Selective DNA-PK inhibition enhances chemotherapy and ionizing radiation activity in soft-tissue sarcomas
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 Overall design: For the CRISPR screen, we utilized the human knockout pooled library GeCKO V2 (Addgene 1000000048), consisting of 122,411 sgRNAs targeting 19,050 genes. In this experiment, 240 million IB114-cas9 cells were infected at a multiplicity of infection (MOI) of 0.3 to achieve a 500X coverage. The cells were then selected for three days at 0.75µg/mL of Puromycin. For the DNAPK sensitivity screen, we treated 30.106  cells with DMSO, and an equal number of cells (30.106) were treated with 40µM of DNAPK inhibitor. The cells were subjected to treatment for 72 hours before collecting them for DNA extraction. Upon comparing the treated cells to the control, the survival rate was approximately 20%. Genomic DNA was extracted using the DNeasy Blood and Tissue kit (Qiagen ID: 69504) following the manufacturer's protocol. The entire genomic DNA was sequenced by Nucleus Biotech. SgRNA was amplified, and Next-Generation Sequencing (NGS) was performed on the Illumina NextSeq platform, targeting 60 million reads.