Hepatic SEL1L-HRD1 ER-associated degradation regulates systemic iron homeostasis via ceruloplasmin.

Iron homeostasis is critical for cellular and organismal function and is tightly regulated to prevent toxicity or anemia due to iron excess or deficiency, respectively. However, subcellular regulatory mechanisms of iron remain largely unexplored. Here, we report that SEL1L-HRD1 protein complex of endoplasmic reticulum (ER)-associated degradation (ERAD) in hepatocytes controls systemic iron homeostasis in a ceruloplasmin (CP)-dependent, and ER stress-independent, manner. Mice with hepatocyte-specific Sel1L deficiency exhibit altered basal iron homeostasis and are sensitized to iron deficiency while resistant to iron overload. Proteomics screening for a factor linking ERAD deficiency to altered iron homeostasis identifies CP, a key ferroxidase involved in systemic iron distribution by catalyzing iron oxidation and efflux from tissues. Indeed, CP is highly unstable and a bona fide substrate of SEL1L-HRD1 ERAD. In the absence of ERAD, CP protein accumulates in the ER and is shunted to refolding, leading to elevated secretion. Providing clinical relevance of these findings, SEL1L-HRD1 ERAD is responsible for the degradation of a subset of disease-causing CP mutants, thereby attenuating their pathogenicity. Together, this study uncovers the role of SEL1L-HRD1 ERAD in systemic iron homeostasis and provides insights into protein misfolding-associated proteotoxicity.

PTEN-induced kinase PINK1 supports colorectal cancer growth by regulating the labile iron pool.

Mitophagy is a cargo-specific autophagic process that recycles damaged mitochondria to promote mitochondrial turnover. PTEN-induced putative kinase 1 (PINK1) mediates the canonical mitophagic pathway. However, the role of PINK1 in diseases where mitophagy has been purported to play a role, such as colorectal cancer, is unclear. Our results here demonstrate that higher PINK1 expression is positively correlated with decreased colon cancer survival, and mitophagy is required for colon cancer growth. We show that doxycycline-inducible knockdown (KD) of PINK1 in a panel of colon cancer cell lines inhibited proliferation, whereas disruption of other mitophagy receptors did not impact cell growth. We observed that PINK KD led to a decrease in mitochondrial respiration, membrane hyperpolarization, accumulation of mitochondrial DNA, and depletion of antioxidant glutathione. In addition, mitochondria are important hubs for the utilization of iron and synthesizing iron-dependent cofactors such as heme and iron sulfur clusters. We observed an increase in the iron storage protein ferritin and a decreased labile iron pool in the PINK1 KD cells, but total cellular iron or markers of iron starvation/overload were not affected. Finally, cellular iron storage and the labile iron pool are maintained via autophagic degradation of ferritin (ferritinophagy). We found overexpressing nuclear receptor coactivator 4, a key adaptor for ferritinophagy, rescued cell growth and the labile iron pool in PINK1 KD cells. These results indicate that PINK1 integrates mitophagy and ferritinophagy to regulate intracellular iron availability and is essential for maintaining intracellular iron homeostasis to support survival and growth in colorectal cancer cells.

Renal-specific loss of ferroportin disrupts iron homeostasis and attenuates recovery from acute kidney injury.

Chronic kidney disease is increasing at an alarming rate and correlates with the increase in diabetes, obesity, and hypertension that disproportionately impact socioeconomically disadvantaged communities. Iron plays essential roles in many biological processes including oxygen transport, mitochondrial function, cell proliferation, and regeneration. However, excess iron induces the generation and propagation of reactive oxygen species, which lead to oxidative stress, cellular damage, and ferroptosis. Iron homeostasis is regulated in part by the kidney through iron resorption from the glomerular filtrate and exports into the plasma by ferroportin (FPN). Yet, the impact of iron overload in the kidney has not been addressed. To test more directly whether excess iron accumulation is toxic to kidneys, we generated a kidney proximal tubule-specific knockout of FPN. Despite significant intracellular iron accumulation in FPN mutant tubules, basal kidney function was not measurably different from wild type kidneys. However, upon induction of acute kidney injury (AKI), FPN mutant kidneys exhibited significantly more damage and failed recovery, evidence for ferroptosis, and increased fibrosis. Thus, disruption of iron export in proximal tubules, leading to iron overload, can significantly impair recovery from AKI and can contribute to progressive renal damage indicative of chronic kidney disease. Understanding the mechanisms that regulate iron homeostasis in the kidney may provide new therapeutic strategies for progressive kidney disease and other ferroptosis-associated disorders.NEW & NOTEWORTHY Physiological iron homeostasis depends in part on renal resorption and export into the plasma. We show that specific deletion of iron exporters in the proximal tubules sensitizes cells to injury and inhibits recovery. This can promote a chronic kidney disease phenotype. Our paper demonstrates the need for iron balance in the proximal tubules to maintain and promote healthy recovery after acute kidney injury.

