Cystine Metabolism Research Articles

A new mechanism of therapeutic effect of stachydrine on heart failure by inhibiting myocardial ferroptosis

Ferroptosis is a novel form of programmed cell death caused by iron-dependent lipid peroxidation and excessive production of ROS. Its morphology is characterized by mitochondrial atrophy, increased mitochondrial membrane density, mitochondrial cristae degeneration and rupture, and unchanged nuclear morphology. Here, we investigated whether a bioactive constituent extracted from the Chinese herb Leonurus japonicus Houtt. (Yimucao), stachydrine, could improve cardiac function by inhibiting myocardial ferroptosis. We found significant morphological features of ferroptosis in a TAC-induced mouse model of heart failure, in which increased lipid peroxidation in cardiac tissue was accompanied by abnormalities in cystine metabolism as well as iron metabolism. The contractile function of adult mouse cardiomyocytes was severely reduced after the occurrence of erastin-induced ferroptosis. We found that in heart failure mice and erastin-induced cardiomyocyte ferroptosis models, stachydrine significantly improved myocardial function, improving mitochondrial morphological features of ferroptosis and associated signaling pathway alterations, including lipid peroxidation levels, cystine metabolism, and iron metabolism. The results of studies on stachydrine provides new inspirations for the treatment of cardiac ferroptosis and chronic heart failure.

Cystathionine γ-Lyase Self-Inactivates by Polysulfidation during Cystine Metabolism.

Cystathionine γ-lyase (CSE) is an enzyme responsible for the biosynthesis of cysteine from cystathionine in the final step of the transsulfuration pathway. It also has β-lyase activity toward cystine, generating cysteine persulfide (Cys-SSH). The chemical reactivity of Cys-SSH is thought to be involved in the catalytic activity of particular proteins via protein polysulfidation, the formation of -S-(S)n-H on their reactive cysteine residues. The Cys136/171 residues of CSE have been proposed to be redox-sensitive residues. Herein, we investigated whether CSE polysulfidation occurs at Cys136/171 during cystine metabolism. Transfection of wild-type CSE into COS-7 cells resulted in increased intracellular Cys-SSH production, which was significantly increased when Cys136Val or Cys136/171Val CSE mutants were transfected, instead of the wild-type enzyme. A biotin-polyethylene glycol-conjugated maleimide capture assay revealed that CSE polysulfidation occurs at Cys136 during cystine metabolism. In vitro incubation of CSE with CSE-enzymatically synthesized Cys-SSH resulted in the inhibition of Cys-SSH production. In contrast, the mutant CSEs (Cys136Val and Cys136/171Val) proved resistant to inhibition. The Cys-SSH-producing CSE activity of Cys136/171Val CSE was higher than that of the wild-type enzyme. Meanwhile, the cysteine-producing CSE activity of this mutant was equivalent to that of the wild-type enzyme. It is assumed that Cys-SSH-producing CSE activity could be auto-inactivated via the polysulfidation of the enzyme during cystine metabolism. Thus, the polysulfidation of CSE at the Cys136 residue may be an integral feature of cystine metabolism, which functions to down-regulate Cys-SSH synthesis by the enzyme.

Guanxining injection alleviates fibrosis in heart failure mice and regulates SLC7A11/GPX4 axis.

