Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the synthesis of deoxyribonucleotides and the target of multiple chemotherapy drugs, including gemcitabine. We previously identified that inhibition of RNR in Ewing sarcoma tumors upregulates the expression levels of multiple members of the activator protein-1 (AP-1) transcription factor family, including c-Jun and c-Fos, and downregulates the expression of c-Myc. However, the broader functions and downstream targets of AP-1, which are highly context- and cell-dependent, are unknown in Ewing sarcoma tumors. Consequently, in this work, we used genetically defined models, transcriptome profiling, and gene-set -enrichment analysis to identify that AP-1 and EWS-FLI1, the driver oncogene in most Ewing sarcoma tumors, reciprocally regulate the expression of multiple extracellular-matrix proteins, including fibronectins, integrins, and collagens. AP-1 expression in Ewing sarcoma cells also drives, concurrent with these perturbations in gene and protein expression, changes in cell morphology and phenotype. We also identified that EWS-FLI1 dysregulates the expression of multiple AP-1 proteins, aligning with previous reports demonstrating genetic and physical interactions between EWS-FLI1 and AP-1. Overall, these results provide novel insights into the distinct, EWS-FLI1-dependent features of Ewing sarcoma tumors and identify a novel, reciprocal regulation of extracellular-matrix components by EWS-FLI1 and AP-1.

Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in the synthesis of deoxyribonucleosides and is required for DNA replication. Multiple types of cancer, including Ewing sarcoma tumors, are sensitive to RNR inhibitors or a reduction in the levels of either the RRM1 or RRM2 subunits of RNR. However, the polypharmacology and off-target effects of RNR inhibitors have complicated the identification of the mechanisms that regulate sensitivity and resistance to this class of drugs. Consequently, we used a conditional knockout (CRISPR/Cas9) and rescue approach to target RRM1 in Ewing sarcoma cells and identified that loss of the RRM1 protein results in the upregulation of the expression of multiple members of the activator protein-1 (AP-1) transcription factor complex, including c-Jun and c-Fos, and downregulation of c-Myc. Notably, overexpression of c-Jun and c-Fos in Ewing sarcoma cells is sufficient to inhibit cell growth and downregulate the expression of the c-Myc oncogene. We also identified that the upregulation of AP-1 is mediated, in part, by SLFN11, which is a replication stress response protein that is expressed at high levels in Ewing sarcoma. In addition, small-molecule inhibitors of RNR, including gemcitabine, and histone deacetylase inhibitors, which reduce the level of the RRM1 protein, also activate AP-1 signaling and downregulate the level of c-Myc in Ewing sarcoma. Overall, these results provide novel insight into the critical pathways activated by loss of RNR activity and the mechanisms of action of inhibitors of RNR. Significance: RNR is the rate-limiting enzyme in the synthesis of deoxyribonucleotides. Although RNR is the target of multiple chemotherapy drugs, polypharmacology and off-target effects have complicated the identification of the precise mechanism of action of these drugs. In this work, using a knockout-rescue approach, we identified that inhibition of RNR upregulates AP-1 signaling and downregulates the level of c-Myc in Ewing sarcoma tumors.

Expression of c-Jun and c-Fos in Ewing sarcoma cells inhibits cell growth and downregulates c-Myc. A, EW8 and TC71 cell lines expressing doxycycline-inducible c-Jun and c-Fos were grown with or without doxycycline for 24 hours. Cell lysates were collected for immunoblotting. B, Colony formation assay for the TO-Jun/Fos cell lines grown with or without doxycycline for 10–12 days. C, Results of GSEA for gene sets downregulated in the EW8-RRM1-KO and TC71-RRM1-KO cell lines after removal of doxycycline for 48 hours. D, The TO-EW8-Fos/Jun and TO-TC71-Fos/Jun cell lines were grown with or without doxycycline for 72 hours. Cell lysates were then collected for immunoblotting. E, The EW8-RRM1-KO and TC71-RRM1-KO cell lines were grown with or without doxycycline for 48 hours. Cell lysates were then collected for immunoblotting. F and G, Ewing sarcoma cell lines were treated with gemcitabine (100 nmol/L) for 24 hours and then cell lysates were collected for immunoblotting. P values were calculated using a two-tailed Student t test. **, P < 0.01; ***, P < 0.001.

Increase in mRNA expression levels of AP-1 family members in RRM1-KO cell lines after removal of doxycycline and loss of the RRM1 protein

Quantification of immunoblots.

Treatment of EW8-RRM1-KO cells with prexasertib.

Dependency of Ewing sarcoma cells on RRM1 and RRM2.

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.

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.

Short-term gemcitabine drug treatment.

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.

Loss of RRM1 activates AP-1 signaling in Ewing sarcoma cells. Volcano plots of DE genes (fold >2, adjusted P value <0.05) in the EW8-RRM1-KO (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.

Gene set enrichment analysis of genes up- and down-regulated in the EW8-RRM1-KO cells after removal of doxycycline.

Alzheimer's disease (AD) is an incurable neurodegenerative disease in which the risk of development increases with age. People with AD are plagued with deficits in their cognition, memory, and basic social skills. Many of these deficits are believed to be caused by the formation of amyloid-β plaques and neurofibrillary tangles in regions of the brain associated with memory, such as the hippocampus. However, one of the early, preclinical symptoms of AD is the loss of olfactory detection and discrimination. To determine if a mouse model of AD expresses the same olfactory dysfunction seen in human AD, 3xTg-AD mice were given a buried food test and, unlike previous studies, compared to their background and parental strains. Results showed that over 52 weeks, the 3xTg-AD mice took significantly longer to find the buried food than the control strains. The olfactory bulbs of the 3xTg-AD mice were removed, sliced, and stained using Congo red for histological analysis. Amyloid deposits were observed predominantly in the granule layer of the olfactory bulb beginning at 13 weeks of age in 3xTg-AD mice, but not in the control strains of mice. Further examination of the buried food test data revealed that 3xTg-AD females had a significantly longer latency to detect the buried food than males beginning at 26 weeks of age. Overall, this study provides further validation of the 3xTg-AD mouse model of AD and supports the idea that simple olfactory testing could be part of the diagnostic process for human AD.