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TNBCTNBCTNBC
TNBC
TNBCTNBC
TNBCTNBC
TNBC
TNBCTNBC
Supplementary Figure 1d
e
f
HDPESTNotch pathway activation(TNBC cases only)
c
a
b
NOTCH1
NOTCH2
NOTCH3
Supplementary Figure 2NOTCH4a
b
Supplementary Figure 3
TCGA-BH-A1FC, ∆exon21-27
Canonical NOTCH1 transcript
34
33
32
31 29 27 25 23 21 19
30 28 26 24 22 20 18
TCGA-A2-A0T0, chr9:139,390,528-139,390,695 deletion
TCGA-A8-A08X, chr9:139,391,422 inter-chromosomal translocation
Canonical NOTCH2 transcript
TCGA-AO-A0J6, chr1:120,458,725-120,458,786 deletion
a
b
34
33
32
31 29 27 25 23 21 19
30 28 26 24 22 20 18
To: chr14:30,447,409
c
TCGA-AN-A0AR, BRD4(exon1)-NOTCH3(exon 26) fusion & chr19:15,270,914-15,272,239 deletion
33 32 31 29 2730 28 26 25
NOTCH3 BRD4EPHX3
TCGA-BH-A1FC TumorTCGA-BH-A1FC Tumor TCGA-BH-A1FC Matched Normala b c
d9q34.3
27 234 28 1
NOTCH1
21 20
234 28 120
Exon 21-27 deletion
S1 S2 S3
Supplementary Figure 4
e f
19p13.13
33 26 25
2 1
33 26
12 1
50Kb
NOTCH3 BRD4
1226bp deletion
a b
c
Supplementary Figure 5
S1 S2 S3
Notch3 wild-type
Mutant Notch3 in TCGA-AN-A0AR
Notch3 p.M1583 Notch3 p.P2067Rfs*84
d
Pfizer Confidential │ 6
TCGA-A2-A0T0 TCGA-A8-A08X TCGA-A8-A0J6
RNA-Seq in tumor
WXS in tumor
WXS in matched normal
a b c
Supplementary Figure 6
a b Supplementary Figure 7
Chromosome Position (MB)
log
2 C
N r
atio
ca
b d
Supplementary Figure 8
e
f g
TGTGAGCAGA CCCGCTGGTT
h
i
Exon 34 Intron 30Junction
CCACTGGGCCCC CTGGGGTGATACCGCAGG
j
k
l
1p12 1q21.1
chr1: 144548815chr1: 120458385
NOTCH234 27 2 1 Intronic
34* 27 2 1
chr1: 1445488151 27 29 31 33
1p12 1q21.1
2 28 30 32 34
1 27 29 31 332 28 30 32
34’
*5’ - - 3’
NOTCH2 Intronic
9q34.3
27 2
27 2
34 3031
34 34*31 3031
1
1
NOTCH1
chr9:139390847chr9:139396004
27 29 31 332 28 30 32 34
9q34.3
NOTCH1
1
5’ - - 3’1 27 29 31 332 28 30 32
3031
34’
*
chr9:139396004
Supplementary Figure 9
TCTCGAGATGGAGATTGGAGATTG
wtdeletion
GCATTCCAACTTTAGCACAAGG
GCATTCCAACTTTAGCACAAGG
CCAGCTACAGCTATGCTGA
AGCGACGTGGGCTCTCCTGGGGC
GGCAGCGACGTGGGCAGGGCGGGGCTCTCCTGGGGC
c
a
b
d
e
f
g
9q34.3
27 234 28 1NOTCH1
34* 28 127*
4q32.3
chr4:166105738
Intergenic
chr9:139390607 chr9:139397768 chr9:139439295
9q34.3 4q32.3
Intergenic
1 27 29 31 332 28 30 32 34
NOTCH1
1 29 31 3328 30 325’ - - 3’
34’
*
chr4:166105738
Alt. splicing chr9:139390682 – chr4:166107169 27’
unsp
liced
splic
ed
Supplementary Figure 10a
b
0 5 10 15 20 25 300
100200300400500600700
Days
Tum
or V
olum
e (m
m^3
) HCC1937
0 5 10 15 20 25 30 35 40 45 500
200
400
600
800
1000
1200Vehicle
PF-03084014
Days
Tum
or V
olum
e (m
m^3
) HBCx5
0 5 10 15 20 25 30 35 400
100200300400500600700800
Days
Tum
or V
olum
e (m
m^3
) HBCx17
0 5 10 15 20 25 30 350
200
400
600
800
1000
1200
Days
Tum
or V
olum
e (m
m^3
) HBCx12B
0 5 10 15 20 25 30 350
