P10.25.a atr inhibition in experimental glioma: discovery and validation of molecular targets for functionally-instructed combination therapies

Abstract BACKGROUND The DNA damage response (DDR) is an important physiological network for the prevention of malignant transformation, e.g. by halting the cell cycle upon DNA damage detection and promoting DNA repair. Glioblastoma are incurable primary brain tumors that display acquired treatment resistance which might also be due to DDR dysregulation. Furthermore, we had recently investigated molecular effects of disrupting the (b)HLH network, a frequently altered and tumor-promoting transcriptional network in glioma. The identified downstream anti-tumor effects included the ataxia telangiectasia and Rad3 related (ATR) pathway. In the present study, we aimed at investigating the anti-glioma effects of ATR inhibition (ATRi) in experimental glioma and exploring potential treatment regimens for forward translation. MATERIAL AND METHODS We assessed ATRi monotherapy in the syngeneic SMA560/VM/Dk mouse model, performed acute cytotoxicity, clonogenic survival assays, cell cycle analyses by flow cytometry and apoptosis induction in vitro. Furthermore, we used RNA sequencing, DigiWest protein profiling analyses and genome-wide CRISPR/Cas9 loss-of function and activation screens. We evaluated synergistic or additive anti-glioma effects of functionally-instructed combination therapies by the Bliss and ZIP synergy models. We xenotransplanted zebrafish embryos with glioma cells to validate most promising combination approaches in vivo. RESULTS ATRi monotherapy induced prolonged latency until the onset of neurological symptoms in the SMA560/VM/Dk mouse model. ATRi reduced viability and clonogenic survival in vitro and ex vivo. Molecular analyses revealed differential patterns of gene expression, e.g. p53 signaling, differing modes of cell cycle and apoptosis regulation, arguing for a profound context specificity of the treatment. Similarly, CRISPR/Cas-9 genome-wide knockout and activation screens revealed distinct genetic vulnerabilities. Based on these RESULTS , we designed a drug library for investigating potential functionally-instructed modifiers of response to ATRi in experimental glioma in vitro, ex vivo and in vivo. CONCLUSION In summary, we present a workflow in experimental glioma for target discovery and validation leading to the design of novel combination therapies. We identify potential combination therapies involving ATRi in experimental glioma that could inform future early phase clinical trials and preclinical studies.