Nrf2 Pathway Research Articles

D-Allulose Reduces Hypertrophy and Endoplasmic Reticulum Stress Induced by Palmitic Acid in Murine 3T3-L1 Adipocytes.

Natural rare sugars are an alternative category of sweeteners with positive physiologic and metabolic effects both in in vitro and animal models. D-allulose is a D-fructose epimer that combines 70% sucrose sweetness with the advantage of an extremely low energy content. However, there are no data about the effect of D-allulose against adipose dysfunction; thus, it remains to be confirmed whether D-allulose is useful in the prevention and in treatment of adipose tissue alterations. With this aim, we evaluated D-allulose's preventive effects on lipid accumulation in 3T3-L1 murine adipocytes exposed to palmitic acid (PA), a trigger for hypertrophic adipocytes. D-allulose in place of glucose prevented adipocyte hypertrophy and the activation of adipogenic markers C/EBP-β and PPARγ induced by high PA concentrations. Additionally, D-allulose pretreatment inhibited the NF-κB pathway and endoplasmic reticulum stress caused by PA, through activation of the Nrf2 pathway. Interestingly, these effects were also observed as D-allulose post PA treatment. Although our data need to be confirmed through in vivo models, our findings suggest that incorporating D-allulose as a glucose substitute in the diet might have a protective role in adipocyte function and support a unique mechanism of action in this sugar as a preventive or therapeutic compound against PA lipotoxicity through the modulation of pathways connected to lipid transport and metabolism.

Abstract LB003: Comprehensive multi-omics analysis unveils NFE2L2/KEAP1 mutations as key drivers of treatment resistance in esophageal squamous cell carcinoma during chemoradiotherapy

Abstract Esophageal squamous cell carcinoma (ESCC) stands as one of the most formidable and lethal cancers, necessitating urgent intervention. Despite extensive research efforts to identify optimal radiotherapy and chemotherapy strategies, complete response rates remain disappointingly low, and the challenge of swift relapse persists. Previous investigations primarily focused on primary tumors, utilizing single-omics approaches to illuminate cellular responses to chemotherapy and immunotherapy. However, our understanding of the mechanistic basis for chemoradiotherapy resistance in ESCC remains severely limited. In this study, we conducted multi-omics profiling of over 100 pre-treatment primary and post-treatment relapse ESCC tumors subjected to chemoradiotherapy from a clinical trial (ESO-Shanghai6; NCT04694391). Our comprehensive approach included whole-exome/genome sequencing (WES/WGS), bulk RNA-sequencing, single-cell RNA sequencing (scRNA-seq), and spatial transcriptome analyses. WES/WGS revealed that tumors with rapid relapse displayed high clonal diversity. Notably, 40% of ESCC relapse patients exhibited NFE2L2/KEAP1 somatic alterations in their primary tumors, correlating with a dismal prognosis. Consistent with these findings, downstream NRF2 signaling targets were upregulated in relapse tumors and radiotherapy-resistant cell lines. Further analysis of scRNA-seq and spatial transcriptome data provided profound insights into the spatial organization of mutation-driven, relapse-associated cell populations and their interactions within the tumor microenvironment (TME). Intriguingly, a subset of epithelial tumor cells characterized by NRF2 pathway activation was highly enriched in relapse tumors. Among immune cells, the infiltration proportions of SPP1+ macrophages and SPP1+TREM2+ macrophages—exhibiting higher myeloid immunosuppressive scores—tended to increase in relapse patients. Crucially, NRF2-signaling activated epithelial cells physically co-localized with SPP1+TREM2+ macrophages exclusively in relapse samples, while SPP1+ macrophages were present in both primary and relapse samples. Interaction analysis suggested that NRF2-signaling activated epithelial cells recruit SPP1+TREM2+ and SPP1+ macrophages, thereby fostering an immunosuppressive TME and promoting post-radiotherapy relapse. In summary, our study provides a comprehensive understanding of the molecular determinants influencing ESCC tumor responses under chemoradiotherapy. NFE2L2/KEAP1 mutations emerge as predictive markers for stratifying ESCC patients, and targeting NRF2 signaling presents a potential avenue to overcome resistance to chemoradiotherapy in this challenging disease. Citation Format: Kuaile Zhao, Hu Chen, Zhenzhen Xun, Jiaying Deng, Yikai Luo, Jinjun Ye, Junqiang Chen, Weiwei Chen, Jun Li, Han Liang. Comprehensive multi-omics analysis unveils NFE2L2/KEAP1 mutations as key drivers of treatment resistance in esophageal squamous cell carcinoma during chemoradiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB003.

Protective effects of sinomenine against dextran sulfate sodium-induced ulcerative colitis in rats via alteration of HO-1/Nrf2 and inflammatory pathway.

