ESTRO 2020 Abstract book

S114 ESTRO 2020

146a expression was significantly downregulated in recurrent GBM tumors compared with the paired primary GBM tumors (p=0.003). Overexpression of miR-146a significantly inhibited tumor cell growth and sensitized patient-derived primary GBM cells to temozolomide (TMZ) treatment in vitro , and showed statistically significant smaller tumor size (p<0.01) and prolonged survival (p=0.001) in vivo . In addition, miR-146a is downregulated in glioma cancer stem cells, and overexpression of miR-146a significantly inhibited glioma cancer stem cell self-renewal using sphere formation assay. We also found that overexpression of miR-146a significantly inhibited NF-κB and ERK activities. Conclusion Our data suggest, for the first time, that miR-146a predicts favorable prognosis for GBM patients and sensitizes primary GBM cells to TMZ treatment in vitro and in vivo through regulating glioma stem cells. Importantly, miR- 146a may prove to be a master switch shutting off NF-κB, ERK, as well as other pathways and may overcome redundancies among these pathways leading to resistance. Funding: R01CA108633, R01CA169368, U10CA180850- 01(NCI), Brain Tumor Funders Collaborative Grant, and The Ohio State University CCC (all to AC), Bohnenn Fund (to PR). OC-0207 Avoidance of DNA replication stress leads to radioresistance in stem cell-like TNBC F. Meyer 1 , A. Engel 1 , B. Riepen 1 , U. Schumacher 2 , A. Dubrovska 3 , C. Petersen 4 , C. Peitzsch 3 , L. Hein 3 , S. Werner 5 , H. Wikmann 5 , K. Rothkamm 1 , K. Borgmann 1 1 University Medical Center Hamburg - Eppendorf UKE, Radiation Biology/Experimental Radiooncology, Hamburg, Germany ; 2 University Medical Center Hamburg - Eppendorf UKE, Institute for Anatomy and Experimental Anatomy, Hamburg, Germany ; 3 Technical University Dresden, OncoRay, Dresden, Germany ; 4 University Medical Center Hamburg - Eppendorf UKE, Clinic for Radiotherapy, Hamburg, Germany ; 5 University Medical Center Hamburg - Eppendorf UKE, Tumorbiology, Hamburg, Germany Purpose or Objective Despite postoperative radiotherapy, triple-negative breast cancer (TNBC) often develops local recurrences and distant metastases. This is attributed to an increased proportion of radiation-resistant tumor stem cells (CSC). CSC are characterized by a more effective DNA damage response and repair processes as well as reduced oxidative stress. However, it is still unclear how DNA repair pathways and the avoidance of oxidative stress determine the radiation resistance of CSC. This project aims to figure out resistance mechanisms of CSC and strategies to overcome their intrinsic therapy resistance Material and Methods The investigations were performed in three isogenic MDA- MB-231 TNBC cell lines, their respective radiation resistant subclones selected by repeated irradiation (10x4Gy). Luminal MCF7 cells served as controls. Expression of Homologous recombination (HR) -related and stem-cell factors was determined and DNA repair in general (53BP1) as well as Replication stress (RPA/yH2AX foci) and HR functionality (RAD51 foci, plasmid reporter assay) were analyzed. Replication processes were examined using the DNA fiber assay, Mitotracker-assays were also performed. The radiosensitizing effect of DNA-repair and S-phase specific inhibitors was analyzed by colony assay and correlated with expression profiles of stem cell markers and DNA repair proteins in the METABRIC database Results An increased expression of the stem cell markers ZEB1 and ALDH1 and a significantly increased activity of ALDH1 was observed in all radioresistant clones. After irradiation, survival in the clonal subpopulation was significantly increased and the number of cellular mitochondria

Material and Methods We used an air liquid interface pseudo-stratified cultures, derived from primary human bronchial epithelial cells (PBECs) of lung cancer patients undergoing lobectomy, and studied the combined effects of irradiation and NOTCH inhibition. We investigated the consequences of blocking NOTCH signaling pathway using the pan-NOTCH γ- secretase inhibitor DBZ alone and when combined with irradiation (2, 4 Gy).In vivo irradiation was also performed to compare the results obtained with human stem cells and murine stem cells. Results We found that basal cells (p63, CK5) cease proliferation at day 21 st and mucous cell differentiation (Muc1/5ac) precedes ciliary differentiation (Ac-Tub) by 5 days, starting at day 10 and both complete at day 28. Proliferation decreases overtime but inhibiting NOTCH both in undifferentiated progenitors and in differentiated cells at day 21st increases TP63+ proliferation alone and even more when combined with radiotherapy. In all the 3 donors NOTCH inhibition increases TP63+basal progenitors and ciliated cells and decreases mucous cells alone and when combined with radiation. In irradiated cultures we observed increased pATM and pCHK2 12h and 24h post irradiation when NOTCH signaling was inhibited. Furthermore, comparable results were confirmed by in vivo irradiation, where the combination of NOTCH inhibition and irradiation increases the percentage of stem cells and ciliated cells when compared to irradiated mice. Conclusion These data support the use of normal patient tissue for predictive toxicity, screening of combination treatments and disclose important novel interactions between NOTCH inhibition and radiotherapy. OC-0206 Epigenetic silencing of miR-146a drives tumor progression and therapeutic resistance in glioblastoma T. Cui 1 , E.H. Bell 1 , J. McElroy 2 , B. Johnson 1 , E. Sebastian 1 , K. Liu 3 , P.M. Gulati 1 , A.P. Becker 1 , M. Geurts 4 , A. Gray 3 , J. Fleming 1 , S. Beyer 1 , M. Venere 1 , J. Haque 1 , Q. Wang 1 , P. Robe 5 , A. Chakravarti 1 1 Arthur G. James Hospital/Ohio State Comprehensive Cancer Center, Department of Radiation Oncology, Columbus, USA ; 2 The Ohio State University Center for Biostatistics, Department of Biomedical Informatics, Columbus, USA ; 3 The Ohio State University College of Medicine, Department of Radiation Oncology, Columbus, USA ; 4 Erasmus MC Cancer Center, Department of Neurology, Utrecht, The Netherlands ; 5 Brain Center Rudolf Magnus- University Medical Center Utrecht, Department of Neurology and Neurosurgery, Utrecht, The Netherlands Purpose or Objective Glioblastomas (GBMs) are the most aggressive primary brain tumors, with an average survival of less than 15 months despite decades of translational investigation. microRNAs (miRNAs) function as post-transcriptional regulators, and are promising novel biomarkers in GBM. The aims of this study are: 1) to investigate novel miRNAs biomarkers that affect tumorigenesis and therapeutic sensitivity, and 2) to study the underlying molecular Total RNAs were isolated from 268 FFPE tumor samples, Nanostring v3 was performed for miRNA expression analysis followed by univariable (UVA) and multivariable (MVA) survival analyses. Cell proliferation, invasion, and apoptosis assays were conducted to define the role of miR- 146a in GBM tumorigenesis and therapeutic response. Results UVA analyses demonstrated that miR-146a is one of the top miRNAs that correlated with better prognosis in GBM patients (p=9.21E-05), which was independent of MGMT promoter methylation by MVA analyses (p<0.001). miR- mechanisms in GBM. Material and Methods