scholarly journals EXTH-04. BLOCKADE OF NRF2/GLUTATHIONE METABOLISM AS A SYNTHETIC LETHALITY APPROACH FOR IDH1-MUTATED GLIOMA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi83-vi83
Author(s):  
Yang Liu ◽  
Yanxin Lu ◽  
Orieta Celiku ◽  
Aiguo Li ◽  
Chunzhang Yang
Keyword(s):  
Tumor Cell ◽  
Metabolic Pathway ◽  
De Novo ◽  
Synthetic Lethality ◽  
Substantial Reduction ◽  
Lower Grade ◽  
Related Factor ◽  
De Novo Synthesis ◽  
Antioxidant Genes ◽  
Cox Regression Analysis

Abstract BACKGROUND Mutations in isocitrate dehydrogenase (IDH1/2) are frequent genetic abnormalities in human malignancies. IDH1/2-mutated cancers are a recently defined disease entity with distinctive patterns of tumor cell biology, metabolism and resistance to therapy. Molecular targeting approaches against this disease cluster remain limited. METHODS We investigated the redox homeostasis in IDH1 mutant-transduced cells and patient-derived brain tumor initiating cells. The importance of antioxidant genes was confirmed through COX regression analysis on a large cohort of lower grade glioma. We investigated the biologic impact of Nuclear factor erythroid 2-related factor 2 (NRF2) on the glutathione de novo synthesis in IDH1-mutated cells. Finally, we evaluated the value of targeting NRF2/glutathione metabolic pathway as a potential synthetic lethality approach for IDH1-mutated cell in vitro and in vivo. RESULTS We discovered that acquisition of cancer-associated IDH1 mutants results in constitutive activation of NRF2-governed cytoprotective pathways through decoupling of NRF2 from its E3 ligase Kelch-like ECH-associated protein 1. NRF2 mediated the transcriptional activation of GCLC, GCLM and SLC7A11, which strengthens the glutathione de novo synthesis, and relieves the metabolic burden derived from IDH1 mutant neomorphic activity. Blockade of the NRF2/glutathione metabolic pathway synergizes with the elevated intrinsic reactive oxygen species, which results in overwhelming oxidative damage in IDH1-mutated cells, as well as a substantial reduction in tumor cell proliferation and xenograft expansion. CONCLUSION Our findings suggest that blockade of the NRF2/glutathione synthetic pathway is a novel targeting strategy for IDH1-mutated malignancies.

scholarly journals EXTH-03. TRIPTOLIDE, A NOVEL THERAPEUTIC AGENT FOR IDH1-MUTATED GLIOMA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi82-vi83
Author(s):  
Di Yu ◽  
Yang Liu ◽  
Guowang Xu ◽  
Chunzhang Yang
Keyword(s):  
Cancer Cells ◽  
De Novo ◽  
Synthetic Lethality ◽  
Redox Homeostasis ◽  
Glutamate Transporter ◽  
Protective Role ◽  
Related Factor ◽  
De Novo Synthesis ◽  
Ros Scavenging ◽  
Idh Mutations

Abstract BACKGROUND Isocitrate dehydrogenase (IDH1/2) mutations are common genetic abnormalities in human malignancies, which result in neomorphic enzyme activity that catalyzes 2-hydroxyglutarate (2-HG) production. While IDH mutations are recognized as critical cancer-associated genetic changes, the therapeutic options for IDH-mutated cancers remain limited. METHODS In the present study, we investigated the reactive oxygen species (ROS) scavenging pathways in patient-derived IDH1-mutated cells. Further, we investigated the protective role of glutathione de novo synthesis for IDH1-mutated cancer cells. Finally, we evaluated triptolide, a diterpenoid epoxide derived from Tripterygium wilfordii, as an experimental therapeutic for IDH1-mutated cells and xenografts. RESULTS We discovered that the neomorphic activity of IDH1 mutant enzyme triggers metabolic depletion and a substantial elevated burden of ROS scavenging in cancer cells. The nuclear factor erythroid 2-related factor 2 (NRF2)-guided antioxidant pathway plays a key role in maintaining redox homeostasis for IDH1-mutated cells. Triptolide serves as a potent inhibitor of NRF2 through enhancing the NRF2 ubiquitination and subsequently proteasomal degradation. Additionally, triptolide compromises the de novo synthesis of glutathione via suppression of NRF2-guided transcription of SLC7A11, which encodes cystine/glutamate transporter (xCT). Reduced availability of glutathione results in overwhelming oxidative damage in DNA and lipid, which translates into severe cytotoxicity and reduced xenograft expansion of IDH1-mutated cells. CONCLUSION Overall our findings highlight triptolide as a valuable synthetic lethality approach for IDH1-mutated malignancies by targeting NRF2-guided redox homeostasis.