Dextran Sulfate Sodium (DSS) induces ulcerative colitis (UC), a type of inflammatory bowel disease (IBD) that leads to inflammation, swelling, and ulcers in the large intestine. The aim of this experimental study is to examine how sinomenine, a plant-derived alkaloid, can prevent or reduce the damage caused by DSS in the colon and rectum of rats. Induction of ulcerative colitis (UC) in rats was achieved by orally administering a 2% Dextran Sulfate Sodium (DSS) solution, while the rats concurrently received oral administrations of sinomenine and sulfasalazine. The food, water intake was estimated. The body weight, disease activity index (DAI), colon length and spleen index estimated. Antioxidant, cytokines, inflammatory parameters and mRNA expression were estimated. The composition of gut microbiota was analyzed at both the phylum and genus levels in the fecal samples obtained from all groups of rats. Sinomenine treatment enhanced the body weight, colon length and reduced the DAI, spleen index. Sinomenine treatment remarkably suppressed the level of NO, MPO, ICAM-1, and VCAM-1 along with alteration of antioxidant parameters such as SOD, CAT, GPx, GR and MDA. Sinomenine treatment also decreased the cytokines like TNF-α, IL-1, IL-1β, IL-6, IL-10, IL-17, IL-18 in the serum and colon tissue; inflammatory parameters viz., PAF, COX-2, PGE2, iNOS, NF-κB; matrix metalloproteinases level such as MMP-1 and MMP-2. Sinomenine significantly (P < 0.001) enhanced the level of HO-1 and Nrf2. Sinomenine altered the mRNA expression of RIP1, RIP3, DRP3, NLRP3, IL-1β, caspase-1 and IL-18. Sinomenine remarkably altered the relative abundance of gut microbiota like firmicutes, Bacteroidetes, F/B ratio, Verrucomicrobia, and Actinobacteria. The results clearly indicate that sinomenine demonstrated a protective effect against DSS-induced inflammation, potentially through the modulation of inflammatory pathways and gut microbiota.

DNA-templated synthesis of surface vacancy-rich bifunctional copper sulfide promotes phototherapy via gene regulation

The current photodynamic performance of CuS nanoparticles (CuS NPs) alone is still greatly challenged by the low quantum yield of reactive oxygen species (ROS). At the same time, the efficiency of photothermal therapy (PTT) and photodynamic therapy (PDT) is reduced due to the self-protective ability of tumor cells when attacked by hyperthermia and ROS. To address this issue, we synthesized DCuxS NPs using customized DNA templates through a surface vacancy-rich engineering strategy. In addition to their inherent photothermal properties, the surface vacancy-rich of DCuxS NPs effectively hinders electron-hole pair complexation, thereby enhancing their photodynamic properties. Furthermore, the tumor-targeting NC3S aptamer and the i-motif DNA/Nrf2 siRNA chimera were attached to both ends of the DNA template. The final NDCuxS@siRNA NPs were highly enriched in tumor cells, while the i-motif undergoes a conformational change to release Nrf2 siRNA upon acidic stimulation for Nrf2 pathway blocking to decrease tumor cell protection effect against ROS and high temperature, creating a favorable tumor microenvironment for enhanced PTT and PDT effects. In this study, we explored the potential of DNA templates for engineering nanomaterials, enhanced the photodynamic properties of DCuxS NPs through a surface vacancy-rich strategy, and exploited the versatility of DNA templates to combine phototherapy with gene regulation, providing a promising approach for clinical tumor treatment.

Oxysophoridine inhibits oxidative stress and inflammation in hepatic fibrosis via regulating Nrf2 and NF-κB pathways

BackgroundHepatic fibrosis (HF) runs through multiple stages of liver diseases and promotes these diseases progression. Oxysophoridine (OSR), derived from Sophora alopecuroides l., is a bioactive alkaloid that has been reported to antagonize alcoholic hepatic injury. However, whether OSR suppresses HF and the mechanisms involved in Nrf2 remain unknown. PurposeSince the dysregulation of inflammation and oxidative stress is responsible for the excessive accumulation of extracellular matrix (ECM) and fibrosis in the liver. We hypothesized that OSR may attenuate HF by inhibiting inflammation and oxidative stress through activating Nrf2 signaling. MethodsIn this study, we employed LPS-stimulated HSC-T6 cells, RAW264.7 cells, and a CCl4-induced C57BL/6 mouse fibrotic model to evaluate its suppressing inflammation and oxidative stress, as well as fibrosis. ResultsThe result showed that OSR significantly reduced α-SMA and TGF-β1 at a low dose of 10 μM in vitro and at a dose of 50 mg/kg in vivo, which is comparable to Silymarin, the only Chinese herbal active ingredient that has been marketed for anti-liver fibrosis. Moreover, OSR effectively suppressed the expression of iNOS at a dose of 10 μM and COX-2 at a dose of 40 μM, respectively. Furthermore, OSR demonstrated inhibitory effects on the IL-1β, IL-6, and TNF-α in vitro and almost extinguished cytokine storm in vivo. OSR exhibited antioxidative effects by reducing MDA and increasing GSH, thereby protecting the cell membrane against oxidative damage and reducing LDH release. Moreover, OSR effectively upregulated the protein levels of Nrf2, HO-1, and p62, but decreased p-NF-κB p65, p-IκBα, and Keap1. Alternatively, mechanisms involved in Nrf2 were verified by siNrf2 interference, siNrf2 interference revealed that the anti-fibrotic effect of OSR was attributed to its activation of Nrf2. ConclusionThe present study provided an effective candidate for HF involved in both activation of Nrf2 and blockage of NF-κB, which has not been reported in the published work. The present study provides new insights for the identification of novel drug development for HF.

