A lymphoblast model for IDH2 gain-of-function activity in d-2-hydroxyglutaric aciduria type II: Novel avenues for biochemical and therapeutic studies

Abstract

The recent discovery of heterozygous isocitrate dehydrogenase 2 (IDH2) mutations of residue Arg 140 to Gln 140 or Gly 140 (IDH2 wt/R140Q, IDH2 wt/R140G) in d-2-hydroxyglutaric aciduria ( D-2-HGA) has defined the primary genetic lesion in 50% of D-2-HGA patients, denoted type II. Overexpression studies with IDH1 R132H and IDH2 R172K mutations demonstrated that the enzymes acquired a new function, converting 2-ketoglutarate (2-KG) to d-2-hydroxyglutarate ( D-2-HG), in lieu of the normal IDH reaction which reversibly converts isocitrate to 2-KG. To confirm the IDH2 wt/R140Q gain-of-function in D-2-HGA type II, and to evaluate potential therapeutic strategies, we developed a specific and sensitive IDH2 wt/R140Q enzyme assay in lymphoblasts. This assay determines gain-of-function activity which converts 2-KG to D-2-HG in homogenates of D-2-HGA type II lymphoblasts, and uses stable-isotope-labeled 2-keto[3,3,4,4- 2H 4]glutarate. The specificity and sensitivity of the assay are enhanced with chiral separation and detection of stable-isotope-labeled D-2-HG by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Eleven potential inhibitors of IDH2 wt/R140Q enzyme activity were evaluated with this procedure. The mean reaction rate in D-2-HGA type II lymphoblasts was 8-fold higher than that of controls and D-2-HGA type I cells (14.4 nmol h −1 mg protein −1 vs. 1.9), with a corresponding 140-fold increase in intracellular D-2-HG level. Optimal inhibition of IDH2 wt/R140Q activity was obtained with oxaloacetate, which competitively inhibited IDH2 wt/R140Q activity. Lymphoblast IDH2 wt/R140Q showed long-term cell culture stability without loss of the heterozygous IDH2 wt/R140Q mutation, underscoring the utility of the lymphoblast model for future biochemical and therapeutic studies.