[Abstract unavailable

BRAF V600E is the most frequent oncogenic protein kinase mutation known. Furthermore, inhibitors targeting ''active'' protein kinases have demonstrated significant utility in the therapeutic repertoire against cancer. Therefore, we pursued the development of specific kinase inhibitors targeting B-Raf, and the V600E allele in particular. By using a structure-guided discovery approach, a potent and selective inhibitor of active B-Raf has been discovered. PLX4720, a 7-azaindole derivative that inhibits B-Raf V600E with an IC50 of 13 nM, defines a class of kinase inhibitor with marked selectivity in both biochemical and cellular assays. PLX4720 preferentially inhibits the active B-Raf V600E kinase compared with a broad spectrum of other kinases, and potent cytotoxic effects are also exclusive to cells bearing the V600E allele. Consistent with the high degree of selectivity, ERK phosphorylation is potently inhibited by PLX4720 in B-Raf V600E -bearing tumor cell lines but not in cells lacking oncogenic B-Raf. In melanoma models, PLX4720 induces cell cycle arrest and apoptosis exclusively in B-Raf V600E -positive cells. In B-Raf V600E -dependent tumor xenograft models, orally dosed PLX4720 causes significant tumor growth delays, including tumor regressions, without evidence of toxicity. The work described here represents the entire discovery process, from initial identification through structural and biological studies in animal models to a promising therapeutic for testing in cancer patients bearing B-Raf V600E -driven tumors.cancer ͉ cell signaling ͉ melanoma ͉ phosphorylation ͉ protein kinases O ncogenic mutations in the BRAF gene (1) correlate with increased severity and decreased response to chemotherapy in a wide variety of human tumors (2-4). Hence, direct therapeutic inhibition of oncogenic B-Raf kinase activity affords an avenue to treat these tumors. The therapeutic approach of targeting oncogenic kinase activity has proved very valuable in oncology (5, 6). Recently, we have described the technique termed scaffold-based drug discovery, a strategy for identifying small molecule inhibitors of cyclic nucleotide phosphodiesterases (7). Here, we describe an expansion of this strategy to discover a scaffold targeting protein kinases, and we report the elaboration of this scaffold into the potent and selective B-Raf V600E inhibitor PLX4720. Because a majority of all melanomas harbor an activating missense mutation (V600E) in the B-Raf oncogene (1), targeted inhibition of the V600E gene product is a particularly rational therapeutic goal in this otherwise therapy-resistant tumor type. Previous generations of B-Raf inhibitors possess Raf inhibitory activity at low nanomolar concentrations (8-13); however, the relative therapeutic efficacy of such inhibitors has been hampered by the lack of bioavailability or by the number of nonspecific targets that are also affected (14, 15). The development of highly specific and effectual inhibitors of the BRAF V600E gene product would provide insight into the true therapeutic rele...

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.

Although >66% of melanomas harbor activating mutations in BRAF and exhibit constitutive activity in the mitogen-activated protein kinase/extracellular signalregulated kinase kinase (MEK)/extracellular signalregulated kinase signaling pathway, it is unclear how effective MEK inhibition will be as a sole therapeutic strategy for melanoma. We investigated the anticancer activity of MEK inhibition in a panel of cell lines derived from radial growth phase (WM35) and vertical growth phase (WM793) of primary melanomas and metastatic melanomas (1205Lu, 451Lu, WM164, and C8161) in a threedimensional spheroid model and found that the metastatic lines were completely resistant to MEK inhibition (U0126 and PD98059) but the earlier stage cell lines were not. Similarly, these same metastatic melanoma lines were also resistant to inhibitors of the phosphatidylinositol 3-kinase/ Akt pathway (LY294002 and wortmannin). Under adherent culture conditions, the MEK inhibitors blocked growth through the induction of cell cycle arrest and up-regulation of p27, but this was readily reversible following inhibitor washout. However, when the phosphatidylinositol 3-kinase and MEK inhibitors were combined, the growth and invasion of the metastatic melanoma three-dimensional spheroids were blocked. Taken together, these results suggest that the most aggressive melanomas are resistant to strategies targeting one signaling pathway and that multiple signaling pathways may need to be targeted for maximal therapeutic efficacy. It is further suggested that BRAF mutational status is not predictive of response to MEK inhibition under three-dimensional culture conditions.

SummaryUnder normal conditions, homeostasis determines whether a cell remains quiescent, proliferates, differentiates, or undergoes apoptosis. In this state of homeostasis, keratinocytes control melanocyte growth and behaviour through a complex system of paracrine growth factors and cell-cell adhesion molecules. Alteration of this delicate homeostatic balance and can lead to altered expression of cell-cell adhesion and cell communication molecules and to the development of melanoma. Melanoma cells escape from this control by keratinocytes through three major mechanisms: (1) down-regulation of receptors important for communication with keratinocytes such as E-cadherin, P-cadherin, desmoglein and connexins, which is achieved through growth factors produced by fibroblasts or keratinocytes; (2) up-regulation of receptors and signalling molecules not found on melanocytes but important for melanomamelanoma and melanoma-fibroblast interactions such as N-cadherin, Mel-CAM, and zonula occludens protein-1 (ZO-1); (3) loss of anchorage to the basement membrane because of an altered expression of the extracellular-matrix binding integrin family. In the current review, we describe the alterations in cell-cell adhesion and communication associated with melanoma development and progression, and discuss how a greater understanding of these processes may aid the future therapy of this disease.

