Oncometabolites: A New Paradigm for Oncology, Metabolism, and the Clinical Laboratory.
Just as analytical measurements of these metabolites are useful in the diagnosis and monitoring of inborn errors of metabolism, increased production of these metabolites in cancer cells makes them ideal potential cancer biomarkers for prognosis, monitoring tumor burden, assessing the effectiveness of treatment, and detecting minimal residual disease and early recurrence. Moreover, the genetic defects that lead to high levels of these metabolites, as well as the downstream signaling pathways driven by them, are attractive targets for therapeutic interventions.
The term oncometabolite was first used for D-2-hydroxyglutarate and subsequently extended to structurally similar and metabolically proximate (Fig. 1) compounds L-2-hydroxyglutarate, succinate, and fumarate. Because of the metabolic proximity and structural similarity, these oncometabolites often affect the same biochemical and molecular pathways to drive cancer progression. The metabolism and cellular effects of oncometabolites have been discussed in recent reviews (6-8). Here we provide a brief review of the tricarboxylic acid (TCA) cycle oncometabolites D-2-hydroxyglutarate, L-2-hydroxyglutarate, succinate, and fumarate, with an emphasis on the role of clinical laboratories in bringing the analysis of oncometabolites into routine clinical practice.
Metabolic Origins of Oncometabolites in Normal and Cancer Cells
The concept of glycolytic fueling of cancer cells known as the Warburg effect (9) has received renewed attention (10). The ability of cancer cells to undergo aerobic glycolysis allows for the production of glycolytic and TCA cycle intermediates that support biosynthetic pathways required for cell growth and proliferation (11). Accordingly, certain cancers are associated with genetic aberrations in TCA cycle enzymes, such as isocitrate dehydrogenase 1 (IDH1), isocitrate dehydrogenase 2 (IDH2), succinate dehydrogenase (SDH), fumarate hydratase (FH), and L-2-hydroxyglutarate dehydrogenase. These enzymes share metabolic proximity in the TCA cycle, and either gain-or loss-of-function mutations in these key TCA cycle enzymes result in overproduction of oncometabolites D-2-hydroxyglutarate, L-2-hydroxyglutarate, succinate, and fumarate (Fig. 1 and Table 1).
D-2-hydroxyglutarate and L-2-hydroxyglutarate are stereoisomers; however, they are produced through distinct biological mechanisms. Although not part of the classic TCA cycle, D-2-hydroxyglutarate and L-2-hydroxyglutarate are byproducts of side reactions that utilize the TCA cycle intermediate, 2-ketoglutarate. In normal cells, D-2-hydroxyglutarate is mainly produced from 2-ketoglutarate through a reaction catalyzed by hydroxyacid-oxoacid transhydrogenase; however, the overproduction of D-2-hydroxyglutarate in cancer is a result of gain-of-function mutations in the genes encoding IDH1 or IDH2. Wild-type isocitrate dehydrogenases catalyze a reversible oxidative decarboxylation reaction converting isocitrate to 2-ketoglutarate. In many cancers, such as gliomas, acute myeloid leukemia, intrahepatic cholangiocarcinoma, and central chondrosarcoma, somatic gain-of-function mutations in IDH1  or IDH2 introduce a neomorphic activity in isocitrate dehydrogenases allowing catalytic reduction of 2-ketoglutarate to D-2-hydroxyglutarate; this leads to markedly increased levels of D-2-hydroxyglutarate in the cancer cells (12-15). Of note, neither the autosomal recessive D-2-hydroxyglutaric aciduria type 1 nor the autosomal dominant D-2-hydroxyglutaric aciduria type 2 has been associated with cancer, suggesting that cellular accumulation of D-2-hydroxyglutarate by itself is not sufficient for oncogenic transformation. L-2-hydroxyglutarate is produced in the mitochondria through the nonspecific activity of malate dehydrogenase catalyzing the reduction of 2-ketoglutarate to L-2-hydroxyglutarate (16). Interestingly, L-2-hydroxyglutaric aciduria, an autosomal recessive inborn error of metabolism caused by loss-of-function mutations of L2HGDH, is associated with brain and extracranial tumors (17, 18). The increased levels of L-2-hydroxyglutarate in a subset of clear cell renal cell carcinomas have been attributed to reduced expression of L-2-hydroxyglutarate dehydrogenase in the cancer cells (19).
SDH and FH participate in sequential reactions in the TCA cycle catalyzing the conversion of succinate to fumarate and fumarate to malate, respectively (Fig. 1). Succinate dehydrogenase is a heterotetrameric complex composed of 4 subunits, SDHA, SDHB, SDHC, and SDHD, and 2 assembly factors, SDHAF1 and SDHAF2 (20). Mutations in the SDH genes have been described in autosomal dominant hereditary paraganglioma, pheochromocytoma, renal carcinoma, thyroid cancer, neuroblastoma, T-cell leukemia, and gastrointestinal stromal tumor (6, 21-28). Heterozygous germline mutations of FH followed by a "second hit" loss of heterozygosity cause hereditary leiomyomatosis and renal cell cancer syndrome (29). FH mutations are also found in a subset of paragangliomas and pheochromocytomas (30), and FH deletions have been described in neuroblastomas (31). On the other hand, homozygous (or compound heterozygous) germline mutations in SDH subunits or FH give rise to autosomal recessive inborn errors of metabolism that have not been associated with cancer predisposition, suggesting that cellular accumulation of succinate or fumarate by itself is not sufficient for oncogenic transformation.
