Abstract
Checkpoint kinase 1 (CHK1) is a central mediator of the DNA damage response (DDR) at the S and G2/M cell cycle checkpoints, and plays a crucial role in preserving genomic integrity. CHK1 overexpression is thought to contribute to cancer aggressiveness, and several selective inhibitors of this kinase are in clinical development for various cancers, including neuroblastoma (NB). Here, we examined the sensitivity of MYCN-amplified NB cell lines to the CHK1 inhibitor PF-477736 and explored mechanisms to increase its efficacy. PF-477736 treatment of two sensitive NB cell lines, SMS-SAN and CHP134, increased the expression of two pro-apoptotic proteins, BAX and PUMA, providing a mechanism for the effect of the CHK1 inhibitor. In contrast, in NB-39-nu and SK-N-BE cell lines, PF-477736 induced DNA double-strand breaks and activated the ataxia telangiectasia mutated serine/threonine kinase (ATM)-p53-p21 axis of the DDR pathway, which rendered the cells relatively insensitive to the antiproliferative effects of the CHK1 inhibitor. Interestingly, combined treatment with PF-477736 and the ATM inhibitor Ku55933 overcame the insensitivity of NB-39-nu and SK-N-BE cells to CHK1 inhibition and induced mitotic cell death. Similarly, co-treatment with PF-477736 and NU7441, a pharmacological inhibitor of DNA-PK, which is also essential for the DDR pathway, rendered the cells sensitive to CHK1 inhibition. Taken together, our results suggest that synthetic lethality between inhibitors of CHK1 and the DDR drives G2/M checkpoint abrogation and could be a novel potential therapeutic strategy for NB.
Highlights
Transformed and untransformed cells respond to threats to genomic integrity, such as double-strand breaks (DSBs), by activating the DNA damage response (DDR), which enables DNA repair at the G1/S, S, and/or G2/M cell cycle checkpoints, thereby allowing progression through the cell cycle
To investigate the sensitivity of human NB cell lines to checkpoint kinase 1 (CHK1) inhibition, we examined the effects of the CHK1i PF-00477736 on the proliferation of four MYCN-amplified NB cell lines: NB-39-nu, SMS-SAN, CHP134, and SK-N-BE [19,20,21,22]
PF-00477736 was originally identified as a potent, selective ATP-competitive small-molecule inhibitor of CHK1 (Ki = 0.49 nM) that potentiates the cytotoxic effect of conventional chemotherapeutic agents in vitro and in vivo [23,24]
Summary
Transformed and untransformed cells respond to threats to genomic integrity, such as double-strand breaks (DSBs), by activating the DNA damage response (DDR), which enables DNA repair at the G1/S, S, and/or G2/M cell cycle checkpoints, thereby allowing progression through the cell cycle. DSBs activate the ATM-CHK2 cascade, whereas single-strand breaks are recognized and repaired by the ATR-CHK1 cascade [1]. CHK1 and CHK2 are serine/threonine kinases that regulate the activity of their target proteins, including p53 and CDC25A–C phosphatases, via phosphorylation. The CDC25 proteins regulate the activity of cyclins and the cyclin-dependent kinases (CDK). CHK1 is principally responsible for initiating repair during S-phase replication (the replication checkpoint) and G2/M (the mitotic entry checkpoint) via phosphorylation of the CDC25 family members and the serine/threonine kinase Wee, whereas CHK2 coordinates repair for S-phase entry (G1 checkpoint) via phosphorylation of p53, which transactivates the CDK inhibitor CDKN1A ( known as p21) [4]
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