Radix et Rhizoma Salviae Miltiorrhizae (Salvia miltiorrhiza Bge., Lamiaceae, Danshen in Chinese) and Chuanxiong Rhizoma (rhizomes of Ligusticum chuanxiong Hort., Apiaceae, Chuanxiong in Chinese) both are important traditional Chinese medicine (TCM) for activating blood and eliminating stasis. Danshen-chuanxiong herb pair has been used for more than 600 years in China. Guanxinning injection (GXN) is a Chinese clinical prescription refined from aqueous extract of Danshen and Chuanxiong at the ratio of 1:1 (w/w). GXN has been mainly used in the clinical therapy of angina, heart failure (HF) and chronic kidney disease in China for almost twenty years. This study aimed to explore the role of GXN on renal fibrosis in heart failure mice and the regulation of GXN on SLC7A11/GPX4 axis. The transverse aortic constriction model was used to mimic HF accompanied by kidney fibrosis model. GXN was administrated by tail vein injection in dose of 12.0, 6.0, 3.0mL/kg, respectively. Telmisartan (6.1mg/kg, gavage) was used as a positive control drug. Cardiac ultrasound indexes of ejection fraction (EF), cardiac output (CO), left ventricle volume (LV Vol), HF biomarker of pro-B type natriuretic peptide (Pro-BNP), kidney function index of serum creatinine (Scr), kidney fibrosis index of collagen volume fraction (CVF) and connective tissue growth factor (CTGF) were evaluated and contrasted. Metabolomic method was employed to analyze the endogenous metabolites changes in kidneys. Besides, contents of catalase (CAT), xanthine oxidase (XOD), nitricoxidesynthase (NOS), glutathione peroxidase 4 (GPX4), the x(c)(-) cysteine/glutamate antiporter (SLC7A11) and ferritin heavy chain (FTH1) in kidney were quantitatively analyzed. In addition, ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to analyze the chemical composition of GXN and network pharmacology was used to predict possible mechanisms and the active ingredients of GXN. The cardiac function indexes of EF, CO and LV Vol, kidney functional indicators of Scr, the degree of kidney fibrosis indicators CVF and CTGF were all relieved to different extent for the model mice treated with GXN. 21 differential metabolites involved in redox regulation, energy metabolism, organic acid metabolism, nucleotide metabolism, etc were identified. Aspartic acid, homocysteine, glycine, and serine, methionine, purine, phenylalanine and tyrosine metabolism were found to be the core redox metabolic pathways regulated by GXN. Furthermore, GXN were found to increase CAT content, upregulate GPX4, SLC7A11 and FTH1 expression in kidney significantly. Not only that, GXN also showed good effect in down-regulating XOD and NOS contents in kidney. Besides, 35 chemical constituents were initially identified in GXN. Active ingredients of GXN-targets-related enzymes/transporters-metabolites network was established to find out that GPX4 was a core protein for GXN and the top 10 active ingredients with the most relevant to renal protective effects of GXN were rosmarinic acid, caffeic acid, ferulic acid, senkyunolide E, protocatechualdehyde, protocatechuic acid, danshensu, L-Ile, vanillic acid, salvianolic acid A. GXN could significantly maintain cardiac function and alleviate the progression of fibrosis in the kidney for HF mice, and the mechanisms of action were related to regulating redox metabolism of aspartate, glycine, serine, and cystine metabolism and SLC7A11/GPX4 axis in kidney. The cardio-renal protective effect of GXN may be attributed to multi-components like rosmarinic acid, caffeic acid, ferulic acid, senkyunolide E, protocatechualdehyde, protocatechuic acid, danshensu, L-Ile, vanillic acid, salvianolic acid A et al.

Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) detection as a rapid and convenient screening test for cystinuria

BackgroundCystinuria is an inborn congenital disorder characterised by a defective cystine metabolism resulting in the formation of cystine stones.The Brand’s test, used for diagnosis, requires dangerous substances, so has been replaced with high-performance liquid chromatography with fluorimetric detection (HPLC-FL). However, this technique requires the use of complex equipment.Infrared spectroscopy, universally used for stone analysis, recently was employed to detect insoluble cystine in urine. The aim of this study is to evaluate Infrared Spectroscopy combined with chemometric analysis as screening method to identify those patients requiring confirmation by HPLC-FL analysis. MethodsWe examined 24 h urine specimens from 57 patients. The quantitative analysis was performed by HPLC-FL. The infrared spectroscopic urine sediment analysis was performed with an ATR accessory (ATR-FTIR). Urine is centrifuged, the supernatant is discarded, and the sediment is dried on to the ATR prism surface.Statistical analysis was performed using a custom-made software developed in MATLAB environment. ResultsThe HPLC-FL determination showed a normal excretion of cystine in 49 samples and an abnormal excretion in the remaining 8 samples.The ATR-FTIR analysis combined with a statistical approach gives a sensitivity of 1.0 and a specificity of 0.82 were obtained. ConclusionsThe introduction of the ATR-FTIR technique in our clinical laboratory setting may reduce time and cost analysis for diagnosis of cystinuria.

α-ketobutyrate links alterations in cystine metabolism to glucose oxidation in mtDNA mutant cells

Pathogenic mutations in the mitochondrial genome (mtDNA) impair organellar ATP production, requiring mutant cells to activate metabolic adaptations for survival. Understanding how metabolism adapts to clinically relevant mtDNA mutations may provide insight into cellular strategies for metabolic flexibility. In this study, we use 13C isotope tracing and metabolic flux analysis to investigate central carbon and amino acid metabolic reprogramming in isogenic cells containing mtDNA mutations. We identify alterations in glutamine and cystine transport which indirectly regulate mitochondrial metabolism and electron transport chain function. Metabolism of cystine can promote glucose oxidation through the transsulfuration pathway and the production of α-ketobutyrate. Intriguingly, activating or inhibiting α-ketobutyrate production is sufficient to modulate both glucose oxidation and mitochondrial respiration in mtDNA mutant cells. Thus, cystine-stimulated transsulfuration serves as an adaptive mechanism linking glucose oxidation and amino acid metabolism in the setting of mtDNA mutations.

Cystine transporter regulation of pentose phosphate pathway dependency and disulfide stress exposes a targetable metabolic vulnerability in cancer.

