SKMEL-19: T1799A (V600E), homozygous GTG -> GAG GTG -> GAG Supplemental Figure 1. Representative Mass Spectrometry and Sanger traces for the SKMEL-19 and SKMEL-32 cell lines. SKMEL-19 harbored a homozygous mutation of BRAF (T1799A, V600E) whereas SKMEL-32 harbored a heterozygous mutation. Supplemental Figure 1 SKMEL-32: T1799A (V600E), heterozygous
SKMEL-19: T1799A (V600E), homozygous GTG -> GAG Supplemental Figure 1. Representative Mass Spectrometry and Sanger traces for the SKMEL-19 and SKMEL-32.
Mar 26, 2015
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SKMEL-19: T1799A (V600E), homozygous
GTG -> GAG
GTG -> GAG
Supplemental Figure 1. Representative Mass Spectrometry and Sanger traces for the SKMEL-19 and SKMEL-32 cell lines. SKMEL-19 harbored a homozygous mutation of BRAF (T1799A, V600E) whereas SKMEL-32 harbored a heterozygous mutation.
Supplemental Figure 1
SKMEL-32: T1799A (V600E), heterozygous
Supplemental Figure 2. PTEN expression in the class of V600EBRAF mutant cell lines. Forty-eight V600EBRAF mutant cell lines (40 melanoma and 8 non-melanoma) were assessed for PTEN and phosphorylated AKT expression. Nine PTEN null cell lines (also shown in Figure 1) were identified and used in subsequent studies. The SKMEL-39 cell line (V600EBRAF, PTEN null) is included in both panels 1 and 2. WM1361A and SKMEL-31 are BRAF wild-type.
Panel 2
SK
ME
L-1
9
SK
ME
L-2
8
SK
ME
L-3
9
SK
ME
L-2
2
SK
ME
L-1
92
SK
ME
L-1
05
SK
ME
L-1
88
SK
ME
L-2
67
SK
ME
L-7
HC
C 3
64
Co
lo20
5
A67
3
DU
447
5
Panel 4
SK
ME
L-1
90
SK
ME
L-2
02
SK
ME
L-7
3
SK
ME
L-3
2
SK
ME
L-4
1
SK
ME
L-9
0
SK
ME
L-2
69
SK
ME
L-3
1
SK
ME
L-2
76
SK
ME
L-2
00
SK
ME
L-2
28
SK
ME
L-2
07
SK
ME
L-2
84
Panel 3
SK
ME
L-1
SK
ME
L-4
0
SK
ME
L-6
4
SK
ME
L-1
33
SK
ME
L-1
78
SK
ME
L-1
96
SK
ME
L-2
22
SK
ME
L-2
32
SK
ME
L-2
38
SK
ME
L-2
39
SK
ME
L-2
44
SK
ME
L-2
75
SK
ME
L-2
83
PTEN
pAkt (S473)
pAkt (T308)
β Actin
PTEN
pAkt (S473)
β Actin
PTEN
pAkt (S473)
β Actin
Panel 1
SK
ME
L-3
7
WM
88
WM
1361
A
Mal
me3
M
A20
58
SK
ME
L-1
1
SK
ME
L-3
9
SK
ME
L-1
00
RK
O-1
NP
A
FR
O
HT
29
PTEN
pAkt (S473)
pAkt (T308)
β Actin
V600E BRAF Non melanoma
BRAF WT melanoma
V600E BRAF melanoma
Supplemental Figure 2
SKMEL19/WTPTEN
SKMEL207/723-724insTTPTEN
GT
T T
1 100 Pten MTAIIKEIVSRNKRRYQEDGFDLDLTYIYPNIIAMGFPAERLEGVYRNNIDDVVRFLDSKHKNHYKIYNLCAERHYDTAKFNCRVAQYPFEDHNPPQLEL Mel207_Ins MTAIIKEIVSRNKRRYQEDGFDLDLTYIYPNIIAMGFPAERLEGVYRNNIDDVVRFLDSKHKNHYKIYNLCAERHYDTAKFNCRVAQYPFEDHNPPQLEL 101 200 Pten IKPFCEDLDQWLSEDDNHVAAIHCKAGKGRTGVMICAYLLHRGKFLKAQEALDFYGEVRTRDKKGVTIPSQRRYVYYYSYLLKNHLDYRPVALLFHKMMF Mel207_Ins IKPFCEDLDQWLSEDDNHVAAIHCKAGKGRTGVMICAYLLHRGKFLKAQEALDFYGEVRTRDKKGVTIPSQRRYVYYYSYLLKNHLDYRPVALLFHKMMF 201 300 Pten ETIPMFSGGTCNPQFVVCQLKVKIYSSNSGPTRREDKFMYFEFPQPLPVCGDIKVEFFHKQNKMLKKDKMFHFWVNTFFIPGPEETSEKVENGSLCDQEI Mel207_Ins ETIPMFSGGTCNPQFVVCQLKVKIYSSNSGPTRREDKFMYFLSSLSRYLCVVISK
T G
A
B
Co
py
nu
mb
er (
log
2)
Chromosome 10 position (mb)
