Nrf2 Target Research Articles

Abstract C126: Translational pharmacokinetic/pharmacodynamic (PK/PD) modeling of novel covalent Kelch-like ECH-associated protein 1 (KEAP1) activators

Abstract Introduction: The KEAP1-nuclear factor erythroid 2-related factor 2 (NRF2) signaling axis is a key homeostatic mechanism for cells to maintain redox balance. In oxidative stress, reactive oxygen species (ROS) modify residues on KEAP1, impairing its binding and ubiquitination of NRF2. This leads to an accumulation and translocation of NRF2 to the nucleus where it increases transcription of genes for antioxidant response [Pillai 2022]. The KEAP1-NRF2 pathway is hijacked in cancers through NRF2 gain of function or KEAP1 loss of function mutations leading to aberrant activation of NRF2. We have discovered small molecule covalent KEAP1 activators that increase Keap1 incorporation into productive E3 complexes with CUL3, promoting the degradation of NRF2. Given the covalent nature of binding, the turnover of KEAP1, the resulting regulation of NRF2 protein level, and NRF2 target gene transcription and translation, the relationship between the small molecule KEAP1 activator plasma PK, tumor PK and KEAP1 target engagement (TE), and resulting PD (NRF2 degradation) is complex. A translational PK/PD model is established to quantitatively describe this for a tool molecule VVD-065 [see Roy 2023], investigate parameters which influence PK/PD relationships, and identify potential efficacious doses to guide development. Methods: The translational PK/PD model includes a physiologically-based PK (PBPK) model to capture available animal PK and predict human plasma PK, a compartmental tumor PK model to describe and predict tumor PK from mouse xenograft experiments, and a mechanistic TE-PD model derived from a published model for binding of KEAP1 and NRF2 and the stabilization and subsequent ubiquitination of the KEAP1-NRF2-drug complex [Liu 2021]. Thus, the translational PK/PD model integrates cumulative available physicochemical properties, in vitro, and preclinical in vivo data of VVD-065 [see Roy 2023]. Model development was undertaken in R software using the mrgsolve package. Results: The established PBPK model successfully recapitulated animal (mouse, rat, dog) PK from physicochemical properties and in vitro ADME and was used to predict human plasma PK. Mouse plasma and tumor PK collected from xenograft experiments was used to construct a compartmental tumor PK model to subsequently predict human tumor PK from simulated human plasma PK. The mechanistic TE-PD model was qualified based on literature, proteomics, and available in vitro and in vivo (xenograft) KEAP1 TE and NRF2 degradation driven by tumor PK. Physiological variability contributing to human PK was incorporated through creation of a virtual population. Sensitivity analyses from the translational PK/PD model revealed tumor PK and KEAP1 turnover as key parameters influencing PD activity. The translational PK/PD model could be used to predict human dosing regimens which maximize KEAP1 TE and resulting NRF2 degradation. Conclusion: A translational PK/PD model is established and can be used to predict efficacious dose regimens and guide clinical development for novel KEAP1 activators. Citation Format: Conrad Housand, Nil Roy, Tine Wyseure, Christie Eissler, Justine Metzger, Michaela Bairlein, Julie O'Brien, Melaminah Williams, Victor Contreras, Marine Garrido, Todd Kinsella, Jenna Goldberg, Matt Patricelli, Peter N. Morcos. Translational pharmacokinetic/pharmacodynamic (PK/PD) modeling of novel covalent Kelch-like ECH-associated protein 1 (KEAP1) activators [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C126.

A New Mouse Strain with a Mutation in the NFE2L2 (NRF2) Gene.

Transcription factor NRF2 is involved in inflammatory reactions, maintenance of redox balance, metabolism of xenobiotics, and is of particular interest for studying aging. In the present work, the CRISPR/Cas9 genome editing technology was used to generate the NRF2ΔNeh2 mice containing a substitution of eight amino acid residues at the N-terminus of the NRF2 protein, upstream of the functional Neh2 domain, which ensures binding of NRF2 to its inhibitor KEAP1. Heterozygote NRF2wt/ΔNeh2 mice gave birth to homozygous mice with lower than expected frequency, accompanied by their increased embryonic lethality and visual signs of anemia. Mouse embryonic fibroblasts (MEFs) from the NRF2ΔNeh2/ΔNeh2 homozygotes showed impaired resistance to oxidative stress compared to the wild-type MEFs. The tissues of homozygous NRF2ΔNeh2/ΔNeh2 animals had a decreased expression of the NRF2 target genes: NAD(P)H:Quinone oxidoreductase-1 (Nqo1); aldehyde oxidase-1 (Aox1); glutathione-S-transferase A4 (Gsta4); while relative mRNA levels of the monocyte chemoattractant protein 1 (Ccl2), vascular cell adhesion molecule 1 (Vcam1), and chemokine Cxcl8 was increased. Thus, the resulting mutation in the Nfe2l2 gene coding for NRF2, partially impaired function of this transcription factor, expanding our insights into the functional role of the unstructured N-terminus of NRF2. The obtained NRF2ΔNeh2 mouse line can be used as a model object for studying various pathologies associated with oxidative stress and inflammation.

