Understanding Cancer Mutations by Genome Editing

Sammanfattning: Mutational analyses of cancer genomes have identified novel candidate cancer genes with hitherto unknown function in cancer. To enable phenotyping of mutations in such genes, we have developed a scalable technology for gene knock-in and knock-out in human somatic cells based on recombination-mediated construct generation and a computational tool to design gene targeting constructs. Using this technology, we have generated somatic cell knock-outs of the putative cancer genes ZBED6 and DIP2C in human colorectal cancer cells. In ZBED6-/- cells complete loss of functional ZBED6 was validated and loss of ZBED6 induced the expression of IGF2. Whole transcriptome and ChIP-seq analyses revealed relative enrichment of ZBED6 binding sites at upregulated genes as compared to downregulated genes. The functional annotation of differentially expressed genes revealed enrichment of genes related to cell cycle and cell proliferation and the transcriptional modulator ZBED6 affected the cell growth and cell cycle of human colorectal cancer cells. In DIP2C-/-cells, transcriptome sequencing revealed 780 differentially expressed genes as compared to their parental cells including the tumour suppressor gene CDKN2A. The DIP2C regulated genes belonged to several cancer related processes such as angiogenesis, cell structure and motility. The DIP2C-/-cells were enlarged and grew slower than their parental cells. To be able to directly compare the phenotypes of mutant KRAS and BRAF in colorectal cancers, we have introduced a KRASG13D allele in RKO BRAFV600E/-/-/ cells. The expression of the mutant KRAS allele was confirmed and anchorage independent growth was restored in KRASG13D cells. The differentially expressed genes both in BRAF and KRAS mutant cells included ERBB, TGFB and histone modification pathways. Together, the isogenic model systems presented here can provide insights to known and novel cancer pathways and can be used for drug discovery.