Combined Inhibition of <scp>ATR</scp> and Ribonucleotide Reductase Induces Synergistic Antineoplastic Activity in Osteosarcoma Cells

BACKGROUND: Osteosarcoma is the most common bone cancer in children and young adults. Its prognosis has not improved significantly since the introduction of the chemotherapy regimen established about 40 years ago, highlighting the need for new therapeutic strategies. AIMS: The present study was undertaken to assess the effectiveness of combined inhibition of two promising drug targets, ATR and ribonucleotide reductase (RNR), in osteosarcoma cells. METHODS AND RESULTS: The ATR inhibitor berzosertib and the RNR inhibitors triapine and didox were tested in TP53 wild-type (U2OS, MG-63) and mutant (SaOS-2) osteosarcoma cell lines. Combination effects were examined by flow cytometric analysis of cell death, loss of the mitochondrial membrane potential and DNA fragmentation as well as by caspase 3/7 activity assay and real-time RT-PCR. The drug interactions were evaluated using combination index analysis. Single treatment with ATR or RNR inhibitors resulted in mild to moderate effects, whereas combined treatment resulted in strong and synergistic effects. ATR and RNR inhibitors cooperated to elicit loss of the mitochondrial membrane potential, to activate caspase 3/7 and to trigger DNA fragmentation, suggesting that the combination of ATR and RNR inhibitors induced an apoptotic form of cell death. The cytotoxic effects were independent of TP53 mutational status. CONCLUSION: Our study demonstrates that combined inhibition of ATR and RNR was effective in osteosarcoma cells. These in vitro findings offer support for investigating in vivo the potential of a combination of ATR and RNR inhibitors as a new treatment strategy for osteosarcoma.