SLC7A11-mediated cystine uptake is critical for maintaining redox balance and cell survival. Here, we show that this comes at a significant cost for cancer cells with high SLC7A11 expression. Actively importing cystine is potentially toxic due to its low solubility, forcing SLC7A11-high cancer cells to constitutively reduce cystine to the more soluble cysteine. This presents a substantial drain on the cellular NADPH pool and renders such cells dependent on the pentose phosphate pathway (PPP). Limiting glucose supply to SLC7A11-high cancer cells results in marked accumulation of intracellular cystine, redox system collapse, and rapid cell death, which can be rescued by treatments that prevent disulfide accumulation. We further show that glucose transporter (GLUT) inhibitors selectively kill SLC7A11-high cancer cells and suppress SLC7A11-high tumor growth. Our results identify a coupling between SLC7A11-associated cystine metabolism and the PPP, and uncover an accompanying metabolic vulnerability for therapeutic targeting in SLC7A11-high cancers.

Cysteine and Cystine Depletion Targets Leukemia Stem Cells

The goal of this project was to identify and target metabolic vulnerabilities of leukemia stem cells (LSCs) to improve therapeutic outcomes for patients with AML. We have previously shown that primary human LSCs reside in a unique metabolic condition characterized by a relatively low oxidative state (termed “ROS-low”) and increased levels of glutathione (Lagadinou et al. Cell Stem Cell, 2013). Cells in this condition are highly dependent on oxidative phosphorylation for survival, in striking contrast to many tumor cells which often rely on glycolysis; indicating that LSCs are governed by distinct metabolic properties. To further elucidate key metabolic properties of LSCs, we measured differences in the global metabolome of ROS-Low LSCs in comparison to ROS-high AML blasts. Our preliminary data demonstrated that ROS-low LSCs have higher levels of amino acids and require amino acid catabolism for survival. We hypothesized that certain individual amino acids may be more important for LSC survival. If true, then targeting specific amino acids may be an avenue towards improved AML therapy.To determine if any individual amino acid is essential for LSC survival, we analyzed AML cells from five patients that were systematically cultured in media lacking one of the twenty amino acids. Cysteine depletion was consistently the most cytotoxic, showing decreased cell viability and colony forming potential of LSCs. We next determined the effect of an engineered human enzyme that selectively degrades cysteine and cystine (AEB3103, Aeglea BioTherapeutics, Inc.) on LSC viability and colony forming potential. We found that AEB3103 treatment decreased viability of LSCs in all AML specimens tested and significantly decreased colony formation (p<0.05). Importantly, AEB3103 treatment did not decrease the frequency or colony forming ability of HSCs in normal mobilized peripheral blood or normal bone marrow.To determine how AEB3103 decreased LSC viability we investigated the metabolic changes that occur upon AEB3103 treatment by mass spectroscopy. We found that AEB3103 treatment decreased the abundance of metabolites involved in glutathione synthesis (cysteine, cystine, glutathione, taurine, and Cys-Gly) in ROS-low LSCs. Tracing of cysteine13C315N in LSCs demonstrated that all detectable heavy cysteine was metabolized to cystine and glutathione. Furthermore, pretreatment with cell permeable glutathione rescued cell viability and colony-forming potential upon AEB3103 treatment. These data suggest that modulation of glutathione is central to the mechanism by which AEB3103 kills LSCs. Glutathione is a well-characterized antioxidant, therefore, we measured ROS levels and the expression of genes known to be expressed upon ROS induction in LSCs. Surprisingly, we did not observe changes in ROS induction or the expression of ROS induced genes upon AEB3103 treatment.Next, we interrogated cellular functions of glutathione that are independent from the role of glutathione as an antioxidant. Glutathione has previously been shown to mediate electron transport chain complex II function via posttranslational glutathione modification, glutathionylation. Therefore, we hypothesized that decreased glutathione levels upon AEB3103 treatment could result in decreased complex II activity and therefore decreased levels of OXPHOS and ATP production. To test this hypothesis, we measured complex II activity, complex II glutathionylation, oxidative phosphorylation, and ATP levels upon AEB3103 treatment. We observed AEB3103 treatment significantly decreased glutathionylation, complex II activity, oxidative phosphorylation, and ATP levels in AML cells (p<0.05). Furthermore, pretreatment with cell permeable glutathione completely reversed the metabolic consequences of AEB3103 treatment. Overall, our data demonstrates that cysteine depletion selectively targets LSCs by decreasing glutathione levels resulting in decreased electron transport chain complex II activity. Furthermore, targeting cysteine and cystine metabolism may be a promising approach to eradicate LSCs. DisclosuresPollyea:AbbVie: Consultancy, Research Funding; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy; Curis: Membership on an entity's Board of Directors or advisory committees.

