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Abstract
The ability to develop a comprehensive panel of treatment predicting factors would significantly improve our ability to stratify patients for cytotoxic or targeted therapies, and prevent patients receiving ineffective treatments. We have investigated if a recently developed genome-wide haploid genetic screen can be used to reveal the critical mediators of response to anticancer therapy. Pancreatic cancer is known to be highly resistant to systemic therapy. Recently epigenetic changes have been shown to be a key determinant in the maintenance of subpopulations of cancer cells with high-level resistance to cytotoxic therapy. We show that in human pancreatic cancer cell lines, treatment with the potent class I histone deacetylase inhibitor, entinostat, synergistically enhances gemcitabine-induced inhibition of cell proliferation and apoptosis. Using a genome-wide haploid genetic screen, we identified deoxycytidine kinase (DCK) as one of the genes with the highest degree of insertional enrichment following treatment with gemcitabine and entinostat; DCK is already known to be the rate-limiting activating enzyme for gemcitabine. Immunoblotting confirmed loss of DCK protein expression in the resistant KBM7 cells. CRISPR/Cas-9 inactivation of DCK in pancreatic cancer cell lines resulted in resistance to gemcitabine alone and in combination with entinostat. We have identified gemcitabine and entinostat as a potential new combination therapy in pancreatic cancer, and in this proof-of-principle study we have demonstrated that a recently developed haploid genetic screen can be used as a novel approach to identify the critical genes that determine treatment response.
Highlights
Loss-of-function genetic screens in model organisms such as yeast, have helped to elucidate many biological processes, but such large-scale gene disruption has not previously been possible in human cells due to the difficulty in generating bi-allelic mutations in diploid cells
Prior to conducting combination studies, we first assessed the single agent antiproliferative activity of the potent class I histone deacetylase (HDAC) inhibitor, entinostat, in pancreatic cancer cells; 6 human pancreatic cancer cell lines (PANC-1, MIA PaCa-2, BxPC-3, CFPAC-1, SUIT2 and SUIT2 Clone 1) were treated with variable concentrations of entinostat (0100μM) for 72 hours and cell viability was assessed by XTT assays
Entinostat caused a dose-dependent decrease in cell proliferation and viability in all cell lines tested (Figure 1). This was associated with a dose-dependent increase in www.impactjournals.com/oncotarget histone H3 acetylation, with no effect on total histone H3 protein levels (Figure 2), confirming that entinostat inhibits the deacetylation activity of the HDACs in pancreatic cancer cell lines
Summary
Loss-of-function genetic screens in model organisms such as yeast, have helped to elucidate many biological processes, but such large-scale gene disruption has not previously been possible in human cells due to the difficulty in generating bi-allelic mutations in diploid cells. While the development of siRNA and shRNA libraries have made it possible to perform analogous lossof-function genetic screens in mammalian cells, RNA interference-based screens suffer from off-target effects, and do not always succeed in completely eliminating gene expression This problem has recently been circumvented following the isolation of a near-haploid human cell line, www.impactjournals.com/oncotarget a derivative of the KBM7 chronic myeloid leukaemia line which is haploid for all chromosomes except chromosome 8 [1]. This has enabled the development of a genomewide loss-of-function screen in human cells, based on insertional mutagenesis of these near haploid cells with a gene-trap retrovirus [2, 3] While this screen has some drawbacks in that it will fail to detect genes essential for cell survival or those that function redundantly, genomewide haploid screening of the KBM7 cell line has been successfully used to identify the host gene products necessary for the cytotoxic effects of several viruses and microbial toxins [2, 4, 5]. We initiated this proof-of-principle study in pancreatic cancer
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