Inhibition of Chk1 with the small molecule inhibitor V158411 induces DNA damage and cell death in an unperturbed S-phase

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.

Chk1 loss creates replication barriers that compromise cell survival independently of excess origin firing

“…It is becoming increasingly clear that sensitivity to Chk1 inhibitors is a consequence of DNA damage in S phase (Koh et al, 2015;Parsels et al, 2016;Sakurikar et al, 2016;Sanjiv et al, 2016;Techer et al, 2016;Wayne et al, 2016). Our work complements these findings, by defining excess origin firing as the predominant mechanism that causes such DNA damage.…”

Endogenous Replication Stress Marks Melanomas Sensitive to CHEK1 Inhibitors In Vivo

“…It is currently thought that CHEK1 inhibition results in S-phase DNA damage, and also overcomes the cell-cycle arrest triggered by the this damage to promote mitotic catastrophe, which is the driver of cell death with CHEK1i treatment (10,11,14,25,26,38,39). In contrast, we found that melanoma cells hypersensitive to GNE-323 died in S-phase, whereas it was the less sensitive cell lines that required transit through mitosis for efficient killing.…”

“…Here we have investigated the hypersensitivity of a panel of melanoma cell lines to the selective and potent CHEK1i GNE-323 and GDC-0575. Comparison of a panel of CHEK1i demonstrated that GDC-0575 was significantly more potent in promoting DNA damage, replication stress and cell death than V158411, LY2603618, and MK-8776 (26). We have previously demonstrated that the selectivity of GNE-323 is based on its inhibition of CHEK1 rather than any significant off-target effect (7).…”

“…Several studies have demonstrated that CHEK1i drives cells into an aberrant mitosis, and this is thought to trigger cell death (10,14,25,26). We have previously reported that CHEK1isensitive melanomas accumulate with S-phase DNA content (7), which was a common feature of the hypersensitive cell lines.…”

The Inhibition of DNA Damage Response Contributes to Cancer Radiotherapy

“…The DDR pathway, a cooperation of complex DNA repair and cell cycle control pathways, has evolved to help cells manage DNA damage burden [8] . IR can produce DNA double-strand breaks (DSBs), which are considered to be lethal DNA lesions within the cells.…”