Compensation, crosstalk and sequestering: the currency of checkpoints in cancer.

Cells are constantly exposed to both endogenous and exogenous stresses that lead directly or indirectly to DNA damage. Cell survival is dependent on two interrelated processes, cell cycle checkpoints and DNA repair; the G1, intra-S and G2 checkpoints block cell cycle progression after DNA damage to provide more time for DNA repair. Cell cycle checkpoints and DNA repair are integrated through a series of signaling networks that involve numerous protein posttranslational modifications (e.g., phosphorylation, acetylation, methylation, ubiquitination) that modulate protein stability, catalytic activity and other properties. Most cancer cells have one or more defects in cell cycle checkpoints and often defects in some aspect of DNA repair. As a consequence, they become addicted to the remaining checkpoint and repair mechanisms. Tumor therapy often involves treatments that cause DNA strand breaks or replication stress. The G1, G2 and intra-S checkpoints are activated in response to DNA strand breaks primarily through the ATM-CHK2 pathway and to DNA replication stresses through the ATR-CHK1 pathway. Thus, disrupting these pathways potentially can enhance the therapeutic effectiveness of cancer treatments. In a recent issue of Cell Cycle, Palii et al. report1 on the interactions between CHK1, PP2A and ATM during the G2 checkpoint response to IR-induced DNA damage (Fig. 1). This analysis was motivated by studies showing that chemical inhibition of CHK1 can enhance chemotherapy for cancer (e.g., see ref. 2), and that chemical inhibition of CHK1 kinase activity does not fully mimic depletion of CHK1 protein. Palii et al. compared and contrasted the effects of CHK1 inhibition in human cell lines by stable depletion with shRNA, transient depletion with siRNA, and chemical inhibition. They found that stable depletion of CHK1 in 293T cells enhanced cellular radiosensitivity in colony formation assays by about 4-fold. As mutation of ATM in ataxia telangiectasia enhances radiosensitivity about 3-fold, this sensitization by CHK1 depletion is remarkable. Depletion or inhibition of CHK1 enhanced phosphorylation at S1981 of ATM after IR, but ATM-dependent G2 checkpoint function was modestly attenuated. This phenomenology was linked to PP2A, a phosphatase that acts on ATM and CHK1 and which is regulated by CHK1.3,4 Depletion or inhibition of CHK1 reduced (sequestered) nuclear PP2A protein levels and enhanced an inhibitory phosphorylation of PP2A on Tyr307. The consequence of inhibition of PP2A was increased basal and IR-induced phosphorylations on the activating S1981 of ATM as well as on S345 and S296 of the remaining CHK1. Cellular growth assays using human mammary epithelial cells with depletion of CHK1 and/or ATM showed that combined depletion of CHK1 and ATM enhanced radiosensitivity, supporting the rationale to use CHK1 inhibitors to increase cancer cure rates using adjuvant radio- and chemotherapies.2,5 Figure 1. Pathways that can contribute to cancer cell survival. Replication stress activates the ATR pathway leading to activation of CHK1 through phosphorylation of Ser345 and Ser317; Ser296 is autophosphorylated and serves as a marker of activated ... The work by Palii and colleagues highlights the cross-talk between different elements of the DNA damage response that can produce compensatory conditions that mitigate cell killing and compromise therapeutic efficacy. While crosstalk between the ATM and ATR pathways has been known for some time, recent proteome-wide analyses of the kinase landscape in the DNA damage response implicate the involvement of dozens of kinases and highlight how extensive and interlaced the pathways in the DNA damage response are (reviewed in ref. 6). Although less is known about the roles of phosphatases, several in addition to the many forms of PP2A, including PP1, PP5 and WIP1 (PP2C) are clearly involved in the DNA damage response. We note, however, that inhibition of PP2A with okadaic acid prior to IR treatment blocked activation of the G2 checkpoint as well as activation of ATR, CHK1 and CHK2,7 indicating that the relationship between ATR, CHK1 and G2 arrest is more complex than shown in Figure 1. Other posttranslational modifications (e.g., acetylation, ubiquitination) also play important, if less well-studied, roles as well. Furthermore, each of the major DNA damage response sensor, transducer and effector enzymes serve cellular functions outside the DNA damage response that also must be understood. Thus, a deeper understanding of the pathways and mechanisms by which the DNA damage checkpoint and repair systems are regulated clearly will be important to developing more effective cancer therapeutics and therapies. The Palii et al. study complements several recent studies which are attempting to dissect these cell survival mechanisms, determine how they can be modulated to selectively effect death in tumor cells and how tumor cells compensate for the loss of these survival mechanisms. For example, a recent report in Cell Cycle by McNeedy et al.8 described the effect of CHK1 inhibition on ATM and DNA-PK activity. As oncologists move to targeted anti-cancer therapies, drug-combination cocktails will be needed to squelch compensatory events that reduce efficacy.