Association of Metal Cations with the Anti-PF4/Heparin Antibody Response in Heparin-Induced Thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is an antibody-mediated immune response against complexes of heparin and platelet factor 4 (PF4). The electrostatic interaction between heparin and PF4 is critical for the anti-PF4/heparin antibody response seen in HIT. The binding of metal cations to heparin induces conformational changes and charge neutralization of the heparin molecule, and cation-heparin binding can modulate the specificity and affinity for heparin-binding partners. However, the effects of metal cation binding to heparin in the context of anti-PF4/heparin antibody response have not been determined. Here, we utilized inductively coupled plasma mass spectrometry (ICP-MS) to quantify 16 metal cations in patient plasma and tested for correlation with anti-PF4/heparin IgG levels and platelet count after clinical suspicion of HIT in a cohort of heparin-treated patients. The average age of the cohort (n = 32) was 60.53 (SD = 14.31) years old, had a mean anti-PF4/heparin antibody optical density [OD405] of 0.93 (SD = 1.21) units, and was primarily female (n = 23). Patients with positive anti-PF4/heparin antibody test results (OD405 ≥ 0.5 units) were younger, had increased weight and BMI, and were more likely to have a positive serotonin release assay (SRA) result compared to antibody-negative patients. We observed statistical differences between antibody-positive and -negative groups for sodium and aluminum and significant correlations of anti-PF4/heparin antibody levels with sodium and silver. While differences in sodium concentrations were associated with antibody-positive status and correlated with antibody levels, no replication was performed. Additional studies are warranted to confirm our observed association, including in vitro binding studies and larger observational cohorts.

Association of Metal Cations with the Anti-PF4/Heparin Antibody Response in Heparin-Induced Thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is an antibody-mediated immune response against complexes of heparin and platelet factor 4 (PF4). The electrostatic interaction between heparin and PF4 is critical for the anti-PF4/heparin antibody response seen in HIT. The binding of metal cations to heparin induces conformational changes and charge neutralization of the heparin molecule, and cation-heparin binding can modulate the specificity and affinity for heparin-binding partners. However, the effects of metal cation binding to heparin in the context of anti-PF4/heparin antibody response have not been determined. Here, we utilized inductively coupled plasma mass spectrometry (ICP-MS) to quantify 16 metal cations in patient plasma and tested for correlation with anti-PF4/heparin IgG levels and platelet count after clinical suspicion of HIT in a cohort of heparin-treated patients. The average age of the cohort (n = 32) was 60.53 (SD = 14.31) years old, had a mean anti-PF4/heparin antibody optical density [OD405] of 0.93 (SD = 1.21) units and was primarily female (n = 23). Patients with positive anti-PF4/heparin antibody test results (OD405 ≥ 0.5 units) were younger, had increased weight and BMI, and were more likely to have a positive serotonin release assay (SRA) result compared to antibody negative patients. We observed statistical differences between antibody positive and negative groups for sodium and aluminum and significant correlations of anti-PF4/heparin antibody levels with sodium and silver. While differences in sodium concentrations were associated with antibody positive status and correlated with antibody levels, no replication was performed. Additional studies are warranted to confirm our observed association, including in vitro binding studies and larger observational cohorts.

The NRF2-p97-NRF2 negative feedback loop.

p97 is a ubiquitin-targeted ATP-dependent segregase that regulates proteostasis, in addition to a variety of other cellular functions. Previously, we demonstrated that p97 negatively regulates NRF2 by extracting ubiquitylated NRF2 from the KEAP1-CUL3-RBX1 E3 ubiquitin ligase complex, facilitating proteasomal destruction. In the current study, we identified p97 as an NRF2-target gene that contains a functional ARE, indicating the presence of an NRF2-p97-NRF2 negative feedback loop that maintains redox homeostasis. Using CRISPR/Cas9 genome editing, we generated endogenous p97 ARE-mutated BEAS-2B cell lines. These p97 ARE-mutated cell lines exhibit altered expression of p97 and NRF2, as well as a compromised response to NRF2 inducers. Importantly, we also found a positive correlation between NRF2 activation and p97 expression in human cancer patients. Finally, using chronic arsenic-transformed cell lines, we demonstrated a synergistic effect of NRF2 and p97 inhibition in killing cancer cells with high NRF2 and p97 expression. Our study suggests dual upregulation of NRF2 and p97 occurs in certain types of cancers, suggesting that inhibition of both NRF2 and p97 could be a promising treatment strategy for stratified cancer patients.

