Small molecule inhibitors of DNA-PKcs and ATM inhibit DNA repair and sensitize cells to ionizing radiation

Abstract

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ATM and DNA-PKcs are members of the phosphatidylinositol (PI) 3-kinase like kinase (PIKK) family of serine/threonine protein kinases. While ATM is a key regulator of the cellular response to ionising radiation and other DNA double strand break inducing treatments, DNA-PK is essential for efficient DNA repair by non homologous end joining (NHEJ). The Northern Institute for Cancer Research, in collaboration with KuDOS Pharmaceuticals, has synthesised and evaluated a series of novel DNA-PK and ATM inhibitors. These include NU7441 (Griffin et al, Proc Amer Assoc Cancer Res, 45:4362, 2004) and KU-55933 (Hickson et al Cancer Res, in press), potent and specific inhibitors of DNA-PK and ATM kinase activities respectively. We demonstrate that both drugs sensitize MCF7 breast carcinoma cells to ionizing radiation. Since DNA-PK is required for efficient NHEJ, inhibition of DNA repair is the expected mechanism for NU7441-induced radiosensitization. We have confirmed this by analyzing radiation induced phosphorylation of histone H2AX in the presence or absence of the inhibitors. The histone variant H2AX is phosphorylated over large chromatin domains at sites of DNA DSBs to form γH2AX foci that are visible by immunofluorescence microscopy. We observed a significant delay in the disappearance of IR-induced γH2AX foci in NU7441 treated MCF7 cells compared to cells irradiated in the absence of drug. Similarly, IR-induced foci were slower to disappear in KU-55933 treated cells, but in this case the γH2AX fluorescence signal was initially weaker than in untreated cells. This is consistent with the reported role of ATM in IR induced phosphorylation of H2AX. Notably, inhibition of DNA-PK had only a small effect on the initial phosphorylation of H2AX but simultaneous treatment with both compounds dramatically reduced initial γH2AX focus formation (15 min after IR). However, γH2AX foci did appear at later times, indicating that another PIKK family member, probably ATR, is able to phosphorylate H2AX to some degree after irradiation. We also observed in most cells an increase in fluorescence intensity from those foci that were remaining after 24 hours, suggesting time dependent increase in the number of phosphorylated H2AX molecules at sites of DSBs. We hypothesise that these novel inhibitors will specifically radio- and chemosensitize breast carcinomas that harbour existing DNA DSB repair defects. With this in mind we intend to compare the radio- and chemosensitizing potential of these drugs in breast carcinoma cell lines harbouring or engineered to contain different DNA damage response deficiencies.