scholarly journals DDRE-08. NRF2/GLUTATHIONE METABOLISM AS A NOVEL THERAPEUTIC TARGET FOR IDH1-MUTATED GLIOMA

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. i8-i8
Author(s):  
Yang Liu ◽  
Di Yu ◽  
Orieta Celiku ◽  
Aiguo Li ◽  
Mioara Larion ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Tumor Cell ◽  
Cell Lines ◽  
Nicotinamide Adenine Dinucleotide ◽  
Cell Biology ◽  
De Novo ◽  
Nicotinamide Adenine ◽  
Glutathione Metabolism ◽  
De Novo Synthesis ◽  
Cox Regression Analysis

Abstract BACKGROUND IDH1-mutated glioma is a recently defined disease entity with distinctive patterns of tumor cell biology, metabolism, and resistance to therapy. Although IDH1 mutations are highly prevalent in patients with WHO II/III glioma, curative molecular targeting approaches remain unavailable for this disease cluster. METHODS In the present study, we investigated the glutathione de novo synthesis pathway through the TCGA patient cohort and patient-derived cell lines with IDH1 mutation. The biologic function of nuclear factor erythroid 2-related factor 2 (NRF2) was analyzed by biochemistry and cell biology assays. Finally, NRF2 inhibitors were evaluated in IDH1-mutated cell lines and preclinical models as an experimental therapy. RESULTS IDH1 mutant neomorphic activity depletes the cellular pools of enzyme cofactors such as nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). The limitation of NAD(P) not only affects the anabolic reactions, but also results in oxidative stress and damages on DNA and protein. Further, we showed that the reprogrammed redox landscape results in constitutive activation of NRF2-governed cytoprotective pathways through the decoupling of NRF2 from its E3 ligase Kelch-like ECH-associated protein 1. NRF2 mediated the transcriptional activation of GCLC, GCLM, and SLC7A11, which not only strengthens the glutathione de novo synthesis, but also relieves the metabolic burden in IDH1-mutated cells. The importance of the glutathione synthesis is further confirmed through COX regression analysis on lower-grade glioma. Blockade of the NRF2/glutathione metabolic pathway synergizes with the elevated intrinsic oxidative stress, which results in overwhelming oxidative damage, as well as a substantial reduction in tumor cell proliferation and xenograft expansion. CONCLUSION We report that the NRF2-guided cytoprotective pathways play pivotal roles in the disease progression of IDH1-mutated glioma. Targeting NRF2 and glutathione metabolism could be novel targeting strategies for IDH1-mutated glioma.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus Induces Nrf2 Activation in Latently Infected Endothelial Cells through SQSTM1 Phosphorylation and Interaction with Polyubiquitinated Keap1

2014 ◽  
Vol 89 (4) ◽  
pp. 2268-2286 ◽  
Author(s):  
Olsi Gjyshi ◽  
Stephanie Flaherty ◽  
Mohanan Valiya Veettil ◽  
Karen E. Johnson ◽  
Bala Chandran ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
De Novo ◽  
Related Factor ◽  
Antioxidant Genes ◽  
Kshv Infection ◽  
Sequestosome 1 ◽  
Nrf2 Activation ◽  
Latently Infected ◽  
Stress Signals

