PFKFB4 interacts with FBXO28 to promote HIF-1α signaling in glioblastoma.

Glioblastoma is a highly aggressive brain tumor for which there is no cure. The metabolic enzyme 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 4 (PFKFB4) is essential for glioblastoma stem-like cell (GSC) survival but its mode of action is unclear. Understanding the role of PFKFB4 in tumor cell survival could allow it to be leveraged in a cancer therapy. Here, we show the importance of PFKFB4 for glioblastoma growth in vivo in an orthotopic patient derived mouse model. In an evaluation of patient tumor samples of different cancer entities, PFKFB4 protein was found to be overexpressed in prostate, lung, colon, mammary and squamous cell carcinoma, with expression level correlating with tumor grade. Gene expression profiling in PFKFB4-silenced GSCs revealed a downregulation of hypoxia related genes and Western blot analysis confirmed a dramatic reduction of HIF (hypoxia inducible factor) protein levels. Through mass spectrometric analysis of immunoprecipitated PFKFB4, we identified the ubiquitin E3 ligase, F-box only protein 28 (FBXO28), as a new interaction partner of PFKFB4. We show that PFKFB4 regulates the ubiquitylation and subsequent proteasomal degradation of HIF-1α, which is mediated by the ubiquitin ligase activity of FBXO28. This newly discovered function of PFKFB4, coupled with its cancer specificity, provides a new strategy for inhibiting HIF-1α in cancer cells.

Pan-mutant IDH1 inhibitor BAY 1436032 for effective treatment of IDH1 mutant astrocytoma in vivo.

Mutations in codon 132 of isocitrate dehydrogenase (IDH) 1 are frequent in diffuse glioma, acute myeloid leukemia, chondrosarcoma and intrahepatic cholangiocarcinoma. These mutations result in a neomorphic enzyme specificity which leads to a dramatic increase of intracellular D-2-hydroxyglutarate (2-HG) in tumor cells. Therefore, mutant IDH1 protein is a highly attractive target for inhibitory drugs. Here, we describe the development and properties of BAY 1436032, a pan-inhibitor of IDH1 protein with different codon 132 mutations. BAY 1436032 strongly reduces 2-HG levels in cells carrying IDH1-R132H, -R132C, -R132G, -R132S and -R132L mutations. Cells not carrying IDH mutations were unaffected. BAY 1436032 did not exhibit toxicity in vitro or in vivo. The pharmacokinetic properties of BAY 1436032 allow for oral administration. In two independent experiments, BAY 1436032 has been shown to significantly prolong survival of mice intracerebrally transplanted with human astrocytoma carrying the IDH1R132H mutation. In conclusion, we developed a pan-inhibitor targeting tumors with different IDH1R132 mutations.

IDH2 Mutations Define a Unique Subtype of Breast Cancer with Altered Nuclear Polarity.

Solid papillary carcinoma with reverse polarity (SPCRP) is a rare breast cancer subtype with an obscure etiology. In this study, we sought to describe its unique histopathologic features and to identify the genetic alterations that underpin SPCRP using massively parallel whole-exome and targeted sequencing. The morphologic and immunohistochemical features of SPCRP support the invasive nature of this subtype. Ten of 13 (77%) SPCRPs harbored hotspot mutations at R172 of the isocitrate dehydrogenase IDH2, of which 8 of 10 displayed concurrent pathogenic mutations affecting PIK3CA or PIK3R1 One of the IDH2 wild-type SPCRPs harbored a TET2 Q548* truncating mutation coupled with a PIK3CA H1047R hotspot mutation. Functional studies demonstrated that IDH2 and PIK3CA hotspot mutations are likely drivers of SPCRP, resulting in its reversed nuclear polarization phenotype. Our results offer a molecular definition of SPCRP as a distinct breast cancer subtype. Concurrent IDH2 and PIK3CA mutations may help diagnose SPCRP and possibly direct effective treatment. Cancer Res; 76(24); 7118-29. ©2016 AACR.

