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UNLABELLED Several chronic inflammatory liver diseases, e.g., chronic hepatitis B or C viral infection and steatohepatitis, have been shown to predispose to the development of hepatocellular carcinoma (HCC). In patients with chronic liver disease, interleukin-6 (IL-6) serum levels are elevated and increase even more when HCC develops. However, the impact and regulatory mechanisms of IL-6 signaling during hepatocarcinogenesis are still poorly defined. Here, we show that gene expression profiles of patients with chromosome 8p loss correlate with increased IL-6 signaling. In addition, the chromosome 8p tumor suppressor genes Src homology 2 domain containing 4A (SH2D4A) and Sorbin and Src homology 3 domain containing 3 (SORBS3) together exerted greater inhibition of cell growth and clonogenicity compared to a single gene. Overexpression of SH2D4A and SORBS3 in HCC cells led to decreased IL-6 target gene expression and reduced signal transducer and activator of transcription 3 (STAT3) signaling. In situ and in vitro coimmunoprecipitation assays revealed that SH2D4A directly interacts with STAT3, thereby retaining STAT3 in the cytoplasm and inhibiting STAT3 transcriptional activity. On the other hand, SORBS3 coactivated estrogen receptor α signaling, leading indirectly to repression of STAT3 signaling. In human HCC tissues, SH2D4A was positively associated with infiltrating regulatory and cytotoxic T-cell populations, suggesting distinct immunophenotypes in HCC subgroups with chromosome 8p loss. Thus, the genetically linked tumor suppressors SH2D4A and SORBS3 functionally cooperate to inhibit STAT3 signaling in HCC. CONCLUSION The chromosome 8p tumor suppressor genes SORBS3 and SH2D4A are physically and functionally linked and provide a molecular mechanism of inhibiting STAT3-mediated IL-6 signaling in HCC cells. (Hepatology 2016;64:828-842).

A seized sample of the synthetic cannabinoid MDMB-CHMICA was studied by means of vibrational and electronic circular dichroism spectroscopy and found to have (S)-configuration by comparison of the experimental spectra with density functional theory calculations. We were able to additionally confirm the absolute configuration was additionally confirmed using X-ray crystallography. Furthermore, the title compound was extracted from five commercially available “Spice-like” herbal mixtures. The extracts were all found to have the same absolute configuration as the seized sample and all analyzed samples were found to be of very high optical purity as judged by chiral high-performance liquid chromatography.

Tetrasubstituted pyrroles can be synthesized in a one-pot procedure from isoxazoles. The process includes the photoinduced in situ formation of acylazirines combined with a subsequent cobalt(II)-catalyzed ring expansion with 1,3-diketones.

Isocitrate dehydrogenase 1 (IDH1), catalyzing oxidative decarboxylation of isocitrate to provide energy for aerobic organisms, is an essential enzyme in the tricarboxylic acid cycle. However, mutant IDH1 (mIDH1) produces oncometabolite D-2-hydroxyglutarate (D2HG) and has recently been confirmed in several types of cancers, particularly in glioma and acute myeloid leukemia. Herein a docking-based virtual screening (VS) of SPECS library was conducted for the allosteric site of mIDH1. The cellular evaluation of the hit compounds led to the identification of FX-03 as a novel selective mIDH1 inhibitor at allosteric site with IC50 values of 55.50 μM and 68.38 μM in HEK-293T cells transfected with IDH1 R132H and IDH1 R132C, respectively. Importantly, FX-03 owned significant selectivity with no inhibition in HEK-293T cells transfected with IDH1 WT. These findings indicate that VS of mIDH1's allosteric site represents a useful strategy for discovery of selective mIDH1 inhibitors and FX-03 deserves further optimization as a lead compound in future study.

Mutations in isocitrate dehydrogenases (IDHs) 1 and 2 frequently occur in acute myeloid leukemia (AML) and result in the production of the oncometabolite d-2-hydroxyglutarate (D2HG). D2HG has been shown to promote leukemogenesis even in the absence of mutated IDH, but the prognostic significance of pretreatment serum D2HG levels in patients with IDH-mutated AML is unclear. We measured D2HG serum levels in 84 patients with IDH-mutated AML treated in the prospective, randomized multicenter AML2003 trial of the German Study Alliance Leukemia. Multivariate Cox regression showed D2HG levels to negatively impact on event-free survival (EFS) as a continuous variable in the entire IDH(mut) cohort (P=0.04), with no effect on overall survival (OS). In a subgroup analysis, the negative impact of D2HG on EFS was found to be restricted to patients with mutations in IDH1 (P=0.003), adjusted for age, leukocyte count, serum lactate dehydrogenase and European LeukemiaNet risk score. We thus conclude that pretreatment D2HG serum levels may yield prognostic information in patients with IDH1-mutated, but not in IDH2-mutated AML, possibly due to different subcellular localizations of IDH1 and IDH2.

