3-hydroxy-L-kynurenamine is an immunomodulatory biogenic amine.

Tryptophan catabolism is a major metabolic pathway utilized by several professional and non-professional antigen presenting cells to maintain immunological tolerance. Here we report that 3-hydroxy-L-kynurenamine (3-HKA) is a biogenic amine produced via an alternative pathway of tryptophan metabolism. In vitro, 3-HKA has an anti-inflammatory profile by inhibiting the IFN-γ mediated STAT1/NF-κΒ pathway in both mouse and human dendritic cells (DCs) with a consequent decrease in the release of pro-inflammatory chemokines and cytokines, most notably TNF, IL-6, and IL12p70. 3-HKA has protective effects in an experimental mouse model of psoriasis by decreasing skin thickness, erythema, scaling and fissuring, reducing TNF, IL-1ß, IFN-γ, and IL-17 production, and inhibiting generation of effector CD8+ T cells. Similarly, in a mouse model of nephrotoxic nephritis, besides reducing inflammatory cytokines, 3-HKA improves proteinuria and serum urea nitrogen, overall ameliorating immune-mediated glomerulonephritis and renal dysfunction. Overall, we propose that this biogenic amine is a crucial component of tryptophan-mediated immune tolerance.

NF-κB Regulation of LRH-1 and ABCG5/8 Potentiates Phytosterol Role in the Pathogenesis of Parenteral Nutrition-Associated Cholestasis.

BACKGROUND AND AIMS: Chronically administered parenteral nutrition (PN) in patients with intestinal failure carries the risk for developing PN-associated cholestasis (PNAC). We have demonstrated that farnesoid X receptor (FXR) and liver X receptor (LXR), proinflammatory interleukin-1 beta (IL-1ß), and infused phytosterols are important in murine PNAC pathogenesis. In this study we examined the role of nuclear receptor liver receptor homolog 1 (LRH-1) and phytosterols in PNAC. APPROACH AND RESULTS: In a C57BL/6 PNAC mouse model (dextran sulfate sodium [DSS] pretreatment followed by 14 days of PN; DSS-PN), hepatic nuclear receptor subfamily 5, group A, member 2/LRH-1 mRNA, LRH-1 protein expression, and binding of LRH-1 at the Abcg5/8 and Cyp7a1 promoter was reduced. Interleukin-1 receptor-deficient mice (Il-1r-/- /DSS-PN) were protected from PNAC and had significantly increased hepatic mRNA and protein expression of LRH-1. NF-κB activation and binding to the LRH-1 promoter were increased in DSS-PN PNAC mice and normalized in Il-1r-/- /DSS-PN mice. Knockdown of NF-κB in IL-1ß-exposed HepG2 cells increased expression of LRH-1 and ABCG5. Treatment of HepG2 cells and primary mouse hepatocytes with an LRH-1 inverse agonist, ML179, significantly reduced mRNA expression of FXR targets ATP binding cassette subfamily C member 2/multidrug resistance associated protein 2 (ABCC2/MRP2), nuclear receptor subfamily 0, groupB, member 2/small heterodimer partner (NR0B2/SHP), and ATP binding cassette subfamily B member 11/bile salt export pump (ABCB11/BSEP). Co-incubation with phytosterols further reduced expression of these genes. Similar results were obtained by suppressing the LRH-1 targets ABCG5/8 by treatment with small interfering RNA, IL-1ß, or LXR antagonist GSK2033. Liquid chromatography-mass spectrometry and chromatin immunoprecipitation experiments in HepG2 cells showed that ATP binding cassette subfamily G member 5/8 (ABCG5/8) suppression by GSK2033 increased the accumulation of phytosterols and reduced binding of FXR to the SHP promoter. Finally, treatment with LRH-1 agonist, dilauroyl phosphatidylcholine (DLPC) protected DSS-PN mice from PNAC. CONCLUSIONS: This study suggests that NF-κB regulation of LRH-1 and downstream genes may affect phytosterol-mediated antagonism of FXR signaling in the pathogenesis of PNAC. LRH-1 could be a potential therapeutic target for PNAC.

Nicotinamide Metabolism Mediates Resistance to Venetoclax in Relapsed Acute Myeloid Leukemia Stem Cells.