Unveiling the therapeutic potential of Dl-3-n-butylphthalide in NTG-induced migraine mouse: activating the Nrf2 pathway to alleviate oxidative stress and neuroinflammation

BackgroundMigraine stands as a prevalent primary headache disorder, with prior research highlighting the significant involvement of oxidative stress and inflammatory pathways in its pathogenesis and chronicity. Existing evidence indicates the capacity of Dl-3-n-butylphthalide (NBP) to mitigate oxidative stress and inflammation, thereby conferring neuroprotective benefits in many central nervous system diseases. However, the specific therapeutic implications of NBP in the context of migraine remain to be elucidated.MethodsWe established a C57BL/6 mouse model of chronic migraine (CM) using recurrent intraperitoneal injections of nitroglycerin (NTG, 10 mg/kg), and prophylactic treatment was simulated by administering NBP (30 mg/kg, 60 mg/kg, 120 mg/kg) by gavage prior to each NTG injection. Mechanical threshold was assessed using von Frey fibers, and photophobia and anxious behaviours were assessed using a light/dark box and elevated plus maze. Expression of c-Fos, calcitonin gene-related peptide (CGRP), Nucleus factor erythroid 2-related factor 2 (Nrf2) and related pathway proteins in the spinal trigeminal nucleus caudalis (SP5C) were detected by Western blotting (WB) or immunofluorescence (IF). The expression of IL-1β, IL-6, TNF-α, Superoxide dismutase (SOD) and malondialdehyde (MDA) in SP5C and CGRP in plasma were detected by ELISA. A reactive oxygen species (ROS) probe was used to detect the expression of ROS in the SP5C.ResultsAt the end of the modelling period, chronic migraine mice showed significantly reduced mechanical nociceptive thresholds, as well as photophobic and anxious behaviours. Pretreatment with NBP attenuated nociceptive sensitization, photophobia, and anxiety in the model mice, reduced expression levels of c-Fos and CGRP in the SP5C and activated Nrf2 and its downstream proteins HO-1 and NQO-1. By measuring the associated cytokines, we also found that NBP reduced levels of oxidative stress and inflammation. Most importantly, the therapeutic effect of NBP was significantly reduced after the administration of ML385 to inhibit Nrf2.ConclusionsOur data suggest that NBP may alleviate migraine by activating the Nrf2 pathway to reduce oxidative stress and inflammation in migraine mouse models, confirming that it may be a potential drug for the treatment of migraine.Graphical

Association between ACE1 and missed abortion: ACE1 promotes H2O2-induced trophoblast cell injury in vitro†.

The aim of this study was to evaluate the role of angiotensin-converting enzyme 1 (ACE1) in H2O2-induced trophoblast cell injury and the potential molecular mechanisms. Oxidative stress was modeled by exposing HTR-8/SVneo cells to 200μM H2O2. Western blot and real-time quantitative PCR methods were used to detect protein and mRNA expression level of ACE1 in chorionic villus tissue and trophoblast HTR-8/SVneo cell. Inhibition of ACE1 expression was achieved by transfection with small interfering RNA. Then flow cytometry, Cell Counting Kit-8, and Transwell assay was used to assess apoptosis, viability, and migration ability of the cells. Reactive oxygen species (ROS) were detected by fluorescent probes, and malondialdehyde (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH) activities were determined by corresponding detection kits. Angiotensin-converting enzyme 1 expression was upregulated in chorionic villus tissue of patients with missed abortion (MA) compared with individuals with normal early pregnancy abortion. H2O2 induced elevated ACE1 expression in HTR-8/SVneo cells, promoted apoptosis, and inhibited cell viability and migration. Knockdown of ACE1 expression inhibited H2O2-induced effects to enhance cell viability and migration and suppress apoptosis. Additionally, H2O2 stimulation caused increased levels of ROS and MDA and decreased SOD and GSH activity in the cells, whereas knockdown of ACE1 expression led to opposite changes of these oxidative stress indicators. Moreover, knockdown of ACE1 attenuated the inhibitory effect of H2O2 on the Nrf2/HO-1 pathway. Angiotensin-converting enzyme 1 was associated with MA, and it promoted H2O2-induced injury of trophoblast cells through inhibiting the Nrf2 pathway. Therefore, ACE1 may serve as a potential therapeutic target for MA.

