Chemosensitization of cancer cells by KU-0060648, a dual inhibitor of DNA-PK and PI-3K

Abstract

DNA double-strand breaks (DSB) are the most cytotoxic lesions induced by topoisomerase II poisons. Nonhomologous end joining (NHEJ) is a major pathway for DSB repair and requires DNA-dependent protein kinase (DNA-PK) activity. DNA-PK catalytic subunit (DNA-PKcs) is structurally similar to PI-3K, which promotes cell survival and proliferation and is upregulated in many cancers. KU-0060648 is a dual inhibitor of DNA-PK and PI-3K in vitro. KU-0060648 was investigated in a panel of human breast and colon cancer cells. The compound inhibited cellular DNA-PK autophosphorylation with IC(50) values of 0.019 μmol/L (MCF7 cells) and 0.17 μmol/L (SW620 cells), and PI-3K-mediated AKT phosphorylation with IC(50) values of 0.039 μmol/L (MCF7 cells) and more than 10 μmol/L (SW620 cells). Five-day exposure to 1 μmol/L KU-0060648 inhibited cell proliferation by more than 95% in MCF7 cells but only by 55% in SW620 cells. In clonogenic survival assays, KU-0060648 increased the cytotoxicity of etoposide and doxorubicin across the panel of DNA-PKcs-proficient cells, but not in DNA-PKcs-deficient cells, thus confirming that enhanced cytotoxicity was due to DNA-PK inhibition. In mice bearing SW620 and MCF7 xenografts, concentrations of KU-0060648 that were sufficient for in vitro growth inhibition and chemosensitization were maintained within the tumor for at least 4 hours at nontoxic doses. KU-0060648 alone delayed the growth of MCF7 xenografts and increased etoposide-induced tumor growth delay in both in SW620 and MCF7 xenografts by up to 4.5-fold, without exacerbating etoposide toxicity to unacceptable levels. The proof-of-principle in vitro and in vivo chemosensitization with KU-0060648 justifies further evaluation of dual DNA-PK and PI-3K inhibitors.

Figure 1. Determination of the cellular specificity of KU-0060648 for DNA-PK-dependent cell survival following exposure to etoposide or doxorubicin

A-B; Clonogenic survival of V3-yac and V3 cells exposed to etoposide alone (solid symbols) or in combination with 1 μM KU-0060648 (open symbols), for 16 hours prior to seeding for colony formation. C-D; Clonogenic survival of M059-Fus-1 and M059J cells exposed to etoposide alone (black bars) or in combination with 1 μM KU-0060648 (white bars), for 16 hours prior to seeding for colony formation. E-F; Clonogenic survival M059-Fus-1 and M059J cells exposed to doxorubicin alone (black bars) or in combination with 1 μM KU-0060648 (white bars), for 16 hours prior to seeding for colony formation. Data are the means ± SD of 3 independent experiments. G; M059J and M059-Fus-1 cells were exposed to 1 μM KU-0060648 (K) or 0.1 μM ZSTK474 (Z) for 1 hour. Cell lysates were prepared and the relative levels of PI-3K-dependent AKT Serine⁴⁷³ phosphorylation determined by western blot.

Figure 2. Growth inhibition, cytotoxicity and chemosensitisation of KU-0060648 in human colon and breast cancer cell lines

A. The effect of KU-0060648 on cell growth (white bars), following 5-day continuous exposure to KU-0060648 was determined as previously described [28]. Cytotoxicity of KU-0060648 (black bars) was determined by the clonogenic survival of cells exposed to KU-0060648 (1 μM) for 16 hours prior to seeding for colony formation. Data are the means ± SD of 3 independent experiments. B. Clonogenic survival of cells exposed to etoposide or doxorubicin in the presence or absence of KU-0060648 (1 μM) for 16 hours, prior to seeding for colony formation. Topoisomerase II poison alone (black bars), Topoisomerase II poison + KU-0060648 (white bars). Data are the means ± SD of 3 independent experiments.

Figure 3. Comparison of PK levels of KU-0060648 and DNA-PK activity within ex vivo SW620 tumour samples following i.v dosing

CD-1 athymic female mice bearing SW620 human tumour xenografts were treated with KU-0060648 at either 2.5 or 25mg/kg i.v. At 1 or 4 hours following compound administration, plasma and tumour tissue were taken. KU-0060648 concentration (white bars) was measured by HPLC and the level of DNA-PK activity (black bars) was determined by measuring the DNA-PK-dependent phosphorylation of a p53 peptide substrate using an ELISA assay. Data are the mean ± SD of 3 replicate mice per time point.

Figure 4. Efficacy of etoposide and KU-0060648 in MCF7 and SW620 sub-cutaneous xenografts

Growth of xenografts is presented as the median relative tumour volume (RTV). Treatment commenced when tumours were palpable (approx. 5mm × 5mm). A. Animals bearing MCF7 tumours (5 /group) were treated with vehicle control (●), KU-0060648 alone (10 mg/kg twice daily × 14, ○ ), etoposide phosphate alone 11.5 mg/kg, daily × 5 , ▲) or KU-0060648 and etoposide (Δ). B. Animals bearing SW620 tumours (5 /group) were treated with vehicle control (●), KU-0060648 alone (10 mg/kg daily × 5, ○ ), etoposide phosphate alone (11.5 mg/kg, daily × 5, ▲) or KU-0060648 and etoposide phosphate (▼ once daily KU-0060648 dosing, Δ twice daily KU-0060648 dosing).