Abstract 3329: The DNA-PK inhibitor, NU7441, sensitises cancer cells to microtubule-targeting agents and modulates vincristine efflux in multidrug resistant cells.
Authors: Mould Emily , Berry Philip , Jamieson David , Hill Christopher , Tan Niu , Elliott Sarah , Durkacz Barbara , Newell David , Willmore Elaine 
Published: August 2014

Microtubules are involved in the formation of the mitotic spindle, and correct separation of the sister chromatids at mitosis is essential for maintaining genomic stability. Microtubule-targeting drugs arrest rapidly-dividing cells and are important chemotherapeutic agents, but the development of multidrug-resistance (MDR) is a serious clinical problem. Along with its role in DNA double-strand break repair, DNA-dependent protein kinase (DNA-PK) has also been shown to play an important role in stabilising spindle formation and preventing mitotic catastrophe (Shang et al, Cancer Res. 2010). In light of this novel finding, we hypothesised that inhibition of DNA-PK would sensitise cells to microtubule-targeting agents. Pilot studies showed that inhibition of DNA-PK by the selective inhibitor, NU7441, caused greater sensitisation to vincristine in multidrug-resistant cells compared to parental cells. Here, we further investigate the role of DNA-PK in response to microtubule-targeting drugs using a novel series of NU7441 analogues, and investigate whether these inhibitors interact with the efflux transporter, P-glycoprotein.

In growth inhibition studies, CCRF-CEM leukaemia cells were sensitised 1.4-fold to vincristine by NU7441 (1μM) whereas their vincristine-resistant derivative CCRF-CEM VCR/R cells were sensitised 8-fold to vincristine. DNA-PK-deficient M059J glioblastoma cells were 2-fold more sensitive to docetaxel than DNA-PK-proficient cells, and NU7441 sensitised only DNA-PK-proficient M059J-Fus1 cells to docetaxel. CCRF-CEM VCR/R cells have a higher basal level of autophosphorylation of DNA-PKcs (Western blotting) than the CCRF-CEM cells and autophosphorylation in response to vincristine was abrogated by NU7441.

To investigate the effects of NU7441 on drug transport, a doxorubicin fluorescence assay showed that in P-glycoprotein-overexpressing canine kidney MDCKII-MDR1 cells, 1μM NU7441 increased doxorubicin cellular fluorescence 16-fold by blocking drug efflux via P-glycoprotein. NU7441 and 3 structurally-related compounds (NU7742 (inactive NU7441 analogue), DRN1 (DNA-PK-inhibitory atropisomeric NU7441 derivative) or DRN2 (DNA-PK non-inhibitory atropisomeric NU7441 derivative)) all increased intracellular vincristine accumulation in the CCRF-CEM VCR/R cells to a level similar to verapamil, as measured by LC-MS. However, the non-inhibitory compound NU7742 was 5-fold less effective at sensitising CCRF-CEM VCR/R cells to vincristine than NU7441 (p=0.008).

The novel finding that DNA-PK inhibition sensitises cells to microtubule-targeting agents suggests that along with its function in DNA repair, this enzyme plays a role in the mitotic spindle checkpoint. Additionally, in multidrug-resistant cells, NU7441 is able to block drug efflux via interaction with P-glycoprotein.

Citation Format: Emily Mould, Philip Berry, David Jamieson, Christopher Hill, Niu Tan, Sarah Elliott, Barbara Durkacz, David Newell, Elaine Willmore. The DNA-PK inhibitor, NU7441, sensitises cancer cells to microtubule-targeting agents and modulates vincristine efflux in multidrug resistant cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3329. doi:10.1158/1538-7445.AM2013-3329