Keap1-Nrf2 System Research Articles

Splicing junction-based classifier for the detection of abnormal constitutive activation of the KEAP1-NRF2 system

The KEAP1-NRF2 system plays a crucial role in responding to oxidative and electrophilic stress. Its dysregulation can cause the overexpression of downstream genes, a known cancer hallmark. Understanding and detecting abnormal KEAP1-NRF2 activity is essential for understanding disease mechanisms and identifying therapeutic targets. This study presents an approach that analyzes splicing patterns by a naive Bayes-based classifier to identify constitutive activation of the KEAP1-NRF2 system, focusing on the higher presence of abnormal splicing junctions as a subproduct of overexpression of downstream genes. Our splicing-based classifier demonstrated robust performance, reliably identifying activation of the KEAP1-NRF2 pathway across extensive datasets, including The Cancer Genome Atlas and the Sequence Read Archive. This shows the classifier’s potential to analyze hundreds of thousands of transcriptomes, highlighting its utility in broad-scale genomic studies and provides a new perspective on utilizing splicing aberrations caused by overexpression as diagnostic markers, offering potential improvements in diagnosis and treatment strategies.

Modular Nanotransporters Deliver Anti-Keap1 Monobody into Mouse Hepatocytes, Thereby Inhibiting Production of Reactive Oxygen Species.

Background/Objectives: The study of oxidative stress in cells and ways to prevent it attract increasing attention. Antioxidant defense of cells can be activated by releasing the transcription factor Nrf2 from a complex with Keap1, its inhibitor protein. The aim of the work was to study the effect of the modular nanotransporter (MNT) carrying an R1 anti-Keap1 monobody (MNTR1) on cell homeostasis. Methods: The murine hepatocyte AML12 cells were used for the study. The interaction of fluorescently labeled MNTR1 with Keap1 fused to hrGFP was studied using the Fluorescence-Lifetime Imaging Microscopy-Förster Resonance Energy Transfer (FLIM-FRET) technique on living AML12 cells transfected with the Keap1-hrGFP gene. The release of Nrf2 from the complex with Keap1 and its levels in the cytoplasm and nuclei of the AML12 cells were examined using a cellular thermal shift assay (CETSA) and confocal laser scanning microscopy, respectively. The effect of MNT on the formation of reactive oxygen species was studied by flow cytometry using 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate. Results: MNTR1 is able to interact with Keap1 in the cytoplasm, leading to the release of Nrf2 from the complex with Keap1 and a rapid rise in Nrf2 levels both in the cytoplasm and nuclei, ultimately causing protection of cells from the action of hydrogen peroxide. The possibility of cleavage of the monobody in endosomes leads to an increase in the observed effects. Conclusions: These findings open up a new approach to specifically modulating the interaction of intracellular proteins, as demonstrated by the example of the Keap1-Nrf2 system.

Specific cancer types and prognosis in patients with variations in the KEAP1-NRF2 system: A retrospective cohort study.

The KEAP1-NRF2 system induces the expression of antioxidant genes in response to various types of oxidative stress. Some cancer cells activate this system, which increases their malignancy through genetic mutations. We performed a retrospective cohort study using the C-CAT database, which contains the gene-panel sequence data from 60,056 cases of diagnosed solid tumors. We analyzed somatic mutations in NRF2 and KEAP1 genes and their associations with clinical outcomes. Variants in the NRF2 gene were clustered in exon 2, which encodes the DLG and ETGE motifs essential for KEAP1 interaction. The NRF2 variants were frequently observed in esophageal and lung squamous cell carcinoma with frequencies of 35.9% and 19.6%, respectively. Among these mutations, the NRF2 variants in the ETGE motif were indicators of a worse prognosis. KEAP1 variants were found in 2.5% of all cases. The variants were frequent in lung cancer and showed a worse prognosis in lung and other types of adenocarcinomas. We then conducted gene expression analysis using TCGA data. While cancers with DLG and ETGE variants were similar in terms of gene expression profiles, there were significant differences between cancers with KEAP1 and NRF2 variants. Our results indicate that genetic alteration of the KEAP1-NRF2 pathway is a major factor in patient prognosis for each cancer type and its genetic variant. Variants in NRF2 and KEAP1 genes can characterize the biological basis of each cancer type and are involved in carcinogenesis, resistance to therapy, and other biological differences.

