Epigenetic and post-translational regulatory networks of ferroptosis in the tumor immune microenvironment

Abstract

Ferroptosis is a non-apoptotic cell death mechanism characterized by iron-dependent membrane lipid peroxidation. The tumor immune microenvironment (TIME) significantly influences ferroptosis sensitivity in both cancer and immune cells. Recent years have witnessed major advances in understanding how multi-level regulatory mechanisms control ferroptosis in tumors, encompassing epigenetic modifications and post-translational protein regulation. Epigenetic mechanisms include DNA methylation, histone modifications, non-coding RNAs, and chromatin remodeling, while post-translational modifications (PTMs) involve phosphorylation, glycosylation, ubiquitination, acetylation, methylation, and lactylation of key ferroptosis proteins. This review examines the intricate relationship between the TIME, ferroptosis, and these dual regulatory networks. We focus particularly on how epigenetic processes and PTMs synergistically control ferroptosis mediators in the TIME, exploring how ubiquitination controls protein stability, and how metabolic modifications like lactylation link cellular metabolism to ferroptosis regulation. These multilevel interactions create a complex regulatory landscape that influences cancer progression, immune evasion, and therapeutic resistance. The crosstalk between epigenetic and post-translational regulation determines ferroptosis susceptibility across different cellular contexts within tumors, with distinct modification patterns observed in cancer cells versus immune infiltrates. Additionally, we discuss emerging therapeutic strategies that simultaneously target both epigenetic and post-translational regulation of ferroptosis, including combination approaches that modulate specific modification enzymes to enhance ferroptosis induction. Understanding these complex multilevel regulatory relationships provides valuable insights for developing novel precision cancer treatment approaches that leverage the therapeutic potential of ferroptosis modulation with potentially significant clinical impact.

Introduction

Epigenetic regulation of cell death mechanisms represents a critical frontier in cancer research, with particular significance for designing novel treatment strategies [1]. Ferroptosis has emerged as a promising therapeutic target due to its distinct iron-dependent, lipid peroxidation-driven mechanism [2]. Crucially, epigenetic modifications—including DNA methylation, histone alterations, non-coding RNA regulation, and chromatin remodeling—substantially influence ferroptosis sensitivity by modulating the expression of key ferroptotic regulators such as solute carrier family 7 member 11 (SLC7A11), GPX4, and ACSL4 [1, 3, 4].

The tumor immune microenvironment (TIME) constitutes a complex ecosystem where cancer cells interact with immune cells, stromal components, and secreted factors. Within this microenvironment, ferroptosis sensitivity is influenced by infiltrating immune cells through direct contact and secreted cytokines, particularly interferon gamma (IFNγ), which downregulates system xc⁻ components and enhances lipid peroxidation. Microenvironmental factors—including hypoxia and metabolite further modulate ferroptotic susceptibility of cancer cells [5]. Immune cells exhibit different sensitivities to ferroptosis, with CD8⁺ T cells being particularly vulnerable due to programmed cell death protein 1 (PD-1) signaling and impaired phospholipid homeostasis [6], while certain macrophages resist ferroptosis through Nrf2-dependent antioxidant systems [7].

Epigenetic programming at the interface of ferroptosis and immune function represents an underexplored paradigm with significant therapeutic implications. Targeting epigenetic regulation of ferroptosis within the TIME presents a promising approach for overcoming cancer therapy resistance [8–10]. This review examines the interplay between epigenetic mechanisms, ferroptotic pathways, and immune cell and stromal cell function within the TIME and explores how these epigenetically regulated ferroptotic events reshape tumor-immune interactions and influence therapeutic outcomes.

Molecular mechanisms of ferroptosis

Ferroptosis differs fundamentally from other cell death forms. This non-apoptotic process relies on iron-dependent accumulation of lipid peroxides, leading to oxidative damage of cellular membranes [2]. Ferroptosis does not involve chromatin condensation, nuclear fragmentation, or membrane blebbing (Fig. 1).

Fig. 1.

Morphological differences between normal and ferroptotic cells

Key regulators and pathways

The biochemical signature of ferroptosis includes redox imbalance and iron accumulation. Elevated lipid peroxidation, particularly of polyunsaturated fatty acid (PUFA) phospholipids, marks this process distinctively [2] (Fig. 2).

Fig. 2.

Core molecular machinery of ferroptosis. This figure illustrates the opposing mechanisms regulating ferroptosis. The left panel shows pro-ferroptotic pathways, where transferrin receptor-mediated iron uptake and PUFA lipid peroxidation via ACSL4 promote ferroptosis. The right panel depicts anti-ferroptotic defenses, including: cystine uptake through system Xc⁻ for GSH synthesis, GPX4-mediated peroxide reduction, FSP1-CoQ10 radical trapping, estrogen receptor (ER)/androgen receptor (AR)-regulated MUFA synthesis, and 7-DHC/DHODH/GPX4 protection of mitochondria

Iron metabolism

Iron catalyzes Fenton reactions that generate reactive oxygen species (ROS), promoting lipid peroxidation. Several proteins regulate iron homeostasis in cells, including transferrin and transferrin receptor (which control iron uptake) [11], ferritin (which handles iron storage) [12], and ferroportin (which manages iron export) [13]. STEAP3 (six-transmembrane epithelial antigen of prostate 3) functions as a metalloreductase in endosomes, reducing Fe³⁺ to Fe²⁺ and facilitating iron availability for cellular processes [14]. Alterations in these proteins affect cellular iron levels and ferroptosis sensitivity. Iron-containing enzymes like lipoxygenases (LOXs) directly contribute to lipid peroxidation during ferroptosis [15]. Beyond therapeutic contexts, environmental toxicants and metabolic stressors can trigger ferroptosis through disruption of cellular iron homeostasis and antioxidant defenses [16].

Lipid peroxidation

The peroxidation of PUFAs in cell membranes is central to ferroptosis execution. Acetyl-CoA carboxylase (ACC) catalyzes the carboxylation of acetyl-CoA to malonyl-CoA (Mal-CoA), which serves as the primary building block for de novo fatty acid synthesis [17]. Acyl-CoA synthetase long-chain family member 4 (ACSL4) enriches membranes with PUFAs by facilitating their incorporation [18]. Lysophosphatidylcholine acyltransferase 3 (LPCAT3) further contributes by incorporating PUFAs into phospholipids. Peroxisomes also play a role in lipid metabolism by processing PUFA-CoA to PUFA-PL through β-oxidation and remodeling pathways [19]. Lipoxygenases catalyze the dioxygenation of PUFAs to generate lipid hydroperoxides [20]. These peroxidized lipids accumulate and eventually cause membrane damage and cell death.

Antioxidant systems

Cells possess multiple defense mechanisms against ferroptosis to maintain redox homeostasis. The system xc⁻ cystine/glutamate antiporter plays a crucial role in this defense network. This system consists of SLC7A11 and SLC3A2 subunits and functions primarily to import cystine needed for glutathione (GSH) synthesis [21]. Recent research has revealed that SLC7A11 also functions as an unconventional H⁺ transporter in lysosomes, as demonstrated in HeLa cells and lung cancer stem cells (A549-derived CD133⁺ cells), suggesting additional protective roles beyond cystine import [22]. GSH subsequently acts as a cofactor for glutathione peroxidase 4 (GPX4), which reduces lipid hydroperoxides and thereby prevents ferroptosis [23]. Inhibition or depletion of system xc⁻, GSH, or GPX4 renders cells highly vulnerable to ferroptosis.

Another protective pathway involves ferroptosis suppressor protein 1 (FSP1), previously known as AIFM2 [24, 25]. This protein reduces coenzyme Q10 to ubiquinol, which functions as a lipophilic radical-trapping antioxidant independently of the GSH-GPX4 axis [24]. Notably, ALDH7A1 has been identified as a critical regulator that protects against ferroptosis by generating membrane NADH and regulating FSP1 activity, as demonstrated in H9c2 rat cardiomyoblasts and primary neonatal rat cardiomyocytes (NRCMs) under myocardial ischemia–reperfusion conditions, thus providing an additional layer of defense [26]. Nuclear factor erythroid 2-related factor 2 (NRF2) also contributes significantly to ferroptosis prevention by orchestrating the transcription of various antioxidant genes involved in GSH synthesis and iron metabolism [27, 28].

Membrane lipid composition plays a decisive role in ferroptosis susceptibility. Stearoyl-CoA desaturase 1 (SCD1) catalyzes the conversion of saturated fatty acids to monounsaturated fatty acids (MUFAs), which are more resistant to peroxidation, thereby conferring ferroptosis protection [29]. ACSL3 preferentially activates MUFAs for phospholipid incorporation, counterbalancing ACSL4-mediated PUFA enrichment [30]. Membrane-bound O-acyltransferases MBOAT1 and MBOAT2 facilitate lipid remodeling by transferring fatty acids to lysophospholipids, modulating membrane PUFA content and ferroptosis sensitivity [31]. Additionally, calcium-independent phospholipase A2β (iPLA2β) selectively removes oxidized PUFA tails from phospholipids, providing a repair mechanism against lipid peroxidation damage [32]. Phospholipids containing two polyunsaturated fatty acyl tails have been demonstrated to significantly promote ferroptosis by serving as preferred substrates for lipid peroxidation [33]. Conversely, 7-dehydrocholesterol functions as an endogenous suppressor of ferroptosis by dictating membrane susceptibility to peroxidation, establishing another important regulatory mechanism for ferroptosis sensitivity [34, 35]. GTP cyclohydrolase 1 (GCH1) catalyzes the rate-limiting step in tetrahydrobiopterin (BH4) biosynthesis, and the GCH1-BH4 axis functions as a potent radical-trapping antioxidant system that protects against ferroptosis independently of GPX4 [36].

GPX4 and dihydroorotate dehydrogenase (DHODH) constitute parallel defense mechanisms against ferroptosis in mitochondria [37]. GPX4 utilizes glutathione to reduce lipid hydroperoxides to non-toxic lipid alcohols, while DHODH reduces ubiquinone to ubiquinol during pyrimidine biosynthesis [24]. Interestingly, recent studies have identified ferroptosis surveillance mechanisms that function independently of GPX4 and are differentially regulated by sex hormones, explaining observed gender differences in ferroptosis susceptibility [38]. GPX4 and DHODH operate in parallel but distinct subcellular compartments with unique CoQ pools. GPX4 functions in both cytoplasm and mitochondria, utilizing glutathione to reduce lipid hydroperoxides across cellular membranes. DHODH specifically localizes to the mitochondrial inner membrane, where it reduces ubiquinone (CoQ₁₀) to ubiquinol during pyrimidine biosynthesis. The mitochondrial CoQ pool is physically separated from the plasma membrane CoQ pool, creating compartment-specific antioxidant systems. Mitochondrial peroxidation becomes rate-limiting when: (1) Mitochondrial GPX4 is depleted while cytoplasmic GPX4 remains active, (2) DHODH is inhibited while FSP1-mediated plasma membrane protection persists, (3) Mitochondria-specific stressors (rotenone, antimycin) overwhelm local defenses. The topology shows GPX4 facing both sides of membranes, DHODH embedded in inner mitochondrial membrane, and FSP1 associated with plasma membrane CoQ pools. Age-related changes in iron homeostasis significantly impact ferroptosis susceptibility, with research demonstrating that aging limits tumorigenesis through reprogramming of iron homeostasis pathways [39]. The resulting ubiquinol functions as a radical-trapping antioxidant that effectively neutralizes lipid peroxides in the mitochondrial inner membrane. These complementary systems prevent the accumulation of toxic lipid peroxidation products that would otherwise trigger ferroptotic cell death. Notably, inhibition of both pathways synergistically induces extensive mitochondrial lipid peroxidation and ferroptosis, representing a potential therapeutic strategy against cancer cells.

Detection methods for ferroptosis

Accurate detection and measurement of ferroptosis are crucial for improving our understanding of this cell death mechanism and its therapeutic targets. The detection of ferroptosis is shown in Box 1.

Box 1 Detection methods for ferroptosis.

1. MORPHOLOGICAL & ULTRASTRUCTURAL ANALYSIS

   • □ Transmission electron microscopy (TEM): mitochondrial shrinkage, increased membrane density, reduced/absent cristae, outer membrane rupture [2].
   • □ Absence of apoptosis markers: no chromatin condensation, nuclear fragmentation, or membrane blebbing.
2. BIOCHEMICAL DETECTION

   • 2.1
     Lipid Peroxidation Assays (Essential)

     • □ BODIPY-C11 (BODIPY 581/591 C11): ratiometric probe for oxidized/reduced lipids by flow cytometry or microscopy [40].
     • □ MDA and 4-HNE: measure aldehydic products by HPLC, immunostaining, or ELISA [41].
     • □ LC–MS/MS lipidomics: identify and quantify oxidized PUFA-phospholipids (PE-AA, PE-AdA, PC-AA) [42].
   • 2.2
     Iron Detection & Quantification (Required)

     • □ Perl's Prussian blue staining: histological detection of ferric iron [43].
     • □ ICP-MS or atomic absorption spectroscopy: quantitative measurement of total cellular iron.
     • □ Calcein-AM or RhoNox-1: fluorescent probes for labile iron pool in live cells.
   • 2.3
     Glutathione & Antioxidant System Analysis

     • □ GSH/GSSG ratio by HPLC: indicator of cellular redox status [44].
     • □ GPX4 activity assay: using tert-butyl hydroperoxide as substrate.
     • □ GPX4 protein levels: Western blot and immunofluorescence for levels and localization [45].
   • 2.4
     Mass Spectrometry-Based Approaches (Advanced)

     • □ Targeted metabolomics: simultaneous quantification of glutathione, cysteine, iron metabolites, lipid peroxides [46].
     • □ Untargeted lipidomics: discovery of novel oxidized lipid species.
3. MOLECULAR & GENETIC DETECTION

   • 3.1
     Gene Expression Analysis

     • □ qPCR or RNA-seq: ferroptosis gene signature (SLC7A11, GPX4, ACSL4, LPCAT3, ALOX15).
     • □ Expected pattern: downregulation of protective genes (GPX4, SLC7A11) or upregulation of pro-ferroptotic genes (ACSL4).
   • 3.2
     Protein Analysis

     • □ Western blot: GPX4, SLC7A11, FSP1, ACSL4, transferrin receptor, ferritin.
     • □ Immunohistochemistry/immunofluorescence: spatial distribution of ferroptosis proteins in tissue.
   • 3.2
     Genetic Epistasis Validation (Definitive)

     • □ GPX4 knockout/knockdown → sensitizes to ferroptosis (rescued by ferroptosis inhibitors).
     • □ SLC7A11 knockout/knockdown → sensitizes to ferroptosis (glutathione depletion).
     • □ ACSL4 knockout/knockdown → protects from ferroptosis (reduced PUFA incorporation).
     • □ Epistatic relationship: ferroptosis inhibitors rescue genetic sensitization.
4. CHEMICAL RESCUE LOGIC (Critical for Specificity)

   • □ Ferroptosis inhibitors MUST rescue (> 70% viability): Ferrostatin-1 (5–10 μM), Liproxstatin-1 (0.5–1 μM), Deferoxamine (50–100 μM), Vitamin E (50–200 μM).
   • □ Non-ferroptotic inhibitors MUST NOT rescue: z-VAD-fmk (apoptosis), Necrostatin-1 (necroptosis), Ac-YVAD-cmk (pyroptosis).
   • □ Parallel testing required: selective rescue by ferroptosis inhibitors is definitive proof.
5. IN VIVO IMAGING (Optional but Recommended)

   • □ MRI: T2-weighted for iron accumulation, CEST-MRI for glutathione levels [47].
   • □ PET: iron-specific tracers (under development for clinical settings).

