prone to developing HNSCC than cSCC com-
pared with mice withp53orTgfbrIIloss (fig.
S4, A to E). Sequencing of the sgRNA target
sites in tumor DNA confirmed frame-shift mu-
tations in all tested tumors (fig. S5). Inducible
activation of thePik3caH1047Roncogene and
Cas9-mediated mutagenesis ofAdam10or
Ripk4in adult mice also led to rapid HNSCC
development (fig. S6, C and D). Loss ofAdam10
orRipk4triggered tumor development even in
wild-type mice with very long latency (fig. S6, A
and B). Thus,Adam10,Ripk4,andAjubaare
strong HNSCC suppressors.
The membrane-anchored metalloproteinase
ADAM10 controls diverse cellular processes
through regulated intramembrane proteoly-
sis of numerous proteins ( 15 – 17 ). To confirm
its role in suppressing HNSCC, we generated
conditionalAdam10fl/fl;Pik3caH1047Rcompound
mutant mice and transduced embryos with
lentiviral Cre. HomozygousAdam10deletion
rapidly induced HNSCC tumors, recapitulat-
ing our CRISPR/Cas9 findings (Fig. 2A and
fig. S7, A to C). Even heterozygousAdam10fl/+;
Pik3caH1047Rmice developed HNSCC, although
with longer latency (Fig. 2A). To assess whether
tumor development was due toAdam10loss of
heterozygosity, we used fluorescence-activated
cell sorting to isolate tumor cells fromAdam10-
homozygous and -heterozygousPik3caH1047R
tumors and from control sgp53;Pik3caH1047R
tumors. Transcriptional profiling and West-
ern blot analysis revealed wild-type Adam10
expression inAdam10-heterozygous tumors,
albeit at a reduced level compared with con-
trol tumors (Fig. 2B and fig. S7D), indicatingthatAdam10functions as a haploinsufficient
tumor suppressor in HNSCC.
Gene set enrichment analysis (GSEA) of
these tumor cell transcriptomes revealed dif-
ferentially expressed gene sets specifically as-
sociated with“Hallmarks of G2M checkpoint”
and“E2F targets”as well as“NOTCH signal-
ing”(Fig. 2C and fig. S8A). NOTCH receptors
are transmembrane proteins that are proteo-
lytically cleaved upon binding to JAG1/2 or
DLL1/3/4 ligands. The cleaved NOTCH intra-
cellular domain (NOTCHIC) enters the nucleus,
binds the DNA-binding protein RBPJ, and reg-
ulates gene expression.Adam10-knockout tumor
cells showed reduced expression of NOTCH
target genes such asHes1andHeyl(fig. S8E),
consistent with ligand-dependent activation of
NOTCH by ADAM10-mediated cleavage ( 14 , 17 ).Loganathanet al.,Science 367 , 1264–1269 (2020) 13 March 2020 2of6
Fig. 1. Direct in vivo CRISPR screen for HNSCC tumor suppressors.
(A) Experimental workflow for pooled in vivo CRISPR screen. A lentiviral
sgRNA library targeting mouse homologs of human HNSCC long tail genes is
introduced into the surface epithelium of mice, and tumors are analyzed by
next-generation sequencing (NGS). (B) Representative images of whole body,
oral cavity, tongue, and palate of newborn Cre-reporter LSL-Confetti mice
transduced with Cre lentivirus. Scale bars, 500mm. (C) Tumor-free survival of
mice transduced with an sgRNA library targeting putative HNSCC genes or
with a control sgRNA library (n> 20 per group;P< 0.0001, log-rank test).
(D) Representative images ofPik3caH1047R;Cas9 mice transduced with the
HNSCC sgRNA library showing multiple, progressively growing tumors in the
oral cavity and tongue. Scale bars, 500mm. (E) Pie chart showing tumor-
suppressor genes with enriched sgRNAs in tumor DNA obtained from the four
different HNSCC mouse models.RESEARCH | REPORT