The NRF2 inducer CDDO-2P-Im provokes a reduction in amyloid β levels in Alzheimer's disease model mice.

Alzheimer's disease (AD) is the most common aetiology of dementia. The transcription factor NF-E2-related factor 2 (NRF2) induces the expression of genes encoding phase II detoxification and antioxidant genes. NRF2 is regulated by Kelch-like ECH-associated protein 1 (KEAP1), and the KEAP1-NRF2 system is the key regulatory system involved in cytoprotection. To examine whether pharmacological induction of NRF2 expression alleviates AD phenotypes in vivo, we employed two AD mouse models, i.e. App NL-G-F/NL-G-F (AppNLGF) and APPV717I::TAUP301L (APP/TAU) mice. As the synthetic oleanane triterpenoid 1-[2-cyano-3,12-dioxooleana-1,9(11-dien-28-oyl)] (CDDO)-4(-pyridin-2-yl)-imidazole (CDDO-2P-Im) exhibits strong NRF2-inducing activity, we treated AD model mice with CDDO-2P-Im. We found that Aβ42 levels were markedly greater in the brains of AppNLGF mice than in those of APP/TAU mice. CDDO-2P-Im treatment significantly decreased Aβ42 levels, but not Aβ40 levels, in APP/TAU mice. Consequently, CDDO-2P-Im also decreased the ratio of Aβ42/Aβ40, a vital marker of amyloid plaque formation. LC-MS/MS analyses revealed that CDDO-2P-Im was delivered to the brains of the APP/TAU mice. CDDO-2P-Im induced the expression of detoxification and antioxidant gene targets of NRF2 and elevated reduced glutathione (GSH) levels in the mouse brain. These results support the notion that CDDO-2P-Im ameliorates AD-related pathologic changes.

The Complex Roles of Redox and Antioxidant Biology in Cancer.

Redox reactions control fundamental biochemical processes, including energy production, metabolism, respiration, detoxification, and signal transduction. Cancer cells, due to their generally active metabolism for sustained proliferation, produce high levels of reactive oxygen species (ROS) compared to normal cells and are equipped with antioxidant defense systems to counteract the detrimental effects of ROS to maintain redox homeostasis. The KEAP1-NRF2 system plays a major role in sensing and regulating endogenous antioxidant defenses in both normal and cancer cells, creating a bivalent contribution of NRF2 to cancer prevention and therapy. Cancer cells hijack the NRF2-dependent antioxidant program and exploit a very unique metabolism as a trade-off for enhanced antioxidant capacity. This work provides an overview of redox metabolism in cancer cells, highlighting the role of the KEAP1-NRF2 system, selenoproteins, sulfur metabolism, heme/iron metabolism, and antioxidants. Finally, we describe therapeutic approaches that can be leveraged to target redox metabolism in cancer.

Resveratrol targeting NRF2 disrupts the binding between KEAP1 and NRF2-DLG motif to ameliorate oxidative stress damage in mice pulmonary infection

