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Meningiomas are among the most frequent intracranial tumors. The secretory variant of meningioma is characterized by glandular differentiation, formation of intracellular lumina and pseudopsammoma bodies, expression of a distinct pattern of cytokeratins and clinically by pronounced perifocal brain edema. Here we describe whole-exome sequencing analysis of DNA from 16 secretory meningiomas and corresponding constitutional tissues. All secretory meningiomas invariably harbored a mutation in both KLF4 and TRAF7. Validation in an independent cohort of 14 secretory meningiomas by Sanger sequencing or derived cleaved amplified polymorphic sequence (dCAPS) assay detected the same pattern, with KLF4 mutations observed in a total of 30/30 and TRAF7 mutations in 29/30 of these tumors. All KLF4 mutations were identical, affected codon 409 and resulted in a lysine to glutamine exchange (K409Q). KLF4 mutations were not found in 89 non-secretory meningiomas, 267 other intracranial tumors including gliomas, glioneuronal tumors, pituitary adenomas and metastases, 59 peripheral nerve sheath tumors and 52 pancreatic tumors. TRAF7 mutations were restricted to the WD40 domains. While KLF4 mutations were exclusively seen in secretory meningiomas, TRAF7 mutations were also observed in 7/89 (8 %) of non-secretory meningiomas. KLF4 and TRAF7 mutations were mutually exclusive with NF2 mutations. In conclusion, our findings suggest an essential contribution of combined KLF4 K409Q and TRAF7 mutations in the genesis of secretory meningioma and demonstrate a role for TRAF7 alterations in other non-NF2 meningiomas.

Isocitrate dehydrogenase 1 (IDH1) is a metabolic enzyme that is frequently mutated in certain cancers, with incidence rates ranging from 7-90% for glioma, chondrosarcoma, intrahepatic cholangiocarcinoma and AML. Wildtype IDH1 (wtIDH1) catalyzes the conversion of isocitrate to α-ketoglutarate (αKG), while tumor-associated mutant IDH1 (mIDH1) catalyzes a rogue reaction: the production of 2-hydroxyglutarate (2-HG) from αKG. 2-HG therefore represents an “oncometabolite” that is believed to play a role in cancer by interfering with αKG-dependent enzymes, which in turn causes hypermethylation of histones/DNA and a block of normal cellular differentiation. Mutant IDH1 is a “driver” oncogene and the inhibition of this altered enzyme will decrease the growth of mIDH1 dependent tumors. We report for the first time the preclinical profile and structure of BAY 1436032, a novel selective mIDH1 inhibitor. An optimization program based on a high throughput screening resulted in the identification of the clinical candidate BAY 1436032 for the treatment of mIDH1 dependent cancer. BAY 1436032 is a double-digit nanomolar and selective pan-inhibitor of the enzymatic activity of various IDH1-R132X mutants in vitro and displayed potent inhibition of 2-HG release (nanomolar range) in patient derived and engineered cell lines expressing different IDH1 mutants. In line with the proposed mode of action, a concentration-dependent lowering of 2-HG was observed in vitro accompanied by differentiation and maturation of mIDH1 tumor cells. Furthermore, BAY 1436032 showed a favourable selectivity profile against wtIDH1/2 and a large panel of off-targets in vitro. To the best of our knowledge we were able to show for the first time single agent in vivo efficacy in mIDH1 patient derived glioma and intrahepatic cholangiocarcinoma solid tumor models with this clinical candidate along with monitoring of intratumoral 2-HG levels as a predictive biomarker. The BBB penetration profile of BAY 1436032 is further supported by preclinical data on in vivo brain-plasma ratios. In conclusion, our data provide in vitro and in vivo proof of concept for BAY 1436032 as a potent and highly selective inhibitor of mutant forms of IDH1. The start of a Phase I study with BAY 1436032 is currently in preparation to determine the safety, tolerability, pharmacokinetics and preliminary anti-tumor and pharmacodynamic biomarker responses in patients with solid tumors. Citation Format: Olaf Panknin, Stefan Pusch, Lena Herbst, Stefan Kaulfuss, Katja Zimmermann, Hartmut Rehwinkel, Roland Neuhaus, Sven Ring, Michael Brüning, Claudia Stark, Katja Prelle, Martin Michels, Michael Jeffers, Holger Hess-Stumpp, Karl Ziegelbauer, Michael Brands, Alwin Krämer, Andreas von Deimling. BAY 1436032: A highly selective, potent and orally available inhibitor of mutant forms of IDH1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2645.

