Data Supplement

Report
BRAFmut
SK-Mel-364 – BRAF(V600E)
SK-Mel-266 – NF1-null
WM3918
MeWo
SK-Mel-217
SK-Mel-113
SK-Mel-103
M308
Malme3M
SK-Mel-239
Mel 304
Mel 364
Malme3M
Malme 3M
SK-Mel-364
Mel 326
SK-Mel-304
NF1
NF1
SK-Mel-173 – NRAS (Q61K)
B.
BRAF(V600E)
SK-Mel-326
WM1382
WM1382
WM1361
WM1361
SK-Mel-187
Mel 187
cKIT
NF1del
MeWo
MeWo
SK-Mel-113
Mel 113
KRASmut
SK-Mel-285
Mel 285
SK-Mel-173
Mel 173
SK-Mel-30
Mel 30
NRAS(Q61K)
SK-Mel-118
Mel 118
NRASmut
A.
NF1
RAS-GTP
Ras GTP
RAS-GTP
Pan-RAS (dark)
RAS
(Dark)
Pan-Ras
KRAS amp
NRAS mut
SK-Mel-217
SK-Mel-30
T 24
NRAS mut
SK-Mel-103
Mewo
Mel 113
C.
NF1-null
HRAS mut
BRAF(V600E)
Pan-RAS (light)
(Light)
HRAS-GTP
Tubulin
NF1-null
Figure S1. Levels of RAS-GTP in melanoma cell lines. A, B, C) Levels of active RAS (RAS-GTP) in melanoma cell lines as a function
of RAS and BRAF mutational status. Cells were harvested 24h after change in media. The T24 bladder cell line shown in panel C is
a bladder cancer cell line with HRAS mutation that was included as a positive control.
A.
Malme3M
(BRAF V600E)
[PD0325901]
Time(hrs)
50 nM
500 nM
0 1 6 24 1 6 24
SK-Mel-239
(BRAF V600E)
50 nM
500 nM
0 1 6 24 1 6 24
SK-Mel-113
(NF1-null)
50 nM
500 nM
0 1 6 24 1 6 24
SK-Mel-103
(NF1-null, NRAS (Q61R))
50 nM
500 nM
0 1 6 24 1 6 24
MeWo
(NF1-null)
50 nM
500 nM
0 1 6 24 1 6 24
SK-Mel-217
(NF1-null; KRAS amp)
50 nM
500 nM
0 1 6 24 1 6 24
pMEK
MEK
pERK
ERK
Cyclin D1
Figure S2. Changes in pERK and cyclin D1 levels as function of time in cells treated with the MEK inhibitor PD0325901.
Melanoma cell lines were treated with 50 or 500nM of the MEK inhibitor PD0325901 for 0, 1, 6 or 24 hours. Changes in pMEK,
MEK, pERK, ERK and cyclin D1 were quantitated by immunoblot.
Nissan MH- NF1 in melanoma
A.
B.
2μM
MK2206
50nM
PD0325901
0
pERK
ERK
1
6
24
1
6
24
50nM
PD0325901
+
2μM
MK2206
1
6
24
Proliferation of SK-Mel-113 on day 5 with drug
1.5
(hours)
Cell Count (x106)
SK-Mel-113
1.0
0.5
pAKT (S473)
0.0
AKT
α-Tubulin
DMSO
50nM PD0325901
2uM MK2206
50nM PD0325901 + 2uM MK2206
Figure S3. Sk-Mel-113 (NF1 null) melanoma cells are resistant to AKT inhibition. A) Changes in pAKT and pERK levels as a
function of time post-treatment with 50 nM PD0325901 (MEK inhibitor), 2μM MK2206 (AKT inhibitor), or the combination. B)
Viable cell count following 5 days of treatment with 50nM PD0325901 (MEK inhibitor), 2μM MK2206 (AKT inhibitor), or the
combination.
Nissan MH- NF1 in melanoma
A
50nM
PD0325901
0
1
6
50nM
trametinib
24
1
6
50nM
PD0325901
0
24
1
6
50nM
trametinib
24
24 (h)
6
1
BRAF(V600E)
SK-Mel-239
SK-Mel-113
NF1 null
MeWo
SK-Mel-103
MEK
pMEK (S217/221)
SK-Mel-239
(BRAF V600E)
B
0
3
10
30
100
SK-Mel-113
(NF1-null)
300
(nM)
0
3
10
30
100
300
(nM)
PD0325901
Trametinib
AZD6244
MEK162
pERK
pERK
PD0325901
C
Trametinib
50
0
-1
0
1
2
50 nM
trametinib
0
0
1
2
% pERK
log (nM AZD6244)
3
6
24
1
6
24
50nM
MEK162
1
6
24
(h)
3
MEK
pERK
100
% pERK
0
2
MEK162
50
-1
1
log (nM Trametinib)
SK-Mel-239
SK-Mel-113
1
1 μM
AZD6244
pMEK
-1
AZD6244
100
MeWo
(NF1-null)
50
0
3
SK-Mel-239
SK-Mel-113
log (nM PD0325901)
0
D
100
SK-Mel-239
SK-Mel-113
% pERK
% pERK
100
SK-Mel-239
SK-Mel-113
ERK
50
0
-1
0
1
2
3
log (nM MEK162)
Figure S4. Variability in induction of pMEK levels following exposure to four allosteric MEK inhibitors. A) pMEK and total MEK
levels were measure by immunoblot in NF1-null or BRAF (V600E) cells following treatment with 50nM PD0325901 or 50nM
trametinib for 0, 1, 6 or 24 hours. B) Change in pERK levels as a function of drug concentration in Sk-Mel-239 and Sk-Mel-113
cells. Cells were treated with PD0325901, trametinib, AZD6244 or MEK162 and collected at 1 hour. C) Quantitation of (B) by
densitometry. Percentages were calculated using the equation 100*([value of dose]-[value of background])/([value of 0nM][value of background]). D) Differences in the induction of pMEK by trametinib, AZD6244 and MEK162 in MeWo (NF1 null) cells.