We previously demonstrated that leukemia stem cells (LSCs) in de novo acute myeloid leukemia (AML) patients are selectively reliant on amino acid metabolism and that treatment with the combination of venetoclax and azacitidine (ven/aza) inhibits amino acid metabolism, leading to cell death. In contrast, ven/aza fails to eradicate LSCs in relapsed/refractory (R/R) patients, suggesting altered metabolic properties. Detailed metabolomic analysis revealed elevated nicotinamide metabolism in relapsed LSCs, which activates both amino acid metabolism and fatty acid oxidation to drive OXPHOS, thereby providing a means for LSCs to circumvent the cytotoxic effects of ven/aza therapy. Genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide metabolism, demonstrated selective eradication of R/R LSCs while sparing normal hematopoietic stem/progenitor cells. Altogether, these findings demonstrate that elevated nicotinamide metabolism is both the mechanistic basis for ven/aza resistance and a metabolic vulnerability of R/R LSCs.

Targeting Glutamine Metabolism and Redox State for Leukemia Therapy.

PURPOSE: Acute myeloid leukemia (AML) is a hematologic malignancy characterized by the accumulation of immature myeloid precursor cells. AML is poorly responsive to conventional chemotherapy and a diagnosis of AML is usually fatal. More effective and less toxic forms of therapy are desperately needed. AML cells are known to be highly dependent on the amino acid glutamine for their survival. These studies were directed at determining the effects of glutaminase inhibition on metabolism in AML and identifying general weaknesses that can be exploited therapeutically. EXPERIMENTAL DESIGN: AML cancer cell lines, primary AML cells, and mouse models of AML and acute lymphoblastic leukemia (ALL) were utilized. RESULTS: We show that blocking glutamine metabolism through the use of a glutaminase inhibitor (CB-839) significantly impairs antioxidant glutathione production in multiple types of AML, resulting in accretion of mitochondrial reactive oxygen species (mitoROS) and apoptotic cell death. Moreover, glutaminase inhibition makes AML cells susceptible to adjuvant drugs that further perturb mitochondrial redox state, such as arsenic trioxide (ATO) and homoharringtonine (HHT). Indeed, the combination of ATO or HHT with CB-839 exacerbates mitoROS and apoptosis, and leads to more complete cell death in AML cell lines, primary AML patient samples, and in vivo using mouse models of AML. In addition, these redox-targeted combination therapies are effective in eradicating ALL cells in vitro and in vivo. CONCLUSIONS: Targeting glutamine metabolism in combination with drugs that perturb mitochondrial redox state represents an effective and potentially widely applicable therapeutic strategy for treating multiple types of leukemia.

Urea Cycle Sustains Cellular Energetics upon EGFR Inhibition in EGFR-Mutant NSCLC.

Mutations in oncogenes and tumor suppressor genes engender unique metabolic phenotypes crucial to the survival of tumor cells. EGFR signaling has been linked to the rewiring of tumor metabolism in non-small cell lung cancer (NSCLC). We have integrated the use of a functional genomics screen and metabolomics to identify metabolic vulnerabilities induced by EGFR inhibition. These studies reveal that following EGFR inhibition, EGFR-driven NSCLC cells become dependent on the urea cycle and, in particular, the urea cycle enzyme CPS1. Combining knockdown of CPS1 with EGFR inhibition further reduces cell proliferation and impedes cell-cycle progression. Profiling of the metabolome demonstrates that suppression of CPS1 potentiates the effects of EGFR inhibition on central carbon metabolism, pyrimidine biosynthesis, and arginine metabolism, coinciding with reduced glycolysis and mitochondrial respiration. We show that EGFR inhibition and CPS1 knockdown lead to a decrease in arginine levels and pyrimidine derivatives, and the addition of exogenous pyrimidines partially rescues the impairment in cell growth. Finally, we show that high expression of CPS1 in lung adenocarcinomas correlated with worse patient prognosis in publicly available databases. These data collectively reveal that NSCLC cells have a greater dependency on the urea cycle to sustain central carbon metabolism, pyrimidine biosynthesis, and arginine metabolism to meet cellular energetics upon inhibition of EGFR. IMPLICATIONS: Our results reveal that the urea cycle may be a novel metabolic vulnerability in the context of EGFR inhibition, providing an opportunity to develop rational combination therapies with EGFR inhibitors for the treatment of EGFR-driven NSCLC.

Mitochondrial redox sensing by the kinase ATM maintains cellular antioxidant capacity.

