Genetic and Proteomic Interrogation of Lower Confidence Candidate Genes Reveals Signaling Networks in β-Catenin-Active Cancers.
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.
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.
Joseph
J
Rosenbluh
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.
Johnathan
J
Mercer
Broad Institute of Harvard and MIT, 415 Main Street, Cambridge, MA 02142, USA.
Yashaswi
Y
Shrestha
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.
Rachel
R
Oliver
Moores Cancer Center and School of Medicine, University of California San Diego, La Jolla, CA 92093, USA.
Pablo
P
Tamayo
Broad Institute of Harvard and MIT, 415 Main Street, Cambridge, MA 02142, USA.
John G
JG
Doench
Broad Institute of Harvard and MIT, 415 Main Street, Cambridge, MA 02142, USA.
Itay
I
Tirosh
Broad Institute of Harvard and MIT, 415 Main Street, Cambridge, MA 02142, USA.
Federica
F
Piccioni
Broad Institute of Harvard and MIT, 415 Main Street, Cambridge, MA 02142, USA.
Ella
E
Hartenian
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.
Heiko
H
Horn
Broad Institute of Harvard and MIT, 415 Main Street, Cambridge, MA 02142, USA.
Lola
L
Fagbami
Broad Institute of Harvard and MIT, 415 Main Street, Cambridge, MA 02142, USA.
David E
DE
Root
Broad Institute of Harvard and MIT, 415 Main Street, Cambridge, MA 02142, USA.
Jacob
J
Jaffe
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.
Kasper
K
Lage
Broad Institute of Harvard and MIT, 415 Main Street, Cambridge, MA 02142, USA.
Jesse S
JS
Boehm
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.
William C
WC
Hahn
D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D000072669:Gene Editing; D015316:Genetic Therapy; D006801:Humans; D009369:Neoplasms; D040901:Proteomics; D000094704:RNA, Guide, CRISPR-Cas Systems; D034741:RNA, Small Interfering; D051176:beta Catenin
D000094704:RNA, Guide, CRISPR-Cas Systems; D034741:RNA, Small Interfering; D051176:beta Catenin
CRISPR-Cas9; RNAi; genetic interactions; protein-protein interactions; β-catenin
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