Objective: Glioma growth is often accompanied by a hypoxic microenvironment favorable for the induction and maintenance of the glioma stem cell (GSC) phenotype. Due to the paucity of cell models of Isocitrate Dehydrogenase 1 mutant (IDH1mut) GSCs, biology under hypoxic conditions has not been sufficiently[for full text, please go to the a.m. URL], 70. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC), Joint Meeting mit der Skandinavischen Gesellschaft für Neurochirurgie

Multiple studies have shown that patients with IDH mutant gliomas survive longer than those with IDH wild type gliomas. However, how the IDH mutation confers a better prognosis, is still largely unclear. To study the role of IDH mutation during glioma initiation and growth, we used a mouse model, which can express mutant Idh1 R132H upon Cre mediated recombination. In vitro, murine Idh1 R132H neural stem cells showed significantly lower proliferation but increased apoptosis rate when compared with normal SVZ derived stem/progenitor cells. We found that Idh1 R132H alone did not generate gliomas in neonatal or adult mice, and only upon crossing Idh1 R132H mice with PtenloxP/loxP;p53loxP/loxP mice, we observed formation of gliomas. In this context, the additional presence of the Idh mutation slowed down tumour growth and increased the rate of apoptosis. We performed RNA and miRNA sequencing on double (Pten/p53) and triple (Idh1/Pten/p53) mutant glioma initiating cells. Gene expression analysis shows a significant enrichment of genes involved in apoptosis and endoplasmic reticulum (ER) stress response in IDH mutant cells. Treatment of the cells with tunicamycin and tharpsigargin, two ER stress inducers, caused significant apoptosis in Idh1 mutant cells but not in Idh1 wt cells. Furthermore, the upregulation of Chop, Atf4 and Gadd34, constituents of the PERK branch (apoptosis), was increased after tunicamycin and tharpsigargin treatment. MiRNA sequencing data showed upregulation of miR-183-5p in Idh1 mutant cells. Subsequent overexpression of miR-183-5p promoted Idh1 wt cells to apoptosis under ER stress. In conclusion, we show here a pathway involved in triggering apoptosis in IDH mutant tumours. Triggering apoptosis with ER-Stress-inducing drugs may in the future provide an option for therapeutic intervention in IDH mutant tumours.

The aggressive nature of glioblastoma (GBM) has been attributed to hyperactivation of receptor tyrosine kinases (RTK) and their downstream signaling pathways including Ras/ERK. Pharmacological agents directed against membrane bound RTKs and cytoplasmic kinases within the Ras/ERK signaling pathway have shown promise pre-clinically, but success is yet to be realized in clinical trials. Because cancer is a disease associated with aberrant gene expression patterns, transcription factors that serve as the convergence points of oncogenic signaling hold great therapeutic promise. However, this has not yet been explored in the context of Ras/ERK signaling in GBM. The HMG-box transcriptional repressor, capicua (CIC), has recently emerged as a conserved nuclear sensor of RTK signaling in Drosophila and mammals. CIC represses EGFR/Ras/ERK pathway-responsive genes, keeping them silent in the absence of signaling. Despite the clear genetic evidence and its connection to one of the most important pathways in cancer, molecular mechanisms governing CIC regulation and its involvement in cancer remain poorly characterized. In GBM, unlike in oligodendroglioma (OD), CIC is not mutated. Surprisingly, we find substantial downregulation of wild type CIC protein despite intact mRNA expression in human GBM tumor samples. Here, we identify the nuclear E3-ubiquitin ligase responsible for the degradation of CIC in response to EGFR/Ras/ERK signaling. Interestingly, common disease-causing CIC variants fail to be ubiquitylated in response to Ras/ERK activation. Creation of a degradation-resistant form of CIC, unresponsive to upstream Ras/ERK signaling, reduced tumor growth and sensitized GBM cells to the effects of Ras/ERK inhibition. Our mechanistic insights into regulation of CIC provides rationale for alternative therapeutic strategies aimed at stabilizing CIC and activating its repressor function in GBM and other Ras/ERK-driven tumors.

Tetrasubstituted imidazoles can be formed in a photochemical one-pot synthesis from aldehydes, α-aminonitriles, and isoxazoles. Condensation of the first two components produces α-(alkylideneamino)nitriles which react under basic conditions with the acylazirines formed in situ by photochemical ring transformation of the isoxazole component. This process includes an unusual cleavage of the C(2)-C(3) bond of the acylazirine. The reaction mechanism was studied by DFT calculations.

The first examples of a photochemically induced vinylogous Nazarov-type cyclization forming a cycloheptadienone core are described. The reaction can be included in a three-step cascade consisting of a photochemical isoxazole-azirine ring contraction, cobalt(II)-catalyzed ring expansion, and the photochemical cyclization. Furthermore, the first representative of the hitherto unknown 1-azatricyclo[2.2.0.0(2,6)]hexanes has been identified as a side product of the azirine formation.

In their recent paper on the degradation of polyethylene by caterpillars of the wax moth Galleria melonella, Bombelli et al.[1] report various experiments, including microscopic and spectroscopic data which the authors believe support the chemical digestion of the polymers by these insects. While the biodegradation of mostly inert artificial polymers is definitely a very interesting research field, we must respectfully disagree with the methodology and conclusions from this paper.