Mitochondria are integral to cellular energy metabolism and ATP production and are involved in regulating many cellular processes. Mitochondria produce reactive oxygen species (ROS), which not only can damage cellular components but also participate in signal transduction. The kinase ATM, which is mutated in the neurodegenerative, autosomal recessive disease ataxia-telangiectasia (A-T), is a key player in the nuclear DNA damage response. However, ATM also performs a redox-sensing function mediated through formation of ROS-dependent disulfide-linked dimers. We found that mitochondria-derived hydrogen peroxide promoted ATM dimerization. In HeLa cells, ATM dimers were localized to the nucleus and inhibited by the redox regulatory protein thioredoxin 1 (TRX1), suggesting the existence of a ROS-mediated, stress-signaling relay from mitochondria to the nucleus. ATM dimer formation did not affect its association with chromatin in the absence or presence of nuclear DNA damage, consistent with the separation of its redox and DNA damage signaling functions. Comparative analysis of U2OS cells expressing either wild-type ATM or the redox sensing-deficient C2991L mutant revealed that one function of ATM redox sensing is to promote glucose flux through the pentose phosphate pathway (PPP) by increasing the abundance and activity of glucose-6-phosphate dehydrogenase (G6PD), thereby increasing cellular antioxidant capacity. The PPP produces the coenzyme NADPH needed for a robust antioxidant response, including the regeneration of TRX1, indicating the existence of a regulatory feedback loop involving ATM and TRX1. We propose that loss of the mitochondrial ROS-sensing function of ATM may cause cellular ROS accumulation and oxidative stress in A-T.

Metabolomics evaluation of early-storage red blood cell rejuvenation at 4°C and 37°C.

BACKGROUND: Refrigerated red blood cell (RBC) storage results in the progressive accumulation of biochemical and morphological alterations collectively referred to as the storage lesion. Storage-induced metabolic alterations can be in part reversed by rejuvenation practices. However, rejuvenation requires an incubation step of RBCs for 1 hour at 37°C, limiting the practicality of providing "on-demand," rejuvenated RBCs. We tested the hypothesis that the addition of rejuvenation solution early in storage as an adjunct additive solution would prevent-in a time window consistent with the average age of units transfused to sickle cell recipients at Duke (15 days)-many of the adverse biochemical changes that can be reversed via standard rejuvenation, while obviating the incubation step. STUDY DESIGN AND METHODS: Metabolomics analyses were performed on cells and supernatants from AS-1 RBC units (n = 4), stored for 15 days. Units were split into pediatric bag aliquots and stored at 4°C. These were untreated controls, washed with or without rejuvenation, performed under either standard (37°C) or cold (4°C) conditions. RESULTS: All three treatments removed most metabolic storage by-products from RBC supernatants. However, only standard and cold rejuvenation provided significant metabolic benefits as judged by the reactivation of glycolysis and regeneration of adenosine triphosphate and 2,3-diphosphoglycerate. Improvements in energy metabolism also translated into increased capacity to restore the total glutathione pool and regenerate oxidized vitamin C in its reduced (ascorbate) form. CONCLUSION: Cold and standard rejuvenation of 15-day-old RBCs primes energy and redox metabolism of stored RBCs, while providing a logistic advantage for routine blood bank processing workflows.

Characterization of rapid extraction protocols for high-throughput metabolomics.

RATIONALE: In the last five years, high-throughput metabolomics has significantly advanced scientific research and holds the potential to promote strides in the fields of clinical metabolomics and personalized medicine. While innovations in the field of flow-injection mass spectrometry and three-minute metabolomics methods now allow investigators to process hundreds to thousands of samples per day, time-sensitive clinical applications, particularly in the emergency department, are limited by a lack of rapid extraction methods. METHODS: Here we characterized the efficacy of fast liquid-liquid extractions for characterization of hydrophilic compounds through ultra-high-pressure liquid chromatography/mass spectrometry. Internal stable-isotope-labeled standards were used to quantitatively characterize markers of energy and oxidative metabolism in human whole blood, plasma and red blood cells - three common matrices of clinical relevance. RESULTS: For all the tested matrices, vortexing time (4-60 min) did not significantly affect extraction yields for the tested hydrophilic metabolites. Coefficients of variations <<20% for all tested compounds, except for the redox-sensitive metabolite cystine (accumulating over time). Internal standards and second extractions confirmed recoveries >80% for all tested metabolites, except for basic amino acids and polyamines, which showed reproducible yields ranging from 50 to 75%. Global profiling and absolute quantitation of 24 metabolites revealed similarities between the plasma and red blood cell metabolomes. CONCLUSIONS: Rapid extraction (~4 min) of hydrophilic compounds is a viable and potentially automatable strategy to perform quantitative analysis of whole blood, plasma and red blood cells for research or clinical applications.