Modulation of the HIF2α-NCOA4 axis in enterocytes attenuates iron loading in a mouse model of hemochromatosis.

Intestinal iron absorption is activated during increased systemic demand for iron. The best-studied example is iron deficiency anemia, which increases intestinal iron absorption. Interestingly, the intestinal response to anemia is very similar to that of iron overload disorders, as both the conditions activate a transcriptional program that leads to a hyperabsorption of iron via the transcription factor hypoxia-inducible factor 2α (HIF2α). However, pathways for selective targeting of intestine-mediated iron overload remain unknown. Nuclear receptor coactivator 4 (NCOA4) is a critical cargo receptor for autophagic breakdown of ferritin and the subsequent release of iron, in a process termed ferritinophagy. Our work demonstrates that NCOA4-mediated intestinal ferritinophagy is integrated into systemic iron demand via HIF2α. To demonstrate the importance of the intestinal HIF2α/ferritinophagy axis in systemic iron homeostasis, whole-body and intestine-specific NCOA4-/- mouse lines were generated and assessed. The analyses revealed that the intestinal and systemic response to iron deficiency was not altered after disruption of intestinal NCOA4. However, in a mouse model of hemochromatosis, ablation of intestinal NCOA4 was protective against iron overload. Therefore, NCOA4 can be selectively targeted for the management of iron overload disorders without disrupting the physiological processes involved in the response to systemic iron deficiency.

Dietary Iron Is Necessary to Support Proliferative Regeneration after Intestinal Injury.

BACKGROUND: Tissue repair and regeneration in the gastrointestinal system are crucial for maintaining homeostasis, with the process relying on intricate cellular interactions and affected by micro- and macro-nutrients. Iron, essential for various biological functions, plays a dual role in tissue healing by potentially causing oxidative damage and participating in anti-inflammatory mechanisms, underscoring its complex relationship with inflammation and tissue repair. OBJECTIVE: The study aimed to elucidate the role of low dietary iron in gastrointestinal tissue repair. METHODS: We utilized quantitative iron measurements to assess iron levels in inflamed regions of patients with ulcerative colitis and Crohn's disease. In addition, 3 mouse models of gastrointestinal injury/repair (dextran sulfate sodium-induced colitis, radiation injury, and wound biopsy) were used to assess the effects of low dietary iron on tissue repair. RESULTS: We found that levels of iron in inflamed regions of both patients with ulcerative colitis and Crohn's disease are elevated. Similarly, during gastrointestinal repair, iron levels were found to be heightened, specifically in intestinal epithelial cells across the 3 injury/repair models. Mice on a low-iron diet showed compromised tissue repair with reduced proliferation. In standard diet, epithelial cells and the stem cell compartment maintain adequate iron stores. However, during a period of iron deficiency, epithelial cells exhaust their iron reserves, whereas the stem cell compartments maintain their iron pools. During injury, when the stem compartment is disrupted, low iron levels impair proliferation and compromise repair mechanisms. CONCLUSIONS: Low dietary iron impairs intestinal repair through compromising the ability of epithelial cells to aid in intestinal proliferation.

Impact of dietary manganese on experimental colitis in mice.

Diet plays a significant role in the pathogenesis of inflammatory bowel disease (IBD). A recent epidemiological study has shown an inverse relationship between nutritional manganese (Mn) status and IBD patients. Mn is an essential micronutrient required for normal cell function and physiological processes. To date, the roles of Mn in intestinal homeostasis remain unknown and the contribution of Mn to IBD has yet to be explored. Here, we provide evidence that Mn is critical for the maintenance of the intestinal barrier and that Mn deficiency exacerbates dextran sulfate sodium (DSS)-induced colitis in mice. Specifically, when treated with DSS, Mn-deficient mice showed increased morbidity, weight loss, and colon injury, with a concomitant increase in inflammatory cytokine levels and oxidative and DNA damage. Even without DSS treatment, dietary Mn deficiency alone increased intestinal permeability by impairing intestinal tight junctions. In contrast, mice fed a Mn-supplemented diet showed slightly increased tolerance to DSS-induced experimental colitis, as judged by the colon length. Despite the well-appreciated roles of intestinal microbiota in driving inflammation in IBD, the gut microbiome composition was not altered by changes in dietary Mn. We conclude that Mn is necessary for proper maintenance of the intestinal barrier and provides protection against DSS-induced colon injury.

