The serine/threonine protein kinase ATM (ataxia-telangiectasia mutated) responds to DNA double strand break (DSB) damage by signalling, via phophorylation events, to key cell cycle and DNA-repair components. Mutation of ATM occurs in the human autosomal recessive disorder ataxia-telangiectasia (A-T), which is characterised by a hypersensitivity to ionising radiation (IR) and aberrant cell cycle control. It has therefore been proposed that inhibition of ATM activity could lead to cellular radio- and chemo-sensitisation. Screening of a combinatorial library based on the non-specific PI 3-kinase inhibitor LY294002, has identified a small molecule ATP competitive inhibitor of ATM, 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (KU-55933). KU-55933 inhibits ATM kinase activity with an IC50 of 10 nM and a Ki of 2.15 nM and is specific for ATM compared to other phosphatidylinositol (PI)-3 kinase related kinase (PIKK) family members e.g. DNA-PK, ATR, mTOR. Cellular inhibition of ATM via KU-55933 has been demonstrated by ablation of the ATM dependent IR induced phosphorylation of serine-15 of p53 and threonine-68 of CHK2. KU-55933 (10 μM) significantly sensitised human tumour cell lines (HeLa, LoVo and SW620) to the cytotoxic effects of IR (cytotoxicity at 2 Gy was increased 5-fold, 16-fold and 7-fold, respectively). KU-55933 also increased the cytotoxicity of DNA DSB inducing chemotherapeutic drugs e.g. the cytotoxicity of 1 μM etoposide was increased 2.6 ± 1.8 –fold in LoVo cells and 13 ± 9.1 –fold in SW620 cells (and the cytotoxicity of 100 nM doxorubicin was increased by 4.1 ± 0.7 and 2.8 ± 0.8 –fold in LoVo and SW620 cells, respectively). Inhibition of ATM by KU-55933 resulted in loss of IR-induced cell cycle arrest and an increase in etoposide-induced G2/M accumulation, consistent with the cell cycle profile observed in cells derived from A-T patients. The cellular specificity of KU-55933 for ATM was demonstrated in A-T derived cell lines. Exposure of A-T cells to KU-55933 did not potentiate the cytotoxic effects of IR and the cell cycle profiles of the A-T cells were unchanged by addition of KU-55933 prior to IR. We conclude that KU-55933 is a specific and potent inhibitor of ATM that can significantly enhance the effects of IR or double-strand break inducing chemotherapeutics at the cellular level. Through targeting the kinase activity of ATM with small molecule ATP competitors, inhibition of ATM function may ultimately improve the efficacy of currently approved clinical protocols for cancer therapy.
[Proc Amer Assoc Cancer Res, Volume 45, 2004]