Issue 9, April 2024

newsletter2024abril

Our quarterly newsletter attempts to provide our latest news and also aims at becoming a forum for analysis of relevant topics on the field of NRF2 and provide comments to some of the most relevant articles published during the quarter. Previous newsletters can be accessed at:

https://benbedphar.org/our-first-newsletter/
https://benbedphar.org/issue-2-abril-2022/
https://benbedphar.org/issue-3-july-2022/
https://benbedphar.org/issue-4-october-2022/
https://benbedphar.org/issue-5-january-2023/
https://benbedphar.org/issue-6-april-2023/
https://benbedphar.org/issue-7-october-2023/
https://benbedphar.org/issue-8-january-2024/

Antonio Cuadrado
Chair of COST Action 20121, BenBedPhar
Autonomous University of Madrid

The effect of a novel compound named PHAR, a protein-protein interaction inhibitor of NRF2/β-TrCP, which induces a mild NRF2 activation in the liver, has been assessed in a mouse model of non-alcoholic steatohepatitis (NASH) and its progression to fibrosis. PHAR effectively activated NRF2 in murine hepatocytes, Kupffer cells, and hepatic stellate cells. The effect of PHAR was analyzed in the STAM mouse model of NASH, based on partial damage of endocrine pancreas and insulin secretion impairment, followed by a high fat diet. Non-invasive analysis using MRI revealed that PHAR protects against liver fat accumulation. Moreover, PHAR attenuated key markers of NASH progression, including liver steatosis, hepatocellular ballooning, inflammation, and fibrosis. Notably, transcriptomic data indicate that PHAR led to upregulation of 3 anti-fibrotic genes (Plg, Serpina1a, and Bmp7) and downregulation of 6 pro-fibrotic (including Acta2 and Col3a1), 11 extracellular matrix remodeling, and 8 inflammatory genes. A separate study conducted in the pancreatic beta cell line INS-1 832/13 and in isolated mouse islets found that PHAR, in combination with electrophilic NRF2 activators that target KEAP1, led to enhanced NRF2 stability, nuclear localization and target gene expression. Overall, these studies suggest that the mild activation of NRF2 via the protein-protein interaction inhibitor PHAR holds promise as a strategy for addressing NASH and its progression to liver fibrosis.

Adapted from:

  • Fernández-Ginés R, Encinar JA, Escoll M, Carnicero-Senabre D, Jiménez-Villegas J, García-Yagüe ÁJ, González-Rodríguez Á, Garcia-Martinez I, M Valverde Á, Rojo AI, Cuadrado A. Specific targeting of the NRF2/β-TrCP axis promotes beneficial effects in NASH. Redox Biol. 2024 Feb; 69:103027. doi: 10.1016/j.redox.2024.103027. Epub 2024 Jan 3. PMID: 38184999.
  • Patibandla C, van Aalten L, Dinkova-Kostova AT, Honda T, Cuadrado A, Fernández-Ginés R, McNeilly AD, Hayes JD, Cantley J, Sutherland C. Inhibition of glycogen synthase kinase-3 enhances NRF2 protein stability, nuclear localisation and target gene transcription in pancreatic beta cells. Redox Biol. 2024 May;71:103117. doi: 10.1016/j.redox.2024.103117. Epub 2024 Mar 7. PMID: 38479223

Ana I Rojo 
Autonomous University of Madrid, Spain
Albena T Dinkova-Kostova 
University of Dundee, United Kingdom
WG2 Leaders, on behalf of authors

https://doi.org/10.1016/j.redox.2023.102978

Iodide plays a pivotal role in thyroid homeostasis due to its crucial involvement in thyroid hormone biosynthesis. Exposure to pharmacological doses of iodide elicits in the thyroid an autoregulatory response to preserve thyroid function, as well as an antioxidant response that is mediated by the Keap1/Nrf2 signaling pathway. The objective of the present study was to investigate the transcriptional response of the thyroid to excess iodide in a background of enhanced Nrf2 signaling. Keap1 knockdown (Keap1KD) mice that have activated Nrf2 signaling were exposed or not to excess iodide in their drinking water for seven days and compared to respective wild-type mice. RNA-sequencing of individual mouse thyroids identified distinct transcriptomic patterns in response to iodide, with Keap1KD mice showing an attenuated inflammatory response, altered thyroidal autoregulation, and enhanced cell growth/proliferative signaling, as confirmed also by Western blotting for key proteins involved in antioxidant, autoregulatory and proliferative responses. These findings underscore novel gene-environment interactions between the activation status of the Keap1/Nrf2 antioxidant response system and the dietary iodide intake, which may have implications not only for the goiter phenotype of Keap1KD mice but also for humans harboring genetic variations in KEAP1 or NFE2L2 or treated with Nrf2-modulating drugs.

