Sarah E. Golding

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

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. [Mol Cancer Ther 2009;8(10):2894–902]

Extracellular Signal-Related Kinase Positively Regulates Ataxia Telangiectasia Mutated, Homologous Recombination Repair, and the DNA Damage Response

The accurate joining of DNA double-strand breaks by homologous recombination repair (HRR) is critical to the long-term survival of the cell. The three major mitogen-activated protein (MAP) kinase (MAPK) signaling pathways, extracellular signal-regulated kinase (ERK), p38, and c-Jun-NH(2)-kinase (JNK), regulate cell growth, survival, and apoptosis. To determine the role of MAPK signaling in HRR, we used a human in vivo I-SceI-based repair system. First, we verified that this repair platform is amenable to pharmacologic manipulation and show that the ataxia telangiectasia mutated (ATM) kinase is critical for HRR. The ATM-specific inhibitor KU-55933 compromised HRR up to 90% in growth-arrested cells, whereas this effect was less pronounced in cycling cells. Then, using well-characterized MAPK small-molecule inhibitors, we show that ERK1/2 and JNK signaling are important positive regulators of HRR in growth-arrested cells. On the other hand, inhibition of the p38 MAPK pathway generated an almost 2-fold stimulation of HRR. When ERK1/2 signaling was stimulated by oncogenic RAF-1, an approximately 2-fold increase in HRR was observed. KU-55933 partly blocked radiation-induced ERK1/2 phosphorylation, suggesting that ATM regulates ERK1/2 signaling. Furthermore, inhibition of MAP/ERK kinase (MEK)/ERK signaling resulted in severely reduced levels of phosphorylated (S1981) ATM foci but not gamma-H2AX foci, and suppressed ATM phosphorylation levels >85% throughout the cell cycle. Collectively, these results show that MAPK signaling positively and negatively regulates HRR in human cells. More specifically, ATM-dependent signaling through the RAF/MEK/ERK pathway is critical for efficient HRR and for radiation-induced ATM activation, suggestive of a regulatory feedback loop between ERK and ATM.

Pro-survival AKT and ERK signaling from EGFR and mutant EGFRvIII enhances DNA double-strand break repair in human glioma cells

The Epidermal growth factor receptor (EGFR) is frequently dysregulated in malignant glioma that leads to increased resistance to cancer therapy. Up-regulation of wild type or expression of mutants such as EGFR variant III (EGFRvIII), the most common EGFR mutation in malignant glioma, is associated with tumor radioresistance and poor clinical outcome. This radioresistance is thought to be the result of a strong cytoprotective response fueled by signaling via AKT and ERK that is heightened by radiation in the clinical dose range. Several groups including ours have shown that this response may modulate DNA repair. Herein, we show that expression of EGFRvIII promoted Pro-survival AKT and ERK signaling from EGFR and mutant EGFRvIII enhances DNA double-strand break repair in human glioma cells γ-H2AX foci resolution, a surrogate for double-strand break (DSB) repair, and thus enhanced DNA repair. Conversely, small molecule inhibitors targeting EGFR, MEK, and the expression of dominant-negative EGFR (EGFR-CD533) significantly reduced the resolution of γ-H2AX foci. When homologous recombination repair (HRR) and non-homologous end joining (NHEJ) were specifically examined, we found that EGFRvIII stimulated and CD533 compromised HRR and NHEJ, respectively. Furthermore, NHEJ was significantly blocked by inhibitors of AKT and ERK signaling pathways. Moreover, expression of EGFRvIII and CD533 increased and reduced, respectively, the formation of phospho-DNA-PKcs and -ATM repair foci, and RAD51 expression levels, indicating that DSB repair is regulated at multiple levels. Altogether, signaling from EGFR and EGFRvIII promotes both HRR and NHEJ that is likely one contributing factor towards the radioresistance of malignant gliomas.