INTRODUCTION: Mutations in isocitrate dehydrogenase (IDH), primarily IDH1R132H, are associated with favourable prognosis and are defining features of oligodendrogliomas and astrocytomas. They produce the oncometabolite 2-hydroxyglutarate which interferes with the TET family of 5’-methylcytosine hydroxylases, and results in a CpG island methylator phenotype. IDH1 mutations are thought to be an early event in the pathogenesis of gliomas, followed by other genetic alterations. It remains to be established how mutant IDH1 contributes to the pathogenesis of human glioma. We previously demonstrated that activation of oncogenic signals in the neurogenic cell population of the subventricular zone (SVZ) in the brain causes tumours and here we use these models to understand the role of mutant IDH1 in brain tumour pathogenesis. MATERIAL AND METHODS: Mice carrying inducible mutations (p53lox/lox, Ptenlox/lox, and IDH1lox-R132H/wt,) were used. Cre mediated recombination of these genes in the neurogenic population in the subventricular zone (SVZ) was achieved by (i) activating the tamoxifen-inducible Cre-ERT2 fusion protein under the control of the promoter/enhancer of GLAST, GFAP, or Nestin, or (ii) by intracranial injection of Adeno virus expressing Cre (Adeno-cre). Furthermore, neural stem cells, isolated from the SVZ, were recombined by Adeno-Cre in vitro and subsequently orthotopically transplanted into the striatum of NOD/SCID mice for in vivo tumour analysis. Finally, human primary glioma cultures of wild-type or mutant IDH1 glioblastomas were xenografted. RESULTS: Whilst the introduction of a mutation of the Pten and p53 genes into the SVZ population results in gliomas, the heterozygous IDH1R132H mutation alone in the same population is not tumorigenic. Similarly, IDH mutant neural stem cells, allografted into the striatum of recipient mice did not generate tumours. Instead, adding the IDH mutation to Pten and p53 mutations resulted in reduced tumour incidence and increased survival. Further, IDH1 mutation reduced the proliferative, migratory, and invasive potential of human or murine glioma-initiating cells in vitro. RNA-seq analysis of murine cells revealed a marked change of global transcriptional expression in IDH1 mutant cells, suggesting an increase in global DNA methylation. Concomitant microRNA-seq analysis identified a miR-335/transmembrane and tetratricopeptide repeat containing 1 (Tmtc1) regulatory axis. miR-335 and its host gene, mesoderm specific transcript (MEST), were significantly down-regulated in IDH1 mutant cells where epigenetic silencing dominated. Tmtc1, a putative target of miR-335, is regulated by DNA methyltransferases (DNMT). We found that IDH1R132H/WT;Pten-/-;p53-/- and IDH1R132H/WT cells had significantly higher Tmtc1 expression, compared to their controls, Pten-/-;p53-/- and non-mutation cells that shared identically low levels of Tmtc1. However, IDH1R132H/WT;Pten-/-;p53-/- cells showed a comparably lower Tmtc1 expression than that in IDH1R132H/WT cells. CONCLUSIONS: Our study demonstrates that IDH1R132H alone is not sufficient to induce tumour formation in vivo. This finding is complemented by the observation that the IDH1 mutation reduces cell proliferation and migration in vitro. Impairment of Tmtc1 expression correlates with the role of IDH1R132H to modulate DNA methylation, which is complementary to its classic commitment to inhibition of DNA demethylases.

Tryptophan catabolism is increasingly recognized as a key and druggable molecular mechanism active in cancer, immune, and glioneural cells and involved in the modulation of antitumor immunity, autoimmunity and glioneural function. In addition to the pivotal rate limiting enzyme indoleamine-2,3-dioxygenase, expression of tryptophan-2,3-dioxygenase (TDO) has recently been described as an alternative pathway responsible for constitutive tryptophan degradation in malignant gliomas and other types of cancer. In addition, TDO has been implicated as a key regulator of neurotoxicity involved in neurodegenerative diseases and ageing. The pathways regulating TDO expression, however, are largely unknown. Here, a siRNA-based transcription factor profiling in human glioblastoma cells revealed that the expression of human TDO is suppressed by endogenous glucocorticoid signaling. Similarly, treatment of glioblastoma cells with the synthetic glucocorticoid dexamethasone led to a reduction of TDO expression and activity in vitro and in vivo. TDO inhibition was dependent on the immunophilin FKBP52, whose FK1 domain physically interacted with the glucocorticoid receptor as demonstrated by bimolecular fluorescence complementation and in situ proximity ligation assays. Accordingly, gene expression profile analyses revealed negative correlation of FKBP52 and TDO in glial and neural tumors and in normal brain. Knockdown of FKBP52 and treatment with the FK-binding immunosuppressant FK506 enhanced TDO expression and activity in glioblastoma cells. In summary, we identify a novel steroid-responsive FKBP52-dependent pathway suppressing the expression and activity of TDO, a central and rate-limiting enzyme in tryptophan metabolism, in human gliomas.

