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

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.

Stemness and Cell Cycle Regulators and Their Modulation by Retinoic Acid in Ewing Sarcoma

“…Thus, our observation that RA induced an increase in CDKN1A and p21 might be interpreted in the context of stemness regulation, although our experiments were limited to these two markers. Induction of CDKN1A/p21 is stimulated by p53, and SK-ES-1 cells harbor a p53 missense mutation which leads to a loss of p53 functioning [42]. However, recent evidence suggests that RA can induce p21 in a p53-independent mechanism in NB4 promyelocytic cells [43], thus a similar process might occur in SK-ES-1 ES cells.…”

Therapeutic Targeting of DNA Repair Pathways in Pediatric Extracranial Solid Tumors: Current State and Implications for Immunotherapy

“…ES cells have demonstrated a particular susceptibility to the inhibition of ribonucleotide reductase, the rate-limiting enzyme in deoxyribonucleotide synthesis [119]. This finding has led to multiple investigations, which have demonstrated the ES susceptibility in in vitro and in vivo models to the combined inhibition of ribonucleotide reductase and either Wee1, ATR, or CHK1 [93,[119][120][121][122]. The combination of Wee1 and PARP inhibition has also shown efficacy in ES cell lines [123], as has the combination of DNA-PK inhibition with PARP inhibition [124].…”