Increased Replication Stress Determines ATR Inhibitor Sensitivity in Neuroblastoma Cells

SIMPLE SUMMARY: Neuroblastoma is a childhood cancer with poor survival and new therapies are urgently needed, especially for high-risk disease. Here, we demonstrate that novel drugs targeting a protein called ATR can specifically kill a type of high-risk neuroblastoma associated with MYCN expression. We show that the mechanism by which this occurs is via increasing the stress on cells when they replicate their DNA. Further, we show that by targeting ATR in combination with other drugs that cause replication stress, we can increase killing of both high-risk MYCN amplified and non amplified neuroblastoma. ABSTRACT: Despite intensive high-dose multimodal therapy, high-risk neuroblastoma (NB) confers a less than 50% survival rate. This study investigates the role of replication stress in sensitivity to inhibition of Ataxia telangiectasia and Rad3-related (ATR) in pre-clinical models of high-risk NB. Amplification of the oncogene MYCN always imparts high-risk disease and occurs in 25% of all NB. Here, we show that MYCN-induced replication stress directly increases sensitivity to the ATR inhibitors VE-821 and AZD6738. PARP inhibition with Olaparib also results in replication stress and ATR activation, and sensitises NB cells to ATR inhibition independently of MYCN status, with synergistic levels of cell death seen in MYCN expressing ATR- and PARP-inhibited cells. Mechanistically, we demonstrate that ATR inhibition increases the number of persistent stalled and collapsed replication forks, exacerbating replication stress. It also abrogates S and G2 cell cycle checkpoints leading to death during mitosis in cells treated with an ATR inhibitor combined with PARP inhibition. In summary, increased replication stress through high MYCN expression, PARP inhibition or chemotherapeutic agents results in sensitivity to ATR inhibition. Our findings provide a mechanistic rationale for the inclusion of ATR and PARP inhibitors as a potential treatment strategy for high-risk NB.