Molecular Cell, Volume 77

Supplemental Information

Paradoxical Role for Wild-Type p53

in Driving Therapy Resistance in Melanoma
Marie R. Webster, Mitchell E. Fane, Gretchen M. Alicea, Subhasree Basu, Andrew V.
Kossenkov, Gloria E. Marino, Stephen M. Douglass, Amanpreet Kaur, Brett L.
Ecker, Keerthana Gnanapradeepan, Abibatou Ndoye, Curtis Kugel, Alexander
Valiga, Jessica Palmer, Qin Liu, Xiaowei Xu, Jessicamarie Morris, Xiangfan Yin, Hong
Wu, Wei Xu, Cathy Zheng, Giorgos C. Karakousis, Ravi K. Amaravadi, Tara C.
Mitchell, Filipe V. Almeida, Min Xiao, Vito W. Rebecca, Ying-Jie Wang, Lynn M.
Schuchter, Meenhard Herlyn, Maureen E. Murphy, and Ashani T. Weeraratna
Supplemental Table 1. Driver mutations in PDX models.
PDX model Mutations
WM4351 BRAFV600E, TERT, MAP3K9, MAP3K9
WM4298 BRAFV600E, BRCA2, KIT
WM4070 BRAFV600K, MAP2K1, ERBB4, TERT
Related to Figure 6 and Figure S6.

Supplemental Table 2. Primers

Oligonucleotides
Wnt5A forward primer ATT CTT GGT GGT CGC TAG GTA This paper N/A
Wnt5A reverse primer CGC CTT CTC CGA TGT ACT GC This paper N/A
TP53 forward 1 primer 1 GTG GAA GGA AAT TTG CGT GT This paper N/A
TP53 reverse 1 primer CCA GTG TGA TGA TGG TGA GG3 This paper N/A
KDM5B forward 1 primer GGA TAC GTG GCG TAA AAT GAA This paper N/A
KDM5B forward 2 primer CAA TGC TGT GGA CCT GTA TGT This paper N/A
KDM5B reverse 2 primer TAC GGA GGG TAT AGT CCC TGG3 This paper N/A
PUMA forward primer GTC CCC TGC CAG ATT TGT G This paper N/A
PUMA reverse primer AGA GGC CGC AGG ACA CTG This paper N/A
TP53 forward mouse primer AAG ATC CGC GGG CGT AA This paper N/A
TP53 reverse mouse primer CAT CCT TTA ACT CTA AGG CCT CAT TC This paper N/A
Cyclophilin A forward GGG TTC CTC CTT TCA CAG AA This paper N/A
Cyclophilin A reverse GAT GCC AGG ACC TGT ATG CT This paper N/A
Related to Star Methods.
 Supplemental Figure 1
A 15 B FS4 Day 5 FS5 Day 5 1205Lu Day 5 WM164 Day 5
a a a
12 a
Wnt5A/18s mRNA

9
6 b b
3 b
b
2
1
0
FS 3

4
W Lu

13

14

W 2
4

12 5

79

16
1
FS

FS

05

FS

FS
M

M
Cell Line
C D E FS4 CTRL
FS4 shWNT5A_2
Slow Cycling

Slow Cycling

Cycling
2.8 Slow Cycling Day 8: CTRL Day 8: shWnt5A_1 8 p=0.03 p=0.04
Wnt5A/18s mRNA
Cycling

Cycling

% of Slow Cycling Cells

2.6
6
2.4
Wnt5A 4
1.1 1 2.7 1 1.0
HSP90 2
FS4 FS5 FS4 FS5
Cell Line 0
6 8
Day
F G H
p=0.002 p< 0.03
Wnt5A High 9.5
p=0.006 Wnt5A High
2.0 8
p=0.03
JARID1B/18s mRNA

p53/18s mRNA

9.0
JARID1B/b-Tubulin

1.5 6 p=0.01
8.5
Wnt5A Low
1.0 4 4 Wnt5A Low

0.5 2
2
0
0.0 0
3
W u

13

14
4

79
L

FS4 FS5 FS13 FS14

FS

FS

05

FS4 FS5 FS13FS14

FS

FS
M
12

Cell Line Cell Line Cell Line

I Cycling J Cycling K FS5

Slow Cycling

2.5 Cycling
Slow Cycling

Slow Cycling 34
1.61 Slow Cycling Slow Cycling 100 p<0.001
32 Cycling
2.0
Cycling

