To extend our findings from mouse to hu-
man cancers, we analyzed 504 HNSCC cases
fromTheCancerGenomeAtlas( 2 ). TheADAM10,
AJUBA, andRIPK4genes were mutated in
0.8%, 7.5%, and 3.0% of tumors, respectively,
a frequency expected for long tail mutations.
However, an additional 12.7% and 11.1% of hu-
man HNSCCs showed heterozygousADAM10
andAJUBAloss, respectively (fig. S16A). Anal-
ysis of overlapping allelic loss segments showed
that chromosome 14 exhibited focal, mono-
allelic losses encompassingAJUBA, whereas
ADAM10exhibited preferential monoallelic
loss of broad chromosome segments (Fig. 4D).
We also found allelic imbalance of heterozy-
gous single nucleotide polymorphisms in the
chromosomal region ofAJUBA,furthercon-
firming heterozygous loss ofAJUBA(fig. S16, B
and C). Mutations and allelic copy number
loss coincided with reduced expression of
ADAM10andAJUBA(Fig.4E).Inaddition,
mutations and allelic loss ofADAM10,AJUBA,orRIPK4showed a trend toward mutual ex-
clusivity with mutations inNOTCH1/2/3re-
ceptors(Fig.4F,fig.S16D,andtableS7).
Altogether, ~27% of HNSCC patients carry
inactivatingNOTCH1/2/3receptor mutations,
and an additional ~40% of HNSCC samples
show inactivating alterations ofADAM10or
AJUBAor amplification of NUMB (Fig. 4F).
Although mutual exclusivity is neither a nec-
essary nor a sufficient condition for genes
involved in the same pathway, our functionalLoganathanet al.,Science 367 , 1264–1269 (2020) 13 March 2020 5of6
Fig. 4. NOTCH target genes and NOTCH pathway mutations in
human tumors.(A) Tumor-free survival forPik3caH1047Rmice transduced
with a lentiviral Cre-sgRNA library targeting genes down-regulated in
Adam10-deficient tumors (P< 0.0001, log-rank test). (B)Piechart
showing top-scoring sgRNAs enriched in tumor DNA from (A). (C)Tumor-free
survival forPik3caH1047R;Cas9 mice transduced with sgRNAs targeting
Itgb5andAmotl2or scrambled control sgRNAs (P< 0.0001, log-rank test).
(D) Patterns of monoallelic minimal deleted regions (MDRs) in
504 HNSCC TCGA samples. (E)ExpressionofADAM10orAJUBAin
tumors carryingADAM10orAJUBAmutations or copy number alterations,
respectively. (F) Genetic alterations ofNOTCHreceptors 1 to 3,ADAM10,
AJUBA,RIPK4,andNUMBin human HNSCC samples (n=504) from
TCGA presented as a cBioPortal OncoPrint displaying a trend toward
mutual exclusivity among mutations.RESEARCH | REPORT