Acquired Resistance to BRAF Inhibition Can Confer Cross-Resistance to Combined BRAF/MEK Inhibition  Kavitha Gowrishankar, Stephanie Snoyman, Gulietta M. Pupo, Therese M. Becker, Richard F. Kefford, Helen Rizos  Journal of Investigative Dermatology  Volume 132, Issue 7, Pages 1850-1859 (July 2012) DOI: 10.1038/jid.2012.63 Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Analysis of the BRAF inhibitor–sensitive MelRMu melanoma cell line. (a) Viability curve for MelRMu cell line treated with increasing concentrations of GSK-BRAFi for 72hours (relative to DMSO-treated controls; mean±SD; n=3). (b) Cell cycle distribution of MelRMu cells treated with either DMSO (0nM) or GSK-BRAFi (100nM) for 72hours. (c) Dual-color flow cytometric Annexin V analysis for apoptosis of MelRMu cells treated with DMSO (0nM) or GSK-BRAFi (100nM) for 72hours. APC, allophycocyanin; PI, propidium iodide. (d) MelRMu cells were treated with 100nM GSK-BRAFi for increasing duration. The effects on extracellular signal–regulated kinase (ERK) activation and cell cycle regulators were determined by immunoblotting. Journal of Investigative Dermatology 2012 132, 1850-1859DOI: (10.1038/jid.2012.63) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Mitogen-activated protein kinase (MAPK) reactivation in cells with acquired resistance to GSK-BRAFi. (a) Viability curves of the parental and isogenic melanoma MelRMu sublines treated with the indicated GSK-BRAFi concentrations for 72hours (relative to DMSO-treated controls; mean±SD; n=3). DR, drug resistant. (b) MelRMu parental cells and BRAF inhibitor–resistant sublines were treated with DMSO (-) or 100nM GSK-BRAFi (+) for 24hours, and the effects on extracellular signal–regulated kinase (ERK) activation and cell cycle regulators were determined by immunoblotting. (c; left panel) Heat map for MEK/ERK activation signature in each of the cell lines treated with DMSO (-) or 100nM GSK-BRAFi (+) for 24hours. Color scale, log2-transformed expression (red, high; green, low) for each gene (row) normalized by the mean of all samples. The probe ID is shown after each gene symbol. (c; right panel) Histograms of mean log2-transformed expression of all transcripts included in the MEK/ERK activation signature (see left panel). MEK, MAPK/ERK kinase. (d) GSK-BRAFi effects on cell cycle distribution and apoptosis. APC, allophycocyanin; PI, propidium iodide. Journal of Investigative Dermatology 2012 132, 1850-1859DOI: (10.1038/jid.2012.63) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 BRAF inhibitor–resistant isogenic MelRMu sublines display distinct mechanisms of acquired resistance. (a) MelRMu parental and BRAF inhibitor–resistant sublines were treated with DMSO (-) or 100nM GSK-BRAFi (+) for 24hours, and the effects on known drug-resistance regulators were determined by immunoblotting. (b) Phosphorylation of platelet-derived growth factor receptor-β (PDGFRβ) was analyzed in the MelRMu parent and DR9 subline using phospho-RTK antibody arrays (left panel) and western immunoblotting (right panel). The positions of phosphorylated EGFR (positive in MelRMu and DR9) and phosphorylated PDGFRβ (negative in MelRMu and DR9) are indicated. DR, drug resistant. (c) Impact of mitogen-activated protein kinase (MAPK) inhibition (100nM GSK-BRAFi and 5nM GSK-MEKi) either alone or in combination with receptor tyrosine kinase inhibition (100nM imatinib mesylate) or phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibition (1μM BEZ235) on cell survival at 72hours after treatment. The inhibitory activity of imatinib and BEZ235 on c-Kit and AKT activation was confirmed in the c-Kit mutant (c-KitW557-K558) melanoma cell line, MelMS. (d) MelRMu and DR4 were transfected with COT1 short hairpin RNA (shRNA; +) or control (-) shRNA. At 24hours after transfection, cells were treated with DMSO (-) or 100nM GSK-BRAFi (+) for an additional 72hours. COT1 silencing (right panel) and the effects on cell death (left panel) were measured 72hours after drug addition. Journal of Investigative Dermatology 2012 132, 1850-1859DOI: (10.1038/jid.2012.63) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Oncogenic N-RAS confers BRAF inhibitor resistance. (a) MelRMu-derived resistant cell lines were transduced with N-RAS no. 1 short hairpin RNA (shRNA; +) or control (-) shRNA. At 5 days after transduction, cells were treated with DMSO (-) or 100nM GSK-BRAFi (+) for an additional 72hours. The effects on phosphorylated extracellular signal–regulated kinase (p-ERK) accumulation were detectable 24hours after exposure to GSK-BRAFi (left panel), and the downstream effects on cell death were measured 72hours after drug addition (right panel). DR, drug resistant. (b) MelRMu parental cell lines were lentivirally transduced with N-RASQ61K or copGFP control, and after 10 days cells were treated with DMSO (-) or 100nM GSK-BRAFi (+). The effects on p-ERK, cell cycle regulators, and N-RAS accumulation (left panel) and apoptosis (right panel) are shown. Journal of Investigative Dermatology 2012 132, 1850-1859DOI: (10.1038/jid.2012.63) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Acquired resistance to BRAF inhibition can dampen MAPK/ERK kinase (MEK) inhibitor sensitivity. (a) Viability curves of the parental and isogenic melanoma MelRMu sublines treated with the indicated GSK-MEKi concentrations for 72hours (relative to DMSO-treated controls; mean±SD; n=2). DR, drug resistant. (b) MelRMu parental cells and BRAF inhibitor–resistant sublines were treated with DMSO (-) or 5nM GSK-MEKi (+) for 24hours, and the effects on extracellular signal–regulated kinase (ERK) activation and cell cycle regulators were determined by immunoblotting. (c) GSK-MEKi effects on cell cycle distribution and apoptosis. APC, allophycocyanin; PI, propidium iodide. (d) MelRMu parental cell lines were lentivirally transduced with N-RASQ61K or copGFP control, and after 10 days cells were treated with DMSO (-) or 5nM GSK-MEKi (+) for 72hours. Percentage of cells in sub-G1 fraction is shown. Journal of Investigative Dermatology 2012 132, 1850-1859DOI: (10.1038/jid.2012.63) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Combined BRAF and MAPK/ERK kinase (MEK) inhibition does not restore sensitivity to mitogen-activated protein kinase (MAPK) inhibition in BRAF inhibitor–resistant cells. (a) The effects of GSK-BRAFi (100nM) and GSK-MEKi (5nM) on cell cycle distribution and apoptosis of MelRMu parental and derived sublines at 72hours after treatment. APC, allophycocyanin; PI, propidium iodide. (b) Activity of combination and single MAPK inhibitors in promoting cell death in MelRMu parental and drug resistant (DR) sublines at 72hours after drug treatment. (c) MelRMu parental cell lines were lentivirally transduced with N-RASQ61K or copGFP control, and after 10 days cells were treated with DMSO (-) or 100nM GSK-BRAFi and 5nM GSK-MEKi (+) for 72hours. Percentage of cells in sub-G1 fraction is shown. (d) MelRMu parental cells and BRAF inhibitor–resistant sublines were treated with DMSO (-) or 100nM GSK-BRAFi and 5nM GSK-MEKi (+) for 24hours, and the effects on extracellular signal–regulated kinase (ERK) activation and cell cycle regulators were determined by immunoblotting. Journal of Investigative Dermatology 2012 132, 1850-1859DOI: (10.1038/jid.2012.63) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions