Role of GPX4 in ferroptosis and its pharmacological implication

Author’s Accepted Manuscript Role of GPX4 in ferroptosis pharmacological implication

and

its

Tobias M. Seibt, Bettina Proneth, Marcus Conrad

www.elsevier.com

PII: DOI: Reference:

S0891-5849(18)31593-4 https://doi.org/10.1016/j.freeradbiomed.2018.09.014 FRB13916

To appear in: Free Radical Biology and Medicine Received date: 8 August 2018 Revised date: 10 September 2018 Accepted date: 12 September 2018 Cite this article as: Tobias M. Seibt, Bettina Proneth and Marcus Conrad, Role of GPX4 in ferroptosis and its pharmacological implication, Free Radical Biology and Medicine, https://doi.org/10.1016/j.freeradbiomed.2018.09.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Role of GPX4 in ferroptosis and its pharmacological implication Tobias M. Seibt1, Bettina Proneth2, Marcus Conrad2* 1

Department of Nephrology, Medizinische Klinik und Poliklinik IV, Klinikum der Universität

München, 80336 München, Germany 2

Helmholtz Zentrum München, Institute of Developmental Genetics, 85764 Neuherberg,

Germany *

Correspondence: Helmholtz Zentrum München, Institute of Developmental Genetics, 85764

Neuherberg, Germany; phone: +49-89-31874608. [email protected]

Abstract Ferroptosis is a non-apoptotic form of cell death characterized by iron-dependent lipid peroxidation and metabolic constraints. Dependence on NADPH/H+, polyunsaturated fatty acid metabolism, and the mevalonate and glutaminolysis metabolic pathways have been implicated in this novel form of regulated necrotic cell death. Genetic studies performed in cells and mice established the selenoenzyme glutathione peroxidase (GPX4) as the key regulator of this form of cell death. Besides these genetic models, the identification of a series of small molecule ferroptosis-specific inhibitors and inducers have not only helped in the delineation of the molecular underpinnings of ferroptosis but they might also prove highly beneficial when tipping the balance between cell death inhibition and induction in the context 1

of degenerative diseases and cancer, respectively. In the latter, the recent recognition that a subset of cancer cell lines including certain triple negative breast cancer cells and those of therapy-resistant high-mesenchymal cell state present a high dependence on this lipid makeup offers unprecedented opportunities to eradicate difficult to treat cancers. Due to the rapidly growing interest in this form of cell death, we provide an overview herein what we know about this field today and its future translational impact. Keywords: Regulated necrosis; GPX4; lipid peroxidation; cysteine metabolism; non-apoptotic cell death; ferritinophagy

1.

Introduction For decades, regulated apoptosis and unregulated, accidental necrosis have been

considered as the two sole cell death routes in multicellular organisms. With the beginning of the 21st century a paradigm shift has been witnessed by researchers in the field with the recognition that many forms of unregulated necrosis in fact do also follow certain regulated cell death routines with distinct molecular patterns and metabolic features [1]. Among these are necroptosis, pyroptosis, netosis, entosis, parthanatos, and cyclophilin D-mediated cell death, with ferroptosis being described for the first time in 2012 [2]. Ferroptosis is characterized by iron-dependent lipid peroxidation and has attracted tremendous interest owing to its unique relevance for a number of pathological conditions including tissue ischemia/reperfusion injuries (IRI), neurodegeneration and cancer [3, 4]. Cells dying by ferroptosis present a distinct bioenergetic signature, are characterized by oncosis 2

(despite unaffected nuclei), and display aberrant mitochondrial morphology including outer membrane

rupture

as

well

as

cellular

disintegration,

therefore

lacking

typical

histomorphological hallmarks of apoptosis and other forms of regulated cell death. Although the final step(s) leading to ferroptotic cell death still remain(s) obscure, the occurrence of specific lipid peroxidation products in a subset of phospholipids directly precedes cellular disintegration and cell death [5]. Therefore, compounds and conditions that hamper the process of lipid peroxidation can be regarded as viable approaches to prevent ferroptosis and related disease. On the molecular level, cysteine availability, glutathione (GSH) biosynthesis and proper functioning of glutathione peroxidase 4 (GPX4) are the heart of keeping ferroptosis in check, whereas conditions that culminate in GPX4 inhibition/destabilization sensitize or even trigger ferroptotic cell death. Recently, additional players involved in polyunsaturated fatty acid (PUFA) metabolism have been added to the list of pro-ferroptotic enzymes, which are ultimately involved in shaping the cellular lipid composition [6-8]. Hence, this review is conceived to provide an overview on what is currently known about the molecular underpinnings, modulators and metabolic constraints as well as the relevance for (patho)physiological conditions in health and disease. We focus here on the mammalian system as almost everything what we have learned about ferroptosis was discovered in this system, being well aware that ferroptosis is not only limited to mammals but has also been described to occur in higher plants and in the unicellular parasite Trypanosoma brucei [9-11].

3

2.

