Patient selection and statistical analysis
Patient samples were obtained from either the Stem Cell and Xenotransplantation Core Facility of the University of Pennsylvania or from the tissue collections of Memorial Sloan-Kettering Cancer Institute. Approval was obtained from the institutional review boards at the University of Pennsylvania (IRB protocol 703185) and Memorial Sloan Kettering Cancer Institute (IRB protocols 95–091 and 06–107), and informed consent was provided according to the Declaration of Helsinki. All samples were collected after de-identification for these studies. For assessing clinical and genetic parameters of IDH1/2 wild-type and mutant AML, 78 serial samples from AML patients referred for molecular testing at Memorial Sloan-Kettering Cancer Center were examined. For 2HG assays, patient samples were obtained from the Stem Cell and Xenotransplantation Core Facility of the University of Pennsylvania. The initial 18 samples were selected from de-identified AML patients aged 50 years or older at diagnosis with lesions determined to have normal cytogenetic status. Cells used for these assays were prepared by Ficoll separation of mononuclear cells (MNCs) from peripheral blood or bone marrow. MNCs were frozen as viable cells in 10% DMSO. Student’s t test and Chi-square analysis were used to analyze data. A p value < 0.05 was considered significant.
Sequence Analysis of IDH1 and IDH2
Genomic DNA was extracted from bone marrow mononuclear cells or from sorted leukemic cells; for samples with less than 70% blasts flow cytometric sorting (FACSAria) was used to isolate blast cells according to leukemic blast immunophenotype before DNA isolation. High-throughput DNA sequence analysis was used to screen for IDH1 and IDH2 mutations. All DNA samples were whole genome amplified using Ø29 polymerase and mutations were validated on unamplified DNA to ensure all mutations were present in the diagnostic sample. Sequencing of IDH1 used primers which cover amino acid residues 41–138 (sense, 5’-TGTGTTGAGATGGACGCCTA-3’; antisense, 5’-GGTGTACTCAGAGCCTTCGC-3’). Sequencing of IDH2 used primers which cover amino acid residues 125–226 (sense, 5’-CTGCCTCTTTGTGGCCTAAG-3’; antisense, 5’-ATTCTGGTTGAAAGATGGCG-3’). Sequence analysis was performed using Mutation Surveyor (SoftGenetics, State College PA) and all mutations were validated by repeat PCR and sequencing on unamplified DNA from the archival sample.
Human IDH2 has 97% homology with pig IDH2, and none of the 13 residues that are different (out of 418 total, excluding the N-terminal mitochondrial signal sequence) are found in the active site. A PDB structure of pig IDH2 is available, with isocitrate in the active site (1LWD) (Ceccarelli et al., 2002
). Based on the highly homologous pig structure, conservative structural models of human IDH2 were built with the CHARMM molecular mechanics package using the CHARMM27 force field. To model wild-type IDH2 with isocitrate, the 13 residues of 1LWD differing between pig and human were first changed to the human sequence. The side chains were then rebuilt, hydrogen atoms added to all residues, the substrate, active site Mn2+
ions, and conserved residues were restrained, and the structure minimized allowing only the changed residues to relax. For the α-ketoglutarate complexes, the substrate conformation/pose was first modeled on isocitrate by removing the β-carboxyl group and replacing it with a hydrogen. The wild-type IDH2-α-ketoglutarate complex was then minimized allowing only the active site residues (as defined in entry 1LWD) and substrate to relax. The R140Q mutant IDH2-α-ketoglutarate complex was subsequently modeled by changing arginine 140 to glutamine, and rebuilding the side chain. The structure was then minimized in two stages. First the mutated residue was allowed to relax, followed by a second relaxation of the active site residues, Mn2+
, and substrate. All modeling images were generated using PyMOL Viewer (DeLano, 2002
Constructing IDH1 and IDH2 mutants
The cDNA clone of human IDH2 (BC009244) was purchased from Invitrogen in pOTB7. Human IDH1 (BC012846.1) was purchased from ATCC in pCMV-Sport6. Standard site-directed mutagenesis techniques were used to generate IDH2 R172K by introducing a g515a change in the IDH2 open reading frame (ORF). IDH1 R132H was made by introducing a g395a base pair change in the IDH1 ORF. Wild-type and mutant sequences were then subcloned into pcDNA3 (Invitrogen) and confirmed by direct sequencing before expression in mammalian cells.
