Ferroptosis is a mode of cell death regulated by iron-dependent lipid peroxidation. Grow- ing evidence suggests ferroptosis induction as a novel anti-cancer modality that could potentially overcome therapy resistance in cancers. The molecular mechanisms involved in the regulation of ferroptosis are complex and highly dependent on context. Therefore, a comprehensive understanding of its execution and protection machinery in each tumor type is necessary for the implementation of this unique cell death mode to target individual cancers.
The first study of ferroptosis in AML was reported in 2015 by Yu et al., who demonstrated that the system xc- inhibitor erastin induces ferroptosis in HL-60 cells in vitro [112]. The cell death was a mixture of ferroptosis and necroptosis, as it was blocked not only by ferrostatin-1 and deferoxamine but also by necrostatin-1 and the knockdown of receptor-interacting protein 3 (RIP3). They also demonstrated the association of autophagy and p38 signaling, the inhibition of which attenuated the anti-leukemia effects of erastin. Later, erastin-induced ferroptosis in HL-60 cells was shown to depend on the cytoplasmic translocation of high mobility group box 1 (HMGB1) from the nucleus, the knockdown of which attenuated ferroptosis in vivo [113]. Recently, Pardieu et al. reported that the xCT gene SLC7A11 is a putative therapeutic vulnerability, especially in NPM1-mutated AML, and a poor prognostic factor [114]. They demonstrated that the system xc- inhibitor sulfasalazine suppressed GSH and induced oxidative stress–dependent cell death partly through ferroptosis, as indicated by its partial rescue by ferrostatin-1. The combination of sulfasalazine with daunorubicin and cytarabine was found to synergistically exert anti-leukemia effects in a patient-derived xenograft model as well as in primary AML cells. A clinical trial of sulfasalazine in combination with intensive chemotherapy in AML patients is being initiated (NCT05580861). K562 chronic myeloid leukemia cells were tested for cysteine depletion-induced ferroptosis [115]. The authors demonstrated that the concomitant inhibition of thioredoxin reductase 1 (TXNRD1) by auranofin leads to ferroptosis in these cells. Interestingly, K562 cells with imatinib resistance (K562/G0 cells) showed higher sensitivity to ferroptosis induced by cysteine depletion.
Hypomethylating agents are used as a standard of care for elderly patients with AML and for patients with myelodysplastic syndromes (MDS) [116]. One hypomethylating agent, decitabine, has been shown to induce ferroptosis and necroptosis in MDS-derived primary cells as well as cell lines [117]. It downregulates GSH and GPX4 while inducing ROS and cell death, which can be blocked by ferrostatin-1, necrostatin-1, and z-VAD-FMK. These data indicate that ferroptosis is at least partially involved in the effect of decitabine.
Wang et al. investigated the anti-leukemia effects observed for the combination of granulocyte-colony stimulating factor (G-CSF) and thrombopoietin (TPO) with low-dose chemotherapy investigated in a phase 2 trial in elderly AML patients [118]. They demonstrate that TPO induces ferroptosis through the suppression of E1A binding protein P300 (EP300)–mediated GPX4 transcription, whereas G-CSF induces pyroptosis through neutrophil elastase, which activates gasdermin D (GSDMD) in AML cells [119].
In 2020, Birsen et al. reported that, independent of its postulated effects against mutant TP53, APR-246 induces ferroptosis in AML cells during the early phases of drug exposure [120]. Ferroptosis induction by APR-246 was later confirmed in esophageal cancer cell lines, which showed an increase in GSH turnover and the suppression of mitochondrial iron-sulfur cluster biosynthesis through NFS1 [121].
