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. Author manuscript; available in PMC: 2010 Oct 1.
Published in final edited form as: Mol Cancer Ther. 2009 Oct 6;8(10):2894–2902. doi: 10.1158/1535-7163.MCT-09-0519

Improved ATM kinase inhibitor KU-60019 radiosensitizes glioma cells, compromises insulin, AKT and ERK prosurvival signaling, and inhibits migration and invasion

Sarah E Golding 1,§, Elizabeth Rosenberg 1,§, Nicholas Valerie 1, Isa Hussaini 2, Mark Frigerio 3, Xiaoling F Cockcroft 3, Wei Yee Chong 3, Marc Hummersone 3, Laurent Rigoreau 3, Keith A Menear 3, Mark O’Connor 3, Lawrence F Povirk 4,7, Timothy van Meter 5, Kristoffer Valerie 1,6,7,*
PMCID: PMC2761990  NIHMSID: NIHMS143737  PMID: 19808981

Abstract

Ataxia telangiectasia (A-T) mutated (ATM) is critical for cell cycle checkpoints and DNA repair. Thus, specific small molecule inhibitors targeting ATM could perhaps be developed into efficient radiosensitizers. Recently, a specific inhibitor of the ATM kinase, KU-55933, was shown to radiosensitize human cancer cells. Herein, we report on an improved analogue of KU-55933 (KU-60019) with Ki and IC50 values half of those of KU-55933. KU-60019 is 10-fold more effective than KU-55933 at blocking radiation-induced phosphorylation of key ATM targets in human glioma cells. As expected, KU-60019 is a highly effective radiosensitizer of human glioma cells. A-T fibroblasts were not radiosensitized by KU-60019 strongly suggesting that the ATM kinase is specifically targeted. Furthermore, KU-60019 reduced basal S473 AKT phosphorylation, suggesting that the ATM kinase might regulate a protein phosphatase acting on AKT. In line with this finding, the effect of KU-60019 on AKT phosphorylation was countered by low levels of okadaic acid, a phosphatase inhibitor, and A-T cells were impaired in S473 AKT phosphorylation in response to radiation and insulin, and unresponsive to KU-60019. We also show that KU-60019 inhibits glioma cell migration and invasion in vitro, suggesting that glioma growth and motility might be controlled by ATM via AKT. Inhibitors of MEK and AKT did not further radiosensitize cells treated with KU-60019 supporting the idea that KU-60019 interferes with prosurvival signaling separate from its radiosensitizing properties. Altogether, KU-60019 inhibits the DNA damage response, reduces AKT phosphorylation and prosurvival signaling, inhibits migration and invasion, and effectively radiosensitizes human glioma cells.

Keywords: DNA repair, radiosurvival, KU-55933

INTRODUCTION

Malignant glioma (MG) and its most aggressive form, glioblastoma multiforme (GBM) are devastating and inevitably lethal cancers of the brain whose victims have a life expectancy of only 12–15 months after diagnosis (1). Standard treatment of glioma is surgery followed by chemoradiation (1). The cellular and molecular biology of glioma is complex and is characterized by a highly invasive and aggressive phenotype, due in part to derailed growth factor-mediated signaling, making these cancers refractory to conventional treatments (2). Thus, there is an urgent need for novel and more effective therapies.

Individuals with the autosomal genetic disease ataxia telangiectasia (A-T) show impaired growth, immune deficiencies, cerebellar degeneration, telangiectasia of the eye, and premature aging (3). At the cellular level, A-T cells are extremely sensitive to ionizing radiation (IR), have impaired G1/S, intra-S, and G2/M checkpoints, and show elevated levels of chromosomal instability (4). The protein mutated in A-T (ATM) and other members of the phosphatidyl-inositol-3′-kinase-related kinase (PIKK) family, including DNA-PKcs and A-T and RAD3-related (ATR), are critical for the cellular response to DNA damage. Serving both complementary and backup roles, the PIKKs control a coordinated defense by the cell at multiple levels including cell cycle checkpoints, DNA double-strand break (DSB) repair, and apoptosis (5, 6), collectively referred to as the DNA damage response (DDR) (7). A critical function of ATM is to act as a protein kinase, phosphorylating an ever increasing number of targets in response to IR. These targets in turn cooperatively orchestrate a global cellular response (8). ATM is believed to regulate DSB repair directly or indirectly through cell cycle checkpoint control, and inhibition or absence of ATM increases radiosensitivity (7, 9). Thus, ATM is an attractive target for tumor radiosensitization.