Roles for Heme Synthesis in the Maintenance of Hematopoietic Stem and Progenitor Cells

Heme is required not only in red blood cells for oxygen transport but also in non-erythroid cells for the proper function of hemoproteins. Cytochrome c is one of the hemoproteins and inevitable for mitochondrial oxidative phosphorylation, suggesting that heme possesses important roles for ATP synthesis and mitochondria maintenance. Heme also can skew the differentiation of hematopoietic stem and progenitor cells (HSPCs) by altering the functions of transcription factors. Recent CRISPR Cas9 dropout screens repeatedly showed that some of the heme synthases, such as CPOX, might be inevitable for the survival and/or proliferation of HSPCs. However, still little is known about the roles for heme synthesis in the maintenance (survival, proliferation and differentiation) of HSPCs. To reveal the roles for heme synthesis in HSPCs in vivo, we developed polyI:C inducible hematopoietic cell specific Cpox knock-out mice ( Cpox f/f; Mx1-Cre). PolyI:C treated Cpox f/f; Mx1-Cre mice (KO mice) showed significant increase in the numbers of hematopoietic stem cells (HSCs) and multipotent stem and progenitor cells (MPP2s and MPP3s) but decrease in the numbers of common myeloid progenitors (CMPs) and some of their progenies (i.e., Gr1 positive cells and platelets). In contrast, its effects on erythroid and lymphoid lineages were limited. This result suggests that heme synthesis inhibition in HSPCs (HSCs, MPP2 and MPP3s) can cause their differentiation arrest. On the other hand, competitive transplantation analysis of LSKs (lineage -sca1 +c-kit +) or HSCs revealed that myelopoiesis were markedly decreased compared to control, suggesting that heme synthesis in HSPCs is important for the proper reconstitution of hematopoiesis. These results indicate that cell intrinsic heme synthesis is inevitable for the maintenance of HSPCs and their lineage specific output in vivo. In depth, we revealed that mitochondrial respiratory activity of Cpox KO HSCs were increased compared to control HSCs, suggesting that alterations of mitochondrial status and/or functions by the inhibition of heme synthesis might contribute to the aberrance in Cpox KO HSPCs. Comprehensive gene expression analysis is now ongoing to support this notion. To further reveal the roles for heme synthesis in human HSPCs, we treated human leukemia cell lines (MV4-11, KG1a and U937) with heme synthesis inhibitors (SA or NMPP). Concentration-dependent cell growth inhibitory effect and cell death were observed with increased apoptosis factors (cleaved caspase 3 and cleaved PARP), suggesting that heme synthesis inhibition triggered apoptosis. We also revealed that CPOX inhibition under doxycycline inducible CRISPR Cas9 system caused cell growth inhibitory effect and cell death in these cells. Second mitochondria-derived activator of caspases (Smac) and cytochrome c were released to cytoplasm, and the inhibitor of apoptosis protein (XIAP) was decreased by heme synthesis inhibitions. Therefore, caspase-3 activation by accumulation of cytoplasmic cytochrome c and Smac might be the inducer of this apoptosis pathway. Comprehensive transcriptomic analysis (RNA-seq) revealed that heme synthesis inhibition induced the release of cytochrome c and Smac from mitochondria into cytoplasm by the transcriptomic upregulation of Oxidative Stress Induced Growth Inhibitor 1 (Osgin1) that is the known target of NRF2. Collectively, this study demonstrated the significance of heme synthesis in the maintenance of HSPCs especially in vivo (mouse) and in vitro (human). Dysfunctions of heme synthesis in HSPCs can cause their aberrance by the alterations in their mitochondrial status which can cause apoptosis and/or differentiation arrest. Considering the metabolic differences (i.e., demands for mitochondrial oxidative phosphorylation and subsequent ATP synthesis) between normal HSPCs and leukemia or unperturbed hematopoiesis and perturbed hematopoiesis (i.e., infections and hematopoietic stem cell transplantations), further analysis to reveal the roles for heme synthesis in the maintenance of HSPCs will pave the way to establish optical approaches in these clinical settings.

Targeting Nrf2 Signaling Pathway in Cancer Prevention and Treatment: The Role of Cannabis Compounds.