Contribution of Amino Acid Metabolism to Hematologic Malignancies

Tumor cells rewire metabolic pathways to meet the high metabolic demands of proliferation, frequently developing auxotrophy to specific amino acid(s) (AAs) required to satisfy protein biosynthesis. Thus specific metabolic inhibitors or AA-depleting enzymes have been developed and tested as cancer therapeutics. For example, depletion of asparagine by bacterial L-asparaginase (ASNase) has proven efficacious against hematologic malignancies, especially leukemia and lymphoma, by starving tumors lacking asparagine synthetase (ASNS). We and others have reported that the glutaminase (GLS) activity of ASNase is required for anticancer activity against ASNS-positive leukemia cell types in vitro.1 In vivo, we have found that durable response to ASNase in pre-clinical models of leukemia also requires glutaminase activity, even against ASNS-negative leukemia models; a glutaminase-deficient mutant of ASNase yielded subsequent leukemia recurrence. We speculate that the underlying anti-leukemia mechanism mediated by ASNase glutaminase activity involves a deeper depletion of asparagine within the tumor microenvironment, since ASNS in nearby cells (adipocytes, mesenchymal stromal cells, etc.) can use glutamine as a precursor for asparagine synthesis. Nevertheless, since L-glutamine depletion is thought to cause the significant side effects of ASNase, enzyme variants with reduced glutaminase coactivity are being developed and tested. Another viable therapeutic strategy involving glutamine starvation via GLS inhibitor has shown significant pre-clinical activity in acute myeloid leukemia (AML) and multiple myeloma (MM) models; this approach is synergistic with hypomethylating agents and BCL2 inhibitors in AML, and with proteasome inhibitors in MM. Recent findings highlight the switch to glutamine metabolism as a metabolic dependency of tyrosine kinase-driven AML, and targeting GLS in conjunction with tyrosine kinase inhibition has been proposed.2 Targeting arginine metabolism has been shown to be another viable therapeutic strategy. Arginine (ARG) depletion using pegylated arginine deaminase (ADI-PEG 20) or pegylated arginase (PEG-ARGase), the 2 critical enzymes of the ARG metabolism/urea cycle, reduced leukemia tumor burden in AML models characterized by low arginosuccinate synthetase (ASS) and high uptake of ARG. However, recently reported Phase I/II clinical trials of recombinant PEG-arginase and of ADI-PEG 20 showed minimal efficacy in relapsed/refractory AML and in solid tumors despite efficient depletion of arginine and low ASS1 expression in tumors, indicating that depletion of arginine alone is insufficient for clinical activity. As a final example of AA metabolic pathways targeted in the treatment of hematologic malignancies, exogenous L-cysteine is required for the synthesis of glutathione for antioxidant cellular defense. In pre-clinical studies, multiple malignancy subtypes were sensitive to cysteine and cystine degradation by an engineered human cyst(e)inase enzyme, including AML, acute lymphocytic leukemia, poor-risk chronic lymphocytic leukemia (CLL), and MM.3 In all therapeutic strategies targeting AA metabolism, the tumor microenvironment may contribute to resistance. For example, bone marrow stromal cells efficiently import cystine, convert it to cysteine, and transport it to CLL cells, facilitating leukemia chemoresistance. Mesenchymal stromal cells and bone marrow adipocytes secrete asparagine and glutamine, respectively, and protect leukemia cells from ASNase cytotoxicity. Recent insights into the immune tumor microenvironment highlight interplay between tumor, AAs, and immune cell functions. Some AAs, such as arginine and glutamine, are essential nutrients for immune cell proliferation and metabolism; excessive tumor consumption of glutamine, or secretion of arginase by myeloid-derived suppressor cells or AML blasts, may deprive immune cells, impair T cell proliferation, and induce immunosuppressive phenotypes. GLS inhibitors that block glutamine consumption and arginase inhibitors that increase plasma arginine, increase availability of their respective target nutrients for immune cells and are, therefore, being explored in ongoing clinical trials as monotherapies and in combination with anti-PD1 blockade.Chan WK, Lorenzi PL, Anishkin A, et al. The glutaminase activity of L-asparaginase is not required for anticancer activity against ASNS-negative cells. Blood. 2014;123:3596-3606.Gallipoli P, Giotopoulos G, Tzelepis K, et al. Glutaminolysis is a metabolic dependency in FLT3(ITD) acute myeloid leukemia unmasked by FLT3 tyrosine kinase inhibition. Blood. 2018;131:1639-1653.Zhang W, Trachootham D, Liu J, et al. Stromal control of cystine metabolism promotes cancer cell survival in chronic lymphocytic leukaemia. Nat Cell Biol. 2012;14:276-286. DisclosuresKonopleva:Stemline Therapeutics: Research Funding. Lorenzi:Erytech Pharma: Consultancy; NIH: Patents & Royalties.