Ethnopharmacological relevanceThe root of Polygonum cuspidatum Sieb. et Zucc (PC), known as 'Huzhang' in the Chinese Pharmacopoeia, has been traditionally employed for its anti-inflammatory, antiviral, antimicrobial, and other biological activities. Polydatin (PD) and its aglycone, resveratrol (RES), are key pharmacologically active components responsible for exerting anti-inflammatory and antioxidant effects. However, its specific targets and action mechanisms remain unclear. Aim of the studyThe equilibrium of the KEAP1-NRF2 system serves as the primary protective response to oxidative and electrophilic stresses within the body, particularly in cases of acute lung injury caused by pathogenic microbial infection. In this study, the precise mechanisms by which RES alleviates oxidative stress damage in conjunction with NRF2 activators are discussed. Materials and methodsThe active components from PC were screened to evaluate their potential to inhibit reactive oxygen species (ROS) and activate antioxidant activity dependent on antioxidant response elements (ARE). RES was evaluated for its potential to alleviate the oxidative stress caused by pathogenic microbial infection. Functional probes were designed to study the RES distribution and identify its targets. A lipopolysaccharide (LPS)-induced oxidative injury model was used to evaluate the effects of RES on the KEAP1-NRF2/ARE pathway in RAW 264.7 cells. The interaction between RES and NRF2 was elucidated using drug-affinity responsive target stability (DARTS), cellular thermal shift assays (CETSA), co-immunoprecipitation (Co-IP), and microscale thermophoresis (MST) techniques. The key binding sites were predicted using molecular docking and validated in NRF2-knockdownand reconstructed cells. Finally, protective effects against pulmonary stress were verified in a mouse model of pathogenic infection. ResultsThe accumulation of RES in lung macrophages disrupted the binding between KEAP1 and NRF2, thereby preventing the ubiquitination degradation of NRF2 through its interaction with Ile28 on the NRF2-DLG motif. The activation of NRF2 resulted in the upregulation of nuclear transcription, enhances the expression of antioxidant genes dependent on ARE, suppresses ROS generation, and ameliorates oxidative damage both in vivo and in vitro. ConclusionThese findings shed light on the potential of RES to mitigate oxidative stress damage caused by pathogenic microorganism-induced lung infections and facilitate the discovery of novel small molecule modulators targeting the KEAP1-NRF2 DLG motif interaction.

Liquid Chromatography/Tandem Mass Spectrometry Analysis of Sophora flavescens Aiton and Protective Effects against Alcohol-Induced Liver Injury and Oxidative Stress in Mice.

In this study, we investigated the hepatoprotective effects of an ethanol extract of Sophora flavescens Aiton (ESF) on an alcohol-induced liver disease mouse model. Alcoholic liver disease (ALD) was caused by the administration of ethanol to male C57/BL6 mice who were given a Lieber-DeCarli liquid diet, including ethanol. The alcoholic fatty liver disease mice were orally administered ESF (100 and 200 mg/kg bw/day) or silymarin (50 mg/kg bw/day), which served as a positive control every day for 16 days. The findings suggest that ESF enhances hepatoprotective benefits by significantly decreasing serum levels of aspartate transaminase (AST) and alanine transaminase (ALT), markers for liver injury. Furthermore, ESF alleviated the accumulation of triglyceride (TG) and total cholesterol (TC), increased serum levels of superoxide dismutase (SOD) and glutathione (GSH), and improved serum alcohol dehydrogenase (ADH) activity in the alcoholic fatty liver disease mice model. Cells and organisms rely on the Kelch-like ECH-associated protein 1- Nuclear factor erythroid 2-related factor 2 (Keap1-Nrf2) system as a critical defensive mechanism in response to oxidative stress. Therefore, Nrf2 plays an important role in ALD antioxidant responses, and its level is decreased by increased reactive oxidation stress (ROS) in the liver. ESF increased Nrf2, which was decreased in ethanol-damaged livers. Additionally, four polyphenol compounds were identified through a qualitative analysis of the ESF using LC-MS/MS. This study confirmed ESF's antioxidative and hangover-elimination effects and suggested the possibility of using Sophora flavescens Aiton (SF) to treat ALD.

KEAP1-NRF2 system regulates age-related spermatogenesis dysfunction.

The average fatherhood age has been consistently increasing in developed countries. Aging has been identified as a risk factor for male infertility. However, its impact on various mechanisms remains unclear. This study focused on the KEAP1-NRF2 oxidative stress response system, by investigating the relationship between the KEAP1-NRF2 system and age-related changes in spermatogenesis. For examination of age-related changes, we used 10-, 30-, 60-, and 90-week-old mice to compare sperm count, sperm motility, and protein expression. For assessment of Keap1 inhibition, 85-week-old C57BL/6J mice were randomly assigned to the following groups: control and bardoxolone methyl (KEAP1 inhibitor). Whole-exome sequencing of a Japanese cohort of patients with non-obstructive azoospermia was performed for evaluating. Sperm count decreased significantly with aging. Oxidative stress and KEAP1 expression in the testes were elevated. Inhibition of KEAP1 in aging mice significantly increased sperm count compared with that in the control group. In the human study, the frequency of a missense-type SNP (rs181294188) causing changes in NFE2L2 (NRF2) activity was significantly higher in patients with non-obstructive azoospermia than in healthy control group. The KEAP1-NRF2 system, an oxidative stress response system, is associated with age-related spermatogenesis dysfunction.