NRF2 controls iron homeostasis and ferroptosis through HERC2 and VAMP8.

Enhancing the intracellular labile iron pool (LIP) represents a powerful, yet untapped strategy for driving ferroptotic death of cancer cells. Here, we show that NRF2 maintains iron homeostasis by controlling HERC2 (E3 ubiquitin ligase for NCOA4 and FBXL5) and VAMP8 (mediates autophagosome-lysosome fusion). NFE2L2/NRF2 knockout cells have low HERC2 expression, leading to a simultaneous increase in ferritin and NCOA4 and recruitment of apoferritin into the autophagosome. NFE2L2/NRF2 knockout cells also have low VAMP8 expression, which leads to ferritinophagy blockage. Therefore, deletion of NFE2L2/NRF2 results in apoferritin accumulation in the autophagosome, an elevated LIP, and enhanced sensitivity to ferroptosis. Concordantly, NRF2 levels correlate with HERC2 and VAMP8 in human ovarian cancer tissues, as well as ferroptosis resistance in a panel of ovarian cancer cell lines. Last, the feasibility of inhibiting NRF2 to increase the LIP and kill cancer cells via ferroptosis was demonstrated in preclinical models, signifying the impact of NRF2 inhibition in cancer treatment.

Decreased autophagosome biogenesis, reduced NRF2, and enhanced ferroptotic cell death are underlying molecular mechanisms of non-alcoholic fatty liver disease.

BACKGROUND AND AIMS: Caloric excess and sedentary lifestyles have led to an epidemic of obesity, metabolic syndrome, and non-alcoholic fatty liver disease (NAFLD). The objective of this study was to investigate the mechanisms underlying high fat diet (HFD)-induced NAFLD, and to explore NRF2 activation as a strategy to alleviate NAFLD. APPROACH AND RESULTS: Herein, we demonstrated that high fat diet (HFD) induced lipid peroxidation and ferroptosis, both of which could be alleviated by NRF2 upregulation. Mechanistically, HFD suppressed autophagosome biogenesis through AMPK- and AKT-mediated mTOR activation and decreased ATG7, resulting in KEAP1 stabilization and decreased NRF2 levels in mouse liver. Furthermore, ATG7 is required for HFD-induced NRF2 downregulation, as ATG7 deletion in Cre-inducible ATG7 knockout mice decreased NRF2 levels and enhanced ferroptosis, which was not further exacerbated by HFD. This finding was recapitulated in mouse hepatocytes, which showed a similar phenotype upon treatment with saturated fatty acids (SFAs) but not monounsaturated fatty acids (MUFAs). Finally, NRF2 activation blocked fatty acid (FA)-mediated NRF2 downregulation, lipid peroxidation, and ferroptosis. Importantly, the HFD-induced alterations were also observed in human fatty liver tissue samples. CONCLUSIONS: HFD-mediated autophagy inhibition, NRF2 suppression, and ferroptosis promotion are important molecular mechanisms of obesity-driven metabolic diseases. NRF2 activation counteracts HFD-mediated NRF2 suppression and ferroptotic cell death. In addition, SFA vs. MUFA regulation of NRF2 may underlie their harmful vs. beneficial effects. Our study reveals NRF2 as a key player in the development and progression of fatty liver disease and that NRF2 activation could serve as a potential therapeutic strategy.