Christina Morgenstern
WG3 Leader​
Karl-Franzens Universität, Austria

NRF2 protects cells from oxidative stress and inflammation by, among others, controlling the expression of several antioxidant and detoxification genes, and is generally considered to be protective against cancer due to its role in reducing oxidative stress and inflammation. However, there is some evidence that NRF2 activation may also promote cancer progression in certain contexts. Specifically, some studies have found that cancer cells can hijack the NRF2 pathway to promote their survival and growth by increasing antioxidant defenses and promoting drug resistance; on the other hand, NRF2 activation in normal cells may help prevent cancer development by reducing oxidative damage and inflammation. These dual roles of NRF2 in cancer are complex and context-dependent, and their implications are not yet fully understood.

ONCTERNAL company (based in San Diego, California, USA) is developing zilovertamab, a humanised monoclonal antibody targeting ROR1, an onco-embryonic antigen expressed on neoplastic cells of a variety of different cancers. ROR1 signaling is associated with epithelial-mesenchymal transition, tumors proliferation and metastasis, and inhibition of ROR1 signaling enhances anti-tumors activity by preventing the activation of NF-κB and NRF2 target genes.

Clinical trials have shown that the humanized anti-ROR1 mAb zilovertamab is safe and effective in inhibiting ROR1 signaling in patients with ROR1-positive leukemia. Zilovertamab is currently being tested in combination with other anti-cancer therapies in clinical trials in several cancer types, including solid tumors, lymphoma and metastatic urothelial carcinoma. Recently, it was reported that sixteen patients with advanced HER2 breast cancer were enrolled in a phase 1b study evaluating zilovertamab in combination with paclitaxel, an antitumor compound. This treatment was well tolerated and effective in inducing a partial response in 6/16 (38%) or stable disease in 6/16 (38%) patients. These results justify further clinical trials targeting ROR1 for treatment and increase interest in the potential clinical use of other NRF2 inhibitors in the future.

Reference

  1. Rebecca A Shatsky, Hemali Batra-Sharma, Teresa Helsten et al. A phase 1b study of zilovertamab in combination with paclitaxel for locally advanced/unresectable or metastatic Her2-negative breast cancer. Clinical Trial Breast Cancer Res. 2024 Feb 26;26(1):32. doi: 10.1186/s13058-024-01782-0.
  2. Elsa Sanchez-Lopez, Emanuela M Ghia , Laura Antonucci , et al. NF-κB-p62-NRF2 survival signaling is associated with high ROR1 expression in chronic lymphocytic leukemia. Cell Death Differ. 2020 Jul;27(7):2206-2216. doi: 10.1038/s41418-020-0496-1. Epub 2020 Jan 28.

Santiago Cuevas
WG4 leader
Principal Investigator Miguel Servet | Molecular Inflammation Group
BioMedical Research Institute of Murcia (IMIB-Arrixaca)

