Article Figures & Data
Figures
- Figure 1.
M14-derived AZ628-resistant (M14BRR) clones display elevated p-ERK1/2 levels and uncoupling of ERK signaling from BRAF. A, dose-response curves of M14 and six M14BRR clones treated with the indicated concentrations of AZ628. The fraction of viable cells is expressed relative to untreated controls. Error bars, SD from the mean. B, immunoblots with the indicated antibodies demonstrating that M14BRR clones exhibit elevated basal activation of ERK1/2 compared with parental M14 cells. Cell lysates from M14 and six independently generated M14BRR clones maintained in the presence of 2 μmol/L AZ628 were collected. C, immunoblots with the indicated antibodies demonstrating that M14BRR cells maintain ERK phosphorylation in the presence of AZ628. Cell lysates from M14 and AZ628-resistant clone (M14BRR2) were collected after 2-h treatment with the indicated concentrations of AZ628.
- Figure 2.
Proliferation of M14 AZ628-resistant (M14BRR) clones is dependent on MEK but not BRAF. A, dose-response curves of M14 and three M14BRR clones treated with the indicated concentrations of the MEK inhibitor U0126. The percentage of viable cells is expressed relative to untreated controls. Error bars, SD from the mean. The A431 cell line survival curve is shown as a negative control. B, AZ628-resistant cells retain sensitivity to U0126. Cell lysates from M14, AZ628-resistant clones, and A431 (negative control) were collected after treatment with the indicated concentrations of AZ628 or U0126 for 2 h. Immunoblotting analysis was performed using antibodies directed against the indicated proteins. C, effective depletion of BRAF protein by shRNA. M14 and M14BRR2 cells were infected with lentivirus containing control (PLKO.1 empty vector) or BRAF-specific shRNA. Cells were puromycin-selected and protein lysates were collected 4 d after the infection. Immunoblotting analysis was performed using antibodies directed against the indicated proteins. D, reduced dependency on BRAF in AZ628-resistant cells. Proliferation assay corresponding to cells in C. Control or BRAF-specific BRAF shRNAs were introduced in A549, M14, and M14BRR2 cells by lentiviral infection and a cell proliferation assay with Syto60 was performed 7 d later. The fraction of viable cells is expressed relative to untreated control. Error bars, SD from the mean.
- Figure 3.
M14 AZ-628–resistant clones express elevated CRAF and exhibit geldanamycin sensitivity. A, increased sensitivity of AZ628-resistant cells to geldanamycin. Survival curves of M14 and AZ628-resistant (M14BRR) clones treated with the indicated concentrations of the indicated drugs. MG132, proteasome inhibitor, nuclear factor-κB inhibitor; KIN001-045, src inhibitor; rapamycin, mTOR inhibitor; PHA-665752, MET kinase inhibitor; sorafenib, multikinase inhibitor; geldanamycin, HSP90 inhibitor. B, AZ628-resistant clones exhibit increased geldanamycin sensitivity. Dose-response of M14 and three M14BRR clones treated with the indicated concentrations of geldanamycin. The fraction of viable cells is expressed relative to untreated controls. Error bars, SD from the mean. C, geldanamycin causes BRAF reduction and CRAF depletion in M14 and AZ628-resistant clones. Cell lysates from M14 and M14BRR2 cells were collected after treatment with the indicated concentrations of geldanamycin for 24 h. Immunoblotting analysis was performed using antibodies directed against the indicated proteins. D, elevated CRAF expression in a subset of AZ628-resistant M14-derived clones. Cell lysates from M14 and six M14BRR clones maintained in the presence of 2 μmol/L of AZ628 were collected. Immunoblotting analysis was performed using antibodies directed against the indicated proteins.
- Figure 4.
