Identification and evaluation of a potent novel ATR inhibitor, NU6027, in breast and ovarian cancer cell lines

Abstract

Background:The ataxia telangiectasia mutated and Rad3-related kinase (ATR) has a key role in the signalling of stalled replication forks and DNA damage to cell cycle checkpoints and DNA repair. It has long been recognised as an important target for cancer therapy but inhibitors have proved elusive. As NU6027, originally developed as a CDK2 inhibitor, potentiated cisplatin in a CDK2-independent manner we postulated that it may inhibit ATR.Methods:Cellular ATR kinase activity was determined by CHK1 phosphorylation in human fibroblasts with inducible dominant-negative ATR-kinase dead expression and human breast cancer MCF7 cells. Cell cycle effects and chemo- and radiopotentiation by NU6027 were determined in MCF7 cells and the role of mismatch repair and p53 was determined in isogenically matched ovarian cancer A2780 cells.Results:NU6027 is a potent inhibitor of cellular ATR activity (IC50=6.7 μ) and enhanced hydroxyurea and cisplatin cytotoxicity in an ATR-dependent manner. NU6027 attenuated G2/M arrest following DNA damage, inhibited RAD51 focus formation and increased the cytotoxicity of the major classes of DNA-damaging anticancer cytotoxic therapy but not the antimitotic, paclitaxel. In A2780 cells sensitisation to cisplatin was greatest in cells with functional p53 and mismatch repair (MMR) and sensitisation to temozolomide was greatest in p53 mutant cells with functional MMR. Importantly, NU6027 was synthetically lethal when DNA single-strand break repair is impaired either through poly(ADP-ribose) polymerase (PARP) inhibition or defects in XRCC1.Conclusion:NU6027 inhibits ATR, impairing G2/M arrest and homologous recombination thus increasing sensitivity to DNA-damaging agents and PARP inhibitors. It provides proof of concept data for clinical development of ATR inhibitors.

Efficient Herpes Simplex Virus 1 Replication Requires Cellular ATR Pathway Proteins

“…Inhibition of ATR signaling also reduces homologous recombination at DSBs in uninfected cells (79,80), as does overexpression of a dominant-negative kinase-dead allele of ATR (81). The viral genome is known to contain nicks and gaps (82), and replication through a nick can cause replication fork stalling, potentially resulting in the generation of a DSB.…”

ATR suppresses apoptosis after UVB light by controlling both translesion synthesis and alternative tolerance pathways

“…The ATR-mediated DNA damage response not only activates cell cycle checkpoints but also regulates replication and different tolerance pathways, and ATR is a vital kinase for protecting the genome against DNA damage that blocks replication. Inhibiting ATR has the potential to protect against UVB-induced carcinogenesis (Conney et al, 2007) and is growing as a potential sensitizer of chemotherapy or radiation, with selective killing of p53-deficient cancer cells (Reaper et al, 2011;Peasland et al, 2011;Prevo et al, 2012;Sultana et al, 2013). Thus, knowledge of the ATR-mediated DNA damage response can also aid in the design of more efficient cancer treatment protocols.…”

Trovafloxacin Enhances Lipopolysaccharide-Stimulated Production of Tumor Necrosis Factor-αby Macrophages: Role of the DNA Damage Response

“…This increase was absent after a 2-hour exposure to TVX. KU55933, a selective ATM inhibitor, and NU6027, an ATR-signaling inhibitor (Peasland et al, 2011), each prevented generation of the pATM/ATR substrate in VEH-or TVX-exposed RAW cells (Fig. 4B), indicating that ATM-and ATR-dependent signaling was activated by TVX.…”

“…Inhibitors KU55933 (Hickson et al, 2004), NU6027 [6-(cyclohexylmethoxy)-5-nitrosopyrimidine-2,4-diamine] (Peasland et al, 2011), and wortmannin (Powis et al, 1994) were dissolved in dimethylsulfoxide at a stock concentration of 10 mM and diluted to final concentrations in 0.5% FBS-containing medium. Inhibitors or an equivalent volume of dimethylsulfoxide vehicle were added at the moment RAW cells were exposed to VEH or TVX, unless noted otherwise.…”