Abstract
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.