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SUPPLEMENTARY FIGURE LEGENDS
Figure S1. (A) Ingenuity Pathway Analysis (IPA) of cellular functions associated with
303 genes that showed synthetic lethality with PLX4720 in a genome-wide siRNA
screen in the MEL624 cell line (FDR corrected, p<0.05). The top ten cellular functions
are shown in the graph. x-axis, cellular functions; y-axis, significance (p<0.05) by the
Fisher’s exact test. (B) Netwalker analysis of the PLX4720-synthetic lethal genes.
Genes associated with mitochondrial activity are labeled with a red asterisk. Inset box
shows the line colors of known gene interactions.
Figure S2. Effect of Selumetinib on the mitochondrial gene expression in high OxPhos
MEL624 cell lines. (A) selumetinib (SEL) treatment for 24 h induced >2-fold increase in
the expression of many oxphos genes in the MEL624 (selumetinib-resistant cell line),
compared to the A375 (selumetinib-sensitive) cell line. Quantile normalized, FDRcorrected (p<0.05) Illumina Beadchip gene expression data is in triplicates and error
bars are standard deviation. (B) Ingenuity iReport analysis of the genes (FDR corrected,
p<0.05) associated with the five electron transport chain enzyme complexes. OxPhos
enzyme complexes constituted by proteins whose gene expression is upregulated >2fold are shown in yellow.
Figure S3. Differential energy consumption and release in selumetinib-sensitive and
resistant melanoma cell lines. (A) Basal OCR and ECAR levels determined by
Seahorse analyzer in the Selumetinib-sensitive and –resistant cell lines. Data is
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representative of quadruplicates. (B) Uptake of glucose from (gray bars) and release of
lactate into (black bars) the media by the sensitive and resistant cell lines. Data is
average of triplicate samples; error bars, standard deviation. SEL, selumetinib. (C)
Basal ATP levels in the cell lysates collected from the selumetinib-sensitive (WM35 and
A375) and -resistant (MEL624 and SKMEL5) cell lysates. Cells were grown in 24 well
plates for 24 hours, harvested, and relative luciferase activity as a measure of relative
ATP levels was determined. The data was adjusted for cell numbers, and is average of
triplicate measurements. Error bars, standard deviation.
Figure S4. Oxygen consumption in MEKi+mTORC1/2i combination-resistant and –
sensitive melanoma cell lines. Fourteen selumetinib-resistant melanoma cell lines were
separated into two groups based on their sensitivity to the combination of
selumetininb+AZD8055. Box plots were drawn showing a resistant group with
combination index, CI>1.0 (Red) and a sensitive group with CI<1.0 (Green).
Significance was determined by T-test.
Figure S5. Cellular effects of FDA approved single agent MEK and BRAF inhibitors
Trametinib and Dabrafenib and their combinations with AZD8055. (A) The MEL624
cells were treated with 10nM Trametinib or 100nM Dabrafenib, or their combination with
250nM of AZD8055 for 72 h. FACS-based PI-cell cycle analysis was performed and the
dead cell (sub-G1 phase) cell populations were plotted in bar graphs. Data shown is
triplicate treatments and error bars are standard deviation. (B) Basal OCR levels in
MEL624 cells treated with the indicated inhibitors for 24 h. Data is representative of
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quadruplicate measurements. (C) Fold changes in PGC1 transcript levels in MEL624
cells after treatment with the indicated inhibitors for 24 h. Real-time qPCR data is
representative of triplicate measurements normalized against GAPDH transcript levels.
Figure S6. RPPA proteomic analysis of protein lysates from three combination-resistant
(1) and three –sensitive (2) BRAF mutant melanoma cell lines following MEK or
mTORC1/2 inhibition. The cells were treated with 0.25M of selumetinib (A) or
AZD8055 (B) in triplicates and incubated for the times shown at the right of the
heatmap. Using supervised hierarchical average linkage clustering, data was clustered
into heatmaps showing fold changes in the levels of upregulated (red) and
downregulated (green) proteins in the inhibitor-treated samples versus dmso controls.
(C) Bar graphs of the normalized RPPA data showing the levels of proteins representing
the targeted effects of selumetinib (P-MAPK), AZD8055 (P-S6), feedback regulation (PAKT_T308), and the apoptosis marker cleaved Caspase 7. The cell lines indicated by
green color are resistant to the combination, while cells in red color are sensitive to the
combination. Data is representative of triplicates. (D) RPPA proteomic analysis of
protein lysates from NRAS mutant cell lines WM3854 and WM1361 treated with
selumetinib or AZD8055 or their combination for 24h. Data represents fold changes in
protein levels between treated and DMSO control samples.