Abstract IA06: Molecular mechanisms of lung cancer development: Between metabolic reprogramming and genomic instability in the field of cancerization

Abstract Airway epithelial cells are prone to assault by risk factors and considered to be the primary cell type involved in the field of cancerization. To investigate risk-associated changes in the bronchial epithelium proteome that may offer new insights into molecular pathogenesis of lung cancer, proteins were identified in the airway epithelial cells of bronchial brushings specimens from risk-stratified individuals by shotgun proteomics. Differential expression of selected proteins was validated by parallel reaction monitoring mass spectrometry in an independent set of individual bronchial brushings. We identified over 300 proteins with a significant trend in expression. Pathway analysis revealed enrichment of carbohydrate metabolic enzymes in high-risk individuals. Glucose consumption and lactate production were increased in human bronchial epithelial BEAS2B cells treated with cigarette smoke condensate for seven months. Increased lipid biosynthetic capacity and net reductive carboxylation were revealed by metabolic flux analyses of [U-13C5] glutamine in this in vitro model, suggesting profound metabolic reprogramming in the airway epithelium of high-risk individuals. These results also pointed towards a fundamental role of glutamine and cysteine metabolism in lung cancer progression. Two amino acid transporters, specifically SLC1A5 and SLC7A11, captured our attention for their high level of expression in lung cancer. These transporters are associated with worse survival in lung cancer. A small-molecular inhibitor of SLC1A5 prevents tumor progression in nude mice, and a glutaminase inhibitor CB-839 exhibits radiosensitization to lung cancer cells, an effect that may be mediated by decreased GSH production. Taken together, these studies provide a rationale for new chemopreventive strategies, selection of patients for surveillance programs, and determining how metabolic reprogramming may contribute genomic instability a requirement to cancer development. Further investigation into how glutamine and cystine metabolism contribute to lung cancer development and progression may lead to novel early-interception strategies in lung cancer. This work is supported by NCI CA186145, CA152662, and DoD W81XWH-11-2-0161. Citation Format: Jamshed Rahman, Xiangming Ji, Chirayu Patel, Jamey Young, Pierre P. Massion. Molecular mechanisms of lung cancer development: Between metabolic reprogramming and genomic instability in the field of cancerization [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr IA06.

A human induced pluripotent stem cell-based in vitro assay predicts developmental toxicity through a retinoic acid receptor-mediated pathway for a series of related retinoid analogues

The relative developmental toxicity potency of a series of retinoid analogues was evaluated using a human induced pluripotent stem (iPS) cell assay that measures changes in the biomarkers ornithine and cystine. Analogue potency was predicted, based on the assay endpoint of the ornithine/cystine (o/c) ratio, to be all-trans-retinoic acid>TTNPB>13-cis-retinoic acid≈9-cis-retinoic acid>acitretin>etretinate>retinol. These rankings correlate with in vivo data and demonstrate successful application of the assay to rank a series of related toxic and non-toxic compounds. The retinoic acid receptor α (RARα)-selective antagonist Ro 41–5253 inhibited the cystine perturbation caused by all-trans-retinoic acid, TTNPB, 13-cis-retinoic acid, 9-cis-retinoic acid, and acitretin. Ornithine was altered independent of RARα in all retinoids except acitretin. These results suggest a role for an RARα-mediated mechanism in retinoid-induced developmental toxicity through altered cystine metabolism.

Abstract IA28: Cancer epigenetic and metabolic mechanisms controlling immunotherapy and chemotherapy

Abstract We have studied the cross-talk between immune cell subsets and tumor/stem cells in the human tumor microenvironment and its impact on tumor immunity and therapy. Our prior research efforts demonstrate that the tumor microenvironment is comprised of immune cells that have been reprogrammed by active tumor-mediated processes to defeat tumor-specific immunity and promote tumor growth in a highly effective manner. These studies have helped define the nature of immune responses in the tumor microenvironment; provide original insights into designing immune therapies to target the immune suppressive mechanisms, including Tregs and inhibitory B7 family members (e.g. PD-L1/PD-1); and treat patients with cancer. Using several human cancers as our models, we demonstrate that Th22 cells and myeloid derived suppressor cells (MDSCs) and macrophages regulate tumor EMT and stemness via epigenetic mechanisms. Furthermore, we found that histone modification and DNA methylation repress the tumor production of Th1-type chemokines and subsequently determine effector T cell trafficking to the tumor microenvironment—affecting tumor progression, therapeutic efficacy of checkpoint blockade and adoptive T cell transfusion, and patient outcome. Thus, epigenetic mechanisms determine tumor immune phenotype (hot vs cold) and tumor biological phenotype (EMT/stem-like vs non EMT/stem like) and shape immunotherapy response. In addition to immunotherapy, we found that effector T cells affect chemoresistance. CD8+ T cells abolish ovarian cancer cisplatin-resistance by altering glutathione and cystine metabolism in fibroblasts. CD8+ T cell-derived IFNγ controls fibroblast glutathione and cysteine through up-regulation of gamma-glutamyl transpeptidase and transcriptional repression of system xc− cystine and glutamate antiporter via the JAK/STAT1 pathway. Thus, effector T cells play a role in cancer chemoresistance. Selective epigenetic reprogramming may alter T cell landscape in cancer and enhance clinical efficacy of cancer immunotherapy and chemotherapy. Citation Format: Weiping Zou. Cancer epigenetic and metabolic mechanisms controlling immunotherapy and chemotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr IA28.