A vaccine targeting mutant IDH1 induces antitumour immunity.

Monoallelic point mutations of isocitrate dehydrogenase type 1 (IDH1) are an early and defining event in the development of a subgroup of gliomas and other types of tumour. They almost uniformly occur in the critical arginine residue (Arg 132) in the catalytic pocket, resulting in a neomorphic enzymatic function, production of the oncometabolite 2-hydroxyglutarate (2-HG), genomic hypermethylation, genetic instability and malignant transformation. More than 70% of diffuse grade II and grade III gliomas carry the most frequent mutation, IDH1(R132H) (ref. 3). From an immunological perspective, IDH1(R132H) represents a potential target for immunotherapy as it is a tumour-specific potential neoantigen with high uniformity and penetrance expressed in all tumour cells. Here we demonstrate that IDH1(R132H) contains an immunogenic epitope suitable for mutation-specific vaccination. Peptides encompassing the mutated region are presented on major histocompatibility complexes (MHC) class II and induce mutation-specific CD4(+) T-helper-1 (TH1) responses. CD4(+) TH1 cells and antibodies spontaneously occurring in patients with IDH1(R132H)-mutated gliomas specifically recognize IDH1(R132H). Peptide vaccination of mice devoid of mouse MHC and transgenic for human MHC class I and II with IDH1(R132H) p123-142 results in an effective MHC class II-restricted mutation-specific antitumour immune response and control of pre-established syngeneic IDH1(R132H)-expressing tumours in a CD4(+) T-cell-dependent manner. As IDH1(R132H) is present in all tumour cells of these slow-growing gliomas, a mutation-specific anti-IDH1(R132H) vaccine may represent a viable novel therapeutic strategy for IDH1(R132H)-mutated tumours.

D-2-Hydroxyglutarate producing neo-enzymatic activity inversely correlates with frequency of the type of isocitrate dehydrogenase 1 mutations found in glioma.

BACKGROUND: IDH mutations frequently occur in diffuse gliomas and result in a neo-enzymatic activity that results in reduction of α-ketoglutarate to D-2-hydroxyglutarate. In gliomas, the frequency of IDH1 mutations in codon 132 increases in the order R132L-R132S-R132G-R132C-R132H with R132H constituting more than 90% of all IDH1 mutations. RESULTS: We determined the levels of D-2-hydroxyglutarate in glioma tissues with IDH1 mutations. D-2-hydroxyglutarate levels increased in the order of R132H-R132C-R132S/R132G/R132L. We expressed and purified IDH1 wild type and mutant protein for biochemical characterization. Enzyme kinetics of mutant IDH protein correlated well with D-2-hydroxyglutarate production in cells with R132H exhibiting the highest and R132L the lowest KM for α-ketoglutarate. Addition of D-2-hydroxyglutarate to the medium of cell lines revealed an inhibitory effect at higher concentrations. Migration of LN229 increased at lower D-2-hydroxyglutarate concentrations while higher concentrations showed no effect. CONCLUSION: These findings may suggest natural selection against the rare IDH1R132 mutations in human glioma due to toxicity caused by high levels of D-2-hydroxyglutarate.

Isocitrate dehydrogenase 1 mutant R132H sensitizes glioma cells to BCNU-induced oxidative stress and cell death.