A grape-supplemented diet prevented ultraviolet (UV) radiation-induced cataract by regulating Nrf2 and XIAP pathways

The purpose of this study is to investigate if grape consumption, in the form of grape powder (GP), could protect against ultraviolet (UV)-induced cataract. Mice were fed with the regular diet, sugar placebo diet, or a grape diet (regular diet supplemented with 5%, 10%, and 15% GP) for 3 months. The mice were then exposed to UV radiation to induce cataract. The results showed that the GP diet dose-dependently inhibited UV-induced cataract and preserved glutathione pools. Interestingly, UV-induced Nrf2 activation was abolished in the groups on the GP diet, suggesting GP consumption may improve redox homeostasis in the lens, making Nrf2 activation unnecessary. For molecular target prediction, a total of 471 proteins regulated by GP were identified using Agilent Literature Search (ALS) software. Among these targets, the X-linked inhibitor of apoptosis (XIAP) was correlated with all of the main active ingredients of GP, including resveratrol, catechin, quercetin, and anthocyanins. Our data confirmed that GP prevented UV-induced suppression of XIAP, indicating that XIAP might be one of the critical molecular targets of GP. In conclusion, this study demonstrated that GP protected the lens from UV-induced cataract development in mice. The protective effects of GP may be attributed to its ability to improve redox homeostasis and activate the XIAP-mediated antiapoptotic pathway.

The Role of NRF2 in Cerebrovascular Protection: Implications for Vascular Cognitive Impairment and Dementia (VCID).

Vascular cognitive impairment and dementia (VCID) represents a broad spectrum of cognitive decline secondary to cerebral vascular aging and injury. It is the second most common type of dementia, and the prevalence continues to increase. Nuclear factor erythroid 2-related factor 2 (NRF2) is enriched in the cerebral vasculature and has diverse roles in metabolic balance, mitochondrial stabilization, redox balance, and anti-inflammation. In this review, we first briefly introduce cerebrovascular aging in VCID and the NRF2 pathway. We then extensively discuss the effects of NRF2 activation in cerebrovascular components such as endothelial cells, vascular smooth muscle cells, pericytes, and perivascular macrophages. Finally, we summarize the clinical potential of NRF2 activators in VCID.

Mechanisms of PM10 Disruption of the Nrf2 Pathway in Cornea.

We have previously shown that PM10 exposure causes oxidative stress and reduces Nrf2 protein levels, and SKQ1 pre-treatment protects against this damage in human corneal epithelial cells (HCE-2). The current study focuses on uncovering the mechanisms underlying acute PM10 toxicity and SKQ1-mediated protection. HCE-2 were pre-treated with SKQ1 and then exposed to 100 μg/mL PM10. Cell viability, oxidative stress markers, programmed cell death, DNA damage, senescence markers, and pro-inflammatory cytokines were analyzed. Nrf2 cellular location and its transcriptional activity were determined. Effects of the Nrf2 inhibitor ML385 were similarly evaluated. Data showed that PM10 decreased cell viability, Nrf2 transcriptional activity, and mRNA levels of antioxidant enzymes, but increased p-PI3K, p-NFκB, COX-2, and iNOS proteins levels. Additionally, PM10 exposure significantly increased DNA damage, phosphor-p53, p16 and p21 protein levels, and β-galactosidase (β-gal) staining, which confirmed the senescence. SKQ1 pre-treatment reversed these effects. ML385 lowered the Nrf2 protein levels and mRNA levels of its downstream targets. ML385 also abrogated the protective effects of SKQ1 against PM10 toxicity by preventing the restoration of cell viability and reduced oxidative stress. In conclusion, PM10 induces inflammation, reduces Nrf2 transcriptional activity, and causes DNA damage, leading to a senescence-like phenotype, which is prevented by SKQ1.

The role of FPR2-mediated ferroptosis in formyl peptide-induced acute lung injury against endothelial barrier damage and protective effect of the mitochondria-derived peptide MOTS-c

BackgroundAcute lung injury (ALI) has garnered significant attention in the field of respiratory and critical care due to its high mortality and morbidity, and limited treatment options. The role of the endothelial barrier in the development of ALI is crucial. Several bacterial pathogenic factors, including the bacteria-derived formyl peptide (fMLP), have been implicated in damaging the endothelial barrier and initiating ALI. However, the mechanism by which fMLP causes ALI remains unclear. In this study, we aim to explore the mechanisms of ALI caused by fMLP and evaluate the protective effects of MOTS-c, a mitochondrial-derived peptide. MethodsWe established a rat model of ALI and a human pulmonary microvascular endothelial cell (HPMVEC) model of ALI by treatment with fMLP. In vivo experiments involved lung histopathology assays, assessments of inflammatory and oxidative stress factors, and measurements of ferroptosis-related proteins and barrier proteins to evaluate the severity of fMLP-induced ALI and the type of tissue damage in rats. In vitro experiments included evaluations of fMLP-induced damage on HPMVEC using cell activity assays, assessments of inflammatory and oxidative stress factors, measurements of ferroptosis-related proteins, endothelial barrier function assays, and examination of the key role of FPR2 in fMLP-induced ALI. We also assessed the protective effect of MOTS-c and investigated its mechanism on the fMLP-induced ALI in vivo and in vitro. ResultsResults from both in vitro and in vivo experiments demonstrate that fMLP promotes the expression of inflammatory and oxidative stress factors, activates ferroptosis and disrupts the vascular endothelial barrier, ultimately contributing to the development and progression of ALI. Mechanistically, ferroptosis mediated by FPR2 plays a key role in fMLP-induced injury, and the Nrf2 and MAPK pathways are involved in this process. Knockdown of FPR2 and inhibition of ferroptosis can attenuate ALI induced by fMLP. Moreover, MOTS-c could protect the vascular endothelial barrier function by inhibiting ferroptosis and suppressing the expression of inflammatory and oxidative stress factors through Nrf2 and MAPK pathways, thereby alleviating fMLP-induced ALI. ConclusionOverall, fMLP disrupts the vascular endothelial barrier through FPR2-mediated ferroptosis, leading to the development and progression of ALI. MOTS-c demonstrates potential as a protective treatment against ALI by alleviating the damage induced by fMLP.

