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. 2023 Sep;149(11):8605-8617.
doi: 10.1007/s00432-023-04804-0. Epub 2023 Apr 25.

Synergistic anticancer activity of combined ATR and ribonucleotide reductase inhibition in Ewing's sarcoma cells

Max-Johann Sturm  1   2 , Julián Andrés Henao-Restrepo  3 , Sabine Becker  1   2 , Hans Proquitté  1 , James F Beck  1 , Jürgen Sonnemann  4   5
Affiliations

Synergistic anticancer activity of combined ATR and ribonucleotide reductase inhibition in Ewing's sarcoma cells

Max-Johann Sturm et al. J Cancer Res Clin Oncol. 2023 Sep.

Abstract

Purpose: Ewing's sarcoma is a highly malignant childhood tumour whose outcome has hardly changed over the past two decades despite numerous attempts at chemotherapy intensification. It is therefore essential to identify new treatment options. The present study was conducted to explore the effectiveness of combined inhibition of two promising targets, ATR and ribonucleotide reductase (RNR), in Ewing's sarcoma cells.

Methods: Effects of the ATR inhibitor VE821 in combination with the RNR inhibitors triapine and didox were assessed in three Ewing's sarcoma cell lines with different TP53 status (WE-68, SK-ES-1, A673) by flow cytometric analysis of cell death, mitochondrial depolarisation and cell cycle distribution as well as by caspase 3/7 activity determination, by immunoblotting and by real-time RT-PCR. Interactions between inhibitors were evaluated by combination index analysis.

Results: Single ATR or RNR inhibitor treatment produced small to moderate effects, while their combined treatment produced strong synergistic ones. ATR and RNR inhibitors elicited synergistic cell death and cooperated in inducing mitochondrial depolarisation, caspase 3/7 activity and DNA fragmentation, evidencing an apoptotic form of cell death. All effects were independent of functional p53. In addition, VE821 in combination with triapine increased p53 level and induced p53 target gene expression (CDKN1A, BBC3) in p53 wild-type Ewing's sarcoma cells.

Conclusion: Our study reveals that combined targeting of ATR and RNR was effective against Ewing's sarcoma in vitro and thus rationalises an in vivo exploration into the potential of combining ATR and RNR inhibitors as a new strategy for the treatment of this challenging disease.

Keywords: ATR; Didox; Ewing’s sarcoma; Ribonucleotide reductase; Triapine; VE821.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
ATRi and RNRi cooperate in inducing cell death in ES cells. One hour after administration of the ATRi VE821, cells were exposed to the RNRi triapine (a) or didox (b) for another 48 h. Cell death was determined by flow-cytometric analysis of PI uptake. Means ± SEM of each three independent measurements are shown
Fig. 2
Fig. 2
The ATRi VE821 and the RNRi triapine cooperate in inducing apoptosis in ES cells. One hour after administration of VE821, cells were exposed to triapine for another 24 h (b) or 48 h (a, c, d, e). c, d, e z-VAD-fmk was applied 1 h before treatment with VE821. a, d Loss of Δψm was determined by flow-cytometric analysis of DiOC6(3) staining. b Caspase 3/7 activity was determined using the fluorogenic substrate Ac-DEVD-AMC; relative caspase 3/7 activities are the ratio of treated cells to untreated cells. c Cell death was determined by flow-cytometric analysis of PI uptake. e sub-G1 cells were determined by flow-cytometric cell cycle analysis of PI-stained ethanol-fixed cells. Means ± SEM of each three independent measurements are shown (*p < 0.05, **p < 0.01, ***p < 0.001)
Fig. 2
Fig. 2
The ATRi VE821 and the RNRi triapine cooperate in inducing apoptosis in ES cells. One hour after administration of VE821, cells were exposed to triapine for another 24 h (b) or 48 h (a, c, d, e). c, d, e z-VAD-fmk was applied 1 h before treatment with VE821. a, d Loss of Δψm was determined by flow-cytometric analysis of DiOC6(3) staining. b Caspase 3/7 activity was determined using the fluorogenic substrate Ac-DEVD-AMC; relative caspase 3/7 activities are the ratio of treated cells to untreated cells. c Cell death was determined by flow-cytometric analysis of PI uptake. e sub-G1 cells were determined by flow-cytometric cell cycle analysis of PI-stained ethanol-fixed cells. Means ± SEM of each three independent measurements are shown (*p < 0.05, **p < 0.01, ***p < 0.001)
Fig. 3
Fig. 3
The ATRi VE821 and the RNRi didox cooperate in inducing apoptosis in ES cells. One hour after administration of VE821, cells were exposed to didox for another 48 h. b, c z-VAD-fmk was applied 1 h before treatment with VE821. a, c Loss of Δψm was determined by flow-cytometric analysis of DiOC6(3) staining. b Cell death was determined by flow-cytometric analysis of PI uptake. Means ± SEM of each three independent measurements are shown (*p < 0.05, **p < 0.01, ***p < 0.001)
Fig. 4
Fig. 4
The ATRi VE821 and the RNRi triapine cooperate in increasing p53 level in p53 wild-type ES cells and in inducing p53 target gene expression in ES cells. One hour after administration of VE821, cells were exposed to triapine for another 24 h. a The abundance of p53 was determined by immunoblotting; the figure is representative of three independent determinations. b CDKN1A and BBC3 expression levels were determined by real-time RT-PCR and normalised to B2M expression levels; relative expression levels are the ratio of treated cells to untreated cells. Means ± SEM of each two independent measurements are shown
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