A cell-based screen identifies ATR inhibitors with synthetic lethal properties for cancer-associated mutations

Genomic Instability Group, Spanish National Cancer Research Centre, Madrid, Spain.
Nature Structural & Molecular Biology (Impact Factor: 13.31). 06/2011; 18(6):721-7. DOI: 10.1038/nsmb.2076
Source: PubMed


Oncogene activation has been shown to generate replication-born DNA damage, also known as replicative stress. The primary responder to replicative stress is not Ataxia-Telangiectasia Mutated (ATM) but rather the kinase ATM and Rad3-related (ATR). One limitation for the study of ATR is the lack of potent inhibitors. We here describe a cell-based screening strategy that has allowed us to identify compounds with ATR inhibitory activity in the nanomolar range. Pharmacological inhibition of ATR generates replicative stress, leading to chromosomal breakage in the presence of conditions that stall replication forks. Moreover, ATR inhibition is particularly toxic for p53-deficient cells, this toxicity being exacerbated by replicative stress-generating conditions such as the overexpression of cyclin E. Notably, one of the compounds we identified is NVP-BEZ235, a dual phosphatidylinositol-3-OH kinase (PI3K) and mTOR inhibitor that is being tested for cancer chemotherapy but that we now show is also very potent against ATM, ATR and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs).