https://doi.org/10.3390/ijms25073683

Adrenergic pathways represent the main channel of communication between the nervous system and the immune system. This cross-talk is required to restore homeostasis after tissue inflammation. During inflammation, blood monocytes migrate within tissue and differentiate into macrophages, which polarize to M1 or M2 macrophages with tissue damaging or reparative properties, respectively. Both monocytes and macrophages express β- adrenergic receptors (β-ARs). Here, we investigated whether the β-AR blocking drug propranolol modulates the monocyte to macrophage differentiation process and further influences macrophages in their polarization toward M1- and M2-like phenotypes. Human monocytes were isolated from peripheral blood and cultured for 6 days with M-CSF in the presence or absence of propranolol and then activated toward a M1 pro-inflammatory state, or a M2 anti-inflammatory/regulatory state (Figure 1A). A chronic exposure of monocytes to propranolol during their differentiation into macrophages in vitro promoted the increase of the M1 markers CD16 and of the M2 markers CD206, CD163 and peroxisome proliferator-activated receptor ɣ expression (Figure 1B). It also increased endocytosis and the release of the anti-inflammatory cytokine IL-10, whereas it reduced physiological reactive oxygen species. Exposure to pro-inflammatory condition of propranolol- differentiated macrophages resulted in an anti-inflammatory promoting effects. At the molecular level, propranolol upregulated the expression of oxidative stress regulators such as the transcription factor nuclear factor E2-related factor 2, heme oxygenase-1 and NAD(P)H Quinone Dehydrogenase 1. By contributing to regulate macrophage activities, propranolol may represent a novel anti-inflammatory and immunomodulating target with relevant therapeutic potential in several inflammatory diseases.

References

  1. Coelho, M.; Soares-Silva, C.; Brandão, D.; Marino, F.; Cosentino, M.; Ribeiro, L. β-Adrenergic modulation of cancer cell proliferation: Available evidence and clinical perspectives. J. Cancer Res. Clin. Oncol. 2017, 143, 275–29.
  2. Cole, S.W.; Nagaraja, A.S.; Lutgendorf, S.K.; Green, P.A.; Sood, A.K. Sympathetic nervous system regulation of the tumour microenvironment. Nat. Rev. Cancer 2015, 15, 563–572.
  3. Cruickshank, J.M. Beta-blockers, plasma lipids, and coronary heart disease. Circulation 1990, 82, II60–II65.
  4. Camejo, G.; Hurt, E.; Thubrikar, M.; Bondjers, G. Modification of low density lipoprotein association with the arterial intima. A possible environment for the antiatherogenic action of beta-blockers. Circulation 1991, 84, VI17–VI22.                                                                                               

Brigitta Buttari
WG5 Leader, on behalf of authors
Istituto Superiore di Sanità, IT

The cytoprotective transcription factor NRF2 regulates the expression of several hundred genes in mammalian cells and is a promising therapeutic target in a number of diseases associated with oxidative stress and inflammation. Hence, an ability to monitor basal and inducible NRF2 signalling is vital for mechanistic understanding in translational studies. Due to some caveats related to the direct measurement of NRF2 levels, the modulation of NRF2 activity is typically determined by measuring changes in the expression of one or more of its target genes and/or the associated protein products. However, there is a lack of consensus regarding the most relevant set of these genes/proteins that best represents NRF2 activity across cell types and species. The authors present the findings of a comprehensive literature search that according to stringent criteria identifies GCLC, GCLM, HMOX1, NQO1, SRXN1 and TXNRD1 as a robust panel of markers that are directly regulated by NRF2 in multiple cell and tissue types. The authors assess the relevance of these markers in clinically accessible biofluids and highlight future challenges in the development and use of NRF2 biomarkers in humans.

Access to the original article: https://pubmed.ncbi.nlm.nih.gov/38643749/

Macrophage polarization towards the M1 phenotype under bacterial product-related exposure (LPS) requires a rapid change in gene expression patterns and cytokine production along with a metabolic rewiring. Metabolic pathways and redox reactions are such tightly connected, giving rise to an area of research referred to as immunometabolism. A role in this context has been paid to the master redox-sensitive regulator Nuclear factor erythroid 2-related factor 2 (Nrf2) and to the 5′-ectonucleotidase CD73, a marker related to macrophage metabolism rearrangement under pro-inflammatory conditions. In this light, a cell model of LPS-stimulated macrophages has been established and nine 4,7-dihydro-4-ethylpyrazolo[l,5-a]pyrimidin-7-ones with a potential anti-inflammatory effect have been administered. This data highlight that two selected compounds (namely, 5 and 8) inhibit the LPS-induced Nrf2 nuclear translocation and ameliorate the activity rate of the antioxidant enzyme catalase. Additionally, the pyridine-containing compound (8) promotes the shift from the pro-inflammatory immunophenotype M1 to the pro-resolving M2 one, by downregulating CD80 and iNOS and by enhancing CD163 and TGFβ1 expression. Most importantly, CD73 is modulated by these compounds as well as the lactate production. This data demonstrate that pyrazolo[l,5-a]pyrimidine derivatives are effective as anti-inflammatory compounds. Furthermore, these pyrazolo[l,5-a]pyrimidines exert their action via CD73-related signaling and modulation of cell metabolism of activated macrophages.
Access to the original article: https://pubmed.ncbi.nlm.nih.gov/38643690/

