The role of DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) kinase in the cellular response to microtubule-targeting drugs

Abstract

DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) kinase are DNA-damage activated kinases that play central roles in the non homologous end joining (NHEJ) and homologous recombination (HR) DNA double strand break repair pathways, respectively. DNA-PK and ATM have both been shown to have roles in addition to DNA repair, involving localisation at the centrosome during mitosis and mitotic regulation. Pilot studies demonstrated that the selective DNA-PK and ATM inhibitors, NU7441 and KU55933, respectively, caused greater sensitisation to DNA-damaging and microtubule-targeting agents in multidrug-resistant cells compared with parental cells. This observation led to the hypothesis that inhibition of DNA-PK and ATM using NU7441 and KU55933, respectively, or loss of DNA-PK function in DNA-PK deficient cells, would sensitise cells to agents that interfere with the formation of the mitotic spindle, e.g. microtubule-targeting agents such as vincristine, docetaxel and paclitaxel. Growth inhibition assays in four different paired parental sensitive and multidrug resistant cell lines (resistant through overexpression of the drug efflux transporter, MDR1) demonstrated that 1 µM NU7441 and 10 µM KU55933 sensitised the multidrug-resistant cells to vincristine and either docetaxel or paclitaxel to a significantly greater extent than in parental cells. Phosphorylation of DNA-PK at a DNA-damage-associated autophosphorylation site (Ser2056) was observed in response to vincristine, which did not cause DNA damage as determined using the COMET assay. Unexpectedly, three MDR1-overexpressing multidrug-resistant cell lines were found to be not only chemo-resistant but also radio-resistant. Investigations into the effects of NU7441 and KU55933 on drug transport demonstrated that in MDR1-overexpressing canine kidney MDCKII-MDR1 cells, 1µM NU7441 significantly increased doxorubicin cellular accumulation, measured by fluorescence microscopy, via an MDR1-dependent mechanism. NU7441 (1 µM), three structurally-related compounds (NU7742 (an inactive NU7441 analogue), DRN1 and DRN2 (DNA-PK-inhibitory and DNA-PK non-inhibitory atropisomeric NU7441 derivatives, respectively)) at 1 µM, and KU55933 at 1 µM and 10 µM, all increased intracellular vincristine accumulation in the MDR1-overexpressing CCRF-CEM VCR/R cells to a level similar to that induced by verapamil, as measured by LC-MS. Growth inhibition and cytotoxicity studies using an isogenic panel of DNA-PK proficient and deficient cell lines, 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)) established that DNA-PK -/- cells were more sensitive to ionising radiation, vincristine and docetaxel, thereby demonstrating a role for DNA-PK in the response of cells to all of these agents. KU55933 (10 µM) caused significant sensitisation in the HCT116 DNA-PK +/+, DNA-PK +/- and DNA PK -/- cells to ionising radiation, vincristine and docetaxel, suggesting an additional role for ATM. The combination of vincristine and ionising radiation was significantly more active in the absence of DNA-PK or following inhibition of DNA-PK. Confocal microscopy studies demonstrated that phosphorylated DNA-PK localised to mitotic structures and that lack or inhibition of DNA-PKcs caused an increase in aberrant mitotic events, such as chromosome misalignment, increases in centrosome number and multipolar spindle formation. Overall, the studies described in this thesis demonstrate that DNA-PK and ATM play a role in mitosis and in the response of cancer cells to microtubule-targeting agents. Dual DNA-PK and MDR1 inhibitors, and dual ATM and MDR1 inhibitors, were identified. These results extend the clinical potential of targeted inhibition of DNA-PK and ATM to use in combination with microtubule-targeting agents