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

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.

Fig. 1. Ewing Sarcoma cells are sensitive to DDK inhibition.

A Ewing Sarcoma cells (A673, RD-ES and SK-ES-1) and the non-Ewing, osteosarcoma cell line U2OS were treated with increasing concentrations of the DDK inhibitors XL413 and TAK-931 for 72 h and cell viability was measured using CCK-8 cell viability reagent. Relative viability was calculated based on DMSO treated cells (n = 3 biological replicates). 2-way ANOVA multiple comparisons, ***p < 0.001, ****p < 0.0001. B Cells were treated with either 0.1% DMSO, 5 µM XL413 (left panel) or 1 µM TAK-931 (right panel) for 48 h. Whole-cell lysates were collected, and western blot was performed for the specified proteins. All original western blot images are included in Supplementary Data. GAPDH was used as a loading control. C Ewing Sarcoma cells were treated with 0.1% DMSO, 5 µM XL413 or 1 µM TAK-931 for 48 h. Cells were stained with anti-Annexin-V (AlexaFluor-488 conjugated) and propidium iodide and analyzed using flow cytometry. Apoptotic cells were designated as Annexin-V (upper right and lower right quadrants) positive cells and were measured using FCS express v7 (n = 3 biological replicates). Bars represent the mean of the data. Unpaired t test, ***p < 0.001, **p < 0.01.

Fig. 2. DDK inhibition disrupts DNA replication and alters cell cycle progression in Ewing sarcoma cells.

A, B U2OS (A) and A673 (B) cells were treated with 1 µM XL413 or 300 nM TAK-931 for the indicated timepoints. Protein lysates were collected, and a western blot was performed to analyze the relative levels of the indicated proteins. 2 mM hydroxyurea (HU) was used as a positive control for replication stress induction. C Cells were treated with 0.1% DMSO, 1 µM XL413 or 300 nM TAK-931 for 8 h followed by a 1-h incubation with 10 µM EdU. Cells were then fixed and stained for EdU and relative EdU intensity was measured using FACS. Representative experiment of 3 biological replicates is shown. D U2OS and A673 cells were treated with either 0.1% DMSO, 1 µM XL413, or 300 nM TAK-931 for 8 h followed by a 1-h incubation with 10 µM EdU. Cells were then fixed and stained for EdU (Alexa-fluor 488) and DNA content (PI) and analyzed using FACS. Representative dot plots of 2 biological replicates are shown. E Quantification of panel D. Specifically, calculations were done by dividing the percentage of cells in the upper right-hand quadrant (late-S phase) by the total number of cells in the upper left and upper right quadrants (total S-phase cells), relative to DMSO treated control cells (n = 2 biological replicates) 2-way ANOVA Dunnett’s multiple comparison test, ****p < 0.0001. Bars represent mean and standard deviation. F Cells were treated with 1 µM XL413 or 300 nM TAK-931 for the indicated timepoints. DNA content was stained with PI and analyzed using FACS. Representative histograms of 3 biological replicates are shown. G Quantification of panel F (n = 3 biological replicates).

Fig. 3. DDK inhibition prolongs S-phase and delays mitotic entry in Ewing sarcoma cells.

A A673 cells were subjected to a double-thymidine block (dTB) and then released into S-phase in the presence of either 0.1% DMSO or 300 nM TAK-931 for the indicated timepoints. DNA content was stained with PI and then analyzed using FACS (n = 3 biological replicates). B A673 cells were subjected to a dTB and then released in the presence of 0.1% DMSO or 300 nM TAK-931 for the indicated timepoints. Protein was collected and a western blot was performed to analyze the protein levels of the indicated proteins. C U2OS and A673 cells were treated with 1 µM XL413 or 300 nM TAK-931 for the indicated timepoints. Protein was collected and a western blot was performed to analyze the protein levels of the indicated proteins. D A673 cells were subjected to a dTB and released into S-phase in the presence of either 0.1% DMSO or 300 nM TAK-931 for the indicated timepoints and protein was collected. E A673 cells were treated with 0.1% DMSO or 300 nM TAK-931 upon dTB release for the indicated timepoints. Cells were fixed and total DNA content was stained with PI. Representative histograms of 3 biological replicates are shown. F Quantification of sub-G1 DNA content from panel D (n = 3 biological replicates) 2-way ANOVA *** p = 0.0002, **** p < 0.0001.

Fig. 4. DDK inhibition causes premature mitotic entry and mitotic catastrophe in Ewing sarcoma cells.

A A673 and U2OS cells were treated with 0.1% DMSO, 1 µM XL413 or 300 nM TAK-931 for 24 h. Cells were then stained for phospho-histone H3 (Serine 10) (pHH3) and DNA (propidium iodide) and analyzed using FACS (representative dot plots of 3 biological replicates). B Quantification of panel A of proportion of pHH3-positive cells that had <4 N DNA content per total pHH3 cells (n = 3 biological replicates) 2-way ANOVA Dunnett’s multiple comparison test ****p < 0.0001. Bars represent mean and standard deviation. C Quantification of panel A of total pHH3 cells (n = 3 biological replicates) 2-way ANOVA Dunnett’s multiple comparison test *p = 0.0117. Bars represent mean and standard deviation. D A673 cells were treated with either 1 µM XL413 or 300 nM TAK-931 for 0, 24 or 48 h. Cells were then fixed and stained for DNA content (DAPI). Mitotic events were termed abnormal if they showed signs of anaphase bridge formation, lagging chromosome(s) during anaphase or anaphase/metaphase events with clear signs of more than 2 poles (n = 2 biological replicates) 2-way ANOVA **p < 0.01. Bars represent mean and standard deviation. Detailed quantitation and images can be found in Fig. S3E A673 cells were treated with 1 µM XL413 or 300 nM TAK-931 for 0, 24, 48 or 72 h. Cells were then fixed and stained for DNA content (DAPI). Micronuclei-containing cells were then quantified (n = 3 biological replicates) 2-way ANOVA ****p < 0.0001. Bars represent mean and standard deviation. F A673 cells were treated with 1 µM XL413 or 300 nM TAK-931 for 0, 24, 48 or 72 h. Cells were then fixed and stained for yH2AX, and total nuclear fluorescence intensity was calculated using FIJI image software. Representative quantification of 2 biological replicates 2way ANOVA, *p < 0.05, ****p < 0.0001. G Cells were treated with 1 µM XL413 (top row) or 300 nM TAK-931 (bottom row) for 0, 24, 48 or 72 h. Total DNA content was stained (PI) and % cells with <2 N DNA content (red) and >4 N DNA content (blue) were quantified using FACS (n = 3 biological replicates) Ordinary one-way ANOVA, *p < 0.05, ***p < 0.001, ****p < 0.0001.