NRF2 Mutations Research Articles

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

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

Redoxhigh phenotype mediated by KEAP1/STK11/SMARCA4/NRF2 mutations diminishes tissue-resident memory CD8+ T cells and attenuates the efficacy of immunotherapy in lung adenocarcinoma

ABSTRACT Metabolism reprogramming within the tumor microenvironment (TME) can have a profound impact on immune cells. Identifying the association between metabolic phenotypes and immune cells in lung adenocarcinoma (LUAD) may reveal mechanisms of resistance to immune checkpoint inhibitors (ICIs). Metabolic phenotypes were classified by expression of metabolic genes. Somatic mutations and transcriptomic features were compared across the different metabolic phenotypes. The metabolic phenotype of LUAD is predominantly determined by reductase-oxidative activity and is divided into two categories: redoxhigh LUAD and redoxlow LUAD. Genetically, redoxhigh LUAD is mainly driven by mutations in KEAP1, STK11, NRF2, or SMARCA4. These mutations are more prevalent in redoxhigh LUAD (72.5%) compared to redoxlow LUAD (17.4%), whereas EGFR mutations are more common in redoxlow LUAD (19.0% vs. 0.7%). Single-cell RNA profiling of pre-treatment and post-treatment samples from patients receiving neoadjuvant chemoimmunotherapy revealed that tissue-resident memory CD8+ T cells are responders to ICIs. However, these cells are significantly reduced in redoxhigh LUAD. The redoxhigh phenotype is primarily attributed to tumor cells and is positively associated with mTORC1 signaling. LUAD with the redoxhigh phenotype demonstrates a lower response rate (39.1% vs. 70.8%, p = 0.001), shorter progression-free survival (3.3 vs. 14.6 months, p = 0.004), and overall survival (12.1 vs. 31.2 months, p = 0.022) when treated with ICIs. The redoxhigh phenotype in LUAD is predominantly driven by mutations in KEAP1, STK11, NRF2, and SMARCA4. This phenotype diminishes the number of tissue-resident memory CD8+ T cells and attenuates the efficacy of ICIs.

ARID1A loss is associated with increased NRF2 signaling in human head and neck squamous cell carcinomas.

Prior to the next generation sequencing and characterization of the tumor genome landscape, mutations in the SWI/SNF chromatin remodeling complex and the KEAP1-NRF2 signaling pathway were underappreciated. While these two classes of mutations appeared to independently contribute to tumor development, recent reports have demonstrated a mechanistic link between these two regulatory mechanisms in specific cancer types and cell models. In this work, we expand upon these data by exploring the relationship between mutations in BAF and PBAF subunits of the SWI/SNF complex and activation of NRF2 signal transduction across many cancer types. ARID1A/B mutations were strongly associated with NRF2 transcriptional activity in head and neck squamous carcinomas (HNSC). Many additional tumor types showed significant association between NRF2 signaling and mutation of specific components of the SWI/SNF complex. Different effects of BAF and PBAF mutations on the polarity of NRF2 signaling were observed. Overall, our results support a context-dependent functional link between SWI/SNF and NRF2 mutations across human cancers and implicate ARID1A inactivation in HPV-negative HNSC in promoting tumor progression and survival through activation of the KEAP1-NRF2 signaling pathway. The tumor-specific effects of these mutations open a new area of study for how mutations in the KEAP1-NRF2 pathway and the SWI/SNF complex contribute to cancer.

Nrf2, A Target for Precision Oncology in Cancer Prognosis and Treatment.

Activating nuclear factor-erythroid 2-related factor (Nrf2), a master regulator of redox homeostasis, has been shown to suppress initiation of carcinogenesis in normal cells. However, this transcription factor has recently been reported to promote proliferation of some transformed or cancerous cells. In tumor cells, Nrf2 is prone to mutations that result in stabilization and concurrent accumulation of its protein product. A hyperactivated mutant form of Nrf2 could support the cancer cells for enhanced proliferation, invasiveness, and resistance to chemotherapeutic agents and radiotherapy, which are associated with a poor clinical outcome. Hence understanding mutations in Nrf2 would have a significant impact on the prognosis and treatment of cancer in the era of precision medicine. This perspective would provide an insight into the genetic alterations in Nrf2 and suggest the application of small molecules, RNAi, and genome editing technologies, particularly CRISR-Cas9, in therapeutic intervention of cancer in the context of the involvement of Nrf2 mutations.