SMAD family member 3 (SMAD3) and SMAD4 repress HIF2α-dependent iron-regulatory genes.

Hypoxia-inducible factor 2α (HIF2α) directly regulates a battery of genes essential for intestinal iron absorption. Interestingly, iron deficiency and overload disorders do not result in increased intestinal expression of glycolytic or angiogenic HIF2α target genes. Similarly, inflammatory and tumor foci can induce a distinct subset of HIF2α target genes in vivo These observations indicate that different stimuli activate distinct subsets of HIF2α target genes via mechanisms that remain unclear. Here, we conducted a high-throughput siRNA-based screen to identify genes that regulate HIF2α's transcriptional activity on the promoter of the iron transporter gene divalent metal transporter-1 (DMT1). SMAD family member 3 (SMAD3) and SMAD4 were identified as potential transcriptional repressors. Further analysis revealed that SMAD4 signaling selectively represses iron-absorptive gene promoters but not the inflammatory or glycolytic HIF2α or HIF1α target genes. Moreover, the highly homologous SMAD2 did not alter HIF2α transcriptional activity. During iron deficiency, SMAD3 and SMAD4 expression was significantly decreased via proteasomal degradation, allowing for derepression of iron target genes. Several iron-regulatory genes contain a SMAD-binding element (SBE) in their proximal promoters; however, mutation of the putative SBE on the DMT1 promoter did not alter the repressive function of SMAD3 or SMAD4. Importantly, the transcription factor forkhead box protein A1 (FOXA1) was critical in SMAD4-induced DMT1 repression, and DNA binding of SMAD4 was essential for the repression of HIF2α activity, suggesting an indirect repressive mechanism through DNA binding. These results provide mechanistic clues to how HIF signaling can be regulated by different cellular cues.

Ferritinophagy is not required for colon cancer cell growth.

Ferritinophagy is a form of selective autophagy responsible for degrading intracellular ferritin, mediated by nuclear receptor coactivator 4 (NCOA4). NCOA4 plays significant roles in systemic iron homeostasis, and its disruption leads to simultaneous anemia and susceptibility to iron overload. The importance of iron colorectal cancer pathogenesis is well studied; however, the role of ferritinophagy in colon cancer cell growth has not been assessed. Disruption of ferritinophagy via NCOA4 knockout leads to only marginal differences in growth under basal and iron-restricted conditions. Moreover, NCOA4 played no significant role in cell death induced by 5-fluorouracil and erastin. Western blotting analysis for ferritin and transferrin receptor 1 found a dose-dependent effect on expression in both proteins in wild-type and NCOA4 knockout cell lines, but further investigation revealed no difference in growth response when treated at both high and low doses. Our data demonstrate a marginal role for ferritinophagy in growth both under normal and cytotoxic conditions in colon cancer cells, as well as a possible compensatory mechanism in colon cancer cells in response to ferroptosis induction.

Computational modeling and analysis of iron release from macrophages.

A major process of iron homeostasis in whole-body iron metabolism is the release of iron from the macrophages of the reticuloendothelial system. Macrophages recognize and phagocytose senescent or damaged erythrocytes. Then, they process the heme iron, which is returned to the circulation for reutilization by red blood cell precursors during erythropoiesis. The amount of iron released, compared to the amount shunted for storage as ferritin, is greater during iron deficiency. A currently accepted model of iron release assumes a passive-gradient with free diffusion of intracellular labile iron (Fe2+) through ferroportin (FPN), the transporter on the plasma membrane. Outside the cell, a multi-copper ferroxidase, ceruloplasmin (Cp), oxidizes ferrous to ferric ion. Apo-transferrin (Tf), the primary carrier of soluble iron in the plasma, binds ferric ion to form mono-ferric and di-ferric transferrin. According to the passive-gradient model, the removal of ferrous ion from the site of release sustains the gradient that maintains the iron release. Subcellular localization of FPN, however, indicates that the role of FPN may be more complex. By experiments and mathematical modeling, we have investigated the detailed mechanism of iron release from macrophages focusing on the roles of the Cp, FPN and apo-Tf. The passive-gradient model is quantitatively analyzed using a mathematical model for the first time. A comparison of experimental data with model simulations shows that the passive-gradient model cannot explain macrophage iron release. However, a facilitated-transport model associated with FPN can explain the iron release mechanism. According to the facilitated-transport model, intracellular FPN carries labile iron to the macrophage membrane. Extracellular Cp accelerates the oxidation of ferrous ion bound to FPN. Apo-Tf in the extracellular environment binds to the oxidized ferrous ion, completing the release process. Facilitated-transport model can correctly predict cellular iron efflux and is essential for physiologically relevant whole-body model of iron metabolism.