ABSTRACTNuclear factor erythroid 2-related factor 2 (Nrf2), the cellular master regulator of the antioxidant response, dissociates from its inhibitor Keap1 when activated by stress signals and participates in the pathogenesis of viral infections and tumorigenesis. Early duringde novoinfection of endothelial cells, KSHV induces Nrf2 through an intricate mechanism involving reactive oxygen species (ROS) and prostaglandin E2 (PGE2). When we investigated the Nrf2 activity during latent KSHV infection, we observed increased nuclear serine-40-phosphorylated Nrf2 in human KS lesions compared to that in healthy tissues. Using KSHV long-term-infected endothelial cells (LTC) as a cellular model for KS, we demonstrated that KSHV infection induces Nrf2 constitutively by extending its half-life, increasing its phosphorylation by protein kinase Cζ (PKCζ) via the infection-induced cyclooxygenase-2 (COX-2)/PGE2 axis and inducing its nuclear localization. Nrf2 knockdown in LTC decreased expression of antioxidant genes and genes involved in KS pathogenesis such as the NAD(P)H quinone oxidase 1 (NQO1), gamma glutamylcysteine synthase heavy unit (γGCSH), the cysteine transporter (xCT), interleukin 6 (IL-6), and vascular endothelial growth factor A (VEGF-A) genes. Nrf2 activation was independent of oxidative stress but dependent on the autophagic protein sequestosome-1 (SQSTM1; p62). SQSTM1 levels were elevated in LTC, a consequence of protein accumulation due to decreased autophagy and Nrf2-mediated transcriptional activation. SQSTM1 was phosphorylated on serine-351 and -403, while Keap1 was polyubiquitinated with lysine-63–ubiquitin chains, modifications known to increase their mutual affinity and interaction, leading to Keap1 degradation and Nrf2 activation. The latent KSHV protein Fas-associated death domain-like interleukin-1β-converting enzyme-inhibitory protein (vFLIP) increased SQSTM1 expression and activated Nrf2. Collectively, these results demonstrate that KSHV induces SQSTM1 to constitutively activate Nrf2, which is involved in the regulation of genes participating in KSHV oncogenesis.IMPORTANCEThe transcription factor Nrf2 is activated by stress signals, including viral infection, and responds by activating the transcription of cytoprotective genes. Recently, Nrf2 has been implicated in oncogenesis and was shown to be activated duringde novoKSHV infection of endothelial cells through ROS-dependent pathways. The present study was undertaken to determine the mechanism of Nrf2 activation during prolonged latent infection of endothelial cells, using an endothelial cell line latently infected with KSHV. We show that Nrf2 activation was elevated in KSHV latently infected endothelial cells independently of oxidative stress but dependent on the autophagic protein sequestosome-1 (SQSTM1), which was involved in the degradation of the Nrf2 inhibitor Keap1. Furthermore, our results indicated that the KSHV latent protein vFLIP participates in Nrf2 activation. This study suggests that KSHV hijacks the host's autophagic protein SQSTM1 to induce Nrf2 activation, thereby manipulating the infected host gene regulation to promote KS pathogenesis.

scholarly journals DDRE-29. DE NOVO PYRIMIDINE SYNTHESIS IS A TARGETABLE VULNERABILITY IN IDH-MUTANT GLIOMA

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. i12-i13
Author(s):  
Diana D Shi ◽  
Adam C Wang ◽  
Michael M Levitt ◽  
Jennifer E Endress ◽  
Min Xu ◽  
...  
Keyword(s):  
Cell Lines ◽  
De Novo ◽  
Synthetic Lethality ◽  
Nucleotide Metabolism ◽  
Metabolic Inhibitor ◽  
Lower Grade ◽  
Nucleotide Synthesis ◽  
Pyrimidine Synthesis ◽  
Pyrimidine Nucleotide ◽  
Idh1 Mutations

