Gastrointestinal tract cancers are some of the deadliest cancers and novel therapeutic options are needed. One hallmark of gastrointestinal cancers is the frequent mutation of tumour suppressor genes resulting in constitutive activation of the Wnt-β-catenin signalling pathway. Wnt-β-catenin signalling is a positive regulator of stem cell self-renewal in normal tissues, but cancer cells may take advantage of it to promote their proliferation and survival. Despite frequent cancer cell reliance on Wnt-β-catenin signalling, no clinical therapeutics currently exist that target it. CRISPR/Cas9 gene editing technology has recently allowed for genome-wide loss- of-function screens in vertebrate cells and may therefore be used to identify novel genetic vulnerabilities in human cancer cells. Genome-wide CRISPR screens were completed in RNF43 mutant pancreatic ductal adenocarcinoma (PDAC) cells lines, which rely on Wnt-β- catenin autocrine signaling for their proliferation. These screens revealed a single Frizzled receptor gene out of ten paralogs, FZD5, to be essential for the proliferation of each RNF43 mutant PDAC line. We generated a synthetic human monoclonal antibody specific to FZD5, and found it effective in reducing the growth of RNF43 mutant pancreatic cancers in vitro and in orthotopic xenograft models. We next identified the homeobox transcription factor CDX2 as a context-dependent fitness gene and Wnt-β-catenin transcriptional target in RNF43 mutant pancreatic cancer cells. CDX2 is uniquely overexpressed in Wnt-FZD5 dependent PDAC cells and transcriptome analysis revealed it may drive an intestinal metaplasia phenotype, and may be a suitable biomarker for Wnt-FZD5 dependency. To further identify novel therapeutic targets we completed synthetic-lethal interaction CRISPR screens in cells in which we engineered APC mutations. APC loss of function mutations cause Wnt-β-catenin pathway activation and occur in 80% of colorectal cancers. As a surrogate model for APC mutant colorectal cancer we engineered APC mutant PDAC cell lines to compare with their parental APC wild-type cell lines. Isogenic CRISPR screens comparing these cell lines identified many synthetic-lethal genes, most notably the Aryl Hydrocarbon Receptor (AHR), as a potential druggable vulnerability. Together this work demonstrates the power of genome-wide CRISPR screens for assessing signalling pathways in cancers, and may lead directly to the development of novel targeted therapeutics.Ph.D
