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 ¼ 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 (PTENÀ). Mechanistically, PLX4720 was found to stimulate AKT signaling in the PTENÀ but not the PTENþ 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 PTENþ (>14-fold) compared with the PTENÀ 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 PTENÀ. Further studies showed that siRNA knockdown of BIM significantly blunted the apoptotic response in PTENþ melanoma cells. Dual treatment of PTENÀ 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. Ó2011 AACR.

Differential scanning calorimetry has been used to examine blends of a poly(ethylene oxide) (PEO), Mn = 300 g/mol, and a poly(methylmethacrylate) (PMMA), Mn = 10,000 g/mol, across the complete composition range. The relatively low molar mass of the PEO minimizes interference from crystallization. In the midrange of composition, ∼25–70% PEO, two broad, but distinct, glass transitions are resolved. These are interpreted as distinct glass transitions of the two components, as anticipated by the self‐concentration model of Lodge and McLeish. The composition dependence of the observed transitions is well described by the self‐concentration approach, using lengthscales of approximately two‐thirds of the Kuhn length. The results are compared with previous measurements on PEO/PMMA blends and other miscible systems. The principal, general conclusion is that one should actually expect two glass transitions in a miscible polymer blend or polymer solution; the rule of thumb that two transitions indicate immiscibility is incorrect. Furthermore, attempts to rationalize two transitions on the basis of incomplete segmental mixing, or other unspecified “nanoheterogeneity,” may not be justified in many cases. © 2006 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 756–763, 2006

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.

Miscible blends containing poly(ethylene oxide) (PEO) have been examined over the entire composition range using differential scanning calorimetry to explore further the reported presence of two glass transitions. Three systems, poly(ethylene oxide)−dimethyl ether (PEO−DME)/poly(methyl methacrylate) (PMMA), PEO/poly(lactide) (PLA), and PEO/poly(vinyl acetate) (PVAc), were chosen in order to study the effects of end-group chemistry, annealing time, and crystallinity on the calorimetric behavior of the blends. The molecular weight of PEO was kept low to minimize the interference due to crystallization. Two distinct glass transitions were observed in the mid-composition range for all three systems. The glass transition temperatures varied smoothly with blend composition between the glass transition temperatures of the two homopolymer components. It was found that the self-concentration model describes the composition dependence of these glass transitions well. Further investigation on selected PEO/PVAc blends showed that annealing time and degree of crystallinity had a little effect on the glass transition behavior. These results confirm that the presence of two glass transitions should not necessarily be taken as an indication of immiscibility.

The mechanisms by which some melanoma cells adapt to BRAF inhibitor therapy are incompletely understood. In the present study, we used mass spectrometry-based phosphoproteomics to determine how BRAF inhibition remodeled the signaling network of melanoma cell lines that were BRAF-mutant and PTEN-null. Short-term BRAF inhibition was associated with marked changes in fibronectin-based adhesion signaling that were PTEN-dependent. These effects were recapitulated through BRAF siRNA knockdown and following treatment with chemotherapeutic drugs. Increased fibronectin expression was also observed in mouse xenograft models as well as specimens from melanoma patients undergoing BRAF inhibitor treatment. Analysis of a melanoma TMA showed loss of PTEN expression to predict for a lower overall survival, with a trend for even lower survival being seen when loss of fibronectin was included in the analysis. Mechanistically, the induction of fibronectin limited the responses of these PTEN-null melanoma cell lines to vemurafenib, with enhanced cytotoxicity observed following the knockdown of either fibronectin or its receptor α5β1 integrin. This in turn abrogated the cytotoxic response to BRAF inhibition via increased AKT signaling, which prevented the induction of cell death by maintaining the expression of the pro-survival protein Mcl-1. The protection conveyed by the induction of fibronectin expression could be overcome through combined treatment with a BRAF and PI3K inhibitor.

No abstract

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.