Mutant V599EB-Raf regulates growth and vascular development of malignant melanoma tumors

Activating mutations of the B-RAF gene are observed in >60% of human melanomas. Approximately 90% of these mutations occur in the activation segment of the kinase domain as a single-base substitution that converts a valine to glutamic acid at codon 599 (V599E) in exon 15. This mutation causes activation of the kinase as well as downstream effectors of the mitogen-activated protein kinase-signaling cascade, leading to melanoma tumor development by an as yet unknown mechanism. In this study, we have identified the role of (V599E)B-Raf in melanoma tumor development by characterizing the mechanism by which this mutant protein promotes melanoma tumorigenesis. Small interfering RNA targeted against B-Raf or a Raf kinase inhibitor (BAY 43-9006) was used to reduce expression and/or activity of (V599E)B-Raf in melanoma tumors. This inhibition led to reduced activity of the mitogen-activated protein kinase-signaling cascade and inhibited tumor development in animals. Targeted reduction of mutant (V599E)B-Raf expression (activity) in melanoma cells before tumor formation inhibited tumorigenesis by reducing the growth potential of melanoma cells. In contrast, reduction of mutant (V599E)B-Raf activity in preexisting tumors prevented further vascular development mediated through decreased vascular endothelial growth factor secretion, subsequently increasing apoptosis in tumors. These effects in combination with reduced proliferative capacity halted growth, but did not shrink the size of preexisting melanoma tumors. Thus, these studies identify the mechanistic underpinnings by which mutant (V599E)B-RAF promotes melanoma development and show the effectiveness of targeting this protein to inhibit melanoma tumor growth.     

Functional analysis of mutations within the kinase activation segment of B-Raf in human colorectal tumors

Mutations in the B-Raf gene have been reported in a number of human cancers, including colorectal carcinoma. More than 80% of the B-Raf mutations were V599E. Although other mutations have been reported, their functional consequences were unclear. Here, we examined the effect of colon tumor-associated B-Raf mutations within the kinase activation segment, including V599E, on extracellular signal-regulated kinase (Erk) and nuclear factor kappaB (NFkappaB) signaling, and on the transformation of NIH3T3 fibroblasts. Among the six mutations examined, only the B-Raf V599E and K600E mutations greatly increased Erk and NFkappaB signaling, and the transformation of NIH3T3 cells. The B-Raf F594L mutation moderately elevated Erk signaling and NIH3T3 transformation, but did not significantly increase NFkappaB signaling. Although the basal kinase activity of the B-Raf T598I mutant was comparable with that of wild-type, its oncogenic Ras-induced kinase activity was decreased to 60% of wild-type activity. The B-Raf D593V and G595R mutants showed severely reduced kinase activity and affected neither NFkappaB signaling nor NIH3T3 transforming activity. These results suggest that the B-Raf activation segment mutations other than V599E reported in colorectal tumors do not necessarily contribute to carcinogenesis by increasing kinase and transforming activities.     

Suppression of BRAF(V599E) in human melanoma abrogates transformation

Activating mutations in the BRAF serine/threonine kinase are found in >70% of human melanomas, of which >90% are BRAF(V599E). We sought to investigate the role of the BRAF(V599E) allele in malignant melanoma. We here report that suppression of BRAF(V599E) expression by RNA interference in cultured human melanoma cells inhibits the mitogen-activated protein kinase cascade, causes growth arrest, and promotes apoptosis. Furthermore, knockdown of BRAF(V599E) expression completely abrogates the transformed phenotype as assessed by colony formation in soft agar. Similar targeting of BRAF(V599E) or wild-type BRAF in human fibrosarcoma cells that lack the BRAF(V599E) mutation does not recapitulate these effects. Moreover, these results are specific for BRAF, as targeted interference of CRAF in melanoma cells does not significantly alter their biological properties. Thus, when present, BRAF(V599E) appears to be essential for melanoma cell viability and transformation and, therefore, represents an attractive therapeutic target in the majority of melanomas that harbor the mutation.     

