P03.10.A IDENTIFYING DNA DAMAGE RESPONSE (DDR) RELATED FACTORS ESSENTIAL FOR RADIOTHERAPY RESPONSE IN GLIOBLASTOMA USING A NOVEL CRISPR/CAS9 LIBRARY, DDRKOL

Abstract

Abstract The standard treatment of glioblastoma, which is the most common and aggressive primary brain tumor, includes combined radiotherapy (IR) and chemotherapy (temozolomide (TMZ)) after surgery. The basic working principle behind radiotherapy is to induce DNA double-strand breaks (DSBs), which is the most lethal form of DNA damage and the primary cause of cell death, and to finally kill cancer cells. Additionally, exposure to IR induces the activation of a multicomponent signal transduction network of DNA damage response (DDR). In this study, we developed a new CRISPR/Cas9-based tool to investigate the role of DDR mechanisms and identify novel genes affecting radiotherapy response in glioblastoma. We generated a focused sgRNA library termed DDRKOL (DNA Damage Response Knock Out Library) targeting the DDR pathway to identify novel modulators of TMZ and radiotherapy response with CRISPR/Cas9 screening in vitro. DDRKOL includes a total of 9000 sgRNAs targeting 888 genes (10 sgRNA/gene) containing all DNA damage-related pathways, curated from Gene Ontology Consortium, 35 sgRNA positive controls targeting cell essential genes, 90 non-targeting control sgRNAs, and 35 internal control genes. The composition and coverage of the library were validated, and DDRKOL was utilized to investigate modulators of TMZ response in U87MG cells. Our future work will involve characterizing different effects of DDRKOL members in radiotherapy response, in different subtypes of gliomas with different genetic BACKGROUND s. Cell response to radiation is important, therefore optimization experiments were performed with different radiation doses in A172, U373, and U373-60Gy cell lines to choose the optimal dose suitable for screening. Accordingly, 2 Gy radiation was chosen for large-scale screens. DDRKOL will serve as an important tool to study not only glioblastoma but also many other diseases that threaten global health. Discovering novel genes that affect the therapy response will be stimulating because it will help understand how to overcome therapy resistance and develop new strategies to design effective therapeutic strategies, which can ultimately translate into clinics.This study is supported by TUBITAK 1001 program (#221S439).