Shared Downstream Effects of Oncometabolites
The structural similarity of D-2-hydroxyglutarate, L-2-hydroxyglutarate, succinate, and fumarate to 2-ketoglutarate provides them the ability to competitively inhibit 2-ketoglutarate-dependent dioxygenases, which are involved in a wide range of biological functions (32).
Members of the 2-ketoglutarate-dependent dioxygenase family include prolyl hydroxylase domain proteins (PHDs), ten-eleven translocation (TET) enzymes, and Jumonji C domain-containing histone lysine demethylases. Under normoxic conditions, PHDs hydroxylate specific proline residues in the hypoxia-inducible factors (HIFs), which are then polyubiquitinated by von Hippel-Lindau E3 ubiquitin ligase complex and degraded by the proteasome. Hypoxia reverses this process, resulting in stabilization of HIFs that are transcription factors for genes involved in angiogenesis, cell growth, and cell migration. Inhibition of PHDs by oncometabolites causes aberrant stabilization of HIFs (termed pseudohypoxia), which then promote angiogenesis and cancer cell growth. TET enzymes hydroxylate 5-methylcytosine in DNA CpG dinucleotides as a step toward cytosine demethylation, whereas lysine demethylases demethylate lysine residues in histones. Inhibition of ten-eleven translocation enzymes and lysine demethylases by oncometabolites leads to aberrant DNA and histone hypermethylation, respectively, effecting epigenetic changes in cancer cells. Indeed, cancers with high levels of oncometabolites have a hypermethylation phenotype (19, 33).
Distinct Downstream Effects of Oncometabolites
In addition to the enzymes and signaling pathways that are shared targets of D-2-hydroxyglutarate, L-2-hydroxyglutarate, succinate, and fumarate, there are other distinct effects elicited by individual oncometabolites.
Fumarate, in particular, appears to be the most multifaceted of these oncometabolites, with wide-ranging effects on cell metabolism and signaling. In addition to producing pseudohypoxia by stabilizing HIFs, a feature shared by the other oncometabolites, fumarate also promotes a pseudohypoxic milieu by noncanonical activation of nuclear factor -B signaling, leading to increased transcription of HIF-1 (34). Fumarate accumulation in cancer cells may also reverse the activity of the urea cycle enzyme argininosuccinate lyase, leading to the production of argininosuccinate from fumarate and arginine.
Argininosuccinate is normally produced by argininosuccinate synthase from citrulline and aspartate. The effect of increased fumarate and reversal of argininosuccinate lyase reaction, therefore, potentially allows aspartate to be diverted from the urea cycle to purine and pyrimidine biosynthesis, favoring tumor growth. Interestingly, the reversal of argininosuccinate lyase activity makes these cells auxotrophic for arginine, which can have therapeutic applications for FH-deficient cancers (35). Another distinct feature of FH-deficient cancer cells is the ability of the accumulated fumarate to induce a posttranslational protein modification called succination. During this process, fumarate reacts with thiol groups of cysteine residues to produce S-(2-succino)-cysteine (36), which has the potential of affecting many proteins, including mitochondrial aconitase and Kelch-like ECH-associated protein-1 (KEAP1) (37). The latter has received attention as an important likely mechanism through which succination promotes tumorigenesis. Succination of KEAP1 removes its repressive effect on nuclear factor (erythroid-derived 2)-like 2 (NRF2). NRF2, a transcription factor, is then free to translocate to the nucleus where it initiates the expression of several genes involved in antioxidation and cytoprotection (38). Additionally, succination of glutathione, as well as direct binding of fumarate to glutathione peroxidase 1, leads to altered reactive oxygen species signaling and implicates oxidative stress-induced senescence as a potential tumor suppressor mechanism for fumarate (39-41).Of note, succination is a process distinct from succinylation. The latter is a recently identified posttranslational modification of proteins including histones, in which succinyl groups are added to lysine residues, an area of investigation in cancer cells with succinate accumulation (42, 43). In addition to promoting angiogenesis through inhibition of PHDs and HIF stabilization, succinate was recently shown to promote angiogenesis through upregulation of vascular endothelial growth factor expression in a HIF-independent mechanism, by binding to (GPCR91)G protein-coupled receptor 91 and consequent activation of signal transducer and activator of transcription 3 (STAT3) and extracellular regulated kinase 1/2 (ERK 1/2) (44). A recently described characteristic unique to D-2-hydroxyglutarate is its ability to induce epithelial-mesenchymal transition and metastatic potential in colorectal cancer cells through trimethylation of histone H3 lysine 4 in the promoter region of ZEB1, the gene encoding zinc finger E-box binding homeobox 1, a master regulator of epithelial-mesenchymal transition (45). Even though L-2-hydroxyglutarate is a stereoisomer of D-2-hydroxyglutarate and a potent inhibitor of 2-ketoglutarate-dependent dioxygenases, the ability to induce epithelial-mesenchymal transition by overexpressing zinc finger E-box binding homeobox 1 (ZEB1) appears to be specific to D-2-hydroxyglutarate (45). These findings suggest divergent oncogenic roles for D-2-hydroxyglutarate and L-2-hydroxyglutarate.
Thus, although there appears to be a common cancer phenotype (hypermethylation, pseudohypoxic signaling) associated with TCA cycle oncometabolites, there is a growing number of examples in which these oncometabolites affect distinct biological mechanisms. Therefore, a comprehensive understanding of the biology of each individual oncometabolite in various cancers can have important diagnostic, prognostic, and therapeutic implications.