BACKGROUND Recently, isocitrate dehydrogenase 1 (IDH1) was identified as a major participant in glioma pathogenesis. At present, the enzymatic activity of the protein has been the main topic in investigating its physiological function, but its signaling pathway allocation was unsuccessful. Interestingly, proteins regulated by phosphoinositide 3-kinase (PI3K)/Akt signaling, are among the top downregulated genes in gliomas associated with high percentage of IDH1 and IDH2 mutations. The aim of this study was to investigate a hypothetical relation between IDH1 and PI3K signaling. METHODS The presence of mutant IDH1 and markers for active PI3K/Akt signaling, present as phosphorylated Akt and podoplanin (PDPN), were investigated in a discovery cohort of 354 patients with glioma. In vitro experiments were used to confirm functional links. RESULTS This study shows an inverse correlation between mutant IDH1 and markers for active PI3K/Akt signaling. In support of a functional link between these molecules, in vitro expression of mutant IDH1 inhibited Akt phosphorylation in a 2-hydroxyglutarate–dependent manner. CONCLUSIONS This study provides patient tumor and in vitro evidence suggesting that mutant IDH1 inhibits PI3K/Akt signaling. Cancer 2014;120:2440–2447. © 2014 American Cancer Society.

There is a lack of relevant prognostic and predictive factors in neurooncology besides mutation of isocitrate dehydrogenase 1, codeletion of 1p/19q and promoter hypermethylation of O 6 -methylguanine-DNA-methyltransferase. More importantly, there is limited translation of these factors into clinical practice. The cancer genome atlas data and also clinical correlative analyses suggest a pivotal role for the epidermal growth factor receptor /protein kinase B/mammalian target of rapamycin (mTOR) pathway in both biology and the clinical course of gliomas. However, attempts to stratify gliomas by activating alterations in this pathway have failed thus far. The tumors of 40 patients with WHO grade II gliomas without immediate postoperative genotoxic treatment and known progression and survival status at a median follow-up of 12.2 years were analyzed for expression of the mTOR complex 2 downstream target N-myc downstream regulated gene (NDRG)1 using immunohistochemistry. Baseline characteristics for NDRG1 absent/low versus moderate/high patients were similar. Time to reintervention was significantly longer in the NDRG1 group (P = 0.026). NDRG1 may become a novel biomarker to guide the decision which WHO°II glioma patients may be followed without postsurgical intervention and which patients should receive genotoxic treatment early on. Validation of this hypothesis will be possible with the observational arm of the RTOG 9802 and the pretreatment step of the EORTC 22033/26032 trials.

The title reaction is incorporated in a three-step cascade transformation providing novel tetracyclic ring structures containing a seven-membered ring.

The NOTCH pathway is an evolutionary conserved signaling pathway that is known to play a pivotal role in physiological liver development and regeneration. Thereby, the NOTCH pathway controls cell fate decisions of bipotential liver progenitor cells promoting a biliary lineage rather than hepatic differentiation. Furthermore, aberrant NOTCH signaling is also a potential driver of liver inflammation, formation and progression of hepatocellular (HCC) and intrahepatic cholangiocarcinoma (iCCA). Previous studies mainly focused on the expression levels of the NOTCH receptors (NOTCH1-4) and their ligands (DLL1/3/4, JAG1/2). However, little is known regarding mutational effects within components of the Notch pathway and their relevance in liver cancer. In a whole exome sequencing approach of 54 human HCC samples, we discovered 19 amino acid-altering mutations in 15 different NOTCH pathway genes which were confirmed by Sanger sequencing to be somatic. In summary, these 15 mutations affected 25.9% of patients (14 out of 54 patients) and presented at least one mutation in one of the included NOTCH pathway components (NOTCH1/3/4, DLL1, DTX1/3/4, HES5, DVL2, LFNG, NUMB, NCSTN, DMXL2, GXYLT2). Consistently, 30% of HCC patients have been reported to contain a tumor-associated hyper-activated NOTCH pathway. Using multiple online tools to predict the effect of the mutations on protein function and tumor relevance, we observed that HES5-Arg31Gly mutation ranked highest. In order to test, whether HES5 is activated by NOTCH in HCC cells, we generated cell lines with inducible expression of NOTCH1 or NOTCH3 intracellular domain and found that the transcription factor HES5 but not HES1 is strongly activated in several HCC cell lines. Furthermore, induction of HES5 in an inducible Hep3B cell line resulted in increased expression of stem cell markers and epithelial-mesenchymal transition markers. In contrary, CCND1, HNF4alpha and HES1 were downregulated. Interestingly, HES5-Arg31Gly failed to modulate the expression of the here tested target genes. The loss of function of HES5-Arg31Gly may be partially explained by lower nuclear translocation of HES5-Arg31Gly protein. In vivo, mouse models using hydrodynamic transduction of transposons expressing MYC and HES5 indicated a tumor suppressive role of HES5, whereas, in AKT-driven liver tumorigenesis, HES5 exhibited oncogenic potential. Consistent with our in vitro experiments, AKT/HES5-driven murine tumors exhibited partial downregulation of HNF4alpha and upregulation of the cholangiocyte marker cytokeratin suggesting a transdifferentiation towards a cholangiocytic phenotype. Thus, we identified somatic mutations in NOTCH pathway genes in 25.9% of HCC patients. Our functional in vitro and in vivo analyses suggest that HES5 has oncogenic or tumor suppressive properties depending on the underlying oncogene or tumor subtype, respectively. Citation Format: Sarah Luiken, Matthias Bieg, Angelika Fraas, Raisatun Sugiyanto, Benjamin Goeppert, Stephan Singer, Stefan Pusch, Arianeb Mehrabi, Thomas Longerich, Peter Schirmacher, Stephanie K. Roessler. NOTCH/HES5 signaling exhibits distinct pro- and anti-tumorigenic roles in liver carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3487.

