Down-regulated NOTCH pathway activity was
also seen in primary keratinocytes isolated
fromAdam10-deficientPik3caH1047Rmice
4 days after birth, before any overt phenotypic
changes could be observed (Fig. 2D and fig. S8,
B to D), indicating that ADAM10 directly ac-
tivates NOTCH.
Given the prominent role of NOTCH signal-
ing in suppressing mouse and human HNSCC
development ( 18 ), we tested whether genet-
ic ablation of canonical NOTCH signaling
components inPik3caH1047Rmice pheno-
copiesAdam10loss. Indeed, sgRNA/Cas9-
mediated mutagenesis ofNotch1,Jag1,and
Jag2or the obligate transcriptional NOTCH
effectorRbpj, as well as conditional deletion of
Notch1andNotch2, caused HNSCC develop-
ment. Targeting the NOTCH ligandDll1or
Adam17, a metalloproteinase implicated in
ligand-independent NOTCH activation that is
required for epidermal barrier function ( 19 , 20 ),
did not trigger tumor development (Fig. 2E;
fig. S9, A and B; and fig. S5D). Finally, we
tested whether restoring NOTCH signaling in
Adam10-deficient mice would block tumor
development. Inducible expression of ectopic
NOTCHICbefore tumor appearance or in es-
tablished tumors resulted in effective tumor
suppression and delayed HNSCC develop-
ment (Fig. 2F and fig. S9, C to F). Thus, loss of
Adam10promotes HNSCC tumorigenesis by
impairing NOTCH signaling.
Our second hit,AJUBA,ismutatedin7%of
HNSCC, 18% of cutaneous SCC, and 2 to 7% of
esophageal SCC cases, but not in other human
cancers ( 3 , 21 ). AJUBA is a scaffold protein
and has been shown to regulate the Hippo
( 22 ), Wnt ( 23 ), and Aurora-A signaling path-
ways ( 24 ) and cell adhesion molecules ( 25 ),
but its role in SCC pathogenesis is unclear ( 1 ).
Ajuba-nullmicewereembryonicallylethal.
However, we found that loss of oneAjubaallele
triggered HNSCC development inPik3caH1047R,
HRasG12V,orK14-HPV16mice but retained ex-
pression of the wild-typeAjubaallele (Fig. 3A
andfig.S10,AtoD).Thesedatasuggestthat
Ajubaalso functions as a haploinsufficient tu-
mor suppressor.
Transcriptional profiling and GSEA of kerat-
inocytes isolated fromsgAjuba;Pik3caH1047R
mice 4 days after birth showed no difference in
Hippo or Wnt pathway activation but signif-
icant down-regulation of“NOTCH signaling”
(fig.S11,AtoD).Tocorroboratethisfinding,we
investigated the effect ofAjubaloss on NOTCH
pathway activation in primary keratinocytes.
We used either the canonical NOTCH ligand
JAG1 or EDTA treatment, which forces disso-
ciation of the heterodimeric NOTCH receptor,
thereby allowing release of NOTCHIC.LossofAjuba impaired ligand- and EDTA-induced
up-regulation of NOTCH target genes such as
Hes1andHey1(Fig. 3B and fig. S11E). To test
whether these changes are directly mediated
by NOTCHIC, we performed Rbpj CUT&RUN
(cleavage under targets and release using nu-
clease) sequencing ( 26 ). Most Rbpj-binding sites
in primary keratinocytes are in enhancers and
promoters, where Rbpj is dynamically recruited
upon NOTCH activation (fig. S12, A and B) ( 27 ).
NOTCH pathway inhibition by pharmacological
ADAM10 inhibition or loss ofAjubamarkedly
impaired Rbpj recruitment to and the expres-
sion of canonical NOTCHtarget genes such as
Hes1,Heyl,andNrarpand other genes such as
Id1,Egfr,Tgfb 1 ,Krt10,andJunB(fig.S12,AtoD,
and table S4). Ten of the 15 primary screening
hits are themselves NOTCH target genes, includ-
ingAjuba,Adam10,Ripk4,Notch1/3,Jag1/2,and
Irf6, indicating the existence of a feed-forward
loop to enhance NOTCH signaling and thus
tumor suppression (figs. S12C and S17 and
table S3).
To investigate Ajuba’s function, we iden-
tified vicinal proteins by proximity-dependent
biotinylation coupled to mass spectrometry
(BioID) in human 293 cells. AJUBA BioID
enriched for Hippo components (LATS1/2,
AMOT, and PTPN14) and several new proximity
interactors including NOTCH1, NOTCH2, andLoganathanet al.,Science 367 , 1264–1269 (2020) 13 March 2020 3of6
Fig. 2. Adam10 suppresses HNSCC development by regulating NOTCH.
(A)Tumor-freesurvivalforAdam10fl/fl,Adam10fl/+,andAdam10+/+;Pik3caH1047R
mice transduced with Cre lentivirus. (B)Adam10mRNA expression levels in
tumors of the indicated genotype. (C) GSEA reveals down-regulation of the NOTCH
pathway inAdam10fl/fl;Pik3caH1047Rtumors. (D)RT-PCRanalysisofNOTCHtarget
gene expression in keratinocytes isolated from postnatal day 4 pups of the indicated
genotype. Data are shown as mean ± SEM (n=3).*P<0.05.(E)Tumor-free
survival forPik3caH1047Rmice transduced with lentiviral Cre-sgRNA targeting the
indicated NOTCH pathway components. (F)NOTCHICexpression rescues loss of
Adam10. Tumor-free survival ofAdam10fl/fl;Pik3caH1047Rmice transduced with
Cre lentiviral constructs that allow for doxycycline-inducible expression of NOTCHIC.
Inducible expression of tdTomato served as a control.RESEARCH | REPORT