Genome-Scale Crispr/Cas9 Screen Identifies Factors Required For Sensitivity To Pyrimidine Nucleoside Analogs

Abstract NUC-1031 (NuCana plc), is a phosphoramidate transformation of gemcitabine, which bypasses cancer cell resistance mechanisms to gemcitabine. This compound showed promising results in clinical studies (phase I/II), demonstrating tumor reduction and durable disease control in patients who were refractory to or had relapsed on gemcitabine. Here, we adopted an unbiased approach to uncover genes and pathways involved in gemcitabine and NUC-1031 resistance. We performed a genome-wide knockdown screen using CRISPR/Cas9 technology in the pancreatic cancer cell line MiaPaCa2. Potential candidates from the screen were validated by gene inactivation using individual or combined single guide (sg)RNA targeting sequences. The effect of gene knockdown on NUC-1031 and gemcitabine resistance was assessed by monitoring cell survival after drug treatment using Celigo cytometry to assess cell number. EC50 values were determined using the resulting survival curves. This screen generated a list of 4 candidate genes potentially involved in NUC-1031 resistance. Two candidates were investigated further: DCK and DCTPP1, both involved in pyrimidine metabolism, especially in the maintenance of the dCMP/dCTP pool. DCK knockdown induced significant resistance to gemcitabine (EC50 350 times higher than in control cells) and a degree of resistance to NUC-1031 (EC50 7 times higher than in control cells). A less robust effect was observed when DCTPP1 was inactivated. DCTPP1 knockdown induced a small decrease in sensitivity to NUC-1031 (EC50 1.5 times higher than in control cells). These results highlight the potential role of dCMP/dCTP pool regulation by DCK and DCTPP1 in sensitivity to pyrimidine nucleoside analogs and may provide a useful biomarker for patient selection. Citation Format: Awa SARR, Jennifer Bré, Peter Mullen, Sarah Blagden, In Hwa Um, David J. Harrison, Paul A. Reynolds. Genome-scale CRISPR/Cas9 screen identifies factors required for sensitivity to pyrimidine nucleoside analogs [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 572.