Cell culture, transfection, and metabolic labeling
293T cells and Bcl-xL-transfected SF188 cells (SF188) were cultured in DMEM (Dulbecco’s modified Eagle’s medium; Invitrogen) with 10% fetal bovine serum (CellGro). For expression of wild-type and mutant IDH1 and IDH2, cells were transfected with Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. For IDH siRNA experiments, cells were transfected with Lipofectamine RNAiMax, with oligonucleotides obtained from Sigma-Proligo. For metabolic labeling experiments, cells were cultured in glutamine-free DMEM supplemented with 4 mM [13C-U]-L-glutamine (Cambridge Isotope) for the 3h prior to metabolite extraction.
Cell lysate based enzyme assays
For IDH2 enzymatic assays, cells were lysed 48 h following transfection, using mammalian protein extraction reagent (Pierce) supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails 1 and 2 (Sigma). Lysates were sonicated and centrifuged at 14,000g at 4 °C. Supernatants were then collected and normalized for total protein concentration. To measure IDH oxidative activity, 0.3 µg of lysate protein was added to 200 µl of an assay solution containing 100 mM Tris-HCl buffer (pH 7.5), 1.3 mM MnCl2, 0.33 mM EDTA, 0.1 mM β-NADP+, and 0.1 mM D-(+)-threo-isocitrate. The increase in 340 nm absorbance (OD340) as a measure of NADPH production was measured every 20 s for 30 min on a SpectraMax 190 spectrophotometer (Molecular Devices). Data are plotted as the mean activity of 3 replicates per lysate averaged among 5 time points centered at every 5 minutes. To measure IDH reductive activity, 3 µg of lysate protein was added to 200 µl of an assay solution containing 100 mM Tris-HCl (pH 7.5), 1.3 mM MnCl2, 0.01 mM β-NADPH, and 0.5 mM α-ketoglutarate. NADPH consumption was measured as the decrease in OD340, with 3 replicates per lysate. For all experiments, OD340 changes in assay buffer lacking lysate protein were measured and subsequently subtracted from the OD340 changes measured in lysate replicates to arrive at final values.
To measure IDH2 levels in cell lysates used for enzymatic assays, aliquots of the same lysates used in activity measurements were separated by SDS-PAGE, transferred to nitrocellulose, probed with IDH2 mouse monoclonal antibody (Abcam, ab55271), and then detected with HRP-conjugated anti-mouse antibody (GE Healthcare, NA931V). For assessing IDH knockdown in siRNA experiments, cells treated in parallel with those used for labeling or proliferative studies were lysed 48 h following transfection in standard RIPA buffer (1% NaDOC, 0.1% SDS, 1% Triton X-100, 0.01 M Tris pH 8.0, 0.14 M NaCl), and then probed with IDH2 antibody as above, IDH1 goat polyclonal antibody (Santa Cruz Biotechnology, sc49996), or IDH3A rabbit polyclonal antibody (Abcam, ab58641). Actin antibody (Santa Cruz, sc1616) was also used to assess equal protein loading of Western blots.