Effector T Cells Abrogate Stroma-Mediated Chemoresistance in Ovarian Cancer

Effector Tcells and fibroblasts are major components in the tumor microenvironment. The means through which these cellular interactions affect chemoresistance is unclear. Here, we show that fibroblasts diminish nuclear accumulation of platinum in ovarian cancer cells, resulting in resistance to platinum-based chemotherapy. We demonstrate that glutathione and cysteine released by fibroblasts contribute to this resistance. CD8(+) Tcells abolish the resistance by altering glutathione and cystine metabolism in fibroblasts. CD8(+) T-cell-derived interferon (IFN)γ controls fibroblast glutathione and cysteine through upregulation of gamma-glutamyltransferases and transcriptional repression of system xc(-) cystine and glutamate antiporter via the JAK/STAT1 pathway. The presence of stromal fibroblasts and CD8(+) Tcells is negatively and positively associated with ovarian cancer patient survival, respectively. Thus, our work uncovers a mode of action for effector Tcells: they abrogate stromal-mediated chemoresistance. Capitalizing upon the interplay between chemotherapy and immunotherapy holds high potential for cancer treatment.

Ferroptosis: A missing puzzle piece in the p53 blueprint?

ABSTRACTRecent evidence indicates that canonical functions of p53 (i.e., apoptosis and growth arrest) are dispensable for p53-mediated tumor suppression. We have uncovered a novel function of p53 that contributes to tumor suppression through regulation of cystine metabolism, reactive oxygen species responses, and ferroptosis. The p53-mediated ferroptotic response via SLC7A11 denotes an extra layer of defense against tumorigenesis in conjunction with other p53 functions.

Çocukluk Çağında Sistinozise Bağlı Posterior Reversibl Ensefalopati Sendromu

Cystinosis is an inherited autosomal recessive disorder, which the defect involves cystinosin, the membrane transport protein playing role in amino acid cystine metabolism. Posterior reversible encephalopathy syndrome (PRES) is a clinic-radiographic entity of heterogeneous etiologies that are grouped together because of similar findings on neuro-imaging and associated symptom complex of headache, vision loss, altered mentation, and seizures. We describe a 10-year-old male patient with nephropathic cystinosis being treated with peritoneal dialysis was admitted to emergency service after generalized tonic clonic seizures at home. Initial brain diffusion weighted magnetic resonance imaging indicated that particularly bilateral parieto-occipital regions revealed restricted diffusion areas and cytotoxic edema elicited neural parenchymal insult. In our case, hypertensive crisis related to ineffective peritoneal dialysis and brain tissue damage due to cystinosis may be able to provide a basis for PRES. Appropriate and prompt treatment is essential to protect the patients from harmful effects of irreversible brain injury.

Physiological Roles and Adverse Effects of the Two Cystine Importers of Escherichia coli.

When cystine is added to Escherichia coli, the bacterium becomes remarkably sensitive to hydrogen peroxide. This effect is due to enlarged intracellular pools of cysteine, which can drive Fenton chemistry. Genetic analysis linked the sensitivity to YdjN, a secondary transporter that along with the FliY-YecSC ABC system is responsible for cystine uptake. FliY-YecSC has a nanomolar Km and is essential for import of trace cystine, whereas YdjN has a micromolar Km and is the predominant importer when cystine is more abundant. Oddly, both systems are strongly induced by the CysB response to sulfur scarcity. The FliY-YecSC system can import a variety of biomolecules, including diaminopimelate; it is therefore vulnerable to competitive inhibition, presumably warranting YdjN induction under low-sulfur conditions. But the consequence is that if micromolar cystine then becomes available, the abundant YdjN massively overimports it, at >30 times the total sulfur demand of the cell. The imported cystine is rapidly reduced to cysteine in a glutathione-dependent process. This action avoids the hazard of disulfide stress, but it precludes feedback inhibition of YdjN by cystine. We conjecture that YdjN possesses no cysteine allosteric site because the isostructural amino acid serine might inappropriately bind in its place. Instead, the cell partially resolves the overaccumulation of cysteine by immediately excreting it, completing a futile import/reduction/export cycle that consumes a large amount of cellular energy. These unique, wasteful, and dangerous features of cystine metabolism are reproduced by other bacteria. We propose to rename ydjN as tcyP and fliY-yecSC as tcyJLN. In general, intracellular metabolite pools are kept at steady, nontoxic levels by a sophisticated combination of transcriptional and allosteric controls. Surprisingly, in E. coli allosteric control is utterly absent from the primary importer of cystine. This flaw allows massive overimport of cystine, which causes acute vulnerability to oxidative stress and is remedied only by wasteful cysteine efflux. The lack of import control may be rationalized by the unusual properties of cysteine itself. This phenomenon justifies the existence of countervailing cysteine export systems, whose purpose is otherwise hard to understand. It also highlights an unexpected link between sulfur metabolism and oxidative damage. Although this investigation focused upon E. coli, experiments confirmed that similar phenomena occur in other species.

