these forms of regulated necrosis,ferroptosis hassparked tremendous interest as it emerges to be the root cause of a number of degenerativediseases and may provide an Achilles heel for barely treatable tumors 3.Ferroptosis was first described as a non-apoptotic form of cell death marked by impairedcystine(i.e.,the oxidized form of cysteine)uptake into cells,glutathione(GSH)depletion andiron-dependent lipid peroxidation 4.However,long before the term ferroptosis was coined,multiple seemingly disparate lines of evidence,obtained by the exploration of variousbiochemical,cellular,and metabolic processes including cysteine/selenium availability,GSHbiosynthesis,iron metabolism,and protection against lipid peroxidation,provided the molecularbackground for how ferroptosis is currently perceived.Several landmark studies in the first de-cade of the 21st century on novel cell-death-inducing small molecules and the in vivo relevanceof the main cellular redox systems in mammals eventually culminated in the recognition offerroptosis as a distinct form of cell death 48.The discoveries that sufficient cystine supplyvia the cystine/glutamate antiporter(alias system xc)and GSH synthesis are prerequisites foroptimal functioning of the selenoenzyme glutathione peroxidase 4(GPX4)and the preventionof lipid peroxidation,established the cyst(e)ine/GSH/GPX4 axis as the key ferroptosis-regulatingsystem in mammals(Figure 1)9,10.Genome-wide genetic screens subsequently uncovered a specific fatty acid ligase,acyl-CoAsynthetase long chain family member 4(ACSL4),to determine cells sensitivity towardHighlightsFerroptosis is a pervasive,disease-relevant metabolic cell death pathway,entirely distinct from other known formsof regulated cell death.Ferroptosisistherootcauseofanumberof degenerative diseases and emergesas a liability for difficult-to-treat tumors.Ferroptosis modulation may unleashunprecedented opportunities for phar-macological intervention.1Institute of Metabolism and Cell Death,Helmholtz Zentrum Mnchen,Ingolstdter Landstr.1,85764Neuherberg,Germany2National Research Medical University,Laboratory of Experimental Oncology,Ostrovityanova 1,Moscow 117997,Russia*Correspondence:marcus.conradhelmholtz-muenchen.de(M.Conrad).Trends in Molecular Medicine,Month 2020,Vol.xx,No.xxhttps:/doi.org/10.1016/j.molmed.2020.08.0101 2020 Elsevier Ltd.All rights reserved.Trends in Molecular MedicineTRMOME 1603 No.of Pages 10ferroptosis 11,12.The role of ACSL4 in ferroptosis is based on its activity to activate long chainpolyunsaturated fatty acids(PUFAs),which,when incorporated into phospholipids,may in-crease the risk of lipid peroxidation 12,13.Although lipid peroxidation and the associated rup-ture of cellular membranes is the hallmark of ferroptosis,it is still intensely debated whether thisprocess is mediated by certain lipoxygenases or by the iron-dependent,radical-mediatedFenton reactionandautoxidationoflipidbilayers 7,10,1417.Either way,inhibitorsoflipidper-oxidation or strategies to lower the abundance of PUFAs in membranes are the most efficientmeans to prevent ferroptosis.Likewise,chelation of iron was repeatedly reported to protectagainst lipid peroxidation and ferroptotic cell death 4,10,albeit that the precise role of iron inferroptosis clearly deserves further investigation.Potential intracellular sources of labile redox-active iron include autophagic degradation of the iron-storage protein ferritin 18,19,hemeoxygenase-1-mediated heme degradation 20,21,and during an iron-starvation response 22.GlossaryAcyl-CoA synthetase long chainfamily member 4(ACSL4):one of fiveclosely related fatty acid ligases thatpreferably activates long PUFAs byligating them with coenzyme A.Chloroacetamide:a reactive group onsmall molecules that can covalentlyinactivate the active-site selenocysteineof selenoproteins.Fenton reaction:describes a chemicalreaction between ferrous iron(Fe2+)andhydrogen peroxide leading to theformation of the highly toxic hydroxylradical(OH.),which can initiate lipidperoxidation and ferroptosis.Ferroptosis suppressor protein 1(FSP1):the second mainstay inferroptosis control,which actsindependently of the GSH/GPX4 axis byregenerating CoQ10.Glutathione(GSH):a tripeptidecomprising glutamate,cysteine,andglycine representing the most abundantintracellular electron donor for GSH-dependent enzymes in mammals(up to10 mM).Glutathione peroxidase 4(GPX4):one of eight GPXs in mammals withunique enzyme activity to efficientlyreduce