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Supplementary Materials and Methods. Supplementary Figure S1: Cell viability in various concentrations of DNA-damaging agents and novel ATR pathway inhibitors. Supplementary Figure S2: Effects of novel and established ATR pathway inhibitors on Chk1 phosphorylation induced by hydroxyurea or cisplatin as assessed by flow cytometry.

Resistance to DNA-damaging chemotherapy is a barrier to effective treatment that appears to be augmented by p53 functional deficiency in many cancers. In p53-deficient cells in which the G1–S checkpoint is compromised, cell viability after DNA damage relies upon intact intra-S and G2–M checkpoints mediated by the ATR (ataxia telangiectasia and Rad3 related) and Chk1 kinases. Thus, a logical rationale to sensitize p53-deficient cancers to DNA-damaging chemotherapy is through the use of ATP-competitive inhibitors of ATR or Chk1. To discover small molecules that may act on uncharacterized components of the ATR pathway, we performed a phenotype-based screen of 9,195 compounds for their ability to inhibit hydroxyurea-induced phosphorylation of Ser345 on Chk1, known to be a critical ATR substrate. This effort led to the identification of four small-molecule compounds, three of which were derived from known bioactive library (anthothecol, dihydrocelastryl, and erysolin) and one of which was a novel synthetic compound termed MARPIN. These compounds all inhibited ATR-selective phosphorylation and sensitized p53-deficient cancer cells to DNA-damaging agents in vitro and in vivo. Notably, these compounds did not inhibit ATR catalytic activity in vitro, unlike typical ATP-competitive inhibitors, but acted in a mechanistically distinct manner to disable ATR–Chk1 function. Our results highlight a set of novel molecular probes to further elucidate druggable mechanisms to improve cancer therapeutic responses produced by DNA-damaging drugs. Cancer Res; 74(24); 7534–45. ©2014 AACR.

An important goal in cancer therapy has been to selectively sensitize cancers to existing drugs that typically work by inducing DNA damage. In particular, many cancers are p53-defective and chemoresistant, thus novel approaches to target p53-defective cancers are needed. One promising strategy to sensitize p53-deficient cells has been to inhibit DNA damage response kinases such as ATM/ATR via ATP-competitive kinase inhibitors. To better understand ATM/ATR activation mechanisms and discover novel probes for the ATM/ATR pathway, we performed a phenotype-based screen of 9,195 small-molecule compounds for inhibitors of hydroxyurea-induced phosphorylation of Chk1, a key ATR substrate. After subsequent biological screens, we selected 4 compounds that inhibited ATR and ATM pathways; 3 were known bioactive agents and 1 was a diversity-oriented-synthetic product. These compounds sensitized p53-deficient cells to diverse DNA-damaging agents. Xenograft experiments were performed on one compound, and it showed synergistic suppression of p53-deficient tumor growth with cisplatin. Importantly, these compounds did not suppress ATR kinase catalytic activity in vitro, unlike typical ATM/ATR kinase inhibitors that are ATP-competitive. To identify molecular targets of one compound, “MARPIN” (ATM and ATR pathway inhibitor), we defined its active site through structure-activity relationship analysis, resulting in synthesis of inactive derivatives of MARPIN. Identification of proteins that specifically bind MARPIN, but not its inactive derivatives, is underway. This phenotype-based chemical genetic screen identified novel ATM/ATR pathway inhibitors that are mechanistically distinct from kinase catalytic inhibitors, and these compounds could serve as probes to identify previously unrecognized druggable targets in DNA damage response pathways. Citation Format: Masaoki Kawasumi, James E. Bradner, Nicola Tolliday, Renee Thibodeau, Heather Sloan, Kay M. Brummond, Paul Nghiem. A chemical genetic screen identifies novel ATM/ATR pathway inhibitors that sensitize p53-deficient cells to DNA-damaging agents without affecting ATR kinase catalytic activity. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B04.