• Minimum Requirements for Confident Ferroptosis Assignment:

  • Fulfill ≥ 4 criteria groups with positive results.
  • Chemical rescue + lipidomics are most definitive (when TEM unavailable).
  • Genetic epistasis provides definitive validation.
  • For mixed phenotypes: use combinatorial inhibitors + pathway-specific markers.

Ferroptosis inhibitors and inducers

Ferroptosis inhibitors

Ferroptosis inhibitors have shown therapeutic potential in neurodegenerative diseases, ischemia–reperfusion injury, and certain cancer contexts where ferroptosis contributes to pathological cell death (Table 1).

Table 1.

Comprehensive overview of ferroptosis inhibitors

Compound name	Target genes/proteins	Mechanism of action	Therapeutic effect	Application	References
Ferrostatin-1 (Fer-1)	Lipid peroxyl radicals	Radical-trapping antioxidant, membrane stabilization	Prevents lipid peroxidation chain reactions	Neurodegeneration, AKI	[48, 49]
Liproxstatin-1 (Lip-1)	Membrane lipids	Lipid peroxyl radical scavenging	Enhanced stability, neuroprotection	CNS disorders, kidney injury	[50, 51]
α-Tocopherol (Vitamin E)	PUFA-containing phospholipids	Lipophilic antioxidant, membrane protection	Prevents PUFA peroxidation	Cardiovascular, neurological	[54, 55]
Deferoxamine (DFO)	Iron (Fe2+/Fe3+)	Iron chelation, prevents Fenton reactions	Reduces catalytic iron availability	Iron overload, ischemia	[52, 53]
Coenzyme Q10	Mitochondrial respiratory chain	Electron transport, antioxidant defense	Maintains membrane integrity	Age-related disorders	[24, 25]
N-Acetylcysteine (NAC)	GSH, GPX4	Glutathione precursor, cysteine donor	Enhances antioxidant capacity	Respiratory, hepatic disorders	[56]
Probucol	Lipid peroxidation cascade	Phenolic antioxidant, membrane stabilizer	Dual cardiovascular/neuroprotection	Atherosclerosis, neurodegeneration	[57]
Idebenone	Mitochondrial complex I	Synthetic CoQ10 analog, enhanced bioavailability	Crosses blood–brain barrier	Neurodegenerative diseases	[24]

Classical ferroptosis inhibitors

Ferrostatin-1 (Fer-1) represents the prototypical ferroptosis inhibitor, functioning as a radical-trapping antioxidant that prevents lipid peroxidation chain reactions [48, 49].

Liproxstatin-1 (Lip-1) emerged as another potent ferroptosis inhibitor with improved stability and bioavailability compared to Fer-1. Lip-1 exhibits superior neuroprotective effects and has been extensively studied in models of neurodegeneration and acute kidney injury [50, 51].

Deferoxamine (DFO) represents a clinically approved iron chelator that prevents ferroptosis by sequestering catalytically active iron species [52, 53].

Natural antioxidant compounds

α-Tocopherol (Vitamin E) serves as the primary lipophilic antioxidant in cellular membranes, directly neutralizing lipid peroxyl radicals and preventing propagation of lipid peroxidation chains [54, 55].

Coenzyme Q10 (CoQ10) functions as both an electron carrier in mitochondrial respiration and a potent lipophilic antioxidant [24, 25].

N-Acetylcysteine (NAC) serves as a precursor to glutathione synthesis, indirectly supporting the GPX4-mediated antioxidant system [56].

Clinically approved agents

Probucol, originally developed as a cholesterol-lowering agent, demonstrates potent ferroptosis inhibition through its phenolic antioxidant properties [57].

Idebenone, a synthetic analog of CoQ10, exhibits enhanced bioavailability and crosses the blood–brain barrier more effectively than its natural counterpart [24].

Ferroptosis inducers

Ferroptosis inducers toward cancer cells makes them attractive therapeutic agents for overcoming treatment resistance (Table 2) [58].

Table 2.

Comprehensive overview of ferroptosis inducers

Compound	Target genes/proteins	Molecular mechanism	Specific ferroptosis effect	Therapeutic indication	Cancer types	References
Erastin	SLC7A11 (System Xc⁻)	Inhibits cystine-glutamate antiporter; depletes intracellular cysteine	Depletes GSH; increases lipid ROS accumulation; triggers mitochondrial dysfunction	Phase I/II trials ongoing; enhances chemotherapy sensitivity	TNBC, Glioblastoma, Pancreatic cancer	[2]
RSL3	GPX4 (Glutathione peroxidase 4)	Covalently binds to GPX4 selenocysteine active site; abolishes enzymatic activity	Blocks lipid peroxide reduction; accumulates lipid ROS to lethal levels	Preclinical models; synergizes with immunotherapy	RCC, Prostate cancer, Melanoma	[59]
ML162	GPX4	Allosteric GPX4 inhibitor; disrupts homodimer formation	Impairs GPX4 enzymatic function; accelerates PUFA-PL oxidation	In vitro validation; potential combination therapy agent	NSCLC, HCC, Ovarian cancer	[59]
Artesunate	Iron homeostasis proteins (TfR1, Ferritin)	Induces intracellular labile iron pool expansion via iron metabolism dysregulation	Enhances Fenton reaction; generates hydroxyl radicals; oxidizes PUFA-PLs	FDA-approved for malaria; repurposing trials in oncology	Leukemia, Colorectal cancer, Lung cancer	[60]
Withaferin A	KEAP1-Nrf2 pathway	Activates KEAP1; suppresses Nrf2-dependent antioxidant response	Depletes GSH and NADPH; sensitizes cells to oxidative stress	Natural compound under preclinical evaluation	Breast cancer, Ovarian cancer	[61]
Dihydroartemisinin (DHA)	Iron metabolism	Cleaves endoperoxide bridge via ferrous iron (Fe2⁺); generates free radicals	Increases lipid peroxidation; disrupts redox homeostasis	Antimalarial drug; phase II trials for solid tumors	HCC, Gastric cancer, Pancreatic cancer	[62]
Sorafenib	System Xc⁻, GPX4	Dual inhibition: blocks cystine uptake and suppresses GPX4 expression	Depletes GSH; impairs lipid ROS detoxification; induces mitochondrial damage	FDA-approved for HCC and RCC; ferroptosis contributes to efficacy	HCC, RCC, Thyroid cancer	[63]
Sulfasalazine	SLC7A11	Competitive inhibitor of System Xc⁻; blocks cystine import	Reduces cysteine availability; depletes GSH; accumulates lipid peroxides	FDA-approved for inflammatory diseases; repurposing for cancer therapy	Glioblastoma, Lymphoma, Lung cancer	[64]

Synthetic ferroptosis inducers

Erastin represents the prototypical ferroptosis inducer. Erastin functions by inhibiting system xc⁻, specifically targeting the SLC7A11 subunit, thereby depleting cellular cystine uptake and subsequent glutathione synthesis [2].

RSL3 (RAS-selective lethal 3) directly targets GPX4, the central regulator of ferroptosis defense.

ML162 functions through a dual mechanism involving both system xc⁻ inhibition and direct induction of lipid peroxidation [59].

Natural ferroptosis inducers

Artesunate, a semi-synthetic derivative of artemisinin, induces ferroptosis through iron-dependent mechanisms involving endoperoxide bridge activation [60].

Withaferin A, a natural steroidal lactone, induces ferroptosis by targeting multiple pathways including NRF2 suppression and direct GPX4 inhibition [61].

Dihydroartemisinin (DHA) represents another artemisinin derivative with potent ferroptosis-inducing properties [62].

Clinical development and applications

Sorafenib, an FDA-approved multi-kinase inhibitor, induces ferroptosis in hepatocellular carcinoma cells through system xc⁻ inhibition and iron accumulation [63].

Sulfasalazine, originally developed for inflammatory bowel disease, inhibits system xc⁻ and induces ferroptosis in various cancer cell lines [64].

Epigenetic mechanisms

Epigenetic regulation represents a sophisticated and dynamic mechanism for modulating gene expression without altering DNA sequences, playing crucial roles in cellular homeostasis, development, and disease pathogenesis. This regulation can be categorized into five primary classifications: DNA methylation, histone modifications, non-coding RNAs, chromatin remodeling, and RNA modifications (Fig. 3).

Fig. 3.

Overview of diverse epigenetic mechanisms controlling gene expression. The figure illustrates five major epigenetic regulatory pathways: DNA methylation (top panel) mediated by DNMT3A/3B with TET-dependent demethylation affecting CpG islands and gene transcription; histone modifications (second panel) showing various PTMs of histone tails with opposing effects of EZH2 (H3K27Me3, repressive) and LSD1 (H3K9Me2, activating); non-coding RNA (third panel) demonstrating how lncRNAs and miRNAs regulate gene expression through chromatin modification, transcription factor recruitment, and mRNA degradation; chromatin remodeling (fourth panel) showing changes in nucleosome positioning and histone modifications (H3K20Me3 vs. H4K16Ac) that alter chromatin accessibility; and RNA modifications (bottom panel) depicting chemical modifications (m5C, ac4C, m6A, Ψ, and A-I editing) distributed across the mRNA structure that influence RNA processing and function. These mechanisms collectively form a complex regulatory network that fine-tunes gene expression patterns

DNA methylation

DNA methylation involves adding methyl groups to cytosine residues, primarily at CpG dinucleotides [65]. DNA methyltransferases (DNMTs) orchestrate this process, with DNMT1 maintaining existing methylation patterns during replication and DNMT3a/3b establishing de novo methylation [66]. Ten-eleven translocation (TET) enzymes mediate active demethylation by oxidizing 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) and subsequent intermediates [67, 68]. In the TIME, DNA methylation patterns can directly influence ferroptosis sensitivity by regulating the expression of iron metabolism genes. However, the role of DNMT1 in ferroptosis regulation appears highly context-dependent, with conflicting results reported across different cancer types. In gallbladder cancer, DNMT1-mediated hypermethylation of the RUNX3 promoter suppresses RUNX3 expression, subsequently reducing ING1-mediated inhibition of SLC7A11, thereby protecting cells from ferroptosis [69]. Conversely, in hepatocellular carcinoma, DNMT1 demonstrates anti-ferroptotic functions by maintaining GPX4 promoter demethylation, thereby sustaining antioxidant capacity [70]. This apparent contradiction may be due to TP53 mutations.

Histone modifications

Histone modifications include various post-translational alterations to histone proteins that influence chromatin structure and gene accessibility, including acetylation, methylation, succinylation, citrullination, lactylation, and crotonylation [71–76]. In oncogenic contexts, enhancer of zeste homolog 2 (EZH2) facilitates H3K27 trimethylation, silencing tumor suppressor genes [77, 78]. LSD1 (Lysine-Specific Demethylase 1) exhibits dual functionality by removing methyl groups from both H3K4 and H3K9 [79, 80]. Within the TIME, histone modifications can regulate ferroptosis-related genes, particularly those involved in glutathione metabolism and lipid peroxidation. Beyond histones, lactylation also modifies non-histone proteins, as discussed in “Hypoxia-induced PTMs” section, creating coordinated regulation of ferroptosis.

Non-coding RNAs

Non-coding RNAs (ncRNAs) regulate gene expression through diverse epigenetic mechanisms [81]. The non-coding RNAs involved in epigenetic regulation mainly include small ncRNAs (miRNAs, siRNAs) and long non-coding RNAs (lncRNAs) that influence chromatin structure and gene activity [82]. LncRNAs influence chromatin structure and gene activity by recruiting chromatin-modifying complexes, binding to transcription factors, and interacting with mRNAs [81, 83, 84]. MicroRNAs typically function post-transcriptionally by binding to complementary sequences in target mRNAs [85, 86]. Within the TIME, specific non-coding RNAs can regulate ferroptotic pathways by modulating iron transporters, lipid metabolism enzymes, and antioxidant systems [87–89].

Chromatin remodeling

Chromatin remodeling involves ATP-dependent alteration of nucleosome positioning, composition, and density to regulate DNA accessibility and transcription [90, 91]. Histone deacetylases (HDACs) regulate acetylation status [92]. Chromatin remodeling factors can impact ferroptosis sensitivity by controlling the accessibility of genes involved in iron homeostasis and lipid peroxidation [92].

RNA modifications

N6-methyladenosine (m6A) is the most prevalent internal mRNA modification, influencing tumor biology through its dynamic regulation by “writers” (methyltransferases), “erasers” (demethylases), and “readers” (proteins that recognize modified RNA). Dysregulation of m6A regulatory components has been linked to oncogenic transformation through altered stability and translation of critical oncogenes and tumor suppressors. Mechanistically, m6A writers (METTL3, METTL14) operate at the epigenetic level—controlling which transcripts are marked—while m6A readers (YTHDF2, YTHDC1) function at the post-transcriptional level—determining marked transcript fate. This dual-layered regulation enables coordinated control of ferroptosis-related gene expression through integration of chromatin states and cellular signaling.

Epigenetic drugs regulating ferroptosis

The therapeutic potential of epigenetic drugs in modulating ferroptosis has emerged as a promising avenue for cancer treatment. These compounds can restore expression of silenced ferroptosis-promoting genes or suppress overexpressed ferroptosis-resistance genes, thereby sensitizing cancer cells to ferroptotic cell death [93, 94] (Table 3).

Table 3.

