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

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.

Listeria monocytogenes TcyKLMN cystine/cysteine transporter facilitates glutathione synthesis and virulence gene expression

Listeria monocytogenes (Lm) is a saprophyte and a human intracellular pathogen. Upon invasion into mammalian cells, it senses multiple metabolic and environmental signals that collectively trigger its transition to the pathogenic state. One of these signals is the tripeptide glutathione, which acts as an allosteric activator of Lm’s master virulence regulator, PrfA. While glutathione synthesis by Lm was shown to be critical for PrfA activation and virulence gene expression, it remains unclear how this tripeptide is synthesized under changing environments, especially in light of the observation that Lm is auxotrophic to one of its precursors, cysteine. Here, we show that the ABC transporter TcyKLMN is a cystine/cysteine importer that supplies cysteine for glutathione synthesis, hence mediating the induction of the virulence genes. Further, we demonstrate that this transporter is negatively regulated by three metabolic regulators: CodY, CymR and CysK, which sense and respond to changing concentrations of branched chain amino acids (BCAA) and cysteine. The data indicate that under low concentrations of BCAA, TcyKLMN is up-regulated, driving the production of glutathione by supplying cysteine, thereby facilitating PrfA activation. These findings provide molecular insight into the coupling of Lm metabolism and virulence, connecting BCAA sensing to cysteine uptake and glutathione biosynthesis, as a mechanism that controls virulence gene expression. This study exemplifies how bacterial pathogens sense their intracellular environment and exploit essential metabolites as effectors of virulence.ImportanceBacterial pathogens sense the repertoire of metabolites in the mammalian niche and use this information to shift into a pathogenic state to accomplish successful infection. Glutathione is a virulence-activating signal that is synthesized by L. monocytogenes during infection of mammalian cells. In this study, we show that cysteine uptake via TcyKLMN drives glutathione synthesis and virulence gene expression. The data emphasize the intimate cross-regulation between metabolism and virulence in bacterial pathogens.

Impact of repeated co-treatment with escitalopram and aripiprazole on the schizophrenia-like behaviors and BDNF mRNA expression in the adult Sprague–Dawley rats exposed to glutathione deficit during early postnatal development of the brain

Background Preclinical and clinical studies have indicated that impaired endogenous synthesis of glutathione during early postnatal development plays a significant role in the pathophysiology of schizophrenia. Moreover, some studies have suggested that antidepressants are able to increase the activity of atypical antipsychotics which may efficiently improve the treatment of negative and cognitive symptoms of schizophrenia. Methods In the present study, we investigated the influence of repeated co-treatment with escitalopram and aripiprazole on the schizophrenia-like behavior and BDNF mRNA expression in adult rats exposed to glutathione deficit during early postnatal development. Male pups between the postnatal days p5–p16 were treated with the inhibitor of glutathione synthesis, BSO (L-buthionine-(S,R)-sulfoximine) and the dopamine uptake inhibitor, GBR 12,909 alone or in combination. Escitalopram and aripiprazole were given repeatedly for 21 days before the tests. On p90–92 rats were evaluated in the behavioral and biochemical tests. Results BSO given alone and together with GBR 12,909 induced deficits in the studied behavioral tests and decreased the expression of BDNF mRNA. Repeated aripiprazole administration at a higher dose reversed these behavioral deficits. Co-treatment with aripiprazole and an ineffective dose of escitalopram also abolished the behavioral deficits in the studied tests. Conclusion The obtained data indicated that the inhibition of glutathione synthesis in early postnatal development induced long-term deficits corresponding to schizophrenia-like behavior and decreased the BDNF mRNA expression in adult rats, and these behavioral deficits were reversed by repeated treatment with a higher dose of aripiprazole and also by co-treatment with aripiprazole and ineffective dose of escitalopram.

Induction of Glutathione Synthesis Provides Cardioprotection Regulating NO, AMPK and PPARa Signaling in Ischemic Rat Hearts

Glutathione (GSH) is essential for antioxidant defence, and its depletion is associated with tissue damage during cardiac ischemia-reperfusion (I/R). GSH is synthesized by the glutamate-cysteine ligase enzyme (GCL) from L-cysteine, which alternatively might be used for hydrogen sulfide production by cystathionine-gamma-lyase (CSE). Here, we have investigated whether in vivo treatment with L-cysteine and an inhibitor of CSE,D,L-propargylglycine (PAG), can modulate cardiac glutathione and whether this treatment can influence heart resistance to I/R in a Langendorff isolated rat hearts model. Pretreatment with PAG + L-cysteine manifested in pronounced cardioprotection, as there was complete recovery of contractile function; preserved constitutive NOS activity; and limited the production of reactive oxygen and nitrogen species in the ischemized myocardium. Cardiac GSH and GSSG levels were increased by 3.5- and 2.1-fold in PAG + L-cysteine hearts and were 3.3- and 3.6-fold higher in PAG + L-cysteine + I/R compared to I/R heart. The cardioprotective effect of PAG + L-cysteine was completely abolished by an inhibitor of GCL, DL-buthionine-(S,R)-sulfoximine. Further analysis indicated diminished fatty acid β-oxidation, increased glucose consumption and anaerobic glycolysis, and promoted OXPHOS proteins and SERCA2 in PAG + L-cysteine + I/R compared to the I/R group. PAG + L-cysteine inhibited PPARα and up-regulated AMPK signalling in the heart. Thus, induction of glutathione synthesis provided cardioprotection regulating NO, AMPK and PPARa signaling in ischemic rat hearts.

Resistance to different anthracycline chemotherapeutics elicits distinct and actionable primary metabolic dependencies in breast cancer

Chemotherapy resistance is a critical barrier in cancer treatment. Metabolic adaptations have been shown to fuel therapy resistance; however, little is known regarding the generality of these changes and whether specific therapies elicit unique metabolic alterations. Using a combination of metabolomics, transcriptomics, and functional genomics, we show that two anthracyclines, doxorubicin and epirubicin, elicit distinct primary metabolic vulnerabilities in human breast cancer cells. Doxorubicin-resistant cells rely on glutamine to drive oxidative phosphorylation and de novo glutathione synthesis, while epirubicin-resistant cells display markedly increased bioenergetic capacity and mitochondrial ATP production. The dependence on these distinct metabolic adaptations is revealed by the increased sensitivity of doxorubicin-resistant cells and tumor xenografts to buthionine sulfoximine (BSO), a drug that interferes with glutathione synthesis, compared with epirubicin-resistant counterparts that are more sensitive to the biguanide phenformin. Overall, our work reveals that metabolic adaptations can vary with therapeutics and that these metabolic dependencies can be exploited as a targeted approach to treat chemotherapy-resistant breast cancer.