CANCER
Rare driver mutations in head and neck
squamous cell carcinomas converge on
NOTCH signaling
Sampath K. Loganathan^1 , Krista Schleicher1,2, Ahmad Malik1,2, Rene Quevedo3,4, Ellen Langille1,2,
Katie Teng^1 , Robin H. Oh1,2, Bhavisha Rathod^1 , Ricky Tsai^1 , Payman Samavarchi-Tehrani^1 ,
Trevor J. Pugh3,4,5, Anne-Claude Gingras1,2, Daniel Schramek1,2*
In most human cancers, only a few genes are mutated at high frequencies; most are mutated at low
frequencies. The functional consequences of these recurrent but infrequent“long tail”mutations
are often unknown. We focused on 484 long tail genes in head and neck squamous cell carcinoma
(HNSCC) and used in vivo CRISPR to screen for genes that, upon mutation, trigger tumor development
in mice. Of the 15 tumor-suppressor genes identified,ADAM10andAJUBAsuppressed HNSCC in a
haploinsufficient manner by promoting NOTCH receptor signaling.ADAM10andAJUBAmutations or
monoallelic loss occur in 28% of human HNSCC cases and are mutually exclusive with NOTCH receptor
mutations. Our results show that oncogenic mutations in 67% of human HNSCC cases converge onto
the NOTCH signaling pathway, making NOTCH inactivation a hallmark of HNSCC.
H
ead and neck squamous cell carcinoma
(HNSCC) is the sixth most common hu-
man cancer, and the 5-year survival rate
is <50% ( 1 ). HNSCC arises in the mucosal
lining of the upper aerodigestive tract
and is tightly linked to tobacco use, alcohol
consumption, and human papillomavirus
(HPV) infection. The most common genetic
alterations in HNSCC affectp53(71%),FAT1
(23%),CDKN2A(22%),PIK3CA(18%),NOTCH1
(17%), andHRAS(6%), followed by a“long tail”
of hundreds of individually rare mutations,
most of which lack biological or clinical vali-
dation ( 2 , 3 ) (fig. S1, A and B).
The long tails of recurrent but rare muta-
tions remain enigmatic in cancers ( 4 , 5 ). It is
difficult to reconcile their apparent positive
selection with their low frequency and im-
mense diversity. The long tail might reflect
the stochasticity of cancer evolution, where
each mutation confers only a small fitness
advantage, but several such low-penetrant
mutations might cooperate and substantially
promote tumor progression ( 6 , 7 ). Some long
tail mutations may be highly penetrant but
simply affect genes that are rarely mutated.
Several mutations may also converge on the
same cellular signaling pathway, which could
explain some of the diversity and low frequen-
cy, highlighting the importance of a pathway-
centric view of the cancer landscape ( 8 , 9 ).
Given that long tail driver mutations can be
the genetic basis for exceptional responses to
therapy ( 10 , 11 ), it is crucial to identify clini-
cally relevant mutations and define their on-
cogenic mechanisms.
To functionally assess HNSCC long tail genes,
we developed a CRISPR screen to identify
genes that, upon mutation, predispose mice
to HNSCC development. We constructed lenti-
viruses that coexpress a single-guide RNA
(sgRNA) and Cre recombinase and used
ultrasound-guided in utero microinjections to
deliver these lentiviruses to the single-layered
surface ectoderm of live mouse embryos ( 12 )
(Fig. 1A). The surface ectoderm generates sev-
eral structures, including the skin epithelium
and oral mucosa. We used multicolor Lox-Stop-
Lox-Confetti mice, which, upon Cre-mediated
excision of the Lox-Stop-Lox (LSL) cassette,
stochastically switch on one of four fluores-
cent proteins, and determined the viral titer
required to generate thousands of discrete
clones within the oral cavity and epidermis
(Fig. 1B).
Next, we tested the efficiency of CRISPR/
Cas9-mediated in vivo mutagenesis (fig. S2A).
We found that LSL-Cas9-GFP;LSL-tdTomato
mice transduced with a Cre lentivirus encoding
a scrambled sgRNA (LV-sgScr-Cre) coex-
pressed green fluorescent protein (GFP) and
tdTomato. By contrast, mice transduced with
sgRNA-Cre lentivirus targeting GFP (LV-sgGFP-
Cre) displayed tdTomato+cells lacking GFP,
demonstrating a knockout efficiency of 85 ±
5% (fig. S2B). Next, we targeted the heme
biosynthesis geneUrod, the loss of which
leads to the accumulation of unprocessed, flu-
orescent porphyrins ( 13 ), which resulted in
bright red fluorescence in the oral mucosa
and skin (fig. S2C), demonstrating efficient
targeting of an endogenous gene.
