Despite aggressive multimodal therapy, including cytotoxic chemotherapy, surgery, and radiation, the prognosis for patients with Ewing sarcoma remains poor, particularly for those with metastatic or relapsed disease. Combining agents that increase DNA replication stress with ATR-CHK1-WEE1 pathway inhibitors, which disrupt the DNA damage response and cell cycle checkpoints, is a promising strategy under clinical investigation in Ewing sarcoma and other cancers. However, the mechanisms by which these drug combinations selectively kill cancer cells under replication stress remain incompletely understood and are often attributed, without strong supporting evidence in many tumor types, to forced mitotic entry. In this study, we show that inhibition of the ATR-CHK1-WEE1 pathway in S-phase-arrested Ewing sarcoma cells triggers rapid apoptosis within 2-4 h, without widespread mitotic entry. This apoptotic response is driven by the activation of cyclin-dependent kinase 1 (CDK1) and is caspase-dependent. We further show that dual targeting of DNA replication and ATR-CHK1-WEE1 signaling in Ewing sarcoma tumors suppresses protein synthesis, and inhibition of protein synthesis prevents cell cycle progression and premature mitotic entry-providing a mechanistic explanation for why aberrant CDK1 activation does not drive mitosis in this context. Moreover, while apoptosis is induced rapidly following drug treatment, the suppression of protein synthesis is prolonged and persists beyond drug removal, suggesting distinct early and late mechanisms of drug-induced toxicity. Collectively, these findings define a unique CDK1- and caspase-dependent apoptotic pathway in response to replication stress and offer new insights into the molecular basis of this therapeutic vulnerability in Ewing sarcoma.

Histone deacetylases (HDAC) regulate diverse pathways in cancer cells. Previously, we identified that Ewing sarcoma tumors, which are caused by a translocation between the EWSR1 and FLI1 genes (EWS::FLI1), are sensitive to drugs that target DNA replication, including the RRM1 and RRM2 subunits of ribonucleotide reductase, and the ATR-checkpoint kinase 1 (CHK1)-WEE1 signaling pathway. In this study, we identified that multiple HDAC inhibitors, including fimepinostat, romidepsin, and panobinostat, downregulate the levels of the RRM1, RRM2, CHK1, and WEE1 proteins in Ewing sarcoma cells and impair DNA replication. Moreover, transcriptome analyses identified that HDAC inhibitors downregulate the expression of multiple components of the prereplication complex, including the minichromosome maintenance complex 2-7 (MCM2-7) proteins and CDT1, that are essential for genomic DNA replication. Additionally, proteomic studies identified that HDAC inhibitors also downregulate the level of the BRD4 protein, a BET bromodomain protein that regulates both the transcriptional program of the EWS::FLI1 oncoprotein and DNA replication. Overall, these results provide novel insight into the molecular mechanisms by which HDAC inhibitors target cancer cells, regulate DNA replication, and inhibit the cellular response to DNA replication stress.Significance: In this work, we identify that HDAC inhibitors broadly disrupt DNA replication and the response to replication stress in Ewing sarcoma cells by downregulating RRM1, RRM2, CHK1, and WEE1. Notably, HDAC inhibitors also reduce MCM2-7 proteins, which are essential for prereplication complex helicase function in replication initiation and elongation, and CDT1, which loads MCM2-7 onto DNA.

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.