Hydrogen: A Rising Star in Gas Medicine as a Mitochondria-Targeting Nutrient via Activating Keap1-Nrf2 Antioxidant System.

The gas molecules O2, NO, H2S, CO, and CH4, have been increasingly used for medical purposes. Other than these gas molecules, H2 is the smallest diatomic molecule in nature and has become a rising star in gas medicine in the past few decades. As a non-toxic and easily accessible gas, H2 has shown preventive and therapeutic effects on various diseases of the respiratory, cardiovascular, central nervous system, and other systems, but the mechanisms are still unclear and even controversial, especially the mechanism of H2 as a selective radical scavenger. Mitochondria are the main organelles regulating energy metabolism in living organisms as well as the main organelle of reactive oxygen species' generation and targeting. We propose that the protective role of H2 may be mainly dependent on its unique ability to penetrate every aspect of cells to regulate mitochondrial homeostasis by activating the Keap1-Nrf2 phase II antioxidant system rather than its direct free radical scavenging activity. In this review, we summarize the protective effects and focus on the mechanism of H2 as a mitochondria-targeting nutrient by activating the Keap1-Nrf2 system in different disease models. In addition, we wish to provide a more rational theoretical support for the medical applications of hydrogen.

NRF2 signaling in cytoprotection and metabolism.

The KEAP1-NRF2 system plays a central role in cytoprotection and defense mechanisms against oxidative stress. Because KEAP1 serves as a biosensor for electrophiles by using its reactive thiols and because NRF2 is a transcriptional factor regulating genes involved in the sulfur-mediated redox reactions, the KEAP1-NRF2 system has been regarded as a sulfur-utilizing cytoprotective mechanism. NRF2 is a key regulator of cytoprotective genes, such as antioxidant and detoxification genes, and also to possess potent anti-inflammatory activity. NRF2 has been recently focused as a regulator for the cellular metabolism and mitochondrial function. Particularly, the NRF2-mediated regulatory mechanisms of metabolites and mitochondria has been considered diverse but has not been fully clarified yet. This review article provides an overview of the molecular mechanisms that regulate NRF2 signaling and its cytoprotective roles, and also highlights NRF2 contribution to the cellular metabolism, particularly in the context of mitochondrial function and newly found sulfur metabolism.

Metabolic reprogramming in Nrf2-driven proliferation of normal rat hepatocytes.

Cancer cells reprogram their metabolic pathways to support bioenergetic and biosynthetic needs and to maintain their redox balance. In several human tumors, the Keap1-Nrf2 system controls proliferation and metabolic reprogramming by regulating the pentose phosphate pathway (PPP). However, whether this metabolic reprogramming also occurs in normal proliferating cells is unclear. To define the metabolic phenotype in normal proliferating hepatocytes, we induced cell proliferation in the liver by 3 distinct stimuli: liver regeneration by partial hepatectomy and hepatic hyperplasia induced by 2 direct mitogens: lead nitrate (LN) or triiodothyronine. Following LN treatment, well-established features of cancer metabolic reprogramming, including enhanced glycolysis, oxidative PPP, nucleic acid synthesis, NAD + /NADH synthesis, and altered amino acid content, as well as downregulated oxidative phosphorylation, occurred in normal proliferating hepatocytes displaying Nrf2 activation. Genetic deletion of Nrf2 blunted LN-induced PPP activation and suppressed hepatocyte proliferation. Moreover, Nrf2 activation and following metabolic reprogramming did not occur when hepatocyte proliferation was induced by partial hepatectomy or triiodothyronine. Many metabolic changes in cancer cells are shared by proliferating normal hepatocytes in response to a hostile environment. Nrf2 activation is essential for bridging metabolic changes with crucial components of cancer metabolic reprogramming, including the activation of oxidative PPP. Our study demonstrates that matured hepatocytes exposed to LN undergo cancer-like metabolic reprogramming and offers a rapid and useful in vivo model to study the molecular alterations underpinning the differences/similarities of metabolic changes in normal and neoplastic hepatocytes.