Comprehensive overview of epigenetic drugs that modulate ferroptosis pathways

Drug name	Target gene/pathway	Molecular function	Epigenetic mechanism	Ferroptosis outcome	Specific ferroptosis mechanism	Cancer type	References
5-Azacytidine	GPX4, SLC7A11	DNA methyltransferase inhibitor (DNMTi)	Promoter demethylation (5mC ↓); reactivates silenced ferroptosis-suppressor genes	Depletes GSH; increases lipid peroxidation	GPX4↓ → impaired lipid ROS detoxification; SLC7A11↓ → reduced cystine uptake	MDS, AML	[94, 95]
Decitabine	SLC7A11, FSP1	DNMTi	Global DNA hypomethylation (5mC ↓); reactivates tumor suppressor genes regulating ferroptosis	Impairs cystine uptake; reduces CoQ10 regeneration	SLC7A11↓ → GSH depletion; FSP1↓ → compromised lipid radical scavenging	AML, CML	[94, 95]
Vorinostat (SAHA)	Nrf2, GPX4	HDAC inhibitor (pan-HDACi)	Histone hyperacetylation (H3K27ac ↑); alters chromatin accessibility at ferroptosis gene loci	Reduces GPX4 activity; increases lipid peroxidation	Nrf2↓ → impaired antioxidant response; GPX4↓ → lipid ROS accumulation	CTCL, Glioblastoma	[96]
Panobinostat	ACSL4, LPCAT3	Pan-HDACi	H3K9ac ↑ at ACSL4 promoter; enhances PUFA-PL synthesis gene transcription	Enhances PUFA-PL synthesis; sensitizes to lipid peroxidation	ACSL4↑, LPCAT3↑ → increased PUFA-PL substrate pool → heightened ferroptosis susceptibility	Multiple myeloma, NSCLC	[94, 95]
GSK126	ACSL4, ALOX15	EZH2 inhibitor	Reduces H3K27me3 at ACSL4/ALOX15 loci; derepresses ferroptosis-promoting genes	Enhances PUFA-PL synthesis; increases lipid peroxidation	ACSL4↑ → PUFA-CoA activation; ALOX15↑ → enzymatic lipid peroxidation	Lymphoma, Melanoma	[10, 97]
EPZ-6438 (Tazemetostat)	GPX4, FSP1	Selective EZH2 inhibitor	H3K27me3 ↓ at ferroptosis-suppressor loci; remodels chromatin to sensitize cells	Reduces GPX4 activity; impairs CoQ10 regeneration	GPX4↓, FSP1↓ → compromised lipid ROS defense systems	INI1-deficient tumors, Epithelioid sarcoma	[10, 97]

Mechanistic classifications

DNA methyltransferase inhibitors

DNA methyltransferase inhibitors represent a major class of epigenetic drugs that can restore expression of ferroptosis-promoting genes silenced by hypermethylation. 5-Azacytidine and decitabine are FDA-approved hypomethylating agents that have demonstrated efficacy in sensitizing cancer cells to ferroptosis by reactivating silenced tumor suppressor genes involved in ferroptotic pathways [94, 95].

Histone deacetylase inhibitors

HDAC inhibitors can modulate ferroptosis sensitivity through multiple mechanisms, including direct effects on ferroptosis gene expression and indirect effects on cellular metabolism and oxidative stress responses. Vorinostat (SAHA) has been shown to enhance ferroptosis by promoting acetylation of histones at ferroptosis-related gene loci [96].

Histone methyltransferase inhibitors

EZH2 inhibitors such as tazemetostat and GSK126 can promote ferroptosis by reducing repressive H3K27me3 marks at ferroptosis-promoting gene promoters. These compounds have shown particular promise in cancers with EZH2 overexpression [10, 97].

PTMs

PTMs represent a fundamental regulatory mechanism that modulates protein function, localization, and stability without altering the genetic code. These reversible modifications enable rapid cellular responses to environmental changes and play essential roles in cellular homeostasis and disease progression. The primary PTMs include phosphorylation, glycosylation, ubiquitination, acetylation, methylation, and lactylation.

Phosphorylation

Phosphorylation involves the addition of phosphate groups to serine, threonine, or tyrosine residues, catalyzed by protein kinases and reversed by phosphatases [98]. This modification serves as a molecular switch that rapidly activates or deactivates protein function and modulates protein interactions. In ferroptosis regulation, phosphorylation controls key enzymes involved in iron metabolism and antioxidant defense systems [99].

Glycosylation

Glycosylation encompasses the attachment of carbohydrate moieties to proteins through N-linked or O-linked modifications [100]. This modification influences protein folding, stability, and trafficking [101]. In ferroptosis regulation, glycosylation modulates iron transporters and antioxidant enzymes that control cellular iron homeostasis [102].

Ubiquitination

Ubiquitination involves the attachment of ubiquitin to lysine residues through E1, E2, and E3 enzymes [103]. Different linkage patterns determine protein fate: K48-linked chains target proteins for degradation while K63-linked chains facilitate signaling [104]. Within ferroptosis pathways, ubiquitination regulates the turnover of iron storage proteins and antioxidant enzymes [105]. A major unresolved question in the field concerns the apparently contradictory effects of GPX4 ubiquitination across different cellular contexts. While K48-linked polyubiquitination typically promotes proteasomal degradation of GPX4 and sensitizes cells to ferroptosis [106], several studies have reported that ubiquitination can paradoxically stabilize GPX4 under certain stress conditions [107]. This discrepancy likely reflects the diversity of ubiquitin chain topologies and their distinct functional outcomes.

Acetylation

Acetylation involves the transfer of acetyl groups to lysine residues, catalyzed by acetyltransferases and reversed by deacetylases [108]. This modification regulates chromatin accessibility and protein function [109]. In ferroptosis regulation, acetylation modulates transcription factors controlling antioxidant gene expression and glutathione synthesis enzymes.

Methylation

Methylation involves the addition of methyl groups to lysine or arginine residues by specific methyltransferases [110]. This modification regulates gene transcription and signal transduction [111]. Within ferroptosis networks, methylation influences iron regulatory proteins and lipid metabolism regulators [112].

Lactylation

Lactylation involves the attachment of lactyl groups derived from lactate to lysine residues [113]. This modification integrates cellular metabolic status with gene expression regulation, particularly in cancer cells with elevated lactate production [114]. In ferroptosis regulation, lactylation links altered metabolism to ferroptosis regulator modulation in the tumor immune microenvironment [115]. Together with histone lactylation “Histone modifications” section), protein lactylation exemplifies how metabolic intermediates bridge epigenetic and PTM regulation “Crosstalk between epigenetic and post-translational regulatory networks in ferroptosis” section

Epigenetic regulation of ferroptosis in the tumor immune microenvironment

Epigenetic modulation of ferroptosis regulators in cancer cells

Epigenetic modifications significantly impact ferroptosis regulation in cancer cells [116] (Fig. 4). DNA methylation patterns of key ferroptosis regulators alter cancer cell fate. For instance, EZH2 epigenetically suppresses atonal bHLH transcription factor 8 (ATOH8) through DNA methylation, protecting cancer cells from ferroptosis by preventing ATOH8-mediated transcriptional repression of stearoyl-CoA desaturase (SCD) [117]. Clear cell ovarian carcinoma exhibits ferroptosis resistance through hypermethylation of the estrogen receptor 1 (ESR1) promoter, leading to dysregulated iron metabolism and enhanced cysteine/glutathione synthesis [118]. DNA methylation-mediated silencing of runt-related transcription factor 3 (RUNX3) contributes to gallbladder cancer progression by inhibiting ferroptotic cell death [69].

Fig. 4.

Epigenetic modulation of ferroptosis regulators in cancer cells. Regulation of ferroptosis in cancer cells through five distinct epigenetic mechanisms: A DNA methylation, B histone modifications, C non-coding RNAs, D chromatin remodeling, and E m6A RNA modifications. Each mechanism controls specific molecular pathways that collectively determine cancer cell susceptibility to ferroptotic cell death within the tumor immune microenvironment

Histone modifications also critically regulate ferroptosis susceptibility. EZH2, frequently overexpressed in cancers, has been implicated in ferroptosis resistance through H3K27me3-mediated silencing of pro-ferroptotic genes [10, 119, 120]. Histone deacetylase inhibitors (HDACis) overcome ferroptosis resistance in colorectal cancer by enhancing H3K27 acetylation at fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5) loci, activating these m6A demethylases and promoting FSP1 mRNA degradation [121]. Moreover, LSD1 inhibition induces ferroptosis by increasing the repressive histone mark H3K9me2 at the activating transcription factor 4 (ATF4) promoter, leading to decreased transcription of ATF4 and reduced expression of SLC7A11 [80].

Non-coding RNAs influence ferroptosis through diverse molecular mechanisms. LncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) functions as a competitive endogenous RNA to sequester miR-362-3p, increasing myo-inositol oxygenase (MIOX) expression, promoting ROS accumulation, and depleting NADPH and GSH levels [122]. LncRNA DACT3-AS1 is secreted by cancer-associated fibroblasts and suppresses gastric cancer progression by targeting the miR-181a-5p/SIRT1 axis to enhance SIRT1-mediated ferroptosis [123]. Hypoxia-responsive lncRNA URB1-AS1 confers sorafenib resistance through induction of ferritin phase separation, iron sequestration, and ferroptosis suppression [12].

Chromatin remodeling impacts ferroptosis sensitivity in cancer cells. The DNA methylation modifier LSH forms a complex with WD repeat domain 76 (WDR76) to epigenetically activate transcription of metabolic genes including GLUT1, SCD1, and FADS2, establishing a protective mechanism against iron-dependent lipid peroxidation and ferroptotic cell death in lung cancer [124]. The Nrf2-L2HG axis modulates histone hypermethylation, altering chromatin structure and accessibility of ferroptosis-related genes [125].

N6-methyladenosine (m6A) modification influences tumor cell fate through bidirectional modulation of ferroptosis sensitivity. The m6A reader protein YTH domain-containing protein 1 (YTHDC1) functions as a tumor progression suppressor by reducing FSP1 expression, enhancing lung cancer cell sensitivity to ferroptotic death [126]. Protein kinase A regulates ferroptosis through phosphorylation of ALKBH5, which controls GPX4 m6A modification [127]. Conversely, tumor cells have evolved mechanisms using m6A modification to inhibit ferroptosis, such as EGFR signaling promoting nuclear retention of ALKBH5, which attenuates m6A modification and protects glioblastoma cells against ferroptotic cell death [128]. In HER2-positive breast cancer, m6A-regulated FGFR4 has been demonstrated to attenuate ferroptotic cell death, significantly contributing to the therapeutic resistance [129]. Hepatocellular carcinoma cells exploit a METTL16-SENP3-LTF axis to confer ferroptosis resistance and facilitate tumorigenesis, demonstrating how m6A writers can orchestrate ferroptosis evasion mechanisms [130]. METTL17 coordinates ferroptosis inhibition and tumorigenesis by regulating mitochondrial translation, highlighting the diverse mechanisms by which m6A-related enzymes reduce ferroptotic vulnerability in colorectal cancer cells [131].

Epigenetic changes in immune cells affecting ferroptosis sensitivity

T cells

T cells can both induce ferroptosis in cancer cells and succumb to ferroptotic death themselves, with the balance determining therapeutic outcomes. Accumulating evidence reveals epigenetic mechanisms—including histone modifications, DNA methylation, and RNA modifications—are essential regulators of T cell ferroptosis sensitivity in the tumor immune microenvironment (TIME) (Fig. 5).

Fig. 5.

Bidirectional regulation of ferroptosis between tumor cells and T cells within the tumor immune microenvironment (TIME). The diagram illustrates three major mechanisms: (1) T cell intrinsic ferroptosis pathways leading to T cell ferroptotic death; (2) Tumor cell-mediated epigenetic regulation of T cell ferroptosis sensitivity; and (3) Bidirectional signaling where tumor cells modulate T cell function while T cell-derived factors simultaneously regulate tumor cell ferroptosis. These interactions collectively shape tumor immune responses and cancer progression within the TIME

Within the TIME, T cell ferroptosis sensitivity is regulated through multiple epigenetic pathways. PD-1 signaling constitutes a central regulatory axis, whereby PD-1 activation in CD8⁺ T cells induces GATA1 binding to the phospholipid phosphatase 1 (Plpp1) promoter, transcriptionally repressing this critical phospholipid homeostasis regulator [6]. This epigenetic suppression disrupts phosphatidylethanolamine and phosphatidylcholine synthesis, ultimately promoting ferroptotic cell death [6].

RNA methylation provides another critical layer of epigenetic control. The RNA N6,2ʹ-O-dimethyladenosine (m6Am) methyltransferase phosphorylated CTD-interacting factor 1 (PCIF1) negatively regulates CD8⁺ T cell antitumor activity by modulating ferroptosis susceptibility. Genetic ablation of PCIF1 elevates m6Am-modified transcripts of ferroptosis suppressor genes (Fth1, Slc3a2) and T cell activation gene Cd69, simultaneously conferring ferroptosis resistance and enhancing CD8⁺ T cell activation [132]. Similarly, neutrophil extracellular traps enhance YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) expression, an m6A reader protein that selectively modifies SLC2A3 transcripts, creating a metabolic environment that inhibits both tumor ferroptosis and CD8⁺ T cell effector functions [133].

GPX4, a master ferroptosis regulator, undergoes epigenetic silencing through ubiquitin-like with PHD and RING finger domains 1 (UHRF1)-mediated promoter hypermethylation [134]. Within T cells, GPX4 maintains metabolic fitness by preventing lipid peroxidation-induced ferroptosis, with the mTORC2-AKT-GSK3β axis preserving memory CD4⁺ T cell persistence by maintaining GPX4 activity and preventing mitochondrial ROS accumulation [135].

Epigenetic mechanisms also regulate tumor cell ferroptosis sensitivity and T cell trafficking. CX3CL1 promoter hypermethylation simultaneously inhibits CD8⁺ T cell infiltration while modulating cancer cell ferroptosis sensitivity [136]. Additionally, epigenetic regulators like heterogeneous nuclear ribonucleoprotein L (HnRNP L) promote immune evasion by concurrently upregulating programmed death-ligand 1 (PD-L1) expression through the YY1/PD-L1 axis and inhibiting ferroptosis [137]. Targeting these regulators enhances ferroptosis sensitivity, increases CD8⁺ T cell infiltration, and synergizes with immune checkpoint blockade therapy.

Tumor-associated macrophages

Tumor-associated macrophages (TAMs) display phenotypic plasticity regulated partly through epigenetic mechanisms controlling ferroptosis susceptibility (Fig. 6). Long non-coding RNAs emerge as pivotal epigenetic regulators, exemplified by FER1L4, which maintains pro-tumorigenic M2 phenotypes through dual epigenetic mechanisms: recruiting DNA methyltransferase DNMT3A to the Nrf2 promoter, inducing DNA methylation that suppresses the Kelch-like ECH-associated protein 1 (KEAP1)-Nrf2 antioxidant pathway [138]; and functioning as a competing endogenous RNA by sponging miR-214-3p, preventing inhibition of glutathione peroxidase 4 (GPX4), thereby preserving its activity and protecting TAMs from lipid peroxidation-induced death [138].