Background: Isocitrate dehydrogenase (IDH) mutations are drivers of different tumors. The IDH1R132 mutation is frequently found in glioma (80%), chondrosarcoma (CS, 45%), intrahepatic cholangiocarcinoma (ICC, 20%), acute myeloid leukemia (AML, 7%), and with low incidences in other solid tumors. Although clinical therapies of mutant IDH inhibition were recently approved for the treatment of AML, few patients benefit from a single therapy with a mutant IDH inhibitor and most of them relapse. Here we report preclinical in vitro and in vivo data on combination therapies of the mIDH1R132 inhibitor BAY 1436032 with standard of care chemotherapies. Methods: in vitro: The sarcoma cell line HT1080 (IDH1R132C), together with two IDH1 wildtype sarcoma cell lines, the SW1353 (IDH2R172S) and SW872 (IDHwt) cell lines, were treated with BAY 1436032 alone and in combination with different standard of care drugs. in vivo: The patient derived xenograft (PDX) ICC model LIXFC2084 (IDH1R132L), the melanoma PDX model MEXF1341 (IDH1R132C) (MEXF1341), the PDX AML model 13PB020 (IDH1R132C) and a sarcoma cell line derived xenograft model HT1080 (IDH1R132C) were included. All models were treated with BAY 1436032 alone and in combination with different standard of care (SOC) drugs. Results: The in vitro treatment with BAY 1436032 unexpectedly increased growth in HT1080 (IDH1R132C) cells with no effect to the control cell lines SW1353 and SW872. In contrast, in vivo, combination of BAY1436032 with everolimus had additive anti-tumor efficacy and was synergistic with cisplatin or the MEK inhibitor refametinib in the treatment of HT1080 xenografts. The combination of BAY 1436032 and refametinib was also additive in the treatment of a melanoma xenograft with IDH1R132C mutation. Another additive efficacy of BAY 1436032 and gemcitabine or cisplatin could be observed in an ICC xenograft model and further in the combination with everolimus in the same model. The combination of BAY 1436032 together with azacitidine in a PDX AML model was also synergistic. Conclusion: These results highlight the combinatorial potential of the mIDH1R132 inhibitor BAY 1436032 with standard of care therapy and the importance of microenvironmental factors, which has to be taken into account when dealing with mutant IDH tumors. Citation Format: Stefan Kaulfuss, Julia Zaman, Anuhar Chaturvedi, Holger Hess-Stumpp, Jennifer Höde, Ricarda Biber, Renan Borowicz, Michael Heuser, Andreas von Deimling, Stefan Pusch. Preclincial in vitro and in vivo combination therapies for mutant IDH1R132 tumors with BAY 1436032 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2182.

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. Multilevel 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 pharmacologic 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. Citation Format: Mirco Friedrich, Lukas Bunse, Theresa Bunse, Edward Green, Tobias Kessler, Stefan Pusch, Katrin Deumelandt, Rafael Carretero, Andreas von Deimling, Francisco J Quintana, Wolfgang Wick, Michael Platten. The oncometabolite R-2-Hydroxyglutarate suppresses the innate immune microenvironment of IDH1-mutated gliomas via aryl hydrocarbon receptor signaling [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr PR15.

A hypoxic microenvironment induces resistance to alkylating agents by activating targets in the mammalian target of rapamycin (mTOR) pathway. The molecular mechanisms involved in this mTOR-mediated hypoxia-induced chemoresistance, however, are unclear. Here we identify the mTOR target N-myc downstream regulated gene 1 (NDRG1) as a key determinant of resistance toward alkylating chemotherapy, driven by hypoxia but also by therapeutic measures such as irradiation, corticosteroids, and chronic exposure to alkylating agents via distinct molecular routes involving hypoxia-inducible factor (HIF)-1alpha, p53, and the mTOR complex 2 (mTORC2)/serum glucocorticoid-induced protein kinase 1 (SGK1) pathway. Resistance toward alkylating chemotherapy but not radiotherapy was dependent on NDRG1 expression and activity. In posttreatment tumor tissue of patients with malignant gliomas, NDRG1 was induced and predictive of poor response to alkylating chemotherapy. On a molecular level, NDRG1 bound and stabilized methyltransferases, chiefly O6-methylguanine-DNA methyltransferase (MGMT), a key enzyme for resistance to alkylating agents in glioblastoma patients. In patients with glioblastoma, MGMT promoter methylation in tumor tissue was not more predictive for response to alkylating chemotherapy in patients who received concomitant corticosteroids.

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.

Tetrasubstituted pyrroles can be synthesized in a one-pot procedure from isoxazoles. The process includes the photoinduced in situ formation of acylazirines combined with a subsequent cobalt(II)-catalyzed ring expansion with 1,3-diketones.