Cycling

1.60 80 Slow Cycling

p21/18s mRNA
p21/HSP90

30
1.59 1.5
p53/HSP90

% of Cells

28 60
p53 1.58
6
1.0 1.0
40
p21 0.9 4 p=0.002
0.5 20 n.s.
HSP90 0.8 2
FS4 FS5 0.7 0.0 0 0
FS4 FS5 FS4 FS5 FS5 1205Lu G0/1 S G2/M
Cell Line Cell Cycle Phase
Cell Line Cell Line
 A

B
5%
0%

15%
10%

FDR
G361
SH4
HS936T
SKMEL1
IGR1
COLO679
COLO792
C32
UACC257
K029AX
Supplemental Figure 2

UACC62
MELHO
SKMEL5
COLO829
COLO849
MALME3M
HMCB
HS939T
WM88
GRM
HS895T
HS852T
RVH421
HS944T
SKMEL31
HS940T
WM2664
A101D
WM1799
COLO800
SKMEL24
HS688AT
HS600T
HT144
WM793
HS934T
MELJUSO
LOXIMVI
A375
WM115
HS695T
HS294T
Negative correlation Positive correlation
TP53

n=206 n=137
expression z-score
WNT5A

cellines

0
1
2

-2
-1
0.5
1.5

-1.5
-0.5
 Supplemental Figure 3
A B
2.0
FS4 p53 pull down
Normalized to GAPDH

FS5 Total Protein

1.5 FS13

FS13
FS14
FS14

FS13
FS14
FS4
FS5

FS4
FS5
1.0
p-MDM2ser395 pMDM2ser395
0.5
p53 p53
0.0
MDM2 MDM4

C D FS4 FS14
3.0 p=0.03 6
8 n.s.

% of Apoptotic Cells
p<0.001
% of Apoptotic Cells
PUMA/18s mRNA

CTRL
2.5
+Dox 6
2.0 4
1.5 4
n.s. 2
0.1 2

0 0
0.0
RL

ox
ox

RL

NA

ox
ox
NA

+D
+D

FS4 FS13

+D
+D
CT

CT

siR
siR

NA

NA
Cell Line
P

P
SP

SP
siR

siR
iA

iA
P

P
SP

SP
iA

iA
E F
5 p=0.08 FS4 FS5 WM793
p=0.02 CTRL
n.s. 0.012 0.05
Go6983 0.8
% of Apoptotic Cells

4 n.s.
Doxorubicin 0.010 Wnt5A/18s mRNA 0.04
Wnt5A/18s mRNA
Wnt5A/18s mRNA

3 Dox +Go 0.6

0.008
0.03
2 0.4 0.006
0.02
0.004
1 0.2 0.01
0.002
0 0.00
0.0 0.000
FS4 FS5
RL

RL

_1

_2
L

_1

_2

_2

Cell Line
TR

5A

5A
5A

5A

CT

CT
5A
C

W
W

nt

sh

sh
sh

sh

W
sh
Supplemental Figure 4
A WM35
****
Young
100 Aged
Proliferation
(Finitial/Fx)

75
****
50 **
25
0
4 6 8 10
Day
B Aged Cond. - - +
Young Cond. - + -
2.5
CTRL + - - DMEM
Normalized to HSP90

2.0 Young Conditioned Media

p53
Aged Conditioned Media
1.5
p21
1.0
Wnt5A
0.5

HSP90 0.0
p53 p21 Wnt5A
WM35

C D
Aged Cond. - - + - - +
2.5 DMEM
Young Cond. - + - - + - Young Conditioned Media
2.0
iASPP/HSP90

CTRL + - - + - - Aged Conditioned Media

iASPP 1.5

p53 1.0
p21 0.5
HSP90 0.0
FS4 WM793 FS4 WM793
Cell Line

E
Young Aged
8 wks > 52 wks

Wnt5A

p53
HSP90

C57BL6 Mice
Supplemental Figure 5

A B
MEKi - - + + - - + + 50

BRAFi - - + + - - + + 40

Weight (g)
Pifithrin-α - + - + - + - + 30 Control
Pifithrin-
p53 20
BRAFi/MEKi
1 0.5 1.9 1.1 1 0.97 1.1 0.5 10 BRAFi/MEKi+Pifithrin-

HSP90 0
20 25 30 35 40 45
FS4 FS5 Day
Supplemental Figure 6

A CTRL BRAF/MEKi

Yumm1.7
CTRL

Yumm1.7
shp53

100 μm

B C Wnt5A
p53 (% of Tumor Cells Positive) Ctrl BRAF/MEKi
Ctrl BRAFi
20% 30%
WM4351
WM4070

100 μm

WM4298
D Wnt5A
Ctrl BRAFi 100 μm

WM4070

100 μm