In addition to delineating the DDR, recent work has established functional interactions between ATM and growth factor-mediated signaling (see (10) for a recent review). Amplification or up-regulation of growth factor receptor tyrosine kinases (RTKs) and loss of the PTEN phosphatase are common events in gliomas and are considered signs of poor prognosis (11). These changes lead to enhanced pro-survival signaling via PI3K/AKT and RAS/RAF/MEK/ERK, resulting in increased proliferation, metastasis, invasion, and radioresistance (7, 11). ATM is known to control insulin-mediated signaling (12–14), which in turn regulates AKT signaling. ATM is also reported to modulate radiation-induced AKT signaling, however the mechanism underlying this response is unclear (14). In addition, our own results have shown that MEK/ERK signaling is modulated by ATM (15). Very recently, several studies demonstrated that DNA-PKcs directly phosphorylates AKT at S473 in response to DNA damage (16–18). Furthermore, ATM phosphorylates DNA-PKcs on T2609 and regulates its function (19), and DNA-PKcs appears to regulate ATM protein levels and activity (20). Collectively, these studies suggest that ATM and DNA-PKcs could perhaps co-regulate DNA damage-induced pro-survival signaling via AKT. Thus, inhibition of ATM signaling may compromise pro-survival signaling in addition to inhibiting more established cell cycle checkpoints and DSB repair targets.

Herein, we report on the radiosensitizing properties of a novel ATM kinase-specific small molecule inhibitor, KU-60019, that is more effective than its predecessor. In addition to establishing its improved radiosensitizing properties we also begin to characterize an ATM-regulated response that controls AKT phosphorylation, which in turn might compromise glioma migration and invasion. Thus, KU-60019 could perhaps be developed into a highly effective cancer drug that not only would work as a radiosensitizer but would also curtail tumor dispersal.

MATERIALS AND METHODS

Chemical synthesis and structure of KU-60019

The chemical synthesis of 2-((2R, 6S)-2, 6-Dimethyl-morpholin-4-yl)-N-[5-(6-morpholin-4-yl-4-oxo-4H-pyran-2-yl)-9H-thioxanthen-2-yl]-acetamide (KU-60019) is described in patent WO/2007/026157 (http://www.wipo.int/portal/index.html.en). The calculated molecular weight of KU-60019 (C30H32N3O5S) is 547.67.

Reagents

Anti-p(S15)-p53, -p(T68)-Chk2, -p(S473)-AKT, -AKT, -p(S136)-BAD, -BAD, -p(S9)GSK3β, and -p(S1981)-ATM antibodies and GST-GSK3β substrate were purchased from Cell Signaling Technology, Inc., Danvers, MA. Anti-γ-H2AX (S139) antibody was from Millipore, Billerica, MA. Anti-β-actin, -p(T202/Y204)-ERK1/2 and -ERK2 antibodies were from Santa Cruz Biotechnology, Santa Cruz, CA. Anti-GSK3β, insulin and okadaic acid were purchased from Sigma Aldrich, St Louis, MO. Matrigel was purchased from BD Biosciences, San Jose, CA. SH-5 was purchased from EMD Biosciences (Gibbstown, NJ), and PD184352 has been described (21). KU-60019, KU-55933, and KU-57788 were all from KuDOS Pharmaceuticals Ltd, Cambridge, UK.

Cell culture and treatments

U87 (22), U1242 and U1242/luc-GFP (23), human malignant glioma cells were cultured in α-MEM medium supplemented with 10% FBS and antibiotics. Human primary GM02270 (normal fibroblasts) and GM05823 (A-T fibroblasts) cells (Coriell Institute for Medical Research, Camden, NJ), were cultured in MEM supplemented with vitamins, minimum-essential amino acids, non-essential amino acids (Invitrogen, Carlsbad, CA), and 15% FBS. Glioma cells were originally obtained from the ATCC and Dr. Allan Yates, Ohio State University, Columbus OH, respectively. Routine characterization includes the ability to form intra-cranial tumors in nude mice, and qRT-PCR (U1242) expression profiling. The cells have not been tested and authenticated by an external service provider. Irradiations were performed using an MDS Nordion Gammacell 40 (ON, Canada) research irradiator with a 137-Cs source delivering a dose rate of 1.05 Gy/min.

Western blotting

Proteins were separated by SDS-PAGE and transferred onto PVDF membranes (BioRad, Hercules, CA) for western blotting as previously described (15).

Cell growth

Cell growth was determined by AlamarBlue® (24). U1242 cells were serially diluted, allowedto attach for 6 h and then exposed to KU-60019 at 3 μM. At days 1, 3 and 5 after seeding, AlamarBlue® (AbD Serotec, Oxford, UK) was added to the medium to the recommended final concentration. Plates were incubated for 1 h at 37°C and fluorescence determined on a FluoroCount plate reader (Packard) (excitation 530 nm, emission 590 nm) and values taken as a measure of cell growth.

Cell survival

Trypan blue/FACS assay. Surviving fractions were calculated by determining the number of live cells in each sample relative to the untreated control sample after trypan blue and flow cytometry. Clonogenic survival. Clonogenic radiosurvival experiments were carried out as described (22, 25). Both procedures and are described in more detail in the supplemental methods.

Migration and invasion assays

Migration and invasion assays were carried out as described (26), and are outlined in more detail in the supplemental methods section.

