ESTRO 38 Abstract book

S1197 ESTRO 38

that METTL3 is a potent target for enhancing therapeutic efficacy in patients with pancreatic cancer. In addition, we performed cDNA expression analysis followed by gene ontology and protein-protein interaction analysis using the Database for Annotation, Visualization, and Integrated Discovery and Search Tool for the Retrieval of Interacting Genes/Proteins databases, respectively. The results demonstrated that METTL3 was associated with mitogen- activated protein kinase cascades, ubiquitin-dependent process and RNA splicing and regulation of cellular process, suggesting functional roles and targets of METTL3. Finally, we confirmed methylation statuses of those genes were changed by METTL3 using MeRIP-Seq. Conclusion The present study demonstrates that METTL3 is associated with chemo- and radioresistance and is a potential therapeutic target of pancreatic cancer. Additionally, our findings suggest several critical pathways, including MAPK cascades, ubiquitin-dependent process, RNA splicing and regulation of cellular process, as possible targets of METTL3. EP-2166 Ro90-7501 is a novel radiosensitizer which inhibits ATM phosphorylation and DNA repair K. Tamari 1 , Z. Li 1 , K. Otani 1 , Y. Takahashi 1 , K. Minami 1 , Y. Seo 2 , O. Suzuki 1 , F. Isohashi 1 , K. Ogawa 1 1 Osaka University Graduate School of Medicine, Radiation Oncology, Suita- Osaka, Japan; 2 Osaka University Graduate School of Medicine, Radiation Oncology, Suita-Osaka, Japan Purpose or Objective To explore new radiosensitizers, we have previously performed a cell-based high throughput screening by using 1280 compounds with irradiation, and found Ro90-7501 as a candidate for radiosensitizer. Ro90-7501 has been reported as an inhibitor of amyloid β42 fibril assembly which causes Alzheimer’s disease. However, the radiosensitizing effect of Ro90-7501 was unknown. The purpose of this study was to validate the radiosensitizing mechanism of Ro90-7501. Material and Methods A human cervical cancer cell line, HeLa, was used for entire experiment. Radiosensitizing effect of Ro90-7501 were validated by clonogenic survival assay and tumor regression assay using female BALB/c nude mice. Flow cytometry was performed to evaluate the difference in apoptosis and cell cycle after irradiation with or without Ro90-7501. Western blot was performed to evaluate the expression of proteins, involved DNA damage response such as pATM, pH2AX, pChk1, and pChk2. Results Clonogenic survival assay showed significant radiosensitizing effects of Ro90-7501 not only in normoxia but also hypoxia. Tumor regression assay in vivo revealed that combination treatment group using radiotherapy with Ro90-7501 (RT+Ro) was significantly reduced tumor volume compared with no treatment group, Ro90-7501 group and radiotherapy group. Ro90-7501 did not induce apparent side effects. Apoptosis significantly increased in RT+Ro group compared with other groups. Cell cycle analysis showed that RT+Ro group increased G2/M arrest. Western blot analysis showed that Ro90-7501 suppressed phosphorylation of ATM, and also suppressed phosphorylation of several downstream proteins such as H2AX, Chk1, Chk2 after irradiation, indicating that DNA repair pathway was inhibited in Ro+RT group. Electronic Poster: Radiobiology track: DNA damage response

Lymphocyte Phenotyping showed that Natural Killer cells defined as CD56+high CD16+ , increased among the follow up with initial values of 0.95% to 1.38% at 6 months. Statistical analysis using Friedman Test (p=0.18) and Wilcoxon test don´t showed significant differences. Regulatory T cells activated defined as (CD4+-CD25+Foxp3 +CD45RA) showed stable values during the follow up (baseline values 4.97% vs. 4.46% at 6 months). No statistical differences were detected. Myeloid-derived suppressor cells (MDSC) CD33+CD11b+CD14-, showed a tendency to lower values during the follow up (basal 62.6% vs 66.1%). No statistical significance was detected. Conclusion High doses of radiation therapy over the lung can provide a systemic effect detected in peripheral blood samples. Even the small sample size, our study shows an increase of stimulatory immune populations with stability or decreasing suppressive populations. EP-2165 m6A RNA modification by METTL3 regulates chemo- and radioresistance in pancreatic cancer cells S. Tatekawa 1,2,3 , M. Konno 2,3 , A. Asai 2,3 , J. Koseki 2 , K. Taketo 1 , H. Ishii 2,3 , K. Ogawa 1 1 Osaka University Graduate School of Medicine, Department of Radiation Oncology, Osaka, Japan; 2 Osaka University Graduate School of Medicine, Medical Data Science, Osaka, Japan; 3 Osaka University Graduate School of Medicine, Cancer Frontier Science, Osaka, Japan Purpose or Objective N6-methyladenosine (m6A) is the most prevalent internal modification of messenger RNA (mRNA) and long noncoding RNA (lncRNA) in the majority of eukaryotes. The m6A modification in RNA can be catalyzed by methyltransferases, or removed by demethylases, which are termed m6A writers and erasers, respectively. m6A mRNA methylation is a gene regulatory mechanism affecting cell differentiation and proliferation through regulation of RNA stabilities, mRNA splicing, microRNA processing and mRNA translation. Especially in cancer, however, it has been mostly unclear what means m6A is dynamically regulated or written by enzymatic components represented by methyltransferase-like 3 (METTL3) and how m6A is significant for each of the numerous genes. We focused on METTL3 in pancreatic cancer, because the prognosis of which is not satisfactory despite the development of multidisciplinary therapies including radiation therapy. Material and Methods To study the roles of m6A mRNA methylation in chemo- and radioresistance, we established METTL3-knockdown pancreatic cancer cell line using short hairpin RNA. We performed proliferation assay, sphere formation assay, chemo- and radiosensitivity assay. For the purpose of searching target genes affected by METTL3, we conducted cDNA expression analysis and anti-m6A antibody methylated RNA immunoprecipitation sequencing (MeRIP- Seq). Results Although there was no difference in morphology and proliferation rate between control and METTL3-depleted cells, METTL3-depleted cells in sphere formation assay showed significantly lower ability than control cells to form spheres. Furthermore, METTL3-depleted cells showed higher sensitivity to anticancer reagents such as gemcitabine, 5-fluorouracil, cisplatin and irradiation through enhancing apoptotic response. Our data suggest Electronic Poster: Radiobiology track: Radiation and tumour metabolism