The KEAP1-NRF2 System in Health and Disease

The KEAP1-NRF2 System in Health and Disease

The KEAP1-NRF2 System and Neurodegenerative Diseases.

Significance: Central nervous system (CNS) diseases are disorders of the brain and/or spinal cord and include neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis. Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor belonging to the cap-n-collar family that harbors a unique basic leucine zipper motif and plays as a master regulator of homeostatic responses. Recent Advances: Kelch-like ECH-associated protein 1 (KEAP1) is an adaptor of the Cullin3 (CUL3)-based ubiquitin E3 ligase that enhances the ubiquitylation of NRF2, which promotes the degradation of NRF2 to suppress its transcriptional activity in the absence of stress. Cysteine residues of KEAP1 are modified under stress conditions, and NRF2 degradation is attenuated, allowing it to accumulate and induce the expression of target genes. This regulatory system is referred to as the KEAP1-NRF2 system and plays a central role in protecting cells against various stresses. NRF2 also negatively regulates the expression of inflammatory cytokine and chemokine genes and suppresses pathological inflammation. As oxidative stress, inflammation, and proteostasis are known to contribute to neurodegenerative diseases, the KEAP1-NRF2 system is an attractive target for the treatment of these diseases. Critical Issues: In mouse models of neurodegenerative diseases, Nrf2 depletion exacerbates symptoms and enhances oxidative damage and inflammation in the CNS. In contrast, chemical or genetic NRF2 activation improves these symptoms. Indeed, the NRF2-activating chemical dimethyl fumarate is now widely used for the clinical treatment of MS. Future Directions: The KEAP1-NRF2 system is a promising therapeutic target for neurodegenerative diseases.

Antioxidant Defense Expressed as Glutathione Status and Keap1-Nrf2 System Action in Relation to Anthropometric Parameters and Body Composition in Young Women with Polycystic Ovary Syndrome.

Metabolic disorders present in women with polycystic ovary syndrome (PCOS) and the associated risk of obesity may result in increased oxidative stress and reproductive failure. Therefore, we evaluated the concentrations of reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione peroxidase (GPx), and reductase (GR), as well as nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associating protein1 (Keap1) in the serum of 56 women with PCOS divided according to the visceral to subcutaneous fat surface ratio (VAT/SAT) and waist-to-hip ratio (WHR) values. Antioxidant parameter levels were measured by competitive inhibition enzyme immunoassay technique. As the VAT/SAT ratio and WHR increased, we observed significantly higher concentrations of GSSG and Keap1 protein and a lower value of the GSSG/GSH ratio (R-index), which is considered an index of cellular redox (p < 0.05). Negative correlations were found between the R-index and body weight, BMI, WHR, subcutaneous and visceral fat surface and the VAT/SAT ratio, and total body fat; positive links were found with fat free mass and total body water. Opposite associations were noted between GSSG level and the aforementioned body composition parameters. Oxidative stress characterized by a depleted reduced-to-oxidized glutathione index is associated with anthropometric and body composition parameters in women with PCOS. In particular, abdominal obesity expressed by the VAT/SAT ratio and/or WHR seems to have a negative impact on glutathione status, which may lead to a disruption of many biological cell processes. The observed negative association of Keap1 with R-index suggests that the elevated oxidative changes dependent on the VAT/SAT ratio may lead to Nrf2 activation to promote antioxidant enzyme expression. Although the GSH/GSSG index as well as the VAT/SAT ratio appear to be good indicators of oxidative status, studies on a larger group of patients should continue to confirm these links among women with PCOS.

Molecular Basis of the KEAP1-NRF2 Signaling Pathway.

