• cancer;
  • clinical trials;
  • transformation;
  • drug delivery;
  • signal transduction;
  • mitogenic cascade;
  • Raf kinases


  1. Top of page
  2. Abstract
  3. Raf kinases: The History
  4. Raf kinase signaling
  5. Raf kinases and mouse cancer models
  6. Raf kinases and human cancer
  7. Raf kinases and cancer drug discovery
  8. Perspectives
  9. Acknowledgements
  10. References

Raf kinase signaling has been thoroughly investigated over the last 20 years. A-Raf, B-Raf and C-Raf, the 3 mammalian members of the Raf family, are involved in a variety of cellular processes such as growth, proliferation, survival, differentiation and transformation. The detection of B-RAF mutations in a wide variety of human cancers, the description of wildtype and mutant B-RAF as tumor antigens in melanoma and the promising outcome of clinical trials evaluating the Raf inhibitor Nexavar® (Sorafenib, BAY 43-9006) have sparked a broad interest in the scientific community. After a short historical detour and an introduction into Raf kinase signaling, we are going to discuss here recent outcomes of Raf kinase research with respect to tumor formation and give an overview on current efforts to develop anticancer therapies interfering with aberrant Raf kinase signaling. © 2006 Wiley-Liss, Inc.

Raf kinases: The History

  1. Top of page
  2. Abstract
  3. Raf kinases: The History
  4. Raf kinase signaling
  5. Raf kinases and mouse cancer models
  6. Raf kinases and human cancer
  7. Raf kinases and cancer drug discovery
  8. Perspectives
  9. Acknowledgements
  10. References

In 1983, the cloning of the acutely transforming replication-defective mouse type C virus 3611-MSV and characterization of its acquired oncogene was reported.1 Since 3611-MSV induces rapidly growing fibrosarcoma in mice, the transduced oncogene was called v-raf, whereas its cellular homologue was named c-raf.1 Shortly thereafter, Bister and coworkers described the cloning of the avian acute leukemia retrovirus Mil Hill No. 2 (MH2).2 MH2 was found to carry a second potential oncogene in addition to v-myc, which was termed v-mil, whereas its cellular counterpart was called c-mil.3 Already a hybridization analysis and the gross comparison of restriction maps of v-raf and v-mil pointed at sequence homologies between these 2 genes.4 By direct sequencing of both genes it was finally proven that 3611-MSV and MH2 have integrated orthologues of the same gene into their genomes.5 In the following we are going to use the nomenclature for Raf kinases as proposed in a recent review from Wellbrock et al.6

In contrast to all other oncogene kinases known at that time, no tyrosine kinase activity was detected in C-Raf.7 Indeed, it was found that C-Raf is a serine/threonine kinase.8, 9 This and the observation that Raf coexists with the Myc oncogene in retroviruses10 led to two novel concepts: (i) There is a tyrosine to serine phosphorylation switch as the growth factor signal enters the cell, and (ii) the mechanistic basis for cooperation between nuclear and cytoplasmic oncogenes as well as for signal transduction from cell surface receptors to the nucleus is the phosphorylation of transcription factor class oncogenes such as Myc.11, 12, 13 Growth factor abrogation and oncogene cooperation studies were performed to corroborate this signaling scheme.14, 15, 16, 17 Additional important early findings were (i) the cooperation between Raf and Myc in growth factor independent proliferation, immortalization and tumor induction,14, 18, 19 leading to the Raf-Myc balance model,20 and (ii) the cell lineage-switch activity of the Raf-Myc oncogene combination.21, 22, 23 The use of a kinase-dead Raf version and its presumed negative autoregulatory N-terminal half as a dominant-negative mutant24, 25, 26 enabled the identification of Raf as the first effector of Ras and positioned Ras at the top of the mitogenic cascade.27 Our finding that constitutive MAP kinase activity in v-raf transformed cells quickly led to the identification of the mitogen-activated kinase kinase MEK as first physiological Raf substrate, thereby completing the description of the classical mitogenic cascade.28, 29, 30

Subsequently, the paralogues A-Raf31, 32, 33 and B-Raf34, 35 were identified. Although all 3 Raf isoforms share considerable sequence similarity,36 they exhibit beneath common also individual functions, which are still far from being fully understood.37, 38 Additional key findings in the early days of Raf kinase research were the demonstration of C-Raf as a mutational target in a lung tumor model suggesting that Raf is a critical effector of Ras,39 the identification of Raf as an apoptosis suppressor cooperating with BCL2 at the outer mitochondrial membrane,40, 41, 42 the dependence of the cellular response to Raf signaling on signaling intensity and context,43, 44 and the ability of Raf to activate the NF-κB transcription factor, a major regulator of inflammatory responses and mesenchymal-epithelial transitions.45, 46

Raf kinase signaling

  1. Top of page
  2. Abstract
  3. Raf kinases: The History
  4. Raf kinase signaling
  5. Raf kinases and mouse cancer models
  6. Raf kinases and human cancer
  7. Raf kinases and cancer drug discovery
  8. Perspectives
  9. Acknowledgements
  10. References

As already described above, the classical mitogenic cascade was originally described as unidirectional highway between extracellular growth factor signals and nuclear transcription events (Fig. 1a). RAS GTPases are activated by the majority of growth factor receptors and bind and recruit Raf to the cell membrane upon activation. The central signaling components Raf, MEK and ERK are then sequentially phosphorylated and activated by each other. More than 70 nuclear and nonnuclear effector molecules of the mitogenic cascade have been identified so far. In addition, Raf kinase signaling in a cascade-independent fashion has been described; for review see Ref.47. This includes the activation of the NF-κB transcription factor,45, 46 the prevention of apoptosis by antagonizing proapoptotic factors such as MST2, the mammalian sterile 20-like kinase,48 ASK1, the apoptosis signal-regulating kinase 1,49 and BAD, the BCL-2-antagonist of cell death,41 and finally the positive regulation of cell migration via the Rho effector kinase Rok-α.50 The regulation of Raf kinase activity is quite complex, far from being fully understood and beyond the scope of this review. For recent comprehensive reviews, please see Refs.6,38,51, 52, 53.

thumbnail image

Figure 1. Raf kinase signaling pathways, (a) Schematic representation of the classical mitogenic cascade. (b) A novel pathway for Raf activation requires heterodimerization of B-Raf and C-Raf to activate MEK. In normal cells this heterodimer formation is Ras dependent, whereas in B-Raf mutant tumor cells heterodimerization is Ras independent. For more details see text.