Imetelstat is a first-in-class telomerase inhibitor currently under a phase 2 clinical trial against AML (NCT05583552), in addition to phase 3 clinical trial against myelodysplastic syndromes and myelofibrosis. A functional genetic screen combined with lipidomics revealed that imetelstat promotes PUFA-containing phospholipid synthesis in ACSL4– and FADS2–dependent manner, leading to AML cell ferroptosis both in vitro and in vivo [122]. However, it has not yet been shown whether the induced ferroptosis is telomerase-dependent or drug-specific off-target effects.
The tyrosine kinase inhibitor Neratinib was approved by the U.S. Food and Drug Administration in 2017 for the treatment of breast cancer, and a phase 1/2 clinical trial of this drug in pediatric patients with relapsed or refractory cancer including leukemia is ongoing (NCT02932280). Recently, neratinib has been shown to induce autophagy-dependent ferroptosis as well as G0/G1 arrest and apoptosis in HL-60 cells [123].
Many natural compounds have been studied for their ability to induce ferroptosis in cancer cells [124]. Dihydroartemisinin is a derivative of Artemisia annua, a plant native to China, and has anti-tumor effects in many types of cancers. This compound has effects against HL-60 cells in vitro and in vivo through the inhibition of mitochondrial oxidative phosphorylation and the activation of AMPK to induce ferritinophagy and ferroptosis [125]. Typhaneoside, a flavonoid extracted from Pollen typhae, has also been reported to induce apoptosis and ferroptosis associated with ferritinophagy in AML cells [126]. Hydnocarpin D is another flavonoid demonstrated to induce apoptosis and ferroptosis through autophagy in ALL cells [127]. Perillaldehyde, the main component of Ammodaucus leucotrichus, downregulates GSH and GPX4 to induce ferroptosis in HL-60 cells and primary AML cells [128]. Glycyrrhetinic acid is a bioactive compound of licorice, and its nanoparticles have been demonstrated to induce ferroptosis in AML cells through GPX4 downregulation [129]. These nanoparticles have synergistic anti-leukemia effects with ferumoxytol, an iron oxide nanoparticle, and programmed cell death ligand 1 (PD-L1) antibody treatment. The isothiocyanate sulforaphane (SFN) is derived from cruciferous vegetables and is known to exert anti-cancer activities. Greco et al. reported that low-dose SFN induces apoptosis whereas high-dose SFN induces ferroptosis in AML cell lines [130]. Interestingly, SFN activates the apoptosis pathway when ferroptosis is impaired, indicating that these two cell death modes could be convertible. Honokiol, a derivative of the magnolia tree, induces cell death in AML cell lines; this cell death was triggered by the upregulation of HO-1 and associated with lipid peroxidation and the alteration of ferroptosis pathway genes [131]. 4-Amino-2-trifluoromethyl-phenyl retinate (ATPR), a retinoid derivative synthesized from all-trans retinoic acid (ATRA), has been shown to activate NCOA4-dependent ferritinophagy and induce ferroptosis in AML in vitro and in vivo [132]. Poricoic acid A, the component of the mushroom Poria cocos, exerts anti-ALL effects in vitro and in vivo through mitochondrial dysfunction and activation of AMPK-mTOR autophagy pathway, leading to apoptosis and ferroptosis in T-ALL cells [133].
ALDH3a2, an enzyme that oxidizes long-chain aliphatic aldehydes, which are byproducts of lipid peroxidation, protects AML cells from oxidative stress. The inhibition of ALDH3a2 induces ferroptosis and exerts synergistic anti-leukemia effects with GPX4 inhibition or standard chemotherapy with cytarabine plus daunorubicin in vivo [134]. In addition, ALDH3a2 is selectively essential in leukemia progenitor cells but not in their normal counterparts, possibly owing to increased oxidative stress in leukemia cells.
Gold nanoparticles have anti-leukemia effects in part through ferroptosis induction. For example, GNR-CSP12 (gold nanorods loaded with chitosan and a 12-mer peptide) can induce ferroptosis through the suppression of global m6A RNA methylation and its combinatorial effects with tyrosine kinase inhibitors or PD-L1 checkpoint inhibitors [135].