Isocitrate dehydrogenase 1 (IDH1) decarboxylates isocitrate to α-ketoglutarate (α-KG) leading to generation of NADPH, which is required to regenerate reduced glutathione (GSH), the major cellular ROS scavenger. Mutation of R132 of IDH1 abrogates generation of α-KG and leads to conversion of α-KG to 2-hydroxyglutarate. We hypothesized that glioma cells expressing mutant IDH1 have a diminished antioxidative capacity and therefore may encounter an ensuing loss of cytoprotection under conditions of oxidative stress. Our study was performed with LN229 cells stably overexpressing IDH1 R132H and wild type IDH1 or with a lentiviral IDH1 knockdown. Quantification of GSH under basal conditions and following treatment with the glutathione reductase inhibitor BCNU revealed significantly lower GSH levels in IDH1 R132H expressing cells and IDH1 KD cells compared to their respective controls. FACS analysis of cell death and ROS production also demonstrated an increased sensitivity of IDH1-R132H-expressing cells and IDH1 KD cells to BCNU, but not to temozolomide. The sensitivity of IDH1-R132H-expressing cells and IDH1 KD cells to ROS induction and cell death was further enhanced with the transaminase inhibitor aminooxyacetic acid and under glutamine free conditions, indicating that these cells were more addicted to glutaminolysis. Increased sensitivity to BCNU-induced ROS production and cell death was confirmed in HEK293 cells inducibly expressing the IDH1 mutants R132H, R132C and R132L. Based on these findings we propose that in addition to its established pro-tumorigenic effects, mutant IDH1 may also limit the resistance of gliomas to specific death stimuli, therefore opening new perspectives for therapy.

A novel, diffusely infiltrative xenograft model of human anaplastic oligodendroglioma with mutations in FUBP1, CIC, and IDH1.

Oligodendroglioma poses a biological conundrum for malignant adult human gliomas: it is a tumor type that is universally incurable for patients, and yet, only a few of the human tumors have been established as cell populations in vitro or as intracranial xenografts in vivo. Their survival, thus, may emerge only within a specific environmental context. To determine the fate of human oligodendroglioma in an experimental model, we studied the development of an anaplastic tumor after intracranial implantation into enhanced green fluorescent protein (eGFP) positive NOD/SCID mice. Remarkably after nearly nine months, the tumor not only engrafted, but it also retained classic histological and genetic features of human oligodendroglioma, in particular cells with a clear cytoplasm, showing an infiltrative growth pattern, and harboring mutations of IDH1 (R132H) and of the tumor suppressor genes, FUBP1 and CIC. The xenografts were highly invasive, exhibiting a distinct migration and growth pattern around neurons, especially in the hippocampus, and following white matter tracts of the corpus callosum with tumor cells accumulating around established vasculature. Although tumors exhibited a high growth fraction in vivo, neither cells from the original patient tumor nor the xenograft exhibited significant growth in vitro over a six-month period. This glioma xenograft is the first to display a pure oligodendroglioma histology and expression of R132H. The unexpected property, that the cells fail to grow in vitro even after passage through the mouse, allows us to uniquely investigate the relationship of this oligodendroglioma with the in vivo microenvironment.

Enzymatic assay for quantitative analysis of (D)-2-hydroxyglutarate.

Levels of (D)-2-hydroxyglutarate [D2HG, (R)-2-hydroxyglutarate] are increased in some metabolic diseases and in neoplasms with mutations in the isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) genes. Determination of D2HG is of relevance to diagnosis and monitoring of disease. Standard detection methods of D2HG levels are liquid-chromatography-mass spectrometry or gas-chromatography-mass spectrometry. Here we present a rapid, inexpensive and sensitive enzymatic assay for the detection of D2HG levels. The assay is based on the conversion of D2HG to α-ketoglutarate (αKG) in the presence of the enzyme (D)-2-hydroxyglutarate dehydrogenase (HGDH) and nicotinamide adenine dinucleotide (NAD(+)). Determination of D2HG concentration is based on the detection of stoichiometrically generated NADH. The quantification limit of the enzymatic assay for D2HG in tumor tissue is 0.44 µM and in serum 2.77 µM. These limits enable detection of basal D2HG levels in human tumor tissues and serum without IDH mutations. Levels of D2HG in frozen and paraffin-embedded tumor tissues containing IDH mutations or in serum from acute myeloid leukemia patients with IDH mutations are significantly higher and can be easily identified with this assay. In conclusion, the assay presented is useful for differentiating basal from elevated D2HG levels in tumor tissue, serum, urine, cultured cells and culture supernatants.

Detection of 2-hydroxyglutarate in formalin-fixed paraffin-embedded glioma specimens by gas chromatography/mass spectrometry.