Download figure to PowerPoint

However, a few general principles are noteworthy. The key feature involves assembly of the cascade at the membrane from pre-existing modules (Ras module, Raf module, KSR module). This process is paralleled by an intricate pattern of phosphorylation and dephosphorylation events leading to conformational changes of signaling molecules. The kinetics of this process depends on the presence of individual Raf isoenzymes and on the engagement of various positive and negative feedback loops. Primarily the phosphorylation status and the localization of Raf kinases determine the association with interacting partners, such as chaperones, other kinases, prolyl isomerases, phosphatases, scaffolding proteins and also lipids and vice versa (Fig. 2). Within this signaling zoo along the mitogenic cascade, there is still more room for novel players. They are definitely more than just additional signaling proteins and contribute significantly to our understanding how Raf kinase signaling really works. Good examples for this conclusion are Prohibitin, which is required for Ras-induced Raf-MEK-ERK activation and epithelial cell migration,54 Shoc2/Sur8, which functions in complex with the catalytic subunit of protein phosphatase 1 as M-Ras effector modulating Raf activity55 and Hyphen, which in Drosophila is part of a novel Raf controlling complex consisting of the scaffold KSR and the Raf binding protein CNK.56

thumbnail image

Figure 2. Raf kinase signaling and its regulation by interacting molecules. Raf kinase signaling is regulated at multiple levels. Besides kinases and phosphatases, also other interacting proteins, such as scaffold proteins and chaperones, regulate the activity and proper intracellular localization of Raf kinases. For more details see text. [Color figure can be viewed in the online issue, which is available at]

Download figure to PowerPoint

In these multiprotein complexes Raf proteins may act as true kinases, but kinase-independent, scaffolding-like functions of Raf kinases have also been described. Work from several groups established that homo- and heterodimerization of Raf kinases clearly exist,57, 58, 59 and that heterodimerization can be Ras induced.59 In addition, it was shown that Raf heterodimerization is regulated by 14-3-3 proteins, mitogens and the Mixed-lineage kinase 3 and is also stabilized by MEK inhibition.59, 60, 61 Marais and coworkers described that heterodimerization is involved in the activation of C-RAF by B-RAF, but that wild-type and mutant B-RAF use different activation mechanisms.62, 63 Whereas wild-type B-RAF activates C-RAF via RAS-induced heterodimerization, mutant B-RAF heterodimerizes with and activates C-RAF in a RAS-independent manner, thereby generating a novel B-RAF > C-RAF > MEK > ERK pathway that is active in normal as well as in transformed cells (Fig. 1b).63 Whether signaling via this pathway just reflects some sort of crossactivation of Raf isoforms or whether it leads to a different set of effector functions in comparison to the classical RAF > MEK > ERK cascade remains to be determined in future.

Raf kinases and mouse cancer models

  1. Top of page
  2. Abstract
  3. Raf kinases: The History
  4. Raf kinase signaling
  5. Raf kinases and mouse cancer models
  6. Raf kinases and human cancer
  7. Raf kinases and cancer drug discovery
  8. Perspectives
  9. Acknowledgements
  10. References

Animal models of human cancer have contributed significant insights into the mechanisms of tumor formation and progression64, 65 and are indispensable tools for drug discovery.66 During the early days of Raf kinase research, infection or transduction of newborn mice with retroviruses or retroviral vectors harbouring either wild-type or mutant Raf kinases were instrumental to prove their transforming potential.18, 67 In another line of experiments mutagen-induced tumor formation in mice was analyzed to detect whether Raf kinases are mutational targets.39, 68 Ethylnitrosourea treatment of mice led to the induction of adenocarcinoma and T-cell lymphoma carrying point mutations in craf in the absence of ras mutations.39 Mutations of b-raf were also detected in ∼20% of chemically induced liver tumors from C3H/He mice.68 In addition, mice have also been used as genetic tools for understanding solid tumor formation.69 When the Sleeping Beauty transposon was used for somatic insertion mutagenesis, accelerated tumor formation in mice predisposed to cancer was noted. Unexpectedly, the cloning of insertion sites revealed that the gene most frequently disrupted by the transposon was b-raf. The resulting truncated B-Raf was lacking N-terminal autoregulatory elements and had transforming properties in focus formation assays.69

The first transgenic mouse tumor model expressing Raf kinases was established in our lab by overexpression of wildtype or constitutive active C-Raf (C-Raf-BXB) under the control of the human surfactant protein-C promoter in type II alveolar pneumocytes.70 All mice expressing C-Raf-BXB developed benign lung adenomas within 4 months of life.71 Raf-induced tumors expanded continuously without any signs of apoptosis.72, 73 Lung tumor formation in vivo was substantially retarded by systemic administration of the MEK inhibitor CI-1040.74 Initially, progression of C-Raf-BXB driven tumors to metastasis was never observed,70 and the stability of this phenotype allowed us to determine the influence of other genes on tumor progression. For example, neither loss of the tumor suppressor p53 nor the cyclin-dependent kinase inhibitor p21 provoked metastasis, although tumor latency was dramatically reduced.23 The loss of the Bcl-2 proto-oncogene greatly retarded lung tumor formation in C-Raf-BXB mice suggesting that bcl-2 is a major susceptibility gene for development of lung cancer in mice and perhaps also in humans.72 The influence of the co-chaperone BAG-1, which is interacting with B-Raf and C-Raf as well as with Bcl-2, was also analyzed on lung tumor formation. In C-Raf-BXB mice, which are heterozygous for bag-1,75 lung adenoma growth was significantly reduced due to increased apoptosis implicating BAG-1 as a critical player in Raf-driven oncogenic transformation.73 Surprisingly, when cell–cell contacts were impaired by conditional expression of dominant-negative E-cadherin, tumor progression from lung adenoma to adenocarcinoma and lymph node metastasis was observed. Moreover, not only loss of cell–cell contacts but also the induction of angiogenesis within the tumor was dramatically enhanced (Ceteci and Rapp, unpublished observations). This provides further experimental support for the angiogenic switch paradigm76 and links for the first time the formation of the adherence complex and the induction of angiogenesis.