Abstract PO-053: Chemical suppression of DHFR induces NRF2 inhibition and tumor regression

Abstract Background: Recurrence in head and neck squamous cell carcinoma (HNSCC) is common due to primary resistance and acquired resistance arising during therapy. Recent studies have highlighted a critical role of NRF2 in radiation (RT) resistance. Constitutive activation of the NRF2 oxidative stress response pathway occurs in 20-30% of primary HNSCC. As a ROS-activated transcription factor that is frequently mutated and active in cancer, NRF2 is central to both intrinsic and acquired determinants of RT resistance. We recently showed that high NRF2 activity is predictive for locoregional failure and poor outcomes following RT in HNSCC. The downstream effectors of NRF2 responsible for RT resistance are poorly defined. Therapeutic strategies to inhibit NRF2 may improve HNSCC patient outcomes, yet remain unproven. Methods: We sought to study NRF2 activation and biology in two model systems: MOC1 syngeneic HNSCC cells and mouse embryonic fibroblasts (MEFs) derived from a NRF2E79Q genetically engineered mouse model. We defined a transcriptional signature associated with an activating NRF2 mutation (NRF2E79Q) and characterized the effect of NRF2 on cell growth and RT response. We also investigated a novel NRF2 inhibitor in the syngeneic mouse MOC1 model. MEFs were isolated from a pregnant mouse, immortalized and selectively infected with 500-800 MOI of Adenovirus Cre-Recombinase for in vitro proliferation and RT assays. Drug studies were performed at 50mM in vitro or 20mg/kg PO daily starting day 7 in vivo. RT was delivered at 0, 4Gy, or 8Gy in vitro or 3Gy daily x 5 days starting day 17 in vivo. Results: RNA isolated from NRF2E79Q MEFs demonstrated activation of numerous oxidative stress pathway transcripts including canonical targets HMOX1, NQO1, BLVRB, and SLC71a11. NRF2E79Q MEFs demonstrated significantly enhanced baseline growth kinetics and RT resistance relative to the NRF2-wildtype line. We recently reported that pyrimethamine—an FDA-approved antiparasitic drug that targets one carbon metabolism— [1.2uM] induced NRF2 ubiquitylation and degradation in vitro and in vivo. Through structure-activity relationship optimization we have produced a novel analogue, [WCDD115; 50nM], with efficacious DHFR suppression and NRF2 inhibition in human and mouse HNSCC cell lines. In vitro, WCDD115 produced growth suppression in the MOC1 cell line which was enhanced in the setting of RT relative to either treatment alone. MOC1-bearing mice treated with WCDD115 or RT as monotherapy had significant reduction in growth kinetics relative to vehicle control. WCDD115 and RT combination therapy produced the greatest effect with significant growth suppression and 25% tumor rejection without weight loss or toxicity. Conclusions: Our study demonstrates that NRF2E79Q mutation is associated with enhanced growth kinetics and RT resistance along with upregulation of oxidative stress pathway targets. Drug targeting with the novel NRF2 inhibitor, WCDD115, has significant tumor suppressive activity in vitro and in vivo and warrants further investigation as a RT-sensitizing agent. Citation Format: Paul Zolkind, Harit Panda, Caroline Krall, Brittany Bowman, Nathan Wamsley, Bernard Weissman, Ben Major. Chemical suppression of DHFR induces NRF2 inhibition and tumor regression [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-053.

Abstract 12: Distinct Nrf2 signaling thresholds mediate lung tumor initiation and progression