Mutations in the isocitrate dehydrogenase (IDH) 1 and 2 genes occur frequently in diffuse astrocytoma and oligodendroglioma. The consecutive amino acid substitutions in the mutant proteins result in a gain of the function to catalyze the reduction of alpha-ketoglutarate to 2-hydroxyglutarate (2HG). So far, all investigated IDH mutations share this gain of function. We here describe a method to detect 2HG levels in archival formalin-fixed paraffin-embedded tumor specimens by stable isotope dilution using gas chromatography followed by mass spectrometry (GC/MS). While 2HG levels are notably decreased during the routine embedding process, preserved amounts are still sufficient to indicate a mutation. Detection of 2HG in archival specimens could make routinely processed tissue accessible for research on 2HG accumulation and may allow studies on correlation with clinical data.

Characterization of R132H mutation-specific IDH1 antibody binding in brain tumors.

Heterozygous point mutations of isocitrate dehydrogenase (IDH)1 codon 132 are frequent in grade II and III gliomas. Recently, we reported an antibody specific for the IDH1R132H mutation. Here we investigate the capability of this antibody to differentiate wild type and mutated IDH1 protein in central nervous system (CNS) tumors by Western blot and immunohistochemistry. Results of protein analysis are correlated to sequencing data. In Western blot, anti-IDH1R132H mouse monoclonal antibody mIDH1R132H detected a specific band only in mutated tumors. Immunohistochemistry of 345 primary brain tumors demonstrated a strong cytoplasmic and weaker nuclear staining in 122 cases. Correlation with direct sequencing of 186 cases resulted in consensus of 177 cases. Genetic retesting of cases with conflicting findings resulted in a match of 186/186 cases, with all discrepancies resolving in favor of immunohistochemistry. Intriguing is the ability of mIDH1R132H to detect single infiltrating tumor cells. The very high frequency and the distribution of this mutation among specific brain tumor entities allow the highly sensitive and specific discrimination of various tumors by immunohistochemistry, such as anaplastic astrocytoma from primary glioblastoma or diffuse astrocytoma World Health Organization (WHO) grade II from pilocytic astrocytoma or ependymoma. Noteworthy is the discrimination of the infiltrating edge of tumors with IDH1 mutation from reactive gliosis.

PCR- and restriction endonuclease-based detection of IDH1 mutations.

Hotspot mutations in codon 132 of the gene encoding isocitrate dehydrogenase 1 (IDH1) have emerged as the most frequent DNA alteration in astrocytomas, oligodendrogliomas and oligoastrocytomas. These mutations have been shown to be of significant diagnostic and prognostic value. So far, assessment of IDH1 mutation relied on DNA sequencing techniques. We generated a set of primers suitable for endonuclease-based detection of hotspot mutations in codon 132 of IDH1. This primer set will allow determining these mutations without the need of DNA sequencing. One set of primer sets is designed to detect the presence or absence of IDH1 mutations in codon 132, while the other primer sets individually recognize the R132H, R132C, R132S, R132G and R132L mutations.

Monoclonal antibody specific for IDH1 R132H mutation.

IDH1 R132H mutations occur in approximately 70% of astrocytomas and oligodendroglial tumors. We developed a mouse monoclonal antibody targeting the IDH1 R132H mutation. Here, we show the high specificity and sensitivity of this antibody on Western blots and tissue sections from formalin fixed paraffin embedded tumor specimens. This antibody is highly useful for tumor classification, in detecting single infiltrating tumor cells and for the characterization of the cellular role of mutant IDH1 protein.

Type and frequency of IDH1 and IDH2 mutations are related to astrocytic and oligodendroglial differentiation and age: a study of 1,010 diffuse gliomas.