Aroylhydrazone Glycoconjugate Prochelators Exploit Glucose Transporter 1 (GLUT1) to Target Iron in Cancer Cells.

Glycoconjugation strategies in anticancer drug discovery exploit the high expression of glucose transporters in malignant cells to achieve preferential uptake and hence attractive pharmacological characteristics of increased therapeutic windows and decreased unwanted toxicity. Here we present the design of glycoconjugated prochelators of aroylhydrazone AH1, an antiproliferative scavenger that targets the increased iron demand of rapidly proliferating malignant cells. The constructs feature a monosaccharide (d-glucose, d-glucosamine, or glycolytic inhibitor 2-deoxy-d-glucose) connected at the C2 or C6 position via a short linker, which masks the chelator through a disulfide bond susceptible to intracellular reduction. Cellular assays showed that the glycoconjugates rely on the GLUT1 transporter for uptake, lead to intracellular iron deprivation, and present antiproliferative activity. Ectopic overexpression of GLUT1 in malignant and normal cells increased the uptake and toxicity of the glycoconjugated prochelators, demonstrating that these compounds are well suited for targeting cells overexpressing glucose transporters and therefore for selective iron sequestration in malignant cells.

Targeted CUL4A inhibition synergizes with cisplatin to yield long-term survival in models of head and neck squamous cell carcinoma through a DDB2-mediated mechanism.

Patients with late-stage and human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) continue to have a very poor prognosis. The development of more effective novel therapies that improve overall survival and overcome drug resistance is an urgent priority. Here we report that HNSCC tumors significantly overexpress NEDD8 and exhibit high sensitivity to the first-in-class NEDD8-activating enzyme (NAE) inhibitor pevonedistat. Additional studies established that disruption of NEDD8-mediated protein turnover with pevonedistat dramatically augmented cisplatin-induced DNA damage and apoptosis in HNSCC models. Further analysis revealed that the specific pevonedistat target CUL4A played an essential role in driving the synergy of the pevonedistat and cisplatin combination. Targeted inhibition of CUL4A resulted in significant downregulation in Damage Specific DNA binding protein 2 (DDB2), a DNA-damage recognition protein that promotes nucleotide excision repair and resistance to cisplatin. Silencing of CUL4A or DDB2 enhanced cisplatin-induced DNA damage and apoptosis in a manner similar to that of pevonedistat demonstrating that targeted inhibition of CUL4A may be a novel approach to augment cisplatin therapy. Administration of pevonedistat to mice bearing HNSCC tumors significantly decreased DDB2 expression in tumor cells, increased DNA damage and potently enhanced the activity of cisplatin to yield tumor regression and long-term survival of all animals. Our findings provide strong rationale for clinical investigation of CUL4A inhibition with pevonedistat as a novel strategy to augment the efficacy of cisplatin therapy for patients with HNSCC and identify loss of DDB2 as a key pharmacodynamic mediator controlling sensitivity to this regimen.

CHML is an NRF2 target gene that regulates mTOR function.

The transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) is often highly expressed in non-small cell lung cancer (NSCLC). Through its target genes, NRF2 enhances cancer progression and chemo/radioresistance, leading to a poorer prognosis in patients with high NRF2 expression. In this study, we identified CHM-like Rab escort protein (CHML; encoding Rep2) as an NRF2 target gene with an antioxidant response element (ARE) in its promoter region (-1622 to -1612). Analysis of patient data curated by The Cancer Genome Atlas (TCGA) and Oncomine databases revealed that CHML mRNA expression was elevated in lung adenocarcinoma (LUAD) patient tumor tissues and correlated with decreased patient survival. Immunohistochemistry (IHC) analysis of normal versus lung cancer patient tissues revealed that Rep2 protein levels were higher in lung tumors compared with normal tissue, which also correlated with increased levels of NRF2. Importantly, siRNA-mediated knockdown of CHML/Rep2 in A549 NSCLC cells decreased their ability to proliferate. Mechanistically, Rep2 mediates mTOR function, as loss of Rep2 inhibited, whereas overexpression enhanced, mTOR translocation and activation at the lysosome. Our findings identify a novel NRF2-Rep2-dependent regulation of mTOR function.

