Abstract
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.
Highlights
Cytosolic and peroxisomal IDH1 contains an Arg132His mutation (IDH1 R132H), while mutant mitochondrial IDH2, contains the analogous Arg172Lys mutation (IDH2 R172K)
The mutant forms of isocitrate dehydrogenase (IDH) catalyze an additional reaction: the NADPH-dependent reduction of KGA to the (R)-enantiomer of 2-hydroxyglutarate [(R)-2HG] (Scheme 1b).[4−6] (R)-2HG has been proposed to be an oncometabolite, which together with the mutant enzyme is capable of driving cancerassociated cellular transformations.[7,8] (R)-2HG is believed to drive transformation, in part, by competitively inhibiting KGAdependent dioxygenase enzymes
In a large proportion of cancers, including low grade gliomas, secondary glioblastoma multiforme, and acute myeloid leukemias, IDH is mutated at a key active site arginine residue
Summary
The approaches developed have allowed photocontrol of nuclear localization sequences,[18] and several enzymes, including kinases,[22] proteases,[23] inteins,[24,25] and cas[9,26] as well as photocontrol of the sites of post-translational modification.[16,19] These approaches have revealed the kinetics of complex signaling pathways with high spatial and temporal resolution[22] and provided approaches to spatially and temporally control tools for proteome and genome editing.[23,26]. The half-life of IDH2(R172PCK)-GFP is approximately 48 h, providing an upper limit on the length of time after photoactivation for which measurements will result from a consistent level of mutant protein (Supplementary Figure S2). There is a lag in measurable 5-hmC depletion (Figure 2c) with respect to the rapid accumulation of 2HG in cells (Figure 2b, Supplementary Figure S3), and 5-hmC levels remain depressed at 48 and 72 h after illumination, when intracellular levels of 2HG have decreased (Figure 2c, Supplementary Figure S3) These observations are consistent with (i) the slow formation of 5-hmC from 5-mC, and (ii) the stability of 5-hmC.[31] At early time points, most of the 5-hmC measured is present before illumination, and it takes time for a measurable fraction of total 5-hmC to result from postillumination synthesis, and for total 5-hmC levels to respond to 2HG levels.
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