So far, only 2 transgenic mouse models with either constitutive77 or conditional78 expression of mutant B-Raf have been published. Constitutive expression of the mutant human V600EB-RAF under the control of the bovine thyroglobulin promoter induced goiter and invasive papillary thyroid cancers (PTC) with tall-cell features, which later transitioned to poorly differentiated carcinomas.77 This resembles closely the phenotype of human PTC and together with the striking histopathologic similarity between mice and human harbouring V600EB-Raf suggests that this mouse cancer model might be useful for the analysis of molecular events leading to dedifferentiation of PTC. In addition, we have generated SPC V600EB-Raf mice and observed cystic lung hyperplasia, signs of inflammation and progression to metastasis, which are further analyzed in ongoing experiments (Goetz and Rapp, unpublished observation). Pritchard and coworkers applied a Cre/lox strategy to generate a conditional knock-in allele of V600EB-Raf.78 Ubiquitous expression of V600EB-Raf resulted in embryonic death before E7.5, whereas expression in adult somatic tissues induced hyperproliferation and bone marrow failure.78

Up to now, 2 Raf-dependent mouse tumor models have been generated that allow a preclinical evaluation of novel therapeutic modalities and potential anticancer drugs. As already mentioned above, the C-Raf BXB mice with constitutive expression of either C-Raf or C-Raf BXB develop lung adenoma70 and have been used to study the in vivo influence of the mitogenic cascade blockers BAY 43-9006 and CI-1040.74 Although both inhibitors reached comparable serum levels in mice, only CI-1040 was able to reduce lung tumor formation, which might be explained by inefficient cellular uptake of BAY 43-9006 or an increased dependency of the Raf-induced lung tumor on MEK activity.74 The lab of Andreeff established a conditional mouse model to study the effects of small molecule inhibitors in vivo using Raf-expressing FDC-P1 hematopoietic cells.79 All 3 Raf isoforms were individually expressed in an N-terminal truncated form and fused to the hormone-binding domain of the estrogen receptor, thereby enabling induction of Raf activity upon β-estradiol addition, as well as to the green fluorescent protein (GFP) allowing immunofluorescence detection. Tumor formation of FDC-P1 cells expressing individual Raf fusion proteins was analyzed in SCID mice. Estradiol pellet implantation into these mice significantly accelerated tumor onset and enhanced tumor growth and disseminated leukemia was observed using bioluminescence imaging.79 Whereas in both animal cancer models the MEK inhibitor CI-1040 reduced proliferation as well as tumor formation in vivo, no obvious effect on apoptosis was observed in our lung model in comparison to the leukemia model, which might be explained by cell-type specific responses.

Raf kinases and human cancer

  1. Top of page
  2. Abstract
  3. Raf kinases: The History
  4. Raf kinase signaling
  5. Raf kinases and mouse cancer models
  6. Raf kinases and human cancer
  7. Raf kinases and cancer drug discovery
  8. Perspectives
  9. Acknowledgements
  10. References

For a long time, it was thought that constitutive activation of the mitogenic cascade,80e.g., by either mutational activation of RAS proteins81 or by overexpression of C-RAF,82 is the main mechanism contributing to tumor formation in humans. A couple of recent publications addressing the mutational status of A-RAF and C-RAF genes in more than 600 cancer cell lines and 500 primary human samples obtained from colorectal and gastric carcinoma, acute leukemias, gliomas, as well as lung, ovarian and testis tumors suggest that mutations in both RAF genes are very rare or nonexistent events.83, 84, 85 The most detailed analysis of 545 cancer cell lines and about 80 primary tumor samples by Marais and coworkers detected no A-RAF mutations and only 4 rare polymorphisms in C-RAF corresponding to a mutational rate of 0.7%.85 The identified 4 C-RAF mutants (P207S, V226I, Q335H, E478K) displayed differences with respect to basal and Ras induced kinase activities but were similar active in standard 3T3 colony formation assays.85

Surprising—but not unexpected—was the discovery of B-RAF mutations in ∼8% of human cancers.86 Initially, as an early outcome of the Cancer Genome Project at the Wellcome Trust Sanger Institute B-RAF mutations were detected in 66% of malignant melanomas and at a lower frequency in a wide range of other human tumors.87 All mutations described there were within or close to the kinase domain of B-RAF and the majority of mutations involved a single base substitution in the kinase domain (T1799A) leading to a V600E amino acid exchange.87 Moreover, mutant V600EB-RAF kinase displayed elevated in vitro kinase activity, constitutive MEK-ERK signaling, as well as enhanced transformation activity in fibroblasts and melanocytes.62, 87, 88, 89, 90 This pivotal discovery started a hunt for B-RAF mutations. In May 2006 the annotated database COSMIC (Catalogue of Somatic Mutations in Cancer) was listing 2,989 B-RAF mutations in 16,120 tumor samples incorporating curated mutation data from 180 publications.91 The tissues represented with the highest mutation frequencies being skin (43% of samples mutated), thyroid (27%), large intestine (15%), ovary (15%) and biliary tract (15%). No B-RAF mutations are so far listed for the urinary tract, testis, prostate, pleura, kidney, genital tract, cervix and bone, but up to now only a smaller number of samples have been analyzed from these tissues.

Although more than 30 B-RAF missense mutations have been published, the mutation most often represented in the database in ∼85% of all entries is the point mutation V600E. The high mutation rate at this position may be biased due to limited hot-spot mutation detection and the question about the predominance of this V600EB-RAF mutation needs to be clarified in future. The crystal structures of the kinase domains of wildtype and V600EB-RAF in complex with the small molecule inhibitor BAY 43-9006 were solved at 2.9 and 3.4 Å resolution, respectively, and provided some insights into the activation and signaling properties of B-RAF wildtype and mutants.62 It was proposed that B-RAF mutants such as the V600EB-RAF mutant, which was classified into a group called “activated mutants” displaying elevated in vitro kinase activities, have no impairment with respect to their overall enzymatic activity and are destabilized in their inactive conformation.86, 92 Another remarkable feature of the V600EB-RAF mutation was unraveled, when human cancer cell lines from various tissues were treated with the MEK inhibitor CI-1040.93 It was shown that the V600EB-RAF mutation—in contrast to the independent Q61RNRAS mutation—is associated with enhanced sensitivity to this drug. This sensitivity was correlated with downregulation of cyclin D, p27 induction and G1 arrest in all and a reduction of RB phosphorylation and increased apoptosis in some of the investigated cell lines with the V600EB-RAF mutation.93 Whether this finding has implications for the treatment of human cancer patients using MEK inhibitors, has to be addressed in future clinical studies with patients with established B-RAF mutation status.52

In radiation-induced thyroid papillary carcinomas another type of B-RAF mutation leading to a novel mode of B-RAF activation was found.94 The paracentric inversion of the long arm of chromosome 7 results in an in-frame fusion of the N-terminus of the A-kinase anchor protein 9 (exons 1–8) and the C-terminal catalytic domain (exons 9–18) of B-RAF. The resulting fusion protein has increased B-RAF kinase activity and is considerably active in NIH 3T3 focus formation assay and tumor formation in nude mice.94 Whether this type of mutational activation is found exclusively in thyroid carcinomas with recent radiation exposure needs to be determined in future. The group of Nikiforov was also successful in describing an additional mechanism of B-RAF activation.95 Using fluorescence in situ hybridization with B-RAF specific and centromeric chromosome 7 probes they were able to detect additional copies of B-RAF or of chromosome 7 in a high percentage follicular thyroid carcinomas. These changes were absent in follicular tumors with RAS mutations and as determined by Western blotting resulted in increased B-RAF protein levels.95 In summary, more and more mechanisms are elucidated contributing to hyperactivation or deregulation of RAF-mediated signaling pathways in human cancer. These are (i) Activating mutations of upstream RAF regulators, (ii) Overexpression of nonmutated RAF proteins due to transcriptional upregulation or due to mutations leading to a net increase of RAF copy numbers and (iii) Mutational activation by either point mutations or chromosomal rearrangements.