Transcription factor NRF2 (NF-E2-related factor 2) is a master regulator of cellular responses against environmental stresses. NRF2 induces expression of detoxification and antioxidant enzymes and suppresses inductions of pro-inflammatory cytokine genes. KEAP1 (Kelch-like ECH-associated protein 1) is an adaptor subunit of CULLIN 3 (CUL3)-based E3 ubiquitin ligase. KEAP1 regulates the activity of NRF2 and acts as a sensor for oxidative and electrophilic stresses. NRF2 has been found to be activated in many types of cancers with poor prognosis. Therapeutic strategies to control NRF2-overeactivated cancers have been considered not only by targeting cancer cells with NRF2 inhibitors or NRF2 synthetic lethal chemicals, but also by targeting host defense with NRF2 inducers. Understanding precise molecular mechanisms how the KEAP1-NRF2 system senses and regulates the cellular response is critical to overcome intractable NRF2-activated cancers.

The KEAP1-NRF2 system and neurodegenerative diseases.

Significance: Central nervous system (CNS) diseases are disorders of the brain and/or spinal cord and include neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS). NF-E2-related factor 2 (NRF2) is a transcription factor belonging to the cap-n-collar (CNC) family that harbors a unique basic leucine zipper motif and plays as a master regulator of homeostatic responses. Recent Advances: Kelch-like ECH-associated protein 1 (KEAP1) is an adaptor of the Cullin3 (CUL3)-based ubiquitin E3 ligase that enhances the ubiquitylation of NRF2, which promotes the degradation of NRF2 to suppress its transcriptional activity in the absence of stress. Cysteine residues of KEAP1 are modified under stress conditions, and NRF2 degradation is attenuated, allowing it to accumulate and induce the expression of target genes. This regulatory system is referred to as the KEAP1-NRF2 system and plays a central role in protecting cells against various stresses. NRF2 also negatively regulates the expression of inflammatory cytokine and chemokine genes and suppresses pathological inflammation. As oxidative stress, inflammation, and proteostasis are known to contribute to neurodegenerative diseases, the KEAP1-NRF2 system is an attractive target for the treatment of these diseases. Critical Issues: In mouse models of neurodegenerative diseases, Nrf2 depletion exacerbates symptoms and enhances oxidative damage and inflammation in the CNS. In contrast, chemical or genetic NRF2 activation improves these symptoms. Indeed, the NRF2-activating chemical dimethyl fumarate (DMF) is now widely used for the clinical treatment of MS. Future Directions: The KEAP1-NRF2 system is a promising therapeutic target for neurodegenerative diseases.

Molecular Docking Analysis of Phytochemicals from Tinospora Cordifolia as Potential Inhibitor against SARS CoV- 2

Abstract: Since the beginning of time, the Indian system of medicine (ISM) and traditional Ayurvedic medicine have employed tinospora cordifolia, also known as giloy. In ayurveda, the plant is known as Rasayana and is highly regarded for boosting the body's defences against specific infectious microorganisms. Of the 40 species, only 4 are found in India. It is a perennial herbaceous vine that is a member of the Menispermaceae genus. It is frequently utilised as a distinctive component of several natural medicines and has been used traditionally for a wide range of illnesses, including fever, vomiting, diabetes, jaundice, anaemia, polyuria, and skin conditions. Instead of treating the disease's cause, plant-based medicine actually balances the body. Due to a lack of effective treatment options, the coronavirus strain (SAR-CoV-2) will become more dangerous. Ayurveda is a style of ancient medicine that offers a holistic approach to treating this illness. The only defence against the coronavirus illness, which is spreading like wildfire, is "immunity." The only way to keep healthy and safe while preparing for the impending epidemic is to build immunity. Good immunity protects us from disease progression and the spread of this dangerous virus in the current situation. Due to its immunomodulatory and antiviral properties, giloy herb gained popularity after the COVID-19 pandemic began. Keap1 keeps Nrf2 in check by securing it in its protein complex. Additionally, Keap1 aids in the ubiquitinationmediated degradation of Nrf2. Keap1 is a down-regulator of Nrf2, in other words. Keap1 must be suppressed and Nrf2 must be released in order to increase the production of biological antioxidants. Since freed Nrf2 is unbound, it moves to the nucleus to activate the antioxidant response element (ARE) found on the genes encoding for antioxidants. Keap1 is a 624 amino acid residue long protein that belongs to the BTB (bric-a-brac, tram-track, and broad-complex) Kelch family. Antioxidant production should therefore be increased in such a situation. We have summarised the function of the Keap1-Nrf2 system in the synthesis of antioxidants in this review article. We also suggest its possible therapeutic application in controlling COVID-19's cytokine storm. This article focuses on molecular docking of KEAP-1-Nrf2, NRF-2 proteins to inhibit COVID- 19 disease.

