Abstract 2399: DNA-PK is activated by microtubule-targeting agents; mechanistic studies using isogenic wild-type and DNA-PK knockout cells

Abstract

Abstract DNA-dependent protein kinase (DNA-PK) is a serine-threonine kinase essential for the double-strand break repair pathway non-homologous end-joining. The DNA-PK inhibitor NU7441 has been shown to sensitise cancer cells to ionising radiation and DNA-damaging agents, both in vitro and in vivo. Recent studies show alternative roles of DNA-PK, including localisation and regulation at the centrosome during mitosis. We therefore hypothesised that targeting DNA-PK would sensitise cells to clinically-used microtubule-targeting agents. We recently discovered that DNA-PK inhibitors sensitise cells to vincristine and docetaxel, and that DNA-PK deficient MO59J glioblastoma cells were 2-fold more sensitive to docetaxel than DNA-PK proficient MO59J-Fus1 cells (Mould E, Proc Ann Meet AACR 2013; 54: #3329). Here, we studied an isogenic panel of DNA-PK proficient and deficient cells to further understand the role of DNA-PK in the cellular response to microtubule-targeting agents. We used the X-MAN isogenic cell line panel of HCT116 cells (Horizon Discovery Ltd) with varying DNA-PK catalytic subunit expression levels (parental DNA-PK +/+, DNA-PK +/-, DNA-PK -/- and DNA-PK -/- cells with PRKDC cDNA re-expression (DNA-PK RE). Since ionising radiation (IR) activates DNA-PK, we used IR-induced DNA-PK activity as a positive control in studies with these cell lines. Growth inhibition and clonogenic survival assays showed the DNA-PK -/- cells were at least 4-fold more radiosensitive than the parental DNA-PK +/+ HCT116 cell line. DNA-PK +/- cells displayed a similar response to IR to the DNA-PK +/+ cells. Western blotting demonstrated that DNA-PK RE cells expressed DNA-PK to a level similar to the DNA-PK +/- cells, and the DNA-PK RE were 2.6-fold more resistant to IR compared to the DNA-PK -/- cells. Cells lacking DNA-PKcs therefore display the expected radiosensitivity. We then analysed the effect of microtubule-targeting agents on this cell line panel. DNA-PK -/- cells were 1.5-fold and 1.7-fold more sensitive to vincristine and docetaxel, respectively, than DNA-PK +/+ cells (growth inhibition assay), which is similar to the level of chemo-sensitisation by the DNA-PK inhibitor, NU7441, in a range of cell lines treated with vincristine or docetaxel. Vincristine treatment (24hrs) resulted in activation of DNA-PK (autophosphorylation at ser 2056) in DNA-PK +/+, DNA-PK +/- and DNA-PK RE cells. These data confirm our previous findings and demonstrate in an isogenic model that DNA-PK plays a role in response of cancer cells to microtubule-targeting agents. Ongoing studies with this cell line panel will investigate changes in expression of DNA-PK downstream targets and other DNA-damage-activated kinases in response to both DNA-damaging and microtubule-targeting agents, complemented with microscopy studies to investigate changes in microtubule dynamics and mitotic spindle formation. Citation Format: Emily V.A. Mould, David R. Newell, Elaine Willmore. DNA-PK is activated by microtubule-targeting agents; mechanistic studies using isogenic wild-type and DNA-PK knockout cells. [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 2399. doi:10.1158/1538-7445.AM2014-2399