P13.16.B TARGETING RBM19 TO ENHANCE RADIOTHERAPY IN GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) treatment faces challenges due to radioresistance. This study focuses on RNA binding motif protein 19 (RBM19), a protein implicated in crucial cellular functions yet underexplored in enhancing radiosensitivity. Our research aims to elucidate the potential of RBM19 in improving radiotherapy outcomes for glioblastoma patients. MATERIAL AND METHODS RBM19 expression in GBM patients and normal brain tissue was analyzed using GEO datasets. The expression of RBM19 and its correlation with patient survival were assessed using clinical and follow-up data from TCGA. In vitro, we manipulated RBM19 expression in U87 and U251 glioblastoma cell lines. The radiosensitivity of cancer cells was determined through colony formation assay, apoptosis, and CCK-8 assay. The comet assay and γH2AX assay were used at different time points to detect the kinetics of DNA damage and repair. Western blot assay was applied to explore the functions of RBM19 on DNA damage response after ionizing radiation, further highlighting its role in DNA damage repair pathway. Co-immunoprecipitation was conducted to study the interaction between RBM19 and CHK1 protein. Xenografts were performed to investigate the in vivo effects of RBM19 on tumor growth. RESULTS Differential gene analysis identified RBM19 as consistently upregulated in GBM across multiple GEO datasets. TCGA data analysis revealed a negative correlation between RBM19 expression and overall and disease-free survival in post-radiotherapy GBM patients. In vitro, alterations in RBM19 expression influenced glioblastoma cell viability post-irradiation, knockdown reduced viability and enhanced radiosensitivity, while overexpression had the opposite effect. Comet assays demonstrated that RBM19 modification affects the extent of DNA damage post-irradiation, with knockdown exacerbating DNA damage. Immunofluorescence for γ-H2AX foci confirmed increased DNA damage in RBM19-knockdown cells. Furthermore, RBM19 knockdown significantly increased apoptosis post-irradiation. Western blot analysis showed that RBM19 influences key proteins in the DNA damage repair pathway, potentially affecting therapeutic resistance in glioblastoma cells. Co-immunoprecipitation found interactions between RBM19 and Checkpoint kinase 1 (CHK1) and ataxia telangiectasia and Rad3-related (ATR), again shedding light on its role in DNA damage repair pathways. In vivo experiments demonstrated that RBM19 knockdown extended survival and reduced tumor volume in irradiated glioblastoma-bearing mice. More DNA damage was observed in RBM19 knockdown xenografts. CONCLUSION RBM19 may serve as a new molecular target for radiosensitization in glioblastoma. This study provides a new direction for research and a potential therapeutic approach for the radiosensitization of clinical glioblastoma treatments.