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.

M

el 1

13

M

ewo

S

K-M

el-1

03

S

K-M

el-2

17

S

K-M

el-3

0

T

24

HRAS-GTP

A. B.

NF1-null

NRAS

mut

KRAS

am

p

NRAS

mut

HRA

S m

utC.

RAS-GTP

SK-M

el-2

39

Mal

me3

M

M30

8

SK-M

el-1

03

SK-M

el-1

13

SK-M

el-2

17

MeW

o

WM

3918

SK-M

el-2

66 –

NF1

-nul

l

SK-M

el-1

73 –

NRA

S (Q

61K)

SK-M

el-3

64 –

BRA

F(V6

00E)

NF1

RAS

Tubulin

NF1-nullBRAF(V600E)

SK-M

el-3

0

SK-M

el-1

73

SK-M

el-1

18

SK-M

el-2

85

SK-M

el-1

13

MeW

o

SK-M

el-1

87

WM

1361

WM

1382

SK-M

el-3

26

SK-M

el-3

04

SK-M

el-3

64

Mal

me3

M

NRAS(Q61K) BRAF(V600E)

NF1

RAS-GTP

Pan-RAS (dark)

Pan-RAS (light)

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.

MeWo(NF1-null)

SK-Mel-103(NF1-null, NRAS (Q61R))

SK-Mel-113(NF1-null)

pERK

ERK

Cyclin D1

pMEK

MEK

Time(hrs)

[PD0325901]

SK-Mel-239(BRAF V600E)

0 1 6 24

50 nM 500 nM

1 6 24

SK-Mel-217(NF1-null; KRAS amp)

Malme3M(BRAF V600E)

0 1 6 24

50 nM 500 nM

1 6 240 1 6 24

50 nM 500 nM

1 6 240 1 6 24

50 nM 500 nM

1 6 240 1 6 24

50 nM 500 nM

1 6 240 1 6 24

50 nM 500 nM

1 6 24

Nissan MH- NF1 in melanoma

A.

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

B.A.

0 1 6 24 1 6 24 1 6 24 (hours)

50nM PD0325901

2μM MK2206

50nM PD0325901

+2μM

MK2206

pAKT (S473)

pERK

α-Tubulin

ERK

AKT

SK-Mel-113

SK-Mel-239

SK-Mel-113

MeWo

SK-Mel-103

0 1 6 24 1 6 24

50nMPD0325901

50nMtrametinib

pMEK (S217/221)

0 1 6 24 1 6 24

50nMPD0325901

50nM trametinib

MEK

BRAF(V600E)

NF1 null

Nissan MH- NF1 in melanoma

pERK pERK

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.

B

A

C D

(h)

PD0325901

Trametinib

AZD6244

MEK162

0 3 10 30 100 300 0 3 10 30 100 300(nM) (nM)

SK-Mel-239(BRAF V600E)

SK-Mel-113(NF1-null)

pMEK

MEK

pERK

ERK

0 1 6 24 1 6 24 1 6 24

50 nM trametinib

1 μM AZD6244

50nMMEK162

MeWo(NF1-null)

(h)

Nissan MH- NF1 in melanoma

M308BRAF 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

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 non-targeting (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.

0 1 6 24 0 1 6 24

No T

RFCT

No si

RNA

siNT siNF1

RasGTP in A375 +/- siNF1 with 2 μM vemurafenib

Nor

mal

ized

RasG

TP le

vels

Hours with Vemurafenib

NF1

RAS-GTP

A B

(hours)

C

* = p<0.01 for each shNF1 vs shNT

pMEK

MEK

pERK

ERK

HA

0 1 6 24 0 1 6 24

HA-NRAS(G12V)Transfection ctl

No DNA

SK-Mel-239BRAF (V600E)

(hours)

Nissan MH- NF1 in melanoma

A375

SK-M

el- 2

39

M30

8

SK-M

el-1

13

RAS-GTP

BRAF(V600E)

NF1-null

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.

A B

C D

A375(BRAF V600E)

2μM vemurafenib

RAS

pMEK

MEK

pERK

ERK

0 1 6 24 (hours)

Nissan MH- NF1 in melanoma

Figure S8. The MAPK pathway and the inhibitors used in the study of NF1-null cells