Abstract KEAP1 negatively regulates the transcription factor NRF2, which regulates the response to oxidative stress. Mutations in the KEAP1-NRF2 pathway occur in up to a third of non-small cell lung cancer (NSCLC) cases, leading to constitutive activation of NRF2 and resistance to therapy in patients. To study the role of NRF2 activation in lung cancer, we developed murine alleles of the KEAP1 and NRF2 mutations found in human NSCLC and performed a thorough interrogation of how these mutations affect tumor initiation and progression. Following targeted activation of these alleles in the lung with adenoviral-Cre, immunohistochemical analysis of Nrf2 and its target Nqo1 revealed that the Keap1R554Q/R554Q mutant was the most activating towards Nrf2, followed by the Nrf2D29H/+ and Keap1R554Q/+ mutants. We found that chronic Nrf2 stabilization by Keap1 or Nrf2 mutation was not sufficient to induce tumorigenesis, even in the absence of tumor suppressors p53 or Lkb1. We also found that constitutive Nrf2 activation promotes lung tumor initiation and early progression of KrasG12D cells to low-grade tumors. To analyze the influence of Keap1/Nrf2 mutation on later progression, we crossed these mutations into the KrasG12D; Trp53fl/fl model of lung adenocarcinoma. Surprisingly, we found that Keap1R554Q/R554Q mutation blocked progression to advanced-grade tumors. We also found that advanced-grade tumors downregulated Nrf2 expression and activity, suggesting that excess Nrf2 activation is detrimental to tumor progression. Next, to test whether the progression block in the Keap1R554Q/R554Q model was dependent on Nrf2, we performed single copy deletion of Nrf2. We found that lowering Nrf2 levels in this model rescued the impairment in tumor progression. Finally, using a NSCLC cell culture system of NRF2 overexpression, we found that high NRF2 levels impaired cell proliferation, viability, and anchorage-independent growth. In our in vitro studies, we observed that NRF2 expression was rapidly downregulated to basal levels to restore proliferation. Our findings indicate that NRF2 promotes lung tumor initiation and early progression but impairs late progression in a dosage-dependent manner. Our preliminary data also suggests that NRF2 hyperactivation is detrimental to tumor cell proliferation. Collectively, our results establish the context-dependence and threshold for NRF2 during the tumorigenic process. Future work will include interrogating the mechanism of NRF2 downregulation in vivo and use of additional cell lines and systems to investigate how NRF2 overexpression can impair tumor cell proliferation. Citation Format: Janine M. DeBlasi, Aimee Falzone, Samantha Caldwell, Yun Pyo Kang, Nicolas Prieto-Farigua, Justin R. Prigge, Edward E. Schmidt, Iok In Christine Chio, Florian A. Karreth, Gina M. DeNicola. Distinct Nrf2 signaling thresholds mediate lung tumor initiation and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 12.

Comparative Analysis of the Mutation and Expression Profile of the Cytoprotective NRF2/ KEAP1/P62/SQSTM1 Signaling Pathway in Different Glioma Subtypes: An <i>In Silico</i> Study

Comparative Analysis of the Mutation and Expression Profile of the Cytoprotective NRF2/ KEAP1/P62/SQSTM1 Signaling Pathway in Different Glioma Subtypes: An <i>In Silico</i> Study

Genetic hyperactivation of Nrf2 causes larval lethality in Keap1a and Keap1b-double-knockout zebrafish

The Keap1–Nrf2 pathway is an evolutionarily conserved mechanism that protects cells from oxidative stress and electrophiles. Keap1 is a repressor of Nrf2 in normal cellular conditions but also a stress sensor for Nrf2 activation. Interestingly, fish and amphibians have two Keap1s (Keap1a and Keap1b), of which Keap1b is the ortholog of mammalian Keap1. Keap1a, on the other hand, is a gene found only in fish and amphibians, having been lost during the evolution to amniotes. We have previously shown that keap1b-knockout zebrafish have increased Nrf2 activity and reduced response to certain Nrf2-activating compounds but that they grow normally to adulthood. This may be because the remaining keap1a suppresses the hyperactivation of Nrf2, which is responsible for the post-natal lethality of Keap1-knockout mice. In this study, we analyzed keap1a;keap1b-double-knockout zebrafish to test this hypothesis. We found that keap1a;keap1b-double-knockout zebrafish, like Keap1-knockout mice, showed eating defects and were lethal within a week of hatching. Genetic introduction of the Nrf2 mutation rescued both the eating defects and the larval lethality, indicating that Nrf2 hyperactivation is the cause. However, unlike Keap1-knockout mice, keap1a;keap1b-double-knockout zebrafish showed no physical blockage of the food pathway; moreover, the cause of death was not directly related to eating defects. RNA-sequencing analysis revealed that keap1a;keap1b-double-knockout larvae showed extraordinarily high expression of known Nrf2-target genes as well as decreased expression of visual cycle genes. Finally, trigonelline or brusatol partially rescued the lethality of keap1a;keap1b-double-knockout larvae, suggesting that they can serve as an in vivo evaluation system for Nrf2-inhibiting compounds.