Statistics

Linear regression, polynomial regression, and unpaired one- or two-tailed t tests were performed as appropriate on triplicate (or more) data sets using GraphPad Prism 3.0 (GraphPad Software, Inc). P values are indicated as; * < 0.05, ** < 0.01, ***<0.001.

RESULTS

KU-60019 is an improved ATM kinase-specific inhibitor

Recently, the novel and specific inhibitor of the ATM kinase, KU-55933 was identified in a screen of a small molecule library based on the relatively non-specific PI3K inhibitor LY294002 (25). KU-55933 has an IC50 of 13 nM and Ki of 2.2 nM in vitro and is highly specific for the ATM kinase using a panel of 60 protein kinases (25). To improve the pharmacokinetics and bioavailability, a new more water-soluble analogue was synthesized that shares many if not all of the KU-55933 structural, pharmacological, and biological effects (see patent WO/2007/026157). KU-60019 is an improved inhibitor of the ATM kinase with an IC50 of 6.3 nM, approximately half that of KU-55933. The IC50 values for DNA-PKcs and ATR are 1.7 and >10 μM, respectively, almost 270-and 1600-fold higher than for ATM (data not shown). KU-60019 has similar if not identical target specificity as KU-55933 with little to no non-specific target effects at 1 μM against a panel of 229 protein kinases (Table S1) with PI3K (p110β/p85α), PI3K (p120γ), and PI3K (p110δ/p85α) inhibited 9, 3, and 27% (data not shown), respectively (Millipore KinaseProfiler™ and PI3-Kinase HTRF™ assay). Notably, mTOR and mTOR/FKBP12 were not inhibited. The chemical structures of KU-60019 and KU-55933 are shown in Fig. 1

Figure 1.

Figure 1

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Chemical structures of KU-60019 and KU-55933.

KU-60019 is a more potent inhibitor of the ATM kinase than KU-55933

To begin determining the relative potency of KU-60019 and KU-55933 to block the ATM kinase in human glioma cells, we assessed the impact on IR-induced phosphorylation of key ATM targets. ATM phosphorylates numerous proteins at specific positions, including p53 at S15, H2AX at S139 (γ-H2AX), and CHK2 at T68 (7, 8). In human U87 glioma cells, KU-55933 completely inhibited phosphorylation of p53 (S15) at 10 μM but not at 3 μM (Fig. 2A, compare lanes 4–6 with 8 and 9), whereas γ-H2AX levels were only partly reduced with 10 μM 1 h after irradiation. By comparison, 3 μM KU-60019 completely inhibited p53 phosphorylation and partial inhibited at 1 μM (Fig. 2A, compare lanes 8 and 9 with 13–15). As with KU-55933, little to no effect on H2AX phosphorylation was seen 1 h after irradiation. Since ATM is believed to phosphorylate H2AX at S139 immediately after irradiation, with DNA-PKcs serving as backup (27, 28), we then examined these responses at both 15 and 60 min after radiation (Fig. 2B). To determine the contribution of DNA-PKcs, we utilized the DNA-PKcs-specific inhibitor KU-57788 (NU7441) (29). As before, KU-60019 at 3 μM completely inhibited p53 phosphorylation 15 min post-IR, whereas inhibiting DNA-PKcs with KU-57788 (2.5 μM) did not (Fig. 2B, compare lanes 5–7). Importantly, even 1 μM of KU-60019 almost completely blocked (>70%) p53 (S15) phosphorylation (Fig. 2B, compare lanes 8 and 9 with 13) suggesting that at the concentration used in the in vitro KinaseProfiler assay (Table S1) almost completely inhibited the DDR in intact cells. As expected, γ-H2AX levels were reduced significantly at 15 min with KU-60019 (Fig. 2B, compare lanes 5 and 6). In addition, when both KU-60019 and KU-57788 were added γ-H2AX levels were reduced even further, close to levels detected in non-irradiated controls (Fig. 2B, compare lanes 6–8). However, at 60 min the combined inhibitory effect of KU-60019 and KU-57788 was reduced as indicated by the increased γ-H2AX levels (compare lanes 8 and 11). These results suggest that ATM is the principal kinase of p53 (S15), and H2AX (S139) phosphorylation at early times after irradiation with DNA-PKcs and ATR serving as complementary and backup kinases, respectively, in agreement with previous reports (27, 28).

Figure 2. KU-60019 is a more effective inhibitor of the ATM kinase than KU-55933.

Figure 2

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(A) U87 cells were treated with KU-55933 or KU-60019 (0, 1, 3, or 10 μM) for 1 h, exposed to 10 Gy of IR and collected for western blot analysis after 1 h. (B) U87 cells were treated with KU-57788 (2.5 μM), KU-60019 (3 μM), or both, exposed to 10 Gy of IR, and collected for western blot analysis after 15 min or 60 min. (C) U1242 cells were treated with KU-60019 (3 μM), exposed to 5 Gy, and collected for western blot analysis after 5, 15, 30, or 60 min. Fold depicts phospho-protein levels normalized to β-actin levels. Drugs remained in the medium throughout the experiments.