Somatic mutations in the IDH1 gene encoding cytosolic NADP+-dependent isocitrate dehydrogenase have been shown in the majority of astrocytomas, oligodendrogliomas and oligoastrocytomas of WHO grades II and III. IDH2 encoding mitochondrial NADP+-dependent isocitrate dehydrogenase is also mutated in these tumors, albeit at much lower frequencies. Preliminary data suggest an importance of IDH1 mutation for prognosis showing that patients with anaplastic astrocytomas, oligodendrogliomas and oligoastrocytomas harboring IDH1 mutations seem to fare much better than patients without this mutation in their tumors. To determine mutation types and their frequencies, we examined 1,010 diffuse gliomas. We detected 716 IDH1 mutations and 31 IDH2 mutations. We found 165 IDH1 (72.7%) and 2 IDH2 mutations (0.9%) in 227 diffuse astrocytomas WHO grade II, 146 IDH1 (64.0%) and 2 IDH2 mutations (0.9%) in 228 anaplastic astrocytomas WHO grade III, 105 IDH1 (82.0%) and 6 IDH2 mutations (4.7%) in 128 oligodendrogliomas WHO grade II, 121 IDH1 (69.5%) and 9 IDH2 mutations (5.2%) in 174 anaplastic oligodendrogliomas WHO grade III, 62 IDH1 (81.6%) and 1 IDH2 mutations (1.3%) in 76 oligoastrocytomas WHO grade II and 117 IDH1 (66.1%) and 11 IDH2 mutations (6.2%) in 177 anaplastic oligoastrocytomas WHO grade III. We report on an inverse association of IDH1 and IDH2 mutations in these gliomas and a non-random distribution of the mutation types within the tumor entities. IDH1 mutations of the R132C type are strongly associated with astrocytoma, while IDH2 mutations predominantly occur in oligodendroglial tumors. In addition, patients with anaplastic glioma harboring IDH1 mutations were on average 6 years younger than those without these alterations.

Analysis of the IDH1 codon 132 mutation in brain tumors.

A recent study reported on mutations in the active site of the isocitrate dehydrogenase (IDH1) gene in 12% of glioblastomas. All mutations detected resulted in an amino acid exchange in position 132. We analyzed the genomic region spanning wild type R132 of IDH1 by direct sequencing in 685 brain tumors including 41 pilocytic astrocytomas, 12 subependymal giant cell astrocytomas, 7 pleomorphic xanthoastrocytomas, 93 diffuse astrocytomas, 120 adult glioblastomas, 14 pediatric glioblastomas, 105 oligodendrogliomas, 83 oligoastrocytomas, 31 ependymomas, 58 medulloblastomas, 9 supratentorial primitive neuroectodermal tumors, 17 schwannomas, 72 meningiomas and 23 pituitary adenomas. A total of 221 somatic IDH1 mutations were detected and the highest frequencies occurred in diffuse astrocytomas (68%), oligodendrogliomas (69%), oligoastrocytomas (78%) and secondary glioblastomas (88%). Primary glioblastomas and other entities were characterized by a low frequency or absence of mutations in amino acid position 132 of IDH1. The very high frequency of IDH1 mutations in WHO grade II astrocytic and oligodendroglial gliomas suggests a role in early tumor development.

Transmembrane domain length of viral K+ channels is a signal for mitochondria targeting.

K(+) channels operate in the plasma membrane and in membranes of organelles including mitochondria. The mechanisms and topogenic information for their differential synthesis and targeting is unknown. This article describes 2 similar viral K(+) channels that are differentially sorted; one protein (Kesv) is imported by the Tom complex into the mitochondria, the other (Kcv) to the plasma membrane. By creating chimeras we discovered that mitochondrial sorting of Kesv depends on a hierarchical combination of N- and C-terminal signals. Crucial is the length of the second transmembrane domain; extending its C terminus by > or = 2 hydrophobic amino acids redirects Kesv from the mitochondrial to the plasma membrane. Activity of Kesv in the plasma membrane is detected electrically or by yeast rescue assays only after this shift in sorting. Hence only minor structural alterations in a transmembrane domain are sufficient to switch sorting of a K(+) channel between the plasma membrane and mitochondria.