Different effects of point mutations within the B-Raf glycine-rich loop in colorectal tumors on mitogen-activated protein/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase and nuclear factor kappaB pathway and cellular transformation

Recently, mutations in the B-Raf gene have been identified in a variety of human cancers, such as melanoma and colorectal carcinoma, and more than 80% of the B-Raf mutations have been V599E. Although other mutations have been reported, their functional consequences are poorly understood. In our earlier study, we demonstrated that colon tumor-associated B-Raf mutations within the kinase activation segment are not necessarily associated with an increase in mitogen-activated protein/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase (MEK/Erk) or nuclear factor kappaB (NFkappaB) signaling activity or in NIH3T3-transforming ability. In this study, we examined the effect of colon tumor-associated mutations within the B-Raf glycine-rich loop (G loop) on MEK/Erk and NFkappaB signaling and on the transformation of NIH3T3 fibroblasts or IEC-6 intestinal epithelial cells. Of the six G loop mutations examined, only the B-Raf G468A significantly increased MEK/Erk and NFkappaB signaling and NIH3T3 transformation. Only this mutation induced transformed phenotypes of IEC-6 cells. In contrast, the B-Raf G468E mutation significantly decreased MEK/Erk signaling and NIH3T3 transformation and had no effect on NFkappaB signaling. The B-Raf F467C mutation moderately elevated MEK/Erk signaling and NIH3T3 transformation. The other three B-Raf mutations, R461I, I462S, and G463E, did not increase MEK/Erk or NFkappaB signaling or NIH3T3 transformation. Except for F467C, none of the tumors with B-Raf mutations examined in this study had K-Ras mutations. These results suggest that some of the B-Raf G loop mutations reported in colorectal tumors do not increase kinase or transforming activities but might contribute to carcinogenesis via other mechanisms or be irrelevant to carcinogenesis.     

RKTG sequesters B-Raf to the Golgi apparatus and inhibits the proliferation and tumorigenicity of human malignant melanoma cells

Raf kinase trapping to Golgi (RKTG) is a newly characterized negative regulator of the Ras-Raf-mitogen-activated and extracellular signal-regulated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK)-signaling pathway via sequestrating Raf-1 to the Golgi apparatus. Among Raf kinase family members, B-Raf is the most frequently mutated gene in human cancers and an activated B-Raf mutation V600E is associated with >60% of human melanomas. Here, we show that RKTG can also bind and translocate B-Raf to the Golgi apparatus. When overexpressed in A375, a human malignant melanoma cell line with B-Raf(V600E), RKTG inhibits ERK activation, cell proliferation and transformation of A375 cells. In addition, the tumorigenicity of the RKTG-expressing A375 cells is suppressed in nude mice. Consistently, cell proliferation rate was reduced in the tumor xenografts in which RKTG was overexpressed. Collectively, our results suggest that RKTG may play a suppressive role in human melanoma that harbors an oncogenic B-Raf mutation via its antagonistic action on B-Raf.     

Exclusion of BRAFV599E as a melanoma susceptibility mutation

Very recently, BRAF mutations were found in about 2/3 of malignant melanomas and at lower frequencies in other human cancers. The BRAF gene codes for a protein in the mitogen-activated protein kinase (MAPK) pathway. All mutations identified to date are within the kinase domain, with a single missense mutation (V599E) accounting for 80%. We investigated the hypothesis that this common somatic BRAF mutation (V599E) would contribute to melanoma predisposition in familial and polygenic malignant melanoma if occurring as a germ-line mutation. We performed comprehensive mutational screening of exon 15 of BRAF using DHPLC (denaturing high-performance liquid chromatography) and DNA sequencing techniques. No V599E mutation could be detected in 172 melanoma patients comprising 46 familial cases, 21 multiple melanoma patients and 106 cases with at least one first-degree relative suffering from other cancers. We therefore conclude that the common somatic BRAF mutation V599E does not contribute to polygenic and familial melanoma predisposition.     