200
400
600
800
1000
1200
Days
Tum
or V
olum
e (m
m^3
) AA0869
0 5 10 15 20 25 300
200
400
600
800
1000
1200
Days
Tum
or V
olum
e (m
m^3
) MDA-MB-231
0 2 4 6 8 10 12 140
200400600800
100012001400
Days
Tum
or V
olum
e (m
m^3
) BT-474
0 2 4 6 8 10 120
200
400
600
800
1000
1200
Days
Tum
or V
olum
e (m
m^3
) HCC1806
Notch W
T
Notch ALT
-100
-50
0
50
100
Tum
or s
ize
(% o
f con
trol
)
p = 0.0485
Tumorregression
Tumor growthinhibition
Supplementary Figure 11a
b
80 KDa
110 KDa
160 KDa
NICD1
MDAMB231
- - + +HBCx14
- - + +PF4014
HCC1599
- - + +
GAPDH40 KDa
c
HBCx14 HCC1599HBCx12B
- - + + - - + + - +- +PF4014
110 KDa
160 KDa
NICD1
GAPDH 40 KDa
110 KDa
160 KDa
NICD1
HCC1937 HBCx14 HCC1599
- + + - -+ +PF4014
GAPDH 40 KDa
-
- + - + - + - + - + - + - + - + - + - + - + - +
HBCx5 HBCx12B HBCx17 HCC1806 BT474 HCC1937 MDAMB231
AA0869 HBCx14 AA1077 MAXF1162
HCC1599
Notch wt Notchmut
0
10
20
30
40
50
60
HES4
Rela
tive
Expr
essio
n
- + - + - + - + - + - + - + - + - + - + - + - +
HBCx5 HBCx12B HBCx17 HCC1806 BT474 HCC1937 MDAMB231
AA0869 HBCx14 AA1077 MAXF1162
HCC1599
Notch wt Notchmut
0
1
2
3
4
5
6
7
8
HEY1
Rela
tive
Expr
essio
n
- + - + - + - + - + - + - + - + - + - + - + - +
HBCx5 HBCx12B HBCx17 HCC1806 BT474 HCC1937 MDAMB231
AA0869 HBCx14 AA1077 MAXF1162
HCC1599
Notch wt Notchmut
0
20
40
60
80
100
120
140
HEY2Re
lativ
e Ex
pres
sion
- + - + - + - + - + - + - + - + - + - + - + - +
HBCx5 HBCx12B HBCx17 HCC1806 BT474 HCC1937 MDAMB231
AA0869 HBCx14 AA1077 MAXF1162
HCC1599
Notch wt Notchmut
0
1
2
3
4
5
6
7
8
HEYL
Rela
tive
Expr
essio
n
Notch wt Notch mutantNotch wt Notch mutant
Notch wt Notch mutant Notch wt Notch mutant
Supplementary Figure 12
PF-4014 PF-4014
PF-4014PF-4014
**
* *
*
**
*
*
- + - + - + - + - + - + - + - + - + - + - + - +
HBCx5 HBCx12B HBCx17 HCC1806 BT474 HCC1937 MDAMB231
AA0869 HBCx14 AA1077 MAXF1162
HCC1599
Notch wt Notchmut
0
1
2
3
4
5
6
7
8
MYCRe
lativ
e Ex
pres
sion
- + - + - + - + - + - + - + - + - + - + - + - +
HBCx5 HBCx12B HBCx17 HCC1806 BT474 HCC1937 MDAMB231
AA0869 HBCx14 AA1077 MAXF1162
HCC1599
Notch wt Notchmut
0
20
40
60
80
100
120
140
160
NRARP
Rela
tive
Expr
essio
n
- + - + - + - + - + - + - + - + - + - + - + - +
HBCx5 HBCx12B HBCx17 HCC1806 BT474 HCC1937 MDAMB231
AA0869 HBCx14 AA1077 MAXF1162
HCC1599
Notch wt Notchmut
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
CCND1
Rela
tive
Expr
essio
n
- + - + - + - + - + - + - + - + - + - + - + - +
HBCx5 HBCx12B HBCx17 HCC1806 BT474 HCC1937 MDAMB231
AA0869 HBCx14 AA1077 MAXF1162
HCC1599
Notch wt Notchmut
0
5
10
15
20
25
30
35
40
NOTCH3
Rela
tive
Expr
essio
n
Notch wt Notch mutant
Notch wt Notch mutant
Notch wt Notch mutant
Notch wt Notch mutant
Supplementary Figure 13
PF-4014 PF-4014
PF-4014PF-4014
*
*
*
* * *
***
*
*
**
*