Abstract 70–90% of lower-grade gliomas and secondary glioblastomas harbor gain-of-function mutations in isocitrate dehydrogenase 1 (IDH1), causing overproduction of the oncometabolite (R)-2-hydroxyglutarate [(R)-2HG]. Although inhibitors of mutant IDH enzymes are effective in other cancers, including leukemia, they have shown guarded efficacy in preclinical and clinical brain tumor studies, thus underscoring the need to identify additional therapeutic targets in IDH mutant glioma. We sought to identify tumor-specific metabolic vulnerabilities induced by IDH1 mutations that could be exploited therapeutically. To uncover such vulnerabilities, we conducted a chemical synthetic lethality screen using isogenic IDH1 mutant and IDH1 wild-type (WT) glioma cell lines and a novel metabolic inhibitor screening platform. We discovered that IDH1 mutant cells are hypersensitive to drugs targeting enzymes in the de novo pyrimidine nucleotide synthesis pathway, including dihydroorotate dehydrogenase (DHODH). This vulnerability is specific because inhibitors of purine nucleotide metabolism did not score in our screen. We validated that the cytotoxicity of pyrimidine synthesis inhibitors is on-target and showed that IDH1 mutant patient-derived glioma stem-like cell lines are also hyperdependent on de novo pyrimidine nucleotide synthesis compared to IDH1 WT lines. To test pyrimidine synthesis dependence of IDH1 mutant gliomas in vivo, we used a brain-penetrent DHODH inhibitor currently undergoing evaluation in leukemia patients, BAY 2402234. We found that BAY 2402234 displays monotherapy activity against gliomas in an orthotopic xenograft model of IDH1 mutant glioma, with an effect size that compared favorably with radiotherapy. We also developed novel genetically engineered and allograft mouse models of mutant IDH1-driven anaplastic astrocytoma and showed that BAY 2402234 blocked growth of orthotopic astrocytoma allografts. Our findings bolster rationale to target DHODH in glioma, highlight BAY 2402234 as a clinical-stage drug that can be used to inhibit DHODH in brain tumors, and establish IDH1 mutations as predictive biomarkers of DHODH inhibitor efficacy.

CSIG-02. NRF2-GUIDED GLUTATHIONE SYNTHESIS IS AN ESSENTIAL METABOLIC PATHWAY IN IN IDH1-MUTATED GLIOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi33-vi33
Author(s):  
Yang Liu ◽  
Di Yu ◽  
Chunzhang Yang
Keyword(s):  
Metabolic Pathway ◽  
Nicotinamide Adenine Dinucleotide ◽  
De Novo ◽  
Redox Homeostasis ◽  
Nicotinamide Adenine ◽  
De Novo Synthesis ◽  
Who Grade ◽  
Glutathione Synthesis ◽  
Synergistic Cytotoxicity ◽  
Idh Mutations

Abstract BACKGROUND Isocitrate dehydrogenase (IDH) mutations are common genetic abnormalities in WHO Grade II/III glioma, which result in the reprogramming of cellular metabolism and redox homeostasis. Many lines of evidence showed that IDH mutations are critical for glioma formation, whereas the therapeutic options for IDH-mutated cancers remain limited. METHODS In the present study, we used the patient-derived glioma cell lines to investigate the role of nuclear factor erythroid 2-related factor 2 (NRF2) governed glutathione de novo synthesis. Further, we evaluated the therapeutic value of NRF2 inhibitors in IDH1-mutated cells and preclinical orthotopic models. RESULTS The neomorphic activity of mutant IDH reprogrammed the metabolic pathways involving enzyme cofactors such as nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). The depletion of NAD(P) in IDH1-mutated cells resulted in elevated oxidative stress and constitutive activation of NRF2-governed cytoprotective pathways through the decoupling of NRF2 from its E3 ligase Kelch-like ECH-associated protein 1 (Keap1). Activation of NRF2 enhanced glutathione synthesis by enhancing the gene transcription of GCLC, GCLM, and SLC7A11, which are the critical for glutathione de novo synthesis. Further, evidence from both in vitro assays and patient cohort indicated that NRF2 governed glutathione synthesis is important for maintaining the redox homeostasis and cell survival, especially in IDH1-mutated glioma. Finally, Blockade of the NRF2/glutathione metabolic pathway exhibited synergistic cytotoxicity with the metabolic stress in IDH1-mutated cells, which results in overwhelming oxidative damage, as well as a substantial reduction in tumor cell proliferation and xenograft expansion. CONCLUSION In this study, we highlighted that NRF2 plays critical roles in the disease progression of IDH1-mutated glioma by prompting glutathione synthesis. Targeting NRF2 governed glutathione metabolism could serve as a valuable synthetic lethality approach for IDH1-mutated malignancies.