The development and progression of cancer are associated with the dysregulation of multiple pathways involved in cell proliferation and survival, as well as dysfunction in redox balance, immune response, and inflammation. The master antioxidant pathway, known as the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, regulates the cellular defense against oxidative stress and inflammation, making it a promising cancer prevention and treatment target. Cannabinoids have demonstrated anti-tumor and anti-inflammatory properties, affecting signaling pathways, including Nrf2. Increased oxidative stress following exposure to anti-cancer therapy prompts cancer cells to activate antioxidant mechanisms. This indicates the dual effect of Nrf2 in cancer cells-influencing proliferation and apoptotic processes and protecting against the toxicity of anti-cancer therapy. Therefore, understanding the complex role of cannabinoids in modulating Nrf2 might shed light on its potential implementation as an anti-cancer support. In this review, we aim to highlight the impact of cannabinoids on Nrf2-related factors, with a focus on cancer prevention and treatment. Additionally, we have presented the results of several research studies that combined cannabidiol (CBD) with other compounds targeting Nrf2. Further studies should be directed toward exploring the anti-inflammatory effects of cannabinoids in the context of cancer prevention and therapy.

Phase 1b/2 Study of Escalating Doses of the NEDD8 Activating Enzyme Inhibitor Pevonedistat Administered in Combination with Standard Induction Therapy (Cytarabine and Idarubicin) in Newly Diagnosed High Risk Acute Myeloid Leukemia

Introduction: Pevonedistat is a potent and selective small-molecule inhibitor of the NEDD8-activating enzyme that has demonstrated clinical activity in acute myeloid leukemia (AML). Preclinical studies suggest that pevonedistat synergistically enhances the activity of cytarabine in AML. We investigated the safety and efficacy of pevonedistat in combination with 7+3 cytarabine and idarubicin induction chemotherapy in adults with high-risk AML. Methods: This open-label, multicenter phase 1b/2 study (NCT03330821) enrolled patients aged ≥18 years with newly diagnosed high-risk AML, defined as therapy-related AML, AML with antecedent myelodysplastic syndrome or AML with myelodysplasia-related changes, the presence of adverse genetic features per ELN 2017 guidelines, or being age ≥55. Eligible patients had an ECOG status of 0-2. Patients were administered pevonedistat on days 1, 3, and 5 of each 28-day cycle in combination with cytarabine 100 mg/m 2 on days 1-7 and idarubicin 12 mg/m 2 on days 1-3. In the phase 1b dose-escalation part of the study, a standard 3+3 design was used to evaluate 2 dose levels of pevonedistat: 15 and 20 mg/m 2. Consolidation consisted of up to 4 cycles of cytarabine (1-3 g/m 2 q12h) on days 1, 3, and 5 or transplant. For pharmacodynamic (PD) assessment, RT-PCR assays were performed on peripheral blood samples to measure expression levels of 7 NRF-2 target genes (ATF3, GCLM, GSR, NQ01, SLC7A11, SRXN1, and TXNRD1). The primary objective for the phase 1b study was to determine the RP2D of pevonedistat in combination with cytarabine and idarubicin. The primary objective for the phase 2 study was to determine the composite complete response rate (CRc; CR+CRi). Secondary objectives included relapse-free survival (RFS), overall survival (OS), and safety and tolerability. Results: A total of 28 patients were enrolled at 5 sites in the US between August 2018 and August 2021. Six patients were enrolled in the phase 1b study and 22 patients in phase 2. Baseline patient characteristics are shown in Table 1. The median age was 61 years (range 47-74). Per the 2017 ELN risk classification, 8 of 28 patients (29%) had favorable risk, 8 (29%) had intermediate risk, and 12 (43%) had adverse risk. All 28 patients were included for safety and response assessments. In phase 1b, none of the 6 patients experienced dose-limiting toxicity at 15 or 20 mg/m 2. Thus, the RP2D of pevonedistat was determined to be 20 mg/m 2. The CRc rate was 64% (95% CI 44-81) with 17 patients achieving CR and 1 patient achieving CRi. Of the 18 patients who achieved CR or CRi, 16 (89%) achieved MRD negativity by multiparameter flow cytometry, and 7 (39%) proceeded to HSCT. The CRc rates for patients in the ELN 2017 favorable, intermediate, and adverse risk groups were 100% (95% CI 63-100), 63% (95% CI 29-89), and 42% (95% CI 18-71), respectively. Eight patients were included in the interim PD analysis. Among patients who achieved CR or CRi (n=6), 4 (ATF3, GSR, SLC7A11, and SRXN1) of the 7 NRF-2 target genes measured were significantly upregulated at 5 hours post-pevonedistat induction compared to baseline. There was no significant increase in NRF-2-regulated gene transcript levels among patients with refractory disease (n=2). The median follow-up was 31.1 months (range 19.1-55.9). The median RFS was 24.4 months (95% CI 7-54.3+) and median OS was 25 months (95% CI 11.9-55.9+). The estimated 1-year and 2-year RFS were 72% (95% CI 52-93) and 55% (95% CI 23-78), respectively. The estimated 1-year and 2-year OS were 68% (95% CI 51-85) and 53% (95% CI 34-72), respectively. The most common grade ≥3 treatment-related adverse events were anemia (61%), febrile neutropenia (25%), and hypophosphatemia (18%). No treatment-related deaths were observed. Conclusion: Pevonedistat in combination with cytarabine and idarubicin was well tolerated among adults with newly diagnosed, high-risk AML and induced deep remissions with promising RFS and OS.

