Photoactivation of Mutant Isocitrate Dehydrogenase 2 Reveals Rapid Cancer-Associated Metabolic and Epigenetic Changes

Isocitrate dehydrogenase is mutated at a key active site arginine residue (Arg172 in IDH2) in many cancers, leading to the synthesis of the oncometabolite (R)-2-hydroxyglutarate (2HG). To investigate the early events following acquisition of this mutation in mammalian cells we created a photoactivatable version of IDH2(R172K), in which K172 is replaced with a photocaged lysine (PCK), via genetic code expansion. Illumination of cells expressing this mutant protein led to a rapid increase in the levels of 2HG, with 2HG levels reaching those measured in patient tumor samples, within 8 h. 2HG accumulation is closely followed by a global decrease in 5-hydroxymethylcytosine (5-hmC) in DNA, demonstrating that perturbations in epigenetic DNA base modifications are an early consequence of mutant IDH2 in cells. Our results provide a paradigm for rapidly and synchronously uncloaking diverse oncogenic mutations in live cells to reveal the sequence of events through which they may ultimately cause transformation.


Reagents
Photocaged lysine 1 was prepared as reported previously. 2 MitoTracker Red CMXRos was purchased from Invitrogen. Western Blot analysis was performed using an antibody against turboGFP (abcam 513). turboGFP is referred to as GFP throughout.

DNA constructs
One shot stbl3 chemically competent Escherichia coli (Invitrogen) were used for cloning steps. IDH2(R172TAG)-GFP and the PCKRS/tRNA CUA pair were cloned into PiggyBac vectors using a similar strategy as described previously. 4,5 The TAG codon was introduced into the IDH2-GFP construct (Origene, RG201152) by site-directed mutagenesis using primers R172TAG_2f/R172TAG_2r and AccuPrime Pfx Supermix (Life Technologies). The region coding IDH2-172TAG-GFP was amplified using primers IDH2_BamHI_15f/tGFP_BamHI_15r. The PCR fragment was inserted into the BamHI site of a PiggyBac targeting vector (PB533A, System Biosciences) using the In-Fusion HD cloning kit (Clontech) resulting in pPB-IDH2-172TAG-GFP. PB533A carries an EF1 promoter and Neo selection marker expressed from an IRES.
The plasmid allowing the stable integration of the orthogonal PCKRS/tRNACUA pair, pPB-4xU6-pylT-PCKRS, was derived from a PiggyBac targeting vector (PB510B1, System Biosciences). PCKRS was cloned into a BamHI site after the CMV promoter. Four copies of a U6-PylT cassette, flanked by SpeI and AvrII restriction sites, were sequentially cloned into the SpeI site of PB510B1. PB510B1 also carries a Puro selection marker driven by an EF1 promoter. pPB-IDH2-172TAG-GFP and pPB-4xU6-pylT-PCKRS were used to generate stable cell lines, as outlined below.
Cell lines were generated following the PiggyBac Transposon System protocol (System Biosciences). 48 h after transfection of the constructs pPB-IDH2-172TAG-GFP, pPB-4xU6-pylT-PCKRS and the PiggyBac Transposase expression vector (System Biosciences) using TransIT-293 (Mirus Bio), stable clones were selected for 3 days with the antibiotics G418 (100 µg/ml) and Puromycin (5 µg/ml) (InvivoGen). 0.2 mM photocaged lysine was added to antibiotic-selected cells for 48 h and single GFP positive stable clones were sorted by flow cytometry and propagated.
For photoactivation experiments, cells were grown in media containing 0.2 mM photocaged lysine 1 for 72 h. One hour prior to light-activation, the media was replaced with fresh DMEM+GlutaMAX+fetal bovine serum without amino acid 1. Photoactivation was carried out for 60 s using a 365 nm LED array positioned under the cell culture plate with a LED power of 9.5 mW per cm 2 .

Flow cytometry
Cells were washed with PBS, detached with 1X trypsin-edta (Gibco) and resuspended in PBS. The GFP fluorescence of samples was analyzed on an Eclipse Flow Cytometry Analyser (iCyt, Sony) equipped with three co-linear lasers (405 nm, 488 nm and 642 nm). GFP fluorescence was measured using the 488 nm laser and 525/50 nm optical filter. Single live cells were gated using SSC/FSC. For single cell sorting during stable cell line generation, a MoFlo Legacy cell sorter (Beckman Coulter) was used. GFP was excited using a 488 nm laser and emission was collected using a 530/40 nm band pass optical filter. Data was processed using FlowJo software.

Immunoblotting
Cells were washed with phosphate buffered saline (PBS) and lysed with RIPA buffer (Sigma) supplemented with protease inhibitor cocktail (Roche). Samples were resolved by SDS-PAGE and analyzed by immunoblotting with anti-turboGFP (Evrogen ab513) after transfer to a nitrocellulose membrane. Image Lab software (Biorad) was used.

Microscopy
Mitochondrial co-localization experiments: cells were grown in Lab-Tek chambered coverglass (Thermo Scientific), in media supplemented with 0.2 mM photocaged lysine 1 for 72 h. They were then incubated with MitoTracker Red CMXRos (Invitrogen) for 30 min, following the manufacturer's protocol. Cells were visualized on a Zeiss LSM 780 inverted confocal microscope with a 40x magnification. Microscope settings: scan resolution 1024x1024, averaging 2, scanning speed 30.98. Images were processed using ImageJ software.

Liquid Chromatography
LC-MS/MS methods were developed by PK/Bioanalytics core facility, Cancer Research UK Cambridge Institute.

Sample preparation
After having washed cells with PBS, cellular metabolites were extracted by addition of 80% aqueous methanol directly to the culture well. 6 Methanolsuspended cells were placed at -80C for a minimum of an hour, thawed, sonicated for one minute and centrifuged. The resulting supernatant was dried and reconstituted in 100 µL of 0.1% formic acid in water, followed by the addition of 50 µl of internal standard working solution. Samples were injected into the LC-MS/MS for quantitative analysis. Calibration standards and isotopically-labeled internal standards (SIL) were included (details given above in reagents section).

Preparation of calibration standards
10 mM stock solutions of 2HG or KGA were prepared in 0.1% formic acid in water. These were diluted in 0.1% formic acid in water to form calibration and quality control standards. Calibration standards ranged from 0.125 to 500 µM.

Chromatographic Conditions
The LC system used consisted of an Agilent 1290 pump with an autoinjector, sample cooler and column heater.
The sample cooler and column temperatures were 4 o C and 40 o C respectively. Analyte separation was