Cyst(e)inase-Rapamycin Combination Induces Ferroptosis in Both In Vitro and In Vivo Models of Hereditary Leiomyomatosis and Renal Cell Cancer.

Renal cell carcinomas associated with hereditary leiomyomatosis and renal cell cancer (HLRCC) are notoriously aggressive and represent the leading cause of death among patients with HLRCC. To date, a safe and effective standardized therapy for this tumor type is lacking. Here we show that the engineered synthetic therapeutic enzyme, Cyst(e)inase, when combined with rapamycin, can effectively induce ferroptosis in HLRCC cells in vivo. The drug combination promotes lipid peroxidation to a greater degree than cysteine deprivation or Cyst(e)inase treatment alone, while rapamycin treatment alone does not induce ferroptosis. Mechanistically, Cyst(e)inase induces ferroptosis by depleting the exogenous cysteine/cystine supply, while rapamycin reduces cellular ferritin level by promoting ferritins' destruction via ferritinophagy. Since both Cyst(e)inase and rapamycin are well tolerated clinically, the combination represents an opportunity to exploit ferroptosis induction as a cancer management strategy. Accordingly, using a xenograft mouse model, we showed that the combination treatment resulted in tumor growth suppression without any notable side effects. In contrast, both Cyst(e)inase only and rapamycin only treatment groups failed to induce a significant change when compared with the vehicle control group. Our results demonstrated the effectiveness of Cyst(e)inase-rapamycin combination in inducing ferroptotic cell death in vivo, supporting the potential translation of the combination therapy into clinical HLRCC management.

Non-canonical NRF2 activation promotes a pro-diabetic shift in hepatic glucose metabolism.

OBJECTIVE: NRF2, a transcription factor that regulates cellular redox and metabolic homeostasis, plays a dual role in human disease. While it is well known that canonical intermittent NRF2 activation protects against diabetes-induced tissue damage, little is known regarding the effects of prolonged non-canonical NRF2 activation in diabetes. The goal of this study was to determine the role and mechanisms of prolonged NRF2 activation in arsenic diabetogenicity. METHODS: To test this, we utilized an integrated transcriptomic and metabolomic approach to assess diabetogenic changes in the livers of wild type, Nrf2-/-, p62-/-, or Nrf2-/-; p62-/- mice exposed to arsenic in the drinking water for 20 weeks. RESULTS: In contrast to canonical oxidative/electrophilic activation, prolonged non-canonical NRF2 activation via p62-mediated sequestration of KEAP1 increases carbohydrate flux through the polyol pathway, resulting in a pro-diabetic shift in glucose homeostasis. This p62- and NRF2-dependent increase in liver fructose metabolism and gluconeogenesis occurs through the upregulation of four novel NRF2 target genes, ketohexokinase (Khk), sorbitol dehydrogenase (Sord), triokinase/FMN cyclase (Tkfc), and hepatocyte nuclear factor 4 (Hnf4A). CONCLUSION: We demonstrate that NRF2 and p62 are essential for arsenic-mediated insulin resistance and glucose intolerance, revealing a pro-diabetic role for prolonged NRF2 activation in arsenic diabetogenesis.

Iron control of erythroid microtubule cytoskeleton as a potential target in treatment of iron-restricted anemia.