A diet of oxidative stress-adapted bacteria improves stress resistance and lifespan in C. elegans via p38-MAPK.

Organisms across taxa face stresses including variable temperature, redox imbalance, and xenobiotics. Successfully responding to stress and restoring homeostasis are crucial for survival. Aging is associated with a decreased stress response and alterations in the microbiome, which contribute to disease development. Animals and their microbiota share their environment; however, microbes have short generation time and can rapidly evolve and potentially affect host physiology during stress. Here, we leverage Caenorhabditis elegans and its simplified bacterial diet to demonstrate how microbial adaptation to oxidative stress affects the host's lifespan and stress response. We find that worms fed stress-evolved bacteria exhibit enhanced stress resistance and an extended lifespan. Through comprehensive genetic and metabolic analysis, we find that iron in stress-evolved bacteria enhances worm stress resistance and lifespan via activation of the mitogen-activated protein kinase pathway. In conclusion, our study provides evidence that understanding microbial stress-mediated adaptations could be used to slow aging and alleviate age-related health decline.

Activation of Intestinal HIF2α Ameliorates Iron-Refractory Anemia.

In clinics, hepcidin levels are elevated in various anemia-related conditions, particularly in iron-refractory anemia and in high inflammatory states that suppress iron absorption, which remains an urgent unmet medical need. To identify effective treatment options for various types of iron-refractory anemia, the potential effect of hypoxia and pharmacologically-mimetic drug FG-4592 (Roxadustat) are evaluated, a hypoxia-inducible factor (HIF)-prolyl hydroxylase (PHD) inhibitor, on mouse models of iron-refractory iron-deficiency anemia (IRIDA), anemia of inflammation and 5-fluorouracil-induced chemotherapy-related anemia. The potent protective effects of both hypoxia and FG-4592 on IRIDA as well as other 2 tested mouse cohorts are found. Mechanistically, it is demonstrated that hypoxia or FG-4592 could stabilize duodenal Hif2α, leading to the activation of Fpn transcription regardless of hepcidin levels, which in turn results in increased intestinal iron absorption and the amelioration of hepcidin-activated anemias. Moreover, duodenal Hif2α overexpression fully rescues phenotypes of Tmprss6 knockout mice, and Hif2α knockout in the gut significantly delays the recovery from 5-fluorouracil-induced anemia, which can not be rescued by FG-4592 treatment. Taken together, the findings of this study provide compelling evidence that targeting intestinal hypoxia-related pathways can serve as a potential therapeutic strategy for treating a broad spectrum of anemia, especially iron refractory anemia.

Recharacterization of RSL3 reveals that the selenoproteome is a druggable target in colorectal cancer.

Ferroptosis is an iron-dependent, non-apoptotic form of cell death resulting from the accumulation of lipid peroxides. Colorectal cancer (CRC) accumulates high levels of intracellular iron and reactive oxygen species (ROS), thereby sensitizing cells to ferroptosis. The selenoprotein glutathione peroxidase (GPx4) is a key enzyme in the detoxification of lipid peroxides and can be inhibited by the compound (S)-RSL3 ([1S,3R]-RSL3). However, the stereoisomer (R)-RSL3 ([1R,3R]-RSL3), which does not inhibit GPx4, exhibits equipotent activity to (S)-RSL3 across a panel of CRC cell lines. Utilizing CRC cell lines with an inducible knockdown of GPx4, we demonstrate that (S)-RSL3 sensitivity does not align with GPx4 dependency. Subsequently, a biotinylated (S)-RSL3 was then synthesized to perform affinity purification-mass spectrometry (AP-MS), revealing that (S)-RSL3 acts as a pan-inhibitor of the selenoproteome, targeting both the glutathione and thioredoxin peroxidase systems as well as multiple additional selenoproteins. To investigate the therapeutic potential of broadly disrupting the selenoproteome as a therapeutic strategy in CRC, we employed further chemical and genetic approaches to disrupt selenoprotein function. The findings demonstrate that the selenoprotein inhibitor Auranofin can induce ferroptosis and/or oxidative cell death both in-vitro and in-vivo. Consistent with this data we observe that AlkBH8, a tRNA-selenocysteine methyltransferase required for the translational incorporation of selenocysteine, is essential for CRC growth. In summary, our research elucidates the complex mechanisms underlying ferroptosis in CRC and reveals that modulation of the selenoproteome provides multiple new therapeutic targets and opportunities in CRC.

