Chk 2 Molecular InteractionMap and Rationale for Chk 2 Inhibitors

To organize the rapidly accumulating information on bioregulatory networks related to the histone g-H2AX-ATM-Chk2-p53-Mdm2 pathways in concise and unambiguous diagrams, we used the molecular interaction map notation (http://discover.nci.nih.gov/min).Molecular interaction maps are particularly useful for networks that include protein-protein binding and posttranslational modifications (e.g., phosphorylation). Both are important for nearly all of the proteins involved in DNA double-strand break signaling. Visualizing the regulatory circuits underlying cellular signaling may help identify key regulatory reactions and defects that can serve as targets for anticancer drugs. DNA double-strand breaks (DSB) are among the most severe genomic lesions. Their repair requires cells to arrest cell cycle progression to avoid further damage during replication or transcription (1–5). Cell cycle arrest may also allow the chromatin to switch from a metabolic (replicative) state to a repair state. DSB can be produced directly by ionizing radiation and certain anticancer drugs that bind to DNA (e.g., bleomycin, neocarcinostatin, topoisomerase II inhibitors, such as etoposide or doxorubicin). DSB can also result from conversion of singlestrand breaks by DNA polymerase collisions or ‘‘replication fork collapse’’ at sites of damaged DNA. Replication DSB occur normally in cancer cells, but their frequency is markedly enhanced by topoisomerase I inhibitors such as the camptothecin derivatives (e.g., topotecan and irinotecan; ref. 6) and DNA alkylating agents. The importance of Chk2, a protein involved in cell cycle arrest due to DSB, is indicated by its conservation in eukaryotes (7–10). However, yeast and vertebrate cells can survive without Chk2, although they are defective in cell cycle checkpoints and apoptosis after irradiation. Chk2 activation by camptothecin treatment has been reported to be defective in several colon carcinoma cell lines from the NCI 60 cell line screen (11). Chk2 inactivation in humans with normal p53 leads to Li-Fraumeni syndrome (12), supporting a genetic connection between Chk2 and p53 and the existence of a Chk2-p53 axis [see the molecular interaction map (MIM) Fig. 1]. Families with Chk2 deficiencies have been identified among patients with increased risk of breast cancer (10, 13, 14). Chk2 functions as a kinase relay for ATM (refs. 9, 15, 16; see MIM), whose gene (ataxia telangiectasia mutant) is mutated in ataxia telangiectasia patients (2). It is generally accepted that DNA damage triggers two main pathways (16). DSB activate the ATM-Chk2 axis, whereas replication lesions activate the ataxia telangiectasia and rad3-related kinase (ATR)–Chk1 axis. However, there is crosstalk between the two pathways and certain lesions activate both. In such cases, the ATM-Chk2 axis is activated first and transiently, whereas the ATR-Chk1 axis is activated secondarily and in a more sustained way (17). The association of Chk1 with replication damage is consistent with the cell cycle–dependent expression of Chk1, which increases during S-phase and peaks in G2. In contrast, Chk2 is expressed throughout the cell cycle, consistent with its broader role in response to DSB produced both during S-phase and independently of replication (18). Although Chk2 shares no sequence homology with Chk1, crosstalk between the Chk2 and Chk1 pathways is also indicated by the observation that several Chk2 substrates (e.g., Cdc25C and p53, highlighted in blue in Fig. 1) are also Chk1 substrates and are phosphorylated by both kinases on the same amino acid residues (9).