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IDH1 R132H mutations are considered to play a key role in the development of low grade gliomas and therefrom derived secondary glioblastomas (GBM). Wild type IDH1 converts isocitrate to α-ketoglutarate (a-KG) while reducing NADP+. IDH1R132H has a neomorphic enzymatic function using a-KG to generate high amounts of the oncometabolite D-2-hydroxyglutarate (D-2-HG). While the effects of D-2-HG have been subject to intense research, D-2-HG independent effects of IDH1R132H on energy homeostasis and redox status are not well studied. Here we demonstrate that IDH1R132H transduction but not D-2-HG alone leads to significantly decreased Krebs cycle metabolite concentrations and proliferation in U87 and the primary GBM cell line HT7606 as well as in immortalized astrocytes SVGp12. Furthermore, IDH1R132H mutation, but not D-2-HG treatment, resulted in a significant drop in NADPH levels in tumor cells (U87 and HT7606), whereas immortalized astrocytes retained normal NADPH levels. Since NAPDH levels can be restored via the reaction of the NAD-kinase, we analyzed NAD levels and enzymes involved in NAD-synthesis in our cell lines. Indeed we found a significant drop of NAD levels and in the activity of the NAD-dependent enzyme sirtuin in IDH1R132H mutant U87 and HT7606 but not in NADPH stable SVGp12-IDH1R132H. Interestingly, there were marked differences in expression of NAD-synthesis enzymes between the different cell-lines. In particular, NAMPT-levels were much higher in U87 and HT7606 then in astrocytes (SVGp12) and significantly decreased in U87-IDH1R132H and HT7606-IDH1R132H. Importantly, we also found decreased levels of NAMPT in primary tumor tissues and patient derived glioma cell lines with IDH1 R132H compared to wild type gliomas. Altogether our results for the first time show that the IDH1 mutation directly affects energy homeostasis and redox status in a cell-type dependent manner. We hypothesize that this leads to a drop in NADPH and NAD-levels during malignant progression, resulting in a disadvantage for proliferating tumor cells. This is in line with the favorable prognosis and good response to chemo- and radiation therapy clinically observed in IDH-mutated gliomas. Our findings suggest that the impaired metabolism in IDH1-mutant tumors might be a promising target for future therapies. Citation Format: Evelin Schrock, Julia Biedermann, Khalil Abou-El-Ardat, Matthias Lehmann, Marina Conde, Mirko Peitzsch, Susan Richter, Ralf Wiedemuth, Matthias Meinhardt, William P. J. Leenders, Christel Herold-Mende, Graeme Eisenhofer, Simone P. Niclou, Leoni Kunz-Schughart, Achim Temme, Barbara Klink. Effects of the IDH1 R132H mutation on redox status and metabolism are cell type dependent but independent from D-2-hydroxyglutarate accumulation. [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 LB-308.

