Heterogeneities in Cell Cycle Checkpoint Activation Following Doxorubicin Treatment Reveal Targetable Vulnerabilities in TP53 Mutated Ultra High-Risk Neuroblastoma Cell Lines.

Linnéa Ödborn Jönsson,Lars Ährlund-Richter,Ximena Lopez-Lorenzo,Nikolas Herold,Shahrzad Shirazi Fard,Ourania N Kostopoulou,Maryam Sahi,Faye Leilah Keller

doi:10.3390/ijms22073664

Abstract

Most chemotherapeutics target DNA integrity and thereby trigger tumour cell death through activation of DNA damage responses that are tightly coupled to the cell cycle. Disturbances in cell cycle regulation can therefore lead to treatment resistance. Here, a comprehensive analysis of cell cycle checkpoint activation following doxorubicin (doxo) treatment was performed using flow cytometry, immunofluorescence and live-cell imaging in a panel of TP53 mutated ultra high-risk neuroblastoma (NB) cell lines, SK-N-DZ, Kelly, SK-N-AS, SK-N-FI, and BE(2)-C. Following treatment, a dose-dependent accumulation in either S- and/or G2/M-phase was observed. This coincided with a heterogeneous increase of cell cycle checkpoint proteins, i.e., phos-ATM, phos-CHK1, phos-CHK2, Wee1, p21Cip1/Waf1, and p27Kip among the cell lines. Combination treatment with doxo and a small-molecule inhibitor of ATM showed a delay in regrowth in SK-N-DZ, of CHK1 in BE(2)-C, of Wee1 in SK-N-FI and BE(2)-C, and of p21 in Kelly and BE(2)-C. Further investigation revealed, in all tested cell lines, a subset of cells arrested in mitosis, indicating independence on the intra-S- and/or G2/M-checkpoints. Taken together, we mapped distinct cell cycle checkpoints in ultra high-risk NB cell lines and identified checkpoint dependent and independent druggable targets.

Highlights

Neuroblastoma (NB) is the most common extracranial solid tumor in early childhood
Cultured cells were exposed to the indicated treatment and analysed after 48 h
Results was performed on SK-N-DZ, Kelly, SK-N-AS, SK-N-FI, and BE(2)-C following mock are presented as percentage cells in each cell cycle phase

Summary

Introduction

Neuroblastoma (NB) is the most common extracranial solid tumor in early childhood. It is a heterogenous neoplasia with clinical presentation ranging from spontaneous regression of metastatic disease to a rapidly progressive course [1]. Among high-risk NB, a subset of approximately 19% is considered to be ultra-high risk, defined as death from disease within 18 months of diagnosis despite intensive treatment [2]. Relapse rates in high-risk NB are as high as 50% despite intensive multimodal treatment and are frequently characterised by therapy resistance and intra-tumor diversity making it difficult to cure [3,4]. Ultra high-risk NB and relapsed NB remain a therapeutic challenge with only limited evidence for efficacious salvage therapies [5]

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Results

Conclusion