Isocitrate dehydrogenase 1 (IDH1) is mutated in various types of human cancer to IDH1 R132H , a structural alteration that leads to catalysis of a-ketoglutarate to the oncometabolite D-2-hydroxyglutarate. In this study, we present evidence that small-molecule inhibitors of IDH1 R132H that are being developed for cancer therapy may pose risks with coadministration of radiotherapy. Cancer cells heterozygous for the IDH1 R132H mutation exhibited less IDH-mediated production of NADPH, such that after exposure to ionizing radiation (IR), there were higher levels of reactive oxygen species, DNA double-strand breaks, and cell death compared with IDH1 wild-type cells. These effects were reversed by the IDH1 R132H inhibitor AGI-5198. Exposure of IDH1 wild-type cells to D-2-hydroxyglutarate was sufficient to reduce IDH-mediated NADPH production and increase IR sensitivity. Mechanistic investigations revealed that the radiosensitivity of heterozygous cells was independent of the well-described DNA hypermethylation phenotype in IDH1-mutated cancers. Thus, our results argue that altered oxidative stress responses are a plausible mechanism to understand the radiosensitivity of IDH1-mutated cancer cells. Further, they offer an explanation for the relatively longer survival of patients with IDH1-mutated tumors, and they imply that administration of IDH1 R132H inhibitors in these patients may limit irradiation efficacy in this setting.

Yeast cells were grown in glucose-limited chemostat cultures and forced to switch to a new carbon source, the fatty acid oleate. Alterations in gene expression were monitored using DNA microarrays combined with bioinformatics tools, among which was included the recently developed algorithm REDUCE. Immediately after the switch to oleate, a transient and very specific stress response was observed, followed by the up-regulation of genes encoding peroxisomal enzymes required for fatty acid metabolism. The stress response included up-regulation of genes coding for enzymes to keep thioredoxin and glutathione reduced, as well as enzymes required for the detoxification of reactive oxygen species. Among the genes coding for various isoenzymes involved in these processes, only a specific subset was expressed. Not the general stress transcription factors Msn2 and Msn4, but rather the specific factor Yap1p seemed to be the main regulator of the stress response. We ascribe the initiation of the oxidative stress response to a combination of poor redox flux and fatty acid-induced uncoupling of the respiratory chain during the metabolic reprogramming phase. INTRODUCTIONAerobic life is associated with the production of reactive oxygen species (ROS) by various metabolic processes. ROS can modify lipids, proteins, and nucleic acids and can particularly cause mutations in DNA, which might contribute to tumor formation. Normally, ROS production is kept at bay by a variety of detoxifying enzymes, some of which derive their reducing power from glutathione (GSH) or thioredoxins (TRXs) (Hohmann and Mager, 1997;Jamieson and Storz, 1997;Grant et al., 1998). However, in certain pathological conditions caused by tissue damage or during treatment with certain pharmaceuticals, this protection fails, probably due to a compromised redox state: [NAD(P)H/ NAD(P)]. Although the mitochondrial respiratory chain is an important source of ROS, peroxisomal metabolism is another contributor in this respect. For instance, in rodents, application of hypolipidemic drugs resulted in enlargement of the peroxisome compartment, and long-term treatment even caused cancer (Lock et al., 1989;Reddy and Mannaerts, 1994).Peroxisomes house a number of oxidative enzymes producing ROS, such as H 2 O 2 , which is formed during the ␤-oxidation of fatty acids (Beevers, 1969;Van den Bosch et al., 1992). In the classic view, the raison d'etre of the organelle is to provide a boundary to keep ROS confined within a compartment where they can be quickly detoxified. Several considerations indicate that this concept may be too simple (Tabak et al., 1999). H 2 O 2 can easily permeate through membranes and loss of peroxisomal catalase remains without symptoms. Is this due to the fact that other detoxifying enzymes come to the rescue? There are indeed suggestions that peroxisomes harbor additional GSH or thioredoxin-dependent detoxifying enzymes (Jeong et al., 1999;Lee et al., 1999b), but it may also be that cytosolic enzymes are recruited. An opportunity to study the role of perox...

Dihydropyrimidine dehydrogenase (DPD) is the initial enzyme acting in the catabolism of the widely used antineoplastic agent 5-fluorouracil (5FU). DPD deficiency is known to cause a potentially lethal toxicity following administration of 5FU. Here, we report novel genetic mechanisms underlying DPD deficiency in patients presenting with grade III/IV 5FU-associated toxicity. In one patient a genomic DPYD deletion of exons 21–23 was observed. In five patients a deep intronic mutation c.1129–5923C>G was identified creating a cryptic splice donor site. As a consequence, a 44 bp fragment corresponding to nucleotides c.1129–5967 to c.1129–5924 of intron 10 was inserted in the mature DPD mRNA. The deleterious c.1129–5923C>G mutation proved to be in cis with three intronic polymorphisms (c.483 + 18G>A, c.959–51T>G, c.680 + 139G>A) and the synonymous mutation c.1236G>A of a previously identified haplotype. Retrospective analysis of 203 cancer patients showed that the c.1129–5923C>G mutation was significantly enriched in patients with severe 5FU-associated toxicity (9.1%) compared to patients without toxicity (2.2%). In addition, a high prevalence was observed for the c.1129–5923C>G mutation in the normal Dutch (2.6%) and German (3.3%) population. Our study demonstrates that a genomic deletion affecting DPYD and a deep intronic mutation affecting pre-mRNA splicing can cause severe 5FU-associated toxicity. We conclude that screening for DPD deficiency should include a search for genomic rearrangements and aberrant splicing.Electronic supplementary materialThe online version of this article (doi:10.1007/s00439-010-0879-3) contains supplementary material, which is available to authorized users.