ATM Kinase Inhibition Preferentially Sensitizes p53-Mutant Glioma to Ionizing Radiation

Purpose: Glioblastoma multiforme (GBM) is the most lethal form of brain cancer with a median survival of only 12 to 15 months. Current standard treatment consists of surgery followed by chemoradiation. The poor survival of patients with GBM is due to aggressive tumor invasiveness, an inability to remove all tumor tissue, and an innate tumor chemo- and radioresistance. Ataxia–telangiectasia mutated (ATM) is an excellent target for radiosensitizing GBM because of its critical role in regulating the DNA damage response and p53, among other cellular processes. As a first step toward this goal, we recently showed that the novel ATM kinase inhibitor KU-60019 reduced migration, invasion, and growth, and potently radiosensitized human glioma cells in vitro. Experimental Design: Using orthotopic xenograft models of GBM, we now show that KU-60019 is also an effective radiosensitizer in vivo. Human glioma cells expressing reporter genes for monitoring tumor growth and dispersal were grown intracranially, and KU-60019 was administered intratumorally by convection-enhanced delivery or osmotic pump. Results: Our results show that the combined effect of KU-60019 and radiation significantly increased survival of mice 2- to 3-fold over controls. Importantly, we show that glioma with mutant p53 is much more sensitive to KU-60019 radiosensitization than genetically matched wild-type glioma. Conclusions: Taken together, our results suggest that an ATM kinase inhibitor may be an effective radiosensitizer and adjuvant therapy for patients with mutant p53 brain cancers. Clin Cancer Res; 19(12); 3189–200. ©2013 AACR.

Dynamic Dependence on ATR and ATM for Double- Strand Break Repair in Human Embryonic Stem Cells and Neural Descendants

The DNA double-strand break (DSB) is the most toxic form of DNA damage. Studies aimed at characterizing DNA repair during development suggest that homologous recombination repair (HRR) is more critical in pluripotent cells compared to differentiated somatic cells in which nonhomologous end joining (NHEJ) is dominant. We have characterized the DNA damage response (DDR) and quality of DNA double-strand break (DSB) repair in human embryonic stem cells (hESCs), and in vitro-derived neural cells. Resolution of ionizing radiation-induced foci (IRIF) was used as a surrogate for DSB repair. The resolution of γ-H2AX foci occurred at a slower rate in hESCs compared to neural progenitors (NPs) and astrocytes perhaps reflective of more complex DSB repair in hESCs. In addition, the resolution of RAD51 foci, indicative of active homologous recombination repair (HRR), showed that hESCs as well as NPs have high capacity for HRR, whereas astrocytes do not. Importantly, the ATM kinase was shown to be critical for foci formation in astrocytes, but not in hESCs, suggesting that the DDR is different in these cells. Blocking the ATM kinase in astrocytes not only prevented the formation but also completely disassembled preformed repair foci. The ability of hESCs to form IRIF was abrogated with caffeine and siRNAs targeted against ATR, implicating that hESCs rely on ATR, rather than ATM for regulating DSB repair. This relationship dynamically changed as cells differentiated. Interestingly, while the inhibition of the DNA-PKcs kinase (and presumably non-homologous endjoining [NHEJ]) in astrocytes slowed IRIF resolution it did not in hESCs, suggesting that repair in hESCs does not utilize DNA-PKcs. Altogether, our results show that hESCs have efficient DSB repair that is largely ATR-dependent HRR, whereas astrocytes critically depend on ATM for NHEJ, which, in part, is DNA-PKcs-independent.

Dynamic inhibition of ATM kinase provides a strategy for glioblastoma multiforme radiosensitization and growth control.

Glioblastoma multiforme (GBM) is notoriously resistant to treatment. Therefore, new treatment strategies are urgently needed. ATM elicits the DNA damage response (DDR), which confers cellular radioresistance; thus, targeting the DDR with an ATM inhibitior (ATMi) is very attractive. Herein, we show that dynamic ATM kinase inhibition in the nanomolar range results in potent radiosensitization of human glioma cells, inhibits growth and does not conflict with temozolomide (TMZ) treatment. The second generation ATMi analog KU-60019 provided quick, reversible and complete inhibition of the DDR at sub-micromolar concentrations in human glioblastoma cells. KU-60019 inhibited the phosphorylation of the major DNA damage effectors p53, H2AX and KAP1 as well as AKT. Colony-forming radiosurvival showed that continuous exposure to nanomolar concentrations of KU-60019 effectively radiosensitized glioblastoma cell lines. When cells were co-treated with KU-60019 and TMZ, a slight increase in radiation-induced cell killing was noted, although TMZ alone was unable to radiosensitize these cells. In addition, without radiation, KU-60019 with or without TMZ reduced glioma cell growth but had no significant effect on the survival of human embryonic stem cell (hESC)-derived astrocytes. Altogether, transient inhibition of the ATM kinase provides a promising strategy for radiosensitizing GBM in combination with standard treatment. In addition, without radiation, KU-60019 limits growth of glioma cells in co-culture with human astrocytes that seem unaffected by the same treatment. Thus, inter-fraction growth inhibition could perhaps be achieved in vivo with minor adverse effects to the brain.