A novel uterine leiomyoma subtype exhibits NRF2 activation and mutations in genes associated with neddylation of the Cullin 3-RING E3 ligase

Uterine leiomyomas, or fibroids, are the most common tumors in women of reproductive age. Uterine leiomyomas can be classified into at least three main molecular subtypes according to mutations affecting MED12, HMGA2, or FH. FH-deficient leiomyomas are characterized by activation of the NRF2 pathway, including upregulation of the NRF2 target gene AKR1B10. Here, we have identified a novel leiomyoma subtype showing AKR1B10 expression but no alterations in FH or other known driver genes. Whole-exome and whole-genome sequencing revealed biallelic mutations in key genes involved in neddylation of the Cullin 3-RING E3 ligase, including UBE2M, NEDD8, CUL3, and NAE1. 3′RNA sequencing confirmed a distinct molecular subtype with activation of the NRF2 pathway. Most tumors displayed cellular histopathology, perivascular hypercellularity, and characteristics typically seen in FH-deficient leiomyomas. These results suggest a novel leiomyoma subtype that is characterized by distinct morphological features, genetic alterations disrupting neddylation of the Cullin 3-RING E3 ligase, and oncogenic NRF2 activation. They also present defective neddylation as a novel mechanism leading to aberrant NRF2 signaling. Molecular characterization of uterine leiomyomas provides novel opportunities for targeted treatment options.

Abstract 921: The Nrf2E79Q activating mutation accelerates growth of pure-small cell lung cancer but not combined small cell lung cancer Tp53floxed/floxed/Cdkn2afloxed/floxed mice

Abstract Inactivating mutations in TP53/P16 are found in most types of non-small cell lung cancer while RB1/P16 losspredominates in small cell lung cancer (SCLC). NRF2 activating mutations are also found frequently in lungcancer, especially in lung squamous cell carcinoma. However, how concurrent mutations in these genes maycontribute to lung cancer development is not fully understood. To address this problem, we compared lung tumordevelopment in a genetically-engineered mouse model (GEMM) with dual Trp53/p16 deficiency, +/- expressionof one of the most common mutations in NRF2 found in human tumors, Nrf2E79Q/+. Surprisingly, both groups,Nrf2E79Q/+ and Nrf2+/+, developed combined-SCLC (C-SCLC): a mixture of SCLC and large cell neuroendocrine(NE) carcinoma along with pure-SCLC (P-SCLC). The appearance of C-SCLC implicates Trp53/p16 double lossin the development of this type of lung cancer. Both groups developed C-SCLC at the same penetrance andincidence; all tumors labeled positive for NE-markers by immunohistochemistry (IHC) including ASCL1, SYP andINSM1. However, C-SCLCs developed by Nrf2E79Q/+ mice did not show NRF2 labeling by IHC, despiterecombination of the Nrf2E79Q/+ allele. In contrast, the Nrf2E79Q/+ mice showed significantly higher incidence of P-SCLC compared to Nrf2+/+ mice. All P-SCLC from Nrf2E79Q/+ mice were NRF2-positive by IHC, while the fewtumors developed by Nrf2+/+ mice were negative. All P-SCLC lesions labeled positive for NE-markers includingASCL1, CGRP & SYP. Both C-SCLCs & P-SCLCs were positive for NKX1.2 (lung cancer-marker) and negativefor P63 (squamous cell-marker). Interestingly, phospho-RB1 was positive for both C-SCLCs & P-SCLCs,suggesting the loss of p16 in our GEMM inactivates Rb1 through loss of inhibition of the cyclin dependent kinases4/6 (CDK4/6). This is the first study showing that the concurrent inactivation of Trp53/p16 in mice drivesdevelopment of C-SCLC. Our study also implicated activation of NRF2 signaling in the progression of SCLC.Our next studies will dissect the mechanisms by which NRF2 activation contributes to the development of SCLCdevelopment. Citation Format: Samera Hamad, Stephanie A. Montgomery, Jeremy M. Simon, Brittany M. Bowman, Kyle B. Spainhower, Ryan M. Murphy, Agnieszka Witkiewicz, Suzanne E. Fenton, Scott H. Randell, Neil Hayes, Erik Knudsen, Trudy G. Oliver, Ben Major, Bernard E. Weissman. The Nrf2E79Q activating mutation accelerates growth of pure-small cell lung cancer but not combined small cell lung cancer Tp53floxed/floxed/Cdkn2afloxed/floxed mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 921.

Abstract No. 365 CT radiomic features associated with NRF2 mutation status in hepatocellular carcinoma

NRF2 mutations in patients with hepatocellular carcinoma (HCC) are associated with poor response to trans-arterial embolization (TAE). We sought to determine if CT radiomic features could predict mutation status.

Abstract No. 510 Transcription signatures underlying aggressive subtypes of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) tumors with NRF2 and ARID1A mutations have more aggressive phenotypes and result in a reduced response to trans-arterial embolization (TAE). Pathway analysis of differentially expressed genes of these tumors may reveal epigenetic modifications that impact embolization response.

