HPB
non-CME
George A. Alyateem, MD
General Surgery Resident
New York Medical College - Metropolitan Hospital Center
Forest Hills, New York, United States
Disclosure information not submitted.
George A. Alyateem, MD
General Surgery Resident
New York Medical College - Metropolitan Hospital Center
Forest Hills, New York, United States
Disclosure information not submitted.
Robert Robey, n/a
Research Scientist
National Cancer Institute, Laboratory of Cell Biology, United States
Disclosure information not submitted.
Michael Gottesman, n/a
Chief, Senior Investigator
National Cancer Institute, Laboratory of Cell Biology, United States
Disclosure information not submitted.
A novel strategy in studying mechanisms of chemotherapy resistance is genome-wide CRISPR screens, in which genes of a particular cell type are either activated, knocked out, or inhibited prior to treatment with a drug of interest. Our objective was to employ all three genome-wide CRISPR screen models in a pancreatic ductal adenocarcinoma cell line to determine potential mechanisms of resistance to gemcitabine and nab-paclitaxel.
Methods:
A genome-wide CRISPR screen was performed. A pancreatic ductal adenocarcinoma cell line (PANC-1) was transduced with a Cas9-expressing lentivirus. Pooled small guide RNAs were then delivered into Cas9-expressing cells to incite a genome-wide genetic modulation. Transcription of a specific gene was either activated (CRISPRa), knocked out (CRISPRko), or inhibited (CRISPRi). PANC-1 cells that underwent a specific genetic modulation were then treated with either gemcitabine or nab-paclitaxel for 10 days. Compared against an untreated control for each technique, genomic DNA was extracted from all cells and analyzed using high-throughput sequencing and bioinformatics analysis. Validation was performed using an apoptosis assay of PANC-1 cells pre-treated with an inhibitor of TTK.
Results:
BUB3 and BUB1B, genes that encode proteins involved in spindle checkpoint function, were identified as genes potentially involved in mechanisms of nab-paclitaxel resistance in the CRISPRko and CRISPRi screens respectively. Cells with BUB3 knocked out or cells with decreased transcription of BUB1B survived at significantly higher levels when treated with nab-paclitaxel compared to control (false discovery rate < 5% in both sets of replicates). No statistically significant mechanisms of resistance were noted for either drug in the CRISPRa screen, nor were any significant mechanisms identified in the gemcitabine group. TTK was also determined to be a top 10 hit for PANC-1 cells treated with nab-paclitaxel in the inactivation screen. TTK, BUB3, and BUB1B work in concert to perform spindle checkpoint functions during mitosis. When PANC-1 cells were pre-treated with a TTK inhibitor, a survival advantage was displayed via an apoptosis assay (33% cell death with pretreatment, compared to 44% cell death without).
Conclusions:
Whole-genome CRISPR screens are a novel approach to studying mechanisms of chemotherapy resistance in cancer. In this study, reduced transcription of BUB3, BUB1B, and TTK were identified as potential mechanisms of resistance in pancreatic ductal adenocarcinoma treated with nab-paclitaxel.