Abstract PR05: Systematic forward genetic screens in haploid human cells reveal new players and regulatory mechanisms in Wnt signaling
Authors: Lebensohn Andres M. , Dubey Ramin , Neitzel Leif R. , Tacchelly Ofelia , Marceau Caleb D. , Bahrami Zahra , Hansen Amanda G. , Ahmed Yashi , Lee Ethan , Carette Jan , Rohatgi Rajat 
Published: May 2016

Wnt signaling is at the core of animal development and regeneration. It controls cell proliferation, differentiation and survival. In humans, defective Wnt signaling leads to several forms of cancer, most notably colorectal cancer (CRC). A convergence of methodological advances enabled us to do a systematic forward genetic analysis of canonical Wnt signaling in human cells, revealing new players and regulatory mechanisms. Using HAP1 cells, a haploid human cell line, we conducted a set of genome-wide screens to interrogate the Wnt pathway under normal and pathological conditions. We constructed a HAP1 line harboring a Wnt-responsive GFP reporter, mutagenized the cells by insertion of a gene trap retrovirus throughout the haploid genome, and sorted for cells exhibiting phenotypes of interest using FACS. By setting appropriate gates we enriched for negative and positive regulators of the pathway. These screens yielded many known players as well as a new transcription factor, TFAP4, required for signaling downstream of β-catenin. Unexpectedly AXIN2, a scaffold in the β-catenin destruction complex, was a prominent hit in the screen for positive regulators. However, the distribution of gene trap insertions in AXIN2 suggested that truncation of the last one or two exons encompassing the DAX domain, as opposed to disruption of the entire AXIN2 gene, was responsible for the observed phenotype. Follow-up analysis of cells in which AXIN1 was eliminated and AXIN2 C-terminal truncations were generated at the single endogenous locus confirmed that this domain is dispensable for destruction complex function. Furthermore, in these cells responsiveness to Wnt was only partially diminished, suggesting that the DAX domain is not essential for transduction of the Wnt signal from the receptor. Instead we show that the C-terminus is involved in regulating Axin2 protein levels. Using CRISPR/CAS9 we then generated haploid cell lines lacking adenomatous polyposis coli (APC), a scaffold in the β-catenin destruction complex, or casein kinase α (CK1α), the priming kinase for β-catenin degradation. These lines have constitutive Wnt signaling activity, and the former recapitulates the APC mutations found in CRC patients. We conducted synthetic genome-wide screens looking for modifiers of these mutations in an otherwise isogenic background. We found that in APC-null cells, deletion of the RNA binding protein SERBP1 reduces β-catenin protein levels significantly, restoring normal levels of Wnt-stimulated signaling. In CK1α-null cells, deletion of the E3 ubiquitin ligase HUWE1 decreases Wnt signaling by over 85% with only a minor reduction of β-catenin protein level. This defect is specific to the CK1α genetic background, since deletion of HUWE1 in APC-null cells has no effect on signaling. These results suggest a destruction complex independent role of CK1α in Wnt signaling, mediated by HUWE1. Our findings are supported by experiments in model organisms. The experimental approaches used in this study are generally applicable to other signaling pathways and more broadly to any cellular process in which a phenotypic readout can be used to enrich for mutant cells. The combination of forward genetics in haploid cells and CRISPR/CAS9-based genome engineering brings to bear on cultured human cells the immense power of genetics traditionally limited to model organisms such as yeast.

This abstract is also presented as Poster B11.

Citation Format: Andres M. Lebensohn, Ramin Dubey, Leif R. Neitzel, Ofelia Tacchelly, Caleb D. Marceau, Zahra Bahrami, Amanda G. Hansen, Yashi Ahmed, Ethan Lee, Jan Carette, Rajat Rohatgi. Systematic forward genetic screens in haploid human cells reveal new players and regulatory mechanisms in Wnt signaling. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr PR05.