IDH1 and IDH2 Mutations In Pediatric Acute Myeloid Leukemia
Authors: Andersson Anna , Miller David , Lynch John , Lemoff Andrew , Cai Zhongling , Pounds Stanley , Radtke Ina , Bing Yan , John Schuetz , Jeffrey Rubnitz , Ribeiro Raul , Raimondi Susana C. , Zhang Jinghui , Shurtleff Sheila , Schulman Brenda , Downing James 
Published: October 2019

Abstract
Abstract 1699
Pediatric de novo acute myeloid leukemia (AML) is a heterogeneous disease that can be divided into clinically distinct subtypes based on the presence of specific chromosomal abnormalities, gene mutations, or morphologic and immunophenotypic features. The best characterized subtypes include leukemias with alterations of the gene encoding the core-binding transcription factor complex, (t(8;21)[AML1- ETO] and inv(16)/t(16;16)[CBFβ-MYH11], rearrangements of the MLL gene on chromosome 11q23, normal cytogenetics, or distinct morphology including acute promyeloctic leukemia with t(15;17)[PML-RARA] and acute megakaryoblastic leukemia (FAB-M7). In AMLs with normal cytogenetics, mutations have also been identified in a number of genes, with alterations in NPM1, FLT3 and CEBPA occurring at an appreciable frequency and influencing therapeutic responses. Recent genome-wide sequencing efforts have led to the identification of a number of new candidate genes involved in the pathogenesis of this disease. Foremost among this list are isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2). IDH1 mutations were initially identified in a whole exome sequencing of glioblastoma multiforme (GBM), but were subsequently shown to be mutated in a variety of myeloid malignancies including up to 16% of adult AMLs with normal cytogenetics. The mutations in both GBM and myeloid malignancies have been heterozygous and restricted to arginine 132 in exon 4 of IDH1, or to either the homologous residue in IDH2, R172, or to a second arginine, R140, also located in its substrate binding pocket. Although the distribution of specific IDH1/IDH2 mutations varies between GBM and AML, each of the analyzed mutations result in a loss of the enzymes ability to catalyze the oxidative carboxylation of isocitrate to a-ketoglutarate (a-KG), coupled with a gain-of-function to catalyze the NADPH-dependent reduction of a–KG to 2-hydroxyglutarate (2-HG). This shift in enzymatic activity results in a dramatic increase in the levels of 2-HG within the leukemic cells; however, how the increase in this metabolite contributes to transformation remains to be determined.
To investigate the frequency of IDH1 and IDH2 mutations in pediatric AML, we sequenced these genes in diagnostic samples from 227 pediatric AML patients. Our analysis identified somatic IDH1/2 mutations in 4% of cases (IDH1 N=3 and IDH2 N=5), with the frequency slightly higher in AMLs with a normal karyotype (7%). IDH1 mutations occurred in codon 132 resulting in replacement of arginine with either cysteine (N=2) or histidine (N=1). By contrast, the mutations in IDH2 did not affect the homologous residue but instead altered codon 140, resulting in replacement of an arginine with either glutamine (N=4) or tryptophan (N=1). Structural modeling studies of IDH2 suggested that the codon 140 mutations should disrupt the enzyme's ability to bind its substrate isocitrate. Consistent with this prediction, enzymology studies showed that recombinant IDH2 R140Q and R140W 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-hydroxyglutarate (2-HG). Analysis of primary leukemic blasts using mass spectrometry confirmed high levels of 2-HG in samples with IDH1/2 mutations. Interestingly, 3/5 leukemias also had FLT3 activating mutations, raising the possibility that these two mutations directly cooperate in leukemogenesis. Defining the biological role of the IDH1/2 mutations in leukemogenesis will benefit by a direct assessment of the biological effect of the mutations on normal murine hematopoietic cell differentiation using both in vitro and in vivo systems.

Disclosures:
No relevant conflicts of interest to declare.