Targeting mutant (V600E) B-Raf in melanoma interrupts immunoediting of leukocyte functions and melanoma extravasation

Polymorphonuclear neutrophils (PMN) facilitate melanoma cell extravasation under dynamic flow conditions by the binding of intercellular adhesion molecule-1 (ICAM-1) on melanoma cells to beta2 integrins on PMNs, which is mediated by endogenously produced chemokine interleukin 8 (IL-8) from the tumor microenvironment. However, little is known about the role of B-Raf, the most mutated gene in malignant melanomas, in this process. In this study, we investigated the functional importance of B-Raf in melanoma extravasation by using short interfering RNA to reduce expression/activity of mutant (V600E)B-Raf in melanoma. Results indicated that knockdown of mutant (V600E)B-Raf inhibited melanoma cell extravasation in vitro and subsequent lung metastasis development in vivo. Mechanistic studies showed that inhibition of (V600E)B-Raf significantly reduced the constitutive secretion of IL-8 from melanoma cells as well as the capacity of endogenous IL-8 production from the melanoma-PMN microenvironment. Furthermore, a reduction in ICAM-1 expression on melanoma cells was detected following mutant (V600E)B-Raf knockdown. Together, these results suggest that targeting mutant (V600E)B-Raf reduces melanoma cell extravasation by decreasing IL-8 production and interrupting ICAM-1-beta2 integrin binding of melanoma cells to the endothelium mediated by PMNs in the microcirculation, which provides a rationale and mechanistic basis for targeting mutant (V600E)B-Raf to inhibit melanoma extravasation and subsequent metastasis development.     

Functional characterization of a BRAF insertion mutant associated with pilocytic astrocytoma

Pilocytic astrocytoma (PA) is emerging as a tumor entity with dysregulated Ras/Raf/MEK/ERK signaling. Common genetic lesions observed in PA, which are linked to aberrant ERK pathway activity, include either NF1 inactivation, KRAS or BRAF gain-of-function mutations. To investigate the mutation spectrum within the proto-oncogene encoding the Ser/Thr-kinase B-Raf in more detail, we analyzed 64 primary tumor samples from children with PA including two patients with neurofibromatosis type 1 (NF1). The well-known BRAF(V600E) mutation was found in 6/64 (9.38%) of our samples. For the first time, we report concomitant presence of a somatic BRAF(V600E) mutation in an NF1 patient indicating that more than one Ras/ERK pathway component can be affected in PA. Furthermore, 2/64 (3.13%) of our samples carried a 3-bp insertion in BRAF resulting in the duplication of threonine 599. This conserved residue is located within the activation segment and, if phosphorylated in a Ras-dependent manner, plays a key role in Raf activation. Here, we demonstrate that this mutant (B-Raf(insT) ) and another B-Raf mutant, which carries two additional threonine residues at this position, display an in vitro kinase activity and cellular MEK/ERK activation potential comparable to those of B-Raf(V600E) . Notably, replacement of threonines by valine residues had similar effects on B-Raf activity, suggesting that the distortion of the peptide backbone by additional amino acids rather than the insertion of additional, potential phosphorylation sites destabilizes the inactive conformation of the kinase domain. We also demonstrate that B-Raf(insT) and B-Raf(V600E) , but not B-Raf(wt) , provoke drastic morphological alterations in human astrocytes.     

SPRY2 is an inhibitor of the ras/extracellular signal-regulated kinase pathway in melanocytes and melanoma cells with wild-type BRAF but not with the V599E mutant