Nrf2 activator Diethyl Maleate attenuates ROS mediated NLRP3 inflammasome activation in murine microglia.

Microglia are the tissue-resident immune cells of the central nervous system. As a part of the innate immune response, NLR Family Pyrin Domain Containing Protein 3 (NLRP3) inflammasome activation leads to cleavage of caspase-1 and triggers secretion of proinflammatory cytokines and may also result in pyroptotic cell death. Inflammasome activation plays a crucial role in inflammatory conditions; aberrant activation of inflammasome contributes to the pathogenesis of neurodegenerative diseases. Diethyl Maleate (DEM) is a promising antiinflammatory chemical to alleviate inflammasome activation. In this study, NLRP3 inflammasome was activated in N9 murine microglia via 1µg/ml LPS (Lipopolysaccharide) for 4h and 5 mM ATP (Adenosine 5'-triphosphate) for 1h, respectively. We demonstrated that 1h pretreatment of DEM attenuated NLRP3 inflammasome activation in microglial cells. Besides, mitochondrial ROS decreased upon DEM pretreatment in inflammasome-induced cells. Likewise, it ameliorated pyroptotic cell death in microglia. DEM is a potent activator of Nrf2 transcription factor, the key regulator of the antioxidant response pathway. Nrf2 has been a significant target to decrease aberrant inflammasome activation through the antioxidant compounds, including DEM. Here, we have shown that DEM increased Nrf2 translocation to the nucleus, resulting in Nrf2 target gene expression in microglia. In conclusion, DEM is a promising protective agent against NLRP3 inflammasome activation.

Abstract 17696: Perm1 Stabilizes Nrf2 via Keap1 Oxidation to Protect the Heart Against Reperfusion Injury

Introduction: Reperfusion after ischemia (IR) produces reactive oxygen species (ROS) that promote myocardial injury. Nrf2 is a transcription factor that promotes transcription of antioxidant enzymes. Keap1 is a negative regulator of Nrf2. When oxidated by ROS, Keap1 releases Nrf2, leading to Nrf2 activation. Perm1 (PPARGC-1 and ESRR-induced regulator, muscle specific 1) is a scaffold or adaptor protein that maintains cellular metabolism. However, whether and how Perm1 protects the heart against IR injury via Nrf2 remains unknown. Methods: We used systemic Perm1 knockout (KO) mice and adeno-associated virus (AAV)-induced cardiac-specific Perm1 overexpression to investigate the pathological role of Perm1 in IR injury. Results: IR-induced myocardial infarction was greater in Perm1KO mice than in control mice. Consistently, infarction was smaller in mice with Perm1 overexpression (Infarction size/area at risk (AAR): AAV-GFP as a control 0.41 and AAV-Perm1 0.28*; p<0.05). Myocardial oxidative stress, evaluated via increases in dityrosine, 4HNE and GSSG/GSH, after IR was greater in Perm1KO but less in Perm1 overexpression mice. Expression of IR-induced Nrf2 target genes, such as catalase, superoxide dismutase 2, heme oxygenase 1, NADH quinone dehydrogenase 1 and thioredoxin 1, was inhibited in Perm1KO but promoted in overexpression mice. The detrimental effects observed in Perm1KO mice were normalized by ML334, an inhibitor of Keap1-Nrf2 interaction (Infarction size/AAR: Wild 0.42; Perm1KO 0.58; Wild with ML334 0.37 and Perm1KO with ML334 0.42*; p<0.05 vs. Perm1KO), suggesting that Perm1 protects the heart via Nrf2. In primary cultured rat cardiomyocytes, Perm1 was bound to Keap1 and promoted its oxidation, thereby stabilizing and activating Nrf2. Perm1-induced Nrf2 activation was verified with reporter gene assays using a consensus binding sequence of Nrf2, termed antioxidant response element. In an in vitro system using recombinant proteins, Perm1 directly bound to Keap1. Conclusions: These results suggest that Perm1 is a novel regulator of cellular redox homeostasis through Nrf2 activation via Keap1 oxidation that protects the heart against IR injury.