ATM-dependent ERK signaling via AKT in response to DNA double-strand breaks.

Ionizing radiation (IR) triggers many signaling pathways primarily originating from either damaged DNA or non-nuclear sources such as growth factor receptors. Thus, to study the DNA damage-induced signaling component alone by irradiation would be a challenge. To generate DNA double-strand breaks (DSBs) and minimize non-nuclear signaling, human cancer cells having bromodeoxyuridine (BrdU) - substituted DNA were treated with the photosensitizer Hoechst 33258 followed by long wavelength UV (UV-A) treatment (BrdU photolysis). BrdU photolysis resulted in well-controlled, dose- dependent generation of DSBs equivalent to radiation doses between 0.2 - 20 Gy, as determined by pulsed-field gel electrophoresis, and accompanied by dose-dependent ATM (ser-1981), H2AX (ser-139), Chk2 (thr-68), and p53 (ser-15) phosphorylation. Interestingly, low levels (≤2 Gy equivalents) of BrdU photolysis stimulated ERK phosphorylation whereas higher (>2 Gy eq.) resulted in ERK dephosphorylation. ERK phosphorylation was ATM-dependent whereas dephosphorylation was ATM-independent. The ATM-dependent increase in ERK phosphorylation was also seen when DSBs were generated by transfection of cells with an EcoRI expression plasmid or by electroporation of EcoRI enzyme. Furthermore, AKT was critical for transmitting the DSB signal to ERK. Altogether, our results show that low levels of DSBs trigger ATM- and AKT-dependent ERK pro-survival signaling and increased cell proliferation whereas higher levels result in ERK dephosphorylation consistent with a dose-dependent switch from pro-survival to anti-survival signaling.

The Influence of Hypoxia and pH on Bioluminescence Imaging of Luciferase-Transfected Tumor Cells and Xenografts

Bioluminescence imaging (BLI) is a relatively new noninvasive technology used for quantitative assessment of tumor growth and therapeutic effect in living animal models. BLI involves the generation of light by luciferase-expressing cells following administration of the substrate luciferin in the presence of oxygen and ATP. In the present study, the effects of hypoxia, hypoperfusion, and pH on BLI signal (BLS) intensity were evaluated in vitro using cultured cells and in vivo using a xenograft model in nude mice. The intensity of the BLS was significantly reduced in the presence of acute and chronic hypoxia. Changes in cell density, viability, and pH also affected BLS. Although BLI is a convenient non-invasive tool for tumor assessment, these factors should be considered when interpreting BLS intensity, especially in solid tumors that could be hypoxic due to rapid growth, inadequate blood supply, and/or treatment.

Mutation of the BRCA1 SQ-cluster results in aberrant mitosis, reduced homologous recombination, and a compensatory increase in non-homologous end joining

Mutations in the breast cancer susceptibility 1 (BRCA1) gene are catalysts for breast and ovarian cancers. Most mutations are associated with the BRCA1 N- and C-terminal domains linked to DNA double-strand break (DSB) repair. However, little is known about the role of the intervening serine-glutamine (SQ) - cluster in the DNA damage response beyond its importance in regulating cell cycle checkpoints. We show that serine-to-alanine alterations at critical residues within the SQ-cluster known to be phosphorylated by ATM and ATR result in reduced homologous recombination repair (HRR) and aberrant mitosis. While a S1387A BRCA1 mutant - previously shown to abrogate S-phase arrest in response to radiation - resulted in only a modest decrease in HRR, S1387A together with an additional alteration, S1423A (BRCA12P), reduced HRR to vector control levels and similar to a quadruple mutant also including S1457A and S1524A (BRCA14P). These effects appeared to be independent of PALB2. Furthermore, we found that BRCA14P promoted a prolonged and struggling HRR late in the cell cycle and shifted DSB repair from HRR to non-homologous end joining which, in the face of irreparable chromosomal damage, resulted in mitotic catastrophe. Altogether, SQ-cluster phosphorylation is critical for allowing adequate time for completing normal HRR prior to mitosis and preventing cells from entering G1 prematurely resulting in gross chromosomal aberrations.

