SUPPLEMENTARY METHODS
Chemical Inhibitors and Reagents
For in vitro studies, MEK (GSK1120212/trametinib, selumetinib) and BRAF (vemurafenib and GSK211436/dabrafenib) inhibitors were purchased from Selleck Chemicals (Houston, TX, USA). The solvent dimethylsulfoxide (DMSO) was obtained from BioShop (Burlington, ON, Canada). All drugs were reconstituted in 100% DMSO to a final concentration of 5–20 mM. For in vivo studies, trametinib, dabrafenib and vemurafenib were purchased from LC labs. These inhibitors were resuspended in PBS + 0.5% hydroxypropyl methylcellulose at a concentration of 2mg/kg (trametinib), 60mg/kg (vemurafenib), 10mg/kg (dabrafenib).
Cell culture, Reagents and siRNA transfections
The following human melanoma cell lines were used: WM2664 (BRAF V600D, NRAS WT) were purchased from the American Type Culture Collection; A375 (BRAF V600E, NRAS WT) and SK-MEL-2 (BRAF WT, NRAS Q61R) were generous gifts from Dr. David Dankort (McGill University); SK-MEL-28 (BRAF V600E, NRAS WT) were a generous gift from Dr. Wilson Miller (Lady Davis Institute for Medical Research); MDA-MB-435 (BRAF V600E, NRAS WT) were obtained from Dr. Morag Park (McGill University). All cells were grown in DMEM medium with 10% FBS, supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, and 2 mmol/L glutamine, for routine cell culture. All cells were incubated in a humidified atmosphere of 95% air and 5% CO2 at 37°C. Transient knockdown of MITF was accomplished by transfection (Lipofectamine 2000, Invitrogen) using 20 nmol/mL of the ON-TARGETplus SMARTpool [pool of four MITF-targeted small interfering RNAs (siRNA), Dharmacon]. An ON-TARGETplus pool of four non-targeting (scrambled) siRNAs was used as a control. The next day, vemurafenib or trametinib was added to the media and protein and RNA lysates were taken 48 hours after inhibitor treatment.
In vivo tumor growth and treatment regimens
Female CD1 nude mice (4-6 weeks old) were purchased from Taconic Farms (Hudson, NY). The animals were housed in facilities managed by the McGill University Animal Resources Centre and all animal experiments were conducted under a McGill University approved Animal Use Protocol in accordance with guidelines established by the Canadian Council on Animal Care. Mice were subcutaneously injected, bilaterally, with 1×106 A375 or WM2664 melanoma cells and monitored until tumors reached a volume 100-150 mm3 (Figs. S8, 4B) or 400 mm3 (Fig. 4D). Tumor-bearing animals were divided into groups that exhibited equivalent average tumor volumes. CDX-011 (2.5 mg/kg) was suspended in PBS and injected via the tail vein and PBS alone was used as a control. Injection of CDX-011, or PBS, was performed on the third day of each cycle of treatment with kinase inhibitors. Trametinib (2 mg/kg) or dabrafenib (10 mg/kg) + Trametinib (2 mg/kg) was given once daily by oral gavage for the first 7 days in cycles of either 14 or 21 days duration, or administered continuously throughout the duration of the experiment. Tumor growth was monitored bi-weekly by caliper measurement for 2-4 cycles. Tumor volumes were calculated using the following formula: πLW2/6, where L is the length and W is the width of the tumor.
Quantitative PCR
RNA was isolated from melanoma cell lines or biopsy samples from patients with melanoma using an RNeasy kit (Qiagen). cDNA was generated from 1 μg of RNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real-time quantitative polymerase chain reaction (RT-qPCR) assays were performed in triplicate using SYBR Green PCR Master Mix (Applied Biosystems) on a Rotor-Gene (Corbett Research) RT-PCR machine. Primer sequences are indicated in Supplementary Table S4. In patient samples, GPNMB and MITF transcript levels were normalized to GAPDH expression, and presented as relative units compared to pretreatment levels. In samples from cell lines, target genes are expressed relative to β-actin. Data acquisition and analysis were performed according to the manufacturer's instructions.