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    • "High TIPIN protein expression in the poor prognosis associated breast tumors There is growing interest in the targeting of DNA replication in anti-tumor strategies (Ma et al., 2012; Toledo et al., 2011). We tried to identify genes encoding proteins involved in replication fork stability and displaying overexpression in TNBC, with the aim of discovering new treatment targets for this BC subgroup. "
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    ABSTRACT: Triple-negative breast cancer (TNBC) is the breast cancer subgroup with the most aggressive clinical behavior. Alternatives to conventional chemotherapy are required to improve the survival of TNBC patients. Gene-expression analyses for different breast cancer subtypes revealed significant overexpression of the Timeless-interacting protein (TIPIN), which is involved in the stability of DNA replication forks, in the highly proliferative associated TNBC samples. Immunohistochemistry analysis showed higher expression of TIPIN in the most proliferative and aggressive breast cancer subtypes including TNBC, and no TIPIN expression in healthy breast tissues. The depletion of TIPIN by RNA interference impairs the proliferation of both human breast cancer and non-tumorigenic cell lines. However, this effect may be specifically associated with apoptosis in breast cancer cells. TIPIN silencing results in higher levels of single-stranded DNA (ssDNA), indicative of replicative stress (RS), in TNBC compared to non-tumorigenic cells. Upon TIPIN depletion, the speed of DNA replication fork was significantly decreased in all BC cells. However, TIPIN-depleted TNBC cells are unable to fire additional replication origins in response to RS and therefore undergo apoptosis. TIPIN knockdown in TNBC cells decreases tumorigenicity in vitro and delays tumor growth in vivo. Our findings suggest that TIPIN is important for the maintenance of DNA replication and represents a potential treatment target for the worst prognosis associated breast cancers, such as TNBC. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
    Molecular oncology 05/2015; 9(8). DOI:10.1016/j.molonc.2015.04.010 · 5.33 Impact Factor
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    • "Proof of principle data that ATR inactivation leads to abrogation of DNA damage-induced G2/M arrest, and sensitization to multiple classes of DNA-damaging agents was generated using genetic approaches, such as knockdown or overexpressing a kinase-dead mutant ATR (ATR-kd) (Nghiem et al., 2001; Cliby et al., 2002). A number of highly potent and selective inhibitors of ATR have recently been identified: NU6027, VE-821, ETP46464, VE-822, AZ-20 and AZD-6738 (Peasland et al., 2011; Reaper et al., 2011; Toledo et al., 2011; Fokas et al., 2012; Foote et al., 2013; Jones et al., 2013). These inhibitors sensitise human cancer cell lines to a variety of DNA-damaging chemotherapy and radiotherapy (Pires et al., 2012; Prevo et al., 2012; Huntoon et al., 2013; Sultana et al., 2013) and some have shown single agent cytotoxic activity in certain cancer cell lines (Foote et al., 2013). "
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    ABSTRACT: ATR, which signals DNA damage to S/G2 cell cycle checkpoints and for repair, is an attractive target in cancer therapy. ATR inhibitors are being developed and a pharmacodynamic assay is needed to support clinical studies. Phosphorylation of ATR targets, Chk1 and H2AX, was evaluated in MCF7 and K562 cells, human volunteer PBMCs and whole blood by Western blot, immunofluorescence microscopy and flow cytometry after DNA damage. The effect of cell cycle phase, ATR knockdown and inhibition on these phosphorylation events was determined. Hydroxyurea, UV and 4NQO induced Chk1 and H2AX phosphorylation in MCF7 and K562 cells. UV/4NQO activation of ATR was detectable in non-cycling cells. Chk1 phosphorylation was reduced by ATR knockdown and reflects ATR activity for 3 h, H2AX phosphorylation after UV/4NQO is ATR-dependent for 1 h but increasingly ATM and DNA-PK-dependent at later time points. In isolated PBMCs both phospho-targets were detectable after UV/4NQO but in PBMCs from whole blood treated with 4NQO only H2AX was detectable. PhosphoChk1 and H2AX are useful biomarkers for ATR inhibition using a variety of immuno-detection methods, but timing may be critical. Importantly, ATR activity is detectable in non-cycling PBMCs allowing them to be used as a surrogate tissue for biomarker measurement. In PBMCs from whole blood treated with 4NQO phosphoH2AX was the most useful biomarker of ATR activity and a clinically viable pharmacodynamic assay for ATR inhibitors has been developed. Copyright © 2014. Published by Elsevier B.V.
    Molecular Oncology 10/2014; 9(2). DOI:10.1016/j.molonc.2014.09.012 · 5.33 Impact Factor
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    • "NVP-BEZ235, PI-103 and LY2940002 are known (with nanomolar IC50 values) to inhibit DNA-activated protein kinase (a PI3K-related kinase family member) [45]. NVP-BEZ235, but not PI-103, inhibits ATM (ataxia telangiectasia, mutated) and ATR (ATM and Rad3-related) protein kinases [46]. PI-103, but not NVP-BEZ235, inhibits mTORC2 [47]. "
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    ABSTRACT: Ocular neovascularization is a common pathology associated with human eye diseases e.g. age-related macular degeneration and proliferative diabetic retinopathy. Blindness represents one of the most feared disabilities and remains a major burden to health-care systems. Current approaches to treat ocular neovascularisation include laser photocoagulation, photodynamic therapy and anti-VEGF therapies: Ranibizumab (Lucentis) and Aflibercept (Eylea). However, high clinical costs, frequent intraocular injections, and increased risk of infections are challenges related with these standards of care. Thus, there is a clinical need to develop more effective drugs that overcome these challenges. Here, we focus on an alternative approach by quantifying the in vivo anti-angiogenic efficacy of combinations of phosphatidylinositol-3-kinase (PI3K) pathway inhibitors. The PI3K/AKT/mTOR pathway is a complex signalling pathway involved in crucial cellular functions such as cell proliferation, migration and angiogenesis. RT-PCR confirms the expression of PI3K target genes (pik3ca, pik3r1, mtor and akt1) in zebrafish trunks from 6 hours post fertilisation (hpf) and in eyes from 2 days post fertilisation (dpf). Using both the zebrafish intersegmental vessel and hyaloid vessel assays to measure the in vivo anti-angiogenic efficacy of PI3K/Akt/mTOR pathway inhibitors, we identified 5 µM combinations of i) NVP-BEZ235 (dual PI3K-mTOR inhibitor) + PI-103 (dual PI3K-mTOR inhibitor); or ii) LY-294002 (pan-PI3K inhibitor) + NVP-BEZ235; or iii) NVP-BEZ235 + rapamycin (mTOR inhibitor); or iv) LY-294002 + rapamycin as the most anti-angiogenic. Treatment of developing larvae from 2-5 dpf with 5 µM NVP-BEZ235 plus PI-103 resulted in an essentially intact ocular morphology and visual behaviour, whereas other combinations severely disrupted the developing retinal morphology and visual function. In human ARPE19 retinal pigment epithelium cells, however, no significant difference in cell number was observed following treatment with the inhibitor combinations. Collectively, these results highlight the potential of combinations of PI3K/AKT/mTOR pathway inhibitors to safely and effectively treat ocular neovascularization.
    PLoS ONE 08/2014; 9(8):e105280. DOI:10.1371/journal.pone.0105280 · 3.23 Impact Factor
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