Increased Replication Stress Determines ATR Inhibitor Sensitivity in Neuroblastoma Cells.

David King,Helen E Bryant,Deborah A Tweddle,Devon Chapman,Harriet E D Southgate,Daniel Harrison,Jessica B Watson,Polly Gravells,Nicola J Curtin,Lindi Chen,Leona Fields,Saskia Roetschke,Daniel Yeomanson

doi:10.3390/cancers13246215

Abstract

Simple SummaryNeuroblastoma 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.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.

Highlights

Neuroblastoma (NB) is a cancer derived from cells of the embryonal neural crest and accounts for 8% of all childhood cancers
We have previously shown that MYCN-amplified cell lines are sensitive to ATR inhibitors (ATRi) [23] and increased sensitivity to checkpoint kinase 1 (CHK1) inhibition in MYCN overexpressing NB cells has been reported [24,25]
We have previously reported that MYCN-amplified cell lines are more sensitive to ATR inhibition than non-MYCN-amplified cell lines, using a panel of NB cell lines with varying genetic abnormalities [23]

Summary

Introduction

Neuroblastoma (NB) is a cancer derived from cells of the embryonal neural crest and accounts for 8% of all childhood cancers. At the time of diagnosis, 50% of patients will have high-risk disease, defined as the presence of metastatic disease in patients >1 year of age or amplification of the MYCN oncogene [1]. The frequency of MYCN amplification increases to around 50% in the high-risk group. MYCN, a member of the MYC family of proto-oncogenes, is a transcription factor that mediates the transcription of genes involved in metabolism, protein biosynthesis, cell cycle regulation, DNA repair, cell adhesion, and the cytoskeleton [7]. It is generally considered that MYCN amplification is an early event in NB development that drives tumorigenic transformation in neural crest-derived cells, resulting in stem cell-like traits, for example, self-renewal, resistance to apoptosis and metabolic changes. Other papers suggest that MYC can interact directly with the pre-replicative complex and that this is the cause of the RS, together with intermediates caused by MYCdriven hyper-transcription [10]

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