The majority of low-grade and secondary high-grade gliomas carry heterozygous hotspot mutations in cytosolic isocitrate dehydrogenase 1 (IDH1) or the mitochondrial variant IDH2. These mutations mostly involve Arg132 in IDH1 and Arg172 or Arg140 in IDH2. Whereas IDHs convert isocitrate to alpha-ketoglutarate (α-KG) with simultaneous reduction of NADP+ to NADPH, these IDH mutants reduce α-KG to D-2-hydroxyglutarate (D-2-HG) while oxidizing NADPH. D-2-HG is a proposed oncometabolite, acting via competitive inhibition of α-KG-dependent enzymes that are involved in metabolism and epigenetic regulation. However, much less is known about the implications of the metabolic stress, imposed by decreased α-KG and NADPH production, for tumor biology. We here present a novel heterozygous IDH1 mutation, IDH1R314C, which was identified by targeted next generation sequencing of a high grade glioma from which a mouse xenograft model and a cell line were generated. IDH1R314C lacks isocitrate-to-α-KG conversion activity due to reduced affinity for NADP+ and differs from the IDH1R132 mutants in that it does not produce D-2-HG. Because IDH1R314C is defective in producing α-KG and NADPH, without concomitant production of the D-2-HG, it represents a valuable tool to study the effects of IDH1-dysfunction on cellular metabolism in the absence of this oncometabolite.

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Patients with malignant gliomas have a poor prognosis with average survival of less than one year. Whereas in other tumor entities the characteristics of tumor metabolism are successfully used for therapeutic approaches, such developments are very rare in brain tumors, notably in gliomas. One metabolic feature characteristic of gliomas, in particular diffuse astrocytomas and oligodendroglial tumors, is the variable content of D-2-hydroxyglutarate (D2HG), a metabolite, which was discovered first in this tumor entity. D2HG is generated in large amounts due to various “gain-of–function” mutations in the isocitrate dehydrogenases IDH-1 and IDH-2. Meanwhile, D2HG has been detected in several other tumor entities including intrahepatic bile-duct cancer, chondrosarcoma, acute myeloid leukemia, and angioimmunoblastic T-cell lymphoma. D2HG is barely detectable in healthy tissue (< 0.1 mM), but its concentration increases up to 35 mM in malignant tumor tissues. Consequently, the “oncometabolite” D2HG has gained increasing interest in the field of tumor metabolism. To facilitate its quantitative measurement without loss of spatial resolution at a microscopical level, we have developed a novel bioluminescence assay for determining D2HG in sections of snap-frozen tissue. The assay was verified independently by photometric tests and liquid chromatography / mass spectrometry (LC/MS). The novel technique allows the microscopically resolved determination of D2HG in a concentration range of 0 – 10 µmol/g tissue (wet weight). In combination with the already established bioluminescence imaging techniques for ATP, glucose, pyruvate, and lactate, the novel D2HG assay enables a comparative characterization of the metabolic profile of individual tumors in a further dimension.

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.

Brain metastases (BM) are frequent and carry a dismal prognosis. BRAF V600E mutations are found in a broad range of tumor types and specific inhibitors targeting BRAF V600E protein exist. We analyzed tumoral BRAF V600E-mutant protein expression using the novel mutation-specific antibody VE1 in a series of 1,120 tumor specimens (885 BM, 157 primary tumors, 78 extra-cranial metastases) of 874 BM patients. In 85 cases, we performed validation of immunohistochemical results by BRAF exon 15 gene sequencing. BRAF V600E protein was expressed in BM of 42/76 (55.3%) melanomas, 1/15 (6.7%) ovarian cancers, 4/72 (5.5%) colorectal cancers, 1/355 (0.3%) lung cancers, 2/6 thyroid cancers and 1/2 choriocarcinomas. BRAF V600E expression showed high intra-tumoral homogeneity and was similar in different tumor manifestations of individual patients. VE1 immunohistochemistry and BRAF exon 15 sequencing were congruent in 68/70 (97.1%) cases, but VE1 immunostaining identified small BRAF V600E expressing tumor cell aggregates in 10 cases with inconclusive genetic results. Melanoma patients with BRAF V600E mutant protein expressing tumors were significantly younger at diagnosis of the primary tumor and at operation of BM than patients with non-mutated tumors. In conclusion, expression of BRAF V600E mutant protein occurs in approximately 6% of BM and is consistent in different tumor manifestations of the same patient. Thus, BRAF V600E inhibiting therapies seem feasible in selected BM patients. Immunohistochemical visualization of V600E-mutant BRAF protein is a promising tool for patient stratification. An integrated approach combining both, VE1 immunohistochemistry and genetic analysis may increase the diagnostic accuracy of BRAF mutation analysis.

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.

Glioblastoma is one of the most aggressive primary brain tumors in adults, with a dismal median overall survival of only 14 months after diagnosis. Glioblastoma stem-like cells (GSCs) are particularly resistant to current therapies, capable of self-renewal and tumour initiation and are hence thought to be major contributors to patient relapse. A kinome and phosphatome wide screen revealed glycolysis gene 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 4 (PFKFB4) as an important candidate gene for GSC survival. Notably, silencing PFKFB4 in an orthotopic xenograft mouse model of glioblastoma completely rid the mice of the tumor. Here we show a brand new function of PFKFB4, independent of its role in glycolysis - namely in the regulation of HIF1α, which is upregulated in GSCs. Gene expression profiling revealed a downregulation in HIF1α target genes in PFKFB4-silenced GSCs, and HIF1α protein levels are also dramatically reduced upon silencing. Mass spectrometric analysis of immunoprecipitated PFKFB4 protein revealed a novel interaction partner, F-box only protein 28 (FBXO28), an E3 ubiqutin ligase. Co-immunoprecipation assays show that FBXO28 forms an SCF multi-protein ubiquitin ligase complex with CUL1 and SKP1. Ubiquitylation studies of HIF1α show that PFKFB4 stabilizes this crucial protein in GSCs by preventing its targeting by FBXO28 for proteasomal degradation. These new findings, coupled with its cancer specific expression in a variety of tumor entities, makes PFKFB4 a compelling target. Citation Format: Emma L. Phillips, Frederic Bethke, Jörg Balss, Stefan Pusch, Stefan Christen, Martina Schnölzer, Antje Habel, David Capper, Andreas Von Deimling, Sarah-Maria Fendt, Peter Lichter, Violaine Goidts. PFKFB4, much more than just a glycolytic gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1906.