Purpose - The institutional structures established by French and British colonists continue to shape the socio-economic landscape of sub-Saharan Africa, potentially contributing to the unequal distribution of child poverty. This study, therefore, has significant relevance as it aims to estimate child poverty disparities within the context of colonial origins in Francophone and Anglophone African countries. Design/methodology/approach - This study utilised data from the Demographic and Health Surveys (DHS) conducted between 2000 and 2019 involving 22 sub-Saharan African states. A rights-based framework was applied based on the SDG-updated Gordon et al. (2003) methodology to measure child poverty. We then compared child poverty disparities between colonial origins using one-way ANOVA tests and risk ratios to quantify the magnitude of these differences. Findings - Critically high estimates of sanitation and housing poverty (above 70% prevalence) were observed in the two colonial settings. Children in Francophone states were 1.4 times (RR = 1.4; 95% CI: 1.1-1.8) more likely to experience higher risks of low medical access, almost twice as likely to experience low vaccine uptake (risk ratios-RR = 1.8; 95% CI: 1.2-2.8) or no formal education (RR = 1.7; 95% CI: 1.1-2.5). All other dimensions were equal between colonial origins. Research limitations/implications - Due to its cross-comparative approach, this study is restricted in making conclusive inferences regarding the causal relationship between colonialism and health/education poverty. Also, given the limited number of countries, particularly Anglophone countries, our sample may not fully represent all French and British ex-colonies. As a follow-up to this study, we intend to repeat this analysis using the harmonised Multiple Indicator Cluster (MIC) surveys, providing a larger number of countries. Originality/value - Despite similar household poverty levels in both colonial settings, Francophone states had lower access to health and education services than Anglophone states. These findings indicate deeper systemic issues that may be rooted in the historical governance styles of centralisation. More effective decentralisation of healthcare and education services in Francophone countries and improved government commitment to addressing household poverty throughout sub-Saharan Africa are imperative.

Novel therapeutic approaches are needed for the treatment of Ewing sarcoma tumors. We previously identified that Ewing sarcoma cell lines are sensitive to drugs that inhibit protein translation. However, translational and therapeutic approaches to inhibit protein synthesis in tumors are limited. In this work, we identified that reactive oxygen species, which are generated by a wide range of chemotherapy and other drugs, inhibit protein synthesis and reduce the level of critical proteins that support tumorigenesis in Ewing sarcoma cells. In particular, we identified that both hydrogen peroxide and auranofin, an inhibitor of thioredoxin reductase and regulator of oxidative stress and reactive oxygen species, activate the repressor of protein translation 4E-BP1 and reduce the levels of the oncogenic proteins RRM2 and PLK1 in Ewing and other sarcoma cell lines. These results provide novel insight into the mechanism of how ROS-inducing drugs target cancer cells via inhibition of protein translation and identify a mechanistic link between ROS and the DNA replication (RRM2) and cell cycle regulatory (PLK1) pathways.

Introduction: Atopic dermatitis, a chronic, pruritic skin disease, affects 10-30% of children and up to 14% of adults in developed countries. ATI-1777, a potent and selective Jak1/3 inhibitor, was designed with multiple sites of metabolism to deliver local efficacy in the skin and limit systemic exposure. In preclinical studies, ATI-1777 selectively inhibited Jak1/3 with limited systemic exposure and without any adverse effects. Primary objective: The primary goal of this study was to assess the preliminary clinical efficacy of ATI-1777 topical solution in adults with moderate or severe atopic dermatitis. Design: ATI-1777-AD-201, a phase 2a, first-in-human, randomized, double-blind, vehicle-controlled, parallel-group study, evaluated the efficacy, safety, tolerability, and pharmacokinetics of ATI-1777 topical solution in 48 participants with atopic dermatitis over 4 weeks. Primary endpoint: The primary endpoint was a reduction of a modified Eczema Area and Severity Index score from baseline. Results: Reduction was significantly greater in the ATI-1777-treated group on day 28 than in vehicle-treated group (percentage reduction from baseline = 74.45% [standard error = 6.455] and 41.43% [standard error = 6.189], respectively [P < .001]). Average plasma concentrations of ATI-1777 were <5% of the half-maximal inhibitory concentration of ATI-1777 for inhibiting Jak1/3. No deaths or serious adverse events were reported. Conclusion: Topical ATI-1777 does not lead to pharmacologically relevant systemic drug exposure and may reduce clinical signs of atopic dermatitis. Trial Registration: The study was registered at ClinicalTrials.gov with the number NCT04598269.

Supplementary Figure 6 shows the effect of drug treatments on mice weights in the xenograft experiments.