Do oral and gut microbiota communicate through redox pathways? A novel asset of the nitrate-nitrite-NO pathway.

Nitrate may act as a regulator of •NO bioavailability via sequential reduction along the nitrate-nitrite-NO pathway with widespread health benefits, including a eubiotic effect on the oral and gut microbiota. Here, we discuss the molecular mechanisms of microbiota-host communication through redox pathways, via the production of •NO and oxidants by the family of NADPH oxidases, namely hydrogen peroxide (via Duox2), superoxide radical (via Nox1 and Nox2) and peroxynitrite, which leads to downstream activation of stress responses (Nrf2 and NFkB pathways) in the host mucosa. The activation of Nox2 by microbial metabolites is also discussed. Finally, we propose a new perspective in which both oral and gut microbiota communicate through redox pathways, with nitrate as the pivot linking both ecosystems.

Exploring the Phytochemical Diversity and Antioxidant Potential of the Vietnamese Smilax glabra Roxb: Insights from UPLC-QTOF-MS/MS and Zebrafish Model Studies.

Research on natural products is growing due to their potential health benefits and medicinal properties. Despite regional variations in phytochemical composition and bioactivity, Smilax glabra Roxb (SGB) has attracted the interest of researchers. Scientists are particularly interested in the Vietnamese SGB variant, which is influenced by biological and environmental factors. Despite geographical differences in phytochemical makeup and bioactivities, SGB remains a fascinating subject in traditional herbal medicine. Using ultra-performance liquid chromatography and quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS), the phytochemicals in Vietnamese SGB extracts were investigated. This study revealed a wide range of phytochemical compounds, including flavonoids, terpenoids, glycosides, alkaloids, organic acids, phenolics, and steroids. Furthermore, utilizing zebrafish as a model organism, we discovered that these extracts have the surprising ability to greatly improve the survival rate of zebrafish larvae exposed to oxidative stress caused by arsenite (NaAsO2) and hydrogen peroxide (H2O2). Notably, our discoveries suggest the occurrence of new antioxidative pathways in addition to the kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, expanding the understanding of the antioxidant properties and potential therapeutic uses of these plants. To summarize, our research findings shed light on the phytochemical composition of Vietnamese SGB, revealing its potential as a natural antioxidant and encouraging further exploration of its underlying mechanisms for future innovative antioxidant therapies.

Abstract 4731: Targeting Lon protease as novel strategy to treat aggressive IDH mutant diffuse malignant astrocytoma

Abstract While isocitrate dehydrogenase (IDH) wild-type and mutant malignant astrocytoma (IDHwt, IDHmt) are diffuse CNS tumors that have a high degree of parallelism with one another, including microvascular proliferation and necrosis, the IDHwt tumors clinically presents with a higher tumor grade and thus poorer prognosis and survival rate. Isocitrate dehydrogenase mutation (IDH) hinders the hypoxia response in gliomas and promotes an advantageous reduction in both tumor growth and treatment resistance. Since recurrence is high, adequate characterization and identification of potential novel molecular targets in IDHmt mutant diffuse glioma is integral for developing new therapeutic strategies for better control of tumor burden and, thus, improved outcomes. Our previous published work has shown that the hypoxia responsive LONP1 mitochondrial protease is involved in glioma growth and treatment resistance and that LONP1 inhibitors can augment the effect of chemotherapy in IDHwt models. Here, we highlight the downstream activity of LONP1 via the NRF2 pathway and how synergistically, along with the IDH mutation, LONP1 overexpression can promote the subsequent proneural to mesenchymal transition (PMT) induced by stress and other tumor microenvironment signals. By characterizing the interplay between LONP1 and IDH mutation in in-vitro models, we demonstrate that oxidative stress modulates both PMT and TMZ drug resistance. When translated to an in-vivo patient-derived, intracranial orthotopic xenograft model, LONP1 overexpression is associated with decreased survival.Based on our findings, future clinical translation includes the possibility of using LONP1 as a novel target to optimize the standard of care treatment in IDH mutant diffuse astrocytoma. Citation Format: Christopher Douglas, Shashi Jain, Naomi Lomeli, Thao Vu, James Pham, Daniela A. Bota. Targeting Lon protease as novel strategy to treat aggressive IDH mutant diffuse malignant astrocytoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4731.

