Presentation on theme: "SKMEL-19: T1799A (V600E), homozygous GTG -> GAG Supplemental Figure 1. Representative Mass Spectrometry and Sanger traces for the SKMEL-19 and SKMEL-32."— Presentation transcript:
SKMEL-19: T1799A (V600E), homozygous 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 V600E BRAF mutant cell lines. Forty-eight V600E BRAF 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 ( V600E BRAF, PTEN null) is included in both panels 1 and 2. WM1361A and SKMEL-31 are BRAF wild-type. Panel 2 SKMEL-19SKMEL-28SKMEL-39SKMEL-22 SKMEL-192SKMEL-105SKMEL-188 SKMEL-267SKMEL-7 HCC 364Colo205 A673DU 4475 Panel 4 SKMEL-190SKMEL-202SKMEL-73SKMEL-32SKMEL-41SKMEL-90SKMEL-269SKMEL-31SKMEL-276SKMEL-200SKMEL-228SKMEL-207SKMEL-284 Panel 3 SKMEL-1SKMEL-40SKMEL-64SKMEL-133SKMEL-178SKMEL-196SKMEL-222SKMEL-232SKMEL-238SKMEL-239SKMEL-244SKMEL-275SKMEL-283 PTEN pAkt (S473) pAkt (T308) β Actin PTEN pAkt (S473) β Actin PTEN pAkt (S473) β Actin Panel 1 SKMEL-37WM88WM1361AMalme3MA2058SKMEL-11SKMEL-39SKMEL-100RKO-1NPAFROHT29 PTEN pAkt (S473) pAkt (T308) β Actin V600E BRAF Non melanoma BRAF WT melanoma V600E BRAF melanoma Supplemental Figure 2
SKMEL19/ WT PTEN SKMEL207/ 723-724insTT PTEN GT TT TG A B Copy number (log2) 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). Copy number (log2) Genome position (10q23.2-q23.31)
SKMEL-133 ( V600E BRAF, PTEN null, RB1 wt) SKMEL-207 ( V600E BRAF, PTEN/RB1 null) Supplemental Figure 4 Supplemental Figure 4. Combined targeting of MEK and PI3K in V600E BRAF, PTEN-null melanoma cell lines. SKMEL-133 ( V600E BRAF, 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.
Malme3M SKMEL-267 SKMEL-1 SKMEL-32 A375 SKMEL-188 SKMEL-11SKMEL-28 WM-88 SKMEL-276SKMEL-228SKMEL-239 SKMEL-41 SKMEL-19 SKMEL-284 SKMEL-178 SKMEL-105 SKMEL-39 SKMEL-207 SKMEL-269 A2058 SKMEL-190 SKMEL-133 pAkt(S473) p16 p27 p130 β Actin RB1 p21 pAkt(T308) Akt PTEN PTEN expressingPTEN null Supplemental Figure 5. RB1 and p16 loss are non-overlapping in melanoma cells lines harboring V600E BRAF mutations. A. Immunoblots of PTEN, pAkt, Akt, p16, p21, p27, RB1 and p130 in 23 melanoma cells lines harboring V600E BRAF 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 ( V600E BRAF, PTEN-null, RB1 wt) were stably infected with retroviral E7, a dysfunctional E721-24 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/vectorMEL39/E7 MEL39/E7 21-24
SKMEL133 ( V600E BRAF, PTEN null, RB1 wt)SKMEL39 ( V600E BRAF, PTEN null, RB1 wt) Supplemental Figure 7 Supplemental Figure 7. Effect of MEK inhibition on the growth of V600E BRAF/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.