Targeting Chk1 in the replicative stress response

In response to DNA damage or interference with DNA synthesis, transformed cells characteristically activate various checkpoints, which together comprise an important component of a complex signaling network that has been termed the DNA damage response (DDR). Checkpoints identified so far are classified in two ways: (1) cell cycle checkpoints: the G₁/S, intra-S or S, and G₂/M checkpoint; or (2) function-specific checkpoints: the DNA damage checkpoint, the replication checkpoint, the mitotic spindle checkpoint, the cytokinesis checkpoint, etc.¹ The observation that transformed cells are characterized by defects in the checkpoint apparatus² has prompted intense efforts to exploit this differential response (relative to normal cells) therapeutically.

Many of these efforts have focused upon the checkpoint kinase Chk1, which, along with Chk2, constitute “distal transducers” within the checkpoint signal transduction pathway. In response to DNA damage [specifically single strand DNA (ssDNA) lesions], Chk1 is activated by the “proximal transducers” ATR and to a certain extent ATM, large PI3 kinase-like proteins which are in turn activated by DNA damage “sensor” proteins in cooperation with “mediator” proteins.¹ Once activated by phosphorylation on Ser317 and Ser345, Chk1 phosphorylates and targets for degradation or cytoplasmic sequestration members of the Cdc25 phosphatase family (e.g., A, B and C), leading to inhibitory phosphorylations of cyclin-dependent kinases (CDKs), most notably cdk1 (p34^cdc2) and cdk2. Inhibition of such CDKs is critical for cell cycle arrest in the face of DNA damage or disruption of the DNA replicative machinery. Conversely, inhibition of Chk1 disables this checkpoint mechanism, allowing cells that have sustained DNA damage to continue their cell cycle traverse inappropriately. This leads to cell death, although the mechanisms by which this event occurs have not been fully elucidated. Notably, recent studies suggest that in addition to its critical role in checkpoint control, Chk1 serves multiple other functions, including direct involvement in cell survival and DNA repair, among others.³

In general, attempts to exploit disruption of Chk1 function have focused on two distinct strategies: potentiation of the lethality of agents that (1) induce DNA damage (e.g., topoisomerase inhibitors⁴ or (2) interfere with DNA replication (e.g., nucleoside analogs).⁵ Attempts to translate such strategies into the clinic have been limited by the toxicities and lack of specificity of available Chk1 inhibitors (e.g., UCN-01).⁶ However, a new generation of more selective Chk1 inhibitors has recently emerged, raising the possibility that newer combination regimens will display enhanced efficacy.

Although the concept of enhancing nucleo-side analog activity by Chk1 inhibition is not entirely new,⁷ the mechanisms underlying such interactions have not yet been fully defined. However, the recent discovery that Chk1 plays a central role in the DNA replication checkpoint induced by replication stress absent exogenous insults⁸ has focused attention on this therapeutic strategy. In the study by McNeely et al., the authors investigated factors contributing to synergism between AZD7762, a new selective and clinically relevant Chk1 inhibitor and the nucleoside analog gemcitabine in various epithelial malignancies. The major finding was that enhanced lethality for the combination very likely involved multiple mechanisms, including reversal of inhibition of replication origin firing and alterations replication fork dynamics, accompanied by DNA damage. In essence, AZD7762 converted gemcitabine-related stalled replication forks into double-strand breaks (DSBs). Significantly, cells with defects in the DNA repair machinery were particularly sensitive to this strategy. Collectively, these findings provide a theoretical foundation for rational attempts to enhance the activity of clinically useful nucleoside analogs by novel and selective Chk1 inhibitors.

The findings described in this study have potentially important implications for the development of second-generation Chk1 inhibitors such as AZD7762, particularly in combination with nucleoside analogs such as gemcitabine which are active against slowly proliferating epithelial tumors. For example, the observation that multiple mechanisms contribute to the lethality of such regimens could explain why a single pharmacodynamic determinant may not predict for activity. Conversely, distal events such as DNA damage induction, reflected by gH2A.X formation, may represent a final endpoint indicative of tumor cell responsiveness. A particularly interesting finding was that cells exhibiting defects in the DNA repair process may be uniquely susceptible to this strategy, analogous to the sensitivity of BRCA1 mutant cells to PARP⁹ and Chk1 inhibitors.¹⁰ Such cells may also display “synthetic lethality” when exposed to regimens combining Chk1 inhibitors with agents that induce DNA damage and disrupt DNA replication. Finally, it will be important to determine if the theoretical promise of this strategy can be realized with newer generation and more selective Chk1 inhibitors that have now entered the clinic.

McNeely S, Conti C, Sheikh T, Patel H, Zabludoff S, Pommier Y, et al. Chk1 inhibition after replicative stress activates a double strand break response mediated by ATM and DNA-dependent protein kinase. Cell Cycle. 2010;9:995–1004. doi: 10.4161/cc.9.5.10935.