The ATM-mediated radiation response was also examined in U1242 glioma cells. Contrary to U87 cells (p53 wild type, and deleted PTEN), U1242 cells express mutant p53 (H175R) and wild-type PTEN (30), and are highly invasive in vivo (23). In these cells, radiation-induced CHK2 phosphorylation (T68) was completely inhibited by KU-60019 at 3 μM up to 1 h after irradiation (Fig. 2C). Moreover, just as with U87 cells, we found that p53 phosphorylation was completely inhibited and γ-H2AX partially inhibited, especially at times ≤15 min. Altogether, KU-60019 is 3- to 10-fold more potent than KU-55933 at blocking radiation-induced phosphorylation of key ATM protein targets in human glioma cells.

KU-60019 is a more potent radiosensitizer than KU-55933

We then examined the relative potency of KU-60019 and KU-55933 at radiosensitizing human glioma cells. Using a novel assay we have developed combining the common Trypan blue viability staining with flow cytometry, we found that KU-55933 and KU-60019 at 10 μM resulted in dose-enhancement ratios (DERs) of 1.6 and 4.4, respectively (Fig. 3A, left and middle). A 10-fold lower concentration of KU-60019 (1 μM) resulted in a DER of 1.7, which was similar to the radiosensitization seen with 10 μM of KU-55933 (Fig. 3A, left). Similarly, KU-60019 at 3 μM radiosensitized U1242 cells to a level between that of U87 cells treated with 1 and 10 μM of KU-60019 (Fig. 3B). To support these results, colony-forming radiosurvival experiments were performed with U87 cells and normal fibroblasts (NFs) (Fig. 3C and S2B). As expected, we found that KU-60019 at 3 μM severely impaired radiosurvival resulting in DERs of 3.0 and 2.8, respectively. Thus, little to no tumor specificity of KU-60019 was noted, as expected. Together, these results show that KU-60019 is approximately 10 times more potent than KU-55933 at radiosensitizing human glioma cells.

Figure 3. KU-60019 radiosensitizes U87 and U1242 human glioma cells and normal but not A-T fibroblasts.

Figure 3

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Cells were treated with KU-60019 or KU-55933 at the indicated concentrations for 1 h prior to IR. Drugs were removed 16 h post IR. Surviving fractions were determined by Trypan blue/FACS assay (A, B and D), or crystal violet staining and colony counting (C). (A) U87 radiosurvival 7 days post-IR; IR +/− KU-55933 at 10 μM (left), IR +/−KU-60019 at 10 μM (middle), or IR +/− KU-60019 at 1 μM (right). (B) U1242 radiosurvival 4 days post-IR; IR +/− KU-60019 at 3 μM. (C) U87 radiosurvival 14 days post-IR; IR +/− KU-60019 at 3 μM. (D) Radiosurvival of normal (NF) or A-T fibroblast 7 days post-IR; IR +/− KU-60019 at 10 μM. Data points; surviving cells are plotted as fraction of control (-IR). Error bars; SEM n = 3. Where error bars are not seen they are obscured by symbols.

KU-60019 specifically targets the ATM kinase to radiosensitize cells

To demonstrate that KU-60019 is specific for ATM, we treated h-TERT-immortalized normal and A-T fibroblasts with KU-60019 prior to IR and determined radiosurvival by Trypan blue/FACS assay (Fig. 3D). As expected, only the NFs were radiosensitized by KU-60019 and not the A-T fibroblasts. This result strongly suggests that KU-60019 is an ATM kinase-specific radiosensitizer.

KU-60019 modulates phosphorylation of AKT at S473

It was recently reported that ATM regulates the phosphorylation of AKT at S473 in response to insulin and IR (14). However, this effect is likely indirect as the S473 site is not a consensus S/T-Q ATM kinase motif (31). DNA-PKcs has been shown to regulate AKT phosphorylation but contrary to ATM, DNA-PKcs directly phosphorylates AKT at S473 in response to DNA damage (16, 17). We previously demonstrated a role for ATM in regulating ERK pro-survival signaling in DSB repair (15), and ERK and AKT signaling are co-regulated to some extent via RAS in response to IR (see (10) for a recent review). Therefore, we tested whether KU-60019 affects S473 AKT phosphorylation in human glioma cells (Fig. 4). After a dose of 5 Gy to U87 cells phospho-AKT (S473) levels increased in a time-dependent manner and peaked at 2.4-fold after 15 min (Fig. 4A, lane 3). KU-60019 almost completely blocked this increase, and in fact, seemed to reduce phosphorylation below the level of an unirradiated control (Fig. 4A, compare lanes 1 and 6). Pooled data from several independent experiments showed that KU-60019 reduced basal AKT S473 phosphorylation by 70% (Fig. S1A). Similar responses were seen with U1242 cells, ie, AKT phosphorylation increased in response to radiation and KU-60019 almost completely abrogated basal and radiation-induced AKT S473 phosphorylation (Fig. 4A). Additionally, we noticed that KU-55933 attenuated radiation-induced AKT T308 phosphorylation (Fig. S1B). A time course of S473 dephosphorylation with KU-60019 alone showed an effect as early as 5 min after the addition of drug (Fig 4B). KU-60019 and KU-55933 also reduced the phosphorylation of (S136) BAD and (S9) GSK3β in vivo and in vitro (Fig. S1C, D), suggesting that AKT activity is reduced.

Figure 4. Radiation-induced and basal AKT (S473) phosphorylation is blocked by KU-60019 in human glioma cells and is impaired in A-T cells.

Figure 4

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(A) U87 or U1242 cells were treated with KU-60019 at 3 μM 1 h prior to irradiation with 5 Gy. Cells were collected at 5, 15, 30 or 60 min after irradiation and processed for western blot analysis. (B) U87 cells were treated with KU-60019 (3 μM) and harvested for western blot analysis after 5, 15, 30, 45 and 60 min. (C) U87 cells were treated with or without KU-60019 (3 μM) and decreasing concentrations of okadaic acid (100, 30, 10, 3 nM) for 1 h, then harvested for western blot analysis. Fold depicts phosphorylated AKT levels compared to untreated control levels (lane 1) normalized to total AKT protein levels. (D) A-T and normal fibroblasts (h-TERT) were exposed to IR (0, 2, 5, or 10 Gy) and collected for western blot analysis after 15 min. Data points; relative phosphorylation levels.

These results show that KU-60019 blocks pro-survival signaling emanating in reduced AKT (S473) phosphorylation in several human glioma cell lines. This response occurs in both p53 wild-type (U87) and mutant (U1242) backgrounds, regardless of PTEN status, and appears independent of DNA damage since AKT phosphorylation levels were below those seen in untreated cells. However, the radiation-induced increase in S473 AKT phosphorylation was not completely inhibited suggesting that phosphorylation (perhaps by DNA-PKcs) still occured (Fig. 4A, compare lanes 6–10). Combined, the results suggest that a protein phosphatase is acting on phosphorylated AKT and that this phosphatase may be under the control of the ATM kinase.

To examine whether a phosphatase could be involved in the response to KU-60019, we treated cells with okadaic acid (OA), a known inhibitor of PP1, PP2A, PP4-6 (32). In the presence of ≥30 nM OA, the effect of KU-60019 on S473 phosphorylation was reduced by >50%. In fact, OA by itself increased AKT S473 phosphorylation >2-fold compared to untreated control (Fig. 4C). These data suggest that an OA-sensitive phosphatase regulated by ATM modulates AKT phosphorylation although we cannot at the present time rule out alternative mechanisms. Further studies will be necessary to identify this putative phosphatase.

Radiation- and insulin-induced AKT phosphorylation is impaired in human A-T fibroblasts

To obtain further indication that AKT signaling is impaired when ATM is inactive, as our observations with KU-60019 suggest, we examined this response in A-T and NFs immortalized with h-TERT. The NFs demonstrated low basal levels of phospho-(S15) p53, which increased after IR (data not shown). On the other hand, A-T (h-TERT) cells did not and also did not produce any radiation-induced S1981 ATM phosphorylation (Fig. S2A). NF-hTERT cells are also radiosensitized by KU-60019 to a similar degree as our glioma lines, demonstrating that KU-60019 is not tumor cell specific (Fig. S2B), as expected. A western blot of extracts from untreated and irradiated A-T and NFs demonstrated that, indeed, S473 AKT phosphorylation increased in NFs after radiation (1.9-fold after 2 Gy), whereas this was not seen with A-T cells (Fig. 4D). In addition, extracts from A-T cells showed basal AKT phosphorylation levels at least 2-fold lower than those from untreated NFs and radiation at any dose did not increase AKT phosphorylation (Fig. 4D). When γ-H2AX levels were examined, a dose-dependent increase (2 to ~12-fold) was observed with NFs whereas the A-T cells only produced a slight increase after 10 Gy (Fig. 4D). Furthermore, a time-dependent increase in AKT phosphorylation was seen as early as 15 min in NFs but not A-T cells (data not shown). All combined, A-T cells are severely impaired in basal and radiation-induced AKT signaling whereas NFs are proficient suggesting that ATM is important for regulating radiation-induced AKT S473 phosphorylation in human cells. Peak AKT phosphorylation occurs at a relatively low dose of radiation (2 Gy) at early times (≤15 min). These observations with A-T and NFs support our findings with KU-60019 in glioma cells and implicate the ATM kinase in regulating AKT phosphorylation.