Immunoblotting
Protein extracts were prepared and immunoblots performed as described previously (1). Antibodies against GPNMB were purchased from R&D systems (Cat. #: AF2550), α-Tubulin antibodies were obtained from Sigma (Cat. #: T5168) and MITF (C5) antibodies were purchased from Thermoscientific (Cat. #: MA5-14146). Antibodies against ERK (Cat. #: 9102), pERK (Cat. #: 9101), and β-actin (Cat. #: 4970) antibodies were obtained from Cell Signaling.
ELISA
For mouse experiments, blood was drawn from mice bearing sub-cutaneous WM2664 tumors on day 0 and 4 of trametinib treatment, and tumor volumes were measured at the time blood samples were collected. Serum was extracted and the ELISA analysis to quantify soluble GPNMB levels performed as per manufacturers instructions using a commercially available kit from R&D systems. Data was represented as soluble GPNMB levels (pg/mL)/tumor volume (mm3). From patients, blood samples were taken prior to initiating treatment and again while on MAPK inhibitor therapy.
SUPPLEMENTARY REFERENCES
1. Rose AA, Annis MG, Dong Z, Pepin F, Hallett M, Park M, et al. ADAM10 releases a soluble form of the GPNMB/Osteoactivin extracellular domain with angiogenic properties. PLoS One. 2010;5:e12093.
2. Lin WM, Baker AC, Beroukhim R, Winckler W, Feng W, Marmion JM, et al. Modeling genomic diversity and tumor dependency in malignant melanoma. Cancer Res. 2008;68:664-73.
SUPPLEMENTARY FIGURE LEGENDS
Supplementary Fig. S1. Heatmap of mRNA expression for individual melanosomal differentiation antigens and a MITF transcriptional signature using a published dataset of melanoma samples (2).
Supplementary Fig. S2. GPNMB expression is induced in multiple BRAF-mutant cells in response to MAPK pathway inhibition. A-B, Immunoblot analysis of GPNMB, MITF, pERK and ERK following treatment of MDA-MB-435 (A) and SK-MEL-28 (B) melanoma cells for 48hr with increasing concentrations of vemurafenib (Vemu), dabrafenib (Dabr), trametinib (Tram) or selumetinib (Selu).
Supplementary Fig. S3. Gene expression of MITF and GPNMB in melanoma cells following MAPK pathway inhibition. RT-qPCR analysis performed on total mRNA isolated from A375 cells treated for 48hrs with vemurafenib (Vemu, 1uM) or trametinib (Tram, 1nM). *, P < 0.0001; **, P < 0.01; ***, P < 0.05.
Supplementary Fig. S4. Dabrafenib and Trametinib stimulate MITF nuclear localization. Cells were treated with 100nM Dabrafenib or 10nM Trametinib in FBS-free media for 24h. Images show MITF (red) and DAPI-stained nuclei (blue). Scale bar = 10μm and applies to all panels.
Supplementary Fig. S5. Correlation of MITF and GPNMB mRNA expression in melanoma samples from patients that were on-treatment or that progressed on therapy with MAPK pathway inhibitors. RT-qPCR data for MITF and GPNMB was expressed as the ratio relative to pre-treatment levels.
Supplementary Fig. S6. Correlation between the expression of individual melanosomal genes with MITF mRNA expression in melanoma samples from patients that were on-treatment or that progressed on therapy with MAPK pathway inhibitors. RT-qPCR data for each gene and MITF was normalized to GAPDH and expressed as the ratio relative to pre-treatment levels.
Supplementary Fig. S7. MAPK pathway inhibition results in elevated levels of soluble GPNMB released from melanoma cells. A, ELISA data measuring the soluble GPNMB extracellular domain (ECD) in the conditioned media (48 hours) from A375 and WM2664 cells following treatment with vehicle (DMSO), vemurafenib (Vemu, 1uM) or trametinib (Tram, 1nM). B, ELISA data measuring soluble GPNMB ECD in the serum of mice bearing sub-cutaneous WM2664 tumors that were divided into vehicle or trametinib (2 mg/kg daily) treated cohorts. The concentration of soluble GPNMB ECD measured in the serum was normalized to the sub-cutaneous tumor volumes, and data are represented as a ratio of the sGPNMB levels on day 4 of treatment to day 0 (pre-treatment) levels.
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