Abstract 14638: NPA7: A Novel Designed Bispecific Peptide That Protects Human Cardiomyocytes Against Oxidative Stress Through the GC-A/Mas Receptor-KEAP1-NRF2 Pathway

Introduction: Excess cardiomyocyte oxidative stress is a major metabolic abnormality in HTN and its progression to HF. Thus, therapies that reduce oxidative stress in the heart may lead to a reduction in CVD related adverse outcomes such as HF and death. Recently, we bioengineered a bispecific peptide NPA7 that co-activates the protective GC-A/cGMP and MasR/cAMP pathways. Previously we showed that NPA7 possesses cardiorenal protective properties. However, the antioxidant effects of NPA7 within the heart is unknown. Hypothesis: NPA7 protects human cardiomyocytes against oxidative stress through the GC-A/Mas Receptor-KEAP1-NRF2 pathway. Methods: HCMs were pretreated with PBS or NPA7 (10 uM) followed by 400 uM hydrogen peroxide (H2O2) to induce oxidative stress. Intracellular ROS was measured by dihydroethidium (DHE) staining and protein levels of KEAP1 and NRF2 via western blot. The ratio of GSH/GSSG were determined by using commercial kit. mRNA levels of G6PD, GCLC, GCLM and GSS in HCMs, which represents antioxidant capacity, were determined by real-time PCR. For knockdown experiments, HCMs were transfected with the siRNA target GC-A and MasR for 24 h and then treated with NPA7 and H2O2 for another 20 h. Results: H2O2 increased DHE staining level, which indicated elevated intracellular ROS levels within HCMs. Notably, treatment with NPA7 decreased DHE level and increased the ratio of GSH/GSSG in HCMs with H2O2. Furthermore, NPA7 significantly increased the mRNA level of NRF2 target genes (G6PD, GCLC, GCLM, GSS) through NRF2 activation and KEAP1 degradation, indicating that NPA7 activates KEAP1-NRF2 pathway, the upstream pathway that regulate antioxidant gene expression, in HCMs. Moreover, these effects were eliminated if GC-A and MasR were silenced by siRNA, thus supporting the protective antioxidant role of both GC-A and MasR. Conclusions: The present study establishes an antioxidant action of NPA7, a novel bispecific designer peptide, on H2O2-induced oxidative stress by the dual targeting of GC-A and MasR through the KEAP1-NRF2 signaling pathway in HCMs. These results advance NPA7 as an innovative therapeutic strategy targeting oxidative stress related to CVD.

Emerging small molecule inhibitors of Bach1 as therapeutic agents: Rationale, recent advances, and future perspectives.

The transcription factor Nrf2 is the master regulator of cellular stress response, facilitating the expression of cytoprotective genes, including those responsible for drug detoxification, immunomodulation, and iron metabolism. FDA-approved Nrf2 activators, Tecfidera and Skyclarys for patients with multiple sclerosis and Friedreich's ataxia, respectively, are non-specific alkylating agents exerting side effects. Nrf2 is under feedback regulation through its target gene, transcriptional repressor Bach1. Specifically, in Parkinson's disease and other neurodegenerative diseases with Bach1 dysregulation, excessive Bach1 accumulation interferes with Nrf2 activation. Bach1 is a heme sensor protein, which, upon heme binding, is targeted for proteasomal degradation, relieving the repression of Nrf2 target genes. Ideally, a combination of Nrf2 stabilization and Bach1 inhibition is necessary to achieve the full therapeutic benefits of Nrf2 activation. Here, we discuss recent advances and future perspectives in developing small molecule inhibitors of Bach1, highlighting the significance of the Bach1/Nrf2 signaling pathway as a promising neurotherapeutic strategy.

Silica-Coated Superparamagnetic Iron Oxide Nanoparticles Ameliorate Leukoaraiosis-Triggered Oxidative Stress via Targeting of Nrf2 to Activate IRS-1/Akt Signals

This study mainly explored the amelioratory effect of silica-coated superparamagnetic iron oxide nanoparticles (SiO4@SPIONs) on leukoaraiosis-associated cognitive dysfunction via targeting of Nrf2 and their potential mechanism. PC12 cell line was given in vitro hypoxia-reoxygenation treatment (OGD/R) and divided into normal group, model group and SiO4@SPIONs treatment group. The cell survival rate, apoptosis rate, levels of ROS (reactive oxygen species), MDA (Malondialdehyde; malonic dialdehyde; Propanedial) and anti-oxidases were monitored. Western-blotting measured the level of Akt and Nrf2. The Akt-targeting antagonist LY294002 was applied for verifying the regulatory effect of SiO4@SPIONs. In comparison with normal cells, model cells exhibited significantly reduced viability, along with significantly elevated apoptosis rate. Meanwhile, model cells also displayed elevated levels of intracellular ROS and MDA, with diminished levels of SOD (Superoxide Dismutase), GSH (glutathione), CAT (Computerized Axial Tomography) and GSH-Px (glutathione peroxidase) which were accompanied by significantly weakened phosphorylation level of Akt. Lastly, model cells also exhibited moderate enhancement of Nrf2 and HO-1 expressions. Moreover, cells in SiO4@SPIONs treatment group exhibited significantly increased viability, along with reduced apoptosis rate. Meanwhile, the cells in the SiO4@SPIONs treatment group also displayed decreased levels of intracellular ROS and MDA, while showing increased levels of SOD, GSH, CAT and GSH-Px, which were accompanied by significantly increased Akt phosphorylation and Nrf2 and HO-1 expressions. The SiO4@SPIONS can ameliorate the leukoaraiosis-triggered oxidative stress via targeting of Nrf2 to activate the IRS-1/Akt signals.