Fig. 6.

Epigenetic modulation of ferroptosis across the immune cells: TAMs, NK Cells, B Cells, TANs and MDSCs

Intercellular communication via exosome-mediated transfer of epigenetic regulators adds another complexity layer. TAM-derived exosomes containing annexin A3 impair ferroptosis in cancer cells by regulating ATF2 ubiquitination and altering ChaC glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1) expression, supporting lymphatic metastasis [139]. Beyond lncRNAs, multiple miRNAs (including miR-214-3p and miR-450b-5p) target ferroptosis-associated genes within the TIME, forming an intricate regulatory network determining TAM fate and influencing tumor progression [93].

Natural killer cells

Natural killer (NK) cell functionality in the TIME is governed by epigenetic programs influencing both their ferroptosis sensitivity and ability to induce ferroptosis in cancer cells (Fig. 6). Nuclear receptor coactivator 4 (NCOA4), a critical ferritinophagy mediator, undergoes regulation through protein arginine methylation via protein arginine methyltransferase 1 (PRMT1) and altered DNA methylation patterns, affecting NK cell cytotoxic capacity [140, 141].

Activation-induced DNA methylation at glutathione peroxidase 4 (GPX4) enhances redox defense mechanisms in NK cells, protecting them during tumor infiltration [142]. DNA methyltransferases epigenetically regulate GPX4 through promoter hypermethylation, while pharmacological DNMT inhibition reverses this pattern and restores expression [143]. NK cells with elevated GPX4 demonstrate resistance to L-kynurenine-induced ferroptosis in gastric cancer microenvironments, preserving their function and enhancing anti-tumor activity [142]. GPX4 inhibitors delivered via hybrid nanovesicles incorporating NK cell-derived extracellular vesicles promote tumor cell ferroptosis through downregulating GPX4 and increasing lipid peroxidation, with NK cell-secreted Fas ligand and IFN-γ enhancing this effect [144].

The other immune cells

B cells

The chromatin remodeling factor helicase, lymphoid-specific (HELLS, also known as LSH) suppresses B cell ferroptosis through DNA methylation maintenance, conferring survival advantage to germinal center B cells during immune responses, while lncRNA NEAT1 promotes ferroptosis in malignant B cells through competing endogenous RNA mechanisms, thereby inhibiting B cell lymphoma proliferation and offering therapeutic opportunities for targeting B cell malignancies (Fig. 6). In germinal center B cells, the chromatin remodeling factor HELLS maintains DNA methylation integrity, potentially regulating ferroptosis sensitivity [145]. In malignant states, lncRNA NEAT1 epigenetically regulates B cells through mechanisms affecting cellular proliferation and ferroptotic sensitivity [146]. These findings establish a mechanistic link between epigenetic regulation and ferroptosis in activated B cells, offering therapeutic opportunities for B cell malignancies that exploit unique epigenetic vulnerabilities while sparing normal B lymphocytes.

Tumor-associated neutrophils

Tumor-associated neutrophils (TANs) display remarkable ferroptosis resistance through GM-CSF-induced upregulation of aconitate decarboxylase 1 (Acod1), which suppress ferroptosis and prolong their immunosuppressive presence in the tumor immune microenvironment (Fig. 6). Acod1 upregulation occurs through the GM-CSF-JAK/STAT5-C/EBPβ signaling pathway, facilitating itaconate production that activates Nrf2-dependent defenses against ferroptosis [147]. The intersection of epigenetics and metabolism in regulating neutrophil ferroptosis represents an important research frontier, as these cells significantly contribute to immunosuppressive microenvironments and promote metastasis.

MDSCs

Myeloid-derived suppressor cells establish immunosuppression within the TIME, with epigenetic mechanisms regulating their relationship with ferroptosis (Fig. 6). Polymorphonuclear MDSCs exhibit unique responses to ferroptotic stress, releasing oxygenated lipids that suppress T-cell function and enhance immunosuppression [148]. DNA methylation patterns influence this process, with PGE2 released during ferroptosis activating DNMT3A in myeloid cells, suppressing immunogenic gene expression [149]. Targeting specific enzymes like DNMT1 or KDM demethylases could potentially reprogram MDSCs to increase ferroptosis susceptibility or diminish immunosuppressive capacity. Hypomethylating agents enhance ferroptosis sensitivity in myeloid cells by demethylating specific promoters and altering expression of key regulators like GPX4 and SLC7A11 [95].

Epigenetic regulation of ferroptosis in stromal cells of the tumor immune microenvironment

Stromal cells within the TIME, particularly cancer-associated fibroblasts and endothelial cells, significantly influence epigenetic regulation of ferroptosis. These stromal components establish an epigenetically regulated microenvironment shielding tumor cells from iron-dependent oxidative damage, promoting treatment resistance through coordinated suppression of ferroptotic pathways (Fig. 7).

Fig. 7.

Epigenetic Modulation of Ferroptosis in stromal cells. The figure illustrates five key relationships: (1) CAF-derived secretory factors regulating cancer cell ferroptosis; (2) CAF-derived extracellular vesicles containing microRNAs/lncRNAs modulating cancer cell ferroptotic sensitivity; (3) CAF intrinsic ferroptosis mechanisms leading to tumor immunosuppression; (4) Reciprocal tumor-CAF interactions where tumor-intrinsic epigenetic alterations shape CAF phenotypes and metabolic reprogramming, influencing ferroptosis vulnerability; and (5) Endothelial cell ferroptosis regulation affecting the tumor vasculature. These diverse mechanisms highlight the complex epigenetic control of ferroptosis across different stromal cell populations within the TIME

Cancer-associated fibroblasts

Cancer-associated fibroblasts (CAFs) within the TIME undergo extensive epigenetic reprogramming that profoundly influences ferroptosis sensitivity in both CAFs themselves and neighboring cancer cells.

CAFs inhibit tumor cell ferroptosis through diverse secretory mechanisms. In glioblastoma, CAF-secreted TSP-4 activates HSF1 in cancer cells, triggering transcriptional upregulation of lncRNA deleted in lymphocytic leukemia 1 (DLEU1), which binds zinc finger protein 36 (ZFP36) and promotes ATF3 mRNA degradation. This epigenetic cascade increases SLC7A11 expression, attenuating erastin-induced ferroptosis and promoting treatment resistance [150]. In triple-negative breast cancer, CAF-produced lactate induces histone lactylation at the ZFP64 promoter, enhancing its expression and driving transcription of ferroptosis-inhibiting genes like GCH1 and ferritin heavy chain 1 (Fth1), conferring chemoresistance through suppression of ferroptotic cell death [151].

CAFs employ exosome-mediated transfer of epigenetic regulators to modulate ferroptosis sensitivity. In pancreatic ductal adenocarcinoma, CAF-derived exosomes containing miR-3173-5p target ACSL4 in cancer cells, inhibiting ferroptosis and promoting gemcitabine resistance [152]. In lung cancer, CAF-secreted exosomes deliver lncRNA ROR1-AS1 to cancer cells, where it interacts with IGF2BP1 to enhance SLC7A11 mRNA stability, inhibiting ferroptosis and promoting tumor growth [153]. Contrary to these protective roles, in gastric cancer, CAF-derived exosomes containing lncRNA DACT3-AS1 enhance oxaliplatin sensitivity by promoting SIRT1-mediated ferroptosis [123].

CAFs themselves undergo ferroptosis regulation, particularly evident in specialized FerroCAFs that accumulate intracellular iron through Hmox1-mediated heme degradation. Elevated iron serves as a cofactor for Kdm6b, an iron-dependent histone demethylase removing repressive H3K27me2/3 marks from target gene promoters [154].

These stromal–epithelial interactions are complemented by tumor-intrinsic alterations reciprocally shaping CAF phenotypes. In pancreatic tumors, SETD2 deficiency drives emergence of distinct ABCA8a-expressing lipid-laden CAF subpopulations through BMP2 signaling activation and H3K27Ac gain [155]. These specialized CAFs supply lipids for tumor cell oxidative phosphorylation, establishing a metabolic symbiosis influencing ferroptosis vulnerability.

Endothelial cell

Ferroptosis significantly impacts endothelial cells within the TIME, with non-coding RNAs emerging as powerful modulators of ferroptotic susceptibility. MicroRNAs exert post-transcriptional control over ferroptosis-related transcripts, with miR-214-3p protecting endothelial cells from erastin-induced ferroptosis by targeting the A20-ACSL4 axis [156]. Within the complex TIME, exosomes released by gastric cancer cells deliver miR-214-3p to vascular endothelial cells, targeting zinc finger protein A20 and inhibiting ferroptosis through negative regulation of ACSL4, which is essential for incorporating polyunsaturated fatty acids into phospholipids for peroxidation [156]. This tumor-endothelium crosstalk demonstrates how cancer cells epigenetically modulate endothelial cell ferroptosis to maintain vascular integrity supporting tumor growth.

Epigenetic regulation of ferroptosis by microenvironmental factors

Hypoxia

Hypoxia critically modulates ferroptosis susceptibility through diverse epigenetic mechanisms (Fig. 8). In oxygen-limited conditions, iron-dependent ferroptotic cell death is regulated via N6-methyladenosine (m6A) RNA modifications, DNA methylation, histone modifications, and non-coding RNAs that collectively orchestrate expression of key ferroptosis regulators.

Fig. 8.

Microenvironmental factors regulation of epigenetic mechanisms controlling ferroptosis in tumor cells. The figure illustrates three key microenvironmental influences: (1) Hypoxic conditions trigger epigenetic modifications that alter cancer cell ferroptosis sensitivity through multiple pathways affecting lipid metabolism and antioxidant capacity; (2) Tumor-associated metabolites induce epigenetic changes that modulate ferroptotic response, particularly through DNA methylation and RNA regulatory mechanisms; and (3) Inflammatory signals within The tumor immune microenvironment reshape the epigenetic landscape controlling ferroptotic cell death through altered transcription factor activity and redox homeostasis. These microenvironmental factors collectively determine tumor cell susceptibility to ferroptosis through diverse epigenetic mechanisms

Hypoxia-induced downregulation of methyltransferase METTL14 reduces m6A modification on SLC7A11 mRNA, preventing YTHDF2-dependent degradation and increasing SLC7A11 protein levels—a critical cystine/glutamate antiporter that inhibits ferroptosis by maintaining glutathione synthesis in HCC cell lines [157]. Lymphocyte-specific helicase (LSH) serves as another pivotal epigenetic regulator under hypoxic conditions, wherein EGLN1 stabilizes c-Myc by promoting HIF-1α degradation, enabling c-Myc to upregulate LSH [124]. Elevated LSH recruits WDR76 to metabolic gene promoters including SCD1 and FADS2, activating their transcription and reducing lipid ROS production and iron accumulation, thereby inhibiting ferroptosis [124]. Additionally, hypoxia induces specific lncRNAs like CBSLR that recruit YTHDF2 to destabilize cystathionine beta-synthase (CBS) mRNA, contributing to ferroptosis resistance in gastric cancer [158]. These epigenetic mechanisms represent promising therapeutic targets for ferroptosis modulation in hypoxia-associated pathologies including cancer, ischemic injuries, and neurodegenerative disorders.

Metabolite

Cellular metabolites critically influence ferroptosis through sophisticated epigenetic regulatory mechanisms (Fig. 8). Tumor metabolic reprogramming exerts profound control over ferroptosis susceptibility by modulating the availability of metabolic intermediates that serve as essential cofactors or substrates for epigenetic-modifying enzymes [159]. Four key metabolites establish this metabolic-epigenetic axis: (1) Acetyl-CoA, generated from glucose and fatty acid metabolism, serves as the obligate substrate for histone acetyltransferases (HATs) that acetylate histones at ferroptosis regulator gene loci (e.g., SLC7A11, GPX4), promoting transcriptionally permissive chromatin states [160, 161]; (2) S-adenosylmethionine (SAM), produced via one-carbon metabolism (folate/methionine cycles), functions as the universal methyl donor for DNA methyltransferases (DNMTs) and histone methyltransferases (HMTs), enabling promoter hypermethylation that silences ferroptosis executors such as ACSL4 [162]; (3) α-Ketoglutarate (α-KG), a TCA cycle intermediate, acts as an essential cofactor for Fe(II)/α-KG-dependent TET DNA demethylases and JmjC histone demethylases, which remove repressive methylation marks and activate ferroptosis defense genes [163]; and (4) NAD+, coupled to cellular energy status, regulates sirtuin deacetylases that modulate histone acetylation and transcription factor activity at ferroptosis-related loci [164]. Cancer cells coordinate glycolysis, TCA cycle flux, and one-carbon metabolism to optimize these cofactor pools, thereby dynamically remodeling the epigenetic landscape governing ferroptosis sensitivity. This metabolic-epigenetic integration provides a mechanistic foundation for understanding how nutrient availability, oncogenic signaling, and metabolic stress converge to regulate ferroptosis through chromatin modifications.

Methyl-donor metabolites directly impact DNA methylation profiles of key ferroptosis regulators. Notably, homocysteine elevation induces site-specific hypermethylation of the GPX4 promoter, transcriptionally silencing this master ferroptosis suppressor and enhancing cellular vulnerability to lipid peroxidation-induced death [165]. The lysine demethylase 3B (KDM3B), responsive to cellular metabolic status, selectively reduces H3K9 methylation at the SLC7A11 promoter, enhancing transcription of this cystine/glutamate antiporter and consequently upregulating glutathione biosynthesis—a central metabolic pathway conferring ferroptosis resistance [166]. Conversely, BRCA1 associated protein-1 (BAP1) and polycomb repressive complex 1 (PRC1) complex deubiquitinate histone H2A at the SLC7A11 promoter region, establishing repressive chromatin architecture that suppresses SLC7A11 expression and promotes ferroptosis through glutathione depletion [167]. LSH, responsive to cellular energetic status, inhibits ferroptosis by recruiting WDR76 to metabolic gene promoters including SCD1 and FADS2, amplifying their expression and consequently preventing lipid ROS generation and iron accumulation [124]. In hepatocellular carcinoma, metabolic reprogramming under hypoxic conditions downregulates METTL14, reducing m6A methylation on SLC7A11 mRNA's 5' UTR and increasing its expression via the YTHDF2 pathway, thereby enhancing glutathione synthesis and conferring ferroptosis resistance [157]. Non-coding RNAs constitute additional epigenetic regulators of ferroptosis-related metabolites. LncRNA LINC00336, responsive to cellular metabolic state, inhibits ferroptosis by decreasing intracellular iron and lipid ROS through ELAVL1 interaction while simultaneously functioning as a competing endogenous RNA by sponging microRNA-6852, thus preserving CBS-mediated ferroptosis inhibition [168]. The microRNA-17–92 cluster, regulated by metabolic signaling pathways, protects cells from erastin-induced ferroptosis by post-transcriptionally downregulating ACSL4, a key enzyme facilitating ferroptosis-promoting lipid metabolism [169]. These metabolite-driven epigenetic mechanisms present significant therapeutic opportunities in cancer. Epigenetic drugs like demethylating agents could potentially restore expression of ferroptosis-promoting genes silenced by hypermethylation [95].