BRAF mutations result in constitutively active BRAF kinase activity and increased extracellular signal-regulated kinase (ERK) signaling and cell proliferation. Initial studies have shown that BRAF mutations occur at a high frequency in melanocytic nevi and metastatic lesions, but recent data have revealed much lower incidence of these mutations in early-stage melanoma, implying that other factors may contribute to melanoma pathogenesis in a wild-type (WT) BRAF context. To identify such contributing factors, we used microarray gene expression profiling to screen for differences in gene expression between a panel of melanocytic and melanoma cell lines with WT BRAF and a group of melanoma cell lines with the V599E BRAF mutation. We found that SPRY2, an inhibitor homologous to SPRY4, which was previously shown to suppress Ras/ERK signaling via direct binding to Raf-1, had reduced expression in WT BRAF cells. Using small interfering RNA-mediated SPRY2 knockdown, we showed that SPRY2 acts as an inhibitor of ERK signaling in melanocytes and WT BRAF melanoma cells, but not in cell lines with the V599E mutation. We also show that SPRY2 and SPRY4 directly bind WT BRAF but not the V599E and other exon 15 BRAF mutants. These data suggest that SPRY2, an inhibitor of ERK signaling, may be bypassed in melanoma cells either by down-regulation of its expression in WT BRAF cells, or by the presence of the BRAF mutation.     

BRAF mutations and RET/PTC rearrangements are alternative events in the etiopathogenesis of PTC

Rearrangement of RET proto-oncogene is the major event in the etiopathogenesis of papillary thyroid carcinoma (PTC). We report a high prevalence of BRAF(V599E) mutation in sporadic PTC and in PTC-derived cell lines. The BRAF(V599E) mutation was detected in 23 of 50 PTC (46%) and in three of four PTC-derived cell lines. The prevalence of the BRAF(V599E) mutation in PTC is the highest reported to date in human carcinomas, being only exceeded by melanoma. PTC with RET/PTC rearrangement as well as the TPC-1 cell line (the only one harboring RET/PTC rearrangement) did not show the BRAF(V599E) mutation. BRAF(V599E) mutation was not detected in any of 23 nodular goiters, 51 follicular adenomas and 18 follicular carcinomas. A distinct mutation in BRAF (codon K600E) was detected in a follicular adenoma. Activating mutations in RAS genes were detected in 15% of FA, 33% of FTC and 7% of PTC. BRAF(V599E) mutation did not coexist with alterations in any of the RAS genes in any of the tumors. These results suggest that BRAF(V599E) mutation is frequent in the etiopathogenesis of PTC. The BRAF(V599E) mutation appears to be an alternative event to RET/PTC rearrangement rather than to RAS mutations, which are rare in PTC. BRAF(V599E) may represent an alternative pathway to oncogenic MAPK activation in PTCs without RET/PTC activation.     

Expression of endogenous oncogenic V600EB-raf induces proliferation and developmental defects in mice and transformation of primary fibroblasts

Mutations of the human B-RAF gene are detected in approximately 8% of cancer samples, primarily in cutaneous melanomas (70%). The most common mutation (90%) is a valine-to-glutamic acid mutation at residue 600 (V600E; formerly V599E according to previous nomenclature). Using a Cre/Lox approach, we have generated a conditional knock-in allele of (V600E)B-raf in mice. We show that widespread expression of (V600E)B-Raf cannot be tolerated in embryonic development, with embryos dying approximately 7.5 dpc. Directed expression of mutant (V600E)B-Raf to somatic tissues using the IFN-inducible Mx1-Cre mouse strain induces a proliferative disorder and bone marrow failure with evidence of nonlymphoid neoplasia of the histiocytic type leading to death within 4 weeks of age. However, expression of mutant B-Raf does not alter the proliferation profile of all somatic tissues. In primary mouse embryonic fibroblasts, expression of endogenous (V600E)B-Raf induces morphologic transformation, increased cell proliferation, and loss of contact inhibition. Thus, (V600E)B-Raf is able to induce several hallmarks of transformation in some primary mouse cells without evidence for the involvement of a cooperating oncogene or tumor suppressor gene.     

