DNA replication stress and translational repression converge to drive CDK1- and caspase-dependent apoptosis in Ewing sarcoma.

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.