Abstract 4287: Whole-genome CRISPR/Cas9 screen of the CHK1 inhibitor prexasertib implicates FAM122A loss as a potential resistance mechanism
Authors: Deraska Peter , Reavis Hunter , Labe Shelby , D'Andrea Alan , Kozono David 
Published: August 2018

Introduction: Phase I clinical trials of the CHK1 inhibitor prexasertib have been completed in non-small cell lung cancer (NSCLC) and other cancers and several Phase I-II trials are currently recruiting. By inhibiting the G2/M and S-phase checkpoints, prexasertib has been shown to cause increased replication stress, DNA damage and premature entry into mitosis, resulting in mitotic catastrophe and cell death. While the inhibitor is showing therapeutic promise, acquired resistance is of concern. Little is known about how cancer cells may become resistant to CHK1 inhibitors. Understanding potential resistance mechanisms may help predict patient responses, design CHK1 therapy combinations and implement means of re-sensitization.

Materials and Methods: NSCLC cell lines A549 and NCI-H460 stably expressing the Cas9 endonuclease were obtained from the Genetic Perturbation Platform at the Broad Institute. Cells were infected with the Brunello v2 lentiviral sgRNA library and grown for 15 days in prexasertib-treated media. Cells were collected pre- and post-drug selection, genomic DNA was extracted, and sgRNA sequences were PCR amplified and Illumina sequenced. Top hits among knockouts that conferred drug resistance were identified by the STARS gene-ranking algorithm. Hit validation was performed with sgRNAs and pooled siRNAs (Qiagen). Prexasertib IC50s were assessed by colony formation and CellTiter-Glo cell viability assays. Cell cycle analysis was performed by propidium iodide flow cytometry. PP2A activity was measured using Ser/Thr phosphatase assay kits (Upstate Biotechnology). Dinaciclib and LB-100 were obtained from Selleck and Apexbio, respectively.

Results: CRISPR knockouts of several individual genes were found to promote resistance of both cell lines to prexasertib with false discovery rates &lt; 0.01%. One top hit codes for a little-known but highly conserved protein FAM122A, which has been shown to inhibit PP2A, which dephosphorylates the Chk1 target protein CDC25C. SiRNA knockdown or sgRNA knockout of FAM122A conferred prexasertib resistance. Similarly, prexasertib-mediated G2/M accumulation was inhibited by FAM122A knockdown. FAM122A silencing increased PP2A activity. FAM122A knockdown mediated resistance was reversed with PP2A B subunit α siRNA knockdown or the PP2A inhibitor LB-100. Dinaciclib, an inhibitor of the CDC25C kinase CDK2, increased prexasertib resistance.

Conclusion: Although CHK1 inhibitors like prexasertib show promise, one mechanism of resistance may be through other cell-cycle regulating compensatory pathways. Identification of FAM122A loss as a potential mechanism for resistance suggests a predictive biomarker and means for re-sensitization of cancer cells to CHK1 inhibitors.

Citation Format: Peter Deraska, Hunter Reavis, Shelby Labe, Alan D'Andrea, David Kozono. Whole-genome CRISPR/Cas9 screen of the CHK1 inhibitor prexasertib implicates FAM122A loss as a potential resistance mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4287.