Inflammatory signals

The interplay between inflammatory signaling and ferroptosis is governed by epigenetic mechanisms that regulate expression of key ferroptosis mediators (Fig. 8). Transcription factors NRF2 and NF-κB function as critical intermediaries linking inflammatory signaling to epigenetic regulation of ferroptosis. NRF2, which orchestrates expression of antioxidant response element-containing genes involved in iron metabolism and glutathione synthesis, undergoes epigenetic regulation through SQSTM1/p62 and KEAP1 [170]. Inflammatory cytokines, particularly IL-6, modulate ferroptosis through the STAT3/GPX4 pathway, influencing lipid peroxidation processes that determine ferroptotic sensitivity [171]. This epigenetic regulatory network governing ferroptosis in inflammatory contexts offers promising opportunities for developing therapeutic approaches targeting epigenetic regulators to modulate ferroptotic cell death in inflammatory diseases and cancer.

PTMs of ferroptosis regulators in the tumor immune microenvironment

PTMs of ferroptosis regulators in cancer cells

PTMs constitute a sophisticated regulatory network that enables cancer cells to rapidly adapt their ferroptosis sensitivity in response to environmental pressures. These dynamic modifications provide temporal and spatial control over ferroptosis mediators, allowing tumor cells to maintain survival advantages within challenging microenvironmental conditions (Fig. 9).

Fig. 9.

PTMs of ferroptosis proteins in the immune microenvironment. The figure illustrates four key regulatory aspects: (1) PTMs of ferroptosis proteins in tumor cells; (2) Regulatory mechanisms in immune cells; (3) Modifications in epithelial cells; and (4) Impact of immune microenvironmental factors on ferroptosis protein PTMs

Phosphorylation emerges as a central mechanism orchestrating ferroptosis resistance through protein stabilization cascades. In glioblastoma, AMPKα1 phosphorylates ZDHHC8 at serine 299, enhancing S-palmitoylation and subsequent stabilization of SLC7A11 [172]. This phosphorylation–palmitoylation coupling maintains glutathione biosynthetic capacity, effectively shielding cancer cells from iron-dependent oxidative damage.

Complementing phosphorylation networks, glycosylation modifications provide additional layers of protection against ferroptotic death. STT3-catalyzed N-glycosylation of CD24 creates a multifaceted shield against ferroptosis by simultaneously reducing glutathione consumption, limiting iron accumulation, and suppressing lipid peroxidation in triple-negative breast cancer [173]. Glycosylation inhibition restores ferroptosis sensitivity and overcomes paclitaxel resistance.

Ubiquitination pathways exert opposing effects on ferroptosis regulators, with E3 ligases serving as critical determinants of cellular fate. TRIM3 exemplifies this regulatory complexity through its K11-linked ubiquitination of SLC7A11 at lysine 37, promoting antiporter degradation and sensitizing cells to ferroptotic death [174]. Cancer cells frequently lose TRIM3 expression, stabilizing SLC7A11 and establishing ferroptosis resistance in non-small cell lung cancer.

The regulatory landscape extends to acetylation modifications that modulate transcriptional control networks. Cold atmospheric plasma treatment induces PCAF-mediated acetylation of HOXB9, destabilizing this transcription factor and disrupting its ability to activate SLC7A11 expression [175]. This mechanism demonstrates therapeutic potential for plasma-based cancer interventions.

Arginine methylation adds another dimension of control through its influence on protein–protein interactions and subsequent degradation pathways. CARM1 methylates ACSL4 at arginine 339, creating a recognition motif for RNF25 E3 ligase binding and promoting ACSL4 degradation [176]. This methylation-ubiquitination cascade reduces cellular capacity for lipid peroxidation, establishing a targetable protective mechanism against ferroptotic death.

Lactylation represents an emerging modification linking metabolic reprogramming to ferroptosis regulation. In the acidic tumor immune microenvironment, NAA10 mediates NSUN2 lactylation at lysine 508, enhancing RNA methyltransferase activity and stabilizing GCLC mRNA through m5C modifications [177]. This connects glycolytic metabolism with glutathione synthesis and ferroptosis resistance.

PTMs of ferroptosis regulators in immune cells

Immune cells employ sophisticated post-translational modification networks to regulate both their own ferroptosis sensitivity and their capacity to induce ferroptotic death in target cells. These modifications enable dynamic responses to inflammatory signals while maintaining immune cell functionality within the tumor immune microenvironment [178].

STAT1 phosphorylation serves as a critical regulatory node connecting interferon signaling to ferroptosis induction. The phosphorylation status of STAT1 determines cancer cell susceptibility to IFNγ-induced ferroptosis through transcriptional control of SLC7A11 and GPX4 expression [179]. The balance between STAT1 phosphorylation and SUMOylation influences both ferroptosis sensitivity and immune checkpoint blockade efficacy.

Glycosylation defects create unexpected links between protein processing and immunogenic cell death. ALG3-deficient cells experience disrupted N-linked glycosylation of SCAP and PD-L1, triggering compensatory SREBP1 activation that leads to lipid hyperperoxidation and immunogenic ferroptosis [173]. This enhances T cell infiltration and anti-PD-1 therapy responses.

The tumor microbiome introduces additional complexity through bacterial-mediated modifications of immune cell ferroptosis pathways. B. parabrevis enhances RORC acetylation, subsequently upregulating neural precursor cell expressed developmentally down-regulated protein 4-like (NEDD4L) expression and promoting ubiquitination-mediated degradation of iron transporters SLC39A14, SLC39A8, and STEAP3 in NK cells [180]. This preserves NK cell cytotoxic function and transforms immunologically “cold” tumors into “hot” microenvironments.

Inflammatory cytokines orchestrate additional PTMs that influence ferroptosis-mediated immune surveillance. IL-1β promotes PCAF-mediated acetylation of nicotinamide nucleotide transhydrogenase (NNT) at lysine 1042, enhancing NADPH production and maintaining iron-sulfur cluster integrity [181]. This enables tumor immune evasion by preventing immunogenic ferroptosis.

PTMs of ferroptosis regulators in stromal cells

Stromal cells within the tumor immune microenvironment undergo distinct PTMs that critically influence vascular integrity, tissue homeostasis, and therapeutic responses through regulation of ferroptosis pathways.

Endothelial cell ferroptosis represents a critical determinant of microvascular stability, with Fundc1 phosphorylation serving as a key regulatory mechanism. Neutrophil extracellular traps induce Fundc1 phosphorylation at tyrosine 18, disrupting mitophagy and causing mitochondrial dysfunction that culminates in ferroptosis-mediated endothelial damage [182]. This creates a pathological cascade linking neutrophil activation to microvascular injury.

Complementary degradation pathways regulate endothelial ferroptosis through targeted protein modifications. N-acetylneuraminic acid facilitates SLC3A2 ubiquitination and P62-mediated autophagic degradation, compromising cystine-glutamate antiporter function and sensitizing endothelial cells to ferroptotic death [183]. This becomes particularly relevant in atherosclerosis progression.

RNA modifications extend post-translational control to the level of mRNA stability and protein expression. NAT10-catalyzed N4-acetylcytidine modification of transferrin receptor (TFRC) mRNA enhances transferrin receptor stability and expression, promoting iron uptake and ferroptosis susceptibility in pulmonary endothelial cells during sepsis [184].

Mesenchymal stromal cells employ distinct epigenetic modifications to regulate ferroptosis sensitivity during inflammatory responses. EZH2-mediated STAT3 methylation suppresses GPX4 expression in dental mesenchymal cells, promoting ferroptosis and exacerbating inflammatory tissue damage during pulpitis [185].

Microenvironmental factor-induced PTMs affecting ferroptosis

Hypoxia-induced PTMs

Hypoxic conditions trigger specific PTMs that determine cellular adaptation versus ferroptotic death [186]. FOXO1 acetylation emerges as a critical regulatory mechanism during ischemia–reperfusion injury, where increased acetylation impairs FOXO1 transcriptional activity and reduces FTH1 expression, sensitizing cardiomyocytes to ferroptosis in primary neonatal mouse cardiomyocytes [187]. The protective circRNA FEACR-NAMPT-Sirt1 axis counteracts this vulnerability by reducing FOXO1 acetylation.

Lactate accumulation under hypoxic conditions creates novel modification opportunities that amplify ferroptosis sensitivity. Hypoxia-induced lactylation of ACSL4 at lysine 83 enhances protein stability and promotes ferroptosis in cardiomyocytes in human nucleus pulposus cells (NPCs) from IDD patients and lung adenocarcinoma cell lines [188].

Metabolite-induced PTMs

Metabolic intermediates serve as both substrates and regulators of PTMs affecting ferroptosis pathways. Substance P exemplifies neuropeptide regulation of ferroptosis through its inhibition of STING phosphorylation via neurokinin 1 receptor (NK1R) signaling, simultaneously suppressing inflammatory cytokine production and TNF-α-induced ferroptosis in colonic epithelial cells [189].

Amino acid metabolism creates additional modification opportunities through the glutamate- Yes-associated protein (YAP) axis. System XC- inhibition leads to glutamate accumulation, activating ADCY10/PKA signaling and promoting glutamine–fructose-6-phosphate transaminase 1 (GFPT1) phosphorylation, which reduces YAP O-GlcNAcylation and enhances Hippo pathway-mediated YAP ubiquitination [190]. This impairs ferritin transcriptional compensation, sensitizing cancer cells to ferroptosis.

Inflammatory signal-induced PTMs

Chronic inflammatory signals orchestrate complex modification networks that link immune cell infiltration with tissue ferroptosis susceptibility. PRMT7-mediated histone mono-methylation at RAP1A regulatory elements promotes monocyte recruitment and subsequent ALOX5 overexpression, generating LTB4 metabolites that trigger ACSL4 expression and ferroptosis in lung epithelial cells [191].

Advanced glycation end products represent pathological modifications that combine metabolic dysfunction with inflammatory signaling to promote ferroptosis. AGEs disrupt SLC7A11/GPX4 antioxidant pathways while activating NF-κB signaling, leading to enhanced production of inflammatory mediators and ferroptosis susceptibility in diabetic periodontitis [192].

Crosstalk between epigenetic and post-translational regulatory networks in ferroptosis

While epigenetic modifications and PTMs are conceptually distinct regulatory mechanisms, accumulating evidence demonstrates their profound functional interdependence in governing ferroptosis within the tumor immune microenvironment (TIME). This crosstalk occurs at multiple hierarchical levels: (1) PTMs directly regulate the activity, stability, and localization of epigenetic enzymes, thereby modulating the epigenetic landscape. For example, PKA phosphorylates METTL14 at S456, enhancing its m6A activity and promoting SLC7A11 mRNA degradation, thereby sensitizing hepatocellular carcinoma cells to ferroptosis [157]. (2) Metabolic intermediates such as lactate and acetyl-CoA serve dual functions as substrates for both histone modifications and protein PTMs. Lactate induces both histone lactylation at the ZFP64 promoter (activating ferroptosis suppressors GCH1/FTH1) in cancer-associated fibroblasts [193] and ACSL4 protein lactylation at K412 (enhancing its stability) in nucleus pulposus cells and lung adenocarcinoma cells [194]. (3) Epigenetically programmed gene expression patterns determine the abundance and activity of PTM machinery components. NRF2 activation transcriptionally upregulates metabolic enzymes producing NADPH, which drives acetyl-CoA and SAM biosynthesis, enhancing substrate availability for acetyltransferases and methyltransferases in a feedforward loop that amplifies ferroptosis resistance [195]. (4) Both regulatory layers converge on shared target proteins critical for ferroptosis sensitivity, creating feedforward and feedback regulatory loops. SLC7A11 expression is controlled by DNA methylation, histone modifications, and m6A modification [157, 196], while its protein stability is regulated by phosphorylation-mediated palmitoylation, TRIM3-mediated ubiquitination, and SUMOylation [197, 198]. Understanding this multi-layered integration is essential for developing therapeutic strategies that effectively modulate ferroptosis in cancer through coordinated targeting of multiple regulatory nodes. A comprehensive overview of how epigenetic and PTM mechanisms coordinately regulate ferroptosis across different cell types within the tumor immune microenvironment is provided in Table 4. The specific regulatory mechanisms governing key ferroptosis effector proteins through both epigenetic and post-translational modifications are detailed in Table 5.

Table 4.