Laboratory Methodologies for Detection and Measurement of Oncometabolites and Their Downstream Effectors
In this section, we will specifically focus on laboratory-based assays for the detection and measurement of oncometabolites and their downstream effectors. It is noteworthy that there are other non-laboratory-based techniques to study oncometabolites, such as magnetic resonance spectroscopy, which can detect total 2-hydroxyglutarate in vivo in IDH-mutated gliomas (46-48).
Pediatric clinical laboratories are typically equipped with hybrid instruments, such as GC-MS, LC-MS, and LC-MS/MS instruments. This set of instruments allows for rapid screening, diagnosis, and follow-up of patients with inborn errors of metabolism, including D-2-hydroxyglutaric aciduria, L-2-hydroxyglutaric aciduria, D,L-2-hydroxyglutaric aciduria, and fumaric aciduria. These same assays can be adapted for quantifying oncometabolites in body fluids, tissues, and cancer cells in culture. For diagnosing and monitoring D-2-hydroxyglutaric aciduria and L-2-hydroxyglutaric aciduria, urine is the sample of choice, as concentrations of both D-2-hydroxyglutarate and L-2-hydroxyglutarate are significantly higher in urine compared with plasma and cerebrospinal fluid (49, 50). The most common method for the quantification of urine organic acids involves ethyl acetate extraction and trimethylsilyl derivatization followed by GC-MS analysis. Because of the high prevalence of IDH1/2 mutations in cancers compared with other TCA cycle aberrations, the most widely studied oncometabolite is D-2-hydroxyglutarate. It is noteworthy that GC-MS assays commonly used for urine organic acid analysis do not distinguish between the stereoisomers of 2-hydroxyglutaric acid. Stereospecific measurements of D-2-hydroxyglutarate and L-2-hydroxyglutarate are highly suitable for adapting to clinical use in patients with IDH-mutated acute myeloid leukemia and other cancers. Separation of 2-hydroxyglutaric acid stereoisomers can be achieved with chiral columns or prechromatography chemical derivatization with chiral chemicals such diacetyl-L-tartaric anhydride (51-57). Although the total 2-hydroxyglutarate levels have been measured in patients with IDH-mutated leukemia, gliomas, and other cancers (15, 58-63), stereospecific measurement of D-2-hydroxyglutarate (Fig. 2) is a more sensitive and specific measure of the activity of mutated IDH genes (64). Indeed, plasma D-2-hydroxyglutarate levels may be more sensitive than conventional DNA sequencing for identification of IDH-mutated acute myeloid leukemia with low blast count (65) and thus may prove to be an excellent assay for detection of minimal residual disease and early recurrence. In patients with IDH-mutated acute myeloid leukemia, total serum 2-hydroxyglutarate concentrations >200 ng/mL (1.35 [micro]mol/L) at complete remission were shown to have a shorter overall survival compared with the patients with total 2-hydroxyglutarate concentrations [less than or equal to] 200 ng/mL (1.35 [micro]mol/L) (58). In a prospective randomized multicenter trial, pretreatment serum D-2-hydroxyglutarate levels negatively correlated with event-free survival in patients with IDH1-mutated acute myeloid leukemia, but not in patients with IDH2-mutated acute myeloid leukemia (66). The stereospecific measurement of D-2-hydroxyglutarate and L-2-hydroxyglutarate also allows for the calculation of the ratio of D-2-hydroxyglutarate to L-2-hydroxyglutarate, which is as sensitive as allele-specific digital PCR for monitoring mutant IDH allele frequencies of 1%, although it is less sensitive than digital PCR for lower mutant allele frequencies (67). An additional advantage of biochemical measurement of D-2-hydroxyglutarate, compared with performing only hotspot genetic analysis for IDH mutations, is that it gives an opportunity to identify other mechanisms for D-2-hydroxyglutarate increases in cancers that are negative for the classic IDH mutations. For example, 2-hydroxyglutarate accumulation in a subset of aggressive breast cancers has been shown to be caused by metabolic reprogramming driven by myelocytomatosis protooncogene (MYC) protein (68). Similarly, D-2-hydroxyglutarate may be produced by reduction of 2-ketoglutarate catalyzed by phosphoglycerate dehydrogenase, the gene for which is amplified in breast cancer and melanoma (69).
These same stereospecific assays can be used to measure L-2-hydroxyglutarate (Fig. 2) (19, 56, 57), although clinical application of L-2-hydroxyglutarate measurement has not yet been realized. Similarly, succinate and fumarate can be easily detected and measured using GC-MS or LC-MS. Current clinical methods for urine organic acid analysis can detect TCA cycle metabolites including succinate and fumarate (70), but their measurement as oncometabolites in body fluids and cancer tissue is not yet in the clinical realm. One study exploring the utility of measuring the succinate-to-fumarate ratio in identifying pheochromocytomas and paragangliomas with SDH mutations reported a diagnostic sensitivity and specificity of 93% and 97%, respectively (71). Measurement of metabolites downstream of the primary TCA cycle, such as argininosuccinate, may also be clinically useful. A pan-oncometabolite assay that measures D-2-hydroxyglutarate, L-2-hydroxyglutarate, succinate, and fumarate, as well as their downstream metabolites, would be an ideal tool for screening and follow-up of cancers with the corresponding metabolic dysregulation.
Another potential method to evaluate oncometabolites or their downstream effectors is immunohistochemistry. As discussed earlier, fumarate activates the KEAP1-NRF2 signaling pathway. The transcription factor NRF2 promotes transcriptional activation of antioxidant proteins including NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 overactivity in cancer cells increases their sensitivity to [beta]-lapachone toxicity and is therefore an attractive therapeutic target in many cancers (72). Both FH loss and NQO1 overexpression in FH-deficient renal cell carcinomas can be detected by immunohistochemistry (Fig. 3). Immunohistochemistry for S-(2-succino)-cysteine can be used to detect succination in FH-deficient renal cell carcinomas; this has been shown to be a sensitive and specific biomarker of FH-mutated tumors (36, 73, 74).