Molecular mechanisms of ferroptotic cell death Mainstay of ferroptosis is the generation of specific phospholipid hydroperoxides in the

presence of catalytically active iron, which is endogenously counteracted by the system xc/GSH/GPX4 axis [2, 12, 13]; consequently, disturbances in any of these protective compartments may result in ferroptotic cell death. The most upstream component of this axis is system xc- [14], a highly specific uptake system for cystine (i.e. the oxidized form of cysteine) and cystathionine at the exchange of glutamate at a 1:1 molar ratio [15, 16] (Fig. 1). xCT (SLC7A11) light chain, which constitutes the substrate-specific subunit of system xc-, is subject to complex transcriptional regulation [17]. While xCT is positively regulated by activating transcription factor 4 (ATF4) under conditions of oxidative stress and cysteine deprivation [18], p53 was recently shown to repress xCT expression leading to cystine starvation and thus increased susceptibility to ferroptosis, thereby possibly contributing to tumor suppression [19, 20] (see also section 5). Once taken up by system xc-, cystine is reduced to cysteine by GSH and/or thioredoxin reductase 1, which in turn is used for GSH biosynthesis [21]. While this route is the most relevant way to cope with the cells’ demand for cysteine in cell culture (where virtually all cysteine is oxidized to cystine), cysteine can also be provided to some extent by the transsulfuration pathway [22], and the neutral amino acid transporter ASC [17]. The latter being most likely the predominant way in the whole organism where the main fraction of plasma and tissue cysteine is found in its reduced form. Since cysteine is the rate-limiting substrate in GSH biosynthesis and GSH is the prevailing antioxidant in mammalian cells, 4

conditions that impede on intracellular cysteine and consequently GSH levels impact directly GPX4 activity and thus predispose to ferroptosis. First identified in 1982 as the second mammalian glutathione peroxidase [23], we and Stockwell’s group demonstrated in 2014 that the selenoperoxidase GPX4 is the key upstream regulator of ferroptosis [12, 13]. The role of GPX4 as the main regulator in the ferroptotic process is based on its unique function to reduce complex hydroperoxides including phospholipid hydroperoxides and cholesterol hydroperoxides to their corresponding counterparts, thereby interrupting the lipid peroxidation chain reaction. In fact, early studies with conditional deletion of Gpx4 in brain and fibroblasts provided initial evidence that neurodegeneration and cell death of hippocampal neurons occurred in non-apoptotic manner entailing massive lipid peroxidation [24]. The promiscuity of GPX4 towards a number of reducing substrates is also reflected by its use of oxidizing substrates. Besides its main cofactor GSH, other low molecular thiols and even protein thiols can be readily exploited by GPX4 [25]. Recently, the molecular role of selenium in form of the 21st amino acid selenocysteine (Sec) in GPX4 has been elucidated. Mice expressing a targeted mutation of the active Sec to Cys (GPX4_U46C) are surprisingly viable (unlike the full knockout of Gpx4 causing early embryonic death [26]) but failed to survive the weaning stage due to severe epileptic seizures [27]. Loss of a specific subset of parvalbumin–positive (PV+) GABAergic interneurons in cortex of homozygous mice was identified as the underlying reason for the seizures, implying that this specific type of inhibitory neurons requires fully functional, selenium containing GPX4 [28]. The reasons for the peculiar sensitivity of PV+ GABAergic interneurons towards cell death 5

are not completely understood and might be of complex nature. For instance, these cells need to migrate and form inhibitory synapses early after birth, which requires a high degree of unsaturation in their membranes. Thus, they might be particularly vulnerable to cell death. Alternatively, these neurons are known to have a particularly high demand for ATP, therefore they might generate a substantial amount of oxygen radicals as a metabolic side product. Remarkably, homozygous GPX4_U46C expressing cells proliferate normally and were as resistant to cytotoxic agents as wildtype cells; however, in stark contrast these cells were exquisitely sensitive to peroxide-induced ferroptosis due to the high susceptibility of Cys containing GPX4 towards peroxide-induced irreversible overoxidation and inactivation of GPX4 in cells [27]. Hence, it emerged that selenium utilization by GPX4 not only allows cells and tissues to become highly resistant to pro-ferroptotic, pathological conditions but perhaps also to exploit hydrogen peroxide to be used as a cellular signaling molecule [29]. While the upstream mechanisms of ferroptosis including cysteine availability, GSH biosynthesis and proper functioning of GPX4 have been well established in the meantime, much less is known about potential downstream events. Genetic screens independently performed in haploid cells and fibroblasts identified acyl-CoA synthetase long-chain family member 4 (ACSL4) as an additional and essential downstream player in the ferroptotic process [6, 8]. ACSL4 is one of a number of fatty acid activating enzymes functioning by esterifying CoA to free fatty acids in an ATP dependent manner. Unlike other family members, ACSL4 shows a high preference towards long chain PUFAs such as arachidonic acid (AA) and adrenic acid (AdA). CRISPR/Cas9-mediated knockout and pharmacological inhibition of ACSL4 by 6