High-grade gliomas (HGGs) are devastating malignancies of the central nervous system, and few treatment options are available for these tumors. In the most malignant form of the disease, glioblastoma multiforme (GBM), over 90% of patients will succumb to their tumor within 5 years after standard of care treatment, consisting of surgery, radiation therapy, and temozolomide chemotherapy. It is now clear that gliomas are molecularly heterogeneous entities, with mutations in tumor suppressors and oncogenes defining many distinct sub-types with important therapy implications. However, almost all HGGs are treated with a limited array of initial therapies, regardless of these molecular differences. Isocitrate dehydrogenase-1 (IDH1), a gene recently found to be mutated in many gliomas, is involved in the conversion of isocitrate to 2-oxoglutarate in cells. The IDH1 R132H mutant enzyme converts 2-oxoglutarate to the oncometabolite (R)-2-hydroxyglutarate (D2HG), which leads to profound metabolic alterations in tumor cells. In addition, recent studies indicate that mutations in IDH1 may also induce altered DSB repair, differential sensitivities to chemo-radiotherapy, and substantial changes in chromatin modifications. Here, we present the creation of a novel astrocyte cell line harboring an engineered heterozygous IDH1 R132H mutation at the endogenous gene locus using CRISPR/Cas9 gene editing. We confirmed expression of the engineered mutation at the protein level, and we have characterized this cell line in a comprehensive panel of functional assays. In particular, we demonstrated that our mutant cell clones secrete high levels of D2HG, and we confirmed that the levels of this oncometabolite can be suppressed with small molecule inhibitors of mutant IDH1. We also characterized the DNA damage response network in IDH1-mutant cells using high-content DNA damage foci assays recently developed by our group, and also in clonogenic survival assays. To our knowledge, this is the first report of an astrocyte cell line harboring an engineered, heterozygous R132H mutation at the endogenous locus. This novel cell line represents a new model system for studying gliomas and has tremendous applications for further cell characterization, mechanistic studies, and drug screening. Citation Format: Nathaniel D. Robinson, Karin R. Purshouse, Nathan R. Fons, Gregory A. Breuer, Stefan Pusch, Andreas von Deimling, Ranjini K. Sundaram, Ranjit S. Bindra. Development and validation of a novel IDH1-mutant astrocyte cell line as a model for high-grade gliomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4277.

The decision to replicate its DNA is of crucial importance for every cell and, in many organisms, is decisive for the progression through the entire cell cycle. A comparison of animals versus yeast has shown that, although most of the involved cell-cycle regulators are divergent in both clades, they fulfill a similar role and the overall network topology of G1/S regulation is highly conserved. Using germline development as a model system, we identified a regulatory cascade controlling entry into S phase in the flowering plant Arabidopsis thaliana, which, as a member of the Plantae supergroup, is phylogenetically only distantly related to Opisthokonts such as yeast and animals. This module comprises the Arabidopsis homologs of the animal transcription factor E2F, the plant homolog of the animal transcriptional repressor Retinoblastoma (Rb)-related 1 (RBR1), the plant-specific F-box protein F-BOX-LIKE 17 (FBL17), the plant specific cyclin-dependent kinase (CDK) inhibitors KRPs, as well as CDKA;1, the plant homolog of the yeast and animal Cdc2+/Cdk1 kinases. Our data show that the principle of a double negative wiring of Rb proteins is highly conserved, likely representing a universal mechanism in eukaryotic cell-cycle control. However, this negative feedback of Rb proteins is differently implemented in plants as it is brought about through a quadruple negative regulation centered around the F-box protein FBL17 that mediates the degradation of CDK inhibitors but is itself directly repressed by Rb. Biomathematical simulations and subsequent experimental confirmation of computational predictions revealed that this regulatory circuit can give rise to hysteresis highlighting the here identified dosage sensitivity of CDK inhibitors in this network., Author Summary In order to grow, multicellular organisms need to multiply their cells. Cell proliferation is achieved through a complex order of events called the cell cycle, during which the nuclear DNA is duplicated and subsequently distributed to the newly forming daughter cells. The decision to replicate the nuclear DNA is in many organisms crucial to progress through the entire cell cycle. Alterations of the cell cycle, especially at the entry point, can cause severe developmental defects and are often causal for maladies, such as cancer. Substantial work in yeast and animals has revealed the regulatory steps controlling S-phase entry. In contrast, relatively little is known about the plant cell cycle despite plants being one of the largest classes of living organisms and despite the importance of plants for human life, for instance as the basis of human nutrition. Our work presents a molecular framework of core cell-cycle regulation for entry into the DNA replication phase in the model plant Arabidopsis. We report here the identification of a regulatory cascade that likely functions in many plant cells and organisms. With this, we also provide an important basis for comparative analyses of cell-cycle control between different eukaryotes, such as yeast and mammals.