Myeloid Hif2α is not essential to maintain systemic iron homeostasis.

Dietary consumption serves as the primary source of iron uptake, and erythropoiesis acts as a major regulator of systemic iron demand. In addition to intestinal iron absorption, macrophages play a crucial role in recycling iron from senescent red blood cells. The kidneys are responsible for the production of erythropoietin (Epo), which stimulates erythropoiesis, whereas the liver plays a central role in producing the iron-regulatory hormone hepcidin. The transcriptional regulator hypoxia-inducible factor (HIF)2α has a central role in the regulation of Epo, hepcidin, and intestinal iron absorption and therefore plays a crucial role in coordinating the tissue crosstalk to maintain systemic iron demands. However, the precise involvement of Hif2α in macrophages in terms of iron homeostasis remains uncertain. Our study demonstrates that deleting Hif2α in macrophages does not disrupt the expression of iron transporters or basal erythropoiesis. Mice lacking Hif2α in myeloid cells exhibited no discernible differences in hemodynamic parameters, including hemoglobin concentrations and erythrocyte count, when compared with littermate controls. This similarity was observed under conditions of both dietary iron deficiency and acute erythropoietic demand. Notably, we observed a significant increase in the expression of iron transporters in the duodenum during iron deficiency, indicating heightened iron absorption. Therefore, our findings suggest that the disruption of Hif2α in myeloid cells does not significantly impact systemic iron homeostasis under normal physiologic conditions. However, its disruption induces adaptive physiologic changes in response to elevated iron demand, potentially serving as a mechanism to sustain increased erythropoietic demand.

Microenvironmental ammonia enhances T cell exhaustion in colorectal cancer.

Effective therapies are lacking for patients with advanced colorectal cancer (CRC). The CRC tumor microenvironment has elevated metabolic waste products due to altered metabolism and proximity to the microbiota. The role of metabolite waste in tumor development, progression, and treatment resistance is unclear. We generated an autochthonous metastatic mouse model of CRC and used unbiased multi-omic analyses to reveal a robust accumulation of tumoral ammonia. The high ammonia levels induce T cell metabolic reprogramming, increase exhaustion, and decrease proliferation. CRC patients have increased serum ammonia, and the ammonia-related gene signature correlates with altered T cell response, adverse patient outcomes, and lack of response to immune checkpoint blockade. We demonstrate that enhancing ammonia clearance reactivates T cells, decreases tumor growth, and extends survival. Moreover, decreasing tumor-associated ammonia enhances anti-PD-L1 efficacy. These findings indicate that enhancing ammonia detoxification can reactivate T cells, highlighting a new approach to enhance the efficacy of immunotherapies.

Protocol for isolation and analysis of small volatile microbiome metabolites from human or mouse samples.

Metabolites are crucial for bidirectional communication between host and microbiome. We describe a protocol for the isolation of organic and aqueous metabolites from mucosal scrapes and feces from mouse and human samples. Although some of the most reactive organic compounds may be lost, this approach generates a functionally reproducible metabolic extract containing both host and microbial compounds appropriate for quantitative mass spectrometry and functional characterization. Our mass spectrometry approach identifies low-abundant and difficult to identify microbially derived metabolites. For complete details on the use and execution of this protocol, please refer to Bell et al. (2021) and Das et al. (2020).

The Role of Ferritin in Health and Disease: Recent Advances and Understandings.

Systemic iron homeostasis needs to be tightly controlled, as both deficiency and excess iron cause major global health concerns, such as iron deficiency anemia, hemochromatosis, etc. In mammals, sufficient dietary acquisition is critical for fulfilling the systemic iron requirement. New questions are emerging about whether and how cellular iron transport pathways integrate with the iron storage mechanism. Ferritin is the intracellular iron storage protein that stores surplus iron after all the cellular needs are fulfilled and releases it in the face of an acute demand. Currently, there is a surge in interest in ferritin research after the discovery of novel pathways like ferritinophagy and ferroptosis. This review emphasizes the most recent ferritin-related discoveries and their impact on systemic iron regulation.