If KU-60019 activates a phosphatase that removes the phosphates on S473 and T308 it would be interesting to see whether stimulation of AKT phosphorylation by insulin would also be affected by KU-60019. Thus, U87 cells were treated with insulin with or without KU-60019 followed by p-AKT western blotting. We noticed a ~50% reduction in AKT phosphorylation with KU-60019 present (Fig. S3A), suggesting that the same ATM-dependent phosphatase acts on AKT regardless of whether stimulation is from radiation, insulin or basal levels of growth. To see whether A-T (h-TERT) cells are also impaired in AKT phosphorylation in response to insulin, we stimulated NF and A-T fibroblasts with insulin +/−KU-60019. We found that A-T cells are impaired in AKT phosphorylation in response to insulin compared to normal fibroblasts (~60% less phosphorylation), in agreement with previous reports (12–14), and that KU-60019 reduced AKT phosphorylation in the NFs (~45%) as it did in glioma cells. However, KU-60019 did not affect S473 phosphorylation in A-T cells in response to insulin, again, suggesting that KU-60019 is specific for the ATM kinase (Fig. S3B). These results demonstrate that insulin signaling emanating in AKT phosphorylation is impaired in A-T cells and that KU-60019 reduces AKT phosphorylation in response to insulin in both glioma cells and NFs. The lack of an effect of KU-60019 on insulin stimulation of AKT phosphorylation in A-T cells is in line with the inability of KU-60019 to radiosensitize A-T cells, and further supports the conclusion that KU-60019 is specific for the ATM kinase.

KU-60019 inhibits migration and invasion of human glioma cells in vitro

Since AKT and ERK signaling regulate glioma migration and invasion perhaps via deregulated RTK-mediated signaling (2), we wanted to determine whether KU-60019 affects these very critical aspects of glioma pathophysiology. Thus, we carried out a migration assay of U87 cells with or without KU-60019 using established in vitro conditions (Fig. 5). We found that KU-60019 inhibited migration of U87 cells ≥70% in a dose-dependent manner (Fig. 5A). Furthermore, invasion is a hallmark of malignant gliomas (2). We found that invasion through matrigel was inhibited ~60% by KU-60019 (Fig. 5B). The U1242 cells show a more invasive phenotype than U87 tumors when grown as orthotopic mouse xenografts ((23), data not shown). Using an alternative in vitro test for migration/motility, the ‘scratch’ or ‘wound healing’ assay, we show that KU-60019 also significantly inhibited U1242 migration by at least 50% (Fig. 5C). In addition, KU-60019 inhibited invasion of U1242 cells by ~60% (Fig. 5D). In line with these findings, we found that KU-60019 at 3 μM suppressed the growth of U1242 cells by ~40% over a 5 day period (Fig. S4). However, the reduction was only noted at later times. Altogether, these results show that KU-60019 inhibits migration/motility, invasion, and to some extent also growth of human glioma cells in vitro.

Figure 5. KU-60019 inhibits migration and invasion of human glioma U87 and U1242 cells in vitro.

Figure 5

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(A) Relative migration of U87-Luc cells through 8 μm pore membranes in the presence of KU-60019 (0, 1, 3, or 10 μM). Quantification of cell numbers was done by determining luciferase activity 6 h post-seeding. Data points; relative luminescence per well. Error Bars; SEM n = 4. RFU = relative fluorescence units. Fold (x) depicts relative migration compared to untreated control (-KU60019). (B) Relative invasion of U87 cells through matrigel-coated inserts +/− KU-60019 at 3 μM. Cells were collected and counted 48 h after seeding. Data points; total number of cells/well, Errors bars; SEM n = 5. Fold (x) depicts relative invasion compared to untreated control. (C) Scratch assay of U1242 cells. Closure of the scratch was measured over time in the presence or absence of KU-60019 at 3 μM. Data points; mean width of scratch. Error Bars; SEM n = 3. Where error bars are not seen they are obscured by symbols. Rate of gap closure was 50% slower in the KU-60019 treated cells at 10 h (p =<0.0005). (D) Relative invasion of U1242 cells through matrigel inserts in the presence or absence of KU-60019 at 3 μM. Cell numbers were determined by ATP luciferase assay after 48 h. Data points; relative luminescence units (RFU) per well. Error Bars; SEM n = 3. Fold (x) depicts relative invasion compared to untreated control (-KU-60019). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns = non-significant.

Inhibition of AKT or MEK/ERK signaling does not enhance KU-60019 radiosensitization

Since AKT and MEK/ERK signaling regulate cell growth and both pathways are frequently up-regulated in cancer cells, both have been explored as potential therapeutic targets with varying levels of success. Drugs inhibiting either the AKT or MEK kinases are cytostatic and synergy with radiation has been inconsistent and appears to be cell-type and cell-state-dependent (33–36). Thus, we wanted to determine whether inhibition of MEK/ERK or AKT signaling enhanced killing beyond inhibition of ATM alone. We reasoned that if KU-60019 indeed inhibits MEK/ERK and AKT signaling as suggested here and in our previous study (15), then no further radiosensitization should be observed when KU-60019 is combined with either a MEK inhibitor or an AKT kinase inhibitor, compared to KU-60019 alone. Thus, U87 cells were treated with PD184352, a highly specific and potent MEK1/2 inhibitor (21), with or without KU-60019, and radiosurvival determined by the Trypan blue/FACS assay. Inhibition of MEK/ERK signaling had only a small but significant effect on IR-induced killing of these cells (Fig. 6A). Importantly, MEK inhibition did not significantly increase killing by IR in combination with KU-60019 compared to KU-60019 alone (Fig. 6A). In fact, when the same response was examined in U1242 cells treatment with PD184352 alone was slightly radioprotective, and again the combined effect of KU-60019 and PD184352 was not enhanced over KU-60019 alone (Fig. S5).