Glioblastoma multiforme (GBM) is the most prevalent malignant primary brain tumor in adults. GBM is classified as primary if it is assumed to have arisen de novo or as secondary if it progressed from lower grade astrocytoma. Previous studies have found that primary and secondary GBMs have distinct molecular and mutational profiles. Both have a grim prognosis with survival times of about a year with therapy. Although much progress in delineating the temporal order of mutations and copy number aberrations in the progression of lower grade gliomas was made in the past years, none of the studies have actively followed individual tumors through their progression. Whereas this method can detect aberrations that are prevalent in gliomas, it can miss events that are important in a subset of gliomas or are necessary for progression. The GlioMath-DD consortium is an interdisciplinary collaboration of several groups at the Technische Universität Dresden (TU-Dresden) aiming to study the progression of gliomas and to come up with a mathematical model for gliomagenesis. The work involves analyzing pairs of gliomas obtained from patients who had presented with a low grade glioma and who later had a recurrence of a higher grade glioma. All tumors were checked by pathologists and then high quality DNA and RNA material extracted and used for analysis. The tumors are analyzed for copy number variations (CNV) by array comparative genome hybridization (aCGH), while gene expression changes and small mutations are analyzed using high-throughput sequencing (RNA- and Exome-seq). The data gleaned from these experiments and from in vitro models of cell growth and spheroid formation will be used by bioinformaticians and mathematicians to infer key signaling networks and formulate a mathematical model of glioma progression. The ultimate aim of this work that spans two and a half years is to create a comprehensive model of glioma promotion and progression. Furthermore, it will pinpoint driver mutations and aberrations that contribute to this progression and eventually isolate biomarkers for diagnosis and therapy. During the conference, we will present preliminary genetic data of our ongoing study. Acknowledgements: The GlioMath-DD project (coordinator: Andreas Deutsch; SAB-Number 100098214) is funded by the European Social Fund (ESF) and the Free State of Saxony Citation Format: Evelin Schröck, Khalil Abou-El-Ardat, Ralf Wiedemuth, Michael Seifert, Alvaro Köhn-Luque, Mirjam Ingargiola, Kristin Stirnnagel, Alexander Krüger, Wolfgang Nagel, Kathrin Geiger, Andreas Beyer, Leoni A. Kunz-Schughart, Gabriele Schackert, Achim Temme, Barbara Klink, Andreas Deutsch. GlioMath-DD: A multidisciplinary approach to study glioma evolution and identify targets for individualized therapies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5341. doi:10.1158/1538-7445.AM2014-5341

Survivin, belonging to the family of Inhibitor of Apoptosis proteins (IAP), is overexpressed in a broad variety of different tumor entities. Besides its role and function in apoptosis, Survivin forms in mitosis with its partners Borealin, inner centromere protein (INCENP) and the enzymatically active member Aurora B kinase the chromosomal passenger complex (CPC). The main function of this complex is sensing and correcting non-bipolar microtubule-kinetochore interaction, regulating chromosome segregation and cytokinesis. Some evidence points to a role of Survivin in connecting mitosis with control of cell cycle arrest. In this study, we assessed the role of Survivin in ensuring chromosomal stability and DNA integrity in HCT116, MCF-7, U87-MG and corresponding isogenic p53-deficient cells. Knock down of Survivin, using retroviral short-hairpin RNAs, caused in all tested cell lines a heavily impaired cytokinesis resulting in polyploidy and poly-merotelic kinetochore spindle assemblies, irrespective of the p53 status. Surprisingly, cells with wild type p53 showed after Survivin RNAi an activation of p53 and accumulation of p21waf/cip and Cyclin D1, indicating G1 arrest. A detailed Flow Cytometry analysis revealed a correlation between the increased p21waf/cip expression and increasing DNA content in Survivin-depleted wild type cells compared to p53-deficient cells. Conversely, BrdU incorporation in wild type cells decreased with DNA content compared to p53-deficient cells with knock down of Survivin. Nonetheless, polyploid wild type cells even with a DNA content larger 4n were still able to incorporate BrdU, indicating transient G1 arrest after Survivin knock down. Furthermore, Survivin RNAi led in all tested cell lines to an induction of DNA lesions and caused a DNA damage response. SKY-analysis revealed numerical aberrations and a significant higher frequency of structural chromosomal aberrations indicative for DNA double strand break repair, only in Survivin-depleted cells. In conclusion, our results highlight the important role of Survivin as a chromosomal passenger protein which in concert with its molecular partners Aurora B, INCENP, and Borealin, controls chromosomal segregation by regulating bipolar spindle attachment and cytokinesis and protects cells from polyploidy and chromosomal instability. Citation Format: Ralf Wiedemuth, Barbara Klink, Evelin Schroeck, Gabriele Schackert, Achim Temme. Survivin safeguards chromosome numbers and protects from aneuploidy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5089. doi:10.1158/1538-7445.AM2014-5089