Before the year 2006 no germline mutations in RAF genes have been described. This was not unforeseen, since our early attempts to generate raf transgenic mice with promoters that are active early during development invariably failed and we had to choose a tissue-specific promoter active at later developmental stages (see above). Unexpectedly, 2 germline mutations, S427GC-RAF and I448VC-RAF, have been identified in 82 samples of patients with therapy-related acute myeloid leukemia.96 Both mutations were located in the kinase domain and able to sustain growth in soft agar assays, but did not induce morphological transformation of NIH 3T3 cells. The authors proposed that these C-RAF mutations constitute a novel tumor-predisposing factor.96 By mutational analysis of individuals with the cardio-facio-cutaneous syndrome, a developmental disorder not associated so far with an increased risk of cancer, 2 groups independently reported 15 different B-RAF missense mutations.97, 98 The majority of these 15 germline mutations had not been detected previously in human tumors and their kinase activities ranged from low (kinase-impaired) to highly elevated (high kinase). Whether these B-RAF mutants also display enhanced sensitivity to MEK inhibitors remains to be established.

So far, only a few studies link the mutational status of B-RAF with disease progression, clinical outcome and patient survival. For example, it was shown that the constitutive activation of the Ras-Raf signaling pathway due to either B-RAF or NRAS mutation is associated with poorer prognosis, i.e., shortened survival, in metastatic melanoma99 and that oncogenic B-RAF mutations rather correlate with progression than initiation of human melanoma.100B-RAF mutations were also correlated with poor survival in microsatellite-stable colon cancers101 and with a poorer clinicopathological outcome for patients with PTC.102 The great majority of publications however do not link the clinical course with the mutational status of B-RAF and due to great variations in sample size and methods used for tumor isolation, mutation detection or statistical analysis many conflicting reports on the impact of B-RAF mutations on tumorigenesis are currently published. Therefore, we are not going to comment these partially conflicting results and refer the reader to some recent excellent reviews on B-RAF mutations in general6, 86, 103 and on overviews focusing on B-RAF and melanoma,104, 105 thyroid106, 107 or ovarian cancer.108

Another rising field of research is to investigate how the immune system deals with B-RAF mutations, or in other words, whether mutated B-RAF is targeted by immune surveillance.109 The immunogenicity of constitutively active V600EB-RAF was successfully demonstrated by the detection of CD8+ responses, which were either HLA-B*2705-restricted110 or HLA-A*0201-restricted111 and of a V600EB-RAF specific CD4+ T-cell response.112 In addition, the loss of the V600EB-RAF genotype in a patient during progression from primary to metastatic melanoma suggested an active immune selection of nonmutated melanoma clones.99 Moreover, B-RAF and V600EB-RAF specific antibodies emerging at late stages of melanoma progression have been discovered.113 Together, these findings make it likely that in future at least advanced stage melanoma patients may benefit from targeting of the V600EB-RAF mutation.

Raf kinases and cancer drug discovery

  1. Top of page
  2. Abstract
  3. Raf kinases: The History
  4. Raf kinase signaling
  5. Raf kinases and mouse cancer models
  6. Raf kinases and human cancer
  7. Raf kinases and cancer drug discovery
  8. Perspectives
  9. Acknowledgements
  10. References

Intense efforts have been taken in the past to develop novel anticancer strategies targeting Raf dependent signaling pathways; for reviews see Refs.114, 115, 116. Considerable clinical progress has been made with the small-molecule inhibitor Nexavar® (Sorafenib, BAY 43-9006), antisense and heat shock protein 90 (HSP90) inhibitors. An overview on the clinical status of different Raf targeting strategies in oncology is given in Figure 3.

thumbnail image

Figure 3. Current status of therapeutic strategies targeting RAF kinases in oncology. 4 general strategies of targeting RAF kinases are shown. The small molecule inhibitor Nexavar® is already approved for the treatment of patients with advanced renal cell carcinoma in the United States, Suisse and Mexico and is currently in clinical trials for the treatment of a wide variety of tumors. The development of the antisense inhibitor ISIS 5132 as anticancer drug has been terminated due to low clinical efficacy. The group of HSP90 and HDAC inhibitors is targeting a whole range of proteins beneath RAF kinases. Other strategies/drugs are currently evaluated at the preclinical stage or in early clinical trails as described. For more details see text.

Download figure to PowerPoint

In 2004, the National Cancer Institute director Andrew von Eschenbach set a ambitious goal for the NCI: the elimination of suffering and death due to cancer by the year 2015.117 A major step towards this direction was the positive evaluation of Nexavar (Sorafenib, BAY 43-9006), a potent small-molecule inhibitor of Raf kinases, in ongoing clinical trials. Sorafenib belongs to the bi-aryl urea class of protein kinase inhibitors118 and its development by Bayer HealthCare and Onyx Pharmaceuticals, Inc. as a C-Raf inhibitor119, 120 was based on the large body of evidence for a role of C-Raf as a major transformation effector of Ras.36 In early biochemical and cellular-based assays, this inhibitor was identified as bonafide C-Raf inhibitor and was demonstrated to reduce tumor cell proliferation in vitro as well as tumor growth in human tumor xenograft models in vivo.121, 122 Upon further characterization BAY 43-9006 was shown not only to inhibit B-Raf and V600EB-Raf in vitro, but also to target efficiently other receptor tyrosine kinases involved in neovascularization and tumor progression, including VEGFR-2, VEGFR-3, Flt-3, c-KIT, PDGFR-α, PDGFR-β and RET.123, 124, 125, 126 Daily oral dosing of BAY 43-9006 demonstrated broad-spectrum antitumor activity in various cancer xenograft models.123 The basis of this antitumor activity was the inhibition of the mitogenic cascade as well as the reduction of neovessel formation demonstrating that BAY 43-9006 is a novel dual action inhibitor targeting tumor cell proliferation as well as tumor angiogenesis.123 Moreover, it was also shown recently that downregulation of the antiapoptotic Bcl-2 family member Mcl-1 by BAY 43-9006 in a MEK/ERK signaling-independent manner contributes to the proapoptotic effects of this inhibitor.127 Additional multiple proapoptotic effects of BAY 43-9006 have been described in human melanoma cells.128 Among them were dephosphorylation of BAD on Ser75 and Ser99 activation of Bak and Bax, downmodulation of Bcl-2 and Bcl-XL levels, downregulation of the mitochondrial transmembrane potential, caspase activation as monitored by PARP cleavage and release of cytochrome c and SMAC from mitochondria. Surprisingly, in some but not all cell lines it was demonstrated by the use of a pan-caspase inhibitor that apoptosis induction by BAY 43-9006 is largely caspase independent and depends primarily on the nuclear translocation of apoptosis inducing factor AIF.128

