Increased Replication Stress Sensitises High Risk Neuroblastoma Cells to ATR and PARP Inhibition ^†

Abstract

Background: Neuroblastoma (NB) is a rare childhood cancer but accounts for 15% of paediatric cancer deaths. MYCN amplification and/or ATM loss through 11q deletion may cause an increase in replication stress (RS) in a substantial proportion (~80%) of high-risk NB. RS creates a dependency on ATR-mediated S and G2 checkpoint control. This study aimed to determine if MYCN amplification or ATM loss identifies cells which are sensitive to ATR inhibition (ATRi). As PARP inhibition causes RS through unrepaired single-strand DNA breaks progressing to replication, we also examined the effect of ATRi on PARP inhibitor (PARPi) cytotoxicity and PARPi-induced RS, cell cycle arrest and homologous recombination repair (HRR) activity. Methods: Cell proliferation in response to 72 h of treatment with the ATR inhibitor, VE-821, was assessed by XTT (Roche) and clonogenic survival assays in a panel of 11 NB cell lines and the effect of VE-821 on growth inhibition caused by the PARPi, Olaparib, in 4 cell lines. CHK1^S345 (a marker of ATR activity) and RPA2^S8 and H2AX^S129 phosphorylation (RS markers) were assessed by Western blotting and immunofluorescent microscopy. HRR was examined by the formation of RAD51 foci by immunofluorescent microscopy. Cell cycle analysis was carried out by flow cytometry. Results: VE-821-induced growth inhibition and cell death was significantly increased in MYCN-amplified cell lines and cell lines with low ATM protein expression (p < 0.05 Mann–Whitney U test). Olaparib (5 µM) treatment increased CHK1^S345 and H2AX^S129 phosphorylation after 24 h of treatment in all cell lines. ATR inhibition prevented CHK1^S345 phosphorylation and reduced Olaparib-induced RAD51 foci formation. VE-821 abrogated Olaparib-induced S and G2 checkpoint arrest. Conclusion: MYCN amplification and low ATM protein expression are determinants of ATRi sensitivity in NB cell lines. ATRi sensitises NB cells to PARPi by abrogating S/G2 checkpoint arrest and impairing HRR.