CIC and FUBP1 mutations have recently been detected in oligodendrogliomas but not in oligoastrocytomas. However, allelic losses in the regions on chromosomal arms 19q and 1p harboring CIC and FUBP1 are a common feature of both, oligodendrogliomas and oligoastrocytomas. To resolve this discrepancy, we analyzed CIC and FUBP1 mutations in a set of primary brain tumors including 18 oligodendrogliomas and 42 oligoastrocytomas. In addition, we analyzed 10 astrocytomas and 16 glioblastomas with allelic losses on 19q as well as a set of 12 medulloblastomas for CIC mutations. CIC mutations were found in 15/18 oligodendrogliomas, 14/42 oligoastrocytomas and 3/10 preselected astrocytomas. With the exception of a single case, all CIC mutations occurred in tumors with combined 1p/19q losses. In contrast to oligodendrogliomas where CIC mutations were always detected along with 1p/19q co-deletion, CIC mutations were only found in 52 % of the 1p/19q co-deleted oligoastrocytomas. FUBP1 mutations were detected in 7/61 tumors, all presenting with CIC mutations. FUBP1 mutations appear to cluster in the DNA binding domain spanning exons 5–14. CIC and FUBP1 mutations exclusively occurred in presence of either IDH1 or IDH2 mutations. Our data confirm CIC and FUBP1 mutations in oligodendrogliomas and demonstrate the presence of these mutations in oligoastrocytomas.

As a consequence of the transient nature of kinase-substrate interactions, the detection of kinase targets, although central for understanding many biological processes, has remained challenging. Here we present a straightforward procedure that relies on the comparison of wild type with activation-loop mutants in the kinase of interest by bimolecular complementation assays. As a proof of functionality, we present the identification and in vivo confirmation of substrates of the major cell-cycle kinase in Arabidopsis, revealing a direct link between cell proliferation and the control of the redox state.

Enzyme-substrate interactions are weak and occur only transiently and thus, a faithful analysis of these interactions typically requires elaborated biochemical methodology. The bimolecular-fluorescence complementation (BiFC) assay, also referred to as split YFP assay, is a powerful and straightforward tool to test protein-protein interactions. This system is commonly used due to many advantages and especially due to its simple ease of use. BIFC relies on the reconstitution of an N-terminal and C-terminal half of YFP into a functional, i.e., fluorescent protein. Noteworthy, the dissociation constant of the two YFP halves is much lower than the association constant leading to a stabilization of the protein-protein interaction to be monitored. Whereas this property is sometimes critical, it also increases the sensitivity of the detection system by stabilizing transient interactions. Here, we exploit this property to detect and monitor interaction between a kinase and its substrate. In particular, we characterize with the BiFC system kinase-variants that show an altered substrate binding.

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.