Astrocytes are glial cells that play key roles in neuroinflammation, which is a common feature in diabetic encephalopathy and aging process. Metformin is an antidiabetic compound that shows neuroprotective properties, including in inflammatory models, but astroglial signaling pathways involved are still poorly known. Interferons α/β are cytokines that participate in antiviral responses and the lack of their signaling increases susceptible to viral infections. Here, the authors investigated the effects of metformin on astrocytes from hypothalamus, a crucial brain region related to inflammatory processes. Astrocyte cultures were derived from interferon α/β receptor knockout (IFNα/βR-/-) and wild-type (WT) mice. Metformin did not change the expression of glial fibrillary acidic protein but caused an anti-inflammatory effect by decreasing pro-inflammatory cytokines (tumor necrosis factor-α and interleukin-1β), as well as increasing gene expression of anti-inflammatory proteins interleukin-10 and Nrf2 (nuclear factor erythroid derived 2 like 2). However, nuclear factor κB p65 and cyclooxygenase 2 were downregulated in WT astrocytes and upregulated in IFNα/βR-/- astrocytes. AMP-activated protein kinase (AMPK), a molecular target of metformin, was upregulated only in WT astrocytes, while sirtuin 1 increased in both mice models. The expression of inducible nitric oxide synthase was decreased in WT astrocytes and heme oxygenase 1 was increased in IFNα/βR-/- astrocytes. Although loss of IFNα/βR-mediated signaling affects some effects of metformin, these results support beneficial roles of this drug in hypothalamic astrocytes. Moreover, paradoxical response of metformin may involve AMPK. Thus, metformin can mediate glioprotection due its effects on age-related disorders in non-diabetic and diabetic encephalopathy individuals.
Access to the original article: https://pubmed.ncbi.nlm.nih.gov/38642825/

The oncogene Aurora kinase A (AURKA) has been implicated in various tumor, yet its role in meningioma remains unexplored. Recent studies have suggested a potential link between AURKA and ferroptosis, although the underlying mechanisms are unclear. This study presented evidence of AURKA upregulation in high grade meningioma and its ability to enhance malignant characteristics. The authors identified AURKA as a suppressor of erastin-induced ferroptosis in meningioma. Mechanistically, AURKA directly interacted with and phosphorylated kelch-like ECH-associated protein 1 (KEAP1), thereby activating nuclear factor erythroid 2 related factor 2 (NFE2L2/NRF2) and target genes transcription. Additionally, forkhead box protein M1 (FOXM1) facilitated the transcription of AURKA. Suppression of AURKA, in conjunction with erastin, yields significant enhancements in the prognosis of a murine model of meningioma. This study elucidates an unidentified mechanism by which AURKA governs ferroptosis, and strongly suggests that the combination of AURKA inhibition and ferroptosis-inducing agents could potentially provide therapeutic benefits for meningioma treatment.
Access to the original article: https://pubmed.ncbi.nlm.nih.gov/38642502/

The Keap1-Nrf2 pathway serves as a central regulator that mediates transcriptional responses to xenobiotic and oxidative stimuli. Recent studies have shown that Keap1 and Nrf2 can regulate transcripts beyond antioxidant and detoxifying genes, yet the underlying mechanisms remain unclear. This research has uncovered that Drosophila Keap1 (dKeap1) and Nrf2 (CncC) proteins can control high-order chromatin structure, including heterochromatin. In this study, the authors identified the molecular interaction between dKeap1 and lamin Dm0, the Drosophila B-type lamin responsible for the architecture of nuclear lamina and chromatin. Ectopic expression of dKeap1 led to an ectopic localization of lamin to the intra-nuclear area, corelated with the spreading of the heterochromatin marker H3K9me2 into euchromatin regions. Additionally, mis-regulated dKeap1 disrupted the morphology of the nuclear lamina. Knocking down of dKeap1 partially rescued the lethality induced by lamin overexpression, suggesting their genetic interaction during development. The discovered dKeap1-lamin interaction suggests a novel role for the Keap1 oxidative/xenobiotic response factor in regulating chromatin architecture.
Access to the original article: https://pubmed.ncbi.nlm.nih.gov/38642177/

