Isocitrate dehydrogenase 1 and 2 (IDH1/2) are key enzymes in cellular metabolism, epigenetic regulation, redox states, and DNA repair. IDH1/2 mutations are causal in the development and/or progression of various types of cancer due to supraphysiological production of d-2-hydroxyglutarate. In various tumor types, IDH1/2-mutated cancers predict for improved responses to treatment with irradiation or chemotherapy. The present review discusses the molecular basis of the sensitivity of IDH1/2-mutated cancers with respect to the function of mutated IDH1/2 in cellular processes and their interactions with novel IDH1/2-mutant inhibitors. Finally, lessons learned from IDH1/2 mutations for future clinical applications in IDH1/2 wild-type cancers are discussed.

Isocitrate dehydrogenase 1 (IDH1) is mutated in various types of human cancer to IDH1R132H, a structural alteration that leads to catalysis of α-ketoglutarate to the oncometabolite D-2-hydroxyglutarate. In this study, we present evidence that small-molecule inhibitors of IDH1R132H that are being developed for cancer therapy may pose risks with coadministration of radiotherapy. Cancer cells heterozygous for the IDH1R132H 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 IDH1R132H 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 IDH1R132H inhibitors in these patients may limit irradiation efficacy in this setting. Cancer Res; 75(22); 4790–802. ©2015 AACR.

Glioblastoma is the most common primary brain tumor with the most dismal prognosis. It is characterized by extensive invasion, migration, and angiogenesis. Median survival is only 15 months due to this behavior, rendering focal surgical resection ineffective and adequate radiotherapy impossible. At this moment, several ion channels have been implicated in glioblastoma proliferation, migration, and invasion. This paper summarizes studies on potassium, sodium, chloride, and calcium channels of glioblastoma. It provides an up-to-date overview of the literature that could ultimately lead to new therapeutic targets.

Immunohistochemistry (IHC) specifically localizes proteins in cells and tissues, but methodologies vary widely. Therefore, we performed a methodological IHC optimization and validation study. First, we compared advantages and disadvantages of cryostat sections versus paraffin sections. Second, we compared and optimized antigen retrieval in paraffin sections using citrate buffer and Tris/EDTA buffer. Third, aminoethyl carbazole (AEC) and 3,3'-diaminobenzidine (DAB) were tested as horseradish peroxidase (HRP) substrates to obtain a water-insoluble coloured end product to visualize antigens. Fourth, secondary antibodies conjugated with either mono-HRP or poly-HRP were compared. The study was performed using serial sections of human tonsil. IHC was performed with primary antibodies against endothelial cell marker CD31, smooth muscle actin (SMA), chemokine stromal-derived factor-1α (SDF-1α) and its receptor C-X-C receptor type 4 (CXCR4), macrophage marker CD68 and proliferation marker Ki67. DAB rather than AEC, and cryostat sections rather than paraffin sections gave optimum staining at highest primary antibody dilutions, whereas tissue morphology in paraffin sections was superior. Loss of antigenicity in paraffin sections by formaldehyde fixation, heat and/or masking of epitopes was counteracted by antigen retrieval but not for all antigens. Two out of six antigens (CD31 and CD68) could not be retrieved irrespective time and type of retrieval. Tris-EDTA was superior to citrate buffer for antigen retrieval. The use of mono-HRP or poly-HRP depended on the affinity of the primary antibody for its antigen. We conclude that IHC methodology optimization and validation are crucial steps for each antibody and each research question.

Glioblastoma is a highly malignant brain tumor for which no cure is available. To identify new therapeutic targets, we performed a mutation analysis of kinase genes in glioblastoma. Database mining and a literature search identified 76 kinases that have been found to be mutated at least twice in multiple cancer types before. Among those we selected 34 kinase genes for mutation analysis. We also included IDH1, IDH2, PTEN, TP53 and NRAS, genes that are known to be mutated at considerable frequencies in glioblastoma. In total, 174 exons of 39 genes in 113 glioblastoma samples from 109 patients and 16 high-grade glioma (HGG) cell lines were sequenced. Our mutation analysis led to the identification of 148 non-synonymous somatic mutations, of which 25 have not been reported before in glioblastoma. Somatic mutations were found in TP53, PTEN, IDH1, PIK3CA, EGFR, BRAF, EPHA3, NRAS, TGFBR2, FLT3 and RPS6KC1. Mapping the mutated genes into known signaling pathways revealed that the large majority of them plays a central role in the PI3K-AKT pathway. The knowledge that at least 50% of glioblastoma tumors display mutational activation of the PI3K-AKT pathway should offer new opportunities for the rational development of therapeutic approaches for glioblastomas. However, due to the development of resistance mechanisms, kinase inhibition studies targeting the PI3K-AKT pathway for relapsing glioblastoma have mostly failed thus far. Other therapies should be investigated, targeting early events in gliomagenesis that involve both kinases and non-kinases.