Wnt5a/Ror2 promotes Nrf2-mediated tissue protective function of astrocytes after brain injury.

Astrocytes, a type of glial cells, play critical roles in promoting the protection and repair of damaged tissues after brain injury. Inflammatory cytokines and growth factors can affect gene expression in astrocytes in injured brains, but signaling pathways and transcriptional mechanisms that regulate tissue protective functions of astrocytes are still poorly understood. In this study, we investigated the molecular mechanisms regulating the function of reactive astrocytes induced in mouse models of stab wound (SW) brain injury and collagenase-induced intracerebral hemorrhage (ICH). We show that basic fibroblast growth factor (bFGF), whose expression is up-regulated in mouse brains after SW injury and ICH, acts synergistically with inflammatory cytokines to activate E2F1-mediated transcription of a gene encoding the Ror-family protein Ror2, a receptor for Wnt5a, in cultured astrocytes. We also found that subsequent activation of Wnt5a/Ror2 signaling in astrocytes results in nuclear accumulation of antioxidative transcription factor Nrf2 at least partly by increased expression of p62/Sqstm1, leading to promoted expression of several Nrf2 target genes, including heme oxygenase 1. Finally, we provide evidence demonstrating that enhanced activation of Wnt5a/Ror2 signaling in astrocytes reduces cellular damage caused by hemin, a degradation product of hemoglobin, and promotes repair of the damaged blood brain barrier after brain hemorrhage.

The Contribution of Hippocampal All-Trans Retinoic Acid (ATRA) Deficiency to Alzheimer's Disease: A Narrative Overview of ATRA-Dependent Gene Expression in Post-Mortem Hippocampal Tissue.

There is accumulating evidence that vitamin A (VA) deficiency contributes to the pathogenesis and progression of Alzheimer's disease (AD). All-trans retinoic acid (ATRA), a metabolite of VA in the brain, serves distinct roles in the human hippocampus. Agonists of retinoic acid receptors (RAR), including ATRA, promote activation of the non-amyloidogenic pathway by enhancing expression of α-secretases, providing a mechanistic basis for delaying/preventing amyloid beta (Aβ) toxicity. However, whether ATRA is actually deficient in the hippocampi of patients with AD is not clear. Here, using a publicly available human transcriptomic dataset, we evaluated the extent to which ATRA-sensitive genes are dysregulated in hippocampal tissue from post-mortem AD brains, relative to age-matched controls. Consistent with ATRA deficiency, we found significant dysregulation of many ATRA-sensitive genes and significant upregulation of RAR co-repressors, supporting the idea of transcriptional repression of ATRA-mediated signaling. Consistent with oxidative stress and neuroinflammation, Nrf2 and NfkB transcripts were upregulated, respectively. Interestingly, transcriptional targets of Nrf2 were not upregulated, accompanied by upregulation of several histone deacetylases. Overall, our investigation of ATRA-sensitive genes in the human hippocampus bolsters the scientific premise of ATRA depletion in AD and that epigenetic factors should be considered and addressed as part of VA supplementation.

Hydrogen sulfide inhibits gene expression associated with aortic valve degeneration by inducing NRF2-related pro-autophagy effect in human aortic valve interstitial cells.

Aortic valve stenosis (AS) is the most common valvular heart disease but there are currently no effective medical treatments that can delay disease progression due to a lack of knowledge of the precise pathophysiology. The expression of sulfide: quinone oxidoreductase (SQOR) and nuclear factor erythroid 2-related factor 2 (NRF2) was decreased in the aortic valve of AS patients. However, the role of SQOR and NRF2 in the pathophysiology of AS has not been found. We investigated the effects of hydrogen sulfide (H2S)-releasing compounds on diseased aortic valve interstitial cells (AVICs) to explain the cellular mechanism of SQOR and elucidate the medical value of H2S for AS treatment. Sodium hydrosulfide (NaHS) treatment increased the expression of SQOR and NRF2 gene and consequently induced the NRF2 target genes, such as NAD(P)H quinone dehydrogenase 1 and cystathionine γ-lyase. In addition, NaHS dose-dependently decreased the expression level of fibrosis and inflammation-related genes (MMP9, TNF-α, IL6) and calcification-related genes (ALP, osteocalcin, RUNX2, COL1A1) in human AVICs. Furthermore, NaHS activated the AMPK-mTOR pathway and inhibited the PI3K-AKT pathway, resulting in a pro-autophagy effect in human AVICs. An NRF2 inhibitor, brusatol, attenuated NaHS-induced AMPK activation and decreased the autophagy markers Beclin-1 and LC3AB, suggesting that the mechanism of action of H2S is related to NRF2. In conclusion, H2S decreased gene expression levels related to aortic valve degeneration and activated AMPK-mTOR-mediated pro-autophagy function associated with NRF2 in human AVICs. Therefore, H2S could be a potential therapeutic target for the development of AS treatment.