lipid peroxides in cellularmembranes thereby preventing lipidperoxidation.Ischemia/reperfusion injury(IRI):a disease condition whereby the oxygensupply to parts of an organ is transientlydisruptedbyocclusionofthe vesselbyablood clotor during surgery.Restorationof the blood flow(i.e.,reperfusion)maycause oxidative stress,massive celldeath,and tissue injury.Lipid peroxidation:oxidativedegradation of lipids,whereby freeradicals steal electrons from the lipids(i.e.,PUFAs)in cellular membranes.Thiscauses the formation of lipid radicals,which can spark the lipid peroxidationchain reaction.If not counteracted byantioxidant enzymes or RTAs,membranes are oxidatively destroyedleading to the rupturing of cells andferroptosis.Necroptosis:the first described formof regulated necrosis uncovered by therecognition that tumor necrosis factoralpha(TNF)induces not only apoptosisand inflammation,but also necroptosisunder specific cell contexts.Necrosis:unregulated forms of celldeath mostly induced by physicalimpacts and chemicaltoxins thatcannotbe prevented by cell-death inhibitors.TrendsTrends inin MolecularMolecular MedicineMedicineFigure 1.The Ferroptosis Signaling Pathway.Ferroptosis is controlled by two main regulatory systems;namely,thecyst(e)ine/glutathione(GSH)/glutathione peroxidase 4(GPX4)and the NAD(P)H/ferroptosis suppressor protein 1(FSP1)/ubiquinone(CoQ10)axis.Ferroptosis prevention via the cyst(e)ine/GSH/GPX4 nexus follows the key steps of cystineuptake and reduction,respectively,GSH biosynthesis,and ultimately the reduction of oxidized phospholipids(PLs)(PL-OOH)to the corresponding alcohols(PL-OH)by GPX4,using GSH as a substrate.Acyl-CoA synthetase long chain family member4(ACSL4)and lysophosphatidylcholine acyltransferase 3(LPCAT3)are directly involved in the incorporation ofpolyunsaturated fatty acids(PUFAs)into cellular membranes,rendering them sensitive toward ferroptosis induction.Thereby,the oxidation of lipid bilayers might occur enzymatically and/or nonenzymatically via auto-oxidation.In the NAD(P)H/FSP1/CoQ10system,the antiferroptotic role of FSP1 is owed to its oxidoreductase activity by reducing extramitochondrial CoQ10to ubiquinol using NAD(P)H/H+.Ubiquinol prevents lipid peroxidation,either directly by reducing lipid radicals(PL-OO)orindirectly using vitamin E(-tocopherol).In addition,the mevalonate pathway provides precursors for the biosynthesis ofCoQ10and squalene;the latter has been shown to contribute to ferroptosis suppression through its antioxidant activity.Abbreviations:GDR,glutathione-disulfide reductase;HMG-CoA,-hydroxy-methylglutaryl-CoA.Trends in Molecular Medicine2Trends in Molecular Medicine,Month 2020,Vol.xx,No.xxIn search of an as-yet-unrecognized ferroptosis resistance mechanisms,two groups indepen-dently discovered ferroptosis suppressor protein-1(FSP1)previously called apoptosis in-ducing factor mitochondria associated 2(AIFM2)as a novel ferroptosis regulator.FSP1provedto be a highlypowerful antiferroptotic enzyme whose overexpression led to complete res-cue from ferroptosis induced by genetic deletion or pharmacological inhibition of GPX4 23,24.The antiferroptotic function of FSP1 is based on its oxidoreductase activity to reduceextramitochondrial ubiquinone(CoQ10)to ubiquinol using NAD(P)H/H+23.Ubiquinol in turnprevents the lipid peroxidation chain reaction,either directly by reducing lipid radicals or indirectlyvia-tocopherol,theprevailing formofvitamin E.Thus,theNAD(P)H/FSP1/CoQ10axisactsinde-pendent of the canonical GPX4-dependent nexusand does not require selenium for properfunc-tioning 25.Besides these two main ferroptosis-suppressing systems,squalene,an intermediate metaboliteof the cholesterol pathway,as well as GTP cyclohydrolase-1 and its metabolic productsdi/tetrahydrobiopterin have been linked to ferroptosis resistance by acting as antioxidants,thuspreventing uncontrolled lipid peroxidation 2628.Conceptual Mechanisms of Ferroptosis in Health and DiseaseIn recent years,ferroptosis has been linked with various pathological conditions,ranging fromdegenerative diseases like ischemia/reperfusion injury(IRI),organ failure,and neurodegen-eration to certain therapy-resistant cancer entities and metastasis formation 3.Most of thesestudies on the tissue-protective role of ferroptosis suppression originated from studies usingtransgenic mice(Box 1 and Figure 2),and the use of ferroptosis-inhibiting compounds.Whilethe physiological implications of ferroptosis