Activating mutations of the serine threonine kinase v-RAF murine sarcoma viral oncogene homolog B1 (BRAF) are frequent in benign and malignant human tumors and are emerging as an important biomarker. Over 95% of BRAF mutations are of the V600E type and specific small molecular inhibitors are currently under pre-clinical or clinical investigation. BRAF mutation status is determined by DNA-based methods, most commonly by sequencing. Here we describe the development of a monoclonal BRAF V600E mutation-specific antibody that can differentiate BRAF V600E and wild type protein in routinely processed formalin-fixed and paraffin-embedded tissue. A total of 47 intracerebral melanoma metastases and 21 primary papillary thyroid carcinomas were evaluated by direct sequencing of BRAF and by immunohistochemistry using the BRAF V600E mutation-specific antibody clone VE1. Correlation of VE1 immunohistochemistry and BRAF sequencing revealed a perfect match for both papillary thyroid carcinomas and melanoma metastases. The staining intensity in BRAF V600E mutated tumor samples ranged from weak to strong. The generally homogenous VE1 staining patterns argue against a clonal heterogeneity of the tumors investigated. Caution is essential when only poorly preserved tissue is available for VE1 immunohistochemical analysis or when tissues with only little total BRAF protein are analyzed. Immunohistochemistry using antibody VE1 may substantially facilitate molecular analysis of BRAF V600E status for diagnostic, prognostic, and predictive purposes.

Enzyme-substrate interactions are weak and occur only transiently and thus, a faithful analysis of these interactions typically requires elaborated biochemical methodology. The bimolecular-fluorescence complementation (BiFC) assay, also referred to as split YFP assay, is a powerful and straightforward tool to test protein-protein interactions. This system is commonly used due to many advantages and especially due to its simple ease of use. BIFC relies on the reconstitution of an N-terminal and C-terminal half of YFP into a functional, i.e., fluorescent protein. Noteworthy, the dissociation constant of the two YFP halves is much lower than the association constant leading to a stabilization of the protein-protein interaction to be monitored. Whereas this property is sometimes critical, it also increases the sensitivity of the detection system by stabilizing transient interactions. Here, we exploit this property to detect and monitor interaction between a kinase and its substrate. In particular, we characterize with the BiFC system kinase-variants that show an altered substrate binding.

Compounds sMesMX 2 (sMes=2,4,6-t-BuC 6 H 2 ; X=Cl, Br; M=Ga, In) were prepared from MX 3 and sMesLi. X-ray structure determinations prove that this species are the first gallium and indium dihalides which are monomeric even in the solid state

In fungi and metazoans, the SCF-type Ubiquitin protein ligases (E3s) play a critical role in cell cycle regulation by degrading negative regulators, such as cell cycle-dependent kinase inhibitors (CKIs) at the G1-to-S-phase checkpoint. Here we report that FBL17, an Arabidopsis thaliana F-box protein, is involved in cell cycle regulation during male gametogenesis. FBL17 expression is strongly enhanced in plants co-expressing E2Fa and DPa, transcription factors that promote S-phase entry. FBL17 loss-of-function mutants fail to undergo pollen mitosis II, which generates the two sperm cells in mature A. thaliana pollen. Nonetheless, the single sperm cell-like cell in fbl17 mutants is functional but will exclusively fertilize the egg cell of the female gametophyte, giving rise to an embryo that will later abort, most likely due to the lack of functional endosperm. Seed abortion can, however, be overcome by mutations in FIE, a component of the Polycomb group complex, overall resembling loss-of-function mutations in the A. thaliana cyclin-dependent kinase CDKA;1. Finally we identified ASK11, as an SKP1-like partner protein of FBL17 and discuss a possible mechanism how SCF(FBL17) may regulate cell division during male gametogenesis.

Entry into mitosis is universally controlled by cyclin-dependent kinases (CDKs). A key regulatory event in metazoans and fission yeast is CDK activation by the removal of inhibitory phosphate groups in the ATP binding pocket catalyzed by Cdc25 phosphatases. In contrast with other multicellular organisms, we show here that in the flowering plant Arabidopsis thaliana, cell cycle control does not depend on sudden changes in the phosphorylation pattern of the PSTAIRE-containing Cdk1 homolog CDKA;1. Consistently, we found that neither mutants in a previously identified CDC25 candidate gene nor plants in which it is overexpressed display cell cycle defects. Inhibitory phosphorylation of CDKs is also the key event in metazoans to arrest cell cycle progression upon DNA damage. However, we show here that the DNA damage checkpoint in Arabidopsis can also operate independently of the phosphorylation of CDKA;1. These observations reveal a surprising degree of divergence in the circuitry of highly conserved core cell cycle regulators in multicellular organisms. Based on biomathematical simulations, we propose a plant-specific model of how progression through the cell cycle could be wired in Arabidopsis.

