FIGURE 2 from Activator Protein-1 (AP-1) Signaling Inhibits the Growth of Ewing Sarcoma Cells in Response to DNA Replication Stress

Loss of RRM1 activates AP-1 signaling in Ewing sarcoma cells. Volcano plots of DE genes (fold >2, adjusted P value A) and TC71-RRM1-KO (B) cell lines in the presence and absence (48 hours) of doxycycline. C, Gene sets (biological processes) enriched in the 412 overlap genes that are upregulated in both the EW8-RRM1-KO and TC71-RRM1-KO cell lines in the absence of doxycycline. D, Results of TFEA performed with the overlap genes that are upregulated in both the EW8-RRM1-KO and TC71-RRM1-KO cell lines in the absence of doxycycline. E, EW8-RRM1-KO cells were treated with different concentrations of doxycycline for 48 hours and then cellular lysates were collected for immunoblotting. F, Doxycycline was removed from the EW8-RRM1-KO and TC71-RRM1-KO cell lines and lysates were collected at different timepoints. G, Doxycycline was removed from the EW8-RRM1-KO and TC71-RRM1-KO cells for 48 hours. Cell lysates were then collected for immunoblotting for total and phosphorylated (Ser73) c-Jun. H, EW8-RRM1-KO and TC71-RRM1-KO cells were grown with or without doxycycline for 48 hours. Subcellular fractionation was then performed and lysates were immunoblotted for c-Jun and markers of the nuclear (α-Tubulin) and cytoplasmic (Lamin A/C) fractions. The parental, unmodified EW8 and TC71 cells were treated with a range of gemcitabine (I) or hydroxyurea doses (J) for 24 hours and then cell lysates were collected for immunoblotting. K, EW8 cells labeled with an AP-1 (luciferase) reporter were used to quantify AP-1 activity when cell lines were treated with gemcitabine or hydroxyurea for 24 hours. TC71 (L) and EW8 (M) cells were engrafted in nude (NCr) mice. After tumors were palpable, the mice were treated with vehicle or gemcitabine (150 mg/kg, intraperitoneal, day 1). On day 2, the mice were sacrificed and the tumors were collected for analysis by immunoblotting. P values were calculated using a two-tailed Student t test or a one-way ANOVA followed by Dunnett multiple comparisons test. **, P < 0.01.</p

Figure S3 from Activator Protein-1 (AP-1) Signaling Inhibits the Growth of Ewing Sarcoma Cells in Response to DNA Replication Stress

Gene set enrichment analysis.</p

FIGURE 4 from Activator Protein-1 (AP-1) Signaling Inhibits the Growth of Ewing Sarcoma Cells in Response to DNA Replication Stress

SLFN11 contributes to the toxicity of RNR inhibition and the upregulation of AP-1. A, Venn diagram demonstrating the overlap between genes upregulated in the EW8-RRM1-KO and TC71-RRM1-KO cells grown in the absence of doxycycline and IEGs. B, EW8-RRM1-KO and TC71-RRM1-KO cells were grown with or without doxycycline for 24 hours, at which point the cells were transfected with control or SLFN11 siRNA and grown for an additional 24 hours. Cell lysates were then collected for immunoblotting. C, EW8-RRM1-KO and TC71-RRM1-KO cells were grown with or without doxycycline for 24 hours, at which point the cells were transfected with control or SLFN11 siRNA and grown for an additional 48 hours. Dead cells were then labeled with propidium iodide and quantified using flow cytometry. EW8 (D) and TC71 (E) cell lines were treated with control or SLFN11 siRNA for 24 hours. Gemcitabine was then added for an additional 24 hours before collecting cellular lysates. P values were calculated using a two-tailed Student t test. ****, P < 0.0001.</p

Figure S2 from Activator protein-1 (AP-1) signaling inhibits the growth of Ewing sarcoma cells in response to DNA replication stress

Treatment of EW8-RRM1-KO cells with prexasertib.</p

Figure S6 from Activator protein-1 (AP-1) signaling inhibits the growth of Ewing sarcoma cells in response to DNA replication stress

Regulation of c-Myc.</p

Figure S5 from Activator protein-1 (AP-1) signaling inhibits the growth of Ewing sarcoma cells in response to DNA replication stress