thiazolidinediones conferred an unparalleled protection against ferroptosis elicited by small molecule ferroptosis inducers or the genetic inactivation of Gpx4 in fibroblasts [6]. An interdisciplinary approach combining redox global phospholipidomics, reverse genetics, bioinformatics and systems biology allowed to pinpoint 15-hydroperoxy-arachidonoyl- and 15hydroperoxy-adrenoyl residues esterified in phosphatidylethanolamines (PE) as proximate signals of the ferroptotic death program [30]. Accordingly, genetic disruption/pharmacological inhibition of ACSL4 prevented the accumulation of these death signals in cells [6, 30]. Although identified in the haploid screen [8], knockout of lysophosphatidylcholine acyltransferase 3 (LPCAT3), an enzyme which preferably re-esterifies polyunsaturated fatty acyl-CoAs into certain lyso-phospholipids, did not yield strong protection against ferroptosis, suggesting cellspecific contexts or compensatory mechanisms by other enzymes of this family of proteins [6].

3.

Importance of iron and lipoxygenase in ferroptosis What remains to be somewhat controversial is the role of iron and lipoxygenases (LOX) in

the death process, specifically in lipid peroxidation. So far, iron chelators have been repeatedly shown to halt ferroptosis (hence its name) induced by chemical or genetic means [2, 13], and overloading of cells with iron indeed sensitizes tumor cells to ferroptosis [31]. Additionally, enzymes/proteins involved in iron metabolism [32], such as transferrin, transferrin receptor 1 and ferroportin [33, 34], as well as heme oxygenase-1 (HO-1) [35, 36] have been implicated in the death process. Ferritinophagy, an autophagic process leading to the degradation of cellular iron storage proteins including the iron binding protein ferritin and 7

the ferritinophagy cargo receptor nuclear receptor coactivator 4 (NCOA4), was recently demonstrated to contribute to ferroptosis by increasing the cellular labile iron pool and increased oxygen radical formation [37]. Freely available iron is thus considered to contribute to the process of lipid peroxidation and cell death by attacking PUFA residues in lipid bilayers in a non-enzymatic manner [5]. Ever since the early recognition that GPX4 counteracts the activity of certain LOX by controlling the so-called cellular peroxide tone (reviewed in [38]), LOX have been surmised to contribute to ferroptotic death by introducing peroxides in fatty acid residues of phospholipids. The enzymatic peroxidation of PUFAs is predominantly catalyzed by LOX, which abstract protons from bis-allylic positions followed by the addition of molecular oxygen. In humans there are six different isoenzymes (ALOXe3/5/12/12/15/15b) harbouring a catalytic active iron - their nomenclature refers to the regional selectivity of hydroperoxide formation [39]. LOX are only able to bind molecular oxygen when iron is oxidized by peroxides to ferric iron [39]. Thus, lowering the peroxide tone of cells by GPX4 may indirectly impact lipoxygenase activity. Early findings indeed suggested that inhibition of 12-lipoxygenase prevents neuronal cell death in HT22 cells induced by GSH depletion and oxidative glutamate toxicity [40], and that lipoxygenase inhibitors rescued Gpx4 knockout induced cell death in mouse embryonic fibroblasts engineered to allow inducible deletion of GPX4 [24]. Conversely, genetic studies performed with Alox15 (the gene encoding 12/15-lipoxygenase in mice) knockout and conditional Gpx4 knockout mice taught us that the double mutant mice fail to rescue from acute renal failure in mice, ferroptosis in fibroblasts [13] and in CD8+ T cells [41], 8

nor does it rescue the early embryonic lethal phenotype of Gpx4-/- embryos [42]. Only subfertility, as observed in mice expressing a dysfunctional GPX4 protein, was rescuable by systemic inactivation of Alox15 [43]. Oxi-lipidomics analysis of cell undergoing ferroptosis and studies with deuterated fatty acids, however, pointed towards a site-specific and thus enzyme-mediated oxidation of PUFA residues [30, 44], thereby contributing to the generation of proximate signals of ferroptotic cell death. Moreover, phosphatidylethanolamine binding protein-1 (PEBP1), a protein with high affinity to phosphatidylethanolamine and known to bind and to inhibit RAF1 kinase activity, was identified to associate and change the substrate specificity of 15-lipoxygenase [45]. This interaction with 15-lipoxygenase seemingly changes the substrate specificity of 15-lipxygenase thus yielding potentially hazardous, peroxidized phophatidylethanolamines, which, when not cleared by GPX4, contribute to ferroptosis. Along the same line, siRNA-mediated knockdown of individual or several lipoxygenases conferred resistance to erastin induced ferroptosis in different cell lines [44], indicating cell type specific contexts. A recent study by Pratt’s group questioned the ostensibly outstanding importance of LOX in the ferroptotic process [46]. While many of the frequently used alleged isoform-selective LOX inhibitors suppress the ferroptotic cell death process not by inhibiting their respective LOX isoform, but rather by acting as unspecific radical trapping agents, overexpression studies with several human LOX isoforms (5-LOX, p12-LOX, and 15-LOX-1) clearly demonstrated that lipid autoxidation is the key driver of ferroptosis. The fact that many of the previous studies with animal models of disease were actually performed with these “specific” inhibitors 9

questions whether these effects are actually mediated by LOX inhibition or by general “antioxidant” effects. In light of the still missing in vivo data that knockout of one or even several LOX isoforms clearly mitigates the effects induced by GPX4 loss, care needs to be taken that LOX indeed play a major role in ferroptosis [47].

