Analysis of a single analyte by mass spectrometry can result in the detection of more than 100 degenerate peaks. These degenerate peaks complicate spectral interpretation and are challenging to annotate. In mass spectrometry-based metabolomics, this degeneracy leads to inflated false discovery rates, data sets containing an order of magnitude more features than analytes, and an inefficient use of resources during data analysis. Although software has been introduced to annotate spectral degeneracy, current approaches are unable to represent several important classes of peak relationships. These include heterodimers and higher complex adducts, distal fragments, relationships between peaks in different polarities, and complex adducts between features and background peaks. Here we outline sources of peak degeneracy in mass spectra that are not annotated by current approaches and introduce a software package called mz.unity to detect these relationships in accurate mass data. Using mz.unity, we find that data sets contain many more complex relationships than we anticipated. Examples include the adduct of glutamate and nicotinamide adenine dinucleotide (NAD), fragments of NAD detected in the same or opposite polarities, and the adduct of glutamate and a background peak. Further, the complex relationships we identify show that several assumptions commonly made when interpreting mass spectral degeneracy do not hold in general. These contributions provide new tools and insight to aid in the annotation of complex spectral relationships and provide a foundation for improved data set identification. Mz.unity is an R package and is freely available at https://github.com/nathaniel-mahieu/mz.unity as well as our laboratory Web site http://pattilab.wustl.edu/software/ .

SummaryGain-of-function mutations in isocitrate dehydroge-nase 1 (IDH1) occur in multiple types of human cancer. Here, we show that these mutations significantly disrupt NADPH homeostasis by consuming NADPH for 2-hydroxyglutarate (2-HG) synthesis. Cells respond to 2-HG synthesis, but not exogenous administration of 2-HG, by increasing pentose phosphate pathway (PPP) flux. We show that 2-HG production competes with reductive biosynthesis and the buffering of oxidative stress, processes that also require NADPH. IDH1 mutants have a decreased capacity to synthesize palmitate and an increased sensitivity to oxidative stress. Our results demonstrate that, even when NADPH is limiting, IDH1 mutants continue to synthesize 2-HG at the expense of other NADPH-requiring pathways that are essential for cell viability. Thus, rather than attempting to decrease 2-HG synthesis in the clinic, the consumption of NADPH by mutant IDH1 may be exploited as a metabolic weakness that sensitizes tumor cells to ionizing radiation, a commonly used anti-cancer therapy.

BackgroundTwo-hydroxyglutarate (2HG) is present at low concentrations in healthy mammalian cells as both an L and D enantiomer. Both the L and D enantiomers have been implicated in regulating cellular physiology by mechanisms that are only partially characterized. In multiple human cancers, the D enantiomer accumulates due to gain-of-function mutations in the enzyme isocitrate dehydrogenase (IDH) and has been hypothesized to drive malignancy through mechanisms that remain incompletely understood. While much attention has been dedicated to identifying the route of 2HG synthesis, the metabolic fate of 2HG has not been studied in detail. Yet the metabolism of 2HG may have important mechanistic consequences influencing cell function and cancer pathogenesis, such as modulating redox potential or producing unknown products with unique modes of action.ResultsBy applying our isotope-based metabolomic platform, we unbiasedly and comprehensively screened for products of L- and D-2HG in HCT116 colorectal carcinoma cells harboring a mutation in IDH1. After incubating HCT116 cells in uniformly 13C-labeled 2HG for 24 h, we used liquid chromatography/mass spectrometry to track the labeled carbons in small molecules. Strikingly, we did not identify any products of 2HG metabolism from the thousands of metabolomic features that we screened. Consistent with these results, we did not detect any significant changes in the labeling patterns of tricarboxylic acid cycle metabolites from wild type or IDH1 mutant cells cultured in 13C-labeled glucose upon the addition of L, D, or racemic mixtures of 2HG. A more sensitive, targeted analysis revealed trace levels of isotopic enrichment (<1 %) in some central carbon metabolites from 13C-labeled 2HG. However, we found that cells do not deplete 2HG from the media at levels above our detection limit over a 48 h time period.ConclusionsTaken together, we conclude that 2HG carbon is not readily transformed in the HCT116 cell line. These data indicate that the phenotypic alterations induced by 2HG are not a result of its metabolic products.

Dyslipidemia and resulting lipotoxicity are pathologic signatures of metabolic syndrome and type 2 diabetes. Excess lipid causes cell dysfunction and induces cell death through pleiotropic mechanisms that link to oxidative stress. However, pathways that regulate the response to metabolic stress are not well understood. Herein, we show that disruption of the box H/ACA SNORA73 small nucleolar RNAs encoded within the small nucleolar RNA hosting gene 3 (Snhg3) causes resistance to lipid-induced cell death and general oxidative stress in cultured cells. This protection from metabolic stress is associated with broad reprogramming of oxidative metabolism that is dependent on the mammalian target of rapamycin signaling axis. Furthermore, we show that knockdown of SNORA73 in vivo protects against hepatic steatosis and lipid-induced oxidative stress and inflammation. Our findings demonstrate a role for SNORA73 in the regulation of metabolism and lipotoxicity.

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Study ObjectiveWe seek to answer the following question: how does consumption of NADPH by mutant IDH2 for 2‐HG synthesis affect other NADPH‐dependent pathways in the mitochondria?MethodsHCT116 IDH2 mutants were incubated in the presence of H2O2 or irradiated with 3,6, or 9 Gy ionizing radiation (IR) and cell viability was assessed with a trypan blue exclusion assay. Stable isotope tracing was performed by incubating HCT116 cells (n=3) with the specified isotope for 12–24 hours, then harvested in ice‐cold methanol. After extracting metabolites from cells in a mixture of methanol, acetonitrile, and water, samples were run on a Thermo QExactive Plus using liquid chromatography/mass spectrometry (LC/MS) with a HILIC column in negative mode.Results and ConclusionIsocitrate dehydrogenase 2 (IDH2) mutants are found in cases of acute myeloid leukemia (AML), glioblastoma, chondrosarcoma, and lymphoma cancers. IDH2 is one of two nicotinamide adenine dinucleotide (NADPH)‐dependent isoforms of IDH. While IDH1 is found exclusively in the cytosol, IDH2 is localized to the mitochondria. Both enzymes are mutated in cancer to produce 2‐hydroxyglutarate (2‐HG) in millimolar amounts. The neomorphic reaction consumes the essential redox factor NADPH. Previous research indicates that IDH1 mutants are dependent upon upregulation of the pentose phosphate pathway (PPP) to supply more NADPH for 2‐HG synthesis. However, this pathway is localized to the cytosol and NADPH is not capable of crossing the mitochondrial membrane to supply the IDH2 reaction. To study the effects of NADPH consumption/production with respect to compartmentalization, we used stable isotope labeling and LC/MS to assess central carbon pathway flux in comparison to IDH1 and WT cells. Our preliminary data suggests that IDH2 mutants may be dependent on compensatory mechanisms to generate essential metabolic factors (such as ME1 and the folate cycle). We conclude that IDH2 mutations lead to metabolic rewiring of the TCA cycle to support the demand of 2‐HG synthesis in the mitochondria.Support or Funding InformationGJP received financial support for this work from the National Institutes of Health Grants R35ES028365 and R21CA191097 as well as the Alfred P. Sloan Foundation, the Pew Scholars Program in the Biomedical Sciences, and the Edward Mallinckrodt, Jr Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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