Characterization of Monocyte-Associated Factor V

1993 ◽  
Vol 70 (02) ◽  
pp. 273-280 ◽  
Author(s):  
Janos Kappelmayer ◽  
Satya P Kunapuli ◽  
Edward G Wyshock ◽  
Robert W Colman
Keyword(s):  
Monoclonal Antibody ◽  
Light Chain ◽  
Peripheral Blood ◽  
De Novo ◽  
Factor V ◽  
Blood Monocytes ◽  
De Novo Synthesis ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Peripheral Blood Monocytes

SummaryWe demonstrate that in addition to possessing binding sites for intact factor V (FV), unstimulated peripheral blood monocytes also express activated factor V (FVa) on their surfaces. FVa was identified on the monocyte surface by monoclonal antibody B38 recognizing FVa light chain and by human oligoclonal antibodies H1 (to FVa light chain) and H2 (to FVa heavy chain) using immunofluorescence microscopy and flow cytometry. On Western blots, partially cleaved FV could be identified as a 220 kDa band in lysates of monocytes. In addition to surface expression of FVa, monocytes also contain intracellular FV as detected only after permeabilization by Triton X-100 by monoclonal antibody B10 directed specifically to the Cl domain not present in FVa. We sought to determine whether the presence of FV in peripheral blood monocytes is a result of de novo synthesis.Using in situ hybridization, no FV mRNA could be detected in monocytes, while in parallel control studies, factor V mRNA was detectable in Hep G2 cells and CD18 mRNA in monocytes. In addition, using reverse transcriptase and the polymerase chain reaction, no FV mRNA was detected in mononuclear cells or in U937 cells, but mRNA for factor V was present in Hep G2 cells using the same techniques. These data suggest that FV is present in human monocytes, presumably acquired by binding of plasma FV, and that the presence of this critical coagulation factor is not due to de novo synthesis.

Platelets Stimulate Thromboplastin Synthesis in Human Endothelial Cells

1983 ◽  
Vol 49 (02) ◽  
pp. 069-072 ◽  
Author(s):  
U L H Johnsen ◽  
T Lyberg ◽  
K S Galdal ◽  
H Prydz
Keyword(s):  
Endothelial Cells ◽  
De Novo ◽  
Umbilical Vein ◽  
Time Dependent ◽  
De Novo Synthesis ◽  
Human Endothelial Cells ◽  
Tumor Promotor ◽  
Coagulation Assay ◽  
Platelet Aggregates ◽  
Umbilical Vein Endothelial Cells

SummaryHuman umbilical vein endothelial cells in culture synthesize thromboplastin upon stimulation with phytohaemagglutinin (PHA) or the tumor promotor 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The thromboplastin activity is further strongly enhanced in a time dependent reaction by the presence of gel-filtered platelets or platelet aggregates. This effect was demonstrable at platelet concentrations lower than those normally found in plasma, it may thus be of pathophysiological relevance. The thromboplastin activity increased with increasing number of platelets added. Cycloheximide inhibited the increase, suggesting that de novo synthesis of the protein component of thromboplastin, apoprotein III, is necessary.When care was taken to remove monocytes no thromboplastin activity and no apoprotein HI antigen could be demonstrated in suspensions of gel-filtered platelets, platelets aggregated with thrombin or homogenized platelets when studied with a coagulation assay and an antibody neutralization technique.

DE NOVO SYNTHESIS OF STEROIDS BY THE TESTES OF THE HUMAN FOETUS AT MIDGESTATION

1971 ◽  
Vol 68 (1_Supplb) ◽  
pp. S135 ◽  
Author(s):  
R. S. Mathur ◽  
N. Wiqvist ◽  
E. Diczfalusy
Keyword(s):  
De Novo ◽  
De Novo Synthesis ◽  
Human Foetus
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