CGK733 does not inhibit ATM or ATR kinase activity in H460 human lung cancer cells

Dear Editor,

The purpose of this letter is to address the activity of CGK733, a small molecule that was reported to inhibit both ATM and ATR kinase activities and block checkpoint signaling with great selectivity [1]. This manuscript was retracted [2]. Nevertheless, CGK733 is marketed as an ATM and ATR kinase inhibitor and several manuscripts have reported that CGK733 has effects on human cells [3–7]. We have investigated the effects of CGK733 in cells exposed to either ionizing radiation (IR) or ultraviolet radiation (UV).

To determine whether CGK733 inhibits ATM kinase activity we examined the well-characterized IR-inducible ATM kinase-dependent phosphorylations on ATM serine 1981 [8] and CHK2 threonine 68 [9]. We treated H460 human lung cancer cells with 10 μM CGK733, 10 μM KU55933 or 1 μM KU60019 and exposed these to 5 Gy γ-rays from a ¹³⁷Cs source. KU55933 and KU60019 are inhibitors of ATM kinase activity [10,11]. We selected H460 because we previously documented the ATM-dependent effects of both KU55933 and KU60019 in these cells [12,13] and we selected a concentration of 10 μM CGK733 because this is the highest dose reported to inhibit the kinase activities of ATM and ATR in human cells.

We observed that 10 μM CGK733 did not inhibit the IR-induced phosphorylation of either ATM serine 1981 or CHK2 threonine 68 one hour following irradiation whereas both 10 μM KU55933 and 1 μM KU60019, which served as positive controls for the experiment, inhibited the IR-induced phosphorylation of both ATM serine 1981 and CHK2 threonine 68 (Fig. 1A). Similar results were obtained using CGK733 purchased from Tocris Bioscience and Sigma–Aldrich.

Fig. 1.

(A) Immunoblots of IR-inducible ATM kinase-dependent phosphorylations on ATM serine 1981 and CHK2 threonine 68. H460 human lung cancer cells were treated with 10 μM CGK733 (Tocris Bioscience), 10 μM KU55933 or 1 μM KU60019 (KuDOS Pharmaceuticals) from 30 min prior to exposure to 5 Gy γ-rays until harvest at 60 min following the insult. Briefly, whole cell extracts were prepared in lysis buffer: 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 50 mM NaF, 1% Tween-20, 0.5% NP40 and 1× protease inhibitor mixture (Roche Applied Science, Indianapolis, IN). Cleared cell extracts were resolved in 3–8% Tris–acetate gels (Invitrogen, Carlsbad, CA) and immunoblotted with rabbit monoclonal anti-ATM 1981S-P antisera (EP1890Y, Epitomics), generic mouse monoclonal anti-ATM antisera (MAT3-4G10/8, Sigma), rabbit polyclonal anti-CHK2 68T-P (2661, Cell Signaling) and mouse monoclonal anti-CHK2 (1C12, Cell Signaling). (B) Immunoblots of the phosphorylation of ATM on serine 1981 and ultraviolet radiation (UV)-induced phosphorylation on CHK1 serine 317. H460 human lung cancer cells were treated with either 10 μM CGK733 or 10 μM ETP-46464 from 30 min prior to exposure to 10 J/m² UV radiation until harvest at 6 h following the insult. Cleared cell lysates were immunoblotted with rabbit polyclonal anti-CHK1 317S-P (2344, Cell Signaling) and mouse monoclonal anti-CHK1 (2G1D5, Cell Signaling).

To determine whether CGK733 inhibits ATR kinase activity we examined the phosphorylation of ATM on serine 1981 and the well-characterized UV-induced phosphorylation on CHK1 serine 317 [14]. We treated H460 cells with 10 μM CGK733 or 10 μM ETP-46464 and exposed these to 10 J/m² UV. ETP-46464 is an inhibitor of ATR kinase activity [15]. We observed that 10 μM CGK733 did not inhibit the UV-induced phosphorylation of CHK1 serine 317 six hours following irradiation whereas 10 μM ETP-46464, which served as a positive control for the experiment, inhibited the UV-induced phosphorylation of CHK1 serine 317 (Fig. 1B). Further, the phosphorylation of ATM serine 1981 was increased in cells incubated in 10 μM ETP-46464 and exposed to 10 J/m² UV. This has been observed previously [15] and is consistent with a model in which inhibition of the kinase activity of ATR causes an accumulation of double-strand DNA breaks, perhaps as a result of the disruption of protein complexes that protect stalled replication from endonucleases.

We conclude that CGK733 does not inhibit ATM or ATR kinase activity in H460 human lung cancer cells.