Anemias of chronic disease and inflammation (ACDI) result from restricted iron delivery to erythroid progenitors. The current studies reveal an organellar response in erythroid iron restriction consisting of disassembly of the microtubule cytoskeleton and associated Golgi disruption. Isocitrate supplementation, known to abrogate the erythroid iron restriction response, induces reassembly of microtubules and Golgi in iron deprived progenitors. Ferritin, based on proteomic profiles, regulation by iron and isocitrate, and putative interaction with microtubules, is assessed as a candidate mediator. Knockdown of ferritin heavy chain (FTH1) in iron replete progenitors induces microtubule collapse and erythropoietic blockade; conversely, enforced ferritin expression rescues erythroid differentiation under conditions of iron restriction. Fumarate, a known ferritin inducer, synergizes with isocitrate in reversing molecular and cellular defects of iron restriction and in oral remediation of murine anemia. These findings identify a cytoskeletal component of erythroid iron restriction and demonstrate potential for its therapeutic targeting in ACDI.

The NRF2-LOC344887 signaling axis suppresses pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible disease characterized by an increase in differentiation of fibroblasts to myofibroblasts and excessive accumulation of extracellular matrix in lung tissue. Pharmacological activation of NRF2 has proved to be a valuable antifibrotic approach, however the detailed mechanisms of how NRF2 mediates antifibrotic function remain unclear. In this study, we found that the antifibrotic function of sulforaphane (SFN), an NRF2 activator, was largely dependent on LOC344887, a long noncoding RNA. Two functional AREs were identified in both the promoter and intron 1 of LOC344887, which defines LOC344887 as a novel anti-fibrotic NRF2 target gene. RNA-seq analysis revealed that LOC344887 controls genes and signaling pathways associated with fibrogenesis. Deletion or downregulation of LOC344887 enhanced expression of CDH2/N-cadherin, as well as a number of other fibrotic genes and blunted the antifibrotic effects of SFN. Furthermore, LOC344887-mediated downregulation of fibrotic genes may involve the PI3K-AKT signaling pathway, as pharmacologic inhibition of PI3K activity blocked the effects of LOC344887 knockdown. Our findings demonstrate that NRF2-mediated LOC344887 upregulation contributes to the antifibrotic potential of SFN by repressing the expression of CDH2 and other fibrotic genes, providing novel insight into how NRF2 controls the regulatory networks of IPF. This study provides a better understanding of the molecular mechanisms of NRF2 activators against pulmonary fibrosis and presents a novel therapeutic axis for prevention and intervention of fibrosis-related diseases.

Advances in hereditary leiomyomatosis and renal cell carcinoma (HLRCC) research.

Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) is an autosomal dominant hereditary cancer syndrome with incomplete penetrance. It is caused by a germline amorphic allele of the FH gene, which encodes the TCA cycle enzyme, fumarate hydratase (FH). HLRCC patients are genetically predisposed to develop skin leiomyomas, uterine fibroids, and the aggressive kidney cancer of type 2 papillary morphology. Loss-of-heterozygocity at the FH locus that cause a complete loss of FH enzymatic function is always detected in these tumor tissues. Molecular pathway elucidation, genomic studies, and systematic genetics screens reported over the last two decades have identified several FH-inactivation driven pathways alterations, as well as rationally conceived treatment strategies that specifically target FH-/- tumor cells. These treatment strategies include ferroptosis induction, oxidative stress promotion, and metabolic alteration. As the fundamental biology of HLRCC continues to be uncovered, these treatment strategies continue to be refined and may one day lead to a strategy to prevent disease onset among HLRCC patients. With a more complete picture of HLRCC biology, the safe translation of experimental treatment strategies into clinical practice is achievable in the foreseeable future.

Differential and overlapping targets of the transcriptional regulators NRF1, NRF2, and NRF3 in human cells.