Figure S7. Correlation of MITF expression with selumetinib and AZD8055 combination
synergy in 14 melanoma cell lines. (A) Scatter plot showing correlation (R2=0.32;
p<0.05) of MITF transcript levels with combination synergy between selumetinib and
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AZD8055, expressed as Combination Index, CI (y-axis). (B) Correlation of MITF levels
with OCR. Colors on individual spheres indicate BRAF and NRAS mutation status the
cell lines. (C/D) Fold changes in the transcript levels of TYR, TRPM1 and DCT genes in
MEL624/WM3854 cell lines after treatment with 0.25M of the indicated inhibitors,
compared to DMSO controls. (E) WM3854 cells in 6 well plates were transfected with a
PGC1 promoter-driven or TRPM1-promoter-driven luciferase reporter plasmid and a
renilla control plasmid, and incubated for 12 h. The cells were washed and treated with
DMSO or 0.25M of the indicated inhibitors in media for 24 h in triplicates. Lysates were
prepared, firefly and renilla luciferase activities were measured and relative luciferase
units (RLU) plotted against treatments.
Figure S8. (A) Effect of selumetinib and AZD8055 on the cellular localization of MITF.
MEL624 cells were treated with either inhibitor or their combination for 24 h, fixed and
probed with MITF antibody and stained with a FITC-labeled secondary antibody. Nuclei
were stained with DAPI and the images documented with immunofluorescence
microscopy. (B & C) Consequence of MITF knockdown on the effect of selumetinib and
AZD8055 on PGC1and TRPM1 reporters. MEL624 cells were transfected with control
siRNA (black bars) or MITF siRNA (grey bars) for 36 h, followed by transfection with (B)
PGC1 or (C) TRPM1 reporter plasmids for 12 h, followed by inhibitor treatments for 24
h. Luciferase reporter measurements were performed as in S7(E). Data is average of
triplicates. Significant differences (p<0.05) between the mock and inhibitor treatments in
the MITF siRNA-transfected cells are indicated by asterisks over the column bars. (D)
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Western blotting analysis showing the efficacy of siRNA knockdown of indicated
proteins and downstream targets P-S6, P-4EBP1 and P-AKT.
Figure S9. Comparative effects of AZD8055 and AZD2014. The MEL624 cells were
treated with DMSO or 0.25M of AZD8055 or AZD2014 for 12 h, followed by protein
lysate preparation and RPPA analysis. (A) Bar graph showing the relative intensity of
RPPA analyzed downstream protein targets of mTORC1/2, namely, P-4EBP1, Pp70S6K, P-S6 and P-AKT after treatment. (B) Supervised hierarchical clustering of 205
proteins and phospho-proteins expressed in MEL624 cells treated with DMSO,
AZD8055 or AZD2014, showing that both inhibitors display similar, target-specific
effects (target proteins in blue outline). (C) MEL624 cells were treated with selumetinib,
AZD8055, AZD2014 or the combination of either mTOR1/2 inhibitor with selumetinib for
24h. Western blotting was performed to detect the levels of the indicated proteins.
Figure S10. The A375 (A) and WM35 (B) cell lines and their selumetinib-resistant
clones were seeded in 96 well plates overnight and treated in triplicates with increasing
concentrations of selumetinib shown under the x-axes. After 72 h incubation, cell
proliferation was determined using Cell Titer Blue reagent and data was plotted as drug
concentration against percentage of cell growth inhibition. (C) Basal, oligomycininhibited (“O”) and FCCP-activated (“F”) OCR of A375, WM35 and their selumetinibresistant clones (“-R1”, “-R2”) determined by Seahorse flux analysis. Data is average of
quadruplicates. (D/E) RPPA analysis of P-MAPK and P-S6 levels in the parental cells
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and their resistant clones, after treatment with DMSO or the indicated inhibitors at
0.25M concentrations.
Figure S11. The proliferation of the A375 (A), its resistant clones –R1 (B) and -R2 (C),
and WM35 (D) and its resistant clones (E, F) in presence of increasing concentrations
of selumetinib, AZD8055 and their combination after 72 h treatment was determined as
described in Figure S8. (G) The A375 and A375-R1 cells were plated at a density of 300
cells/well in 6-well plates and incubated for a period of 30 days. The cells were
replenished with fresh growth media containing dmso (mock) or 0.25M of the indicated
inhibitors, once every four days for a period of 30 days. The colonies formed in the wells
were stained using crystal violet, washed and counted. Data is representative of
triplicates.
Figure S12. (A) Ratio of PGC1 and MITF gene expression at the time of disease
progression versus pre-treatment in the MGH cohort of patients treated with singleagent vemurafenib (MGH-25) or with dabrafenib + trametinib (all others). Expression
levels in pre-treatment and progression samples were normalized to that of GAPDH,
and fold changes (Disease Progression:Pre-treatment) are shown as average of
triplicates. (B) PGC1 expression in 917 cell lines of the Cancer Cell Line Encyclopedia
(CCLE) representing 23 cancer types (www.broadinstitute.org/software/cprg/?q=node/11).
Normalized PGC1 expression levels from the gene expression array data was plotted
for the alphabetically arranged cancer types, with PGC1 levels represented in an
ascending order for each cancer type. Inset shows the PGC1 levels in the 59
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melanoma cell lines in the CCLE separated by their BRAF/NRAS mutation status.
Wt/Wt represent melanomas without mutations in these two genes.
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