WEE1 Inhibition Augments CDC7 (DDK) Inhibitor–induced Cell Death in Ewing Sarcoma by Forcing Premature Mitotic Entry and Mitotic Catastrophe

Ewing sarcoma is an aggressive childhood cancer for which treatment options remain limited and toxic. There is an urgent need for the identification of novel therapeutic strategies. Our group has recently shown that Ewing cells rely on the S-phase kinase cell division cycle 7 (CDC7) DBF4-dependent kinase (DDK) to maintain replication rates and cell viability and that DDK inhibition causes an increase in the phosphorylation of CDK1 and a significant delay in mitotic entry. Here, we expand on our previous findings and show that DDK inhibitor–induced mitotic entry delay is dependent upon WEE1 kinase. Specifically, WEE1 phosphorylates CDK1 and prevents mitotic entry upon DDK inhibition due to the presence of underreplicated DNA, potentially limiting the cytotoxic effects of DDK inhibition. To overcome this, we combined the inhibition of DDK with the inhibition of WEE1 and found that this results in elevated levels of premature mitotic entry, mitotic catastrophe, and apoptosis. Importantly, we have found that DDK and WEE1 inhibitors display a synergistic relationship with regards to reducing cell viability of Ewing sarcoma cells. Interestingly, the cytotoxic nature of this combination can be suppressed by the inhibition of CDK1 or microtubule polymerization, indicating that mitotic progression is required to elicit the cytotoxic effects. This is the first study to display the potential of utilizing the combined inhibition of DDK and WEE1 for the treatment of cancer. We believe this will offer a potential therapeutic strategy for the treatment of Ewing sarcoma as well as other tumor types that display sensitivity to DDK inhibitors. SIGNIFICANCE: Ewing sarcoma is an aggressive pediatric cancer for which chemotherapy is very intense and often results in acute toxicities. Here, we have found that the combined inhibition of CDC7 kinase (DDK) and WEE1 kinase forces high levels of mitotic errors and synergistic cell death, specifically in Ewing sarcoma cells. This combination has the potential to provide a highly efficacious and minimally toxic treatment strategy for patients with Ewing sarcoma.