As in other eukaryotes, progression through the cell cycle in plants is governed by cyclin-dependent kinases. Phosphorylation of a canonical Thr residue in the T-loop of the kinases is required for high enzyme activity in animals and yeast. We show that the Arabidopsis thaliana Cdc2+/Cdc28 homolog CDKA;1 is also phosphorylated in the T-loop and that phosphorylation at the conserved Thr-161 residue is essential for its function. A phospho-mimicry T161D substitution restored the primary defect of cdka;1 mutants, and although the T161D substitution displayed a dramatically reduced kinase activity with a compromised ability to bind substrates, homozygous mutant plants were recovered. The rescue by the T161D substitution, however, was not complete, and the resulting plants displayed various developmental abnormalities. For instance, even though flowers were formed, these plants were completely sterile as a result of a failure of the meiotic program, indicating that different requirements for CDKA;1 function are needed during plant development.

Like other solid tumors, glioblastomas (GB) feature vast hypoxic areas that are thought to promote tumor growth, angiogenesis and even drug resistance and hence facilitate an aggressive growth and recurrence after treatment. Elucidation of hypoxia-signalling and identification of the major regulators of hypoxia-dependent chemoprotection seems logical for the optimization of current treatment regimens. Using comparative 2D gel electrophoresis and mass spectrometry analysis, we here identify N-myc downstream regulated gene 1 (NDRG1) as upregulated by chronic sublethal hypoxia in GB cell lines. Immunohistochemical analysis of human astrocytoma specimens shows a marked increase in expression from WHO °II to °IV and a preferential perinecrotic localization of NDRG1 in GB. In vitro, besides the hypoxia-driven induction of NDRG1, which is mediated by Hif1α and Hif2α, NDRG1 is also induced by irradiation. NDRG1 limits glioma cell invasiveness in vitro and reduces proliferation in vitro and in vivo. Furthermore NDRG1 acts antiangiogenic with reduced endothelial cell migration and sprouting, and interferes with the expression of multiple angiogenesis-related signalling molecules. Most interestingly NDRG1 protects from temozolomide (TMZ)-induced G2/M-arrest and subsequent reduction of tumor cell proliferation. Analyzing pairs of GB tissue samples of relapsed glioblastoma patients reveals a dramatic increase of NDRG1-positive cells suggesting a treatment-related selection for NDRG1 expressing populations and a potential role for NDRG1 in the recurrence of GB. In line with these findings, quantifying the NDRG1 status in tissue specimens of the UKT05 GB trial, which looked at a dose-intensified TMZ therapy plus standard radiotherapy, reveals a negative correlation between NDRG1 status and overall survival. In the present work we identify hypoxia-driven NDRG1 as a modulator of tumor growth, invasion, angiogenesis and as a novel mediator of chemoresistance towards TMZ in GB. Our findings shed light on the role of hypoxia in adaption to chemotherapeutic treatment and introduce NDRG1 as a promising candidate for targeted therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4736. doi:1538-7445.AM2012-4736

The 1,4-dithia-2,6-diaza-3,5-diborinanes 1a-d react with elemental sodium with formation of the 1,3-diaza-2,4-diboretidines 2a-d. By use of more sodium in case of 1 d or 3,5-bis(diethylamino)- 2-cyclohexyl-6-trimethylsilyldiborinane, the 1,3-thiaza-2,4-diboretidines 3 were formed. 3.5-Dimethyl-2,6-bis(trimethylsilyl)-1,4-dithia-2,6-diaza-3,5-diborinane gives the borazine 4, The reaction of di-t-butyl-sulfurdiimide with 2,6-di-t-butyl-3,5-dimethyl-1,4-dithia-2,6-diaza-3,5-diborinane leads by ring contraction to the four-membered ring system 5. 1H, 11B, 13C NMR and mass spectra are reported and discussed.