Akt3 and mutant V600E B-Raf cooperate to promote early melanoma development

B-Raf is the most mutated gene in melanoma; however, the mechanism through which it promotes early melanomas remains uncertain. Most nevi contain activated (V600E)B-Raf but few develop into melanoma, and expression in melanocytes is inhibitory with low protein levels present in surviving cells, suggesting unknown cooperative oncogenic events are necessary for melanoma development. Because many melanomas have (V600E)B-Raf and active Akt3, it is possible that these proteins cooperatively facilitate melanocyte transformation. In this study, Akt3 is shown to phosphorylate (V600E)B-Raf to lower its activity as well as that of the downstream mitogen-activated protein kinase (MAPK) pathway to levels promoting early melanoma development. Expression of active Akt3 in early melanoma cells containing (V600E)B-Raf reduced MAPK signaling and promoted anchorage-independent growth. Furthermore, expression of both (V600E)B-Raf and active Akt3 in melanocytes promoted a transformed phenotype. Mechanistically, aberrant Akt3 activity in early melanomas serves to phosphorylate Ser(364) and Ser(428) on (V600E)B-Raf to reduce activity of (V600E)B-Raf to levels that promote rather than inhibit proliferation, which aids melanocytic transformation. Inhibition of (V600E)B-Raf or Akt3 in advanced melanoma cells in which both pathways were active reduced anchorage-independent growth and tumor development in a cooperatively acting manner. Inhibition of Akt3 alone in these cells led to increased MAPK signaling. In summary, these results suggest that activating B-Raf mutations initially promote nevi development, but the resulting high, intense activation of the MAPK pathway inhibits further tumor progression requiring Akt3 activation to bypass this barrier and aid melanoma development.     

Targeting V600EB-Raf and Akt3 using nanoliposomal-small interfering RNA inhibits cutaneous melanocytic lesion development

Most events promoting early melanoma development are yet to be identified, but deregulation of the B-Raf and Akt3 signaling cascades is an important regulator of this process. Approximately 90% of normal moles and approximately 60% of early invasive cutaneous melanomas contain a T1799A B-Raf mutation ((V600E)B-Raf), leading to 10 times higher enzyme activity and constitutive activation of the mitogen-activated protein kinase pathway. Furthermore, approximately 70% of melanomas have elevated Akt3 signaling due to increased gene copy number and PTEN loss. Therefore, targeting (V600E)B-Raf and Akt3 signaling is necessary to prevent or treat cutaneous melanocytic lesions. Agents specifically targeting these proteins are needed, having fewer side effects than those inhibiting both normal and mutant B-Raf protein or targeting all three Akt isoforms. In this study, a unique nanoliposomal-ultrasound-mediated approach has been developed for delivering small interfering RNA (siRNA) specifically targeting (V600E)B-Raf and Akt3 into melanocytic tumors present in skin to retard melanoma development. Novel cationic nanoliposomes stably encapsulate siRNA targeting (V600E)B-Raf or Akt3, providing protection from degradation and facilitating entry into melanoma cells to decrease expression of these proteins. Low-frequency ultrasound using a lightweight four-cymbal transducer array enables penetration of nanoliposomal-siRNA complex throughout the epidermal and dermal layers of laboratory-generated or animal skin. Nanoliposomal-mediated siRNA targeting of (V600E)B-Raf and Akt3 led to a cooperatively acting approximately 65% decrease in early or invasive cutaneous melanoma compared with inhibition of each singly with negligible associated systemic toxicity. Thus, cationic nanoliposomes loaded with siRNA targeting (V600E)B-Raf and Akt3 provide an effective approach for targeted inhibition of early or invasive cutaneous melanomas.     

Histone deacetylase inhibitor FK228 suppresses the Ras-MAP kinase signaling pathway by upregulating Rap1 and induces apoptosis in malignant melanoma

