Use of a genome-wide haploid genetic screen to identify the critical mediators of response to novel combination therapy with gemcitabine and entinostat in pancreatic cancer: a proof-of-principle study
Session type: Poster sessions
Pancreatic cancer is highly resistant to chemotherapy. Recently epigenetic changes were revealed to be a key determinant in the maintenance of subpopulations of cancer cells with high-level resistance to cytotoxic therapy. We have thus investigated the effect of the potent class I histone deacetylase inhibitor, entinostat, on gemcitabine sensitivity in vitro. Recently a genome-wide haploid genetic screening method has been developed and successfully used to identify the host factors necessary for the cytotoxic effects of several viruses and microbial toxins. We hypothesised that this haploid genetic screening approach could also be used to reveal the critical mediators of response to anticancer therapies.
Six human pancreatic cancer cell lines were treated with gemcitabine and entinostat and cell proliferation and apoptosis were assessed. Haploid KBM7 cells mutagenized with a retroviral gene-trap vector were then exposed to gemcitabine and entinostat and resistant clones expanded as a pool. High throughput sequencing was used to map insertions in the resistant population to the human genome. Genes with the highest degree of insertional enrichment were selected for validation using the CRISPR/Cas9 gene editing system.
Entinostat treatment synergistically enhanced gemcitabine-induced inhibition of cell proliferation and apoptosis in all the cell lines tested. Deoxycytidine kinase (DCK) was identified as one of the genes with the highest degree of insertional enrichment from the haploid genetic screen; 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. In this proof-of-principle study we have also shown that a haploid genetic screening approach can be used to identify the critical mediators of response.