Background: Mutations in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) are frequently found in patients with acute myeloid leukemia (AML) and several other tumors. Mutant IDH1 produces R-2-hydroxyglutarate (R-2HG), which induces histone and DNA hypermethylation through inhibition of epigenetic regulators, and leads to a block in differentiation to promote tumorigenesis. Methods: We developed a novel, highly active oral pan-IDH1 inhibitor, BAY-1436032, for clinical evaluation. Its inhibitory potency was evaluated in primary human AML cells in vitro for the five major IDH1R132 mutation types and in two patient derived AML xenograft (PDX) models in vivo, in which BAY-1436032 cleared leukemic blasts in peripheral blood and prolonged survival by induction of differentiation and inhibition of leukemia stem cell self-renewal. Results: R-2HG production by mutant IDH1 was effectively inhibited in patient derived AML cells with all reported IDH1R132 mutations ex vivo by BAY-1436032 with an IC50 between 3 to 16 nM. AML cells cultured ex vivo showed morphologic differentiation and marked upregulation of the myeloid differentiation markers CD14 and CD15. For in vivo experiments, human AML cells from two patients were transplanted into sublethally irradiated NSG mice. After stable engraftment at 17 (PDX1) or 90 (PDX2) days post transplantation, mice were treated with BAY-1436032 orally every day at a dose of 150 mg/kg or vehicle for 100-150 days (n=10 per group). The R/S-2HG ratio in serum was reduced to near normal levels by BAY-1436032. Leukemic cell counts in peripheral blood constantly increased in control mice, while leukemic cells declined from day 30 of BAY-1436032 treatment onwards with morphologic and immunophenotypic evidence of differentiation (Figure). Importantly, all BAY-1436032 treated PDX1 mice survived until the end of treatment at 150 days. In contrast, vehicle-treated mice died with a median latency of 91 days (range 70-95, P To assess the effect of BAY-1436032 on leukemic stem cell self-renewal we treated PDX1 mice with 150 mg/kg BAY-1436032 or vehicle for 4 weeks and performed a limiting dilution transplantation experiment in secondary recipient mice. LSC frequency was 100-fold lower in BAY-1436032 treated compared to control mice. Gene expression profiling showed that stemness associated genes were downregulated, while genes associated with myeloid differentiation like PU.1 and CEBPA were upregulated upon treatment with BAY-1436032. In addition, cell cycle progression was slowed and E2F transcription factors concomitantly inhibited. In accordance with gene expression profiling results, methylation of the PU.1 promoter decreased, while E2F1 promoter methylation increased upon treatment with BAY-1436032. Finally, histone trimethylation levels at residues H3K4, H3K9, H3K27, and H3K36 decreased in both IDH1R132C and IDH1R132H mutant AML cells but not in IDH1 wildtype cells upon BAY-1436032 treatment. Conclusion: In summary, the novel oral pan-mutant IDH1 inhibitor BAY-1436032 is active against all IDH1R132 mutation types and shows strong anti-leukemic activity in two independent AML PDX mouse models. Clinical development is ongoing with a first in man study with BAY-1436032 in IDH1 mutant solid tumors. * M. Heuser and L. Herbst contributed equally to this article #A. Krämer and A. Chaturvedi share senior authorship Figure Human leukemic cells in peripheral blood of mice treated with BAY-1436032. ** P Disclosures Heuser: Tetralogic: Research Funding; BerGenBio: Research Funding; Karyopharm Therapeutics Inc: Research Funding; Bayer Pharma AG: Research Funding; Celgene: Honoraria; Novartis: Consultancy, Research Funding; Pfizer: Research Funding. Pusch:German Cancer Research Center: Patents & Royalties: WO2013/127997A1. Kaulfuss:Bayer Pharma AG: Employment. Panknin:Bayer Pharma AG: Employment. Zimmermann:Bayer Pharma AG: Employment, Patents & Royalties: WO2015/121210 . Toschi:Bayer Pharma AG: Employment. Neuhaus:Bayer Pharma AG: Employment, Patents & Royalties: WO2015/121210. Haegebarth:Bayer Pharma AG: Employment, Equity Ownership. Rehwinkel:Bayer Pharma AG: Employment, Equity Ownership, Patents & Royalties: WO2015/121210. Hess-Stumpp:Bayer Pharma AG: Employment. Bauser:Bayer Pharma AG: Employment. Ho:Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. von Deimling:German Cancer Research Center: Patents & Royalties: IDH1R132H mutant specific antibody H09; BRAF V600E mutant specific antibody VE1; BAY-1436032 patent.