are still being unraveled,it is believed that protec-tion from ferroptosis is an evolutionary requirement,which arose from the incorporation ofPUFAs into cellular membranes to support the formation of complex organisms,neuronalnetworks,and mammalian development 29.Tumor cells,by contrast,exploit PUFA-enriched environments and associated changes in cellular plasticity to switch to a therapy-resistant state or to undergo epithelialmesenchymal transition,which represents an attractivevulnerability for therapeutic intervention 25.To date,compelling evidence exists for therelevance of ferroptosis in animal models of disease,although proof-of-concept studies inpatients remain in their infancy.Polyunsaturated fatty acids(PUFAs):fatty acids containing morethanonedoublebondintheirbackbone.Radical-trapping antioxidants(RTAs):type of small molecule-compounds including alpha-tocopherol,liproxstatin,and ferrostatin that can stopthe process of lipid peroxidation at thelevel of lipid radicals.Selenocysteine:the 21stproteinogenic amino acid,differing fromits analog cysteine only by thereplacement of sulfur with selenium.Selenoprotein:a small family of 25dedicated proteins in humans thatcontain the 21st,rare amino acidselenocysteine.System xc:a heterodimeric cystine/glutamate amino acid transportercomprising xCT(SLC7A11)and 4F2(SLC3A2),importingcystineincellswhilereleasing one molecule of glutamate.Ubiquinone(CoQ10):also known ascoenzyme Q10;a lipophilic metabolitederived from the mevalonate pathwaythat usually functions in themitochondrial electron transport chain.Extramitochondrial CoQ10regeneratedby FSP1 acts like a strong RTA andregenerates vitamin E to prevent lipidperoxidation.Box 1.Tissue-Specific Susceptibilities toward Ferroptosis in MiceSince the discovery of ferroptosis as a distinctive form of necrotic cell death,there has been an extensive search for boththe molecular and the metabolic determinants that determine cells sensitivity toward ferroptosis.While it is still far frombeing understood why certain tissues or even cells in a given tissue are susceptible to ferroptosis,transgenic mouse stud-ies have been instrumental in delineating ferroptosis-sensitive versus-resistant tissues(see Figure 2 in main text).SinceGPX4 is crucial for early embryonic development 75,tissue-specific Gpx4-knockout studies unveiled that certain neuro-nal subpopulations,including hippocampal neurons 7,glutamatergic neurons and parvalbumin-positive interneurons ofthe cortex 29,76,cerebellar Purkinje cells 77,and motoneurons 46,are strictly dependent on functional GPX4,whilecertain neuronal subpopulations of the hypothalamus and dopaminergic neurons are resistant to GPX4 loss 78.Outsidethe brain,proximal kidney tubular cells are the most ferroptosis-sensitive cell type,since the inducible Gpx4 knockoutcauses acute kidney injury resembling that of delayed graft function as occurs intermittently during organ transplantation10.Also,hemostasis ofCD8-positiveTcells,reticulocytematuration,andinnate-likeB1 andmarginalzoneB cellsrequireGPX4 expression 65,79,80.Notably,in certain tissues Gpx4 deficiency can be bypassed by dietary vitamin E levels,which can be remarkably high in most chows used for mouse experiments.For instance,endothelium-specific Gpx4-knockout mice kept under regular dietary conditions are phenotypically normal,while under low vitamin E endothelial celldeath causes thrombosis,multiple microinfarcts,and death in mice 81.A similar finding has been reported for hepato-cyte-specific GPX4-null mice 82.Hence,these findings warrant careful planning when interrogating the in vivo relevanceof ferroptosis not only in(patho)physiological contexts,but also when assessing the in vivo pharmacological potential ofnovel ferroptosis modulators.Trends in Molecular MedicineTrends in Molecular Medicine,Month 2020,Vol.xx,No.xx3Ferroptosis as the Key Driver of IRI and Organ FailureIRI underlies several disorders,including stroke,cardiac infarction,and other conditions wherethebloodflowintotheorganisinterruptedforagivenperiodoftime(e.g.,duringsurgicalinterven-tion or organ transplantation)3.In this context,ferroptosis inhibitors have been shown to suc-cessfully protect against IRI in various organs including the liver 10,kidney 30,intestine 31,and lung 32,during myocardial infarction 33,34,and in stroke 35(Figure 2).Additionaltissue-protective effects of ferroptosis inhibitors have been reported in models of acute renalfailure