Association of Nrf2 expression and mutation with Weiss and Helsinki scores in adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is a rare malignant tumor. Genetic abnormalities that may represent therapeutic targets and prognostic factors in ACC remain unclear. Besides being one of the main cellular defense mechanisms that regulates antioxidant pathways for detoxifying reactive oxygen species (ROS), the transcription factor nuclear factor erythroid 2–related factor 2 (Nrf2) promotes tumor proliferation by increasing metabolic activity. In surgical specimens from 12 cases of nonmetastatic ACCs and nine cases of benign adrenocortical adenoma (ACA), we investigated gene mutation and protein expressions for Nrf2 and the preoperative maximum standard glucose uptake (SUVmax) on [18F]fluorodeoxy‐glucose positron emission tomography. Three of five ACCs with a Weiss score of 7 to 9 were Nrf2 mutants; these ACCs had higher expression of Nrf2 and higher preoperative SUVmax. The other seven ACCs had a Weiss score of 3 to 6; these seven ACCs and all the ACAs were non‐Nrf2 gene mutants. Patients with a Weiss score of 7 to 9 and Nrf2 mutant ACC had shorter overall survival. Based on Helsinki scoring, three ACCs with a Helsinki score greater than 17 had Nrf2 mutants, higher expression of Nrf2, higher preoperative SUVmax, and shorter overall survival. Our findings indicate that Nrf2 activation and the associated increase in metabolism play roles in ACC, in particular in ACC with a Weiss score of 7 to 9 and a Helsinki score of greater than 17.

Escaping KRAS: Gaining Autonomy and Resistance to KRAS Inhibition in KRAS Mutant Cancers.

Simple SummaryWhile KRAS is a driver oncogene, tumor cells can acquire mutant KRAS independency by activating pathways that functionally substitute for mutant KRAS. These KRAS-independent tumor cells exhibit a mesenchymal phenotype, readily primed for potential metastasis. The activation of YAP and/or RSK-mTOR pathways and mutations in LKB1, KEAP1, and/or NRF2 are associated with mutant KRAS autonomy. These alterations rewire survival signaling and metabolic processes originally governed by mutant KRAS. The presence of KRAS-independent cells is associated with the heterogeneity of KRAS mutant cancers, as well as variable responses to therapies. Notably, KRAS G12C-specific inhibitors appear to be effective only in tumors dependent on mutant KRAS for their survival. Therefore, determining KRAS dependency will be critical for selecting patients who should be treated with mutant-specific inhibitors. Furthermore, elucidating underlying mechanisms of KRAS autonomy is crucial towards developing optimal treatment strategies for KRAS-independent tumors.Activating mutations in KRAS are present in 25% of human cancers. When mutated, the KRAS protein becomes constitutively active, stimulating various effector pathways and leading to the deregulation of key cellular processes, including the suppression of apoptosis and enhancement of proliferation. Furthermore, mutant KRAS also promotes metabolic deregulation and alterations in the tumor microenvironment. However, some KRAS mutant cancer cells become independent of KRAS for their survival by activating diverse bypass networks that maintain essential survival signaling originally governed by mutant KRAS. The proposed inducers of KRAS independency are the activation of YAP1 and/or RSK-mTOR pathways and co-mutations in SKT11 (LKB1), KEAP1, and NFE2L2 (NRF2) genes. Metabolic reprogramming, such as increased glutaminolysis, is also associated with KRAS autonomy. The presence or absence of KRAS dependency is related to the heterogeneity of KRAS mutant cancers. Epithelial-to-mesenchymal transition (EMT) in tumor cells is also a characteristic phenotype of KRAS independency. Translationally, this loss of dependence is a cause of primary and acquired resistance to mutant KRAS-specific inhibitors. While KRAS-dependent tumors can be treated with mutant KRAS inhibitor monotherapy, for KRAS-independent tumors, we need an improved understanding of activated bypass signaling pathways towards leveraging vulnerabilities, and advancing therapeutic options for this patient subset.

Nrf2 Mutation/Activation Is Dispensable for the Development of Chemically Induced Mouse HCC.