Ferroptosis as a p53-mediated activity during tumour suppression

Although p53-mediated cell-cycle arrest, senescence and apoptosis serve as critical barriers to cancer development, emerging evidence suggests that the metabolic activities of p53 are also important. Here we show that p53 inhibits cystine uptake and sensitizes cells to ferroptosis, a non-apoptotic form of cell death, by repressing expression of SLC7A11, a key component of the cystine/glutamate antiporter. Notably, p53(3KR), an acetylation-defective mutant that fails to induce cell-cycle arrest, senescence and apoptosis, fully retains the ability to regulate SLC7A11 expression and induce ferroptosis upon reactive oxygen species (ROS)-induced stress. Analysis of mutant mice shows that these non-canonical p53 activities contribute to embryonic development and the lethality associated with loss of Mdm2. Moreover, SLC7A11 is highly expressed in human tumours, and its overexpression inhibits ROS-induced ferroptosis and abrogates p53(3KR)-mediated tumour growth suppression in xenograft models. Our findings uncover a new mode of tumour suppression based on p53 regulation of cystine metabolism, ROS responses and ferroptosis.

Novel Cystine Ester Mimics for the Treatment of Cystinuria-induced Urolithiasis in a Knockout Mouse Model

To assess the effectiveness of l-cystine dimethyl ester (CDME), an inhibitor of cystine crystal growth, for the treatment of cystine urolithiasis in an Slc3a1 knockout mouse model of cystinuria. CDME (200 μg per mouse) or water was delivered by gavage daily for 4 weeks. Higher doses by gavage or in the water supply were administered to assess organ toxicity. Urinary amino acids and cystine stones were analyzed to assess drug efficacy using several analytical methods. Treatment with CDME led to a significant decrease in stone size compared with that of the water group (P = .0002), but the number of stones was greater (P = .005). The change in stone size distribution between the 2 groups was evident by micro computed tomography. Overall, cystine excretion in urine was the same between the 2 groups (P = .23), indicating that CDME did not interfere with cystine metabolism. Scanning electron microscopy analysis of cystine stones from the CDME group demonstrated a change in crystal habit, with numerous small crystals. l-cysteine methyl ester was detected by ultra-performance liquid chromatography-mass spectrometer in stones from the CDME group only, indicating that a CDME metabolite was incorporated into the crystal structure. No pathologic changes were observed at the doses tested. These data demonstrate that CDME promotes formation of small stones but does not prevent stone formation, consistent with the hypothesis that CDME inhibits cystine crystal growth. Combined with the lack of observed adverse effects, our findings support the use of CDME as a viable treatment for cystine urolithiasis.

Abstract 5395: High-throughput cysteine concentration determination by specific fluorescence sensors in cell culture medium