The discovery of oncometabolites has initiated a new paradigm in cancer biology that has the potential to positively affect the treatment of patients. This includes the discovery of new therapeutic targets that exploit vulnerabilities of cancer cells, such as their dependence on oncometabolites (e.g., inhibition of mutant isocitrate dehydrogenases that produce D-2-hydroxyglutarate) (75, 76), as well as downstream targets of the oncometabolites (e.g., metabolic and epigenetic modulators) (7, 77). Targeted therapeutic approaches, however, would have to consider the overall effects of genetic mutations that produce oncometabolites. For instance, IDH-mutated cancer cells have not only high levels of D-2-hydroxyglutarate but also concurrent low levels of reduced NADPH, which is consumed during the reduction of 2-ketoglutarate to D-2-hydroxyglutarate. This may make the IDH-mutated cancer cells sensitive to conventional chemotherapy and radiation via increased generation of reactive oxygen species (78). This sensitivity of IDH-mutated cancer cells to conventional chemotherapy and radiation may be inadvertently reversed by the small molecule inhibitors of mutant isocitrate dehydrogenases (79). Regardless, as this field progresses, clinical laboratories will play an essential role in patient treatment by developing and validating novel methods to measure oncometabolites and their downstream effectors, thus harnessing their potential as effective biomarkers.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.
Acknowledgment: The authors thank Ms. Crysten Timbes for editorial help.
(1.) Struys EA, Salomons GS, Achouri Y, Van Schaftingen E, Grosso S, Craigen WJ, et al. Mutations in the D-2-hydroxyglutarate dehydrogenase gene cause D-2-hydroxyglutaric aciduria. Am J Hum Genet 2005;76: 358-60.
(2.) Kranendijk M, Struys EA, van Schaftingen E, Gibson KM, Kanhai WA, van der Knaap MS, et al. IDH2 mutations in patients with D-2-hydroxyglutaric aciduria. Science 2010;330:336.
(3.) Rzem R, Veiga-da-Cunha M, Noel G, Goffette S, Nassogne MC, Tabarki B, et al. A gene encoding a putative FAD-dependent L-2-hydroxyglutarate dehydrogenase is mutated in L-2-hydroxyglutaric aciduria. Proc Natl Acad Sci U S A 2004;101:16849-54.
(4.) Nota B, Struys EA, Pop A, Jansen EE, Fernandez Ojeda MR, Kanhai WA, et al. Deficiency in SLC25A1, encoding the mitochondrial citrate carrier, causes combined D-2-and L-2-hydroxyglutaric aciduria. Am J Hum Genet 2013;92:627-31.
(5.) Gellera C, Uziel G, Rimoldi M, Zeviani M, Laverda A, Carrara F, DiDonato S. Fumarase deficiency is an autosomal recessive encephalopathy affecting both the mitochondrial and the cytosolic enzymes. Neurology 1990;40: 495-9.
(6.) Yang M, Soga T, Pollard PJ. Oncometabolites: linking altered metabolism with cancer. J Clin Invest 2013; 123:3652-8.
(7.) Nowicki S, Gottlieb E. Oncometabolites: tailoring our genes. FEBS J 2015;282:2796-805.
(8.) Sciacovelli M, Frezza C. Oncometabolites: unconventional triggers of oncogenic signalling cascades. Free Radic Biol Med 2016;100:175-81.
(9.) Warburg O. On the origin of cancer cells. Science 1956; 123:309-14.
(10.) Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144:646-74.
(11.) DeBerardinis RJ, Lum JJ, Hatzivassiliou G, Thompson CB. The biology of cancer: metabolic reprogramming fuels cell growth and proliferation. Cell Metab 2008;7: 11-20.
(12.) Dang L, White DW, Gross S, Bennett BD, Bittinger MA, Driggers EM, et al. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature 2009; 462:739-44.
(13.) Ward PS, Patel J, Wise DR, Abdel-Wahab O, Bennett BD, Coller HA, et al. The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate. Cancer Cell 2010;17:225-34.
(14.) Amary MF, Bacsi K, Maggiani F, Damato S, Halai D, Berisha F, et al. IDH1 and IDH2 mutations are frequent events in central chondrosarcoma and central and periosteal chondromas but not in other mesenchymal tumours. J Pathol 2011;224:334-43.
(15.) Borger DR, Goyal L, Yau T, Poon RT, Ancukiewicz M, Deshpande V, et al. Circulating oncometabolite 2-hydroxyglutarate is a potential surrogate biomarker in patients with isocitrate dehydrogenase-mutant intrahepatic cholangiocarcinoma. Clin Cancer Res 2014;20: 1884-90.
(16.) Rzem R, Vincent MF, Van Schaftingen E, Veiga-da-Cunha M. L-2-hydroxyglutaric aciduria, a defect of metabolite repair. J Inherit Metab Dis 2007;30:681-9.
(17.) Aghili M, Zahedi F, Rafiee E. Hydroxyglutaric aciduria and malignant brain tumor: a case report and literature review. J Neurooncol 2009;91:233-6.
(18.) Rogers RE, Deberardinis RJ, Klesse LJ, Boriack RL, Margraf L, Rakheja D. Wilms tumor in a child with L-2-hydroxyglutaric aciduria. Pediatr Dev Pathol 2010;13: 408-11.
