Abstract: Background Canine oral melanoma carries a poor prognosis, and there is a need for additional therapeutic approaches. Genome-wide knockout screening using the CRISPR-Cas9 system is now a well-established technique for identifying molecular vulnerabilities and genes underpinning treatment resistance in vitro in human molecular oncology, but this has not previously been applied to dogs.
Hypothesis/Objectives Genome-wide CRISPR knockout screening can identify potential novel molecular targets in cultured canine oral melanoma cells.
Animals Commercially-available oral melanoma cell lines derived from 3 dogs.
Methods A first-in-species canine genome-wide CRISPR knockout library was created allowing inactivation of 20,230 genes in parallel. This was introduced into canine melanoma cell lines CMGD2, CMGD5 and CML-10, and control non-neoplastic cells (fibroblasts), via a lentiviral vector. Integrated guide RNAs (sgRNAs) were sequenced as a proxy for gene knockout, and compared between baseline and surviving cells after 14-21 days of culture, to determine genes conditionally essential to melanoma cell survival, using statistical algorithm MAGeCK.
Results Efficacy of the technique in canine cells was confirmed by consistent depletion of sgRNAs targeting known universally essential genes in human cells. Genes with statistically-significant negative enrichment (FDR< 0.05), without evidence of universal essentiality in normal cells, included potentially targetable genes implicated in other cancers, involved in biological pathways including Wnt signalling, growth hormone signalling and the hypusination pathway.
Conclusions and clinical importance Genes identified as conditionally essential could be targets for novel therapeutics. Top candidates will be subject to in vitro validation using available inhibitors, to determine suitability for further translational investigation.
