1 Supplemental Figure 1 SKMEL-19: T1799A (V600E), homozygous GTG -> GAGSKMEL-32: T1799A (V600E), heterozygousGTG -> GAGSupplemental 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.
2 Supplemental Figure 2 Panel 1 Panel 2 Panel 3 Panel 4 SKMEL-19SKMEL-28SKMEL-39SKMEL-22SKMEL-192SKMEL-105SKMEL-188SKMEL-267SKMEL-7HCC 364Colo205A673DU 4475Panel 4SKMEL-190SKMEL-202SKMEL-73SKMEL-32SKMEL-41SKMEL-90SKMEL-269SKMEL-31SKMEL-276SKMEL-200SKMEL-228SKMEL-207SKMEL-284Panel 3SKMEL-1SKMEL-40SKMEL-64SKMEL-133SKMEL-178SKMEL-196SKMEL-222SKMEL-232SKMEL-238SKMEL-239SKMEL-244SKMEL-275SKMEL-283PTENpAkt (S473)pAkt (T308)β ActinPanel 1SKMEL-37WM88WM1361AMalme3MA2058SKMEL-11SKMEL-100RKO-1NPAFROHT29V600E BRAF Non melanomaBRAF WT melanomaV600E BRAF melanomaSupplemental 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.
3 Supplemental Figure 3 A B C SKMEL19/WTPTEN SKMEL207/723-724insTTPTEN Copy number (log2)Genome position (10q23.2-q23.31)Copy number (log2)Chromosome 10 position (mb)SKMEL190Supplemental 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 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).
4 (V600EBRAF, PTEN null, RB1 wt) Supplemental Figure 4SKMEL-133(V600EBRAF, PTEN null, RB1 wt)SKMEL-207(V600EBRAF, PTEN/RB1 null)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 PD (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.
5 Supplemental Figure 5 PTEN expressing PTEN null SKMEL-267 Malme3M WM-88A375SKMEL-41SKMEL-19SKMEL-284SKMEL-178SKMEL-105SKMEL-39SKMEL-207SKMEL-269A2058SKMEL-190SKMEL-133PTENpAkt(T308)pAkt(S473)Aktp16p21p27RB1p130β ActinSupplemental 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.
6 Supplemental Figure 6 A B MEL39/vector MEL39/E7 MEL39/E721-24 Supplemental Figure 6. Loss of RB1 function confers resistance to PLX 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.
7 Supplemental Figure 7SKMEL39 (V600EBRAF, PTEN null, RB1 wt)SKMEL133 (V600EBRAF, PTEN null, RB1 wt)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 PD (25 mg/kg/day by oral gavage on days 1-5 each week) or vehicle only as control. Error bars represent standard error.