4.

Ferroptosis Modulators With the recognition that the small molecule erastin targets system xc- and thereby

specifically triggers this form of death and that ferrostatin-1 prevents erastin-induced ferroptosis [2], it has become evident that ferroptosis is a druggable pathway with a number of tractable nodes that can be modulated in two ways: Ferroptosis inducers can be used to kill malignant cells, whereas ferroptosis inhibitors are applicable in diseases characterized by early cell and tissue loss [3, 5]. Ferroptosis modulators described herein are summarized in Table 1 and Table 2. a) Ferroptosis inducing mechanisms Compounds that interfere with system xc- include erastin and its analogues causing cysteine deprivation, GSH depletion, endoplasmic reticulum stress and cell death [2, 48, 49] (Table 1). Other ways to inhibit system xc- are millimolar concentrations of L-glutamate, sulfasalazine, diaryl-isoxazoles [50] or sorafenib [51], just to name a few [52]. L-buthionine sulfoximine (BSO) is a long known irreversible and highly specific inhibitor of γglutamylcysteine synthetase, the enzyme catalyzing the rate-limiting step in GSH synthesis, 10

therefore resulting in GSH depletion [53]. (1S,3R)-RSL-3 (RSL3) is the first described GPX4 inhibitor [12]. Since RSL3 contains an electrophilic chloroacetamide, it seems to covalently interact with the nucleophilic active site Sec of GPX4 leading to irreversible inactivation of the enzyme [44]. In fact, cells expressing the GPX4_U46C variant are by at least one order of magnitude less sensitive to RSL3 than wildtype GPX4 expressing cells [27]. Other direct and indirect GPX4 inhibitors include FIN56 [54], ML162 and ML210 [12, 55], as well as the 1,2dioxolane FINO2 [56]. FINO2, however, as recently been shown to effectively oxidize ferrous iron independent of ALOX activity in vitro, thereby promoting Fenton-type chemistry [57], resulting in a more widespread array of oxidized phospholipids than that induced by GPX4 inhibition. Compared to ferroptosis induction via erastin or RSL3, the iron chelator deferoxamine had a much more protective effect when HT-1080 cells were treated with FINO2. Interestingly, protein expression of iron transition (transferrin receptor, TFR), regulatory (iron-responsive element-binding protein 2, IREB2) and storage proteins (ferritin light chain 1, FTL1) remained unchanged, arguing for a critical involvement of the cellular labile iron pool [57]. Beyond these, untargeted and redox-active small molecules have been reported, such as artemisinin derivatives [55, 58-60], that specifically induce ferroptosis. Additionally, siramesine, a lysosome disrupting agent, and lapatinib, a tyrosine kinase inhibitor, elicit ferroptosis in a synergistic manner in various breast cancer cell lines by altering iron regulation [61]. Various groups recently reported different techniques and approaches that enable Fenton reaction-based killing of tumor cells. For instance, overloading of tumor cells with iron using ultrasmall

nanoparticles,

functionalized

with 11

melanoma-targeting

peptides,

caused

widespread ferroptosis in cultured cancer cells and tumor regression in tumor bearing mice, which could be abrogated by liproxstatin-1 [31]. Alternatively, linoleic acid hydroperoxide tethered on iron oxide nanoparticles allowed to trigger cell death not only in the glioblastoma cell line U87MG in vitro but also in tumor bearing mice, in a mechanism involving the generation of toxic amounts of singlet oxygen [62]. Similarly, PLGA (Polylactide-co-glycolide) particles loaded with a toxic H2O2/Fe3O4 cargo facilitated the effective killing of HeLa cells in a temporal manner using ultrasound-induced hydroxyl radical generation and associated cell death [63]. Packaging p53 expressing plasmids in so-called metal−organic networks (MONp53) and treatment of HT1080 tumor cells and tumor carrying mice with MON-p53 elicited ferroptotic cancer cell death in vitro and in addition led to tumor growth inhibition, reduced metastases and an increased overall survival benefit of tumor bearing mice [64]. Moreover, the natural phytochemical withaferin derived from Withania somnifera roots was shown to trigger ferroptosis by a dual mechanism [65]. On one hand it acts like a type 2 ferroptosis inducing agent (FIN) by direct binding to Cys107 of GPX4, thereby leading to degradation of GPX4, and on the other hand by inducing a number of genes including HO-1, when cells were treated with a medium dose of withaferin. HO-1 upregulation in turn leads to increased heme degradation and consequently a robust increase of the cellular iron labile pool [65]. Hence, the increasing repertoire of different ferroptosis inducers including small molecules, peptides, natural compounds and nanoparticle-based vehicles will prove highly beneficial in designing novel anti-cancer strategies.

