Abstract 2744: The novel and contrasting roles of ATM and DNA-PK in the response of cells to microtubule-targeting anticancer agents

Abstract

Abstract Ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) are DNA-damage activated kinases that play vital roles in the DNA double-strand break repair pathways homologous recombination (HR) and non-homologous end joining (NHEJ), respectively. ATM and DNA-PK have both been shown to have roles in addition to DNA repair, including localising at the centrosome during mitosis and mitotic regulation. This latter role led to the hypothesis that inhibition of ATM and DNA-PK, using the small molecule inhibitors KU55933 and NU7441, respectively, will sensitise cells to agents that interfere with formation of the mitotic spindle, e.g. microtubule-targeting agents. Here we investigated the sensitisation effects of the ATM inhibitor KU55933 to microtubule-targeting agents in paired sensitive and multidrug-resistant cells. In growth inhibition studies, KU55933 (10μM) sensitised CCRF-CEM leukaemia cells 4-fold to vincristine, and sensitised vincristine-resistant CCRF-CEM VCR/R cells 40-fold to vincristine. We have previously shown that inhibition of DNA-PK sensitises parental and multidrug-resistant (MDR) cells to microtubule-targeting agents and that DNA-PK is activated in response to these agents (Mould E, Proc Ann Meet AACR 2013; 54: #3329). The DNA-PK inhibitor increased vincristine-induced caspase 3/7 activation 2-fold after 24 hours, similar to the increase seen in combination with the positive control mitoxantrone. In contrast, KU55933 did not increase vincristine-induced caspase 3/7-mediated apoptosis, indicating a different mechanism of increased cell death. Vincristine-induced DNA-PK autophosphorylation (ser2056) (Western blotting) was abrogated by NU7441. However, the microtubule-targeting agents had no effect on ATM autophosphorylation (ser1981). Ongoing studies include confocal microscopy to determine localisation of ATM and DNA-PK during spindle formation, and the effects of microtubule-targeting agents and ATM/DNA-PK inhibitors on localisation, to further elucidate the novel roles of these kinases. We have also demonstrated that 1μM NU7441 induced a 2.1-fold (p=0.04) increase in vincristine accumulation in MDR1-overexpressing cells and that NU7441 is a dual DNA-PK MDR1 inhibitor. Due to the increased sensitisation observed in MDR cells, the effect of KU55933 on drug accumulation was investigated. LC-MS studies demonstrated that both 1μM and 10μM KU55933 increased intracellular vincristine accumulation 1.5-fold and 2.7-fold, respectively, in MDR1-overexpressing cells, indicating that KU55933 acts as a dual ATM and MDR1 inhibitor. These results highlight the complexity of the roles of ATM and DNA-PK and extend the therapeutic potential of targeting these kinases in combination with chemotherapeutic agents. Citation Format: Emily V.A. Mould, Stephanie E.A. Burnell, Philip L. Berry, David R. Newell, Elaine Willmore. The novel and contrasting roles of ATM and DNA-PK in the response of cells to microtubule-targeting anticancer agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2744. doi:10.1158/1538-7445.AM2014-2744