Regulation of anti-inflammatory and antioxidant responses by methanol extract of Mikania cordata (Burm. f.) B. L. Rob. leaves via the inactivation of NF-κB and MAPK signaling pathways and activation of Nrf2 in LPS-induced RAW 264.7 macrophages

Mikania cordata (Burm. f.) B.L. Rob. has been traditionally used in tropical countries throughout Asia and Africa to treat gastric ulcers, dyspepsia, and dysentery. However, the mechanisms responsible for its anti-inflammatory and antioxidant activities are not fully understood. Therefore, this study sought to investigate the anti-inflammatory and antioxidant effects of methanol extracts of M. cordata (MMC) on inflammation and oxidative stress in lipopolysaccharide (LPS)-stimulated murine RAW 264.7 macrophages and elucidate its underlying regulatory mechanism. MMC significantly suppressed the production of nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-stimulated RAW 264.7 macrophages by downregulating the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) at both the mRNA and protein levels. Moreover, MMC effectively reduced the mRNA expression levels and production of pro-inflammatory cytokines, including interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α). These suppressive effects of MMC on pro-inflammatory mediators and cytokines were mediated through the inhibition of transforming growth factor beta-activated kinase 1 (TAK1), which subsequently blocked the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs). MMC also upregulated the nuclear factor erythroid-2-related factor 2 (Nrf2) by inducing the degradation of Kelch-like ECH-related protein 1 (Keap1), an Nrf2-specific E3 ligase. Accordingly, MMC enhanced Nrf2 target gene expression of anti-oxidative regulators such as heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1). However, it had minimal effect on the DPPH radical scavenging capacity in vitro. Collectively, these findings demonstrate that MMC holds promise as a potential therapeutic agent for alleviating inflammation-related diseases and oxidative stress.

NRF2 is essential for iron-overload stimulated osteoclast differentiation through regulation of redox and iron homeostasis.

Iron overload enhances osteoclastic bone resorption and induces osteoporosis. Excess iron is highly toxic. The modulation of redox and iron homeostasis is critical for osteoclast differentiation under iron-overload condition. Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that regulates the cellular defense against oxidative stress and iron overload through the expression of genes involved in anti-oxidative processes and iron metabolism. Our studies demonstrated that NRF2 activation was suppressed during osteoclast differentiation. Under iron-overload condition, NRF2 and its mediated antioxidant and iron metabolism genes were activated by reactive oxygen species (ROS), which enhanced antioxidant capability. NRF2 mediated the upregulation of iron exporter ferroportin 1 (FPN1) and iron storage protein ferritin, contributing to decreased levels of intracellular iron. Nfe2l2 knockout induced oxidative stress and promoted osteoclast differentiation under normal condition, but induced ferroptosis under iron-overload condition. Nfe2l2 knockout alleviated iron overload induced bone loss by inhibiting osteoclast differentiation. Our results suggest that NRF2 activation is essential for osteoclast differentiation by enhancing antioxidant capability and reducing intracellular iron under iron-overload condition. Targeting NRF2 to induce ferroptosis could be a potential therapy for the treatment of iron-overload induced osteoporosis.

Hepatocyte-specific deletion of small heterodimer partner protects mice against acetaminophen-induced hepatotoxicity via activation of Nrf2.

Acetaminophen (APAP) overdose stands as the primary cause of acute liver failure in the United States. APAP hepatotoxicity involves hepatic glutathione (GSH) depletion and mitochondrial damage. To counteract the toxicity of APAP, the nuclear factor erythroid 2 like 2 (Nrf2) activates the expression of genes responsible for drug detoxification and GSH synthesis. In this study, we present evidence that the elimination of hepatocyte small heterodimer partner, a critical transcriptional repressor for liver metabolism, results in Nrf2 activation and protects mice from APAP-induced acute liver injury. Initial investigations conducted on wildtype (WT) mice revealed a swift downregulation of Shp mRNA within the first 24 h after APAP administration. Subsequent treatment of hepatocyte-specific Shp knockout (ShpHep-/-) mice with 300 mg/kg APAP for 2 h exhibited comparable bioactivation of APAP with that observed in the WT controls. However, a significant reduction in liver injury was observed in ShpHep-/- after APAP treatment for 6 and 24 h. The decreased liver injury correlated with a faster recovery of GSH, attributable to heightened expression of Nrf2 target genes involved in APAP detoxification and GSH synthesis. Moreover, in vitro studies revealed that SHP protein interacted with NRF2 protein, inhibiting the transcription of Nrf2 target genes. These findings hold relevance for humans, as overexpression of SHP hindered APAP-induced NRF2 activation in primary human hepatocytes. In conclusion, our studies have unveiled a novel regulatory axis involving SHP and NRF2 in APAP-induced acute liver injury, emphasizing SHP as a promising therapeutic target in APAP overdose-induced hepatotoxicity.

