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Supplemental Figure 1. Characterization of the resistant cell lines. Supplemental Figure 2. Quantification of the cell invasion from figure 1D. Supplemental Figure 3. Model showing ligand dependent and independent ephrin signaling. Supplemental Figure 4. Acute drug treatment does not induce ligand independent ephrin signaling. Supplemental Figure 5. Quantification of matrigel invasion data from Figure 2E. Supplemental Figure 6. knockdown of EphA2 does not resensitize to BRAF inhibition. Supplemental Figure 7. PI3K and AKT inhibitors block AKT signaling. Supplemental Figure 8. Removal of drug increases cell doubling time. Supplemental Figure 9. Removal of drug reverses the resistance phenotype. Supplemental Figure 10. Removal of drug reverses EphA2 signaling and melanoma invasion. Supplemental Figure 11. Time course of EphA2 induction following chronic BRAF inhibition Supplemental Table 1. Levels of EphA2 staining in primary and metastatic melanoma lesions Supplemental Table 2. Increased EphA2 staining observed in melanoma patient specimens on BRAF inhibitor trial Supplemental Table 3. Incidence of new metastases in patients treated with either vemurafenib or dacarbazine.

The HSP90 inhibitor XL888 is effective at reversing BRAF inhibitor resistance in melanoma, including that mediated through acquired NRAS mutations. The present study has investigated the mechanism of action of XL888 in NRAS-mutant melanoma. Treatment of NRAS-mutant melanoma cell lines with XL888 led to an inhibition of growth, G2–M phase cell-cycle arrest, and the inhibition of cell survival in three-dimensional spheroid and colony formation assays. In vitro, HSP90 inhibition led to the degradation of ARAF, CRAF, Wee1, Chk1, and cdc2 and was associated with decreased mitogen-activated protein kinase (MAPK), AKT, mTOR, and c-jun NH2 kinase (JNK) signaling. Apoptosis induction was associated with increased BIM expression and a decrease in the expression of the prosurvival protein Mcl-1. The critical role of increased BIM and decreased Mcl-1 expression in the survival of NRAS-mutant melanoma cell lines was shown through siRNA knockdown and overexpression studies. In an animal xenograft model of NRAS-mutant melanoma, XL888 treatment led to reduced tumor growth and apoptosis induction. Important differences in the pattern of client degradation were noted between the in vivo and in vitro studies. In vivo, XL888 treatment led to degradation of CDK4 and Wee1 and the inhibition of AKT/S6 signaling with little or no effect observed upon ARAF, CRAF, or MAPK. Blockade of Wee1, using either siRNA knockdown or the inhibitor MK1775, was associated with significant levels of growth inhibition and apoptosis induction. Together, these studies have identified Wee1 as a key target of XL888, suggesting novel therapeutic strategies for NRAS-mutant melanoma. Mol Cancer Ther; 12(6); 901–12. ©2013 AACR.

PDF file - 295K, Supplemental Figure 1: XL888 (0.3 μM) treatment induces the nuclear accumulation of FOXO3a. Supplemental Figure 2: XL888 decreases Mcl-1 mRNA expression in NRAS mutant melanoma. Supplemental Figure 3: BAK is not required for XL888-mediated apoptosis. Supplemental Figure 4: The BH3 family protein inhibitor ABT-737 enhances the pro-apoptotic effects of XL888 in the WM1366 and WM1361A cell lines. Supplemental Figure 5: The effects of XL888 (300nM) and ABT-737 (1 μM) on the viability of NRAS mutant melanoma spheroids. Supplemental Table 1: List of heat shock proteins used on the LC-MRM by UniPROT ID and gene name.

Purpose: The clinical use of BRAF inhibitors is being hampered by the acquisition of drug resistance. This study shows the potential therapeutic use of the HSP90 inhibitor (XL888) in six different models of vemurafenib resistance.Experimental Design: The ability of XL888 to inhibit growth and to induce apoptosis and tumor regression of vemurafenib-resistant melanoma cell lines was shown in vitro and in vivo. A novel mass spectrometry–based pharmacodynamic assay was developed to measure intratumoral HSP70 levels following HSP90 inhibition in melanoma cell lines, xenografts, and melanoma biopsies. Mechanistic studies were carried out to determine the mechanism of XL888-induced apoptosis.Results: XL888 potently inhibited cell growth, induced apoptosis, and prevented the growth of vemurafenib-resistant melanoma cell lines in 3-dimensional cell culture, long-term colony formation assays, and human melanoma mouse xenografts. The reversal of the resistance phenotype was associated with the degradation of PDGFRβ, COT, IGFR1, CRAF, ARAF, S6, cyclin D1, and AKT, which in turn led to the nuclear accumulation of FOXO3a, an increase in BIM (Bcl-2 interacting mediator of cell death) expression, and the downregulation of Mcl-1. In most resistance models, XL888 treatment increased BIM expression, decreased Mcl-1 expression, and induced apoptosis more effectively than dual mitogen-activated protein–extracellular signal–regulated kinase/phosphoinositide 3-kinase (MEK/PI3K) inhibition.Conclusions: HSP90 inhibition may be a highly effective strategy at managing the diverse array of resistance mechanisms being reported to BRAF inhibitors and appears to be more effective at restoring BIM expression and downregulating Mcl-1 expression than combined MEK/PI3K inhibitor therapy. Clin Cancer Res; 18(9); 2502–14. ©2012 AACR.

CCR Translation for This Article from The HSP90 Inhibitor XL888 Overcomes BRAF Inhibitor Resistance Mediated through Diverse Mechanisms

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Supplementary Tables 1-2, Figures 1-12 from PTEN Loss Confers BRAF Inhibitor Resistance to Melanoma Cells through the Suppression of BIM Expression

This study addresses the role of PTEN loss in intrinsic resistance to the BRAF inhibitor PLX4720. Immunohistochemical staining of a tissue array covering all stages of melanocytic neoplasia (n = 192) revealed PTEN expression to be lost in >10% of all melanoma cases. Although PTEN expression status did not predict for sensitivity to the growth inhibitory effects of PLX4720, it was predictive for apoptosis, with only limited cell death observed in melanomas lacking PTEN expression (PTEN−). Mechanistically, PLX4720 was found to stimulate AKT signaling in the PTEN− but not the PTEN+ cell lines. Liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM) was performed to identify differences in apoptosis signaling between the two cell line groups. PLX4720 treatment significantly increased BIM expression in the PTEN+ (>14-fold) compared with the PTEN− cell lines (four-fold). A role for PTEN in the regulation of PLX4720-mediated BIM expression was confirmed by siRNA knockdown of PTEN and through reintroduction of PTEN into cells that were PTEN−. Further studies showed that siRNA knockdown of BIM significantly blunted the apoptotic response in PTEN+ melanoma cells. Dual treatment of PTEN− cells with PLX4720 and a PI3K inhibitor enhanced BIM expression at both the mRNA and protein level and increased the level of apoptosis through a mechanism involving AKT3 and the activation of FOXO3a. In conclusion, we have shown for the first time that loss of PTEN contributes to intrinsic BRAF inhibitor resistance via the suppression of BIM-mediated apoptosis. Cancer Res; 71(7); 2750–60. ©2011 AACR.