Disruption of wild-type IDH1 suppresses D-2-hydroxyglutarate production in IDH1-mutated gliomas

Abstract

Point mutations at Arg132 of the cytoplasmic NADP(+)-dependent isocitrate dehydrogenase 1 (IDH1) occur frequently in gliomas and result in a gain of function to produce the "oncometabolite" D-2-hydroxyglutarate (D-2HG). The mutated IDH1 allele is usually associated with a wild-type IDH1 allele (heterozygous) in cancer. Here, we identify 2 gliomas that underwent loss of the wild-type IDH1 allele but retained the mutant IDH1 allele following tumor progression from World Health Organization (WHO) grade III anaplastic astrocytomas to WHO grade IV glioblastomas. Intratumoral D-2HG was 14-fold lower in the glioblastomas lacking wild-type IDH1 than in glioblastomas with heterozygous IDH1 mutations. To characterize the contribution of wild-type IDH1 to cancer cell D-2HG production, we established an IDH1-mutated astrocytoma (IMA) cell line from a WHO grade III anaplastic astrocytoma. Disruption of the wild-type IDH1 allele in IMA cells by gene targeting resulted in an 87-fold decrease in cellular D-2HG levels, showing that both wild-type and mutant IDH1 alleles are required for D-2HG production in glioma cells. Expression of wild-type IDH1 was also critical for mutant IDH1-associated D-2HG production in the colorectal cancer cell line HCT116. These insights may aid in the development of therapeutic strategies to target IDH1-mutated cancers.

Figure 1

IDH1^R132H/− gliomas have lower D-2HG than IDH1^R132H/WT gliomas. A, Two anaplastic astrocytomas WHO grade III (A3) which progressed to glioblastomas WHO grade IV (sGBM) demonstrate loss of the wild type IDH1 allele and decreased levels of D-2HG. Mutant IDH1^R132H is CAT (His); wild type IDH1 is CGT (Arg). B, D-2HG levels in human gliomas with wild type IDH1 (WT/WT), heterozygous IDH1 mutation (R132H/WT), or IDH1 mutation with loss of wild type IDH1 (R132H/−). LGG, lower grade glioma WHO grade II or III.

Figure 2

Establishment of an IDH1 mutated anaplastic astrocytoma WHO grade III cell line (IMA). A, Representative sequencing chromatograms for IDH1 codon 132 in gDNA and cDNA. Both the primary tumor and IMA are heterozygous for wild type (CGT) and mutant (CAT) alleles coding for an Arginine (R) to Histidine (H) change at amino acid residue 132. B, Bright field image of adherent IMA cells. C, D-2HG level in lysates of IMA (IDH1^R132H/WT), two IDH1 wild type allele knocked out subclones KO-1 and KO-2 (IDH1^R132H/−), and HCT116, HOG and 293 cells which do not contain IDH1 mutations. *D-2HG was significantly higher in IMA for pairwise comparisons with each other cell line (P<0.0001 for each, Student's t test). Mean ± s.d. are shown from lysates collected in triplicate.

Figure 3

Targeted knockout of wild type IDH1 in IMA. A, Targeting vector used to disrupt expression of one allele of IDH1. The targeting vector contains a splice acceptor (SA), internal ribosomal entry sequence (IRES), neomycin selectable marker (neo), and a polyadenylation site (pA), all flanked by right and left homology arms (LHA and RHA, respectively) and inverted terminal repeats (ITR). B, Diagnostic PCR confirms homologous integration of the targeting vector into the IDH1 genomic locus for clones KO-1 and KO-2, but not for the parental IMA cells. Diagnostic PCR primer pairs P1 and P2 each employ one primer that anneals within the targeting vector and a second primer that is outside the homology region to specifically amplify DNA that has incorporated the targeting vector. C, IDH1 R132 gDNA sequencing shows that the targeting vector disrupted the IDH1^WT allele and not the IDH1^R132H allele in KO-1 and KO-2. The sequence of PCR product P3 reflects the genotype of both the intact and targeted alleles, while P4 reflects the genotype of the intact allele only. D, cDNA sequencing confirms that KO-1 and KO-2 only express IDH1^R132H, and not IDH1^WT.

Figure 4

Efficient D-2HG production in IDH1 mutant cells requires wild type IDH1. A, KO-1 (IDH1^R132H/−) cells were infected with lentivirus expressing wild type IDH1^WT or IDH1^R132H. After one month of selection, cell lysates were prepared for western blots, using cells infected with empty vector (EV) lentivirus as a control. B, D-2HG levels in lysates from indicated KO-1 (IDH1^R132H/−) cells with ectopic expression of vector control, wild type IDH1^WT, or IDH1^R132H. C, KO-1 (IDH1^R132H/−) cells were treated with the indicated concentrations of cell permeable octyl-αKG for 20 hours, followed by measurement of cellular αKG and D-2HG levels. Mean ± s.d. are shown for samples analyzed in triplicate and are representative of two independent experiments. *P<0.05, **P<0.0001, Student's t test.