Abstract
Chk1 kinase is a critical component of the DNA damage response checkpoint and Chk1 inhibitors are currently under clinical investigation. Chk1 suppresses oncogene-induced replication stress with Chk1 inhibitors demonstrating activity as a monotherapy in numerous cancer types. Understanding the mechanism by which Chk1 inhibitors induce DNA damage and cancer cell death is essential for their future clinical development. Here we characterize the mechanism by which the novel Chk1 inhibitor (V158411) increased DNA damage and cell death in models of human cancer. V158411 induced a time- and concentration-dependent increase in γH2AX-positive nuclei that was restricted to cells actively undergoing DNA synthesis. γH2AX induction was an early event and correlated with activation of the ATR/ATM/DNA-PKcs DNA damage response pathways. The appearance of γH2AX positive nuclei preceded ssDNA appearance and RPA exhaustion. Complete and sustained inhibition of Chk1 kinase was necessary to activate a robust γH2AX induction and growth inhibition. Chk1 inhibitor cytotoxicity correlated with induction of DNA damage with cells undergoing apoptosis, mitotic slippage and DNA damage-induced permanent cell cycle arrest. We identified two distinct classes of Chk1 inhibitors: those that induced a strong increase in γH2AX, pChk1 (S317) and pRPA32 (S4/S8) (including V158411, LY2603618 and ARRY-1A) and those that did not (including MK-8776 and GNE-900). Tumor cell death, induced through increased DNA damage, coupled with abrogation of cell cycle checkpoints makes selective inhibitors of Chk1 a potentially useful therapeutic treatment for multiple human cancers.
Highlights
The human genome experiences environmentally and endogenously generated DNA damage on a daily basis
Chk1 inhibition by V158411, in HT29 cells, increased the fraction of cells staining positive for pannuclear γH2AX, a marker of DNA double strand breaks (DSBs), rather than γH2AX foci as was observed with the DNA damaging agents gemcitabine and camptothecin (Figure 1A)
There is a growing realization, that Chk1 inhibitors may possess anti-tumor efficacy when administered as a single agent with pre-clinical anti-tumor activity observed in a range of cancer models [25, 28, 30, 34, 35]
Summary
The human genome experiences environmentally and endogenously generated DNA damage on a daily basis. Oncogene activation and/or the loss of tumor suppressor proteins drive tumor cell proliferation resulting in increased replication stress. In pre-cancerous lesions, increased replication stress is an important contributor to genome instability resulting in the increased acquisition of genomic changes and cancer development [1,2,3]. The uncontrolled replication burst that occurs in tumor cells following dysregulation of cell cycle controls results in replication fork stalling and DNA double-strand breaks (DSBs) arising from fork collapse [4,5,6]. Sustained activation of the DDR in precancerous lesions leads to apoptosis and/or senescence preventing cancer development [7,8,9]. The acquisition of additional mutations allows cells to evade these checkpoints resulting in transformation and carcinogenesis
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