Abstract 1252: AI-derived predictions improve identification of real-world cancer driver mutations

Abstract AI-based variant effect predictors (VEPs), such as AlphaMissense, which is based on the protein structure prediction method AlphaFold, have gained significant attention for their potential to predict pathogenic effects of mutations. However, the utility of these methods for identifying pathogenic mutations in cancer remains unknown. We sought to quantify the prevalence of variants of unknown significance (VUSs) across the pan-cancer, multi-institution AACR GENIE cohort (N=160,965 samples) and test the utility of VEPs (SIFT, PolyPhen, MutationAssessor, REVEL, CADD, and AlphaMissense), using publicly available mutation annotations from each method, for 1) Annotating known pathogenic somatic cancer variants across the GENIE cohort, using known benign single nucleotide polymorphisms as negative controls 2) Identifying VUSs associated with overall survival (OS) in two cohorts of patients with non-small cell cancer (NSCLC), the MSK-IMPACT NCSLC (N=8,690) and non-MSK AACR GENIE Biopharma Collaborative NSCLC (N=977) cohorts, and 3) Identifying VUSs associated with known genomic patterns of mutual exclusivity in the MSK-IMPACT NSCLC cohort. In the GENIE cohort, 79% of mutations identified were VUSs according to an FDA-recognized molecular knowledge database, with a wide range of VUS frequencies across genes. Among VEPs trained without prior human-annotated knowledge, CADD and AlphaMissense had the highest AUROCs (0.940, 95%CI 0.937-0.943 and 0.917, 95%CI 0.913-0.922 respectively) for predicting known cancer drivers, demonstrating the power of protein structure modeling or functional genomic data for enhancing cancer drivers prediction. All VEPs identified known driver mutations in tumor suppressor genes more accurately than oncogenes, highlighting the challenge in identifying gain-of-function mutations. We tested whether VUSs reclassified as pathogenic by VEPs are associated with prognosis using inverse probability of treatment-weighted Cox’s propensity hazard models controlling for tumor mutational burden, treatment history, and other factors. VUSs reclassified as pathogenic by AlphaMissense and other VEPs in KEAP1 and SMARCA4 correlated with worse OS, comparable to that of known oncogenic mutations in those genes. Patients with tumors containing VUSs annotated as benign had similar OS to patients without analogous driver mutations. Finally, VUSs reclassified as oncogenic by AlphaMissense in genes within the RTK/RAS and NRF2 pathways followed expected patterns of mutual exclusivity, further suggesting biological validity. Despite not being trained to predict somatic effects in cancer, AI-derived mutation annotations can broaden the subset of annotated pathogenic variants in cancer and contribute to a more complete understanding of cancer genetics. Real-world outcomes and alteration patterns are additional benchmarks to consider when assessing the utility of VEPs in cancer. Citation Format: Thinh N. Tran, Chris Fong, Karl Pichotta, Anisha Luthra, Ronglai Shen, Yuan Chen, Michele Waters, Susie Kim, Gregory Riely, Debyani Chakravarty, Nikolaus Schultz, Justin Jee. AI-derived predictions improve identification of real-world cancer driver mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1252.

Incidence of type 2 diabetes, cardiovascular disease and chronic kidney disease in patients with multiple sclerosis initiating disease-modifying therapies: Retrospective cohort study using a frequentist model averaging statistical framework.

Researchers are increasingly using insights derived from large-scale, electronic healthcare data to inform drug development and provide human validation of novel treatment pathways and aid in drug repurposing/repositioning. The objective of this study was to determine whether treatment of patients with multiple sclerosis with dimethyl fumarate, an activator of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, results in a change in incidence of type 2 diabetes and its complications. This retrospective cohort study used administrative claims data to derive four cohorts of adults with multiple sclerosis initiating dimethyl fumarate, teriflunomide, glatiramer acetate or fingolimod between January 2013 and December 2018. A causal inference frequentist model averaging framework based on machine learning was used to compare the time to first occurrence of a composite endpoint of type 2 diabetes, cardiovascular disease or chronic kidney disease, as well as each individual outcome, across the four treatment cohorts. There was a statistically significantly lower risk of incidence for dimethyl fumarate versus teriflunomide for the composite endpoint (restricted hazard ratio [95% confidence interval] 0.70 [0.55, 0.90]) and type 2 diabetes (0.65 [0.49, 0.98]), myocardial infarction (0.59 [0.35, 0.97]) and chronic kidney disease (0.52 [0.28, 0.86]). No differences for other individual outcomes or for dimethyl fumarate versus the other two cohorts were observed. This study effectively demonstrated the use of an innovative statistical methodology to test a clinical hypothesis using real-world data to perform early target validation for drug discovery. Although there was a trend among patients treated with dimethyl fumarate towards a decreased incidence of type 2 diabetes, cardiovascular disease and chronic kidney disease relative to other disease-modifying therapies-which was statistically significant for the comparison with teriflunomide-this study did not definitively support the hypothesis that Nrf2 activation provided additional metabolic disease benefit in patients with multiple sclerosis.

