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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

ABSTRACT 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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    • "f the DNA damage response ( DDR ) ( Rieder and Maiato , 2004 ; Brown and Baltimore , 2000 ; Lam et al . , 2004 ; Lö ffler et al . , 2006 ; Zachos and Gillespie , 2007 ) . A defective DDR would compromise the ability of the cell to repair DNA damage , thereby leading to an enhancement in the levels of DNA strand breaks ( Syljuå sen et al . , 2005 ; Toledo et al . , 2011b ) . Evaluation of DNA strand breaks by monitoring gH2AX staining revealed a signifi - cant accumulation of gH2AX - positive cells upon knockdown of WT - H - Ras in the DLD1 - K - Ras Mut cells and a panel of K - Ras mutant ( Mut ) pancreatic cancer cells ( Panc - 1 , AsPC - 1 , PL45 , and MIA PaCa - 2 ) ( Figures 2A – 2C ) . In contrast"
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    • "Comparable numbers of cells treated with either the ATM inhibitor KU55933 or DMSO control displayed visible HA-LSD1 stripes overlapping with the pH2AX signal upon DNA damage (Fig. 3, a and c). There was also no change in LSD1 recruitment using an ATR inhibitor (Toledo et al., 2011; Fig. S1 g). At the concentrations used, both inhibitors were indeed functional (Fig. S2 a). "
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    • "These observations suggest that the transient association of FANCD2 with MCM proteins is a general response to replication stress signaling induced by oncogenic transformation or by replication-blocking agents. To test whether ATR-Chk1 signaling was necessary for FANCD2 to target MCM proteins, we used ETP-46464, a specific inhibitor of ATR (Toledo et al., 2011). ATR inhibition partially reduced the phosphorylation of MCM2 on Ser108 and completely abolished the phosphorylation of Chk1 on Ser345 (Figure 3C, upper panel). "
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