BAY 43-9006 rapidly advanced in clinical trials and was awarded fast track status by the US Food and Drug Administration (FDA) in 2004. In the middle of 2005, 59 clinical trials were sponsored by either the National Cancer Institute or Bayer/Onyx for the use of Sorafenib as single or as combination agent as treatment for a wide variety of solid and lymphoid tumors, including Phase III clinical trials for advanced renal carcinoma, advanced malignant melanoma and primary hepatic cancer.129, 130 In February 2006 the initiation of a randomized, double-blind, placebo-controlled Phase III clinical trial studying Sorafenib administered in combination with the chemotherapeutic agents carboplatin and paclitaxel in patients with non-small cell lung cancer (NSCLC) was announced. At the 2005 Annual ASCO Meeting Sorafenib was reported to double progression-free survival in advanced renal cell carcinoma by the BAY 43-9006 TARGETs Clinical Trial Group.131 In December 2005 Sorafenib was finally approved by the FDA for the treatment of patients with advanced renal cell carcinoma and is now marketed as Nexavar in the US. In the meantime, Nexavar was also approved in Suisse and Mexico; EU wide approval is expected in the second half of 2006.

Early experiments using vector-driven antisense expression of full-length C-RAF already demonstrated that antisense inhibition is a valuable approach to reduce significantly the proliferation and tumorigenesis of transformed cell lines.132 The antitumor activity of ISIS 5132, a 20-nucleotide phosphorothioate 2′ deoxynucleotide targeting the 3′ untranslated region of C-RAF mRNA, was very promising at the early preclinical stage.133, 134 However, no major clinical benefits were observed in several Phase I135, 136, 137 or Phase II clinical trials.138, 139, 140, 141 In addition, the use of ISIS 13650, a second generation antisense oligonucleotide with further modifications in the sugar moiety targeting the same C-RAF sequence as ISIS 5132, has not been superior in certain preclinical settings.142 LErafAON, a C-RAF antisense oligodeoxyribonucleotide applied as a liposome-encapsulated formulation, which is supposed to increase the stability and the cellular uptake of the antisense inhibitor,143 is currently evaluated in several Phase I clinical trials as monotherapy as well as in combination with either radiation or chemotherapy.144, 145 Moreover, additional antisense approaches are currently tested for antitumor activity in preclinical models. So far, depletion of either B-RAF or mutant V600EB-RAF by small-interfering RNAs (siRNAs) reduced proliferation and invasiveness of melanoma cell lines,146, 147, 148 and also the growth and vascular development of malignant melanoma tumors.149 Reduction of in vivo tumor growth by application of C-RAF siRNA was also reported in xenograft models of human prostate150 and also breast cancer.151

The benzoquinone ansamycin Geldanamycin (GA) was originally isolated as compound with antifungal activities and was later found to reduce oncogene-dependent proliferation of tumor cells; for review see Ref.152. This is primarily due to binding and inhibition of a major cellular chaperone, HSP90, which prevents the conformational maturation of HSP90 client proteins and promotes their proteasomal degradation.153 More than 50 client proteins of HSP90, including Raf kinases, Akt/PKB, ErbB2 and Met, have been identified. The therapeutic selectivity of HSP90 inhibitors in tumor versus normal cells was explained by increased HSP90 expression and the concomitant formation of a supersensitive multichaperone complex in cancer cells.154 Due to severe hepatotoxicity in animal studies GA was not further clinically evaluated,155 whereas 17-AAG (17-allylamino-17-demethoxygeldanamycin), a GA derivate with a significantly reduced toxicity, entered several Phase I and now also Phase II clinical trials with encouraging results.156 Interestingly, it was shown recently that wild-type and mutant B-RAF proteins are also HSP90 client proteins and are targeted to ubiquitin-dependent proteolysis by 17-AAG.157 The V600EB-RAF protein and most of the other mutant B-RAF kinases were 17-AAG hypersensitive in comparison to nonmutated B-RAF.157, 158 17-DMAG, a second HSP90 inhibitor with higher solubility and antitumor efficacy,159, 160 is currently tested on patients with solid tumors and lymphomas in 3 Phase I clinical trials sponsored by the NCI. Although HSP90 inhibitors are promising, the usefulness of HSP90 inhibitors for cancer therapy is discussed controversially. It was shown recently that inhibition of HSP90 by GA or 17-AAG concomitantly induced massive expression of HSP40, HSP70 and HSP90 proteins.161 Overexpression of HSP family members, such as the well-recognized antiapoptotic factor HSP70, in turn contributes to drug resistance and a poor response to combination-chemotherapy regimens thereby helping tumor cells to survive.162 The group of Bhalla used pretreatment with K562, a benzylidine lactam HSP70 inhibitor, in tissue culture to block HSP70 induction which was accompanied by an increase of the antitumor activity of 17-AAG.163 On the other hand, increased HSP70 levels may promote the formation of stable complexes with tumor antigens, which upon release from the cell may lead to the activation of antigen-presenting cells thereby breaking tolerance to tumor antigens and eliciting CD8+ tumor-specific responses.164 More basic and clinical research is clearly needed to find out for which type of tumors mono- or combination therapies with HSP90 inhibitors are beneficial.

Another set of inhibitors, which is currently under active clinical development as anticancer drug, is the heterogeneous group of histone deacetylase (HDAC) inhibitors.165, 166 Although histones are the primary targets of HDACs and transcriptional reactivation of “dormant” tumor-suppressor genes is clearly one major mechanism of action, other cellular targets and pleiotropic effects have been described.167 In context with Raf kinases, HDAC inhibitors seem to have 2 ways of action: (i) in human multiple myeloma C-RAF mRNA expression was substantially reduced in response to treatment with the HDAC inhibitor SAHA168 and (ii) in human leukemia cell lines it was shown that HDAC inhibitors such as LAQ824 induce acetylation and inactivation of HSP90 which in turn promotes degradation of HSP90 client proteins including C-RAF.169, 170, 171 SAHA is currently in Phase II/III trials for the treatment of advanced cutaneous T-cell lymphoma and relapsed diffuse large B-cell lymphoma, whereas LAQ824 is in Phase I.