Abstract Cysteine is a sulfur-containing amino acid that can be biosynthesized in human (1). It is not stable in free solution and can be auto-oxidized to cystine. Cystine is more stable and can travel freely through the blood (2). After intracellular accumulation by different transporters, especially Xc-transporter (3, 4), cystine will be rapidly reduced to cysteine which can then be used to synthesis protein or glutathione (GSH) synthesis5. Cysteine may also release from the cells by Alanine-Serine-Cysteine transporter (3). The accumulation of cysteine can feed other cells with low expression or mutation of Xc-transporter. It has been reported that stromal cells can promote cancer cell survival by the control of cystine metabolism to cysteine(5). Cysteine concentration distribution now may affect cell survival and cell-cell signaling (2, 5). It is still hard to study the total cysteine concentration in different culture system or in vivo in a high-through put way. Traditional LC-MS methods are not only cumbersome and time consuming, it also hard to process different samples in parallel. Here we report a series of novel fluorescent sensors which have great selectivity and sensitivity towards cysteine. Using these sensor compounds, we can monitor cysteine level in situ in cell culture system. Upon screening different culture medium, we found cysteine concentrations are different with cell type and culture time. We envision our fluorescent probes can be used to elucidate cysteine's function in cell survival as well as signal transduction between cells. 1. Lehmann, A.; Hagberg, H.; Orwar, O.; Sandberg, M., Cysteine sulphinate and cysteate: mediators of cysteine toxicity in the neonatal rat brain? Eur J Neurosci 1993, 5, (10), 1398-412. 2. Li, L.; Lim, J.; Jacobs, M. D.; Kistler, J.; Donaldson, P. J., Regional differences in cystine accumulation point to a sutural delivery pathway to the lens core. Invest Ophthalmol Vis Sci 2007, 48, (3), 1253-60. 3. Scopelliti, A. J.; Ryan, R. M.; Vandenberg, R. J., Molecular determinants for functional differences between alanine-serine-cysteine transporter 1 and other glutamate transporter family members. J Biol Chem 288, (12), 8250-7. 4. Huang, Y.; Dai, Z.; Barbacioru, C.; Sadee, W., Cystine-glutamate transporter SLC7A11 in cancer chemosensitivity and chemoresistance. Cancer Res 2005, 65, (16), 7446-54. 5. Zhang, W.; Trachootham, D.; Liu, J.; Chen, G.; Pelicano, H.; Garcia-Prieto, C.; Lu, W.; Burger, J. A.; Croce, C. M.; Plunkett, W.; Keating, M. J.; Huang, P., Stromal control of cystine metabolism promotes cancer cell survival in chronic lymphocytic leukaemia. Nat Cell Biol 14, (3), 276-86. Citation Format: Hong Guan. High-throughput cysteine concentration determination by specific fluorescence sensors in cell culture medium. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5395. doi:10.1158/1538-7445.AM2014-5395

P39 Protoporphyrin modification by biological thiols and hydrogen sulfide

Sulfur metabolism furnishes cells with important metabolic compounds, among which glutathione (GSH), cysteine (Cys) and hydrogen sulfide (H2S) [1] , [2] . GSH is important in protecting cells against oxidative injuries and its intracellular redox couple varies within cells depending on their biological status. Cys is a precursor of several important macromolecules and participates in redox-based signaling via post-translational modifications (PTMs). H2S is a small “gaseous” molecule, which exerts important signaling functions in the vascular and neuronal systems and might signal through S-sulfhydration and HSNO formation [3] . As a result of their crucial role in cellular physiology and the reactivity of their SH group, sulfur metabolism is tightly controlled in response to environmental changes and defects affecting sulfur metabolic compounds and redox-based signaling are often associated with severe pathologies [e.g. [4] , [5] , [6] , [7] ]. In here we pursue the efforts in understanding the biology of sulfur compounds at the interface of chemistry and biology. We thus seek to explore in vitro the various effects elicited by RSH/H2S under normal versus pathological conditions. To “mimic” pathological conditions, we employed trace elements whose acute toxicity is well-known and are often used as tumor biomarkers, e.g. iron or copper. As model we chose a heme-containing constituent of the innate immune system, catalase (Cat) that has been reported to be inactivated due to sulfur compounds homeostasis deregulation, e.g. in animals bearing tumors due to aberrancy in cystine metabolism or in animal models of chronic hyperhomocysteinemia [8] , [9] , [10] , [11] . Our results indicate for the first time the peculiar formation of sulfcatalase in the presence of RSH alone. Furthermore, the observed IC50 greatly decreased in the presence of trace elements and felt within the physiological thiol concentrations, elucidating the aforementioned inactivation of Cat due to sulfur compounds homeostasis deregulation. In contrast, in the presence of H2S, we observed a new PTM of Cat. This latter is responsible for Cat activation in the presence of trace elements and physiological H2S concentrations.

Stromal control of cystine metabolism promotes cancer cell survival in chronic lymphocytic leukaemia

Tissue stromal cells interact with leukemia cells and profoundly affect their viability and drug sensitivity. Here we show a biochemical mechanism by which bone marrow stromal cells modulate the redox status of chronic lymphocytic leukemia (CLL) cells and promote cellular survival and drug resistance. Primary CLL cells from patients exhibit limited ability to transport cystine for glutathione (GSH) synthesis due to a low expression of Xc- transporter, while bone marrow stromal cells effectively import cystine and convert it to cysteine, which is then released into the microenvironment for uptake by CLL cells to promote GSH synthesis. The elevated GSH enhances leukemia cell survival and protects them from drug-induced cytotoxicity. Furthermore, disabling this protective mechanism significantly sensitizes CLL cells to drug treatment in stromal environment. This stromal-leukemia interaction is critical for CLL cell survival and represents a key biochemical pathway for effectively targeting leukemia cells to overcome drug resistance in vivo.