3-Bromofluoranthene (3-BrFlu) is the secondary metabolite of fluoranthene, which is classified as a polycyclic aromatic hydrocarbon, through bromination and exists in the fine particulate matter of air pollutants. Endothelial dysfunction plays a critical role in the pathogenesis of cardiovascular and vascular diseases. Little is known about the molecular mechanism of 3-BrFlu on endothelial dysfunction in vivo and in vitro assay. In the present study, 3-BrFlu included concentration-dependent changes in ectopic angiogenesis of the sub-intestinal vein and dilation of the dorsal aorta in zebrafish. Disruption of vascular endothelial integrity and up-regulation of vascular endothelial permeability were also induced by 3-BrFlu in a concentration-dependent manner through pro-inflammatory responses in vascular endothelial cells, namely, SVEC4-10 cells. Generation of pro-inflammatory mediator PGE2 was induced by 3-BrFlu through COX2 expression. Expression of COX2 and generation of pro-inflammatory cytokines, including TNFα and IL-6, were induced by 3-BrFlu through phosphorylation of NF-κB p65, which was mediated by phosphorylation of MAPK, including p38 MAPK, ERK and JNK. Furthermore, generation of intracellular ROS was induced by 3-BrFlu, which is associated with the down-regulated activities of the antioxidant enzyme (AOE), including SOD and catalase. We also found that 3-BrFlu up-regulated expression of the AOE and HO-1 induced by 3-BrFlu through Nrf-2 expression. However, the 3-BrFlu-induced upregulation of AOE and HO-1 expression could not be revised the responses of vascular endothelial dysfunction. In conclusion, 3-BrFlu is a hazardous substance that results in vascular endothelial dysfunction through the MAPK-mediated-NFκB pro-inflammatory pathway and intracellular ROS generation.
Access to the original article: https://pubmed.ncbi.nlm.nih.gov/38635053/

As a unique iron-dependent non-apoptotic cell death, Ferroptosis is involved in the pathogenesis and development of many human diseases and has become a research hotspot in recent years. However, the regulatory role of ferroptosis in the gut-liver-brain axis has not been elucidated. This paper summarizes the regulatory role of ferroptosis and provides theoretical basis for related research. The authors searched PubMed, CNKI and Wed of Science databases on ferroptosis mediated gut-liver-brain axis diseases, summarized the regulatory role of ferroptosis on organ axis, and explained the adverse effects of related regulatory effects on various diseases. According to the summary, the main way in which ferroptosis mediates the gut-liver-brain axis is oxidative stress, and the key cross-talk of ferroptosis affecting signaling pathway network is Nrf2/HO-1. However, there were no specific marker between different organ axes mediate by ferroptosis. This study illustrates the main ways and key cross-talk of ferroptosis mediating the gut-liver-brain axis, providing a basis for future research.
Access to the original article: https://pubmed.ncbi.nlm.nih.gov/38631669/

Mitochondrial fatty acid oxidation (mtFAO) plays an important role in hepatic energy metabolism. Severe mtFAO injury leads to nonalcoholic fatty liver disease (NAFLD) and liver failure. Several drugs have been withdrawn owing to safety issues, such as induction of fatty liver disease through mtFAO disruption. For instance, the antimicrobial triclocarban (TCC), an environmental contaminant that was removed from the market due to its unknown safety in humans, induces NAFLD in rats and promotes hepatic FAO in mice. Therefore, there are no consistent conclusions regarding the effects of TCC on FAO and lipid droplet accumulation. The authors hypothesized that TCC induces lipid droplet accumulation by inhibiting mtFAO in human hepatocytes. Here, the authors evaluated mitochondrial respiration in HepaRG cells to investigate the effects of TCC on fatty acid-driven oxidation in cells, electron transport chain parameters, lipid droplet accumulation, and antioxidant genes. The results suggest that TCC increases oxidative stress gene expression (GCLM, p62, HO-1, and NRF2) through lipid droplet accumulation via mtFAO inhibition in HepaRG cells. The results of the present study provide further insights into the effect of TCC on human NAFLD through mtFAO inhibition, and further in vivo studies could be used to validate the mechanisms.
Access to the original article:https://pubmed.ncbi.nlm.nih.gov/38631510/

