GSE158338
CHK1 Inhibitor Blocks Phosphorylation of FAM122A and Promotes Replication Stress
While effective anti-cancer drugs targeting the CHK1 kinase are advancing in the clinic, drug resistance is rapidly emerging.  Here, we demonstrate that CRISPR-mediated knockout of the little-known gene FAM122A confers cellular resistance to CHK1 inhibitors and cross-resistance to ATR inhibitors.  Knockout of FAM122A results in activation of PP2A-B55a, a phosphatase which dephosphorylates the WEE1 protein and rescues WEE1 from Ubiquitin-mediated degradation.  The resulting increase in WEE1 protein expression reduces replication stress, activates the G2/M checkpoint, and confers cellular resistance to CHK1 inhibitors.  Interestingly, in tumor cells with oncogene-driven replication stress, CHK1 can directly phosphorylate FAM122A, leading to activation of the PP2A-B55a phosphatase and increased WEE1 expression.  A combination of a CHK1 inhibitor plus a WEE1 inhibitor can overcome CHK1 inhibitor resistance of these tumor cells, thereby enhancing anti-cancer activity.  The FAM122A expression level in a tumor cell can serve as a useful biomarker for predicting CHK1 inhibitor sensitivity or resistance. Overall design: Prexasertib-resistant cells were generated by first culturing with graded concentrations of prexasertib for one week and surviving cells in the drug concentrations of IC90 or IC80 were harvested. These cells were then cultured in the IC50 drug concentrations for 1-2 weeks and were gradually adapted to higher doses of drug for almost 2-3 months. The resistant cells were then maintained in a medium without the drug. The experiment was performed in duplicates. Total RNAs were extracted and sequenced on an Illumina NovaSeq 6000. RNA-sequence data were analyzed using the VIPER snakemake pipeline. Differential gene expression analysis was performed by DESeq2 and limma, comparing the mRNA expression levels of prexasertib-resistant cells to those of the parent cells.