Abstract 1466: Tobacco carcinogen impacts redox pathway to facilitate pancreatic ductal adenocarcinoma (PDAC) initiation and progression

Abstract The 5-year survival rate for pancreatic ductal adenocarcinoma (PDAC) is only 10%. PDAC is projected to soon become the second leading cause of cancer death. Tobacco is one of the main risk factors for PDAC. Incidence of PDAC increases 2-fold among tobacco users, and 20-35% of PDAC cases are attributed to smoking. However, the specific pathways activated by tobacco carcinogens remain elusive.This research project addresses the impact of carcinogens in pancreatic cancer etiology. We hypothesize that carcinogens in tobacco increase reactive oxygen species (ROS), which elevates the Nrf2 redox pathway to facilitate PDAC initiation and progression. Specifically, we focus on two major subclasses of carcinogens: the heavy metal cadmium (Cd) and the polycyclic aromatic hydrocarbon (PAH) benzo(a)pyrene (BaP). Using mouse pancreatic ductal organoid cultures, we found that acute carcinogen exposure increases Nrf2 activity, which is sustained after "intermediate" exposure (5 passages). Intriguingly, chronic exposure (10 passages) downregulates Nrf2 activity, which has been shown to be associated with acquired metastatic potential. Furthermore, we found an upregulation in NFkB and EGFR signaling after intermediate exposure, which are involved in microenvironment remodeling and increased proliferative potential. Overall, we demonstrate that acute carcinogen exposure elevates ROS levels and activates the Nrf2 pathway, whereas prolonged exposure upregulates NFkB and EGFR pathways. Furthermore, we have established a novel carcinogenesis model using 3D organoid culture to recapitulate each step in pancreatic tumorigenesis. Collectively, this project contributes to our understanding of tobacco-related PDAC pathogenesis. Citation Format: Xiaoxue Lin, McKenna Stamp, Alexandra Fowler, Chelsea Bottomley, Shira Okhovat, Kristina Peck, Dannielle Engle. Tobacco carcinogen impacts redox pathway to facilitate pancreatic ductal adenocarcinoma (PDAC) initiation and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1466.

Tetramethylpyrazine Antagonizes the Subchronic Cadmium Exposure-Induced Oxidative Damage in Mouse Livers via the Nrf2/HO-1 Pathway.

Hepatic oxidative stress is an important mechanism of Cd-induced hepatotoxicity, and it is ameliorated by TMP. However, this underlying mechanism remains to be elucidated. To investigate the mechanism of the protective effect of TMP on liver injuries in mice induced by subchronic cadmium exposure, 60 healthy male ICR mice were randomly divided into five groups of 12 mice each, namely, control (CON), Cd (2 mg/kg of CdCl2), Cd + 100 mg/kg of TMP, Cd + 150 mg/kg of TMP, and Cd + 200 mg/kg of TMP, and were acclimatized and fed for 7 d. The five groups of mice were gavaged for 28 consecutive days with a maximum dose of 0.2 mL/10 g/day. Except for the control group, all groups were given fluoride (35 mg/kg) by an intraperitoneal injection on the last day of the experiment. The results of this study show that compared with the Cd group, TMP attenuated CdCl2-induced pathological changes in the liver and improved the ultrastructure of liver cells, and TMP significantly decreased the MDA level (p < 0.05) and increased the levels of T-AOC, T-SOD, and GSH (p < 0.05). The results of mRNA detection show that TMP significantly increased the levels of Nrf2 in the liver compared with the Cd group as well as the HO-1 and mRNA expression levels in the liver (p < 0.05). In conclusion, TMP could inhibit oxidative stress and attenuate Cd group-induced liver injuries by activating the Nrf2 pathway.

Abstract 2028: The triterpenoid CDDO-Methyl ester requires Nrf2 to decrease lung tumor burden and to protect against the toxicity of chemotherapy in experimental lung cancer