Re-Sensitization of Azacytidine-Resistant MDS/AML Cells Via Modulation of KEAP1-NRF2 Redox Pathway

Introduction: The hypomethylation therapy by the cytidine analogue pro-drug 5-azacytidine (AZA) significantly extends the life expectancy of patients with myeloid malignancies, as shown in randomized trials in patients with myelodysplastic syndromes (MDS). Although AZA induces more durable hematologic responses, the rate and duration of remissions are insufficient, and most patients eventually progress on therapy, highlighting the need to better understand the mechanisms of drug resistance. Our and others’ previous data suggest that AZA resistance is the result of the interplay between multiple molecular mechanisms [Minařík L. et al. 2022]. Recent evidence highlights the important role of oxidative/reductive (redox) homeostasis in the development of resistance [Pollyea, et al. 2018, Jones CL. et al. 2019, Gu, X. et al. 2021]. Redox homeostasis is controlled by redox sensor Kelch like associated protein 1 (KEAP1), whose oxidation of cysteine under elevated oxidative stress stabilizes the transcription factor Nuclear factor erythroid 2-related factor 2 (NRF2), leading to cytoprotection against oxidative stress. In this work we aimed to investigate the role of the KEAP1-NRF2 pathway in the AZA efficacy. Methods: We derived AZA-resistant clones (AZA-R) from the human OCI-M2 MDS/acute myeloid leukemia (AML) cell line. The IC50 of AZA-R estimated by WST assay ranged from 4-16 µM. We applied CM2-H2DCFDA and ThiolTracker fluorescent probes to determine the oxidative state and glutathione levels (GSH) of AZA-sensitive (AZA-S) and AZA-R cells. To evaluate functional impact of altered redox homeostasis in AZA-R, we applied a quantitative mass spectrometry-based proteomic approach to identify protein targets of oxidative modifications in AZA-S and AZA-R after AZA treatment. The consequence of altered redox homeostasis was determined by RNA sequencing of AZA-S and AZA-R cells after 24h treatment with 1 µM AZA. The findings were functionally validated. Results: We showed that the redox state of AZA-S was increased by AZA, while the basal oxidative levels of AZA-R cells were 2-fold higher than those of AZA-S cells and were not further altered by AZA. Similarly, AZA induced GSH levels in AZA-S but not in AZA-R cells. Treatment of AZA-S with N-acetylcysteine (NAC) ameliorated oxidative stress and alleviated AZA toxicity, suggesting that AZA-induced oxidative stress is a key factor in its cytotoxic effect. Conversely, a reduction in GSH levels in these cells led to an increase in oxidative state, which was accompanied by enhanced sensitivity to AZA. Proteomic analysis showed that AZA treatment of AZA-S cells resulted in altered cysteine oxidation of 20% of cysteine proteins (578 out of 2853). Resistance to AZA was associated with oxidation state of 14% of cysteine proteins, among which the key redox sensor KEAP1 was significantly differently oxidized between AZA-R and AZA-S. Transcriptomic profiling revealed that 75 out of 478 NRF2 target genes were differentially expressed between AZA-S and AZA-R. NRF2 protein levels were significantly lower in the nucleus of AZA-R MDS/AML myeloblasts by Western blotting. These data indicate a loss of the dynamic response of AZA-R cells to oxidative stress by the KEAP1-NRF2 system. Therefore, we aimed to activate NRF2 in AZA-R by treatment with KEAP1 inhibitors (iKEAP1). We found that NRF2 activation in these cells modifies the redox balance and restores the sensitivity of AZA-R cells to AZA, presumably by restoring the antioxidant properties of the cells. Finally, we transplanted AZA-S and AZA-R cells into immunodeficient mice in order to validate our data in vivo. Generated Cell line-Derived Xenograft (CDX) AZA-R models were treated with iKEAP and indeed iKEAP1 markedly prolonged event-free survival and overall survival of MDS/AML mice upon AZA treatment. Conclusions: We demonstrate that the mechanism of AZA resistance involves the cellular adaptation to oxidative stress and that modulation of the KEAP1-NRF2 cellular antioxidant response pathway can efficiently re-sensitize AZA-resistant cells in vitro. We also demonstrated our findings of enhanced AZA efficacy by KEAP1 inhibition in vivo in an experimental CDX mouse model. This preliminary evidence supports the initiation of a randomized clinical trial to block KEAP1-NRF2 pathway in patients with high-risk-MDS progressing on AZA therapy.