Integrative epigenetic-PTM regulation of ferroptosis by cell type

Cell type	Cancer type/cell line	Epigenetic writer	Epigenetic eraser	Epigenetic reader	PTM enzyme	Ferroptosis effector	Direction	Compartment	References
Cancer Cells	Hepatocellular carcinoma (HepG2, Huh7)	DNMT1	TET1/2	–	–	GPX4	−	Nucleus → Cyto/Mito	[70]
Cancer Cells	Gallbladder cancer (GBC-SD, NOZ)	DNMT1, DNMT3A	TET2	–	–	RUNX3 → SLC7A11	−	Nucleus → PM	[69]
Cancer Cells	Colorectal cancer (HCT116, SW480)	EZH2 (H3K27me3)	KDM6A/B	–	–	SLC7A11, ATOH8	−	Nucleus → PM	[117]
Cancer Cells	Lung cancer (A549, H1299)	YTHDC1	FTO, ALKBH5	YTHDF2	–	FSP1	−	Cytoplasm	[126]
Cancer Cells	Glioblastoma (U87, LN229)	METTL3	ALKBH5	–	–	GPX4	±	Cytoplasm	[128]
Cancer Cells	Breast cancer (MCF-7, MDA-MB-231, HER2 +)	METTL14	FTO	YTHDF1	m6A	FGFR4	−	Cytoplasm	[129]
T Cells	CD8 + TILs (MC38 tumor model, murine)	–	–	–	GATA1 (TF)	PLPP1	−	Nucleus → ER/PM	[6]
T Cells	CD8 + T cells (primary murine)	PCIF1 (m6Am writer)	–	–	m6Am	FTH1, SLC3A2	−	Cytoplasm	[132]
T Cells	CD4 + memory T cells (primary murine)	–	SIRT1/3	–	Deacetylation	GPX4	+	Cyto/Mito	[135]
TAMs	M2-like TAMs (THP-1 derived, tumor co-culture)	DNMT3A (via lncRNA FER1L4)	–	–	DNA methylation	NRF2 (via KEAP1)	−	Nucleus	[138]
TAMs	TAMs (THP-1, RAW264.7)	–	–	–	miR-214-3p (FER1L4 sponge)	GPX4	+	Cytoplasm	[138]
NK Cells	Primary human NK cells	PRMT1	–	–	Arginine methylation	NCOA4	±	Cyto → Mito	[141]
NK Cells	NK cells (gastric cancer microenvironment)	DNMTs	–	–	DNA methylation	GPX4	−	Nucleus → Cyto	[142]
CAFs	CAFs from glioblastoma (patient-derived)	–	–	–	ZFP36 (RBP)	ATF3 → SLC7A11	+	Cyto (intercellular)	[150]

Direction symbols: −  = inhibits ferroptosis, +  = promotes ferroptosis, ±  = context-dependent

PM plasma membrane, Cyto cytoplasm, Mito mitochondria, ER endoplasmic reticulum, TF transcription factor, RBP RNA-binding protein, GBM glioblastoma

Table 5.

Epigenetic and PTM regulation of key ferroptosis effector proteins

Target	Regulatory type	Specific mechanism	Effect on target & ferroptosis	Model/cell type	Refs
SLC7A11	DNA Methylation	DNMT1 hypermethylation of RUNX3 promoter	Suppresses RUNX3 → reduces ING1-mediated inhibition of SLC7A11 → increased SLC7A11 expression → enhanced cystine import → anti-ferroptotic	Gallbladder cancer	[69]
SLC7A11	DNA Methylation	DNMT1 maintains GPX4 promoter demethylation	Context: In HCC, maintains antioxidant capacity including SLC7A11-related GSH synthesis pathway → anti-ferroptotic	Hepatocellular carcinoma	[70]
SLC7A11	Histone Modification	LSD1 inhibition increases H3K9me2 at ATF4 promoter	Decreased ATF4 transcription → reduced SLC7A11 expression → impaired cystine uptake → pro-ferroptotic	Cancer cells (type not specified)	[80]
SLC7A11	Non-coding RNA	LncRNA NEAT1 sequesters miR-362-3p	Indirect: increases MIOX → promotes ROS, depletes NADPH/GSH → affects SLC7A11-cystine-GSH axis → pro-ferroptotic	Liver cancer	[122]
SLC7A11	Non-coding RNA	CAF-secreted lncRNA DACT3-AS1 → miR-181a-5p/SIRT1	Enhances SIRT1-mediated ferroptosis (affects SLC7A11-related antioxidant pathways) → pro-ferroptotic	Gastric cancer	[123]
SLC7A11	Chromatin Remodeling	LSH-WDR76 activates GLUT1, SCD1, FADS2	Protective lipid metabolism → indirectly supports SLC7A11-mediated antioxidant capacity → anti-ferroptotic	Lung cancer	[124]
SLC7A11	RNA Modification (m6A)	Hypoxia → METTL14↓ → reduced m6A on SLC7A11 mRNA	Prevents YTHDF2 degradation → increased SLC7A11 protein → enhanced cystine import, GSH synthesis → anti-ferroptotic	HCC cell lines	[157]
SLC7A11	Phosphorylation-Palmitoylation	AMPKα1 phosphorylates ZDHHC8 (Ser299)	Enhances S-palmitoylation → stabilizes SLC7A11 at plasma membrane → maintains cystine uptake, GSH biosynthesis → anti-ferroptotic	Glioblastoma	[172]
SLC7A11	Ubiquitination	TRIM3 K11-linked ubiquitination at Lys37	Promotes SLC7A11 degradation → reduced cystine import → GSH depletion → pro-ferroptotic	Non-small cell lung cancer	[174]
SLC7A11	Protein Function	SLC7A11 as H⁺ transporter in lysosomes	Additional protective role in lysosomal pH regulation → affects cellular redox balance → anti-ferroptotic	HeLa cells, A549-CD133 + cells	[22]
GPX4	DNA Methylation	DNMT1 maintains GPX4 promoter demethylation	Sustains GPX4 transcription → maintains lipid hydroperoxide reduction → anti-ferroptotic	Hepatocellular carcinoma	[70]
GPX4	DNA Methylation	UHRF1 recruits DNMT1 → GPX4 promoter hypermethylation	Transcriptional silencing of GPX4 → loss of lipid peroxide detoxification → pro-ferroptotic	Pulmonary fibrosis model; T cells	[134]
GPX4	DNA Methylation	Homocysteine → GPX4 promoter hypermethylation	Transcriptionally silences GPX4 → enhanced lipid peroxidation vulnerability → pro-ferroptotic	Model not specified	[165]
GPX4	DNA Methylation	Activation-induced methylation in NK cells	Enhances GPX4 expression → strengthens redox defense → protects NK cells during tumor infiltration → anti-ferroptotic	NK cells (gastric cancer)	[142]
GPX4	Non-coding RNA	LncRNA FER1L4 sponges miR-214-3p	Prevents miR-214-3p inhibition of GPX4 → preserves GPX4 activity → maintains lipid peroxide reduction → anti-ferroptotic	Tumor-associated macrophages	[138]
GPX4	RNA Modification (m6A)	PKA phosphorylates ALKBH5 → controls GPX4 m6A	Modulates GPX4 mRNA stability/translation → affects GPX4 protein levels → context-dependent ferroptosis	Model not specified	[127]
GPX4	Ubiquitination	K48-linked polyubiquitination	Targets GPX4 for proteasomal degradation → loss of lipid peroxide detoxification → pro-ferroptotic	MEFs, multiple cancer cells	[106]
GPX4	Ubiquitination	CST1 recruits OTUB1 (deubiquitinase)	Prevents GPX4 degradation → stabilizes GPX4 protein → maintains antioxidant defense → anti-ferroptotic	Gastric cancer	[199]
GPX4	Phosphorylation	STAT1 phosphorylation (IFNγ signaling)	Regulates transcriptional control of GPX4 expression → modulates cancer cell ferroptosis susceptibility	Multiple cancer cells	[179]
GPX4	Signaling Pathway	mTORC2-AKT-GSK3β axis maintains GPX4 activity	Preserves GPX4 function → prevents mitochondrial ROS → maintains memory CD4 + T cell persistence	Memory CD4 + T cells (murine)	[135]
DHODH	Enzymatic Function	DHODH reduces ubiquinone to ubiquinol	Generates ubiquinol in mitochondrial inner membrane → radical-trapping antioxidant → neutralizes lipid peroxides → anti-ferroptotic (parallel to GPX4)	Multiple models	[37]
DHODH	Parallel Defense	DHODH operates independently of GPX4-GSH axis	Redundant ferroptosis protection → dual inhibition (DHODH + GPX4) synergistically induces mitochondrial lipid peroxidation → pro-ferroptotic	Cancer cells (in vitro + in vivo)	[37]
DHODH	Hormonal Regulation	Ferroptosis mechanisms differentially regulated by sex hormones	Explains gender differences in ferroptosis susceptibility → DHODH activity may be hormonally modulated	Multiple models	[38]
DHODH	Subcellular Localization	DHODH in mitochondrial inner membrane	Produces ubiquinol specifically in mitochondria → compartment-specific ferroptosis defense distinct from cytoplasmic GPX4	Structural/biochemical studies	[24, 37]
PD-1–GATA1–PLPP1	Immune Checkpoint	PD-1 activation in CD8 + TILs	Initiates signaling → induces GATA1 binding to PLPP1 promoter (step 1)	CD8 + TILs (MC38 tumor, murine)	[6]
PD-1–GATA1–PLPP1	Epigenetic Repression	GATA1 binds PLPP1 promoter	Transcriptional repression → suppresses PLPP1 expression → disrupts phospholipid homeostasis (step 2)	CD8 + TILs (murine)	[6]
PD-1–GATA1–PLPP1	Lipid Metabolism	PLPP1 suppression disrupts PE/PC synthesis	Impaired phosphatidylethanolamine/phosphatidylcholine synthesis → accumulation of peroxidation-prone lipids → pro-ferroptotic in CD8 + T cells (step 3)	CD8 + TILs (murine)	[6]
PD-1–GATA1–PLPP1	Therapeutic Relevance	PD-1–GATA1–PLPP1 axis as target	Anti-PD-1 therapy aims to block axis → rescue T cells from ferroptosis → enhance anti-tumor immunity (clinical validation needed)	MC38 model; human validation pending	[6]

Epigenetic and post-translational modifications drive ferroptosis inducer resistance

Aberrant epigenetic modifications and PTMs orchestrate ferroptosis resistance through multiple mechanisms [199, 200]. Ubiquitination dynamics stabilize anti-ferroptotic proteins: CST1 recruits OTUB1 to prevent GPX4 degradation in gastric cancer, while HDLBP-lncFAL axis inhibits TRIM69-mediated FSP1 degradation in hepatocellular carcinoma [199]. DNA hypermethylation silences ferroptosis-sensitizing genes, exemplified by FSP1 promoter methylation in acute lymphoblastic leukemia and FADS1/ELOVL5 silencing in gastric cance. m6A modification confers resistance via METTL3-mediated SLC7A11 mRNA stabilization in lung adenocarcinoma and FGFR4 accumulation in HER2-resistant breast cancer. ncRNA networks buffer ferroptosis: LINC00239 stabilizes NRF2 by sequestering KEAP1 in colorectal cancer [199].

Therapeutic strategies to overcome resistance include combining DNMT inhibitors (azacitidine), m6A modulators (STM2457), deubiquitinase inhibitors, and ferroptosis inducers [199–201]. These reversible modifications represent tractable targets for resensitizing therapy-resistant cancers.

Clinical studies of ferroptosis

The translation of ferroptosis research from bench to bedside has gained significant momentum, with numerous clinical studies investigating the therapeutic potential of targeting ferroptotic pathways in various diseases. These studies span multiple therapeutic areas, including oncology, neurology, and cardiology, reflecting the broad pathophysiological relevance of ferroptosis [133, 134]. In this context, therapeutic strategies targeting ferroptosis have garnered substantial attention from researchers and clinicians, with an increasing number of ferroptosis modulators entering clinical trials (Table 6, sourced from ClinicalTrials.gov).

Table 6.

