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Cell Syst. 2016 Sep 28;3(3):302-316.e4. doi: 10.1016/j.cels.2016.09.001.

Genetic and Proteomic Interrogation of Lower Confidence Candidate Genes Reveals Signaling Networks in β-Catenin-Active Cancers.

Author information

1
Broad Institute of Harvard and MIT, 415 Main Street, Cambridge, MA 02142, USA; Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, 450 Brookline Avenue, Boston, MA 02215, USA.
2
Broad Institute of Harvard and MIT, 415 Main Street, Cambridge, MA 02142, USA; Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
3
Broad Institute of Harvard and MIT, 415 Main Street, Cambridge, MA 02142, USA.
4
Moores Cancer Center and School of Medicine, University of California San Diego, La Jolla, CA 92093, USA.
5
Broad Institute of Harvard and MIT, 415 Main Street, Cambridge, MA 02142, USA; Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, 450 Brookline Avenue, Boston, MA 02215, USA. Electronic address: william_hahn@dfci.harvard.edu.

Abstract

Genome-scale expression studies and comprehensive loss-of-function genetic screens have focused almost exclusively on the highest confidence candidate genes. Here, we describe a strategy for characterizing the lower confidence candidates identified by such approaches. We interrogated 177 genes that we classified as essential for the proliferation of cancer cells exhibiting constitutive β-catenin activity and integrated data for each of the candidates, derived from orthogonal short hairpin RNA (shRNA) knockdown and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated gene editing knockout screens, to yield 69 validated genes. We then characterized the relationships between sets of these genes using complementary assays: medium-throughput stable isotope labeling by amino acids in cell culture (SILAC)-based mass spectrometry, yielding 3,639 protein-protein interactions, and a CRISPR-mediated pairwise double knockout screen, yielding 375 combinations exhibiting greater- or lesser-than-additive phenotypic effects indicating genetic interactions. These studies identify previously unreported regulators of β-catenin, define functional networks required for the survival of β-catenin-active cancers, and provide an experimental strategy that may be applied to define other signaling networks.

KEYWORDS:

CRISPR-Cas9; RNAi; genetic interactions; protein-protein interactions; β-catenin

PMID:
27684187
PMCID:
PMC5455996
DOI:
10.1016/j.cels.2016.09.001
[Indexed for MEDLINE]
Free PMC Article

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