Histone deacetylase (HDAC) inhibitors are expected to be effective for refractory cancer because their mechanism of action differs from that of conventional antineoplastic agents. In this study, we examined the effect of the HDAC inhibitor FK228 on malignant melanoma, as well as its molecular mechanisms. FK228 was highly effective against melanoma compared with other commonly used drugs. By comparing the gene expression profiles of melanoma cells and normal melanocytes, we defined a subset of genes specifically upregulated in melanoma cells by FK228, which included Rap1, a small GTP-binding protein of the Ras family. The expression of Rap1 mRNA and protein increased in FK228-treated melanoma cells in both a dose- and a time-dependent manner. A decrease in the phosphorylation of c-Raf, MEK1/2, and ERK1/2 was accompanied by an increase in Rap1 expression in both FK228-treated and Rap1-overexpressing cells. Inhibition of Rap1 upregulation by small interfering RNA (siRNA) abrogated the induction of apoptosis and suppression of ERK1/2 phosphorylation in FK228-treated melanoma cells. These results indicate that the cytotoxic effects of FK228 are mediated via the upregulation of Rap1. Furthermore, we found that Rap1 was overexpressed and formed a complex with B-Raf in melanoma cell lines with a V599E mutation of B-Raf. The siRNA-mediated abrogation of Rap1 overexpression increased the viability of these cells, suggesting that Rap1 is also an endogenous regulator of Ras-MAP kinase signaling in melanomas.     

Differential sensitivity of melanoma cell lines with differing B-Raf mutational status to the new oncogenic B-Raf kinase inhibitor UI-152

Activating mutations in B-Raf kinase are common in malignant melanoma, an aggressive tumor of neuroectodermal origin. In the present study, the antiproliferative effect of the new oncogenic B-Raf targeting drug UI-152 on two types of melanoma cell lines with differing B-Raf mutational status was examined, and the underlying mechanisms were investigated. In cellular assays, UI-152 displayed high selectivity for tumor cells bearing B-Raf(V600E), showing more than 1000-fold higher inhibition of their proliferation than wild-type B-Raf-bearing cells. As expected, UI-152 completely abolished MEK-ERK phosphorylation in A375P cells harboring B-Raf(V600E). In SK-MEL-2 cells expressing B-Raf(WT), UI-152 caused the paradoxical activation of the MAPK pathway but to a much lesser extent than that observed of other oncogenic B-Raf inhibitors. These data suggest that UI-152 may be a more ideal B-Raf inhibitor capable of preserving potency against oncogenic B-Raf while minimizing the paradoxical activation of MAPK signaling. In addition, we showed that UI-152 treatment of A375P cells simultaneously induced cellular autophagy and apoptosis. However, autophagy inhibition with 3-methyladenine and inhibition of apoptosis by overexpression of the X-linked inhibitor of apoptosis failed to rescue melanoma cells from UI-152-induced cell death, implying that apoptosis and autophagy may cooperate in the induction of cell death in UI-152-treated cells. Collectively, our data suggest that UI-152 may be an effective B-Raf inhibitor and a potential therapeutic strategy for B-Raf(WT) and Ras mutant melanoma.     

Inhibition of growth and invasive ability of melanoma by inactivation of mutated BRAF with lentivirus-mediated RNA interference

Oncogenic mutations of molecules involved in the mitogen-activated protein kinase (MAPK) pathways provide signals mediating both tumor growth and invasion in various cancers including melanomas. BRAF somatic mutations, found in 66% of melanomas, have NIH3T3 transforming ability with the elevated kinase activity in vitro. We attempted to mediate RNA interference (RNAi) with HIV lentiviral vectors specific for either wild type or the most frequently mutated form of BRAF (V599E) in 10 melanoma cell lines, and found that RNAi inhibited the growth of most melanoma cell lines in vitro as well as in vivo, which was accompanied by decrease of both BRAF protein and ERK phosphorylation. Interestingly, the mutated BRAF (V599E)-specific siRNA inhibited the growth and MAPK activity of only melanoma cell lines with this mutation. Furthermore, BRAF RNAi inhibited matrigel invasion of melanoma cells accompanied with a decrease of matrix metalloproteinase activity and beta(1) integrin expression. These results clarify that the mutated BRAF (V599E) is essentially involved in malignant phenotype of melanoma cells through the MAPK activation and is an attractive molecular target for melanoma treatment. The lentivirus-mediated RNAi specific for oncogenic mutations may be a powerful technique for gene therapy of cancer.     