12

b) Ferroptosis inhibiting mechanisms Ferroptosis inhibition presents the opposite and highly promising way by protecting from cell loss and tissue deterioration in the context of degenerative diseases. Ferrostatin-1 was the first described small molecule ferroptosis inhibitor preventing erastin-induced cell death [2] (Table 2). A phenotypic screening campaign in cells undergoing Gpx4 knockout-induced ferroptosis led to the identification of liproxstatin-1 as the first in vivo efficacious ferroptosis inhibitor. Liproxstatin-1 showed IC50 values in the low nanomolar range in vitro, significantly prolonged survival in a genetic model of acute renal failure, as well as ameliorated hepatic ischemia/reperfusion injury in mice [13]. In the same study, it was shown that necrostatin-1, the first published necroptosis and receptor interacting protein kinase 1 (RIPK1) inhibitor [66], also prevents ferroptosis as a side effect, suggesting that a fraction of previously reported in vivo experiments with positive outcomes subscribed to necrostatin-1 might be due to ferroptosis inhibition rather than necroptosis prevention. Recently, the mode of action of liproxstatins has been elucidated indicating that these molecules act as superior radical trapping antioxidants by breaking the autoxidation chain reaction [67]. Unlike Nature’s most efficacious ferroptosis inhibitor vitamin E (tocopherols and tocotrienols), which is consumed during the reduction of lipid hydroperoxides, liproxstatins are regenerated during this process and can trap multiple peroxyl radicals per molecule as also described for nitroxide-based compounds described further below. As aforementioned, lipoxygenase inhibitors such as zileuton, baicalein, LOXBlock-1-3, MK886, PD146176, BWA4C etc. have shown anti-ferroptotic effects, although care should be 13

taken when using these inhibitors as most of them are either not specific or even show generalized anti-oxidant effects [3, 13]. The same holds true for U0162, a widely used MEK1/2 inhibitor initially thought to be involved in ferroptosis, until it was shown that it confers nonspecific antioxidant activity [33]. Inhibition of the metabolic glutaminolysis pathway with the glutaminase inhibitor “compound 968” or amino-oxyacetic acid, a pan-transaminase inhibitor, demonstrated anti-ferroptotic effects both in fibroblasts and in isolated hearts subjected to ischemia-reperfusion injury [33]. The discovery of ACSL4 as an important downstream player of ferroptosis and its druggability with thiazolidinediones (TZD) [68], a class of compounds that was previously known to activate the peroxisome proliferator-activated gamma receptor (PPARγ)

therefore used for the treatment of diabetes mellitus type 2, opens another

opportunity to intervene with the ferroptosis pathway. Treatment of cells with the TZD rosiglitazone yielded the same lipidomic signature as observed in ACSL4 knockout cells and more importantly, TZDs prevented both accumulation of lipid hydroperoxides and ferroptosis induced by GPX4 inhibition/genetic disruption [6]. Besides these, phenoxazines [69] and a number of nitroxide-based compounds (untargeted or mito-targeted) including XJB-5-131, JP4-039 [70], 2,2,6,6-tetramethylpiperidin- N-oxyl (TEMPO), N-arylnitroxide, N, Ndiarylnitroxide and

phenoxazine- N-oxyl have been described as highly promising anti-

ferroptotic agents [71]. Thereby, the nitroxides reduce peroxyl radicals with high rate constants in a catalytic manner. Regeneration of nitroxides involves two steps, first hydride transfer from the substrate to the oxoammonium ion form of nitroxide and second H-atom transfer from the resultant hydroxylamine to a peroxyl radical.

14

An alternative approach using structure- and computation-based methods identified an allosteric site in GPX4 that allowed to discover the first GPX4 allosteric activators [72]. By increasing the enzyme activity of GPX4, these molecules suppress ferroptosis triggered by erastin and cholesterol hydroperoxides, and in addition dampen NFkB activation. None of the reported GPX4 activators presented reducing or iron chelating activity, therefore these molecules might be developed as promising cyto-protective and anti-inflammatory agents. Hence, the identification of an ever growing list of ferroptosis inducers and inhibitors as well as the unraveling of their mode of actions have greatly helped to illuminate the molecular mechanisms and the therapeutic potential of ferroptosis.

5.

(Patho)physiological Relevance Our current understanding of the relevance of ferroptotic cell death in physiologic and

pathophysiologic contexts mainly stems from investigations using small molecule ferroptosis modulators and genetic mouse/cell models for key regulators of ferroptosis (Tables 1, 2). While ferroptosis has been explored originally in the context of small molecules specifically eliciting cancer cell death by the Stockwell laboratory [2, 73], our contribution to the field began with the early recognition that the inducible deletion of the key ferroptosis regulator GPX4 in mice causes a novel non-apoptotic form of cell death in fibroblasts, neuronal cultures and in pyramidal cells of the hippocampus [24]. Later on, it was demonstrated by both groups that ferroptosis is not only limited to cancer cell death [12], but that it is also a highly relevant form of cell death in adult kidney tubular cells [13]. 15

