IDH1 and IDH2 mutations in pediatric acute leukemia

Abstract

To investigate the frequency of isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) mutations in pediatric acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL), we sequenced these genes in diagnostic samples from 515 patients (227 AMLs and 288 ALLs). Somatic IDH1/IDH2 mutations were rare in ALL (N=1), but were more common in AML, occurring in 3.5% (IDH1 N=3 and IDH2 N=5), with the frequency higher in AMLs with a normal karyotype (9.8%). The identified IDH1 mutations occurred in codon 132 resulting in replacement of arginine with either cysteine (N=3) or histidine (N=1). By contrast, mutations in IDH2 did not affect the homologous residue but instead altered codon 140, resulting in replacement of arginine with either glutamine (N=4) or tryptophan (N=1). Structural modeling of IDH2 suggested that codon 140 mutations disrupt the enzyme's ability to bind its substrate isocitrate. Accordingly, recombinant IDH2 R140Q/W were unable to carry out the decarboxylation of isocitrate to α-ketoglutarate (α-KG), but instead gained the neomorphic activity to reduce α-KG to R(-)-2-hydroxyglutarete (2-HG). Analysis of primary leukemic blasts confirmed high levels of 2-HG in AMLs with IDH1/IDH2 mutations. Interestingly, 3/5 AMLs with IDH2 mutations had FLT3-activating mutations, raising the possibility that these mutations cooperate in leukemogenesis.

Figure 1

Genomic structure of IDH1 and IDH2 and structural model of the location of R140 and R172 in the substrate-binding pocket of IDH2. (a) Illustration of the exon/intron structure of IDH1 and IDH2. The location within exon 4 of codon R132 in IDH1 and codon R140 in IDH2 are marked by arrows, and the surrounding nucleotide sequence and encoded amino acids are highlighted. Codon R132 in IDH1 and the homologous residue R172 in IDH2 are shown in red and codon 140 in IDH2 is shown in blue. (b) Model of the positions of the R140 and R172 amino acids in the IDH2 substrate binding pocket. The two protomers in the IDH2 homodimer are illustrated in green and purple, with the nitrogen atoms in amino acids R140 and R172 shown in blue. The bound substrate, isocitrate, is depicted with carbon atoms as yellow sticks and oxygen atoms in red. The manganese ion is shown as a grey sphere. Salt-bridges and hydrogen bonds are shown as dashed lines.

Figure 2

Enzymatic analysis of the IDH1 and IDH2 mutant proteins. The activity of recombinant IDH1 and IDH2 proteins to catalyze the NADP⁺-dependent oxidative decarboxylation of isocitrate to α-KG using 30uM Isocitrate and 100uM NADP are shown in panels a and b, respectively, and their ability to catalyze the NADPH-dependent reduction of α–KG to 2-HG using 0.5 mM α-KG and 100uM NADPH are shown in c and d, respectively. All graphs are based on triplicate measurements with the mean ± standard deviations expressed as a relative level compared to WT:WT homodimers, with the latter set as 100%. The SD in panel c and d are < 0.03 and are thus below the resolution of the figure. (e) The intracellular level of 2-HG was measured by liquid chromatography/mass spectrometry in pediatric AML cells from primary diagnostic bone marrow samples. The data for mutant IDH1/IDH2 includes two leukemia samples containing IDH1 mutations (one with R132H and one with R132C, ▴), and two containing the R140Q IDH2 mutations (◆). The wild-type IDH1/IDH2 data was generated using two pediatric AML samples that lacked mutations in either IDH1 or IDH2 (●).