ESTRO 38 Abstract book

S345 ESTRO 38

data provides new RNA-based predictors of chemoresistance, as well as potential targets to counteract this resistance in GBM. SP-0655 Irradiation and targeted inhibition of the PI3K/AKT and MAPK pathways in glioma P. Sminia 1 , R. Narayan 1 , A. Gasol 1 , F. Cornelissen 2 , J. Theys 3 , T. Lagerwei 4 , E. De Vries 5 , F. Bikhezar 1 , A. Denkova 6 , R. De Kruijff 6 , B. Slotman 1 , L. Stalpers 7 , B. Baumert 8 , B. Westerman 2 1 VU University Medical Center, Radiation Oncology, Amsterdam, The Netherlands ; 2 VU University Medical Center, Neurosurgery- Neuro-Oncology Research Group, Amsterdam, The Netherlands; 3 Maastricht University Medical Center, Radiation Oncology, Maastricht, The Netherlands; 4 VU University Medical Center, Neurosurgery- Neuro-Oncology Research, Amsterdam, The Netherlands ; 5 VU University Medical Center, Molecular Cell Biology and Immunology, Amsterdam, The Netherlands ; 6 Delft University of Technology, Radiation Science and Technology, Delft, The Netherlands; 7 Academic Medical Center Amsterdam, Radiation Oncology, Amsterdam, The Netherlands; 8 University Hospital Bonn, Radiation Oncology, Bonn, Germany Abstract text Commonly defective core signaling pathways in glioma contributing to radioresistance include the tumor protein p53 (TP53) pathway, the retinoblastoma (RB) pathway and receptor tyrosine kinase (RTK) pathway. Combined treatment of irradiation, the key component of the current standard treatment of glioblastoma (GBM) patients, with drugs that selectively target these pathways, might improve the outcome of therapy. Studies were performed on GBM cell lines and patient derived primary cultures grown as monolayer and multicellular spheroids with endpoints cell proliferation, clonogenic cell survival, spheroid growth rate, - volume reduction and – time to regrow. We investigated the single agent efficacy and radiosensitizing potential of a panel of novel targeted small molecule drugs including MK2206, RAD001, BEZ235, MLN0128 and MEK162. Effects on cell cycle distribution and expression of key target proteins were evaluated. Out of the panel of drugs, both the allosteric AKT inhibitor MK2206 and MEK162 (binimetinib), which is an allosteric inhibitor of MEK1/2, were found to act a radiosensitizer. MK2206 delayed the growth of spheroids and sensitized to both irradiation and temozolomide via reduced phosphorylation of Thr308 and Ser473 residues of AKT. MEK162 was found to down-regulate and dephosphorylate the cell cycle checkpoint proteins CDK1/CDK2/WEE1 and DNA damage response proteins p-ATM/p-CHK2. When combined with radiation this led to a prolonged DNA damage signal. Next, the combination of MEK162 (50 mg/kg) and irradiation (3 x 2 Gy) was studied on orthotopic GBM8 brain tumor xenografts. The data showed a significantly reduced growth rate, increased growth delay and prolonged survival time of the animals. In addition, RNA expression of responsive cell cultures correlated to mesenchymal stratification of patient expression data. However, the delivery of MEK162 to the tumor site, like most chemical compounds, is abrogated by the blood-brain barrier (BBB). First in vitro data on BBB crossing demonstrated effective passaging of the drug when loaded in polymeric nanocarriers. In conclusion, both the AKT inhibitor MK2206 and the MAPK inhibitor MEK162 demonstrated radiosensitizing potential in GBM spheroids in vitro and in orthotopic GBM xenografts in vivo. We identified a patient subgroup that might benefit from MEK inhibition combined with radiotherapy. This combinatorial treatment approach opens a novel therapeutic avenue for GBM patients. Supported by the Dutch Cancer Foundation (KWF), grant

clinical trials. One contributing factor is thought to be the failure of many therapeutics to target glioblastoma stem- like cells (GSC) which exhibit DNA damage response (DDR) activation and enhanced DNA double strand break (DSB) repair. Although DDR activation and radiation resistance in GSC were documented many years ago the underlying reasons for this have remained enigmatic. Elucidation and understanding of this phenomenon would have significant implications for efforts to increase the efficacy of radiotherapy in the clinic. In this presentation we will provide an overview of DDR activation observed in GSC and explore its implications for radiation resistance. We will describe a unique DNA replication phenotype in GSC that predisposes them to high levels of DNA replication stress as an underlying mechanism for DDR activation and discuss possible sources of elevated replication stress in this cell population. Finally, we will examine targeting of DNA replication stress response as a promising therapeutic and radiation sensitisation strategy for clinical translation. SP-0653 Tumor cell connections causing radiation resistance F. Winkler 1 1 University of Heidelberg, Neurology, Heidelberg, Germany Abstract text The recent discovery of ultra-long and thin membrane protrusions of glioma cells, called tumor microtubes (TMs), has added to our understanding of these incurable tumors (Osswald et al., Nature 2015). Astrocytoma (including glioblastoma) cells extend these highly functional structures to colonize the brain, and to interconnect to one large communicating multicellular network. Glioma cells integrated into this TM network resist the cytotoxic effects of radiotherapy and chemotherapy. This talk will cover how our understanding of the disease "glioma" has changed, and how novel therapies are developed to tackle this basic cellular mechanism of resistance against radiotherapy, chemotherapy, and surgery. SP-0654 Transcriptional response to temozolomide in Glioblastoma reveals critical role of long non-coding RNAs S. Niclou 1 , S. Fritah 1 , M. Sarmini 1 , W. Jiang 2 , A. Muller 1 , M. Dieterle 1 , R. Mitra 2 , A. Golebiewska 1 , Z. Zhao 2 , F. Azuaje 1 1 Luxembourg Institute of Health, Department Of Oncology, Luxembourg, Luxembourg; 2 Vanderbilt University, Bioinformatics, Nashville, USA Abstract text Resistance to the alkylating agent temozolomide (TMZ) is a major cause of Glioblastoma (GBM) recurrence and dismal prognosis. To date, the epigenetic regulation of the O6-methylguanine methyltransferase (MGMT) promoter is the only TMZ-predictive marker with clinical relevance, but there is little knowledge of regulatory transcriptional pathways involved in therapy resistance. Here, we provide a comprehensive overview of the transcriptional response to TMZ in patient-derived cellular GBM models, with a special focus on the contribution of non-coding RNAs as novel markers of chemoresistance. Using RNA-Seq and small RNA-seq, we have uncovered a complex transcriptional response to TMZ and identified a subset of long non-coding RNAs (lncRNAs) mediating regulatory circuits. Interestingly, lncRNAs were mapped to processes linked to drug sensitivity, cell cycle regulation and/or developmental pathways. Among these lncRNAs, we characterized a novel lncRNA termed RADAR (RNA associated with DNA Damage and Replication) and show that RADAR is a chromatin-associated lncRNA involved in cell cycle control, apoptosis and sister chromatid cohesion. Altogether, this integrative analysis of RNA-seq