Abstract The NRF2 cytoprotective pathway, a drug target for many inflammation-related diseases, regulates immune cell function. While the anti-inflammatory nature of NRF2 activation protects healthy cells from malignant transformation, cancer cells can utilize the pathway to promote resistance to anti-cancer drugs. Up to 30% of human lung adenocarcinomas acquire mutations in either NFE2L2 or its negative regulator KEAP1 which result in constitutive activation of the NRF2 pathway. However, despite our knowledge of the important immunomodulatory effects of NRF2, these effects are not well characterized in the context of cancer. With NRF2 activators now approved for clinical use, it is critical to understand the impact of these drugs in cancer. Triterpenoids including CDDO-methyl ester (CDDO-Me, also known as bardoxolone methyl) are potent pharmacological NRF2 activators with demonstrated anti-cancer activity in preclinical models. In an early-stage preclinical model of lung cancer, CDDO-Me significantly decreased tumor burden in a dose- and Nrf2-dependent manner and improved immune signatures within the tumor microenvironment. However, most human lung cancers are not diagnosed until more advanced stages. To test CDDO-Me in an established tumor intervention model, lung tumors were initiated with vinyl carbamate in A/J WT and Nrf2 knockout (KO) mice. Tumors were allowed to develop for 8 weeks post initiation, after which mice were fed either a vehicle control diet or CDDO-Me (50-100 mg/kg of diet) for an additional 8-12 weeks, alone or in combination with carboplatin and paclitaxel (C/P) at 50 mg/kg and 15 mg/kg, respectively, by IP injection every other week. CDDO-Me significantly (p &amp;lt; 0.05) decreased surface lung tumor counts 35-71% in a Nrf2-dependent manner, and C/P significantly (p &amp;lt; 0.001) reduced tumor burden 53-59% regardless of Nrf2 status. The combination of CDDO-Me + C/P reduced tumor burden in WT lungs by 84% (p &amp;lt; 0.05), more than either agent alone. Nrf2 KO mice had an approximately two-fold increase (p &amp;lt; 0.001) of surface tumors compared to WT mice in both studies, regardless of treatment. C/P reduced tumor burden similarly in Nrf2 WT and Nrf2 KO mice. Interestingly, the combination of CDDO-Me + C/P increased T cell infiltration into the lungs of WT mice, but this change was not observed in groups treated with either agent alone. Importantly, CDDO-Me did not decrease the efficacy of C/P but protected WT mice from mortality and weight loss and lowered white and red blood cell counts. These studies suggest that NRF2 activation in advanced lung cancers can still decrease tumor burden by favorably modulating the immune microenvironment and, importantly, complements the anti-tumor efficacy of conventional chemotherapy while decreasing drug toxicity. Citation Format: Jessica Ann Moerland, Karen T. Liby. The triterpenoid CDDO-Methyl ester requires Nrf2 to decrease lung tumor burden and to protect against the toxicity of chemotherapy in experimental lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2028.

Abstract 4507: Discovery of a smart molecular glue: A small-molecular compound selectively degrades Nrf2 in KEAP1-mutated tumor cells, by restoring the broken KEAP1 mutant/Nrf2 interaction

Abstract Overcoming therapeutic resistance without incurring prohibitive normal tissue toxicity is a great challenge in anticancer therapies. Transcription factor NRF2 is a master regulator of cellular protective response, which provides protective effects in normal tissues. KEAP1, the endogenous NRF2 inhibitor, binds NRF2 and redirect it towards proteasome-dependent degradation. KEAP1/NRF2 interaction is therefore critical for maintaining NRF2 at a basal level. Accumulated studies revealed cancer cells could hijack NRF2 pathway to confer drug resistance. A number of clinically-relevant KEAP1 mutations were shown to disrupt the KEAP1/NRF2 interaction, leading to elevated NRF2 level and confer drug resistance. Here, by structure-based drug design approach, we discovered a small-molecule NRF2 inhibitor, R16, which selectively binds KEAP1 mutants and restores their NRF2-inhibitory function in tumor cells. We performed in silico screening against the NCI-open database. Top-ranking candidates were initially evaluated by ARE-luc reporter assays, leading to discovery of R16. Effect of R16 in restoring interaction between KEAP1 mutants and NRF2 were evaluated by BRET2 assays. Among 14 KEAP1 mutations that result in disruption of KEAP1/NRF2 interaction, R16 is active against 11 of them. A variety of assays, including tryptophan quenching, cellular target engagement, BRET2 and GST-pull down assays, indicated that R16 selectively engage KEAP1 mutants and restore their interaction with NRF2, leading to NRF2 degradation. R16 at 0.5 uM substantially sensitizes KEAP1-mutated tumor cells to cisplatin and gefitinib, with no obvious effect in wild-type KEAP1 cells. Importantly, R16 showed significant in-vivo efficacy in sensitizing A549 xenograft bearing KEAP1 G333C mutation to cisplatin, while it has no effect on xenograft with wild-type KEAP1. KEAP1-mutated tumors are emerging as a sub-group with severe resistance to currently available therapies. Here, we have identified a KEAP1 mutant-selective NRF2 inhibitor, which potently sensitizes KEAP1-mutated cells/tumors to anticancer agents, with little effects on the WT-KEAP1. Our work demonstrated it is feasible to restore the NRF2-inhibitory function of KEAP1 mutants, by targeting the mutants with compounds to repair the disrupted KEAP1 mutant/NRF2 interactions. The KEAP1 mutant-selectivity is critical, as NRF2 is a master regulator of cellular defense response in WT-KEAP1 normal tissues. Citation Format: Xiaohong Tian, Tahar Aboulkassim, Qiang Liu, Mark Hancock, Jian Hui Wu, Gerald Batist. Discovery of a smart molecular glue: A small-molecular compound selectively degrades Nrf2 in KEAP1-mutated tumor cells, by restoring the broken KEAP1 mutant/Nrf2 interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4507.