Cellular organic acids were extracted as previously described (Bennett et al., 2008
). Briefly, after gentle removal of culture medium from proliferating cells, or freezing medium from frozen viable AML samples, cells were rapidly quenched with 80% methanol, chilled to −80°C, and then incubated at −80°C for 15 min. Extracts were subsequently transferred and centrifuged at 14,000g for 20 min. at 4°C. The organic acid pool in the supernatant was further purified by drying under nitrogen gas, redissolving in deionized water, and then elution from an AG-1 ×8 100–200 anion exchange resin (Bio-Rad) in 3 N HCl after washing with five column volumes. For media analysis, culture medium from transfected cells 24–48 h following transfection was collected and diluted five-fold with methanol. After centrifugation at 14,000g for 20 min at 4 °C to remove precipitated protein, supernatants were dried under nitrogen gas, and organic acids were purified as described above.
Gas chromatography-mass spectrometry (GC-MS) analysis
After drying the HCl eluate, samples were redissolved in a 1:1 mixture of acetonitrile and N-methyl-N-tert
-butyldimethylsilyltrifluoroacetamide (MTBSTFA; Regis) and heated for 1 h at 60°C to derivatize prior to GC-MS analysis. Samples were injected into an Agilent 7890A GC with an HP-5MS capillary column, connected to an Agilent 5975C Mass selective detector operating in splitless mode using electron impact ionization with ionizing voltage of −70 eV and electron multiplier set to 1060 V. GC temperature was started at 100 °C for 3 min, ramped to 230 °C at 4 °C/min and held for 4 min, then ramped to 300 °C and held for 5 min. Mass range of 50–500 amu was recorded at 2.71 scans/sec. Isotopic enrichment in citric acid was monitored using ions at m/e−
463 and 464 for citrate +4 and citrate +5 (containing 4 and 5 13
C-enriched atoms, respectively), formed through loss of a t
-butyl (−57 amu) and t
-butyldimethylsilanol (−132 amu) from the molecular ion tetra-TBDMS-citric acid (648 amu). Isotopomer distributions were simultaneously corrected for naturally occurring heavy isotopes of all elements in each mass fragment using a correction matrix as previously described (Weckwerth, 2007
). Identification of the 2HG metabolite peak was confirmed using standards obtained from Sigma. 2HG and glutamate signal intensities were quantified by integration of peak areas.
Liquid chromatography-mass spectrometry (LC-MS)
Organic acids from cellular extracts were purified as described above, followed by evaporation to dryness under nitrogen. After redissolving samples in deionized water, citrate was detected on two different LC-MS approaches, both of which gave comparable results. In both cases, LC separation was by reversed phase chromatography using tributylamine as an ion pairing agent (Lu et al., 2008
; Luo et al., 2007
) with ionization by negative electrospray at 23 kV. The first MS approach used a Thermo Discovery Max triple quadrupole mass spectrometer in multiple reaction monitoring mode, with citrate quantified using the reaction 191 → 87 at 20 eV. Additional reactions for every possible labeled form of citrate were also monitored using variations of the same transition. Reactions used to monitor other TCA components have been described previously (Bajad et al., 2006
). The second MS approach used a Thermo Exactive Orbitrap mass spectrometer operated at 100,000 mass resolving power, with citrate and its isotope-labeled forms quantified based on extracted ion chromatograms at their exact masses.
Most cancer-associated enzyme mutations result in either catalytic inactivation or constitutive activation. Here we report that the common feature of IDH1 and IDH2 mutations observed in AML and glioma is the acquisition of an enzymatic activity not shared by either wild-type enzyme. The product of this neomorphic enzyme activity can be readily detected in tumor samples and we show that tumor metabolite analysis can identify patients with tumor-associated IDH mutations. Using this method, we discovered a 2HG-producing IDH2 mutation, IDH2 R140Q, that was present in 9% of serial AML samples. Overall, IDH1 and IDH2 mutations were observed in over 23% of AML patients.
- All IDH mutations reported in cancer share a common neomorphic enzymatic activity.
- Both wild-type IDH1 and IDH2 are required for cell proliferation.
- IDH2 R140Q mutations occur in 9% of AML cases.
- Overall, IDH2 mutations appear more common than IDH1 mutations in AML.