Abstract PR03: Genome-wide screens for Wnt signaling in human haploid cells

• Published in: Molecular cancer therapeutics Vol. 12; no. 5_Supplement; p. PR03
• Main Authors: Lebensohn, Andres, Hughes, Casey, Marceau, Caleb, Rohatgi, Rajat, Carette, Jan
• Format: Journal Article
• Language: English
• Published: 01.05.2013
• ISSN: 1535-7163; 1538-8514
• DOI: 10.1158/1535-7163.PMS-PR03

Abstract Wnt signaling is at the core of human development and stem cell biology. Defective Wnt signaling leads to several forms of cancer, most notably colorectal cancer. Using HAP1 cells, a human haploid cell line, we conducted a set of genome-wide forward genetic screens to identify new Wnt pathway components. We constructed a HAP1 line harboring a GFP reporter driven by the Wnt-responsive TCF promoter, we mutagenized the cells by insertion of a gene-trap retrovirus throughout the haploid genome, and we sorted the mutagenized population for cells exhibiting phenotypes of interest using FACS. In order to find 1. negative regulators, 2. positive regulators and 3. feedback modulators of the pathway, we sorted for 1. cells expressing GFP in the absence of Wnt, 2. cells with reduced GFP expression after strong Wnt stimulation and 3. cells with enhanced GFP expression after mild Wnt stimulation, respectively. Deep sequencing of amplified genomic DNA flanking the retroviral insertion sites in the sorted cell populations revealed genes whose disruption may have led to the phenotype of interest. Among the most frequently disrupted genes (some with over 150 unique retroviral insertions) we found many of the known components of the canonical Wnt pathway, including LRP6, casein kinase I, APC, β-catenin, DOT1-L, TCF4 and the recently discovered negative feedback regulator ZNRF3. Unexpectedly, the screen for positive regulators revealed retroviral insertions in AXIN2 (a scaffold protein found in the β-catenin destruction complex) predicted to yield a C-terminally truncated form that appears to act as a dominant negative. We are currently assessing hits not previously known to be involved in Wnt signaling. This abstract is also presented as Poster A40. Citation Format: Andres Lebensohn, Casey Hughes, Caleb Marceau, Rajat Rohatgi, Jan Carette. Genome-wide screens for Wnt signaling in human haploid cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr PR03.