Clinical trials targeting ferroptosis in human diseases

Disease category	Specific disease/cancer type	Trial ID	Intervention/agent	Mechanism/target	Phase/status	Key outcomes
Neurodegenerative	Parkinson's Disease	NCT00943748	Deferiprone	Iron chelator, inhibits lipid peroxidation and ferroptosis	Phase II, Completed	Reduced SN iron, slowed motor handicap progression
	Parkinson's Disease	NCT01539837	Deferiprone	Iron chelator, ferroptosis inhibitor	Phase II, Completed	Reduced brain iron deposition, improved motor symptoms
	Parkinson's Disease	NCT02655315	Deferiprone	Iron chelator	Phase II, Large multicenter ongoing	Evaluating global effect in PD patients (European trial)
	Parkinson's Disease	NCT03204929	Cu(II)ATSM	Radical scavenger, prevents lipid peroxidation	Phase I, Completed	Positive effect in early idiopathic PD
	Parkinson's Disease	NCT02212678	N-acetyl cysteine (NAC)	Antioxidant, GSH precursor, ferroptosis inhibitor	Clinical Trial	Beneficial effects on PD symptoms
	Parkinson's Disease	NCT02445651	N-acetyl cysteine (NAC)	Antioxidant, GSH precursor	Clinical Trial	Improved oxidative stress markers
	Parkinson's Disease	NCT02424708	Glutathione (GSH)	Direct antioxidant, ferroptosis inhibitor	Clinical Trial	Beneficial effects observed
	Parkinson's Disease	IRCT201604035623N73	Vitamin E (α-tocopherol)	Lipid peroxidation inhibitor, ferroptosis inhibitor	Clinical Trial	Antioxidant neuroprotection
	Parkinson's Disease	NCT01892176	Coenzyme Q10 (CoQ10)	Mitochondrial antioxidant, ferroptosis inhibitor	Clinical Trial	Showed beneficial effects
	Alzheimer's Disease	NCT03234686	Deferiprone	Iron chelator, reduces oxidative stress and ferroptosis	Phase II, Ongoing	Cognitive protection (under investigation)
	Amyotrophic Lateral Sclerosis (ALS)	NCT02164253	Deferiprone	Iron chelator, neuroprotection	Phase II, Completed	Neuroprotective effects observed
	Amyotrophic Lateral Sclerosis (ALS)	NCT03293069	Deferiprone	Iron chelator	Phase II, Ongoing	Under investigation
	Amyotrophic Lateral Sclerosis (ALS)	NCT02870634	Cu(II)ATSM	Radical scavenger	Phase I, Completed	Well-tolerated
	Amyotrophic Lateral Sclerosis (ALS)	NCT03136809	Cu(II)ATSM	Radical scavenger	Phase I, Ongoing	Under investigation
	Amyotrophic Lateral Sclerosis (ALS)	NCT04082832	Cu(II)ATSM	Radical scavenger	Phase II, Ongoing	Under investigation
	Amyotrophic Lateral Sclerosis (ALS)	NCT04313166	Cu(II)ATSM	Radical scavenger	Phase II, Ongoing	Under investigation
Cancer	Hepatocellular Carcinoma (HCC)	NCT00105443	Sorafenib	Multi-kinase inhibitor, induces ferroptosis via System Xc- and GPX4 inhibition	Phase III, Approved	OS 10.7 vs 7.9 months (placebo), SHARP trial
	Hepatocellular Carcinoma (HCC)	NCT03434379	Atezolizumab + Bevacizumab	Immunotherapy + anti-angiogenic induced ferroptosis	Phase III, Approved	FDA approved for unresectable HCC (IMbrave150)
	Hepatocellular Carcinoma (HCC)	NCT03535259	Sorafenib	GPX4 inactivation via GSH depletion	Phase II, Completed	Evaluated in Chinese population
	Renal Cell Carcinoma (RCC)	Multiple trials	Sorafenib	Induces ferroptosis, System Xc- inhibitor, GSH depletion	FDA-approved	Significant survival benefit in advanced RCC
	Renal Cell Carcinoma (RCC)	NCT02811861	Lenvatinib + Pembrolizumab	TKI-induced ferroptosis + immunotherapy	Phase III, Approved	FDA approved for first-line treatment (CLEAR trial)
	Renal Cell Carcinoma (RCC)	NCT03141177	Nivolumab + Cabozantinib	Immunotherapy + TKI ferroptosis induction	Phase III, Approved	mPFS 16.4 months, FDA approved (CheckMate 9ER)
	Glioblastoma (GBM)	NCT04205357	Sulfasalazine + Stereotactic Radiosurgery	System Xc- inhibition + radiation-induced ferroptosis	Phase I	Safety and feasibility demonstrated
	Glioblastoma (GBM)	ACTRN12610000915055	Carboplatin + Bevacizumab	DNA damage + apoptosis (also affects ferroptosis pathway)	Phase II, Completed	Combination therapy evaluated
	Glioblastoma (GBM)	NCT03212742	Olaparib + TRAIL	PARP inhibitor sensitizes to death receptor apoptosis	Phase I/IIa, Recruiting	Induces multiple cell death pathways
	Glioma	NCT01577966	Buthionine sulfoximine	GPX4 inactivation via GSH depletion (Class I FINs)	Phase I, Completed	Well-tolerated, induces ferroptosis
	Glioma	NCT00002730	Buthionine sulfoximine	GPX4 inactivation via GSH depletion	Phase I, Completed	Safety profile established
	Breast Cancer (Metastatic)	NCT00764036	Artesunate	Iron-dependent ROS generation, ferroptosis induction	Phase I, Completed	Promising activity in metastatic breast cancer
	Breast Cancer	NCT03093129	Artesunate	Iron-dependent ROS, ferroptosis inducer	Phase II, Recruiting	Under investigation
	Breast Cancer	NCT00416403	Fluvastatin	HMG-CoA reductase inhibitor, GPX4 inactivation	Phase II, Completed	Induces ferroptosis
	Breast Cancer	NCT00188669	Pentoxifylline	Induces apoptosis via NF-κB decrease	Phase II, Terminated	Early termination
	Breast Cancer	NCT00028639	Bortezomib	Proteasome inhibitor, NF-κB pathway	Phase II, Completed	Completed evaluation
	Breast Cancer	NCT00689195	Withaferin A	GPX4 inactivation/depletion (Class II/III FINs)	Phase II, Unknown	Status unknown
	Non-Small Cell Lung Cancer (NSCLC)	NCT01928576	Azacitidine + Entinostat + Nivolumab	Epigenetic therapy enhances ferroptosis + immunotherapy	Phase II	Promising results in metastatic NSCLC
	Non-Small Cell Lung Cancer (NSCLC)	NCT02512172	Romidepsin/5-Azacitidine + Pembrolizumab	Epigenetic modulation + immunotherapy	Phase II	Evaluated combination therapy
	Non-Small Cell Lung Cancer (NSCLC)	NCT02559778	Statins	Reduce selenoproteins (GPX4) and CoQ10 biosynthesis	Phase II, Recruiting	Under investigation
	Non-Small Cell Lung Cancer (NSCLC)	NCT03247088	Statins	Ferroptosis induction via GPX4 reduction	Phase I/II, Recruiting	Ongoing evaluation
	Non-Small Cell Lung Cancer (NSCLC)	NCT04383938	APR-246 (Eprenetapopt)	GSH depletion, thioredoxin inhibition, p53 targeting	Phase I, Completed	Evaluated in multiple cancers
	Small Cell Lung Cancer (SCLC)	Multiple	Navitoclax	Dual Bcl-2/Bcl-xL inhibitor	Phase I/II	Under investigation
	Acute Myeloid Leukemia (AML)	NCT03263936	Decitabine + Vorinostat	Epigenetic modulation, sensitizes to ferroptosis	Phase I	Well-tolerated, effective in R/R AML
	Acute Myeloid Leukemia (AML)	NCT03971019	Statins	Reduce GPX4, induce ferroptosis	Phase III	Large scale evaluation
	Acute Myeloid Leukemia (AML)	NCT04383938	APR-246 (Eprenetapopt)	GSH depletion, p53 targeting	Phase I, Completed	Multiple cancer types
	Acute Myeloid Leukemia (AML)	NCT03913949	APG-2575	Selective Bcl-2 inhibitor	Phase I, Recruiting	AML and NHL
	Lymphoma (NHL)	NCT02079740	Navitoclax	Dual Bcl-2/Bcl-xL inhibitor	Phase I/II, Recruiting	Under evaluation
	Lymphoma (NHL)	NCT02143401	Navitoclax	Bcl-2/Bcl-xL inhibitor	Phase I, Active	Ongoing
	Lymphoma (NHL)	NCT02603445	S55746 (BCL201)	Selective Bcl-2 inhibitor	Phase I, Completed	Completed evaluation
	Lymphoma/Multiple Myeloma	NCT05493800	MIT-001	Ferroptosis inhibitor, prevents oral mucositis	Phase II, Active	Ongoing, launched Aug 2022
	Multiple Myeloma	NCT02920697	S55746 (BCL201)	Selective Bcl-2 inhibitor	Phase I, Completed	Safety and efficacy
	Multiple Myeloma	NCT02098161	LCL161	IAP inhibitor, SMAC mimetic	Phase II, Completed	Apoptosis induction
	Myelodysplastic Syndromes (MDS)	NCT03745716	Eprenetapopt	Targets p53-mutant cells, induces ferroptosis	Phase III, Completed	Evaluated in MDS
	Myelodysplastic Syndromes (MDS)	NCT02098343	Sulfasalazine	System Xc- inhibitor, depletes GPX4	Phase I/II, Completed	Ferroptosis induction
	Colorectal Cancer	NCT02353026	Artesunate	GPX4 inactivation via GSH depletion, ferroptosis	Phase I, Completed	Well-tolerated, promising
	Colorectal Cancer	NCT04098744	Artesunate	Ferroptosis inducer	Phase II, Recruiting	Stage II/III CRC
	Colorectal Cancer	NCT02098161	LCL161	IAP inhibitor	Phase II, Completed	Apoptosis pathway
	Colorectal Cancer	NCT04229992	Magnesium glycinate	Modulates necroptosis and inflammation	Not applicable, Active	Under investigation
	Colorectal Adenoma	NCT02965703	Aspirin	MLKL-mediated necroptosis	Phase II, Active	Ongoing
	Pancreatic Ductal Adenocarcinoma (PDAC)	Multiple	Sorafenib	GPX4 inactivation, ferroptosis induction	Clinical studies	Evaluated efficacy
	Pancreatic Cancer	NCT03681951	Pembrolizumab + GSK3145095	RIPK1 inhibitor + immunotherapy	Phase II, Terminated	Early termination
	Ovarian Cancer	NCT02269293	Selinexor	Decreases NF-κB, induces apoptosis	Phase I, Completed	Completed evaluation
	Ovarian Cancer (High-grade)	NCT04383938	Eprenetapopt	p53 targeting, GSH depletion	Phase I/II, Completed	Evaluated combination
	Bladder Cancer	NCT04383938	APR-246 (Eprenetapopt)	GSH depletion, ferroptosis	Phase I, Completed	Multiple cancer types
	Cervical Cancer	NCT02633098	Artesunate	Ferroptosis induction	Phase II, Recruiting	Under investigation
	Head and Neck Squamous Cell Carcinoma	NCT03803774	Birinapant + IMRT	IAP inhibitor + radiation therapy	Phase I, Recruiting	Combination therapy
	Osteosarcoma	NCT01464606	Actinomycin-D + Doxorubicin + Topotecan + Bleomycin	Multiple mechanisms including ferroptosis	Not applicable, Active	Multi-agent chemotherapy
	Neuroblastoma	NCT00064350	Statins	Reduce GPX4 and CoQ10	Phase II, Completed	Completed evaluation
	Neuroblastoma	NCT02340845	Bromelain	Upregulation of ACSL4, ferroptosis inducer	Phase II, Unknown	Status unknown
	Solid Tumors (Advanced)	NCT03595059	ABBV-155	Bcl-xL inhibitor	Phase I, Recruiting	Pan-cancer evaluation
	Solid Tumors	NCT04214860	Eprenetapopt	p53 targeting, ferroptosis	Phase I, Completed	Broad cancer types
Cardiovascular	Cardiac Iron Overload	NCT01254227	Deferoxamine (DFO)	Iron chelator, inhibits iron overload and ferroptosis	Phase II, Completed	Improved cardiac function
	Iron Overload Cardiomyopathy	NCT00800761	Deferiprone	Iron chelator, prevents ferroptosis	Phase IV, Completed	Reduced cardiac iron deposition
	Heart Defects, Congenital	NCT02519335	Dexrazoxane	Inhibits iron overload	Phase I, Terminated	Safety concerns, terminated
	Heart Failure	NCT03676296	Puerarin	Reduces ROS and NOX4 production	Phase II, Completed	Cardioprotective effects
	Coronary Artery Disease	NCT00554203	Sulfasalazine	System Xc- inhibitor	Not applicable, Completed	Evaluated cardiovascular effects
	Myocardial Infarction	NCT00390832	Erythropoietin	Activates PI3K/Akt pathway, anti-ferroptosis	Phase III, Completed	Cardioprotection evaluated
	Acute Ischemic Stroke	NCT00821821	MCI-186 (Edaravone)	Free radical scavenger, inhibits ferroptosis	Phase II, Completed	Neuroprotective in stroke
	Ischemic Stroke	NCT03320018	Minocycline	Alters mitochondrial membrane, reduces apoptosis	Phase II/III, Recruiting	Neuroprotection

HCC Hepatocellular carcinoma, RCC renal cell carcinoma, AML acute myeloid leukemia, NHL non-Hodgkin lymphoma, NSCLC non-small cell lung cancer, SCLC small cell lung cancer, ALS amyotrophic lateral sclerosis, GBM glioblastoma, MDS myelodysplastic syndromes, PDAC pancreatic ductal adenocarcinoma, CRC colorectal cancer, SN substantia nigra, R/R relapsed/refractory, OS overall survival, mPFS median progression-free survival, GPX4 glutathione peroxidase 4, GSH glutathione, ROS reactive oxygen species, CoQ10 coenzyme Q10, TKI tyrosine kinase inhibitor, SRS stereotactic radiosurgery, IAP inhibitor of apoptosis proteins, SMAC second mitochondria-derived activator of caspases, IMRT intensity-modulated radiation therapy, FINs ferroptosis-inducing compounds, DFO deferoxamine, NAC N-acetyl cysteine

Challenges in clinical translation of epigenetic and PTM-targeting therapies

Despite the promising preclinical results, several challenges hinder the clinical translation of epigenetic and PTM-targeting therapies for ferroptosis modulation. First, off-target effects remain a major concern, as many epigenetic modifiers and PTM enzymes regulate multiple cellular processes beyond ferroptosis. For instance, epigenetic drugs HDAC inhibitors modulate transcription factors (p53, NF-κB), structural proteins (α-tubulin), and metabolic enzymes (GAPDH, PKM2) that extend beyond ferroptosis-related genes, potentially leading to unintended transcriptional reprogramming [202]. Additionally, certain kinase inhibitors exhibit higher binding affinity for abundant cellular kinases (e.g., mTOR, MAPK, AMPK) compared to ferroptosis-regulatory kinases, resulting in preferential off-target engagement [203] that diminishes their efficacy in modulating GPX4 or SLC7A11 phosphorylation and thereby compromising ferroptosis induction [204]. Second, bioavailability of many small-molecule inhibitors is limited due to poor solubility, rapid metabolism, and inadequate tissue penetration, particularly across the blood–brain barrier [205]. Third, patient stratification represents a critical challenge, as ferroptosis sensitivity varies significantly based on tumor genetics, metabolic status, and immune infiltration patterns [206]. Identifying predictive biomarkers such as GPX4 expression levels [207], iron status [208], and specific epigenetic signatures will be essential for selecting patients most likely to benefit from these therapies.

Targeted ferroptosis therapy

Targeted ferroptosis therapy represents a paradigm shift in cancer treatment, leveraging the unique metabolic vulnerabilities of malignant cells to selectively induce iron-dependent cell death. This therapeutic approach exploits the distinct iron metabolism, redox homeostasis, and lipid composition differences between cancer cells and normal tissues [209, 210].

Therapeutic strategies for ferroptosis induction

System xc⁻ inhibition

Targeting the cystine/glutamate antiporter system xc⁻ represents one of the most established approaches to ferroptosis induction. Sulfasalazine, an FDA-approved anti-inflammatory drug, inhibits system xc⁻ by competing with cystine for binding sites, thereby depleting cellular glutathione and sensitizing cells to ferroptosis [211]. Clinical studies have demonstrated the feasibility of repurposing sulfasalazine for cancer treatment, with manageable side effects and evidence of anti-tumor activity.

Erastin, the prototypical ferroptosis inducer, functions through potent and selective inhibition of system xc⁻. While erastin itself has limited clinical utility due to poor pharmacokinetic properties, numerous erastin derivatives and analogs have been developed with improved drug-like characteristics [212]. These second-generation compounds maintain the ferroptosis-inducing activity while offering enhanced stability, bioavailability, and tumor selectivity.

Direct GPX4 inhibition

GPX4 represents the master regulator of ferroptosis defense, making it an attractive therapeutic target. RSL3 (RAS-selective lethal 3) functions as a direct, covalent inhibitor of GPX4 by binding to its active site selenocysteine residue [59]. This mechanism of action makes RSL3 particularly effective at inducing ferroptosis even in cells with functional system xc⁻.

ML162, another direct GPX4 inhibitor, has shown promising results in preclinical models of cancer treatment. The compound demonstrates selectivity for cancer cells over normal cells, likely due to differences in baseline oxidative stress and antioxidant capacity [213]. However, the development of clinically viable GPX4 inhibitors remains challenging due to the essential role of GPX4 in normal cellular homeostasis.

Iron metabolism targeting

Modulation of cellular iron levels provides another avenue for ferroptosis induction. Iron supplementation using compounds such as ferric ammonium citrate (FAC) or iron nanoparticles can promote ferroptosis by increasing the availability of catalytic iron for Fenton chemistry [214]. However, systemic iron loading carries significant risks, necessitating targeted delivery approaches.

Transferrin-conjugated iron delivery systems have been developed to selectively increase iron levels in cancer cells that overexpress transferrin receptors. These targeted approaches minimize systemic iron toxicity while effectively inducing ferroptosis in malignant cells [11].