B-Raf(V600E) signaling deregulates the mitotic spindle checkpoint through stabilizing Mps1 levels in melanoma cells

The B-Raf(V600E) mutant, found in 65% of human melanomas, drives constitutive activation of the extracellular signal-regulated kinase (ERK) pathway and is implicated in tumorigenesis. Recently, we showed that B-Raf is important for spindle formation and the mitotic spindle checkpoint arrest. In this study, we demonstrate that B-Raf(V600E) signaling deregulates the spindle checkpoint as a consequence of stabilizing monopolar spindle 1 (Mps1) levels in human melanoma cells. Upon introducing the B-Raf(V600E) mutant into wild-type B-Raf melanoma cells, Mps1 protein and activity increased 3- and 10-fold, respectively. In addition, Mps1 became hyperphosphorylated, which correlated with stabilization of Mps1 protein levels. In contrast, reduction of B-Raf by RNAi or inactivation of ERK by the MEK inhibitor U0126 resulted in a precipitous decline in Mps1 levels. Together, these results suggest that B-Raf signaling through ERK regulates the stability of Mps1. Finally, B-Raf(V600E) expression induces a mitotic delay due to promoting robust activation of the mitotic spindle checkpoint. These effects were dependent on the induction of Mps1 levels by oncogenic B-Raf(V600E) as shown by depleting Mps1 with short interfering RNA. Collectively, our findings implicate a new mechanism through which B-Raf(V600E) exerts its oncogenic effects in melanoma.     

B-raf exon 15 mutations are common in primary melanoma resection specimens but not associated with clinical outcome

Downstream of Ras, the serine/threonine kinase B-raf has recently been reported to be mutated, among other carcinomas, in a majority of melanoma cell lines with a preponderance of mutations within the kinase domain including the activating V599E transition. We therefore investigated a representative number of 50 primary melanoma resection specimens for the presence of mutations within the activation segment (exon 15) of the B-raf kinase domain. Applying polymerase chain reaction and single-strand conformation polymorphism gel electrophoresis, followed by DNA cloning and sequencing, we found 19 cases (38%) to harbor somatic B-raf exon 15 mutations. With respect to the B-raf protein sequence, the V599E mutation was predicted in 63% of these positive melanomas, followed in frequency by the V599K transition (31%). Detection of B-raf exon 15 mutations or prediction of the activating mutation V599E were not statistically associated with the risk for subsequent metastasis in the follow-up of patients. Altogether, the B-raf oncogene is affected in a substantial subset of melanoma resection specimens. As B-raf alterations possibly affect melanocyte-specific pathways controlling proliferation and differentiation, activation of this oncogene may contribute to the development of melanoma.     

Immunogenicity of constitutively active V599EBRaf

Activating BRAF somatic missense mutations within the kinase domain are present in 60-66% of melanomas. The vast majority of these represent a single substitution of glutamate for valine (V599E). Here, we demonstrate spontaneous HLA-B*2705-restricted cytotoxic T-cell responses against an epitope derived from (V599E)BRaf. These T-cell responses were mutation specific as the corresponding epitope derived from wild-type BRaf was not recognized. The loss of the (V599E)BRAF genotype during progression from primary to metastatic melanoma in patients with (V599E)BRaf specific T-cell responses suggests an active immune selection of nonmutated melanoma clones by the tumor-bearing host.     

A novel class of specific Hsp90 small molecule inhibitors demonstrate in vitro and in vivo anti-tumor activity in human melanoma cells

Hsp90 is required for conformational maturation and stability of numerous key signaling proteins (clients) involved in cell proliferation and transformation. Here we describe two novel Hsp90 inhibitors, PF-4470296 and PF-3823863, demonstrate a differential sensitivity in B-Raf mutant (V600E) versus wild-type protein degradation. These two inhibitors inhibit proliferation and anchorage-independent growth, and abolish in vivo xenograft tumor growth in melanoma cells regardless of B-Raf mutation status. Mutant B-Raf protein and other Hsp90 clients, such as cMet, ErbB2, C-Raf, and AKT, are degraded in cells and xenograft tumors. Our results indicate that Hsp90 inhibitors induce anti-tumor activity in melanoma cells and are likely to show therapeutic benefit in melanoma patients by collaboratively targeting multiple pathways.