The untapped potential of targeting NRF2 in neurodegenerative disease.

Since its initial discovery almost three decades ago, the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) has been shown to regulate a host of downstream transcriptional responses and play a critical role in preventing or promoting disease progression depending on the context. Critically, while the importance of proper nuclear factor erythroid 2-related factor 2 function has been demonstrated across a variety of pathological settings, the ability to progress NRF2-targeted therapeutics to clinic has remained frustratingly elusive. This is particularly true in the case of age-related pathologies, where nuclear factor erythroid 2-related factor 2 is a well-established mitigator of many of the observed pathogenic effects, yet options to target this pathway remain limited. Along these lines, loss of nuclear factor erythroid 2-related factor 2 function has clearly been shown to enhance neuropathological outcomes, with enhancing nuclear factor erythroid 2-related factor 2 pathway activation to prevent neurodegenerative/neurological disease progression continuing to be an active area of interest. One critical obstacle in generating successful therapeutics for brain-related pathologies is the ability of the compound to cross the blood brain barrier (BBB), which has also hampered the implementation of several promising nuclear factor erythroid 2-related factor 2 inducers. Another limitation is that many nuclear factor erythroid 2-related factor 2 activators have undesirable off-target effects due to their electrophilic nature. Despite these constraints, the field has continued to evolve, and several viable means of targeting nuclear factor erythroid 2-related factor 2 in a neuropathological context have emerged. In this perspective, we will briefly discuss the key findings and promising therapeutic options that have been discovered to date, as well as highlight emerging areas of NRF2-neurodegeneration research that provide hope for successfully targeting this pathway in the future.

Retracted: Silencing MR-1 Protects against Myocardial Injury Induced by Chronic Intermittent Hypoxia by Targeting Nrf2 through Antioxidant Stress and Anti-Inflammation Pathways.

[This retracts the article DOI: 10.1155/2022/3471447.].

The cell-specific roles of Nrf2 in acute and chronic phases of ischemic stroke.

Ischemic stroke refers to the sudden loss of blood flow in a specific area of the brain. It is the fifth leading cause of mortality and the leading cause of permanent disability. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) controls the production of several antioxidants and protective proteins and it has been investigated as a possible pharmaceutical target for reducing harmful oxidative events in brain ischemia. Each cell type exhibits different roles and behaviors in different phases post-stroke, which is comprehensive yet important to understand to optimize management strategies and goals for care for stroke patients. In this review, we comprehensively summarize the protective effects of Nrf2 in experimental ischemic stroke, emphasizing the role of Nrf2 in different cell types including neurons, astrocytes, oligodendrocytes, microglia, and endothelial cells during acute and chronic phases of stroke and providing insights on the neuroprotective role of Nrf2 on each cell type throughout the long term of stroke care. We also highlight the importance of targeting Nrf2 in clinical settings while considering a variety of important factors such as age, drug dosage, delivery route, and time of administration.

Targeted Proteomic Quantitation of NRF2 Signaling and Predictive Biomarkers in HNSCC

The NFE2L2 (NRF2) oncogene and transcription factor drives a gene expression program that promotes cancer progression, metabolic reprogramming, immune evasion, and chemoradiation resistance. Patient stratification by NRF2 activity may guide treatment decisions to improve outcome. Here, we developed a mass spectrometry-based targeted proteomics assay based on internal standard-triggered parallel reaction monitoring to quantify 69 NRF2 pathway components and targets, as well as 21 proteins of broad clinical significance in head and neck squamous cell carcinoma (HNSCC). We improved an existing internal standard-triggered parallel reaction monitoring acquisition algorithm, called SureQuant, to increase throughput, sensitivity, and precision. Testing the optimized platform on 27 lung and upper aerodigestive cancer cell models revealed 35 NRF2 responsive proteins. In formalin-fixed paraffin-embedded HNSCCs, NRF2 signaling intensity positively correlated with NRF2-activating mutations and with SOX2 protein expression. Protein markers of T-cell infiltration correlated positively with one another and with human papilloma virus infection status. CDKN2A (p16) protein expression positively correlated with the human papilloma virus oncogenic E7 protein and confirmed the presence of translationally active virus. This work establishes a clinically actionable HNSCC protein biomarker assay capable of quantifying over 600 peptides from frozen or formalin-fixed paraffin-embedded archived tissues in under 90min.