High-risk neuroblastomas with MYCN amplification (MNA) have a very poor outcome, making the search for new therapeutic approaches for these patients an absolute priority. Despite initial encouraging results obtained with PARP inhibitors in neuroblastoma preclinical models, the expression of PARPs and the biochemical consequences of their inhibition on the DNA damage response (DDR) were not characterized. Analysis of multiple datasets in R2 indicates that high PARP1/2 expression is significantly associated with more aggressive tumors and with poor survival in primary neuroblastomas, suggesting it is a potential and previously unrecognized prognostic factor. In vitro, inhibition of PARP activity via olaparib impaired neuroblastoma proliferation with slightly different effects between MNA and MYCN single copy (MNSC) cells. However, olaparib induced accumulation of DNA double strand breaks, a typical DDR including H2AX and p53 phosphorylation, and appearance of heavily disorganized and fragmented nuclei, suggestive of mitotic catastrophe, in a MYCN-dependent fashion. Time-lapse microscopy studies revealed that PARP inhibition elongates cell cycle and mitosis duration irrespective of MYCN expression, while an unsuccessful mitosis culminating in mitotic catastrophe only occurs in MNA cells. An higher rate of 53BP1 foci, micronuclei and anaphase bridges in olaparib-treated MYCN-overexpressing cells -compared to MNSC- indicated that mitotic catastrophe is most likely due to the attempt to undergo mitosis despite the presence of damaged or incompletely replicated DNA. In fact, despite a strong activation of the ATR-CHK1-CDC25A checkpoint, olaparib-treated MYCN-overexpressing cells transiently delay S-phase completion, but subsequently progress through mitosis and eventually undergo mitotic catastrophe. Consistently, CHK1 inhibition further accelerates progression in G2/M and strongly potentiates this phenotype, while CDK1 inhibition impedes mitotic entry and abrogates mitotic catastrophe.
These data emphasize the pivotal role of PARPs in controlling MYCN-dependent replication stress and support the introduction of PARP and CHK1 inhibitors in therapeutic approaches for neuroblastomas with high MYC activity.