Background & AimsActivation of the kelch-like ECH-associated protein 1 (Keap1)–nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway has been associated with metabolic reprogramming in many tumors, including hepatocellular carcinoma (HCC). However, the contribution of Nrf2 mutations in this process remains elusive. Here, we investigated the occurrence of Nrf2 mutations in distinct models of mouse hepatocarcinogenesis.MethodsHCCs were generated by experimental protocols consisting of the following: (1) a single dose of diethylnitrosamine (DEN), followed by repeated treatments with the nuclear-receptor agonist 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene; (2) repeated treatments with 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene alone; (3) a single dose of DEN followed by exposure to a choline-deficient L-amino acid–defined diet; and (4) a single dose of DEN with no further treatment. All of these protocols led to HCC development within 28–42 weeks. Activation of the Keap1-Nrf2 pathway was investigated by analyzing the presence of Nrf2 gene mutations, and the expression of Nrf2 target genes. Metabolic reprogramming was assessed by evaluating the expression of genes involved in glycolysis, the pentose phosphate pathway, and glutaminolysis.ResultsNo Nrf2 mutations were found in any of the models of hepatocarcinogenesis analyzed. Intriguingly, despite the described cooperation between β-catenin and the Nrf2 pathway, we found no evidence of Nrf2 activation in both early dysplastic nodules and HCCs, characterized by the presence of up to 80%–90% β-catenin mutations. No HCC metabolic reprogramming was observed either.ConclusionsThese results show that, unlike rat hepatocarcinogenesis, Nrf2 mutations do not occur in 4 distinct models of chemically induced mouse HCC. Interestingly, in the same models, metabolic reprogramming also was minimal or absent, supporting the concept that Nrf2 activation is critical for the switch from oxidative to glycolytic metabolism.

New insights into the mechanism of Keap1-Nrf2 interaction based on cancer-associated mutations

AimsKeap1-Nrf2 signaling pathway is one of the most important antioxidant signaling pathways, and its abnormal activation is related to cancer metastasis and drug resistance. Many studies have shown Keap1 and Nrf2 mutations are closely associated with cancer occurrence. However, few studies focus on Keap1-Nrf2 binding characteristics of cancer-associated mutations. The study investigated the molecular mechanism between Keap1/Nrf2 mutations and cancer. Main methodsWe have determined the crystal structure of the Keap1-Kelch domain with Nrf2 25-mer peptide. What's more, we clarified the molecular effects of Nrf2Thr80 and Nrf2Pro85 on the binding of Keap1 by the method isothermal titration calorimetry (ITC), differential scanning fluorimetry (DSF) and electrophoretic mobility shift assay (EMSA). Especially, we confirmed the effect of Thr80 and Pro85 mutations on Keap1/Nrf2 signaling pathway in HEK293T cells by RT-PCR and western blot (WB). Finally, we verified the effect of six cancer-related high-frequency somatic mutations Keap1G364C, Keap1D422N, Keap1R470C, Keap1G480W, Keap1E493Q and Keap1R601L on binding with Nrf2 through ITC experiments. Key findingsNrf2Thr80 and Nrf2Pro85 play a vital role in the Keap1-Nrf2 interaction. Mutant or modification at position Thr80 will disrupt the interaction. Especially, Nrf2Thr80 and Nrf2Pro85 mutations activate the expression of cytoprotective genes in HEK293T cells. As for Keap1, except G364C, the binding affinity of other cancer-related mutants to Nrf2 hardly changed, which means that Keap1 mutants can activate Nrf2 without disrupting the binding to Nrf2. SignificanceThe study provides new insight into Keap1/Nrf2 signaling pathway and cancer.

Abstract 2939: Activation of NRF2 signaling accelerates development of small cell lung cancer in mice

Abstract Activating mutations in NRF2 frequently occur in human lung cancers, especially the E79Q mutation. However, how the activating mutation contributes to lung tumor development is not well understood. We used a new genetically engineered mouse model (GEMM), LSL-Nrf2E79Q/+ with Cre-inducible expression of this mutation within the endogenous Nrf2 locus, to understand its contribution to lung tumor development. We compared tumor development in p53fl/fl;p16fl/fl;LSL-Nrf2+/+ (WT) versus p53fl/fl;p16fl/fl;Nrf2E79Q/+ (Het) mice after intranasal instillation of Adenoviral Cre. While we did not observe a significant difference in lung adenocarcinoma (LUAC) between the Het and WT mice, the Het mice showed significantly higher frequencies of bronchial neuroendocrine tumors that resembled small cell lung carcinomas (SCLC) compared to WT. Additionally, the latency of SCLC lesions was lower in Het mice compared to WT. Surprisingly, LUACs in both groups were negative for NRF2 by IHC labeling despite recombination of the mutant Nrf2 allele. While all lesions in the WT and Het mice displayed positive ASCL1 and CGRp (NE markers) IHC labeling, only the SCLCs in Het mice expressed NRF2. This is the first study showing activation of Nrf2E79Q mutation drives development of small cell lung carcinomas. Interestingly, our data also suggest that the E79Q NRF2 activating mutation may inhibit the progression of LUAD. Citation Format: Samera Hamad, Stephanie Montgomery, Brittany Bowman, Ryan Murphy, Scott Randell, Trudy Oliver, D. Neil Hayes, M. Ben Major, Bernard Weissman. Activation of NRF2 signaling accelerates development of small cell lung cancer in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2939.