Non-coding RNAs have also been reported to be involved in ferroptosis regulation in AML cells. A nuclear long non-coding RNA (lncRNA), LINC00618, is downregulated in AML, and its induction by vincristine treatment activates ferroptosis and apoptosis through SLC7A11 downregulation and BAX upregulation as well as caspase-3 cleavage [136]. The circular RNA circKDM4C, which is also downregulated in AML, sequesters the microRNA hsa-let-7b-5p and upregulates its downstream target, p53 [137]. The induction of circKDM4C causes AML cells to undergo ferroptosis, possibly through the downregulation of GPX4 and FTH1.
Recent discoveries of various non-apoptotic RCD modes, including ferroptosis, have expanded the potential modalities to induce death in cancer cells, especially in cancers resistant to conventional therapies targeting apoptosis mechanisms. A comprehensive understanding of the complex regulatory mechanisms of RCD and its involvement in cancer pathophysiology is necessary to develop RCD modes different from apoptosis into advanced cancer therapies.
While more than 10 years of extensive research has provided us with a large body of knowledge about ferroptosis, one needs to be cautious as most of the current mechanistic insights are based on in vitro models, whose environments differ from those in vivo. Components of the in vivo environment that likely affect ferroptosis regulation include oxygen; trace metals, including iron and selenium; and various metabolites such as amino acids and fatty acids. In addition, interactions with the tumor immune microenvironment (i.e., macrophages and immune cells) and even between cancer cells have been shown to affect cell vulnerability to ferroptosis, a complexity that is difficult to recapitulate in culture models. In fact, mesenchymal stem cells (MSCs) protect leukemia cells from oxidative stress through upregulation of GSH [138][139], while its significance in ferroptosis protection remains to be elucidated. Therefore, the in vitro findings must be validated in vivo to obtain a precise understanding of the mechanisms regulating ferroptosis, for which further development of specific and stable in vivo drugs to induce ferroptosis is of urgent need. The lack of definitive markers of ferroptosis is an obstacle in studying ferroptosis in vivo and in clinical settings in humans. Hence, direct evidence of ferroptosis in cancer patients treated with anti-cancer agents including ferroptosis inducers is lacking so far [140].
Since leukemia is characterized by increased oxidative stress and iron overload, one can speculate that leukemia cells are vulnerable to ferroptosis, suggesting a therapeutic potential. However, studies focusing on ferroptosis as a therapeutic modality for leukemia are limited, as discussed here. Given the physiological roles of ferroptosis, the therapeutic windows of ferroptosis induction should be carefully determined. A recent study demonstrated the potential vulnerability of hematopoietic stem cells to ferroptosis [141], warranting precise monitoring of normal hematopoiesis upon anti-cancer treatments involving ferroptosis. Taken together, implementing the concept of ferroptosis into leukemia therapeutics requires fine-tuning ferroptosis induction in normal versus malignant cells, which should be tested and established through in vivo studies and ultimately clinical trials.
In addition to understanding the underlying molecular mechanisms of ferroptosis, some of which may be specific to leukemia cells, identifying potential biomarkers that predict sensitivity or resistance to ferroptotic stimuli is critical for the development of this unique cell death mode as anti-leukemia therapy. Advancements in single-cell analysis technologies will help in the identification of potential vulnerabilities of leukemia stem/progenitor cells with specific genetic or molecular phenotypes that can be targeted for ferroptosis induction. Pursuing synthetic lethal interactions would be one promising strategy to efficiently target those populations while minimizing potential toxicity to normal cells [142]. Lastly, recent studies have revealed the interconnectivity and plasticity of different RCD pathways including ferroptosis that could compensate each other upon cell death stimuli (detailed review elsewhere [143]). Understanding the interactions as well as the molecular mechanisms of individual RCD pathways would facilitate the development of combination therapies to overcome resistance to various RCD mechanisms.