Genome-scale CRISPR/Cas9 screen determines factors modulating sensitivity to ProTide NUC-1031

Awa Sarr,Jennifer Bré,Tsz Huen Chan,Paul A Reynolds,David J Harrison,In Hwa Um,Peter Mullen

doi:10.1038/s41598-019-44089-3

Awa Sarr, Jennifer Bré + Show 5 more

Open Access

https://doi.org/10.1038/s41598-019-44089-3

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Journal: Scientific Reports	Publication Date: May 21, 2019
Citations: 11	License type: open-access

Affiliation: University of St Andrews

Abstract

Gemcitabine is a fluoropyrimidine analogue that is used as a mainstay of chemotherapy treatment for pancreatic and ovarian cancers, amongst others. Despite its widespread use, gemcitabine achieves responses in less than 10% of patients with metastatic pancreatic cancer and has a very limited impact on overall survival due to intrinsic and acquired resistance. NUC-1031 (Acelarin), a phosphoramidate transformation of gemcitabine, was the first anti-cancer ProTide to enter the clinic. We find it displays important in vitro cytotoxicity differences to gemcitabine, and a genome-wide CRISPR/Cas9 genetic screening approach identified only the pyrimidine metabolism pathway as modifying cancer cell sensitivity to NUC-1031. Low deoxycytidine kinase expression in tumour biopsies from patients treated with gemcitabine, assessed by immunostaining and image analysis, correlates with a poor prognosis, but there is no such correlation in tumour biopsies from a Phase I cohort treated with NUC-1031.

Highlights

NUC-1031 (Acelarin), a phosphoramidate transformation of gemcitabine, is the first anti-cancer ProTide to enter the clinic[1]
In order to investigate the effect of NUC-1031 and gemcitabine on dCMP/dCTP pool regulation, cytotoxicity assays for NUC-1031 and gemcitabine were carried out on MiaPaCa2 pancreatic cancer cells and A2780 ovarian cancer cells
Despite its widespread use as a mainstay of chemotherapy in patients with pancreatic, ovarian, lung, breast and biliary tract cancers, amongst others, the fluoropyrimidine gemcitabine achieves responses in less than 10% of patients with metastatic pancreatic cancer and has a very limited impact on overall survival due to intrinsic and acquired resistance

Summary

Introduction

NUC-1031 (Acelarin), a phosphoramidate transformation of gemcitabine, is the first anti-cancer ProTide to enter the clinic[1]. Deficiency of the activating enzyme DCK led to acquired gemcitabine resistance in a human ovarian carcinoma cell line exposed to increasing levels of gemcitabine in vitro[19] and patients with pancreatic cancer who express low levels of DCK have significantly poorer overall survival than those with high levels[20]. Pre-clinical data show the increased lipophilicity of NUC-1031 enables it to circumvent hENT1-mediated transmembrane transport and, once inside the cell, the phosphoramidate protective group is cleaved off by esterases, releasing dFdCMP which is rapidly converted to dFdCDP and dFdCTP, bypassing the rate-limiting step of phosphorylation by DCK. NUC-1031 avoids CDA-mediated catabolism, preventing dFdU accumulation[22,23,24] Both nucleotide synthesis and degradation are important in maintaining the dNTP pool and substrate cycling by 5ʹ‐nucleotidases and nucleoside kinases represent points of control[25]

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