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.

BACKGROUND Glioma therapy is challenged by the invasive nature of glioma resulting in tumor recurrence and treatment resistance. Lysosomal protein transmembrane 5 (LAPTM5) was identified to inhibit invasion by screening for invasion-associated genes in glioma. The aim of this study was to decipher the function of LAPTM5 in glioblastoma and its interaction with the CD40 receptor which was shown to be highly expressed in up to 40% of glioblastoma. METHODS LAPTM5 expression was correlated with clinical outcome of glioma patients. Knockdown of LAPTM5 was performed in different glioma cell lines to analyze the impact on clonogenicity, invasiveness, sensitivity to temozolomide chemotherapy and tumorigenicity in-vitro and in-vivo in a subcutaneous xenograft mouse model. Expression array was used to elucidate the underlying pathways. CD40 knockdown and overexpression was induced to prove the crosstalk of LAPTM5 and CD40. RESULTS LAPTM5 expression correlated with better overall survival in high grade glioma patients and acted as a tumor suppressor in CD40 positive glioblastoma cells. LAPTM5 inhibited CD40-mediated NFκB activation resulting in anti-invasive, anti-clonogenic and temozolomide sensitizing effects in-vitro and in-vivo. Vice-versa, knockdown of LAPTM5 enhanced tumorigenicity by activation of the NFκB pathway which was overcome by NFκB inhibition. Importantly, CD40 expression was required for LAPTM5-mediated tumor suppressive activity. CONCLUSION LAPTM5 conveyed tumor suppressive and temozolomide sensitizing effects in CD40-positive glioblastoma by inhibition of CD40-mediated NFκB activation and thereby might provide a reasonable biomarker for sensitivity to temozolomide in CD40-positive glioblastoma.

BACKGROUND: IDH1-mutated gliomas are associated with less abundant and phenotypically skewed innate and adaptive immune cell infiltrates compared to IDH1 wild-type tumors. Despite this, the most frequent mutation - IDH1R132H - represents a clonal shared neoantigen and mutations in IDH are associated with a more favorable prognosis. While the tumor cell-intrinsic consequences of the oncometabolite R-2-hydroxyglutarate (R-2-HG) accumulating in IDH1-mutated gliomas as a result of a neomorphic enzymatic function, are well-characterized, potential direct paracrine effects of R-2-HG influencing the glioma immune microenvironment remain incompletely understood. AIM: This study aimed at characterizing the impact of the oncometabolite R-2-HG on the innate immune microenvironment of IDH1-mutated gliomas. METHODS AND RESULTS: By means of comprehensive analyses of expression datasets from human gliomas and syngeneic murine tumor models as well as transporter studies we demonstrate that R-2-HG is imported by both microglia and macrophages via SLC family transporters and suppresses their function in a paracrine manner. Functional analyses of microglia and macrophages indicate an R-2-HG-driven induction of tolerogenicity as evidenced by accumulation of IL10 and TGFβ and suppression of MHC-II expression, which results in impaired activation of antigen-specific T cells and activation of immune checkpoint molecules. Multi-level signature profiling of human tumor-infiltrating as well as primary immune cells was complemented by reporter gene assays and pathway analyses and revealed that R-2-HG activates the cytosolic transcription factor aryl hydrocarbon receptor (AHR), a key immunomodulatory target of immunosuppressive tryptophan metabolism. By means of knockout models, the observed immunosuppressive phenotype was shown to be AHR-dependent. Functional relevance of R-2-HG-mediated, AHR-driven impairment of myeloid cell immunity was demonstrated in vivo by pharmacological AHR inhibition, increasing the efficacy of checkpoint blockade. CONCLUSION: R-2-HG impairs antitumor immunity in IDH1-mutated gliomas by activating the AHR in innate immune cells, thus suppressing the innate immune microenvironment by compromising antigen presentation and activation of antigen-specific T cells. This, together with recent findings on inhibitory effects on T cell immunity, represents a novel mechanism of immune evasion of an immunogenic driver mutation and opens a novel therapeutic approach to IDH1-mutated gliomas.