Proliferation of AZ628-resistant M14 clones is dependent on CRAF. A, down-regulation of CRAF in AZ628-resistant clones results in reduced p-ERK1/2. M14 and M14BRR2 cells were infected with a lentivirus control (PLKO.1 empty vector) or a virus expressing CRAF-specific shRNA. Cells were puromycin-selected and protein lysates were collected 4 d after the infection. Immunoblotting analysis was performed using antibodies directed against the indicated proteins. B, AZ628-resistant M14 cells are dependent on CRAF. Cell viability assay corresponding to A. Control or CRAF-specific shRNAs were introduced into A549, M14, M14BRR2, and M14BRR8 cells, and cell proliferation assays with Syto60 were performed 5 d later. The fraction of cells relative to untreated controls is expressed. Error bars, SD from the mean. C, CRAF levels in AZ628-resistant cells vary proportionately to the concentration of AZ628 in which cells are maintained. Cell lysates from M14BRR2 cells growing in the indicated concentrations of AZ628 for several passages were collected. Immunoblotting analysis was performed using antibodies directed against the indicated proteins.
- Figure 5.
CRAF overexpression can confer resistance to RAF inhibition A, CRAF cDNA or control (pBABE empty vector) were introduced into M14 parental cells. Cell lysates were collected and immunoblotting analysis was performed using antibodies directed against CRAF and t-ERK 1/2 (loading control). B, M14 cells expressing exogenous CRAF exhibit reduced sensitivity to AZ628. Dose-response curves corresponding to cells in A. M14 + CRAF and M14 + pBABE control vector cells were treated with the indicated concentrations of AZ628. The fraction of viable cells is expressed relative to untreated controls. Error bars, SD from the mean. C, M14 cells expressing exogenous CRAF exhibit reduced suppression of ERK1/2 activation after AZ628 treatment. Cell lysates from M14 + pBABE control vector and M14 + CRAF were collected after treatment with the indicated concentrations of AZ628 for 2 h. Immunoblotting analysis was performed using antibodies directed against the indicated proteins.
- Figure 6.
CRAF overexpression can confer insensitivity to RAF inhibition. A, left, AZ628 insensitivity among three BRAF mutant tumor cell lines. Three AZ628-insensitive BRAF mutant cell lines (A2058, Sw1417, and Wm1552C) were treated with 0.2 μmol/L AZ628, and a proliferation assay with Syto60 was performed 72 h later. The fraction of viable cells is expressed relative to untreated controls. Error bars, SD from the mean. Right, AZ628 fails to suppress p-ERK1/2 in Wm1552C cells. Cell lysates from three AZ628-insensitive cell lines were collected after treatment with the indicated concentrations of AZ628 for 2 h. Immunoblotting analysis was performed using antibodies directed against p-ERK1/2 and t-ERK1/2 (loading control). B, left, CRAF levels are relatively high in Wm1552C cells. Cell lysates from M14 and three AZ628-insensitive cell lines were collected. Immunoblotting analysis was performed using antibodies directed against the indicated proteins. Middle, Wm1552C cells exhibit geldanamycin sensitivity. M14 and three AZ628-insensitive cell lines (Wm1552C, A2058, Sw1417) were treated with 0.1 μmol/L geldanamycin, and a proliferation assay with Syto60 was performed 72 h later. The percentage of viable cells is expressed relative to untreated controls. Error bars, SD from the mean. Right, geldanamycin treatment causes CRAF depletion and suppresses ERK1/2 activation in Wm1552C cells. Cell lysates from Wm1552C were collected after treatment with the indicated concentrations of geldanamycin for 24 h. Immunoblotting analysis was performed using antibodies directed against the indicated proteins. C, left, CRAF depletion by shRNA suppresses ERK1/2 activation in Wm1552C cells. Wm1552C cells were infected with a lentivirus control (PLKO.1 empty vector) or a virus expressing CRAF-specific shRNA. Cells were puromycin-selected, and protein lysates were collected 4 d after the infection. Immunoblotting analysis was performed using antibodies directed against the indicated proteins. Middle, CRAF depletion by shRNA inhibits cell growth in Wm1552C cells. Cell viability corresponding to cells in left panel. Control or CRAF-specific shRNAs were introduced in Wm1552C cells and a cell proliferation assay with Syto60 was performed 5 d later. The fraction of viable cells is expressed relative to untreated controls. Error bars, SD. Right, immunoblots demonstrating that AZ628 effectively suppresses ERK phosphorylation in two different melanoma cell lines that harbor activated NRAS alleles.
Additional Files
Supplementary Data, Montagut, et al.
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Volume 68, Issue 12
