DNA-dependent kinase (DNA-PK) is a nuclear serine/threonine protein kinase complex that is a key component of the non-homologous end joining (NHEJ) process. DNA-PK plays an important role in the cellular response to DNA damage through the detection and repair of DNA double strand breaks (DSB). Cancer therapies such as ionising radiation (IR) or topoisomerase II inhibitors (doxorubicin) generate DSB which can be repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). It can therefore be hypothesised a DNA-PK inhibitor would potentiate the activity of these agents. We have developed a highly potent and selective inhibitor of DNA-PK, AZD7648, which inhibits IR-induced DNA-PK S2056 auto phosphoryalation with an IC50 = 92 nM in A549 non-small cell lung cancer (NSCLC) cells. AZD7648 is a potent radiosensitiser where treatment in combination with IR led to a concentration-dependent reduction of the colony survival capacity of A549 and H1299 NSCLC cells (DEF37 at 100 nM = 1.7 and 2.5, respectively). In A549 cells, AZD7648 (≥1 µM) in combination with 2Gy IR for 48 hours led to a significant accumulation of cells arrested in the G2/M of the cell cycle, a 4-fold increase in micronuclei formation, and 3-fold induction of γH2AX, pATM S1981 and 53BP1 foci formation compared with IR alone. AZD7648 was also found to combine synergistically with doxorubicin in a panel of ovarian and triple negative breast cancer (TNBC) cell lines in cell growth inhibition assays when applying the Loewe additivity model (synergy scores 4 - 35). In vivo the combination of AZD7648 with IR (5x 2Gy) induced tumour regression in H1299 and A549 NSCLC xenografts in a dose-dependent manner (84 and 11% regression respectively), while monotherapy treatment only achieved tumour growth inhibition. In these two models the increased activation by IR of three primary DNA-PK pharmacodynamic markers, pDNAPK (S2056), pRPA32 (S4/8) and γH2AX, was inhibited by AZD7648 treatment (70-90% inhibition 2 h after IR + AZD7648). Similarly, liposomal doxorubicin (2.5 mg/kg weekly) in combination with AZD7648 (37.5 mg/kg bid) induced tumour regressions in the BT474c ER+ breast cancer xenograft model and in a TNBC PDX model (63% and 33% regression respectively), while monotherapy treatments only achieved tumour growth inhibition. These data confirm that DNA-PK inhibition with AZD7648 enhances the efficacy of a range of DSB inducing agents in vitro and in vivo, providing a clear rationale for its clinical investigation.
Citation Format: Jacqueline H. Fok, Antonio Ramos-Montoya, Neil James, Mercedes Vazquez-Chantada, Valeria Follia, Ankur Karmokar, Anna Staniszewska, Lenka Oplustil O’Connor, Emma Dean, Simon J. Hollingsworth, Barry R. Davies, Elaine B. Cadogan. AZD7648, a potent and selective inhibitor of DNA-PK, potentiates the activity of ionising radiation and doxorubicin in vitro and causes tumour regression in xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3512.