Figure 6. AKT and MEK/ERK signaling are subsets of the ATM signaling network.

Figure 6

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U87 cells were treated with KU-60019 (1 μM), and/or (A) PD184352 (3 μM), (B) SH-5 (10 μM) for 2 h prior to IR (4 Gy). Drugs were removed 16 h post-IR. Surviving fractions were determined by Trypan blue/FACS assay 7 days post-IR. Data points; number of surviving cells were plotted as fraction of control (-IR). Error bars; SEM n = 3. Fold (x) depicts relative changes in surviving fractions as compared to drug treated control (-IR). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns = non-significant. # indicates no significant difference between untreated cells and KU-60019 alone or in combination with PD184352; p > 0.5 (A), or SH-5; p > 0.5 (B). (C) Western blot analysis of extracts from U87 cells treated with SH-5 (10 or 20 μM) for 2 h, and then exposed to 5 Gy. Cells were collected for western blotting 15 min post-IR. Fold depicts relative changes in AKT S473 phosphorylation compared to untreated control (-SH-5, -IR) after normalizing to total AKT expression levels.

We then examined a possible synergy between ATM and AKT inhibition. Cells were treated with SH-5, a pan-inhibitor of AKT that binds to AKT and blocks its activation (37). However, when the effects on radiation survival were determined, SH-5 had little effect on IR-induced cell killing and was, in fact, radioprotective in both U87 and U1242 cells. When SH-5 was combined with KU-60019 and IR, again no additive effect was observed with KU-60019 in either U87 or U1242 cells (Fig. 6B, and S5B). However, we found that SH-5 was able to block AKT phosphorylation by 80% in U87 cells (Fig. 6C). Collectively, these data show that KU-60019 radiosensitization does not increase in combination with inhibitors of either MEK/ERK or AKT signaling that by themselves have little to no effect on glioma cell radiosurvival, suggesting that the effects of KU-60019 on MEK/ERK and AKT signaling on radiosensitization are minor compared to the effects on classical DDR targets and separate from the effects of KU-60019 alone on glioma cell migration, invasion and growth.

DISCUSSION

In this report we have demonstrated that the ATM kinase-specific inhibitor KU-60019 is about 10 times more effective than its predecessor KU-55933 at radiosensitizing human glioma cells. Our results show unambiguously that the phosphorylation of key intra-cellular targets of the ATM kinase, including p53, H2AX and CHK2 is inhibited or completely abrogated in the presence of low micromolar concentrations of KU-60019. We show that the target for KU-60019 is ATM since normal but not A-T fibroblasts are radiosensitized. Furthermore, 1 μM of KU-60019 almost completely blocked the DDR in cells, a concentration that had no or very little effect on 229 kinases in vitro further supporting KU-60019 ’s specificity for the ATM kinase. We also show that KU-60019 reduces phosphorylation of AKT at S473 in glioma and NFs but not A-T cells regardless of whether the cells are irradiated, stimulated with insulin or during normal growth. Our results with KU-60019 agree with those from previous studies demonstrating that A-T cells and normal cells with ATM expression knocked down by siRNA displayed impaired AKT phosphorylation in response to insulin and radiation (13, 14).

Recently, a study by Bozulic et al. reported that DNA-PKcs phosphorylates AKT at S473 in response to radiation thereby regulating cell survival through p53 and p21 transcriptional control and cell cycle checkpoints that modulate apoptosis (17). In the same study, an experiment addressed whether ATM was also important for this DNA damage response involving DNA-PKcs, but no such involvement was noted (17). However, only ATM −/− mouse embryonic fibroblasts were used and not human cells. We have tested normal and ATM −/− MEFs for the ability to modulate S473 AKT phosphorylation in response to radiation and we also did not find any difference in the AKT response (data not shown). However, our results presented herein using human fibroblasts and glioma cells suggest show that ATM is important for controlling AKT phosphorylation. Thus, there could be a difference between how human and mouse cells control DNA damage-induced signaling that influences AKT signaling. There are numerous examples that human and mouse cells process and repair DNA damage differently (38). Importantly, our findings suggest that ATM signaling counteracts AKT phosphorylation by DNA-PKcs (17), and other kinases acting on AKT in response insulin.

