Abstract 2040: The Mre11 nuclease is critical for sensitivity of cells to Chk1 inhibition

Abstract

Abstract The Chk1 kinase is required for the arrest of cell cycle progression when DNA is damaged, thereby providing time for cells to repair their DNA and recover. Chk1 also stabilizes stalled replication forks. As a consequence, many Chk1 inhibitors have been developed and tested for their potential to enhance DNA damage-induced tumor cell killing. However, inhibition of Chk1 alone, without any additional exogenous agent, can be cytotoxic. Using the novel Chk1 inhibitor MK-8776 (previously known as SCH900776), we screened a panel of cell lines and found that some are killed rapidly at low concentrations while other cell lines continue to grow in the presence of much higher concentrations. Understanding the underlying mechanisms of this sensitivity is critical for defining which patients might respond best to therapy with Chk1 inhibitors. We have investigated the mechanism of sensitivity in U2OS osteosarcoma cells. Upon incubation with MK-8776, single-stranded DNA regions (ssDNA) and double-strand breaks (DSB) begin to appear within 6 h. These DSB have been attributed to the structure-specific DNA endonuclease, Mus81. The Mre11/Rad50/Nbs1 (MRN) complex is known to be responsible for the resection of DSB to ssDNA. However, we show that inhibition of the Mre11 nuclease activity leads to a decrease not only in the amount of ssDNA following Chk1 inhibition, but also in the formation of DSB, suggesting that DSB arise as a consequence of ssDNA formation. These findings were corroborated by the discovery that Mre11-deficient ATLD1 cells are highly resistant to MK-8776 and form neither ssDNA nor DSB following treatment, but once complimented with exogenous Mre11, the cells accumulate both ssDNA and DSB when incubated with MK-8776. Inhibition of Chk1 also leads to aberrant activation of Cdc25A and Cdk2, and our findings suggest that Mre11 provides the link between Cdk2 activation and Mus81. We propose that the variable sensitivity of cell lines to Chk1 inhibition reflects defects in this pathway. These data highlight a novel role for Mre11 in the production of DSB and may help define which tumors are more sensitive to MK-8776 alone or in combination with DNA damaging agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2040. doi:1538-7445.AM2012-2040