Purpose The clinical use of BRAF inhibitors is being hampered by the acquisition of drug resistance. This study demonstrates the potential therapeutic utility of the HSP90 inhibitor (XL888) in 6 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 demonstrated 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 performed 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 3D 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 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 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.

Emergence of clinical resistance to BRAF inhibitors, alone or in combination with MEK inhibitors, limits clinical responses in melanoma. Inhibiting HSP90 offers an approach to simultaneously interfere with multiple resistance mechanisms. Using the HSP90 inhibitor, AT13387, which is currently in clinical trials, we investigated the potential of HSP90 inhibition to overcome or delay the emergence of resistance to these kinase inhibitors in melanoma models. In vitro, treating vemurafenib-sensitive cells (A375 or SK-MEL-28) with a combination of AT13387 and vemurafenib prevented colony growth under conditions where vemurafenib treatment alone generated resistant colonies. In vivo, when AT13387 was combined with vemurafenib in a SK-MEL-28, vemurafenib-sensitive model, no regrowth of tumors was observed over 5 months, although 2 out of 7 tumors in the vemurafenib monotherapy group relapsed in this time. Together these data suggest that the combination of these agents can delay the emergence of resistance. Cell lines with acquired vemurafenib resistance, derived from these models (A375R, SK-MEL-28R) were also sensitive to HSP90 inhibitor treatment; key clients were depleted, apoptosis was induced and growth in 3D-culture was inhibited. Similar effects were observed in cell lines with acquired resistance to both BRAF and MEK inhibitors (SK-MEL-28RR, WM164RR, 1205LuRR). These data suggest that treatment with an HSP90 inhibitor, such as AT13387, is a potential approach for combatting resistance to BRAF and MEK inhibition in melanoma. Moreover, frontline combination of these agents with an HSP90 inhibitor could delay the emergence of resistance, providing a strong rationale for clinical investigation of such combinations in BRAF-mutated melanoma.

The evolution of cancer therapy into complex regimens with multiple drugs requires novel approaches for the development and evaluation of companion biomarkers. Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) is a versatile platform for biomarker measurement. In this study, we describe the development and use of the LC-MRM platform to study the adaptive signaling responses of melanoma cells to inhibitors of HSP90 (XL888) and MEK (AZD6244). XL888 had good anti-tumor activity against NRAS mutant melanoma cell lines as well as BRAF mutant cells with acquired resistance to BRAF inhibitors both in vitro and in vivo. LC-MRM analysis showed HSP90 inhibition to be associated with decreased expression of multiple receptor tyrosine kinases, modules in the PI3K/AKT/mammalian target of rapamycin pathway, and the MAPK/CDK4 signaling axis in NRAS mutant melanoma cell lines and the inhibition of PI3K/AKT signaling in BRAF mutant melanoma xenografts with acquired vemurafenib resistance. The LC-MRM approach targeting more than 80 cancer signaling proteins was highly sensitive and could be applied to fine needle aspirates from xenografts and clinical melanoma specimens (using 50 g of total protein). We further showed MEK inhibition to be associated with signaling through the NFB and WNT signaling pathways, as well as increased receptor tyrosine kinase expression and activation. Validation studies identified PDGF receptor ␤ signaling as a potential escape mechanism from MEK inhibition, which could be overcome through combined use of AZD6244 and the PDGF receptor inhibitor, creno- Despite excitement about the development of targeted therapy strategies for cancer, few cures have been achieved. In patients with BRAF mutant melanoma, treatment with small molecule BRAF inhibitors typically follows a course of response and tumor shrinkage followed by eventual relapse and resistance (mean progression-free survival is ϳ5.3 months) (1). Resistance to BRAF inhibitors is typically accompanied by reactivation of the MAPK signaling pathway, an effect mediated through activating mutations in NRAS and MEK1/2, genomic amplification of BRAF, increased expression of CRAF and Cot, and the acquisition of BRAF splice-form mutants (2-5). There is also evidence that increased PI3K/AKT signaling, resulting from the genetic inactivation of PTEN and NF1 and increased receptor tyrosine kinase (RTK) 1 signaling, may be involved in acquired BRAF inhibitor resistance (5-7). Many of the signaling proteins implicated in the escape from BRAF inhibitor therapy are clients of heat shock protein (HSP)-90 (8). Preclinical evidence now indicates that HSP90 inhibitors can overcome acquired and intrinsic BRAF inhibitor resistance, and clinical trials have been initiated to evaluate the BRAF/HSP90 combination in newly diagnosed patients (8, 9).Although targeted therapy strategies have been promising in BRAF mutant melanoma, few options currently exist for the 15-20% of melanoma patients whose tumors harbor activating NRAS mutations (10). Alth...

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 3D 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 MAPK, AKT, mTOR and JNK signaling. Apoptosis induction was associated with increased BIM expression and a decrease in the expression of the pro-survival protein Mcl-1. The critical role of increased BIM and decreased Mcl-1 expression in the survival of NRAS mutant melanoma cell lines was demonstrated 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.