Meanwhile a myriad of studies using tissue- and cell type-specific deletion of GPX4 have provided evidence that various tissues/cells, such as distinct neuronal cell populations including glutamatergic neurons of the forebrain [24], Purkinje cells of the cerebellum [74] and motor neurons [75], photoreceptor cells [76], kidney tubular cells [13], CD8+ T cells [41], endothelial cells [77], hepatocytes [78] and sperm cells [79] in principle can undergo ferroptosis. Just to stress here: Although a number of these studies have been performed long before the term “ferroptosis” was introduced in 2012, and instead terms other than ferroptosis such as oxytosis/non-apoptotic cell death/lipidoxytosis were used [80], they ultimately describe a similar if not the same phenomenon. In vivo studies with ferroptosis-specific inhibitors, such as liproxstatins and ferrostatins, either alone or in combination with inhibitors targeting alternative cell death pathways proved to mitigate tissue injury associated with transient ischemia/reperfusion (I/R) in liver [13] and kidney [81], oxalate crystal-induced acute kidney injury [81], intracerebral hemorrhage [82], ischemic stroke [83], or in genetic models of GPX4 deficiency in kidney [13] and brain [84], again highlighting the pharmacological amenability of this death pathway. It is also worth mentioning that in some tissues GPX4 deficiency can be compensated by dietary vitamin E supplementation (e.g. in endothelial cells, hepatocytes and CD8+ T cells) [77, 78], whereas other tissues are obligatory dependent on a functional GPX4/GSH system. In neurodegeneration, such as Parkinson`s (PD) disease, GSH depletion, nigral iron accumulation and lipid peroxidation have been frequently described [85-90]. Iron chelation significantly reduced neuronal damage and improved motor functions in a murine model of PD 16

[91]. First clinical trials revealed promising results for low dose iron chelation as future therapeutic considerations for early stages of PD [91]. Hence, multicenter, parallel-group, placebo-controlled, randomized clinical trials are now recruiting PD patients to evaluate the ability of the iron chelator deferiprone to slow down disease progression in early stages of PD (ClinicalTrials.gov Identifier: NCT02655315; NCT02728843). Besides acute and chronic degenerative diseases, ferroptotic cancer cell death is the second major area which can be harnessed to combat difficult to treat cancer entities [3]. In fact, a number of small molecules ferroptosis inducers (see above) have been described in the last few years that can specifically trigger this form of cell death at least in a cellular context. Yet, in many cases their in vivo efficacy remains to be verified as some of them target nodes of the ferroptosis cascade such as system xc- and GPX4 that might be bypassed in vivo by other transporters, antioxidants etc. at least in some tumor contexts [92], or that require high amounts of the inducers [12]. As discussed above, in this context the reported tumor suppressing function of p53 by repressing expression of SLC7A11 und thus depleting cells of cysteine the might be of substantial relevance [19, 20]. On the other hand, stabilization of p53 has been demonstrated to attenuate erastin-induced ferroptotic cell death in human HT-1080 fibrosarcoma cells and primary mouse embryonic fibroblasts (but not in non-cancerous IMR90 human fetal lung fibroblasts endogenously expressing p53) by transcriptional upregulation of target gene CDKN1A encoding for the cell-cycle arrest protein p21 [93]. Yet, inhibition of ferroptosis is presumably achieved by preserving the intracellular GSH pool rather than pure cell-cycle arrest as reported in this study [93]. P53 also inhibits dipeptidyl-peptidase-4 (DPP4) 17

activity and DPP4 promotes lipid peroxidation and ferroptotic cell death induction in p53deficient human colorectal cancer cells [94]. With the recent discovery that there is a clear correlation between ACSL4 expression and sensitivity to ferroptosis induction in a subset of triple negative breast cancer cells [6], new possibilities for the establishment of ferroptosis-based anticancer strategies in ACSL4 positive tumor entities may open. Remarkably, a recent study found that therapy-resistanceassociated high-mesenchymal state cancer cells are highly dependent on GPX4 (unlike their non-transformed mesenchymal counterparts) and treatment with respective GPX4 inhibitors direct and indirect ones - rendered these tumor cells highly vulnerable to this form of death [95]. This dependency seems to rely on the fatty acid composition of lipid bilayers because ACSL4 knockdown confers resistance of high-mesenchymal state cancer cells towards ferroptosis induction as previously shown for luminal-type triple negative breast cancer cells [6, 95]. Another lever to efficiently eradicate tumors including ovarian cancer might rely on their high addiction to iron [34]. In fact, ultrasmall silica-based nanoparticles, functionalized with melanoma-targeting peptide, efficiently triggered ferroptosis not only in cell culture but also in tumor bearing mice, both of which could be fully inhibited by liproxstatin-1. The underlying molecular mechanisms were associated with cellular iron overload as these particles had a high propensity for adsorbing iron [31]. As described above, exploiting Fenton-based chemistry in killing tumor cells by delivering engineered (and activatable) nanoparticles carrying toxic peroxide/iron cargos to tumors is indeed a highly active, upcoming field of 18

