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

*

*

*

* * *

***

*

*

**

*