(19.) Shim EH, Livi CB, Rakheja D, Tan J, Benson D, Parekh V, et al. L-2-Hydroxyglutarate: an epigenetic modifier and putative oncometabolite in renal cancer. Cancer Discov 2014;4:1290-8.
(20.) Van Vranken JG, Na U, Winge DR, Rutter J. Protein-mediated assembly of succinate dehydrogenase and its cofactors. Crit Rev Biochem Mol Biol 2015;50:168-80.
(21.) Williamson SR, Eble JN, Amin MB, Gupta NS, Smith SC, Sholl LM, et al. Succinate dehydrogenase-deficient renal cell carcinoma: detailed characterization of 11 tumors defining a unique subtype of renal cell carcinoma. Mod Pathol 2015;28:80-94.
(22.) Hao HX, Khalimonchuk O, Schraders M, Dephoure N, Bayley JP, Kunst H, et al. SDH5, a gene required for flavination of succinate dehydrogenase, is mutated in paraganglioma. Science 2009;325:1139-42.
(23.) Baysal BE. A recurrent stop-codon mutation in succinate dehydrogenase subunit B gene in normal peripheral blood and childhood T-cell acute leukemia. PloS One 2007;2:e436.
(24.) Baysal BE, Ferrell RE, Willett-Brozick JE, Lawrence EC, Myssiorek D, Bosch A, et al. Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma. Science 2000;287:848-51.
(25.) Matyakhina L, Bei TA, McWhinney SR, Pasini B, Cameron S, Gunawan B, et al. Genetics of carney triad: recurrent losses at chromosome 1 but lack of germline mutations in genes associated with paragangliomas and gastrointestinal stromal tumors. J Clin Endocrinol Metab 2007;92:2938-43.
(26.) Niemann S, Muller U. Mutations in SDHC cause autosomal dominant paraganglioma, type 3. Nat Genet 2000;26:268-70.
(27.) Astuti D, Latif F, Dallol A, Dahia PL, Douglas F, George E, et al. Gene mutations in the succinate dehydrogenase subunitSDHBcause susceptibility to familial pheochromocytoma and to familial paraganglioma. Am J Hum Genet 2001;69:49-54.
(28.) Bardella C, Pollard PJ, Tomlinson I. SDH mutations in cancer. Biochim Biophys Acta 2011;1807:1432-43.
(29.) Tomlinson IP, Alam NA, Rowan AJ, Barclay E, Jaeger EE, Kelsell D, et al. Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell cancer. Nat Genet 2002; 30:406-10.
(30.) Castro-Vega LJ, Buffet A, De Cubas AA, Cascon A, Menara M, Khalifa E, et al. Germline mutations in FH confer predisposition to malignant pheochromocytomas and paragangliomas. Hum Mol Genet 2014;23:2440-6.
(31.) Fieuw A, Kumps C, Schramm A, Pattyn F, Menten B, Antonacci F, et al. Identification of a novel recurrent 1q42.2-1qter deletion in high risk MYCN single copy 11q deleted neuroblastomas. Int J Cancer 2012;130: 2599-606.
(32.) Xiao M, Yang H, Xu W, Ma S, Lin H, Zhu H, et al. Inhibition of alpha-KG-dependent histone and DNA demethylases by fumarate and succinate that are accumulated in mutations of FH and SDH tumor suppressors. Genes Dev 2012;26:1326-38.
(33.) Waterfall JJ, Killian JK, Meltzer PS. The role of mutation of metabolism-related genes in genomic hypermethylation. Biochem Biophys Res Commun 2014;455:16-23.
(34.) Shanmugasundaram K, Nayak B, Shim EH, Livi CB, Block K, Sudarshan S. The oncometabolite fumarate promotes pseudohypoxia through noncanonical activation of NF-[kappa]B signaling. J Biol Chem 2014;289:24691-9.
(35.) Zheng L, Mackenzie ED, Karim SA, Hedley A, Blyth K, Kalna G, et al. Reversed argininosuccinate lyase activity in fumarate hydratase-deficient cancer cells. Cancer Metab 2013;1:12.
(36.) Bardella C, El-Bahrawy M, Frizzell N, Adam J, Ternette N, Hatipoglu E, et al. Aberrant succination of proteins in fumarate hydratase-deficient mice and HLRCC patients is a robust biomarker of mutation status. J Pathol 2011; 225:4-11.
(37.) Yang M, Ternette N, Su H, Dabiri R, Kessler BM, Adam J, et al. The succinated proteome of FH-mutant tumours. Metabolites 2014;4:640-54.
(38.) Kinch L, Grishin NV, Brugarolas J. Succination of Keap1 and activation of Nrf2-dependent antioxidant pathways in FH-deficient papillary renal cell carcinoma type 2. Cancer Cell 2011;20:418-20.
(39.) Sullivan LB, Martinez-Garcia E, Nguyen H, Mullen AR, Dufour E, Sudarshan S, et al. The proto-oncometabolite fumarate binds glutathione to amplify ROS-dependent signaling. Mol Cell 2013;51:236-48.
(40.) Jin L, Li D, Alesi GN, Fan J, Kang HB, Lu Z, et al. Glutamate dehydrogenase 1 signals through antioxidant glutathione peroxidase 1 to regulate redox homeostasis and tumor growth. Cancer Cell 2015;27:257-70.
(41.) Zheng L, Cardaci S, Jerby L, MacKenzie ED, Sciacovelli M, Johnson TI, et al. Fumarate induces redox-dependent senescence by modifying glutathione metabolism. Nat Commun 2015;6:6001.