NRF2 DLG Domain Mutations Identified in Japanese Liver Cancer Patients Affect the Transcriptional Activity in HCC Cell Lines.

Geographically, East Asia had the highest liver cancer burden in 2017. Besides this, liver cancer-related deaths were high in Japan, accounting for 3.90% of total deaths. The development of liver cancer is influenced by several factors, and genetic alteration is one of the critical factors among them. Therefore, the detailed mechanism driving the oncogenic transformation of liver cells needs to be elucidated. Recently, many researchers have focused on investigating the liver cancer genome and identified somatic mutations (MTs) of several transcription factors. In this line, next-generation sequencing of the cancer genome identified that oxidative stress-related transcription factor NRF2 (NFE2L2) is mutated in different cancers, including hepatocellular carcinoma (HCC). Here, we demonstrated that NRF2 DLG motif mutations (NRF2 D29A and L30F), found in Japanese liver cancer patients, upregulate the transcriptional activity of NRF2 in HCC cell lines. Moreover, the transcriptional activity of NRF2 mutations is not suppressed by KEAP1, presumably because NRF2 MTs disturb proper NRF2-KEAP1 binding and block KEAP1-mediated degradation of NRF2. Additionally, we showed that both MTs upregulate the transcriptional activity of NRF2 on the MMP9 promoter in Hepa1-6 and Huh7 cells, suggesting that MT derived gain-of-function of NRF2 may be important for liver tumor progression. We also found that ectopic overexpression of oncogenic BRAF WT and V600E increases the transcriptional activity of NRF2 WT on both the 3xARE reporter and MMP9 promoter. Interestingly, NRF2 D29A and L30F MTs with oncogenic BRAF V600E MT synergistically upregulate the transcription activity of NRF2 on the 3xARE reporter and MMP9 promoter in Hepa1-6 and Huh7 cells. In summary, our findings suggest that MTs in NRF2 have pathogenic effects, and that NRF2 MTs together with oncogenic BRAF V600E MT synergistically cause more aberrant transcriptional activity. The high activity of NRF2 MTs in HCC with BRAF MT warrants further exploration of the potential diagnostic, prognostic, and therapeutic utility of this pathway in HCC.

Молекулярные механизмы развития рака легкого

Рак легкого является многоэтапным процессом с участием генетических факторов и изменений в эпигенетическом регулировании клеточного цикла. Амплификация генов, сверхэкспрессия, точечные мутациии или перестройки ДНК могут привести к постоянной позитивной регуляции митогенных сигналов роста, вследствие чего усиливается онкогенная сигнализация в клетках. В данном обзоре рассматриваются нарушения в 3 сигнальных путях (RAS-RAF-MEK-ERK, PI3K-Akt-mTOR и Keap1-Nrf2) при раке легкого. Причинами неконтролируемого роста и пролиферации клеток являются мутации EGFR, KRAS и аномальное слияние EML4-ALK, вследствие чего активируются 2 основных пути внутриклеточной сигнализации RAS-RAF-MEK-ERK и PI3K-Akt-mTOR. Канцерогенез при раке легкого может быть вызван генетическими аберрациями в любых компонентах пути PI3K-Akt-mTOR, его отрицательных регуляторах, взаимосвязанных путях и передаче сигналов через рецепторы RTK. Нарушения сигнального пути Keap1-Nrf2, обнаруженные при раке легкого, вызваны соматическими мутациями, потерей гетерозиготности или метилированием ДНК в промоторной области Keap1 и мутациями Nrf2, что приводит к увеличению накопления Nrf2. Опухоли с высоким уровнем Nrf2 связаны с плохим прогнозом из-за радио- и химиорезистентности и агрессивной пролиферации раковых клеток. Гиперактивность Nrf2 помогает автономным злокачественным клеткам выдерживать высокие уровни эндогенных активных форм кислорода (АФК) и избегать апоптоза. В этой связи важным подходом для профилактики рака является усиление активности Nrf2, тогда как для лечения рака желательно ингибирование Nrf2. Lung cancer is a multistep process involving genetic factors and changes in the epigenetic regulation of cell cycle. Gene amplification, overexpression, point mutations or DNA rearrangements may lead to continuous positive regulation of mitogenic growth signals resulting in enhancement of oncogenic cell signaling. This review focuses on disorders of three signaling pathways (RAS-RAF-MEK-ERK, PI3K-Akt-mTOR, and Keap1-Nrf2) in lung cancer. The causes for the uncontrolled cell growth and proliferation are EGFR and KRAS mutations and the abnormal EML4-ALK merging, which activate two major pathways of intracellular signaling, RAS-RAF-MEK-ERK and PI3K-Akt-mTOR. Carcinogenesis in lung cancer may result from genetic aberrations in any component of the PI3K-Akt-mTOR pathway, its negative regulators, interconnected pathways, and the signal transduction through RTK receptors. The disorders of the Keap1-Nrf2 signaling pathway found in lung cancer are caused by somatic mutations, loss of heterozygosity or DNA methylation in the Keap1 promoter region and Nrf2 mutations, which lead to an increase in the accumulation of Nrf2. Tumors with high Nrf2 levels are associated with a poor prognosis due to their radio- and chemoresistance and aggressive proliferation of cancer cells. The Nrf2 hyperactivity helps autonomous malignant cells withstand high levels of endogenous reactive oxygen species (ROS) and evade apoptosis. In this regard, enhancing the Nrf2 activity is an important approach to prevention of cancer while Nrf2 inhibition is desirable for its treatment.

