Mutations in the metabolic enzyme isocitrate dehydrogenase (IDH) were recently found in ∼80% of WHO grade II-IV gliomas. These mutations inhibit the enzyme's ability to convert isocitrate to α-ketoglutarate and, instead, confer a novel gain-of-function resulting in the conversion of α-ketoglutarate to 2-hydroxglutarate. However, the fundamental mechanism(s) by which these mutations affect glioma cell growth remain unclear. IDH1 and 2 function as homodimers in the cytosol and mitochondria, respectively. Dimerization is mediated by interactions between the two clasp domains of each IDH protein and dimeric IDH contains two active sites each composed of amino acid residues from both subunits. Thus, the dimerization of IDH is vital for its enzymatic role within the cell. Interestingly, IDH mutations have been observed as heterozygous events in glioma cells suggesting that the cells require the wild-type IDH protein in order to function. To examine and visualize the interaction between wild-type and mutant IDH proteins, we employed the protein-protein interaction assay: Bimolecular Fluorescence Complementation (BiFC). This approach is based on the formation of a fluorescent complex though the association of two fragments of a fluorescent reporter protein fused to two proteins of interest. An interaction between the proteins facilitates association between the two fragments to produce a bimolecular fluorescent complex. Since IDH1 has a C-terminal peroxisomal targeting sequence, the N-terminal 172 amino acid residues of Venus (VN) were fused to the N-terminus of wild-type IDH1 and residues 155-238 amino acids of Venus (VC) were fused to the N-terminus of mutant IDH1. IDH2, however, has an N-terminal mitochondrial targeting sequence and thus the N-terminal and C-terminal fragments of Venus were fused to the C-terminus of wild-type and mutant IDH2, respectively. 293FT cells were transfected with each fluorescent reporter construct and counterstained with nuclear and cytosolic markers to confirm complex formation and cellular localization. Approximately 60% of the cells showed evidence of IDH complex formation. We confirmed localization to the cytosol/peroxisomes and mitochondria for wild-type and mutant IDH1 and IDH2 heterodimers, respectively, and observed that, in the case of IDH1, wild-type and mutant heterodimers demonstrate physiologically normal activity compared to inactive mutant IDH1 homodimers. Conversely, we observed no significant difference between wild-type and mutant IDH2 heterodimers when compared to mutant IDH2 homodimers. This data suggests that mutant IDH1 requires wild-type IDH1 in order to be enzymatically active while mutant IDH2 does not require wild-type IDH2. To determine the effect of wildtype and mutant IDH heterodimer formation on glioma cell growth, we plan to assess the tumorigenic potential of IDH1 heterodimers either alone or in concert with other known glioma genetic abnormalities in vivo.
Citation Format: Gemma L. Robinson, Matthew R. Guthrie, Marytheresa Ifediba, Matthew W. VanBrocklin, Sheri L. Holmen. Defining the role of mutant isocitrate dehydrogenase in malignant glioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2438. doi:10.1158/1538-7445.AM2014-2438