A new generation, clinical-stage DNA-PK inhibitor, M3814, potently and selectively blocks one of the two major pathways for repair of DNA double strand breaks (DSB) and synergizes with ionizing radiation (IR) and DSB-inducing chemotherapy. We show that by inhibiting DNA-PK catalytic activity in the presence of DNA DSBs, M3814 simultaneously suppresses DNA repair and a negative regulatory signal to ATM, leading to enhanced activation of the ATM dependent signaling, including CHK2 and p53-dependent cell cycle arrest. Combination treatment of proliferating p53 wild-type cancer cells (A549, A375, H460) with a single dose of ionizing radiation (2-5Gy) and sustained exposure to M3814 induced a complete G1/S and G2/M cell cycle block. Within 4-7 days of treatment cells acquired a typical senescence phenotype with large/flat morphology and β-Gal staining. Live cell imaging and BrdU labeling in A549 cells demonstrated that this phenotype is not reversible following M3814 removal, in contrast to a fully reversible senescence-like phenotype caused by selective p53 activation by MDM2 inhibitor Nutlin-3a. Isogenic p53-null A549 cells lost the ability to fully arrest their cell cycle, confirming the role of p53 in senescence induction. Analysis of mRNA from IR/M3814 induced senescent A549 and A375 cells by the Nanostring PanCancer Immune profiling panel revealed activation of a large group of genes from several immune response pathways, including interferon, cytokine/chemokine, and complement. Eighteen genes were commonly upregulated >3-150 fold compared to controls. These substantial changes in gene expression were built gradually and correlated with the development of senescence phenotype. Several proteins from the induced subset were measured in the cell media (MesoScale Discovery) and confirmed that they are secreted by senescent cells in the absence of M3814. Culture media from M3814-induced senescent cells showed increased immunomodulatory effect on human PBMC-derived immune cells, suggesting increased probability for clearance of IR+M3814 induced senescent cancer cells by the immune system.

Citation Format: Yige Guo, Qing Sun, Xiaohong Liu, Janusz Puc, Frank Czauderna, Frank Zenke, Andree Blaukat, Lyubomir T. Vassilev. Pharmacological DNA-PK inhibition induces ATM/p53 dependent premature senescence with immunomodulatory phenotype in irradiated cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 982.