SKMEL190
Supplemental Figure 3
C
Supplemental Figure 3. Loss of PTEN expression in melanoma cell lines was the result of mutation or focal deletion. A. Sanger sequencing of cDNA generated from the SKMEL-207 identifying a homozygous two base pair insertion c.723_724insTT in the PTEN mRNA (shown) and corresponding exon sequencing data (not shown). This insertion results in early truncation of the protein. SKMEL-19 expresses wild type PTEN and is shown for comparison. B. Probe level and segmentation data for the SKMEL-133 and SKMEL-39 cell lines showing a focal deletion of the PTEN gene in SKMEL-133. SKMEL-39 cells are copy number neutral at the PTEN locus. C. Probe-level and segmentation data for the SKMEL-190 cell line from both Agilent 244K- and 1M-feature arrays (gray and blue/red respectively).
Co
py
nu
mb
er (
log
2)
Genome position (10q23.2-q23.31)
SKMEL-133 (V600EBRAF, PTEN null, RB1 wt)
SKMEL-207 (V600EBRAF, PTEN/RB1 null)
Supplemental Figure 4
Supplemental Figure 4. Combined targeting of MEK and PI3K in V600EBRAF, PTEN-null melanoma cell lines. SKMEL-133 (V600EBRAF, PTEN null, RB1 wt) cells and SKMEL-207 (V600E BRAF, PTEN null, RB1 null) cells were treated with either the MEK inhibitor PD0325901 (50 nM), the PI3 kinase inhibitor PI-103 (2.5 µM) or the combination. Induction of cell death was determined by FACS following 6, 24 and 48 hours of drug exposure. Error bars represent standard deviation of triplicate experiments.
pAkt(S473)
p16
p27
p130
β Actin
RB1
p21
pAkt(T308)
Akt
PTEN
PTEN expressing PTEN null
Supplemental Figure 5. RB1 and p16 loss are non-overlapping in melanoma cells lines harboring V600EBRAF mutations. A. Immunoblots of PTEN, pAkt, Akt, p16, p21, p27, RB1 and p130 in 23 melanoma cells lines harboring V600EBRAF mutations. Two cell lines, SKMEL-207 and A2058, expressed no RB1 protein. Both of these cell lines expressed wild-type p16.
Supplemental Figure 5
Supplemental Figure 6
A
B
Supplemental Figure 6. Loss of RB1 function confers resistance to PLX4720. A. SKMEL-39 cells (V600EBRAF, PTEN-null, RB1 wt) were stably infected with retroviral E7, a dysfunctional E7∆21-24 mutant or empty vector. IC50 values for the RAF inhibitor PLX4720 were determined by Alamar Blue assay on Day 5 of drug treatment. B. S Phase changes of the same cells as in A after 2.5 μM PLX4720 treatment for 48 hours. The error bars represent the standard deviation of three experiments.
MEL39/vector MEL39/E7 MEL39/E721-24
SKMEL133 (V600EBRAF, PTEN null, RB1 wt)SKMEL39 (V600EBRAF, PTEN null, RB1 wt)
Supplemental Figure 7
Supplemental Figure 7. Effect of MEK inhibition on the growth of V600EBRAF/PTEN-null xenograft models. Established SKMEL-39 (A) and SKMEL-133 (B) xenografts were treated with PD0325901 (25 mg/kg/day by oral gavage on days 1-5 each week) or vehicle only as control. Error bars represent standard error.
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