CDC7 kinase (DDK) inhibition disrupts DNA replication leading to mitotic catastrophe in Ewing sarcoma

Anna Woloszynska,Jennie R. Sims,Joseph Kuechle,Miranda L. Lynch,Joyce E. Ohm,Jeremy F. Kane,Jeffrey C. Martin,Ajay Gupta,Lingqiu Gao,Tamara J. Hagoel,Thomas Melendy

doi:10.1038/s41420-022-00877-x

Abstract

Ewing sarcoma is the second most common bone malignancy in children and adolescents. In recent years, a large body of evidence has emerged that suggests Ewing tumors harbor large amounts of replication stress (RS). CDC7, also known as DDK (DBF4-dependent kinase), is a serine/threonine kinase that is involved in a diverse array of cellular functions including the regulation of DNA replication initiation and activation of the RS response. Due to DDK’s diverse roles during replication, coupled with the fact that there is an increased level of RS within Ewing tumors, we hypothesized that Ewing sarcoma cells would be particularly vulnerable to DDK inhibition. Here, we report that DDK inhibition resulted a significant reduction in cell viability and the induction of apoptosis, specifically in Ewing sarcoma cells. Treatment with DDK inhibitors dramatically reduced the rate of replication, prolonged S-phase, and led to a pronounced increase in phospho-CDC2 (Y15), indicating delay of mitotic entry. The induction of cell death corresponded to mitotic exit and G1 entry, suggesting improper mitotic progression. In accordance with this, we find that DDK inhibition caused premature mitotic entry resulting in mitotic abnormalities such as anaphase bridges, lagging chromosomes, and cells with >2 poles in Ewing sarcoma cells. This abnormal progression through mitosis resulted in mitotic catastrophe as evidenced by the formation of micronuclei and induction of DNA damage. Together, these findings suggest that DDK activity is required for the faithful and timely completion of DNA replication in Ewing cells and that DDK inhibition may present a viable therapeutic strategy for the treatment of Ewing sarcoma.

Highlights

Ewing sarcoma is a rare childhood sarcoma that mainly presents in the bone [1, 2]
DDK inhibition causes cell death in Ewing sarcoma cells Due to the vital role that DDK plays during DNA replication, we hypothesized that Ewing sarcoma cells would be highly sensitive to its inhibition
We found that Ewing sarcoma lines were significantly more sensitive to both DDK inhibitors as compared to the U2OS osteosarcoma cell line (Fig. 1A)

Summary

Introduction

Ewing sarcoma is a rare childhood sarcoma that mainly presents in the bone [1, 2]. It is characterized by a recurrent chromosomal translocation between the RNA-binding protein, EWSR1, and the ETS transcription factor, FLI1 [3]. Treatment of Ewing sarcoma has remained largely unchanged for several decades, consisting of surgical resection and the use of alkylating agents, such as Ifosfamide and Cyclophosphamide, and topoisomerase poisons, mainly Etoposide and Doxorubicin [4]. Ewing tumors have been previously shown to harbor elevated levels of endogenous DNA damage, elevated levels of replication stress (RS) and sensitivity to ATR [5, 6] and CHK1 inhibitors [7, 8] This endogenous replication stress has recently been attributed to the formation of R-loops due to an EWS-FLI1-dependent promotion of CDK9-mediated RNA Pol-II activation [9]. Together these observations indicate that Ewing sarcoma cells are dependent on their ability to successfully manage high levels of replication stress

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Results

Conclusion