HPB
George A. Alyateem, MD
General Surgery Resident
New York Medical College - Metropolitan Hospital Center
Forest Hills, New York, United States
Disclosure: Disclosure information not submitted.
Chemoresistance in pancreatic ductal adenocarcinoma creates a challenge for clinicians. 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. The primary objective of this project was to employ genome-wide CRISPR screens using activation, knockout, and inhibition 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 (Figure 1). 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.
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.
Conclusions:
Whole-genome CRISPR screens are a novel approach to studying mechanisms of chemotherapy resistance in cancer. In this study, reduced transcription of BUB3 and BUB1B were identified as potential mechanisms of resistance in pancreatic ductal adenocarcinoma treated with nab-paclitaxel.