α-(alkylideneamino)nitriles generated in situ from α-aminonitriles and aldehydes react with acylaziridines formed in situ by photochemical ring transformation of isoxazoles.

For a targeted cancer vaccine to be effective, the antigen of interest needs to be naturally processed and presented on MHC by the target cell or an antigen-presenting cell (APC) in the tumor stroma. The presence of these characteristics is often assumed based on animal models, evaluation of antigen-overexpressing APCs in vitro, or assays of material-consuming immune precipitation from fresh solid tissue. Here, we evaluated the use of an alternative approach that uses the proximity ligation assay (PLA) to identify the presentation of an MHC class II-restricted antigen in paraffin-embedded tissue sections from patients with brain tumors. This approach required a specific antibody directed against the epitope that was presented. We used an antibody that specifically binds an epitope of mutated isocitrate dehydrogenase type 1 (IDH1R132H), which is frequently expressed in gliomas and other types of tumors. In situ PLA showed that the IDH1R132H epitope colocalizes with MHC class II in IDH1R132H-mutated glioma tissue. Moreover, PLA demonstrated colocalization between the class II epitope-containing melanoma antigen New York esophageal 1 and MHC class II. Collectively, our data suggest that PLA may be a useful tool to acquire information on whether an antigen is presented in situ, and this technique has potential to guide clinical studies that use antigen-specific cancer immunotherapy.

Recent studies have shown that cyclin-dependent kinase (CDK) inhibitors can have a tremendous impact on cell cycle progression in plants. In animals, CDK inhibitors are tightly regulated, especially by posttranslational mechanisms of which control of nuclear access and regulation of protein turnover are particularly important. Here we address the posttranslational regulation of INHIBITOR/INTERACTOR OF CDK 1 (ICK1)/KIP RELATED PROTEIN 1 (KRP1), an Arabidopsis (Arabidopsis thaliana) CDK inhibitor. We show that ICK1/KRP1 exerts its function in the nucleus and its presence in the nucleus is controlled by multiple nuclear localization signals as well as by nuclear export. In addition, we show that ICK1/KRP1 localizes to different subnuclear domains, i.e. in the nucleoplasm and to the chromocenters, hinting at specific actions within the nuclear compartment. Localization to the chromocenters is mediated by an N-terminal domain, in addition we find that this domain may be involved in cyclin binding. Further we demonstrate that ICK1/KRP1 is an unstable protein and degraded by the 26S proteasome in the nucleus. This degradation is mediated by at least two domains indicating the presence of at least two different pathways impinging on ICK1/KRP1 protein stability.

Astrocytic brain tumours, including glioblastomas, are incurable neoplasms characterized by diffusely infiltrative growth. Here we show that many tumour cells in astrocytomas extend ultra-long membrane protrusions, and use these distinct tumour microtubes as routes for brain invasion, proliferation, and to interconnect over long distances. The resulting network allows multicellular communication through microtube-associated gap junctions. When damage to the network occurred, tumour microtubes were used for repair. Moreover, the microtube-connected astrocytoma cells, but not those remaining unconnected throughout tumour progression, were protected from cell death inflicted by radiotherapy. The neuronal growth-associated protein 43 was important for microtube formation and function, and drove microtube-dependent tumour cell invasion, proliferation, interconnection, and radioresistance. Oligodendroglial brain tumours were deficient in this mechanism. In summary, astrocytomas can develop functional multicellular network structures. Disconnection of astrocytoma cells by targeting their tumour microtubes emerges as a new principle to reduce the treatment resistance of this disease.

Recently, isocitrate dehydrogenase 1 (IDH1) was identified as a major participant in glioma pathogenesis. At present, the enzymatic activity of the protein has been the main topic in investigating its physiological function, but its signaling pathway allocation was unsuccessful. Interestingly, proteins regulated by phosphoinositide 3-kinase (PI3K)/Akt signaling, are among the top downregulated genes in gliomas associated with high percentage of IDH1 and IDH2 mutations. The aim of this study was to investigate a hypothetical relation between IDH1 and PI3K signaling.The presence of mutant IDH1 and markers for active PI3K/Akt signaling, present as phosphorylated Akt and podoplanin (PDPN), were investigated in a discovery cohort of 354 patients with glioma. In vitro experiments were used to confirm functional links.This study shows an inverse correlation between mutant IDH1 and markers for active PI3K/Akt signaling. In support of a functional link between these molecules, in vitro expression of mutant IDH1 inhibited Akt phosphorylation in a 2-hydroxyglutarate-dependent manner.This study provides patient tumor and in vitro evidence suggesting that mutant IDH1 inhibits PI3K/Akt signaling.