(42.) Zhang Z, Tan M, Xie Z, Dai L, Chen Y, Zhao Y. Identification of lysine succinylation as a new post-translational modification. Nat Chem Biol 2011;7:58-63.
(43.) Jiang S, Yan W. Succinate in the cancer-immune cycle. Cancer Lett 2017;390:45-7.
(44.) Mu X, Zhao T, Xu C, Shi W, Geng B, Shen J, et al. Oncometabolite succinate promotes angiogenesis by upregulating VEGF expression through GPR91-mediated STAT3 and ERK activation. Oncotarget 2017;8:13174-85.
(45.) Colvin H, Nishida N, Konno M, Haraguchi N, Takahashi H, Nishimura J, et al. Oncometabolite D-2-hydroxyglurate directly induces epithelial-mesenchymal transition and is associated with distant metastasis in colorectal cancer. Sci Rep 2016;6:36289.
(46.) Choi C, Ganji SK, DeBerardinis RJ, Hatanpaa KJ, Rakheja D, Kovacs Z, et al. 2-Hydroxyglutarate detection by magnetic resonance spectroscopy in IDH-mutated patients with gliomas. Nat Med 2012;18:624-9.
(47.) Elkhaled A, Jalbert LE, Phillips JJ, Yoshihara HA, Parvataneni R, Srinivasan R, et al. Magnetic resonance of 2-hydroxyglutarate in IDH1-mutated low-grade gliomas. Sci Transl Med 2012;4:116ra5.
(48.) Andronesi OC, Kim GS, Gerstner E, Batchelor T, Tzika AA, Fantin VR, et al. Detection of 2-hydroxyglutarate in IDH-mutated glioma patients by in vivo spectral-editing and 2D correlation magnetic resonance spectroscopy. Sci Transl Med 2012;4:116ra4.
(49.) Gibson KM, ten Brink HJ, Schor DS, Kok RM, Bootsma AH, Hoffmann GF, Jakobs C. Stable-isotope dilution analysis of D-and L-2-hydroxyglutaric acid: application to the detection and prenatal diagnosis of D-and L-2-hydroxyglutaric acidemias. Pediatr Res 1993;34:277-80.
(50.) Kranendijk M, Struys EA, Salomons GS, Van der Knaap MS, Jakobs C. Progress in understanding 2-hydroxyglutaric acidurias. J Inherit Metab Dis 2012; 35:571-87.
(51.) Struys EA, Jansen EE, Verhoeven NM, Jakobs C. Measurement of urinary D-and L-2-hydroxyglutarate enantiomers by stable-isotope-dilution liquid chromatography-tandem mass spectrometry after derivatization with diacetyl-L-tartaric anhydride. Clin Chem 2004;50:1391-5.
(52.) Rashed MS, AlAmoudi M, Aboul-Enein HY. Chiral liquid chromatography tandem mass spectrometry in the determination of the configuration of 2-hydroxyglutaric acid in urine. Biomed Chromatogr 2000;14:317-20.
(53.) Svidrnoch M, Pribylka A, Bekarek V, Sevcik J, Smolka V, Maier V. Enantioseparation of d,l-2-hydroxyglutaric acid by capillary electrophoresis with tandem mass spectrometry--fast and efficient tool for d-and l-2-hydroxyglutaracidurias diagnosis. J Chromatogr A 2016;1467:383-90.
(54.) Calderon C, Horak J, Lammerhofer M. Chiral separation of 2-hydroxyglutaric acid on cinchonan carbamate based weak chiral anion exchangers by high-performance liquid chromatography. J Chromatogr A 2016;1467:239-45.
(55.) Cheng QY, Xiong J, Huang W, Ma Q, Ci W, Feng YQ, Yuan BF. Sensitive determination of onco-metabolites of D-and L-2-hydroxyglutarate enantiomers by chiral derivatization combined with liquid chromatography/ mass spectrometry analysis. Sci Rep 2015;5:15217.
(56.) Rakheja D, Boriack RL, Mitui M, Khokhar S, Holt SA, Kapur P. Papillary thyroid carcinoma shows elevated levels of 2-hydroxyglutarate. Tumour Biol 2011;32: 325-33.
(57.) Rakheja D, Mitui M, Boriack RL, Deberardinis RJ. Isocitrate dehydrogenase 1/2 mutational analyses and 2-hydroxyglutarate measurements in Wilms tumors. Pediatr Blood Cancer 2011;56:379-83.
(58.) DiNardo CD, Propert KJ, Loren AW, Paietta E, Sun Z, Levine RL, et al. Serum 2-hydroxyglutarate levels predict isocitrate dehydrogenase mutations and clinical outcome in acute myeloid leukemia. Blood 2013;121: 4917-24.
(59.) Wang JH, Chen WL, Li JM, Wu SF, Chen TL, Zhu YM, et al. Prognostic significance of 2-hydroxyglutarate levels in acute myeloid leukemia in China. Proc Natl Acad Sci U S A 2013;110:17017-22.
(60.) Janin M, Mylonas E, Saada V, Micol JB, Renneville A, Quivoron C, et al. Serum 2-hydroxyglutarate production in IDH1-and IDH2-mutated de novo acute myeloid leukemia: a study by the Acute Leukemia French Association group. J Clin Oncol 2014;32:297-305.
(61.) Lombardi G, Della Puppa A, Zagonel V. Urine 2-hydroxyglutarate in glioma. Oncologist 2016;21: 1026.
(62.) Lombardi G, Corona G, Bellu L, Della Puppa A, Pambuku A, Fiduccia P, et al. Diagnostic value of plasma and urinary 2-hydroxyglutarate to identify patients with isocitrate dehydrogenase-mutated glioma. Oncologist 2015;20:562-7.
