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.
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.
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.
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.
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.
Abstract
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.
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.
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 >=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.
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