Reuterin in the healthy gut microbiome suppresses colorectal cancer growth through altering redox balance.

Microbial dysbiosis is a colorectal cancer (CRC) hallmark and contributes to inflammation, tumor growth, and therapy response. Gut microbes signal via metabolites, but how the metabolites impact CRC is largely unknown. We interrogated fecal metabolites associated with mouse models of colon tumorigenesis with varying mutational load. We find that microbial metabolites from healthy mice or humans are growth-repressive, and this response is attenuated in mice and patients with CRC. Microbial profiling reveals that Lactobacillus reuteri and its metabolite, reuterin, are downregulated in mouse and human CRC. Reuterin alters redox balance, and reduces proliferation and survival in colon cancer cells. Reuterin induces selective protein oxidation and inhibits ribosomal biogenesis and protein translation. Exogenous Lactobacillus reuteri restricts colon tumor growth, increases tumor reactive oxygen species, and decreases protein translation in vivo. Our findings indicate that a healthy microbiome and specifically, Lactobacillus reuteri, is protective against CRC through microbial metabolite exchange.

NCOA4-Mediated Ferritinophagy Is a Pancreatic Cancer Dependency via Maintenance of Iron Bioavailability for Iron-Sulfur Cluster Proteins.

Pancreatic ductal adenocarcinomas (PDAC) depend on autophagy for survival; however, the metabolic substrates that autophagy provides to drive PDAC progression are unclear. Ferritin, the cellular iron storage complex, is targeted for lysosomal degradation (ferritinophagy) by the selective autophagy adaptor NCOA4, resulting in release of iron for cellular utilization. Using patient-derived and murine models of PDAC, we demonstrate that ferritinophagy is upregulated in PDAC to sustain iron availability, thereby promoting tumor progression. Quantitative proteomics reveals that ferritinophagy fuels iron-sulfur cluster protein synthesis to support mitochondrial homeostasis. Targeting NCOA4 leads to tumor growth delay and prolonged survival but with the development of compensatory iron acquisition pathways. Finally, enhanced ferritinophagy accelerates PDAC tumorigenesis, and an elevated ferritinophagy expression signature predicts for poor prognosis in patients with PDAC. Together, our data reveal that the maintenance of iron homeostasis is a critical function of PDAC autophagy, and we define NCOA4-mediated ferritinophagy as a therapeutic target in PDAC. SIGNIFICANCE: Autophagy and iron metabolism are metabolic dependencies in PDAC. However, targeted therapies for these pathways are lacking. We identify NCOA4-mediated selective autophagy of ferritin ("ferritinophagy") as upregulated in PDAC. Ferritinophagy supports PDAC iron metabolism and thereby tumor progression and represents a new therapeutic target in PDAC. See related commentary by Jain and Amaravadi, p. 2023. See related article by Ravichandran et al., p. 2198. This article is highlighted in the In This Issue feature, p. 2007.

HIF-2α activation potentiates oxidative cell death in colorectal cancers by increasing cellular iron.

Hypoxia is a hallmark of solid tumors that promotes cell growth, survival, and metastasis and confers resistance to chemo and radiotherapies. Hypoxic responses are largely mediated by the transcription factors hypoxia-inducible factor 1α (HIF-1α) and HIF-2α. Our work demonstrates that HIF-2α is essential for colorectal cancer (CRC) progression. However, targeting hypoxic cells is difficult, and tumors rapidly acquire resistance to inhibitors of HIF-2α. To overcome this limitation, we performed a small molecule screen to identify HIF-2α-dependent vulnerabilities. Several known ferroptosis activators and dimethyl fumarate (DMF), a cell-permeable mitochondrial metabolite derivative, led to selective synthetic lethality in HIF-2α-expressing tumor enteroids. Our work demonstrated that HIF-2α integrated 2 independent forms of cell death via regulation of cellular iron and oxidation. First, activation of HIF-2α upregulated lipid and iron regulatory genes in CRC cells and colon tumors in mice and led to a ferroptosis-susceptible cell state. Second, via an iron-dependent, lipid peroxidation-independent pathway, HIF-2α activation potentiated ROS via irreversible cysteine oxidation and enhanced cell death. Inhibition or knockdown of HIF-2α decreased ROS and resistance to oxidative cell death in vitro and in vivo. Our results demonstrated a mechanistic vulnerability in cancer cells that were dependent on HIF-2α that can be leveraged for CRC treatment.