Combination therapy approaches

Ferroptosis and immunotherapy

The combination of ferroptosis induction with immunotherapy has emerged as a particularly promising therapeutic strategy. Polycomb repressive complex 2 (PRC2), particularly EZH2-mediated H3K27me3, simultaneously suppresses ferroptosis executors (ACSL4, ALOX15) while promoting PD-L1 transcription through epigenetic silencing of negative regulators, creating dual therapy resistance [215, 216]. Conversely, EZH2 inhibitors (tazemetostat) restore both ferroptosis sensitivity and reduce PD-L1 expression, mechanistically explaining synergy with anti-PD-1 antibodies. At the PTM level, SIRT1 deacetylase modifies both NRF2 (enhancing its transcriptional activity and ferroptosis resistance) and PD-L1 (stabilizing protein levels and immune evasion), representing a common regulatory node [217]. Furthermore, NRF2—the master antioxidant transcription factor conferring ferroptosis resistance—directly transactivates PD-L1 through antioxidant response elements in its promoter, mechanistically coupling metabolic stress responses with immune checkpoint upregulation [218]. This shared regulatory architecture explains why ferroptosis inducers that deplete GSH and inactivate NRF2 simultaneously reduce PD-L1 expression, enhancing T cell-mediated killing. These molecular links establish that ferroptosis and immune evasion are not independent processes but rather coordinately regulated through convergent epigenetic and PTM pathways, providing a clear mechanistic foundation for combination strategies.

Ferroptosis and radiotherapy

Radiation therapy combined with ferroptosis induction demonstrates enhanced therapeutic efficacy through multiple mechanisms. Radiation-induced DNA damage can deplete cellular antioxidant systems, rendering cells more susceptible to ferroptotic death [219]. Conversely, ferroptosis inducers can sensitize cancer cells to radiation by compromising their DNA repair capacity and increasing oxidative stress.

Preclinical studies have demonstrated that combining ferroptosis inducers with radiation therapy can overcome radioresistance in various cancer types. The optimal sequencing and dosing of these combinations are subjects of ongoing clinical investigation [220, 221].

Ferroptosis and chemotherapy

Conventional chemotherapeutic agents can be combined with ferroptosis inducers to enhance treatment efficacy and overcome drug resistance. Many chemotherapy drugs induce oxidative stress and deplete antioxidant systems, creating a cellular environment conducive to ferroptosis [222]. For example, cisplatin treatment can reduce glutathione levels and GPX4 activity, sensitizing cells to ferroptosis induction.

The combination of ferroptosis inducers with targeted therapies has also shown promise. EGFR inhibitors combined with ferroptosis inducers have demonstrated enhanced activity in lung cancer models, with the EGFR inhibition disrupting survival signaling while ferroptosis induction provides direct cytotoxic effects [223].

Precision medicine approaches

Biomarker-driven patient selection

The success of targeted ferroptosis therapy depends critically on identifying patients whose tumors are most likely to respond to treatment. Ferroptosis susceptibility biomarkers including low GPX4 expression, high transferrin receptor levels, and elevated ACSL4 expression have been associated with improved response to ferroptosis-inducing therapies [224].

Genomic biomarkers such as mutations in p53, KEAP1, and NRF2 can also predict ferroptosis sensitivity. p53 mutations that impair antioxidant gene expression may sensitize tumors to ferroptosis, while NRF2 activation through KEAP1 mutations may confer resistance [225].

Metabolic profiling

Metabolomic approaches to assess tumor ferroptosis susceptibility are being developed for clinical applications. Tumor levels of glutathione, cysteine, and iron-containing metabolites can provide information about baseline ferroptosis vulnerability [226]. Additionally, lipid profiling to assess polyunsaturated fatty acid content and membrane composition may predict ferroptosis sensitivity.

Resistance mechanisms and overcoming strategies

Adaptive resistance

Cancer cells can develop resistance to ferroptosis through various adaptive mechanisms. Upregulation of antioxidant systems, including increased GPX4 expression, enhanced glutathione synthesis, and activation of NRF2 signaling, represents common resistance mechanisms [227]. Understanding these adaptive responses is crucial for developing strategies to overcome or prevent resistance.

Metabolic reprogramming, including increased fatty acid desaturation and altered lipid composition, can also confer ferroptosis resistance. Cancer cells may reduce their content of polyunsaturated fatty acids or increase monounsaturated fatty acid synthesis to reduce membrane susceptibility to peroxidation [228].

Combination strategies to overcome resistance

Rational combination approaches can overcome ferroptosis resistance by targeting multiple pathways simultaneously. Combining system xc⁻ inhibitors with GPX4 inhibitors can prevent adaptive upregulation of alternative defense pathways [229]. Similarly, combining ferroptosis inducers with inhibitors of antioxidant response pathways, such as NRF2 inhibitors, can enhance and sustain ferroptotic cell death.

Clinical translation challenges and future directions

Cell-type-specific targeting within the tumor immune microenvironment

A fundamental challenge in ferroptosis-based cancer therapy lies in achieving selective induction in malignant cells while preserving anti-tumor immune cells. This is particularly critical as CD8⁺ T cells demonstrate heightened ferroptosis susceptibility due to PD-1 signaling and impaired phospholipid homeostasis [230], while certain immunosuppressive cells exhibit resistance through Nrf2-dependent antioxidant systems [147, 231].

Several strategies are emerging to address this challenge. Tumor antigen-directed delivery using antibody–drug conjugates (ADCs) targeting tumor-specific markers (HER2, EGFR, CD19) can selectively deliver ferroptosis inducers to cancer cells while sparing immune infiltrates [232]. Tumor immune microenvironment-responsive nanoparticles that release drugs under specific pH, hypoxia, or enzymatic conditions (e.g., MMP activity) achieve spatial selectivity [233]. Temporal modulation—administering ferroptosis inducers after immune checkpoint blockade when T cells have expanded—can minimize impact on anti-tumor immunity. Additionally, metabolic vulnerability-based targeting exploits cancer cells’ heightened dependence on specific pathways (e.g., glutaminolysis in c-Myc-driven tumors) for selective ferroptosis induction [234].

Predictive biomarkers for patient stratification

Robust predictive biomarkers are essential for rational patient selection. Protein-based biomarkers combining low GPX4 with high ACSL4 expression predict ferroptosis sensitivity across multiple cancer types [235]. Immunohistochemical scoring integrating GPX4, SLC7A11, and FSP1 identifies “ferroptosis-vulnerable” tumors [236–238]. Metabolomic biomarkers including baseline GSH/GSSG ratios and elevated PUFA-containing phospholipids indicate ferroptosis susceptibility [239]. Genetic biomarkers such as TP53 mutations and KEAP1/NRF2 alterations modulate ferroptosis sensitivity [240]. Epigenetic signatures including DNA methylation patterns at ferroptosis regulator promoters provide complementary predictive information [3].

Safety challenges and mitigation strategies

Ferroptosis inducers pose potential toxicity risks to highly metabolic normal tissues, particularly liver, kidneys, and heart, which exhibit intrinsic vulnerability due to elevated mitochondrial activity, active lipid metabolism, and high iron utilization [241, 242]. Cardiac tissue is similarly vulnerable, as doxorubicin-induced cardiotoxicity is partially mediated through ferroptosis, demonstrating the risk of myocardial lipid peroxidation [243]. Ferroptosis inducers cause acute liver damage characterized by elevated ALT/AST levels, increased lipid peroxidation, and reduced GPx4 expression, which can be significantly reduced by ferrostatin-1 treatment [244].

Several mitigation strategies can minimize normal tissue toxicity: (1) Tumor-Targeted Delivery: Nanoparticles have been developed that stay inactive in normal tissues but become active in the acidic tumor environment, selectively killing tumor cells while minimizing harm to normal tissues [245]; (2) Protective Co-treatment: Liproxstatin-1 can protect multiple organs by reducing liver enzyme levels (ALT), kidney markers (creatinine), and heart damage markers (troponin T) without compromising anti-tumor effects [246]; (3) Real-Time Monitoring: Regular monitoring of liver enzymes (ALT/AST), kidney function (creatinine), and heart damage markers (troponin T) allows early detection of toxicity and timely dose adjustment [246]. These approaches are essential for safely using ferroptosis-targeted therapies in cancer treatment.

Advanced delivery systems as solutions

Sophisticated delivery platforms address multiple challenges including tumor selectivity, systemic toxicity, and pharmacokinetic limitations. Nanoparticle systems such as iron oxide nanoparticles provide dual functionality (iron for Fenton chemistry plus drug delivery), while mesoporous silica nanoparticles and liposomes improve tumor accumulation and reduce off-target effects [247, 248].

Stimuli-responsive systems release cargo specifically in tumor tissues: pH-responsive carriers exploit tumor acidity [233], redox-responsive systems are activated by elevated tumor glutathione [249], and enzyme-responsive systems leverage tumor-associated proteases (e.g., MMPs) [250]. Dual-responsive platforms combining multiple triggers provide enhanced selectivity.

Antibody–drug conjugates targeting tumor antigens enable receptor-mediated internalization in cancer cells [232]. Exosome-based delivery offers biocompatibility and barrier-crossing capability [249]. Combination delivery platforms co-delivering ferroptosis inducers with immunotherapy agents (e.g., RSL3 plus anti-PD-L1) achieve synergistic effects [251]. Several nanoparticle-based ferroptosis therapies have entered early-phase clinical development, including carbon nanoparticle-loaded iron formulations for intratumoral injection in solid tumors (NCT06048367).

Multi-omics technologies for deciphering ferroptosis heterogeneity

The integration of multi-omics technologies is revolutionizing ferroptosis research within the tumor immune microenvironment (TIME). Single-cell RNA sequencing (scRNA-seq) has identified ferroptosis-resistant cancer cell subpopulations with upregulated GPX4 and SLC7A11 expression, revealing intratumoral heterogeneity that explains therapy resistance [252]. Spatial transcriptomics combined with multiplexed imaging maps ferroptosis regulator expression across different tumor regions, demonstrating that hypoxic niches harbor ferroptosis-resistant cells with elevated FSP1 and DHODH [25]. Lipidomics profiling has revealed striking heterogeneity in PUFA-containing phospholipids (e.g., arachidonoyl-PE, adrenoyl-PE) across tumor cell subpopulations, with ACSL4-high cells accumulating ferroptosis-prone lipid species [30]. Integrated multi-omics approaches combining genomics, transcriptomics, and metabolomics have identified predictive signatures; for instance, tumors with TP53 mutations, high SLC7A11 expression, and elevated GSH levels exhibit ferroptosis resistance [213]. These technologies enable precision patient stratification and rational therapy design for ferroptosis-based cancer treatment.

Conclusion and future perspectives

The interface between epigenetic regulation, ferroptosis, and the tumor immune microenvironment (TIME) represents an emerging frontier in cancer research with significant therapeutic implications. This review has examined how diverse epigenetic mechanisms modulate ferroptosis sensitivity within the complex TIME and explored their impact on cancer progression and treatment response (Fig. 10).

Fig. 10.

Comprehensive overview of epigenetic and post-translational regulation of ferroptosis in the tumor immune microenvironment

Epigenetic modifications dynamically regulate expression of key ferroptosis mediators, including SLC7A11, GPX4, and ACSL4, thereby modulating cancer cell susceptibility to ferroptotic cell death. These regulatory processes respond continuously to TIME components, including hypoxia, metabolites, inflammatory cytokines, and cellular interactions. The bidirectional relationship between epigenetic regulation and the TIME establishes a complex network determining ferroptosis outcomes in cancer.

The therapeutic potential of targeting epigenetic regulation of ferroptosis within the TIME is increasingly recognized. Epigenetic modulators, including DNMT inhibitors, HDAC inhibitors, and BET inhibitors, have demonstrated efficacy in enhancing ferroptosis sensitivity in cancer cells. Combination strategies incorporating these epigenetic drugs with ferroptosis inducers and immunotherapeutic agents have exhibited synergistic effects in preclinical models, offering promising approaches for improving cancer treatment.

Translating these findings to clinical applications faces several challenges. Epigenetic alterations exhibit marked heterogeneity across cancer types and within individual tumors, necessitating personalized approaches. Development of reliable biomarkers for ferroptosis sensitivity and response to epigenetic therapies is essential for patient selection and treatment optimization. Additionally, addressing potential off-target effects of epigenetic drugs requires development of more selective agents.

Future research should focus on developing epigenetic modulators with enhanced selectivity and potency, identifying cancer-specific epigenetic vulnerabilities enabling selective ferroptosis induction in malignant cells while sparing normal tissues, and exploring rational combination strategies targeting multiple aspects of ferroptosis regulation and the TIME. Advanced technologies including artificial intelligence, single-cell epigenomics, spatial transcriptomics, and multi-omics integration will provide unprecedented insights into the complex interactions between epigenetic mechanisms, ferroptosis, and the TIME, guiding development of innovative therapeutic approaches [253].

The epigenetic regulation of ferroptosis within the TIME represents a promising frontier in cancer research. Understanding these complex interactions provides valuable insights for developing novel treatment strategies that harness ferroptosis’s therapeutic potential through epigenetic modulation. As research advances, we anticipate emerging therapeutic approaches that effectively target epigenetic regulation of ferroptosis within the TIME, ultimately improving cancer patient outcomes.

Abbreviations

ACSL4

Acyl-CoA synthetase long-chain family member 4

ATF4

Activating transcription factor 4

DHODH

Dihydroorotate dehydrogenase

FTH1

Ferritin heavy chain

FSP1

Ferroptosis suppressor protein 1

GCH1

GTP cyclohydrolase 1

GPX4

Glutathione peroxidase 4

GSH

Glutathione

KEAP1

Kelch-like ECH-associated protein 1

LPCAT3

Lysophosphatidylcholine acyltransferase 3

LSD1

Lysine-specific demethylase 1

NRF2

Nuclear factor erythroid 2-related factor 2

PD-1

Programmed cell death protein 1

PD-L1

Programmed death-ligand 1

PTGS2

Prostaglandin-endoperoxide synthase 2

SCD1

Stearoyl-CoA desaturase 1

SIRT1

Sirtuin 1

SLC3A2

Solute carrier family 3 member 2

SLC7A11

Solute carrier family 7 member 11

STAT3

Signal transducer and activator of transcription 3

TFRC

Transferrin receptor

Author contributions

LD planned, designed, and wrote the majority of the manuscript. YZ, JL and CH helped collecting information. JL and XC planned and guided the project and wrote the manuscript. All authors reviewed the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (82473945), Taizhou University Medicine Special Project (2023XY01), Taizhou Anti-Cancer Association Cancer special research project (TACA2025-A02).

Data availability

Not applicable.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

The content of this manuscript has not been previously published and is not under consideration for publication elsewhere.

Competing interests

The authors declare no competing interests.