In human glioma cells, but also in NFs and several other human cancer cell lines (data not shown), KU-60019 reduces S473 AKT phosphorylation regardless of radiation or insulin stimulation. Our findings are in line with the negative regulation of an as of yet unidentified phosphatase acting on p-S473 AKT and p-T308, that is regulated by ATM, ie, ATM indirectly regulates or needs to directly phosphorylate this phosphatase to keep it inactive. As to the nature of this phosphatase – this is presently unknown. However, there are several candidates able to dephosphorylate AKT, including protein phosphatase 1, protein phosphatase 2A, PTEN, and PHLPP (37, 39–42). Alternatively, ATM could potentially influence DNA-PKcs activity via T2609 phosphorylation but that is less likely since we still see radiation-induced increases in S473 AKT phosphorylation with KU-60019 present (Fig. 4A). This putative phosphatase is inhibited by low concentrations of OA suggesting it could be PP1, PP2A, or PP4-6 (32), however, more studies are needed to reveal its identity. Although our results are reminecent of an ATM-regulated phosphatase we cannot at this time rule out alternative mechanisms. Altogether, our results support a role for ATM in regulating AKT phosphorylation, not by increasing phosphorylation but by reducing basal, radiation- and insulin-induced AKT phosphorylation via a counter-acting protein phosphatase.

In an extension of our findings regarding a possible role for ATM in regulating pro-survival AKT and ERK signaling, we also demonstrated that KU-60019 has profound effects on glioma cell migration/motility and invasion in vitro. This finding is not surprising since both AKT and ERK have been associated with these processes in a variety of different cell types (for review see (11)). The inhibition of basal AKT phosphorylation that we observe with KU-60019 regardless of whether cells are irradiated or not is expected to affect growth, and indeed a slight effect on cell growth was seen after several days in the presence of KU-60019. Both MEK/ERK and AKT have been shown to be up-regulated in malignant gliomas and associated with radioresistance and poor prognosis. Inhibitors of both have been explored as potential radiosensitizers with mixed results (33–36). Inhibition of either target is known to reduce tumor growth but synergy or additive effect with radiation have not been consistent (for review see (10)). Our results suggest that in conjunction with KU-60019, neither MEK/ERK nor AKT inhibition is capable of further increasing IR-induced cell death over KU-60019 alone. Our findings suggest that that KU-60019 inhibits both AKT and MEK/ERK pro-survival signaling in addition to the phosphorylation of its better characterized DDR protein targets. The observed additional effects of inhibiting ATM on pro-survival signaling, migration and invasion, possibly via MEK/ERK and AKT signaling, is an exciting new finding. From a therapeutic standpoint, the inhibition of glioma growth, migration and invasion in vitro by low, but yet radiosensitizing concentrations of KU-60019, could perhaps translate into better control of tumor dispersal in vivo, a hallmark of malignant glioma (2).

In summary, we have demonstrated herein that KU-60019 is a specific and much improved ATM kinase inhibitor able to radiosensitize human glioma cells in the low micromolar range. Radiosensitization is likely caused by the ability of KU-60019 to inhibit the plethora of ATM phosphorylation targets and upset cell cycle checkpoints, reduce DNA repair, and increase cell death. Furthermore, our results suggest that KU-60019 alone (without radiation) inhibits glioma motility and invasion perhaps acting on the AKT and MEK/ERK pro-survival signaling pathways. Further pre-clinical testing will address whether some or all of these in vitro effects are also seen in vivo and whether KU-60019 could be developed into an effective and safe radiosensitizer of malignant glioma.

Supplementary Material

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Acknowledgments

Supported by NIH P01CA72955 (KV), NIH T32CA085159 (SEG), the American Brain Tumor Association (SEG), and a Massey Cancer Center Pilot grant (KV).

The Massey Cancer Center Flow Cytometry and Imaging Facility are supported in part by NIH grant P30CA16059. We thank Graeme Smith and Andrew Slade at KuDOS Pharamaceuticals Ltd for reagents and helpful discussions.

Abbreviations

A-T

ataxia telangiectasia

ATM

A-T mutated

ATR

A-T and RAD3-related

CNS

central nervous system

DER

dose enhancement ratio

DDR

DNA damage response

DNA-PKcs

DNA-dependent protein kinase catalytic subunit

DSB

double-strand break

ERK

extra-cellular signal regulated kinase

FACS

fluorescence assisted cell sorting

GBM

glioblastoma multiforme

IR

ionizing radiation

MAPK

mitogen-activated protein kinase

MEK

MAPK-kinase

MG

malignant glioma

NHEJ

non-homologous end-joining

OA

okadaic acid

PI3K

phosphatidyl-inositol-3′-kinase

PIKK

PI3K-related kinase

PTEN

phosphatase and tensin homologue deleted on chromosome 10

RTK

receptor tyrosine kinase

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Supplementary Materials

1
NIHMS143737-supplement-1.pdf (1.1MB, pdf)
2
NIHMS143737-supplement-2.doc (157KB, doc)
3
NIHMS143737-supplement-3.doc (51.5KB, doc)

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