(63.) Fathi AT, Sadrzadeh H, Comander AH, Higgins MJ, Bardia A, Perry A, et al. Isocitrate dehydrogenase 1 (IDH1) mutation in breast adenocarcinoma is associated with elevated levels of serum and urine 2-hydroxyglutarate. Oncologist 2014;19:602-7.
(64.) Struys EA. 2-Hydroxyglutarate is not a metabolite; D-2-hydroxyglutarate and L-2-hydroxyglutarate are! Proc Natl Acad Sci U S A 2013;110:E4939.
(65.) McGehee E, Rakheja D, Oliver D, Chen W, Boriack R, Collins RH Jr. The importance of plasma D-2HG measurement in screening for IDH mutations in acute myeloid leukaemia. Br J Haematol 2016;173:323-6.
(66.) Balss J, Thiede C, Bochtler T, Okun JG, Saadati M, Benner A, et al. Pretreatment d-2-hydroxyglutarate serum levels negatively impact on outcome in IDH1-mutated acute myeloid leukemia. Leukemia 2016;30: 782-8.
(67.) Wiseman DH, Struys EA, Wilks DP, Clark CI, Dennis MW, Jansen EE, et al. Direct comparison of quantitative digital PCR and 2-hydroxyglutarate enantiomeric ratio for IDH mutant allele frequency assessment in myeloid malignancy. Leukemia 2015;29:2421-3.
(68.) Terunuma A, Putluri N, Mishra P, Mathe EA, Dorsey TH, Yi M, et al. MYC-driven accumulation of 2-hydroxyglutarate is associated with breast cancer prognosis. J Clin Invest 2014;124:398-412.
(69.) Fan J, Teng X, Liu L, Mattaini KR, Looper RE, Vander Heiden MG, Rabinowitz JD. Human phosphoglycerate dehydrogenase produces the oncometabolite D-2-hydroxyglutarate. ACS Chem Biol 2015;10:510-6.
(70.) Jones PM, Bennett MJ. Urine organic acid analysis for inherited metabolic disease using gas chromatography-mass spectrometry. Methods Mol Biol 2010;603:423-31.
(71.) Richter S, Peitzsch M, Rapizzi E, Lenders JW, Qin N, de Cubas AA, et al. Krebs cycle metabolite profiling for identification and stratification of pheochromocytomas/ paragangliomas due to succinate dehydrogenase deficiency. J Clin Endocrinol Metab 2014;99:3903-11.
(72.) Reinicke KE, Bey EA, Bentle MS, Pink JJ, Ingalls ST, Hoppel CL, et al. Development of beta-lapachone prodrugs for therapy against human cancer cells with elevated NAD(P)H:quinone oxidoreductase 1 levels. Clin Cancer Res 2005;11:3055-64.
(73.) Chen YB, Brannon AR, Toubaji A, Dudas ME, Won HH, Al-Ahmadie HA, et al. Hereditary leiomyomatosis and renal cell carcinoma syndrome-associated renal cancer: recognition of the syndrome by pathologic features and the utility of detecting aberrant succination by immunohistochemistry. Am J Surg Pathol 2014;38:627-37.
(74.) Llamas-Velasco M, Requena L, Adam J, Frizzell N, Hartmann A, Mentzel T. Loss of fumarate hydratase and aberrant protein succination detected with S-(2-succino)-cysteine staining to identify patients with multiple cutaneous and uterine leiomyomatosis and hereditary leiomyomatosis and renal cell cancer syndrome. Am J Dermatopathol 2016;38:887-91.
(75.) Yen K, Travins J, Wang F, David MD, Artin E, Straley K, et al. AG-221, a first-in-class therapy targeting acute myeloid leukemia harboring oncogenic IDH2 mutations. Cancer Discov 2017;7:478-93.
(76.) Pusch S, Krausert S, Fischer V, Balss J, Ott M, Schrimpf D, et al. Pan-mutant IDH1 inhibitor BAY 1436032 for effective treatment of IDH1 mutant astrocytoma in vivo. Acta Neuropathol 2017;133:629-44.
(77.) Morin A, Letouze E, Gimenez-Roqueplo AP, Favier J. Oncometabolites-driven tumorigenesis: from genetics to targeted therapy. Int J Cancer 2014;135:2237-48.
(78.) Shi J, Sun B, Shi W, Zuo H, Cui D, Ni L, Chen J. Decreasing GSH and increasing ROS in chemosensitivity gliomas with IDH1 mutation. Tumour Biol 2015;36: 655-62.
(79.) Molenaar RJ, Botman D, Smits MA, Hira VV, van Lith SA, Stap J, et al. Radioprotection of IDH1-mutated cancer cells by the IDH1-mutant inhibitor AGI-5198. Cancer Res 2015;75:4790-802.
Rebecca R.J. Collins, [1,2] Khushbu Patel, [1,2] William C. Putnam,  Payal Kapur,  and Dinesh Rakheja [1,2,4] *
 Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX;  Department of Pathology and Laboratory Medicine, Children's Health, Dallas, TX;  Office of Clinical and Translational Research, Texas Tech University Health Sciences Center, Dallas, TX;  Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX.
* Address correspondence to this author at: Department of Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9073. Fax 214-456-0779; e-mail firstname.lastname@example.org.
Received March 15, 2017; accepted September 19, 2017.