Nissan MH- NF1 in melanoma
M308
BRAF V600E, NF1 Q1070*
(89% / 243)
Figure S5. Exon-capture deep sequencing via IMPACT of the NF1 gene in M308 shows a nonsense Q1070* mutation.
Nissan MH- NF1 in melanoma
Levels of RasGTP in A375 with 2uM vemurafenib
with or without NF1
1
6
24
HA-NRAS(G12V)
0
1
6
(hours)
24
pMEK
10
5
0
0
MEK
24
0
6
Transfection ctl
No DNA
siNF1
siNT2
15
1
SK-Mel-239
BRAF (V600E)
Normalized RasGTP levels
B
RasGTP in A375 +/- siNF1
with 2 μM vemurafenib
20
RasGTP normalized to
no transfection control time 0
A
Hours with Vemurafenib
No siRNA
No TRFCT
Hours with Vemurafenib
pERK
siNT
0
ERK
1
siNF1
6
24
0
1
6
24 (hours)
NF1
HA
RAS-GTP
A375
A375 shNF1 with Vemurafenib
C
*
shNT
shNF1 #2 + DOX
shNF1 #4 + DOX
shNF1#6 + DOX
*
% growth
% growth
*
50
0
shNF1 with Vemurafenib
100
100
50
shNT
shNF1 #2 + DOX
shNF1 #4 + DOX
shNF1#6 + DOX
* = p<0.01 for each shNF1 vs shNT
*
*
*
0
2000
4000
[Vemurafenib]
6000
0
0
2000
4000
6000
[Vemurafenib]
shNF1 #2
2 ug/mL doxycycline
-
+
shNF1 #4
-
+
shNF1 #6
-
+
NF1
Figure S6. Expression of activated RAS is sufficient to confer resistance to vemurafenib in Sk-Mel-239 (BRAF V600E) cells. A)
SK-Mel-239 cells were transfected with constitutively active NRAS (G12V) or a no-DNA transfection control. Cells were then
treated with 2μM vemurafenib for 0, 1, 6, or 24 hours. B) Quantification of activated RAS (RAS-GTP) in a BRAF (V600E) mutant
melanoma cell line (A375) following knockdown of NF1 by siRNA and treatment with 2μM vemurafenib. Controls include nontargeting (siNT), no transfection (no TRFCT), and no siRNA. RAS-GTP values normalized to no-siRNA control. C) Total cell counts
following 4 days of treatment with increasing concentrations of vemurafenib (nM) following NF1 knockdown with three different
shRNAs. shRNA targeting NF1 or a NT control were expressed under a tet-on promoter. Values shown represent percent DMSO
control. Error bars are SEM, n=3.
Nissan MH- NF1 in melanoma
A375
2uMA375
PLX4032
A
B
A375 with Vemurafenib
(BRAF V600E)
00
11
66 24
24
(h)
(hours)
Cell count (millions)
2μM vemurafenib
2.0
pMEK
pMEK
MEK
pERK
pERK
MEK
0nM
2nM
20nM
200nM
2000nM
5000nM
1.5
1.0
0.5
0.0
ERK
ERK
Day 0
Day 3
Day 5
Days
% growth
A375
100
SK-Mel-239 (BRAF(V600E)/NF1WT)
A375 (BRAF(V600E)/NF1WT)
M308 (BRAF(V600E)/NF1-null)
50
SK-Mel-113
IC50 curve of BRAF mutant cell lines with Vemurafenib
M308
D
SK-Mel- 239
C
RAS-GTP
RAS
BRAF(V600E)
NF1-null
0
0
1
2
3
4
log ([PLX4032]) (nM)
Figure S7. Sensitivity of A375 (BRAF V600E) cells to vemurafenib. A) A375 cells were treated with 2μM vemurafenib for 0, 1, 6,
or 24 hours and downstream RAF effectors were assessed by immunoblot. B) A375 cells were treated with increasing doses of
vemurafenib and cell numbers were quantitated at 0, 3, or 5 days. Error bars are SEM, n=3. C) BRAF (V600E) mutant M308, A375
and SK-Mel-239 cells were treated with increasing doses of vemurafenib for 5 days, and percent cell growth was plotted as a
function of drug concentration. D) Quantitation of active RAS levels in A375, SK-Mel-239, M308 and SK-Mel-113.
Nissan MH- NF1 in melanoma
Figure S8. The MAPK pathway and the inhibitors used in the study of NF1-null cells
Nissan MH- NF1 in melanoma

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