MRE11 and ATM AKTivate pro-survival signaling

Whether during normal cell growth or after genotoxic stress, there is a continuous battle between survival and death that determines cell fate. Tumor cells have acquired enhanced anti-apoptotic and pro-survival properties that make them resistant to cancer therapy. It is well-established that AKT is hyperactivated in many cancers, and that this protects against cell killing in response to DNA damaging agents, including ionizing radiation (IR). A plethora of events are triggered in response to IR, including the DNA damage response (DDR), apoptosis/death as well as pro-survival signaling via ERK and PI3K/AKT.1 Many laboratories have reported that IR activates AKT (seen as increased levels of AKT phosphorylation at S473 and T308). ATM (ataxia telangiectasia mutated), the master regulator of the DDR, in part controls insulin and IR-induced phosphorylation of AKT,2,3 and AKT has been shown to regulate DSB repair in a number of different cell systems.4-8 However, it is unclear how the DDR signal originating from DSBs is transduced to AKT. The article by Fraser et al. published in the July 1st issue of Cell Cycle reports that MRE11 promotes the accumulation of phosphorylated AKT (S473) at DSBs via ATM.9 They demonstrated that AKT is important for conferring radioresistance and for promoting the ligation of linearized plasmids in vitro, suggesting that AKT might facilitate the resealing of DSBs, thereby enhancing cell survival. Using IR, nuclear UV laser micro-irradiation or an inducible restriction endonuclease to induce DSBs, they showed that pAKT (S473), but not pAKT (T308) or total AKT, accumulated in the vicinity of IR-induced DSB and colocalized with γH2AX and pATM (S1981). Knowing that MRE11-RAD50-NBS1 (MRN) is positioned upstream of ATM, they knocked down MRE11 expression and showed that pAKT (S473) foci formation was dependent on MRE11. However, MRE11 did not require the MRE11 endonuclease domain, suggesting that DNA resection was not necessary to attract pAKT to repair centers. They went on to show that the histone ubiquitin ligase RNF168 was also required for DSB-induced pAKT localization. However, DNA-PKcs, PI3K and ATR were not. These results demonstrate that DSBs activate a signaling cascade that directly promotes a PI3K-independent pathway of AKT phosphorylation that is dependent on MRE11-ATM-RNF168 signaling. Altogether, the authors suggest that ubiquitin-dependent and DNA-PKcs-independent non-homologous end joining (NHEJ) repair is the target for pAKT. What remains unclear is whether the global effects resulting from AKT knockdown on radiosurvival can be fully accounted for by the relatively small fraction of total AKT that localized to DSBs as pAKT (S473). Furthermore, is NHEJ specifically influenced by AKT, or is homologous recombination repair (HRR) also affected? Since resection was not necessary for attracting pAKT to repair foci, this might argue against a role for HRR. As the kinetics of foci removal was not closely examined in the study (except for a single 24 h time point), it is difficult to say whether the presence of pAKT at DSBs influences repair in vivo. Another question is the role of other AKT isoforms. Only AKT1 was manipulated by RNAi. It would be interesting to know whether AKT2 and AKT3, two other very similar AKT isoforms that provide overlapping and back-up roles to AKT1, influence pAKT foci localization and repair. Most commercially available antibodies against pAKT (S473) also recognize phosphorylated AKT2 (S474) and AKT3 (S472). Thus, the roles of AKT2 and AKT3 in this response, if any, should be explored further. The results by Fraser et al. are complementary to recent findings by Khalil et al.,10 who demonstrated that DSBs resulting from BrdU photolysis or endonuclease electroporation triggered an ATM-dependent, pro-survival signaling cascade. This cascade required functional AKT in order to transmit the signal to MEK/ERK and promote cell proliferation. Collectively, conclusions from these two studies add weight to the notion that after low levels of DNA damage pro-survival signaling might be yet another arm of the DDR regulated by ATM. Thus, AKT could play the role of gatekeeper, somehow weighing cues from the DDR and growth factor signaling, to make balanced decisions together with ATM as to whether a cell will repair its DNA and live or be unable to and die.