The nuclear factor (erythroid 2)-like (NRF) transcription factors are a subset of cap'n'collar transcriptional regulators. They consist of three members, NRF1, NRF2, and NRF3, that regulate the expression of genes containing antioxidant-response elements (AREs) in their promoter regions. Although all NRF members regulate ARE-containing genes, each is associated with distinct roles. A comprehensive study of differential and overlapping DNA-binding and transcriptional activities of the NRFs has not yet been conducted. Here, we performed chromatin immunoprecipitation (ChIP)-exo sequencing, an approach that combines ChIP with exonuclease treatment to pinpoint regulatory elements in DNA with high precision, in conjunction with RNA-sequencing to define the transcriptional targets of each NRF member. Our approach, done in three U2OS cell lines, identified 31 genes that were regulated by all three NRF members, 27 that were regulated similarly by all three, and four genes that were differentially regulated by at least one NRF member. We also found genes that were up- or down-regulated by only one NRF member, with 84, 84, and 22 genes that were regulated by NRF1, NRF2, and NRF3, respectively. Analysis of the ARE motifs identified in ChIP peaks revealed that NRF2 prefers binding to AREs flanked by GC-rich regions and that NRF1 prefers AT-rich flanking regions. Thus, sequence preference, likely in combination with upstream signaling events, determines NRF member activation under specific cellular contexts. Our analysis provides a comprehensive description of differential and overlapping gene regulation by the transcriptional regulators NRF1, NRF2, and NRF3.

Genome-Wide CRISPR Screen Reveals Autophagy Disruption as the Convergence Mechanism That Regulates the NRF2 Transcription Factor.

The nuclear factor (erythroid 2)-like 2 (NRF2 or NFE2L2) transcription factor regulates the expression of many genes that are critical in maintaining cellular homeostasis. Its deregulation has been implicated in many diseases, including cancer and metabolic and neurodegenerative diseases. While several mechanisms by which NRF2 can be activated have gradually been identified over time, a more complete regulatory network of NRF2 is still lacking. Here we show through a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screen that a total of 273 genes, when knocked out, will lead to sustained NRF2 activation. Pathway analysis revealed a significant overrepresentation of genes (18 of the 273 genes) involved in autophagy. Molecular validation of a subset of the enriched genes identified 8 high-confidence genes that negatively regulate NRF2 activity irrespective of cell type: ATG12, ATG7, GOSR1, IFT172, NRXN2, RAB6A, VPS37A, and the well-known negative regulator of NRF2, KEAP1 Of these, ATG12, ATG7, KEAP1, and VPS37A are known to be involved in autophagic processes. Our results present a comprehensive list of NRF2 negative regulators and reveal an intimate link between autophagy and NRF2 regulation.

RPA1 binding to NRF2 switches ARE-dependent transcriptional activation to ARE-NRE-dependent repression.

NRF2 regulates cellular redox homeostasis, metabolic balance, and proteostasis by forming a dimer with small musculoaponeurotic fibrosarcoma proteins (sMAFs) and binding to antioxidant response elements (AREs) to activate target gene transcription. In contrast, NRF2-ARE-dependent transcriptional repression is unreported. Here, we describe NRF2-mediated gene repression via a specific seven-nucleotide sequence flanking the ARE, which we term the NRF2-replication protein A1 (RPA1) element (NRE). Mechanistically, RPA1 competes with sMAF for NRF2 binding, followed by interaction of NRF2-RPA1 with the ARE-NRE and eduction of promoter activity. Genome-wide in silico and RNA-seq analyses revealed this NRF2-RPA1-ARE-NRE complex mediates negative regulation of many genes with diverse functions, indicating that this mechanism is a fundamental cellular process. Notably, repression of MYLK, which encodes the nonmuscle myosin light chain kinase, by the NRF2-RPA1-ARE-NRE complex disrupts vascular integrity in preclinical inflammatory lung injury models, illustrating the translational significance of NRF2-mediated transcriptional repression. Our findings reveal a gene-suppressive function of NRF2 and a subset of negatively regulated NRF2 target genes, underscoring the broad impact of NRF2 in physiological and pathological settings.

A catalogue of somatic NRF2 gain-of-function mutations in cancer.

