Purpose: Patients with advanced soft-tissue sarcomas (STSs) exhibit a poor prognosis and have few therapeutic options. DNA-dependent protein kinase catalytic subunit (DNA-PK) is a multifunctional serine—threonine protein kinase that plays a crucial role in DNA double-strand damage repair via nonhomologous end joining (NHEJ). Experimental design: To investigate the therapeutic potential of DNA-PK targeting in STS, we first evaluated the prognostic value of DNA-PK expression in two large cohorts of patients with STS. We then used the potent and selective DNA-PK inhibitor AZD7648 compound to investigate the antitumor effect of the pharmacological inhibition of DNA-PK in vitro via MTT, apoptosis, cell cycle, and proliferation assays. In vivo studies were performed with patient-derived xenograft models to evaluate the effects of AZD7648 in combination with chemotherapy or ionizing radiation on tumor growth. The mechanisms of sensitivity and resistance to DNA-PK inhibition were investigated by using a genome-wide CRISPR-Cas9 positive screen. Results: DNA-PK overexpression is significantly associated with poor prognosis in patients with sarcomas. Selective pharmacological inhibition of DNA-PK strongly synergizes with radiation- and doxorubicin-based regimen in sarcoma models. By using a genome-wide CRISPR-Cas9 positive screen, we identified genes involved in sensitivity to DNA-PK inhibition. Conclusion: DNA-PK inhibition deserves clinical investigation to improve response to current therapies in patients with sarcoma.