Understanding the human immune response to cancer is a prerequisite for the development of effective cancer immunotherapies.172, 173 As already mentioned above, a couple of early preclinical studies have evaluated the usefulness of B-RAF as target for immunotherapy. In melanoma patients harboring the V600EB-RAF mutation, a 29-mer B-RAF peptide incorporating the V600E mutation was used for in vitro stimulation of lymphocytes, generating MHC class II-restricted CD4+ T cells specific for this peptide as well as for melanoma cells expressing V600EB-RAF.112 A spontaneous HLA-B*2705-restricted cytotoxic T-cell response against an epitope derived from V600EB-RAF was demonstrated for the first time in melanoma patients.110 Moreover, in this publication also the loss of the V600EB-RAF genotype during progression from primary to metastatic melanoma in patients with V600EB-RAF specific T-cell responses was shown. This suggests an active immune selection of nonmutated melanoma clones by the tumor-bearing host.110 Finally, the screening of sera of 148 patients with advanced melanoma revealed that in ∼9% of sera B-RAF/V600EB-RAF specific antibodies are present demonstrating a humoral response of the immune system.113 In summary, further studies investigating cell-mediated and humoral responses are clearly needed to validate B-RAF/V600EB-RAF as a specific target for immunotherapy.


  1. Top of page
  2. Abstract
  3. Raf kinases: The History
  4. Raf kinase signaling
  5. Raf kinases and mouse cancer models
  6. Raf kinases and human cancer
  7. Raf kinases and cancer drug discovery
  8. Perspectives
  9. Acknowledgements
  10. References

The new facets of Raf kinases that have been discovered over the past 5 years are really fascinating. However, a number of pressing questions need to be addressed in near future in more detail.

  • 1
    What is still missing is an in vitro reconstituted system with highly purified components that allows us to study the signaling properties of wildtype and mutant Raf kinases. This might not only clarify the impact of individual phosphorylation events or binding partners, but also help us together with the determination of Raf kinase structure at higher resolution to design better Raf kinase inhibitors. In addition, an in vitro reconstituted system would be instrumental in verifying and fine-tuning the pathway predictions recently obtained by computational and mathematical modeling of the mitogenic cascade174; for a recent comprehensive review see Ref.175.
  • 2
    Although Nexavar®/Sorafenib has certainly blockbuster potential, lessons learned on resistance development induced by several inhibitors in clinical use suggest that there is better no halt in the development of novel Raf kinase inhibitors. Several companies are actively pursuing this task (see overview Table I) and try to generate novel RAF mutation- or isoform-specific inhibitors in the light of the importance of the V600EB-RAF mutation. At the 97th AACR Annual Meeting in April 2006 the Chiron Corporation introduced the novel RAF inhibitor CHIR-265 and announced also the initiation of a Phase I clinical trial for melanoma patients.177, 178, 179 At the same meeting Plexxikon Inc. introduced the B-RAF specific inhibitor PLX4032.180 Although a couple of other companies, such as Sunesis, Exelixis and ArQule, are currently broadcasting the successful development of B-RAF selective inhibitors, there have been no data published on these so far.
    In the past, small-molecule Raf kinase inhibitors were primarily targeting the catalytic active site of these enzymes, whereas there have been no reports on allosteric inhibitors. This class of inhibitors offers an alternative approach to the inhibition of protein activities in particular for proteins undergoing conformational changes during their activation cycle, as previously shown for the N-WASP inhibitor Wiskostatin.189 Another way to go is the design of therapeutic approaches that specifically disrupt specific protein–protein interactions,190 in which Raf kinases are participating. If indeed isoform-exclusive interactions between individual Raf kinases and interacting proteins exist (Fig. 2), this might certainly help to bypass the missing isoform-specific selectivity of current active-site Raf inhibitors. A particularly interesting interaction to target in this respect is the interaction between C-Raf and Prohibitin, a highly conserved protein that was recently shown to be indispensable for RAS-induced activation of the mitogenic cascade and epithelial cell migration.54
  • 3
    Whereas the detection of Raf-specific mutations is a top priority for oncologists nowadays, a systematic mutational analysis of genes encoding proteins negatively regulating the mitogenic cascade, such as RKIP, Spred and Sprouty, is still missing. So far, only reduced expression levels at certain stages of tumor development imply these proteins as potential tumor or metastasis suppressor genes. Another interesting point of future investigations is the functional analysis of microRNAs targeting Raf genes during normal development and tumor formation. As predicted from miRNA target databases as miRBase,191 all 3 isoforms are targeted by specific miRNAs (A-RAF:miR-412, B-RAF:miR-29a/b/c, C-RAF:miR-19a/b/c/d).
  • 4
    The functional consequences of RAF mutations in human tumors have to be addressed using state-of-the art genomic and proteomic approaches. Two recent reports described the detection of V600EB-RAF specific gene expression patterns by microarray analyses in PTC192 and colorectal cancer193 and discussed the relevance of specific oncogene pathway signatures for diagnosis, clinical disease management and target discovery for directed therapies. Statements on the impact of B-RAF mutations on tumor establishment and progression absolutely require large clinical studies with patients being stratified for their B-RAF mutational status and with correctly staged tumor samples of a statistically significant amount of patients.
  • 5
    Another neglected branch of investigation is the functional analysis of the role Raf kinases may have in stem cell biology and epigenetic regulation. Although by genetic analyses of transgenic and knock-out mice we have a gross understanding of the roles Raf kinases play during organismal development and during differentiation of multiple cell lineages, there are currently no published data associating Raf kinases directly to stem cell renewal or early stem cell differentiation. An indirect hint that Raf kinases might play a role in these processes is derived from the observation that hypermitogenic signaling via Raf kinases targets Bmi-1, a Polycomb family transcriptional repressor, which is required for the maintenance of stem cells in multiple tissues including the nervous and hematopoietic system.194 We have previously described a proliferative switch that links Raf signals to the phosphorylation and release of Bmi-1 from repressive chromatin complexes.195 Whereas low intensity Raf signals are required for normal proliferative responses, high intensity Raf signals induce an antiproliferative response that may include delayed cell cycle progression and subsequent differentiation, apoptosis or senescence.195 Since Bmi-1 is an epigenetic chromatin modifier that is also linked to cancer development it might be of interest to investigate whether the Raf-Bmi-1 connection has an impact on tumorigenesis. This might offer the possibility to manipulate cancer or cancer stem cells in a way that may lead either to apoptosis or prevents uncontrolled proliferation and differentiation.
Table I. Small Molecule Raf Kinase Inhibitors1
SubstanceChemical classIC50 (nM)RemarksDeveloped byReferences
  • 1

    Overview on selected small molecule Raf inhibitors. Compound activity in general is measured in vitro by their ability to inhibit RAF-mediated phosphorylation of kinase dead MAP kinase kinase MEK. IC50 values are presented to give an estimate of the in vitro activity of these inhibitors on C-RAF, B-RAF and V600EB-RAF.