Previously published online at DOI: 10.1373/clinchem.2016.267666
 Nonstandard abbreviations: OMIM, Online Mendelian Inheritance in Man; TCA, tricarboxylic acid; IDH, isocitrate dehydrogenase; SDH, succinate dehydrogenase; FH, fumarate hydratase; PHD, prolyl hydroxylase domain protein; HIF, hypoxia-inducible factor; NRF2, nuclear factor (erythroid-derived 2)-like 2; NQO1, NAD(P)H:quinone oxidoreductase 1.
 Human Genes: IDH1, isocitrate dehydrogenase 1; IDH2, isocitrate dehydrogenase 2; L2HGDH, L-2-hydroxyglutarate dehydrogenase; SDH, succinate dehydrogenase; ZEB1, zinc finger E-box binding homeobox 1; FH, fumarate hydratase; SDHA, succinate dehydrogenase complex flavoprotein subunit A; SDHB, succinate dehydrogenase complex iron sulfur subunit B; SDHC, succinate dehydrogenase complex subunit C; SDHD, succinate dehydrogenase complex subunit D; SDHAF1, succinate dehydrogenase complex assembly factor 1; SDHAF2, succinate dehydrogenase complex assembly factor 2.
Caption: Fig. 1. TCA cycle oncometabolites. The metabolic locations of the oncometabolites D-2-hydroxyglutarate, L-2-hydroxyglutarate, succinate, and fumarate are shown in the TCA cycle. The structures of the oncometabolites highlight their similarity to each other and to 2-ketoglutarate. Schematic representation of the shared and distinct biologic effects of the oncometabolites is in orange.
Caption: Fig. 2. Chromatograms showing levels of D-2-hydroxyglutarate and L-2-hydroxyglutarate. Markedly increased D-2-hydroxyglutarate in plasma of a patient with IDH-mutated acute myeloid leukemia (A). Plasma sample of a patient with IDH-mutated acute myeloid leukemia was evaluated by LC-MS/MS after extraction followed by derivatization with diacetyl-L-tartaric anhydride as described previously (51, 56, 57). Moderately increased L-2-hydroxyglutarate in tissue extract of a clear cell renal cell carcinoma (B). Tissue sample of a clear cell renal cell carcinoma was evaluated by LC-MS/MS after extraction followed by derivatization with diacetyl-L-tartaric anhydride as reported previously (19). Normal levels ofD-2-hydroxyglutarate and L-2-hydroxyglutarate in a plasma sample (C). In our laboratory, we use this assay clinically for measuringD-2-hydroxyglutarate and L-2-hydroxyglutarate in plasma or serum of patients with acute myeloid leukemia. The reference ranges that we have established are as follows: D-2-hydroxyglutarate, 18-263 ng/mL (0.12-1.78 [micro]mol/L); L-2-hydroxyglutarate, 6-147 ng/mL (0.04-0.99 [micro]mol/L); total D,L-2-hydroxyglutarate, 60-375 ng/mL (0.41-2.53 [micro]mol/L); and ratio of D-2-hydroxyglutarate to L-2-hydroxyglutarate, 0.2-3.8. Peak 1, L-2-hydroxyglutarate; Peak 2, L-2-hydroxyglutarate-d4 (internal standard); Peak 3, D-2-hydroxyglutarate-d4 (internal standard); Peak 4, D-2-hydroxyglutarate. Note that the y axis scales are different in each chromatogram.
Caption: Fig. 3. Photomicrographs of an FH-mutated renal cell carcinoma. Hematoxylin and eosin-stained section at 200x original magnification shows the classic histologic features of FH-mutated renal cell carcinoma that include prominent nucleoli and perinucleolar clearing (highlighted in the inset at 400x original magnification) (A). Immunostain for fumarate hydratase (200x original magnification) shows absent staining in the cancer cells (B). The staining is preserved in the stromal and endothelial cells between the tumor cells. Immunostain for NQO1 (200x original magnification) shows diffuse and strong cytoplasmic staining in cancer cells (C).
Table 1. TCA cycle oncometabolites. Oncometabolite Genes Associated malignancies D-2-hydroxyglutarate IDH1 Glioma, acute myeloid leukemia, IDH2 intrahepatic cholangiocarcinoma, central chondrosarcoma L-2-hydroxyglutarate L2HGDH Brain tumors, renal cell carcinoma Succinate SDHA Paraganglioma, pheochromocytoma, SDHB renal cell carcinoma, thyroid SDHC cancer, neuroblastoma, T-cell SDHD leukemia, gastrointestinal SDHAF1 stromal tumor SDHAF2 Fumarate FH Hereditary leiomyomatosis and renal cell cancer syndrome, paraganglioma, pheochromocytoma, neuroblastoma Oncometabolite Genes Downstream effects References D-2-hydroxyglutarate IDH1 DNA and histone (12-15, 19, IDH2 hypermethylation, HIF 33, 45) stabilization with pseudohypoxia response, epithelial mesenchymal transition L-2-hydroxyglutarate L2HGDH DNA and histone (17-19, 33) hypermethylation, HIF stabilization with pseudohypoxia response Succinate SDHA DNA and histone hyperme- (6, 19, SDHB thylation, HIF stabi- 21-28, SDHC lization with pseudo- 33, SDHD hypoxia response 42-44) SDHAF1 succinylation SDHAF2 Fumarate FH DNA and histone hyperme- (19, 29-31, thylation, HIF stabili- 33, zation and increased 34-41) transcription with pseudohypoxia response, succination, nucleotide synthesis, redox signaling
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|Author:||Collins, Rebecca R.J.; Patel, Khushbu; Putnam, William C.; Kapur, Payal; Rakheja, Dinesh|
|Date:||Dec 1, 2017|
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