Identification and characterization of somatic mutations in cancer have important prognostication and treatment implications. Genes encoding the Nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcription factor and its negative regulator, Kelch-like ECH-associated protein 1 (KEAP1), are frequently mutated in cancer. These mutations drive constitutive NRF2 activation and correlate with poor prognosis. Despite its apparent significance, a comprehensive catalogue of somatic NRF2 mutations across different tumor types is still lacking. Here, we catalogue NRF2 mutations in The Cancer Genome Atlas (TCGA) database. 226 unique NRF2-mutant tumors were identified from 10,364 cases. NRF2 mutations were found in 21 out of the 33 tumor types. A total of 11 hotspots were identified. Of these, mutation to the R34 position was most frequent. Notably, R34 and D29 mutations were overrepresented in bladder, lung, and uterine cancers. Analyses of corresponding RNA sequencing data using a de novo derived gene expression classifier showed that the R34 mutations drive constitutive NRF2 activation with a selection pressure biased against the formation of R34L. Of all R34 mutants, R34L conferred the least degree of protein stabilization, suggesting a pro-tumor NRF2 half-life threshold. Our findings offer a comprehensive catalogue of NRF2 mutations in cancer that can help prognostication and NRF2 research.

Fumarate hydratase inactivation in hereditary leiomyomatosis and renal cell cancer is synthetic lethal with ferroptosis induction.

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a hereditary cancer syndrome characterized by inactivation of the Krebs cycle enzyme fumarate hydratase (FH). HLRCC patients are at high risk of developing kidney cancer of type 2 papillary morphology that is refractory to current radiotherapy, immunotherapy and chemotherapy. Hence, an effective therapy for this deadly form of cancer is urgently needed. Here, we show that FH inactivation (FH-/- ) proves synthetic lethal with inducers of ferroptosis, an iron-dependent and nonapoptotic form of cell death. Specifically, we identified gene signatures for compound sensitivities based on drug responses for 9 different drug classes against the NCI-60 cell lines. These signatures predicted that ferroptosis inducers would be selectively toxic to FH-/- cell line UOK262. Preferential cell death against UOK262-FH-/- was confirmed with 4 different ferroptosis inducers. Mechanistically, the FH-/- sensitivity to ferroptosis is attributed to dysfunctional GPX4, the primary cellular defender against ferroptosis. We identified that C93 of GPX4 is readily post-translationally modified by fumarates that accumulate in conditions of FH-/- , and that C93 modification represses GPX4 activity. Induction of ferroptosis in FH-inactivated tumors represents an opportunity for synthetic lethality in cancer.

The Roles of NRF2 in Modulating Cellular Iron Homeostasis.

SIGNIFICANCE: Iron and oxygen are intimately linked: iron is an essential nutrient utilized as a cofactor in enzymes for oxygen transport, oxidative phosphorylation, and metabolite oxidation. However, excess labile iron facilitates the formation of oxygen-derived free radicals capable of damaging biomolecules. Therefore, biological utilization of iron is a tightly regulated process. The nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcription factor, which can respond to oxidative and electrophilic stress, regulates several genes involved in iron metabolism. Recent Advances: The bulk of NRF2 transcription factor research has focused on its roles in detoxification and cancer prevention. Recent works have identified that several genes involved in heme synthesis, hemoglobin catabolism, iron storage, and iron export are under the control of NRF2. Constitutive NRF2 activation and subsequent deregulation of iron metabolism have been implicated in cancer development: NRF2-mediated upregulation of the iron storage protein ferritin or heme oxygenase 1 can lead to enhanced proliferation and therapy resistance. Of note, NRF2 activation and alterations to iron signaling in cancers may hinder efforts to induce the iron-dependent cell death process known as ferroptosis. CRITICAL ISSUES: Despite growing recognition of NRF2 as a modulator of iron signaling, exactly how iron metabolism is altered due to NRF2 activation in normal physiology and in pathologic conditions remains imprecise; moreover, the roles of NRF2-mediated iron signaling changes in disease progression are only beginning to be uncovered. FUTURE DIRECTIONS: Further studies are necessary to connect NRF2 activation with physiological and pathological changes to iron signaling and oxidative stress. Antioxid. Redox Signal. 00, 000-000.