  • 2

    Only a range of IC50 values for all three Raf kinases was given.

  • 3

    For SB-590885 data from a fluorescent ligand displacement assay have been incorporated and expressed as Kd (nM). Poor solubility in water may prevent the further clinical development of certain drugs. Abbreviations used: n.d., not determined; HTS, high-throughput screen.

Nexavar®/BAY 43-9006/SorafenibDiphenyl urea62238Approved for patients with advanced renal cell carcinomaBayer/Onyx122,123,176
CHIR-265Substituted benzazole3–602Clinical trial initiatedChiron177, 178, 179
PLX4032Not publishedn.d.10031Clinical trial planned in late 2006Plexxikon180
X-6-(3 acetamidophenyl) pyrazinesDi-substituted pyrazinesn.d.<800n.d.Initial results of HTS screenCentre for Cancer Therapeutics, Sutton, UK181
Several compounds3,5, Di-substituted pyridinesn.d.n.d.> 500Initial results of HTS screenCentre for Cancer Therapeutics, Sutton, UK182
SB-590885 (33)Triarylimidazolen.d.0.33n.d.Initial report on synthesis, activity and selectivityGlaxoSmithKline183
AAL881Isoquinoline430940220Preclinical evaluationNovartis184
LBT613Isoquinoline120200210Preclinical evaluationNovartis185
Omega-carboxypyridylDiphenyl urea50–100n.d.n.d.Increased solubility in waterBayer186
Compound 2Benzylidene oxindole9n.d.n.d.Weak activity in cell linesGlaxoSmithKline187
ZM 336372Benzamide10100n.d.In vitro inhibitor, but in vivo activator of C-RAFAstraZeneca188
L-779450Triarylimidazole1.410n.d.Poorly soluble in aqueous systemsMerck187,188


