PARP inhibition causes replication stress in preclinical models of high risk neuroblastoma and synergises with inhibition of ATR


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Harriet Southgate1,Nicola J Curtin1,Deborah A Tweddle1
1Newcastle University, Newcastle, UK

Abstract

Background

Neuroblastoma (NB) is the commonest extra-cranial malignant solid tumour of childhood and one of the most difficult to cure. Around 50% of high-risk NBs have MYCN oncogene amplification (MNA) that promotes rapid DNA replication, leading to errors and replication stress (RS). Cells with RS are acutely dependent on the DNA damage sensor kinase ATR. PARP inhibition results in unrepaired single strand DNA breaks progressing to replication, further increasing RS. We hypothesise that combining PARP and ATR inhibition will lead to greater cytotoxicity due to increased RS.

Aim:

To assess synergism between PARP inhibition and ATR inhibition in high risk NB cell lines and to measure RS. 

Method

Human NB cell lines: SHSY5Y and SKNAS (non-MNA), and NGP and N20_R1 (MNA). The PARPi olaparib and the ATRi VE-821 were used. CHK1S345 and H2AXS129 phosphorylation was assessed using Western blotting to determine ATR activity and RS respectively.  RS was also determined by gH2AX foci formation using immunofluorescent microscopy. Cytotoxicity was assessed by XTT cell proliferation assay (Roche) and colony formation assay.  

Results

Olaparib (5 µM) treatment increased CHK1S345 and H2AXS129 phosphorylation after treatment for 24 hours in all cell lines. H2AXS129 phosphorylation was further increased with the addition of VE-821 (1 µM). ATR inhibition prevented CHK1S345 phosphorylation, as expected.  The number of gH2AX foci exhibited in the cell lines by immunofluorescence increased after treatment with olaparib (1 µM) which was further increased with the addition of VE-821 (1 µM). In cytotoxicity assays, combination index analysis (Calcusyn) showed that ATR inhibition by VE-821 is synergistic with olaparib at sub lethal concentrations (<1 µM) (CI value 0.04-0.89), although this effect is lost at higher concentrations.

Conclusion

ATR inhibition by VE-821 is synergistic with olaparib at sub lethal concentrations (<1 µM) and further increases the replication stress caused by PARP inhibition.