Endogenous Replication Stress Marks Melanomas Sensitive to CHEK1 Inhibitors In Vivo

Abstract

Checkpoint kinase 1 inhibitors (CHEK1i) have single-agent activity and Here, we have investigated the molecular basis of this activity. We have assessed a panel of melanoma cell lines for their sensitivity to the CHEK1i GNE-323 and GDC-0575 and The effects of these compounds on responses to DNA replication stress were analyzed in the hypersensitive cell lines. A subset of melanoma cell lines is hypersensitive to CHEK1i-induced cell death , and the drug effectively inhibits tumor growth In the hypersensitive cell lines, GNE-323 triggers cell death without cells entering mitosis. CHEK1i treatment triggers strong RPA2 hyperphosphorylation and increased DNA damage in only hypersensitive cells. The increased replication stress was associated with a defective S-phase cell-cycle checkpoint. The number and intensity of pRPA2 Ser4/8 foci in untreated tumors appeared to be a marker of elevated replication stress correlated with sensitivity to CHEK1i. CHEK1i have single-agent activity in a subset of melanomas with elevated endogenous replication stress. CHEK1i treatment strongly increased this replication stress and DNA damage, and this correlated with increased cell death. The level of endogenous replication is marked by the pRPA2Ser4/8 foci in the untreated tumors, and may be a useful marker of replication stress .

Combined use of subclinical hydroxyurea and CHK1 inhibitor effectively controls melanoma and lung cancer progression, with reduced normal tissue toxicity compared to gemcitabine

“…Cells were labelled with 10 lM ethynyl-2 0 -deoxyuridine (EdU) in complete media for 2 h and then harvested and the cells fixed with 4% formaldehyde. EdU was labelled using CLICK chemistry with Alex-a488-azide and DNA with 4 0 ,6-diamidino-2-phenylindole (DAPI), and then, the percentage of EdU-positive cells was quantified using high-content imaging as described in Oo et al (2018).…”

“…The combination with gemcitabine has been trialled in patients with limited success, with dose-limiting toxicities being a major shortcoming of this combination (Daud et al, 2015;Infante et al, 2017). Here, we have investigated the effects of subclinical concentrations of gemcitabine and HU in NSCLC and melanoma cell lines and TS cultures, and in normal skin fibroblasts to compare the effects of these RNR inhibitors on synergy with the CHK1i GDC-0575 (Oo et al, 2018).…”

“…S3. The sensitivity of indicated melanoma lines to GNE-323, a closely related CHEK1 inhibitor (Oo et al, 2018) or the ATR inhibitor VE-821, with or without 0.2 mM HU for 72 h. Fig. S4.…”

“…The relatively small proportion of tumours with high levels of endogenous replication stress can be targeted by CHK1 inhibitors (CHK1i) as a single agent to selectively destroy these tumours (Brooks et al, 2013;Oo et al, 2018;Sakurikar et al, 2016). CHK1i also synergise potently with replication stress inducers in vitro (Xiao et al, 2013).…”

Novel CHK1 inhibitor MU380 exhibits significant single-agent activity in TP53-mutated chronic lymphocytic leukemia cells

“…for combination with chemotherapy. Nevertheless, some of them showed interesting pre-clinical single-agent activity against diverse cancer types, including breast and ovarian cancer, 14 smallcell lung cancer, 15 colorectal cancer, 16 neuroblastoma, 17 melanoma, 18 MYC driven lymphoma, 19 and leukemia. 20,21 Currently, several CHK1 inhibitors are undergoing evalution in clinical trials focusing on solid tumors and hematologic malignancies.…”