research [96]. Another highly promising approach might be cystathionine-γ-lyase (CGL, cyst(e)inase)-mediated systemic deletion of cyst(e)ine, the building block of GSH biosynthesis [97]. In fact, administration of cyst(e)inase to cell, monkey and tumor bearing mice caused a robust deletion of L-cysteine and concomitant increases in cellular oxygen radical formation. This strategy proved highly efficient in suppressing the growth of prostate carcinoma allografts as well as tumor xenografts (breast and prostate) and robustly prolonged the survival of a leukemic B cell tumor model in vivo. In an era of personalized medicine and patient-centered cancer treatment, ferroptosis inducing strategies could also augment existing kinase inhibitor, antibody- and autoimmunebased therapies. Melanoma skin cancer originates from epidermal, pigment-producing melanocytes and advanced metastatic stages of the disease are currently associated with very poor prognosis. Highly plastic melanoma skin cancer cells potentially evade targeted immunotherapy by dedifferentiation, subsequently becoming more susceptible to ferroptosisinducing agents, such as erastin, RSL3, ML162 and ML210 [98]. Along the same line, acquired drug resistance of therapy-resistant tumor cells of so-called “persister” cells was recently described to lead to a dependence on a functional GSH/GPX4 axis along a broad panel of cancer cells lines [99]. And in lung adenocarcinomas cysteine desulfurase (NFS1), an enzyme that removes sulfur from cysteine thereby providing inorganic sulfur, was found to be positively selected as this cancer entity is highly dependent on iron-sulfur cluster biosynthesis [100]. Consequently, shRNA-mediated suppression of NFS1 not only limits iron–sulfur cluster

19

availability particularly under elevated oxygen tensions, but also predisposes MDA-MB-231 cells to ferroptosis. Hence, it emerges that metabolic constraints including iron handling, cysteine/GSH and polyunsaturated fatty acid metabolism constitute an Achilles heel for certain tumor entities, thereby providing previously unrecognized starting points for combating cancer.

6.

Concluding Remarks and Future Considerations During the last few years, substantial progress has been made in the understanding of the

molecular and metabolic underpinnings of ferroptotic cell death. Studies performed in transgenic mice further allowed us to pinpoint which cells and tissues are in principle prone to succumb to ferroptosis. This knowledge will be of utmost importance when interrogating the aetiopathologies and upstream events possibly leading to ferroptosis in relevant disease scenarios, such as neuronal demise in amyotrophic lateral sclerosis (ALS) and Huntington’s disease (HD), as well as in IRI in the context of organ transplantation, stroke and cardiac infarction. What remains entirely unanswered is the potential cross-talk of cells dying by ferroptosis with the immune system. Yet, it can be anticipated that similar to other immunologically non-silent forms of non-apoptotic cell death, such as necroptosis, cellular contents and factors released by ferroptotic cells may have a strong pro-inflammatory impact [101]. If so, it would be highly interesting to see if oxidatively modified lipids or its breakdown products might ignite such a pro-inflammatory auto-amplification loop [102]. 20

The implementation of animal models in the study of ferroptosis will become even more relevant in future studies as the redox environment between (cell) culture conditions and a whole organism such as the mouse vary substantially. This is best illustrated by the fact that mice with targeted loss of SLC7A11 are fully viable [103], whereas any type of cells isolated from these knockout mice die within 24 hours upon isolation. The loss of SLC7A11 can thus be easily bypassed by other transport systems in vivo as approx. half of cysteine is still present in its reduced form in plasma and extracellular compartment. By stark contrast, in tissue culture conditions virtually all cysteine is readily oxidized to cystine whereupon it can only be taken up by system xc-. This might be of utmost importance when considering novel anticancer strategies based on system xc- inhibition. The same holds true for vitamin E content (and probably selenium, iron etc.) in cell culture serum and equally important in animal diets, as vitamin E does not only prevent cells from succumbing to ferroptosis but was also shown to compensate for the conditional loss of GPX4 in endothelial cells and hepatocytes in vivo [77, 78]. Another important factor that deserves consideration is the fatty acid composition of serum, which, in light of the role of ACSL4 in ferroptosis, may also have a significant impact on the outcome in different experimental settings. Beyond these considerations and the growing number of experimental data obtained in cells and mice, it remains to be formally shown that ferroptosis indeed contributes to tissue deterioration in human pathologies. Although robust data have been generated in various animal models of disease suggesting a widespread contribution of this form of death in tissue, the establishment of robust biomarkers specific for ferroptosis and/or the clinical 21

development of ferroptosis-specific modulators are imperative in the study of ferroptosis in human contexts.

Acknowledgements This work was in part supported by the FöFoLe program by LMU Munich (to T.S.), the Deutsche Forschungsgemeinschaft (DFG) CO 291/5-2, the Human Frontier Science Program (HFSP) RGP0013, the Helmholtz Validation Funds (Helmholtz Association of German Research Centres), the German Federal Ministry of Education and Research (BMBF) through the Joint Project Modelling ALS Disease In Vitro (MAIV, 01EK1611B) and the VIP+ program NEUROPROTEKT (03VP04260).