CeO2-x nanorods are functional mimics of natural haloperoxidases. They catalyze the oxidative bromination of phenol red to bromophenol blue and of natural signaling molecules involved in bacterial quorum sensing. Laboratory and field tests with paint formulations containing 2 wt% of CeO2-x nanorods show a reduction in biofouling comparable to Cu2 O, the most typical biocidal pigment.

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.

The mutant IDH1 protein, which is expressed in a large fraction of human gliomas, is shown to be immunogenic; mutant-specific immune responses can be detected in patients with IDH1 mutated gliomas and generated in mice and are shown to treat established IDH1 mutant tumours in a syngeneic MHC humanized mouse model in a CD4 T-cell-dependent manner. Isocitrate dehydrogenase type 1 (IDH1) point mutations are associated with certain slow-growing gliomas and other tumours. This study in a humanized syngeneic tumour mouse model shows that IDH1(R132H), the mutant IDH1 protein most commonly expressed in gliomas, is immunogenic, capable of inducing a human MHC class II-restricted spontaneous and functionally relevant immune response. These findings suggest that some patients with gliomas with a high prevalence of the IDH1(R132H) mutation may benefit from a tumour vaccine based on the IDH1(R132H) antigen. Monoallelic point mutations of isocitrate dehydrogenase type 1 (IDH1) are an early and defining event in the development of a subgroup of gliomas1,2,3 and other types of tumour4,5,6. 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)7,8, genomic hypermethylation9,10,11, genetic instability and malignant transformation12. 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 cells13,14. 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 gliomas15, a mutation-specific anti-IDH1(R132H) vaccine may represent a viable novel therapeutic strategy for IDH1(R132H)-mutated tumours.

Tryptophan catabolism is increasingly recognized as a key and druggable molecular mechanism active in cancer, immune, and glioneural cells and involved in the modulation of antitumor immunity, autoimmunity and glioneural function. In addition to the pivotal rate limiting enzyme indoleamine-2,3-dioxygenase, expression of tryptophan-2,3-dioxygenase (TDO) has recently been described as an alternative pathway responsible for constitutive tryptophan degradation in malignant gliomas and other types of cancer. In addition, TDO has been implicated as a key regulator of neurotoxicity involved in neurodegenerative diseases and ageing. The pathways regulating TDO expression, however, are largely unknown. Here, a siRNA-based transcription factor profiling in human glioblastoma cells revealed that the expression of human TDO is suppressed by endogenous glucocorticoid signaling. Similarly, treatment of glioblastoma cells with the synthetic glucocorticoid dexamethasone led to a reduction of TDO expression and activity in vitro and in vivo. TDO inhibition was dependent on the immunophilin FKBP52, whose FK1 domain physically interacted with the glucocorticoid receptor as demonstrated by bimolecular fluorescence complementation and in situ proximity ligation assays. Accordingly, gene expression profile analyses revealed negative correlation of FKBP52 and TDO in glial and neural tumors and in normal brain. Knockdown of FKBP52 and treatment with the FK-binding immunosuppressant FK506 enhanced TDO expression and activity in glioblastoma cells. In summary, we identify a novel steroid-responsive FKBP52-dependent pathway suppressing the expression and activity of TDO, a central and rate-limiting enzyme in tryptophan metabolism, in human gliomas.

Non-central nervous system hemangiopericytoma (HPC) and solitary fibrous tumor (SFT) are considered by pathologists as two variants of a single tumor entity now subsumed under the entity SFT. Recent detection of frequent NAB2-STAT6 fusions in both, HPC and SFT, provided additional support for this view. On the other hand, current neuropathological practice still distinguishes between HPC and SFT. The present study set out to identify genes involved in the formation of meningeal HPC. We performed exome sequencing and detected the NAB2-STAT6 fusion in DNA of 8/10 meningeal HPC thereby providing evidence of close relationship of these tumors with peripheral SFT. Due to the considerable effort required for exome sequencing, we sought to explore surrogate markers for the NAB2-STAT6 fusion protein. We adopted the Duolink proximity ligation assay and demonstrated the presence of NAB2-STAT6 fusion protein in 17/17 HPC and the absence in 15/15 meningiomas. More practical, presence of the NAB2-STAT6 fusion protein resulted in a strong nuclear signal in STAT6 immunohistochemistry. The nuclear reallocation of STAT6 was detected in 35/37 meningeal HPC and 25/25 meningeal SFT but not in 87 meningiomas representing the most important differential diagnosis. Tissues not harboring the NAB2-STAT6 fusion protein presented with nuclear expression of NAB2 and cytoplasmic expression of STAT6 proteins. In conclusion, we provide strong evidence for meningeal HPC and SFT to constitute variants of a single entity which is defined by NAB2-STAT6 fusion. In addition, we demonstrate that this fusion can be rapidly detected by STAT6 immunohistochemistry which shows a consistent nuclear reallocation. This immunohistochemical assay may prove valuable for the differentiation of HPC and SFT from other mesenchymal neoplasms.