Gemcitabine resistant cell lines.</p

Figure S2 from Activator Protein-1 (AP-1) Signaling Inhibits the Growth of Ewing Sarcoma Cells in Response to DNA Replication Stress

Treatment of EW8-RRM1-KO cells with prexasertib.</p

FIGURE 5 from Activator Protein-1 (AP-1) Signaling Inhibits the Growth of Ewing Sarcoma Cells in Response to DNA Replication Stress

HDAC inhibitors decrease the level of the RRM1 protein and increase expression of c-Jun in Ewing sarcoma cells. A, EW8 and TC71 cell lines were treated with the dual PI3K-HDAC inhibitor fimepinostat for 24 hours and then cell lysates were collected for immunoblotting. B, Dose–response curves for Ewing sarcoma cell lines treated with different concentrations of fimepinostat for 72 hours. Cell viability was assessed using the AlamarBlue Fluorescence Assay. The results are representative of two independent experiments. Error bars represent mean ± SD of three technical replicates. C, Ewing sarcoma (EW8 and TC71) and osteosarcoma (U2OS) cell lines were treated with the HDAC inhibitors panobinostat or romidepsin for 24 hours and then cell lysate was collected for immunoblotting. D, log2 fold change (FC) in RRM1 mRNA in EW8 and TC71 cells treated with panobinostat or romidepsin for 24 hours. The results are representative of two independent experiments. Error bars represent the mean ± SD of three technical replicates. E, EW8 and TC71 cells were treated with panobinostat, romidepsin, or fimepinostat for 24 hours and then cell lysates were collected for immunoblotting. F, Ewing sarcoma cell lines were treated with romidepsin for 24 hours and then cell lysates were collected for immunoblotting.</p

FIGURE 1 from Activator Protein-1 (AP-1) Signaling Inhibits the Growth of Ewing Sarcoma Cells in Response to DNA Replication Stress

Conditional knockout of RRM1 causes cell-cycle arrest, DNA damage, and apoptosis in Ewing sarcoma cells. A, Schematic illustrating the approach used to knockout the endogenous RRM1 gene in Ewing sarcoma cells that express an exogenous and doxycycline-inducible RRM1 transgene that is codon optimized and resistant to targeting by CRISPR/Cas9. B, EW8-TO-RRM1 and EW8-RRM1-KO cells were treated with different concentrations of doxycycline for 72 hours and then cellular lysates were collected for immunoblotting. C, Doxycycline was removed from the EW8-RRM1-KO cells for 24–72 hours and cellular lysates were collected for immunoblotting. D, Growth assay for EW8-RRM1-KO cells with and without doxycycline. Cell viability was assessed at different timepoints using the AlamarBlue Fluorescence Assay. The results are representative of two independent experiments. Error bars represent mean ± SD of three technical replicates. E, Representative cell-cycle analysis of EW8-RRM1-KO cells growing with or without of doxycycline for 48 hours. F, Doxycycline was removed from the EW8-RRM1-KO and TC71-RRM1-KO cell lines for 72 hours and then cellular lysates were collected for immunoblotting. G, Doxycycline was removed from the EW8-RRM1-KO and TC71-RRM1-KO cell lines and lysates were collected at different time points. H, Doxycycline was removed from the EW8-RRM1-KO and TC71-RRM1-KO cell lines for different amounts of time and then dead cells were labeled with propidium iodide and quantified using flow cytometry. I, Colony formation assay for the EW8-RRM1-KO and TC71-RRM1-KO cell lines in the presence or absence of doxycycline for 10–12 days. J, Doxycycline was removed from the EW8-RRM1-KO cell line for 48 hours and then cellular lysates were collected for immunoblotting for markers of DNA replication stress. K, EW8-RRM1-KO cells were grown with or without doxycycline for 24 hours, at which point the cells were treated with vehicle (DMSO), prexasertib (CHK1 inhibitor), or berzosertib (ATR inhibitor) for an additional 24 hours. Cell lysates were then collected for immunoblotting. P values were calculated using a two-tailed Student t test. ****, P < 0.0001.</p

Figure S3 from Activator protein-1 (AP-1) signaling inhibits the growth of Ewing sarcoma cells in response to DNA replication stress

Gene set enrichment analysis.</p