NUMB (fig. S13A). NUMB recruits the E3 ubi-
quitin ligase ITCH to NOTCH, thereby facilita-
ting NOTCH ubiquitination and degradation
( 28 ). In accordance with the BioID results,
FLAG-tagged Ajuba coimmunoprecipitated
(co-IP) endogenous Lats1, Notch1, Notch2, and
Numb from primary mouse keratinocytes
(fig. S13B and Fig. 3C). The reciprocal co-IP also
showed that endogenous Notch1 and Notch2
interacted with endogenous Ajuba (fig. S13C).
Next, we explored how Ajuba regulates
NOTCH signaling. NOTCH activation increased
the interaction between Ajuba and Numb but
reduced binding between AJUBA and cleaved
NOTCH1IC(Fig. 3C), implicating a dynami-
cally regulated process. To test functional
relevance, we genetically ablated Ajuba in
primary keratinocytes and analyzed Notch-
Numb-Itch complex formation. Loss of Ajuba
resulted in (i) increased association between
Numb and Notch1, (ii) aberrant recruitment
of Itch to Notch1, and (iii) concomitant in-
creased Notch1 ubiquitination, all of which
was reversed by reexpressing Ajuba (Fig. 3D
and fig. S13, D and E). Consistent with these
findings, nuclear accumulation of NOTCH1IC
and NOTCH2ICafter receptor activation was
markedly reduced inAjuba-knockout cells,
which was reversed upon reexpressing Ajuba
(Fig. 3E and fig. S14, A to D). Last, we tested
whether Numb, which is amplified in ~37%
of HNSCC patients, could promote HNSCC
development. Indeed, overexpression of Numb
inPik3caH1047Rmice triggered rapid HNSCC
and cSCC development (fig. S14E). These data
suggest that Ajuba binds and sequesters Numb,
thereby promoting NOTCH signaling and
HNSCC tumor suppression.
Our third hit,Ripk4(Receptor Interacting
Protein Kinase 4), was reported to be a NOTCH
target gene ( 29 ), further implicating NOTCH
signaling in HNSCC. Indeed, NOTCH pathway
activation led to increased Rbpj CUT&RUN
footprints in promoter and enhancer regions
ofRipk4and transcriptional up-regulation of
Ripk4(figs. S11E and S12, B and C). Con-
ditionalRipk4fl/fl;Pik3caH1047Rbut not heter-
ozygousRipk4fl/+;Pik3caH1047Rcompound
mutant mice formed tumors upon lentiviral
Cre transduction, corroborating our CRISPR
results (fig. S15A). Ripk4 promotes the differ-
entiation of oral and epidermal keratinocytesby phosphorylating and activating the Irf6
(Interferon Regulatory Factor 6) transcrip-
tion factor ( 30 ). Irf6 also scored in our screen
and constitutes another direct NOTCH target
gene (table S3 and fig. S12C). These data in-
dicate that the Ripk4–Irf6 axis is an impor-
tant downstream effector of NOTCH.
To identify additional downstream NOTCH
targets that suppress HNSCC, we generated
a sgRNA library targeting 80 genes down-
regulated uponAdam10ablation (fig. S15, B
andC,andtableS6).TransducedPik3caH1047R;
Cas9micedevelopedtumorsenrichedfor
sgRNAs targeting the integrin subunitItgb5,
the Hippo component Angiomotin-Like 2
(Amotl2), Endonuclease Domain Containing 1
(Endod1), and Sushi Domain Containing 2
(Susd2) (Fig. 4, A and B, and fig. S15D). Inde-
pendent sgRNAs targetingItgb5orAmotl2
confirmed our findings and induced highly
aggressive SCC in thePik3caH1047Rmice (Fig.
4C and figs. S5E and S15E). CUT&RUN sequenc-
ing and real-time polymerase chain reaction
(RT-PCR) confirmed thatItgb5orAmotl2are
direct NOTCH target genes (Fig. 2D and fig.
S12C) ( 27 ).Loganathanet al.,Science 367 , 1264–1269 (2020) 13 March 2020 4of6
Fig. 3. Ajuba is a new NOTCH regulator.(A) Tumor-free survival of Ajuba+/−
and Ajuba+/+mice inPik3caH1047R,HRasG12V, or K14-HPV16 mouse backgrounds.
(B) RT-PCR results showing effects of CRISPR/Cas9-mediated ablation ofAjuba
on EDTA activation of the canonical NOTCH targetsHes1andHey1in primary
mouse keratinocytes. Data are shown as means ± SEM (n= 3). *P< 0.05.
(C) Co-IP of HA-Ajuba with endogenous Numb, full-length Notch1 (300 kDa), and
NOTCH1IC(120 kDa) in primary keratinocytes, which changes upon NOTCH pathway
activation (30 min EDTA; 15 min recovery). (D) Co-IP of endogenous Notch1 with
Numb and Itch1 in wild-type (WT), Ajuba-knockout (KO), and Ajuba-overexpressing
(KO+OE) primary keratinocytes. (E) Genetic ablation of Ajuba impairs nuclear
accumulation of NOTCHIC. Immunofluorescence of Ajuba WT and KO as well as
Ajuba-overexpressing (OE) WT and KO primary keratinocytes is shown. Cells were
treated with EDTA (30 min) to stimulate NOTCH receptor activation, allowed to
recover for 15 min (EDTA rec), and stained for NOTCH1. Scale bar, 50mm.RESEARCH | REPORT