MK-8776, a novel Chk1 inhibitor, exhibits an improved radiosensitizing effect compared to UCN-01 by exacerbating radiation-induced aberrant mitosis

Abstract

Checkpoint kinase 1 (Chk1) is an evolutionarily conserved serine/threonine kinase that plays an important role in G2/M checkpoint signaling. Here, we evaluate the radiosensitizing effects of a novel selective Chk1 inhibitor MK-8776, comparing its efficacy with a first-generation Chk1 inhibitor UCN-01, and attempt to elucidate the mechanism of radiosensitization. In a clonogenic survival assay, MK-8776 demonstrated a more pronounced radiosensitizing effect than UCN-01, with lower cytotoxicity. Importantly, radiosensitization by MK-8776 can be achieved at doses as low as 2.5 Gy, which is a clinically applicable irradiation dose. MK-8776, but not UCN-01, exacerbated mitotic catastrophe (MC) and centrosome abnormalities, without affecting repair kinetics of DNA double strand breaks. Furthermore, live-cell imaging revealed that MK-8776 significantly abrogated the radiation-induced G2/M checkpoint, prolonged the mitotic phase, and enhanced aberrant mitosis. This suggests that Chk1 inhibition by MK-8776 activates a spindle assembly checkpoint and increases mitotic defects in irradiated EMT6 cells. In conclusion, we have shown that, at minimally toxic concentrations, MK-8776 enhances radiation-induced cell death through the enhancement of aberrant mitosis and MC, without affecting DNA damage repair.

Bromodomain and extraterminal domain inhibition synergizes with WEE1‐inhibitor AZD1775 effect by impairing nonhomologous end joining and enhancing DNA damage in nonsmall cell lung cancer

“…Cells that displayed signs of aberrant nuclei, such as micronuclei, multilobular nuclei or fragment nuclei, were regarded as cells undergoing mitotic catastrophe as previously reported (Fig. a ) . The combined JQ1 and AZD1775 treatment considerably increased mitotic catastrophe relative to that after each single treatment (Fig.…”

“…5a). 28 The combined JQ1 and AZD1775 treatment considerably increased mitotic catastrophe relative to that after each single treatment (Fig. 5b).…”

“…Analysis of mitotic catastrophe was performed as previously reported . For immunofluorescence staining, cells were treated with specified compounds for 24 hr.…”

“…Analysis of mitotic catastrophe was performed as previously reported. 27,28 For immunofluorescence staining, cells were treated with specified compounds for 24 hr. Cells were fixed using 4% paraformaldehyde for 20 min at 4 C and permeabilized with PBS containing 0.5% Triton X-100 for 10 min at 4 C. Cells were incubated with Blocking One Histo (Nacalai Tesque, Kyoto, Japan) for 5 min at room temperature to block nonspecific antibody-binding sites.…”

Exploiting DNA Replication Stress for Cancer Treatment

“…Accordingly, ATR inhibition initiates widespread DNA synthesis from normally dormant replication origins, generating enough ssDNA to exhaust the cellular pools of RPA and depleting the nucleotide pools necessary for DNA synthesis (9,11). ATR and CHK1 inhibition also promotes premature entry into mitosis despite the presence of under-replicated DNA, leading to chromosome fragmentation (54,55). Given their mechanisms of action, ATR and CHK1 inhibitors synergize with compounds that induce replication stress, and increase the toxicity of nucleoside analogues in ovarian, lung, and pancreatic cancer (56-60), platinum-based agents in lung, gastric, ovarian, bladder, and breast cancer (50,61,62) and topoisomerase inhibitors in breast, colon, and brain tumors (63,64).…”