To determine whether this approach can
reveal genetic interactions, we recapitulatedcooperation between oncogenic phosphatidyl-
inositol 3-kinase (PI3K)/Akt signaling and loss
of tumor suppressors such as transforming
growth factor-breceptor II (TgfbrII)orp53
( 14 ). Cas9-mediated ablation ofTgfbrIIor
p53in conditionalPik3caH1047Rmice (LSL-
Pik3caH1047R;LSL-Cas9) triggered rapid for-
mation of HNSCC tumors, whereas littermates
transduced with scrambled control sgRNAs
remained asymptomatic (fig. S2, D and E).
Because of the transduction method used,
these mice simultaneously developed HNSCC
as well as cutaneous SCC (cSCC). The latter
tumors are genetically, histologically, and path-
ologically related to HNSCC ( 3 ).
Next, we generated a lentiviral sgRNA li-
brarytomutatethemousehomologsof484re-
currentbutinfrequenthumanHNSCClongtail
genes and control libraries containing 418 non-
targeting sgRNAs or 414 randomly picked
genes (four sgRNAs per gene) (figs. S1 and
S2F and tables S1 and S2) ( 2 ). To identify
tumor-suppressor genes that cooperate with
known HNSCC oncogenic driver mutations,
we transduced these sgRNA libraries into
LSL-Cas9 mice that harbor (i) a conditional
Pik3caoncogene (LSL-Pik3caH1047R); (ii) a con-
ditionalHRasoncogene (LSL-HRasG12V);
(iii) a conditional, dominant-negative p53
mutation (LSL-p53R270H); or (iv) epithelium-
specific expression of the HPV16-E6/E7 onco-
genes (K14-HPV16). Within a year, none of the
mice transduced with the control libraries
developed tumors, highlighting thatPik3caH1047R,
HRasG12V,p53R270H, and HPV16-E6/E7 on
their own are insufficient to initiate SCC. By
contrast, all mice transduced with the long
tail sgRNA library developed multiple HNSCC
and cSCC tumors within weeks (Fig. 1, C and D,
and fig. S3, A and B).Pik3caH1047R;Cas9mice
transduced with an sgRNA library targeting
215 breast cancer long tail genes did not de-
velop tumors over a 4-month observation
period (fig. S2G and table S2), indicating the
existence of strong, HNSCC-specific long tail
tumor-suppressor genes.
To identify these tumor-suppressor genes,
we determined sgRNA representation in 205
mouse tumors. Most tumors showed strong
enrichment for a singlesgRNA compared with
tumor-adjacent, phenotypically normal epithe-
lium (fig. S3C). Fifteen genes showed enrich-
ment of two or more independent sgRNAs in
multiple tumors, withAdam10,Ripk4,and
Ajubabeing the most prevalent hits, followed
byNotch2andNotch3(Fig. 1E, fig. S3D, and
table S3). Although most genes scored in all
oncogenic backgrounds,Ripk4, for example,
did not surface in HPV-mutant mice. For val-
idation of our top hits, we injectedPik3caH1047R;
Cas9andHRasG12V;Cas9mouse embryos with
Adam10,Ajuba,orRipk4sgRNAs that were not
presentintheinitiallibrary.Thesemicerapidly
developed invasive SCC tumors but were moreRESEARCH
Loganathanet al.,Science 367 , 1264–1269 (2020) 13 March 2020 1of6
(^1) Centre for Molecular and Systems Biology, Lunenfeld-
Tanenbaum Research Institute, Mount Sinai Hospital, Toronto,
Ontario, Canada.^2 Department of Molecular Genetics,
University of Toronto, Toronto, Ontario, Canada.^3 Princess
Margaret Cancer Centre, University Health Network, Toronto,
Ontario, Canada.^4 Department of Medical Biophysics,
University of Toronto, Toronto, Ontario, Canada.^5 Ontario
Institute for Cancer Research, Toronto, Ontario, Canada.
*Corresponding author. Email: [email protected]