DDK inhibition disrupts replication leading to mitotic catastrophe in Ewing sarcoma

Abstract

Ewing sarcoma is the second most common bone malignancy in children and adolescents. Patients with upfront metastatic or recurrent disease have poor outcomes with 5-year survival rates of <30%. CDC7, also known as DDK (DBF4-dependent kinase), is a serine-threonine kinase that, in coordination with its activation subunit ASK (or DBF4), is involved in a diverse array of cellular functions including the regulation of DNA replication initiation and activation of the replication stress response. Due to DDKs diverse roles during replication, coupled with an increased level of genomic instability and R-loop-mediated replication stress within Ewing sarcoma cells, we hypothesized that Ewing sarcoma cells would be particularly vulnerable to DDK inhibitors. Here, we show that treatment with two selective DDK inhibitors, TAK-931 and XL413, results in apoptosis and a significant reduction in cell viability in EWS-FLI1-harboring Ewing sarcoma cell lines. We show that low dose DDK inhibition in Ewing sarcoma cells causes an accumulation of cells in late-S phase with a reduced replication capacity. There is also evidence of premature mitotic entry indicating an inability to properly complete DNA replication in a timely manner upon DDK inhibition. Also, there is a significant increase in the formation of micronuclei and other aberrant mitotic structures upon DDK inhibition in Ewing sarcoma cells indicating a failure to properly progress through S-phase followed by improper mitotic entry/progression, resulting in mitotic catastrophe. Interestingly, we observed minimal signs of mitotic accumulation, despite clear evidence of replication and mitotic stress, suggesting a failure to properly enforce the mitotic checkpoint. Together, these results suggest that Ewing sarcoma cells rely on the activity of DDK to maintain cell viability and suggest that DDK inhibition may prove to be a viable therapeutic strategy for patients with Ewing sarcoma.