Constitutive activation of MEK-ERK signaling is often found in melanomas. Here, we identify a mechanism that links ERK with JNK signaling in human melanoma. Constitutively active ERK increases c-Jun transcription and stability, which are mediated by CREB and GSK3, respectively. Subsequently, c-Jun increases transcription of target genes, including RACK1, an adaptor protein that enables PKC to phosphorylate and enhance JNK activity, enforcing a feed-forward mechanism of the JNK-Jun pathway. Activated c-Jun is also responsible for elevated cyclin D1 expression, which is frequently overexpressed in human melanoma. Our data reveal that, in human melanoma, the rewired ERK signaling pathway upregulates JNK and activates the c-Jun oncogene and its downstream targets, including RACK1 and cyclin D1.

Recent studies have shown that there is a considerable heterogeneity in the response of melanoma cell lines to MEK and BRAF inhibitors. In the current study, we address whether dysregulation of cyclin-dependent kinase 4 (CDK4) and/or cyclin D1 contribute to the BRAF inhibitor resistance of melanoma cells. Mutational screening identified a panel of melanoma cell lines that harbored both a BRAF V600E mutation and a CDK4 mutation: K22Q (1205Lu), R24C (WM39, WM46, and SK-Mel-28), and R24L (WM902B). Pharmacologic studies showed that the presence of a CDK4 mutation did not alter the sensitivity of these cell lines to the BRAF inhibitor. The only cell line with significant BRAF inhibitor resistance was found to harbor both a CDK4 mutation and a CCND1 amplification. Array comparative genomic hybridization analysis showed that CCND1 was amplified in 17% of BRAF V600E -mutated human metastatic melanoma samples, indicating the clinical relevance of this finding. As the levels of CCND1 amplification in cell lines are lower than those seen in clinical specimens, we overexpressed cyclin D1 alone and in the presence of CDK4 in a drug-sensitive melanoma line. Cyclin D1 overexpression alone increased resistance and this was enhanced when cyclin D1 and CDK4 were concurrently overexpressed. In conclusion, increased levels of cyclin D1, resulting from genomic amplification, may contribute to the BRAF inhibitor resistance of BRAF V600E -mutated melanomas, particularly when found in the context of a CDK4 mutation/ overexpression. [Mol Cancer Ther 2008;7(9):2876 -83]

During the process of oncogenic transformation, melanoma cells escape from normal growth-control mechanisms and acquire the ability to invade surrounding tissues and organs. The Ras/Raf/MEK/ERK pathway is a major pathway involved in the control of growth signals, cell survival and invasion. Melanomas are known to harbour activating mutations of both Ras and BRAF, suggesting that the downstream effector ERK may be playing a major role in the oncogenic behaviour of these tumours. The past few years have seen a growth in the understanding of the role of ERK and the MAP kinase pathway in melanoma. The aim of the current review is to assess the role of ERK in melanoma behaviour and to determine whether modulation of these kinases could offer new therapeutic opportunities. © 2003 Wiley-Liss, Inc. The Ras/Raf/MEK/ERK pathway Extracellular signals, generated through the interaction of ligands with their respective receptor tyrosine kinases, are transmitted to the interior of the cell via the switching of the Ras-family GTPases from the inactive GDP-bound state to the active GTPbound state. In its GTP-bound state, Ras activates a number of downstream signalling cascades involved in controlling cell growth and behaviour. One such Ras-activated pathway is a family of protein serine/threonine protein kinases known as the mitogen activated protein (MAP) kinase cascade (see extensive review). 1,2 Initially, Ras interacts with and activates the serine/threonine protein kinase Raf, 3 which exists in 3 isoforms: A-Raf, B-Raf and C-Raf1. Once active, Raf, serine phosphorylates 2 further kinases, MEK1 and MEK2, 4,5 which in turn tyrosine/threonine phosphorylates extracellular-signal regulated kinase (ERK) 1 and ERK2 (Fig. 1). 6 Upon activation, the ERKs either phosphorylate a number of cytoplasmic targets or migrate to the nucleus, 7 where they phosphorylate and activate a number of transcription factors such as c-Fos, Elk-1. 8 The aberrant activation of MAP kinase pathways are implicated in the growth and pathologic behaviour of cancer cells. Activating mutations of Ras, Raf and MEK 9,10 are able to oncogenically transform fibroblasts in vitro. Factors driving ERK: the role of Ras, B-Raf and extracellular growth factorsThere is a growing body of evidence suggesting that activation of the Ras/Raf/MEK/ERK pathway may be involved in the oncogenic behaviour of melanoma. In vitro studies have demonstrated that melanoma cell lines and tumour tissues have high constitutive ERK activity. [11][12][13][14][15] The high constitutive ERK activity in melanoma is most likely a consequence of mutations in upstream components of the MAP kinase pathway. Activating mutations of Ras are found in 25% of all cancers, 16 with some, such as pancreatic and colon cancer, harbouring mutation rates of 90% and 50%, respectively. Ras mutations have been identified in 9 -15% of melanomas. [17][18][19][20] Most of the Ras mutations are at Codon-61 of N-Ras, with K-Ras and H-Ras mutations being relatively rare. 17,19,21 However, in addition to Ras, Raf-famil...