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Fig. 1. Metabolic pathways that contribute to ferroptosis. Metabolic pathways, such as cysteine and glutathione metabolism, iron handling, glutaminolysis, the mevalonate pathway as well as polyunsaturated fatty acid metabolism essentially impinge on the ferroptotic process. Well-established ferroptosis inducers and inhibitors are marked with red and green boxes, respectively (for a more comprehensive list of ferroptosis modulators see tables 1 and 2) (Abbreviations: ACSL4, acyl-CoA synthetase long-chain family member 4; AOA, aminooxyacetate;

BSO,

L-buthionine

sulfoximine;

CBS,

cystathionine-beta-synthase;

CGL,

cystathionine gamma-lyase; Cys, L-cysteine, Cys-Cys, cystine; FDFT1, farnesyl-diphosphate farnesyltransferase 1; FA, fatty acid; FA-CoA, fatty acid-CoA; Fe, iron; γ-GCS, γglutamylcysteine synthetase; GPX4, glutathione peroxidase 4; Gln, L-glutamine; GLS, glutaminase; GLS2, glutaminase 2; GSH, reduced glutathione; GSSG, di-glutathione; GSR, glutathione-disulfide reductase; Glu, L-glutamate; HMG-CoA, 3-hydroxy-3-methylglutarylcoenzyme A; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; HO-1, heme oxygenase 1; α31

KG, α-ketoglutarate; LPCAT3, lysophosphatidylcholine acyltransferase 3; LOX, lipoxygenase; NCOA4,

nuclear

receptor

coactivator

4;

PE,

phosphatidyl-ethanolamine;

PE-OOH,

phosphatidyl-ethanolamine hydroperoxide; PE-OH, phosphatidyl-ethanolamine hydroxide; Sec, selenocysteine; TA, transaminases; TCA cycle, tricarboxylic acid cycle; TFRC, transferrin receptor).

Table 1. Summary of frequently used ferroptosis inducers Compound

Mode of action

Erastin

System xc inhibitor

References

-

[2]

-

[2, 104]

Sulfasalazine

System xc inhibitor

Sorafenib

System xc inhibitor

-

[48, 105] -

Diaryl-isoxazoles

Non-competitive system xc inhibitors

[50]

BSO

γGCS inhibitor,

[12, 13, 24]

depletion of GSH RSL3

GPX4 inhibitor

[12]

FIN56

induces degradation of GPX4

[54]

ML162

GPX4 inhibitor

[12, 55]

ML210

GPX4 inhibitor

[12, 55]

Artemisinin derivatives

GPX4 inhibitors

[58-60]

FINO2

GPX4 inhibition and iron oxidation

[56, 57]

Siramesine/Lapatinib

Synergistic effect on ferroptosis induction through increase in intracellular iron concentration

[61]

Nanoparticle-based vehicles

Cargoes include polyunsaturated fatty acids, peroxides and iron and combinations thereof leading to iron overloading and peroxide-mediated cancer cell death

[31, 62-64]

Engineered human cyst(e)inase

Systemic depletion of L-cyst(e)ine

[97]

Withaferin

Triggers GPX4 degradation and HO-1 upregulation

[65]

Abbreviations: BSO: L-buthionine sulfoximine; RSL3: (1S,3R)-RSL-3; FIN: ferroptosis inducing compound; γGCS: γglutamylcysteine synthetase; GPX4: glutathione peroxidase 4; HO-1: heme oxygenase-1

32

Table 2. Summary of frequently used ferroptosis inhibitors Compound

Mode of action

Reference

Liproxstatin-1

Catalytic RTA, prevention of lipid peroxidation

[13]

Ferrostatin-1

Catalytic RTA, prevention of lipid peroxidation

[2]

Necrostatin-1

RIPK1 inhibitor; mechanism of ferroptosis inhibition unknown

[13, 66]

Vitamin E

Lipophilic antioxidant compensating GPX4 loss

[24]

Compound 968

Glutaminase inhibitor

[33]

Amino-oxyacetic acid

Glutaminase inhibitor

[33]

Rosiglitazone/Troglitazone/

ACSL4 inhibitor

[6]

Phenoxazin

Catalytic RTA, prevention of lipid peroxidation

[69]

Nitroxide-based compounds

Catalytic RTA, prevention of lipid peroxidation

[70, 71]

Allosteric GPX4 activators

Increases GPX4 activity

[72]

Pioglitazone

Abbreviations: ACSL4: acyl-CoA synthetase long-chain family member 4; GPX4: glutathione peroxidase 4; RIPK1: receptor interacting protein kinase 1; RTA: radical-trapping antioxidant

Highlights -

Ferroptosis may underlie several pathologies including neurodegeneration and cancer

- Ferroptosis presents a number of tractable nodes for pharmacological intervention - The degree of unsaturation of lipid bilayers determines ferroptosis sensitivity - The final steps of ferroptosis execution remain to be solved

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