Z Gastroenterol 2015; 53 - KG194
DOI: 10.1055/s-0035-1559220

A kinome wide CRISPR/Cas9 screen uncovers novel resistance factors to MEK inhibition in colorectal cancer

T Zhan 1, D Brüggemann 1, F Heigwer 1, M Breinig 1, J Winter 1, S Leible 1, M Ebert 2, M Boutros 1
  • 1Deutsches Krebsforschungszentrum Heidelberg, Signalwege und funktionelle Genomik, Heidelberg, Deutschland
  • 2Medizinische Fakultät Mannheim, Universität Heidelberg, Universitätsmedizin Mannheim, II. Medizinische Klinik, Mannheim, Deutschland

Einleitung: The Ras/MEK/ERK pathway is frequently mutated in colorectal cancer (CRC) and KRAS mutations present a major therapeutic challenge. Attempts to inhibit downstream effectors of KRAS including MEK revealed only modest clinical efficiency. The mechanisms by which CRC with KRAS mutation acquires resistance to MEK inhibition are not completely understood. Pooled CRISPR/Cas9 screens offer a novel high-throughput method for functional characterization of genes by generating knockouts and can be used to identify mechanisms of drug resistance.

Methodik: A pooled, kinome-wide knockout library of KRAS mutant CRC cells was generated using CRISPR/Cas9 technology. A total of 604 kinases and phosphatases were included in the library, covered by 20 sgRNA designs per gene. These knockout cells were treated with the MEK inhibitor Trametinib (GSK-1120212) or DMSO for 12 days and the relative sgRNA abundance of each treatment group was quantified by deep sequencing.

Ergebnis: A comprehensive pipeline for generation of pooled knockout libraries from in silico sgRNA design to statistical analysis and data presentation was established and successfully tested with this screen. After treatment with Trametinib, sgRNAs of several upstream kinases of key cellular pathway were significantly enriched, which indicates that knockout of those genes are beneficial for cell survival under MEK treatment. Candidate genes were specific for MEK inhibition, as treatment of the same cell library with the PLK1 inhibitor Volasertib yielded a completely different set of enriched and depleted genes.

Schlussfolgerung: High-coverage CRISPR/Cas9 knockout screens are a powerful tool for discovery of drug resistance mechanisms. Candidate genes mediating resistance to MEK inhibition in CRC will be further characterized and results presented.