Abstract PO033: Regulation of immune checkpoint proteins in NSCLC adenocarcinoma

Abstract Lung cancer is the leading cause of cancer-related deaths in both men and women. Approximately 85% of lung cancers are non-small cell lung cancer (NSCLC), with adenocarcinoma (adenoCa) being the main subtype. Well-defined mutational signatures of various lung cancer subtypes have been exploited for targeted therapies, but cancer cells often acquire resistance to these treatments, which could be mediated by the inflammatory tumor microenvironment (TME). Inflammation within the TME is known to promote cancer progression and therapy resistance. The inflammatory cytokine, interleukin-1 beta (IL-1β) is elevated in the TME of several cancers and correlates with poor outcomes. In NSCLC patients, serum IL-1β is elevated relative to normal subjects; within the same patient, IL-1β is elevated in the tumor relative to adjacent non-tumor tissue. Significantly, the Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS) showed that blockade of IL-1 signaling could drastically reduce lung cancer incidence and mortality. Interestingly, we observed that NSCLC adenoCa cell lines H1838, H2228, H2347, HCC4150 and HCC827 have significantly higher basal expression of the Type 1 IL-1 receptor (IL-1R1), relative to immortalized normal Human Bronchial Epithelial Cell (HBEC-3KT) and Human Small Airway Epithelial Cell (HSAEC-1KT) lines, suggesting that adenoCa cells are primed to respond to IL-1 signaling. Cancer cells can promote immunosuppression through the expression of immune checkpoint proteins (ICPs). Previously, we identified several ICPs, including programmed death-ligand 1 (PD-L1), tryptophan 2,3-dioxygenase 2 (TDO2), indoleamine 2, 3-dioxygenase 1 (IDO1) and growth differentiation factor 15 (GDF15), that were developmentally upregulated by transcription factor nuclear factor erythroid 2-related factor 2 (NRF2/NFE2L2) in human fetal lung epithelial cells during differentiation in culture. Importantly, genomic studies have revealed that >20% of lung adenoCa harbor mutations in NRF2 or its inhibitor KEAP1, which cause NRF2 activation. Lung adenoCa with signatures of increased NRF2 activation manifest reduced immune cell infiltration, suggesting that increased expression of NRF2 might create a more permissive immune cell environment, resulting in evasion and tolerance to the host immune system. Given that IL-1β induces ROS which can activate NRF2, we hypothesized that IL-1β present in the TME may enhance expression of NRF2 and ICPs in NSCLC adenoCa. We found that increased expression of ICPs in adenoCa cell lines H1838, H2228, H2347, HCC4150 and HCC827 compared to HBEC-3KT and HSAEC-1KT cells was correlated with significantly increased expression of NRF2. When these adenoCa cell lines were treated with IL-1β, we observed a marked induction in NRF2 and ICP mRNA and protein levels. Taken together our studies indicate that NSCLC adenoCa cells are primed to respond to IL-1 signaling which induces NRF2 and ICPs to promote tumor cell survival through immunosuppression. Citation Format: Afshan Fathima Nawas, Ritu Mishra, Carole R. Mendelson. Regulation of immune checkpoint proteins in NSCLC adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO033.