Cullin RING E3 ligases (CRL) have emerged as key regulators of disease-modifying pathways and therapeutic targets. Cullin3 (Cul3)-containing CRL (CRL3) has been implicated in regulating hepatic insulin and oxidative stress signaling. However, CRL3 function in liver pathophysiology is poorly defined. Here, the authors report that hepatocyte Cul3 knockout results in rapid resolution of steatosis in obese mice. However, the remarkable resistance of hepatocyte Cul3 knockout mice to developing steatosis does not lead to overall metabolic improvement but causes systemic metabolic disturbances. Liver transcriptomics analysis identifies that CRL3 inactivation causes persistent activation of the nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant defense pathway, which also reprograms the lipid transcriptional network to prevent TG storage. Furthermore, global metabolomics reveals that NRF2 activation induces numerous NAD+-consuming aldehyde dehydrogenases to increase the cellular NADH/NAD+ ratio, a redox imbalance termed NADH reductive stress that inhibits the glycolysis-citrate-lipogenesis axis in Cul3 knockout livers. As a result, this NRF2-induced cellular lipid storage defect promotes hepatic ceramide accumulation, elevates circulating fatty acids, and worsens systemic insulin resistance in a vicious cycle. Hepatic lipid accumulation is restored, and liver injury and hyperglycemia are attenuated when NRF2 activation and NADH reductive stress are abolished in hepatocyte Cul3/Nrf2 double-knockout mice. The resistance to hepatic steatosis, hyperglycemia, and NADH reductive stress are observed in hepatocyte Keap1 knockout mice with NRF2 activation. In summary, this study defines a critical role of CRL3 in hepatic metabolic regulation and demonstrates that the CRL3 downstream NRF2 overactivation causes hepatic metabolic maladaptation to obesity and insulin resistance.
Access to the original article: https://pubmed.ncbi.nlm.nih.gov/38630726/

Arsenic is highly toxic to the human bladder. In the present study, the authors established a human bladder epithelial cell line that closely mimics normal human bladder epithelial cells by immortalizing primary uroplakin 1B-positive human bladder epithelial cells with human telomerase reverse transcriptase (HBladEC-T). The uroplakin 1B-positive human bladder epithelial cell line was then used to evaluate the toxicity of seven arsenicals (iAsV, iAsIII, MMAV, MMAIII, DMAV, DMAIII, and DMMTAV). The cellular uptake and metabolism of each arsenical was different. Trivalent arsenicals and DMMTAV exhibited higher cellular uptake than pentavalent arsenicals. Except for MMAV, arsenicals were transported into cells by aquaglyceroporin 9 (AQP9). In addition to AQP9, DMAIIIand DMMTAV were also taken up by glucose transporter 5. Microarray analysis demonstrated that arsenical treatment commonly activated the NRF2-mediated oxidative stress response pathway. ROS production increased with all arsenicals, except for MMAV. The activating transcription factor 3 (ATF3) was commonly upregulated in response to oxidative stress in HBladEC-T cells: ATF3 is an important regulator of necroptosis, which is crucial in arsenical-induced bladder carcinogenesis. Inorganic arsenics induced apoptosis while MMAV and DMAIII induced necroptosis. MMAIII, DMAV, and DMMTAV induced both cell death pathways. In summary, MMAIII exhibited the strongest cytotoxicity, followed by DMMTAV, iAsIII, DMAIII, iAsV, DMAV, and MMAV. The cytotoxicity of the tested arsenicals on HBladEC-T cells correlated with their cellular uptake and ROS generation. The ROS/NRF2/ATF3/CHOP signaling pathway emerged as a common mechanism mediating the cytotoxicity and carcinogenicity of arsenicals in HBladEC-T cells.
Access to the original article:https://pubmed.ncbi.nlm.nih.gov/38630284/

Joana Miranda
Faculty of Pharmacy, University of Lisbon
Portugal

Summary