Pancreatic cancer (PC) is an aggressive disease with high lethality rate due to multiple resistance mechanisms. We used in vitro CRISPR/Cas9 genetic drop-out screening to identify genes involved in the regulation of PC cell line sensitivity to platinum chemotherapy drugs.
We used two sgRNA libraries: 1) enriched for genes regulating cell cycle and nuclear proteins genes (CC, 50 000 sgRNA targeting 4 716 genes); 2) genome-wide (GW, 90 000 sgRNA targeting 18 164 genes). We performed screens in MIA PaCa-2 cells expressing doxycycline-inducible Cas9. Cells were treated with established IC30 of oxaliplatin (1 uM) or cisplatin (3 uM) for 9 cell divisions (12 days). Genomic DNA was extracted and sgRNA-containing regions were amplified and barcoded by PCR for further analysis by NGS. Statistical analysis for sgRNA enrichment or depletion was performed using R package comparing cells treated with the drugs vs. vehicle in the presence of Cas9/doxycycline.
We identified 755 genes which significantly changed in cisplatin or oxaliplatin-treated cells (FDR 5%, p < 0.05). Candidate genes (n = 130) were further selected if at least 2 sgRNA per gene showed more than 2-fold change vs. vehicle. Among the 130 genes, 16 were known platinum sensitivity regulators involved in the double stranded break DNA repair pathway; 11 genes were positive platinum sensitivity regulators as their inactivation reduced sensitivity; 119 genes were negative platinum sensitivity regulators as their inactivation increased sensitivity. Gene Ontology analysis of the 130 candidate genes allowed us to identify regulators of cell cycle (n = 46), DNA replication and repair (n = 43), cellular compromise (n = 15), cellular assembly and organization (n = 35) and cell morphology (n = 48). Analysis of protein-protein interaction network showed that the majority of the hits (n = 74) are directly involved into cell cycle regulation and DNA repair processes.
We identified 130 candidate genes potentially involved in the modulation of platinum resistance most of which are regulating the cell cycle and DNA repair which is in keeping with the known DNA damaging mechanisms of action of platinum chemotherapy.
Clinical trial identification
Legal entity responsible for the study
Kazan Federal University.
The work is performed according to the Russian Government Program of Competitive Growth of Kazan Federal University.
All authors have declared no conflicts of interest.