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Combined inhibition of ribonucleotide reductase and WEE1 induces synergistic anticancer activity in Ewing's sarcoma
Judy Ziener1,2, Julián Andrés Henao-Restrepo3, Johanna Leonhardi1,2
1Department of Paediatric and Adolescent Medicine, Jena University Hospital, Friedrich Schiller University Jena, Jena, Germany.
Background:
Ewing's sarcoma is a childhood bone and soft tissue cancer with poor prognosis. Treatment outcomes for Ewing's sarcoma patients have improved only modestly over the past decades, making the development of new treatment strategies paramount. In this study, the combined targeting of ribonucleotide reductase (RNR) and WEE1 was explored for its effectiveness against Ewing's sarcoma cells.
Methods:
The RNR inhibitor triapine and the WEE1 inhibitors adavosertib and ZN-c3 were tested in p53 wild-type and p53 mutant Ewing's sarcoma cells. The combination of adavosertib with the PARP inhibitors olaparib and veliparib was tested for comparison. Combinatorial effects were determined by flow cytometric analyses of cell death, loss of mitochondrial membrane potential and DNA fragmentation as well as by caspase 3/7 activity assay, immunoblotting and real-time RT-PCR. The drug interactions were assessed using combination index analysis.
Results:
RNR and WEE1 inhibitors were weakly to moderately effective on their own, but highly effective in combination. The combination treatments were similarly effective in p53 wild-type and p53 mutant cells. They synergistically induced cell death and cooperated to elicit mitochondrial membrane potential decay, to activate caspase 3/7 and to trigger DNA fragmentation, evidencing the induction of the apoptotic cell death cascade. They also cooperated to boost CHK1 phosphorylation, indicating augmented replication stress after combination treatment. In comparison, the combination of adavosertib with PARP inhibitors produced weaker synergistic effects.
Conclusion:
Our findings show that combined inhibition of RNR and WEE1 was effective against Ewing's sarcoma in vitro. They thus provide a rationale for the evaluation of the potential of combined targeting of RNR and WEE1 in Ewing's sarcoma in vivo.
Insights
Combining ribonucleotide reductase (RNR) and WEE1 inhibitors shows significant promise for treating Ewing sarcoma. This novel combination therapy effectively induces cancer cell death, offering a new strategy for this challenging childhood cancer.
Area of Science:
- Oncology
- Cancer Biology
- Pharmacology
Background:
- Ewing sarcoma is a pediatric bone and soft tissue cancer with a poor prognosis.
- Current treatment outcomes for Ewing sarcoma have seen limited improvement, necessitating novel therapeutic strategies.
- This study investigates the combined inhibition of ribonucleotide reductase (RNR) and WEE1 as a potential treatment for Ewing sarcoma.
Purpose of the Study:
- To evaluate the efficacy of combined RNR and WEE1 inhibition against Ewing sarcoma cells.
- To compare the synergistic effects of RNR and WEE1 inhibitors with combinations involving PARP inhibitors.
- To assess the impact of these combinations on cell death pathways and replication stress.
Main Methods:
- Tested RNR inhibitor (triapine) and WEE1 inhibitors (adavosertib, ZN-c3) in Ewing sarcoma cell lines (p53 wild-type and mutant).
- Assessed combinatorial effects using assays for cell death, mitochondrial membrane potential, DNA fragmentation, caspase 3/7 activity, and gene expression.
- Utilized combination index analysis to determine drug interactions and compared with adavosertib and PARP inhibitor combinations.
Main Results:
- RNR and WEE1 inhibitors showed moderate efficacy alone but high effectiveness in combination, irrespective of p53 status.
- Combination treatments synergistically induced apoptosis, evidenced by increased cell death, mitochondrial dysfunction, caspase activation, and DNA fragmentation.
- Combined inhibition led to increased CHK1 phosphorylation, indicating enhanced replication stress, with weaker synergy observed for adavosertib-PARP inhibitor combinations.
Conclusions:
- Combined inhibition of RNR and WEE1 demonstrates significant in vitro efficacy against Ewing sarcoma.
- These findings support the further investigation of combined RNR and WEE1 targeting as a potential in vivo therapeutic strategy for Ewing sarcoma.
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