Establishment of Neh2-Cre:tdTomato reporter mouse for monitoring the exposure history to electrophilic stress.

Cells are often exposed to exogenous and endogenous redox disturbances and exert their protective mechanisms in response to stimuli. The KEAP1-NRF2 system plays pivotal roles in counteracting oxidative damage. Due to the transient nature of NRF2 activation, the identification of cells in which NRF2 is activated in response to systemic stimuli is sometimes not easy. To examine the electrophilic stress response at a single-cell resolution, we aimed to develop a new reporter mouse in this study. A cell-tracing strategy exploiting Cre recombinase-mediated activation of a reporter gene was chosen for stable detection of reporter expression instead of real-time monitoring of the cellular response. We established a transgenic mouse line expressing the Neh2-Cre recombinase fusion protein. As Neh2 is an amino-terminal domain of NRF2 that serves as a degron and mediates KEAP1-dependent degradation and electrophile-inducible stabilization, Neh2-Cre was expected to be activated in response to electrophiles. The Neh2-Cre transgenic mouse was crossed with the ROSA26-loxP-stop-loxP-tdTomato reporter mouse (ROSA-LSL-tdTomato mouse). The compound mutant reporter mice exhibited accumulation of tdTomato-positive cells in various organs after repeated administration of CDDO-Im, one of the NRF2-inducing electrophiles. The mice were also successfully used for the detection of cells that experienced a cisplatin-induced electrophilic stress response.

The KEAP1-NRF2 System and Esophageal Cancer.

Simple SummaryNRF2-activated or NRF2-addicted cancers show high incidence, especially in esophageal squamous cell carcinoma (ESCC). ESCC with high NRF2 expression is largely resistant to the current major treatments for ESCC and therefore shows a very poor prognosis. In order to develop effective treatments for NRF2-addicted esophageal cancers, the elucidation and understanding of the mechanistic basis of NRF2 function in NRF2-addicted cancer cells are critically important. This review summarizes the current knowledge of the KEAP1-NRF2 system and proposes three distinct approaches for the treatment of NRF2-addicted ESCC.NRF2 (nuclear factor erythroid 2-related factor 2) is a transcription factor that regulates the expression of many cytoprotective genes. NRF2 activation is mainly regulated by KEAP1 (kelch-like ECH-associated protein 1) through ubiquitination and proteasome degradation. Esophageal cancer is classified histologically into two major types: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). ESCC harbors more genetic alterations in the KEAP-NRF2 system than EAC does, which results in NRF2 activation in these cancers. NRF2-addicted ESCC exhibits increased malignancy and acquisition of resistance to chemoradiotherapy. Therefore, it has been recognized that the development of drugs targeting the KEAP1-NRF2 system based on the molecular dissection of NRF2 function is important and urgent for the treatment of ESCC, along with efficient clinical screening for NRF2-addicted ESCC patients. Recently, the fate of NRF2-activated cells in esophageal tissues, which was under the influence of strong cell competition, and its relationship to the pathogenesis of ESCC, was clarified. In this review, we will summarize the current knowledge of the KEAP1-NRF2 system and the treatment of ESCC. We propose three main strategies for the treatment of NRF2-addicted cancer: (1) NRF2 inhibitors, (2) synthetic lethal drugs for NRF2-addicted cancers, and (3) NRF2 inducers of the host defense system.