Purpose Data on treatments for male breast cancer patients are limited owing to rarity and underrepresentation in clinical trials. The real-world POLARIS study gathers data on palbociclib use for the treatment of hormone receptor–positive/human epidermal growth factor receptor 2–negative (HR+/HER2–) advanced breast cancer (ABC) in female and male patients. This sub-analysis describes real-world palbociclib treatment patterns, clinical outcomes, and quality of life (QoL) in male patients. Methods POLARIS is a prospective, noninterventional, multicenter, real-world study of patients with HR+/HER2– ABC receiving palbociclib. Assessments included medical record reviews, patient QoL questionnaires (European Organisation for Research and Treatment of Cancer Quality-of-Life Questionnaire–Core 30), site characteristics questionnaires, and physician treatment selection surveys. Variables included demographics, disease history, global health status/QoL, clinical assessments and adverse events. Analyses were descriptive in nature. For clinical outcomes, real-world tumor responses and progression were determined by physician assessment in routine clinical practice. Real-world progression-free survival (rwPFS) was described using the Kaplan–Meier method. Results At data cutoff, 15 male patients were enrolled (median age, 66 years). Nine patients received palbociclib as a first-line treatment and 6 as a second-line or later treatment. Patients received a median of 20 cycles of palbociclib. Neutropenia was experienced by 2 patients and grade ≥ 3 adverse events were reported in 11 patients. Global health status/QoL scores remained generally consistent during the study. One patient (6.7%) achieved a complete tumor response, 4 (26.7%) a partial response, and 8 (53.3%) stable disease. Median rwPFS was 19.8 months (95% CI, 7.4–38.0). Median follow-up duration was 24.7 months (95% CI, 20.0–35.7). Conclusion This real-world analysis showed that palbociclib was well tolerated and provides preliminary data on treatment patterns and outcomes with palbociclib in male patients with HR+/HER2– ABC, helping inform the use of palbociclib in this patient subgroup. Trial identifier NCT03280303.

Abstract: Rhabdomyosarcoma (RMS) is one of the most common soft tissue sarcomas in children. This lesion is classically included in the generic group of “small round blue cell tumors” along with other entities that share similar microscopic features. Although the head and neck region is a frequent site for primary tumors, cutaneous metastases of RMS involving this anatomical location are rare in the pediatric population. We report a case of a 12-year old girl previously diagnosed with a primary alveolar RMS involving the left maxillary sinus, presenting with a metastatic lesion on the skin of the left temple area. Along with a brief review of the previous case reports on the topic, we highlight the initial immunohistochemistry panel useful for diagnosing this tumor.

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.

Supplementary Figure 3 shows 4E-BP1 mRNA and protein levels in cell lines.

Supplementary Figure 6 shows the effect of RRM2 knockdown in Ewing sarcoma cells, as well as RT-qPCR data for cells treated with AZD1775 and LY2603618.

Supplementary Figure 2 shows the effects RO-3306 on apoptosis and DNA damage.

Supplementary Figure 5 shows a Loewe matrix plot for VX-970 and AZD1775.

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.

S1. There is differential susceptibility to P-AscH- across cell lines. S2. Bolus addition of hydrogen peroxide depletes intracellular ATP and NAD+ in MIA PaCa-2 and PANC-1 cells. S3. Pharmacological ascorbate depletes intracellular NADH in MIA PaCa-2 cells, but not in PANC-1, 339, and H6c7 cells. S4. Pharmacological inhibition of PARP1 by olaparib enhances the cytotoxicity of P-AscH- in MIA PaCa-2 cells. S5. A combination of P-AscH- and prexasertib synergistically inhibits cell proliferation of MIA PaCa-2 cells. Supplementary Table S1. Normal pancreatic cells are more resistant to P-AscH- than pancreatic cancer cells. Supplementary Table S2. The ED50's for depletion of ATP and NAD+ correlate with kcell for the removal of H2O2. Supplementary Table S3. The gene target and primer pairs used for QPCR.

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

Supplementary Figure 5 shows the effects of prexasertib on markers of apoptosis.

Supplementary Figure 8 shows the effect of gemcitabine on the phosphorylation of 4E-BP1 in non-Ewing sarcoma cell lines.

Quantification of immunoblots.