  1. Top of page
  2. Abstract
  3. Raf kinases: The History
  4. Raf kinase signaling
  5. Raf kinases and mouse cancer models
  6. Raf kinases and human cancer
  7. Raf kinases and cancer drug discovery
  8. Perspectives
  9. Acknowledgements
  10. References
  • 1
    Rapp UR, Goldsborough MD, Mark GE, Bonner TI, Groffen J, Reynolds FH,Jr, Stephenson JR. Structure and biological activity of v-raf, a unique oncogene transduced by a retrovirus. Proc Natl Acad Sci USA 1983; 80: 421822.
  • 2
    Jansen HW, Patschinsky T, Bister K. Avian oncovirus MH2: molecular cloning of proviral DNA and structural analysis of viral RNA and protein. J Virol 1983; 48: 6173.
  • 3
    Jansen HW, Ruckert B, Lurz R, Bister K. Two unrelated cell-derived sequences in the genome of avian leukemia and carcinoma inducing retrovirus MH2. EMBO J 1983; 2: 196975.
  • 4
    Jansen HW, Lurz R, Bister K, Bonner TI, Mark GE, Rapp UR. Homologous cell-derived oncogenes in avian carcinoma virus MH2 and murine sarcoma virus 3611. Nature 1984; 307: 2814.
  • 5
    Sutrave P, Bonner TI, Rapp UR, Jansen HW, Patschinsky T, Bister K. Nucleotide sequence of avian retroviral oncogene v-mil: homologue of murine retroviral oncogene v-raf. Nature 1984; 309: 858.
  • 6
    Wellbrock C, Karasarides M, Marais R. The RAF proteins take centre stage. Nat Rev Mol Cell Biol 2004; 5: 87585.
  • 7
    Rapp UR, Reynolds FH,Jr, Stephenson JR. New mammalian transforming retrovirus: demonstration of a polyprotein gene product. J Virol 1983; 45: 91424.
  • 8
    Mark GE, Rapp UR. Primary structure of v-raf: relatedness to the src family of oncogenes. Science 1984; 224: 2859.
  • 9
    Moelling K, Heimann B, Beimling P, Rapp UR, Sander T. Serine- and threonine-specific protein kinase activities of purified gag-mil and gag-raf proteins. Nature 1984; 312: 55861.
  • 10
    Sutrave P, Jansen HW, Bister K, Rapp UR. 3′-Terminal region of avian carcinoma virus MH2 shares sequence elements with avian sarcoma viruses Y73 and SR-A. J Virol 1984; 52: 7035.
  • 11
    Rapp UR, Cleveland JL, Storm SM, Beck TW, Huleihel M. Transformation by raf and myc oncogenes. Princess Takamatsu Symp 1986; 17: 5574.
  • 12
    Roberts TM, Kaplan D, Morgan W, Keller T, Mamon H, Piwnica-Worms H, Druker B, Cohen B, Schaffhausen B, Whitman M, Cantley L, Raap UR, et al. Tyrosine phosphorylation in signal transduction. Cold Spring Harb Symp Quant Biol 1988; 53: 16171.
  • 13
    Rapp UR, Heidecker G, Huleihel M, Cleveland JL, Choi WC, Pawson T, Ihle JN, Anderson WB. Raf family serine/threonine protein kinases in mitogen signal transduction. Cold Spring Harb Symp Quant Biol 1988; 53: 17384.
  • 14
    Blasi E, Mathieson BJ, Varesio L, Cleveland JL, Borchert PA, Rapp UR. Selective immortalization of murine macrophages from fresh bone marrow by a raf/myc recombinant murine retrovirus. Nature 1985; 318: 66770.
  • 15
    Rapp UR, Bonner TI, Moelling K, Jansen HW, Bister K, Ihle J. Genes and gene products involved in growth regulation of tumor cells. Recent Results Cancer Res 1985; 99: 22136.
  • 16
    Rapp UR, Cleveland JL, Brightman K, Scott A, Ihle JN. Abrogation of IL-3 and IL-2 dependence by recombinant murine retroviruses expressing v-myc oncogenes. Nature 1985; 317: 4348.
  • 17
    Rapp UR, Bonner TI, Cleveland JL. The raf oncogene. In: Gallo RC, Stehelin D, Varnier OE, eds. Retroviruses and human pathology. Clifton, New Jersey: Humana Press, 1986, 44972.
  • 18
    Rapp UR, Cleveland JL, Fredrickson TN, Holmes KL, Morse HC,III, Jansen HW, Patschinsky T, Bister K. Rapid induction of hemopoietic neoplasms in newborn mice by a raf(mil)/myc recombinant murine retrovirus. J Virol 1985; 55: 2333.
  • 19
    Troppmair J, Potter M, Wax JS, Rapp UR. An altered v-raf is required in addition to v-myc in J3V1 virus for acceleration of murine plasmacytomagenesis. Proc Natl Acad Sci USA 1989; 86: 99415.
  • 20
    Rapp UR, Bruder JT, Troppmair J. Role of the raf signal transduction pathway in Fos/Jun regulation and determination of cell fates. In: Angel PE, Herrlich PA, eds. The FOS and JUN families of transcription factors. Boca Raton: CRC Press, 1994, 22147.
  • 21
    Principato M, Klinken SP, Cleveland JL, Rapp UR, Holmes KL, Pierce JH, Morse HC,III. In vitro transformation of murine bone marrow cells with a v-raf/v-myc retrovirus yields clonally related mature B cells and macrophages. Curr Top Microbiol Immunol 1988; 141: 3141.
  • 22
    Principato M, Cleveland JL, Rapp UR, Holmes KL, Pierce JH, Morse HC,III, Klinken SP. Transformation of murine bone marrow cells with combined v-raf-v-myc oncogenes yields clonally related mature B cells and macrophages. Mol Cell Biol 1990; 10: 35628.
  • 23
    Fedorov LM, Papadopoulos T, Tyrsin OY, Twardzik T, Goetz R, Rapp UR. Loss of p53 in craf-induced transgenic lung adenoma leads to tumor acceleration and phenotypic switch. Cancer Res 2003; 63: 226877.
  • 24
    Heidecker G, Huleihel M, Cleveland JL, Kolch W, Beck TW, Lloyd P, Pawson T, Rapp UR. Mutational activation of c-raf-1 and definition of the minimal transforming sequence. Mol Cell Biol 1990; 10: 250312.
  • 25
    Kolch W, Cleveland JL, Rapp UR. Role of oncogenes in the abrogation of growth factor requirements of hemopoietic cells. In: Paukovits WR, ed. Growth regulation and cancerogenesis. Boca Raton: CRC Press, 1991, 279303.
  • 26
    Bruder JT, Heidecker G, Rapp UR. Serum-, TPA-, and Ras-induced expression from Ap-1/Ets-driven promoters requires Raf-1 kinase. Genes Dev 1992; 6: 54556.
  • 27
    Zhang XF, Settleman J, Kyriakis JM, Takeuchi-Suzuki E, Elledge SJ, Marshall MS, Bruder JT, Rapp UR, Avruch J. Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1. Nature 1993; 364: 30813.
  • 28
    Kyriakis JM, App H, Zhang XF, Banerjee P, Brautigan DL, Rapp UR, Avruch J. Raf-1 activates MAP kinase-kinase. Nature 1992; 358: 41721.
  • 29
    Howe LR, Leevers SJ, Gomez N, Nakielny S, Cohen P, Marshall CJ. Activation of the MAP kinase pathway by the protein kinase raf. Cell 1992; 71: 33542.
  • 30
    Dent P, Haser W, Haystead TA, Vincent LA, Roberts TM, Sturgill TW. Activation of mitogen-activated protein kinase kinase by v-Raf in NIH 3T3 cells and in vitro. Science 1992; 257: 14047.
  • 31
    Huleihel M, Goldsborough M, Cleveland J, Gunnell M, Bonner T, Rapp UR. Characterization of murine A-raf, a new oncogene related to the v-raf oncogene. Mol Cell Biol 1986; 6: 265562.
  • 32
    Huebner K, Rushdi AA, Griffin CA, Isobe M, Kozak C, Emanuel BS, Nagarajan L, Cleveland JL, Bonner TI, Goldsborough MD, Croce CM, Raap UR. Actively transcribed genes in the raf oncogene group, located on the X chromosome in mouse and human. Proc Natl Acad Sci USA 1986; 83: 39348.
  • 33
    Beck TW, Huleihel M, Gunnell M, Bonner TI, Rapp UR. The complete coding sequence of the human A-raf-1 oncogene and transforming activity of a human A-raf carrying retrovirus. Nucleic Acids Res 1987; 15: 595609.
  • 34
    Ikawa S, Fukui M, Ueyama Y, Tamaoki N, Yamamoto T, Toyoshima K. B-raf, a new member of the raf family, is activated by DNA rearrangement. Mol Cell Biol 1988; 8: 26514.
  • 35
    Sithanandam G, Kolch W, Duh FM, Rapp UR. Complete coding sequence of a human B-raf cDNA and detection of B-raf protein kinase with isozyme specific antibodies. Oncogene 1990; 5: 177580.
  • 36
    Daum G, Eisenmann-Tappe I, Fries HW, Troppmair J, Rapp UR. The ins and outs of Raf kinases. Trends Biochem Sci 1994; 19: 47480.
  • 37
    Hagemann C, Rapp UR. Isotype-specific functions of Raf kinases. Exp Cell Res 1999; 253: 3446.
  • 38
    Chong H, Vikis HG, Guan KL. Mechanisms of regulating the Raf kinase family. Cell Signal 2003; 15: 4639.
  • 39
    Storm SM, Rapp UR. Oncogene activation: c-raf-1 gene mutations in experimental and naturally occurring tumors. Toxicol Lett 1993; 67: 20110.
  • 40
    Cleveland JL, Troppmair J, Packham G, Askew DS, Lloyd P, Gonzalez-Garcia M, Nunez G, Ihle JN, Rapp UR. v-Raf suppresses apoptosis and promotes growth of interleukin-3-dependent myeloid cells. Oncogene 1994; 9: 221726.
  • 41
    Wang HG, Rapp UR, Reed JC. Bcl-2 targets the protein kinase Raf-1 to mitochondria. Cell 1996; 87: 62938.
  • 42
    Wang HG, Takayama S, Rapp UR, Reed JC. Bcl-2 interacting protein, BAG-1, binds to and activates the kinase Raf-1. Proc Natl Acad Sci USA 1996; 93: 70638.
  • 43
    Woods D, Parry D, Cherwinski H, Bosch E, Lees E, McMahon M. Raf-induced proliferation or cell cycle arrest is determined by the level of Raf activity with arrest mediated by p21Cip1. Mol Cell Biol 1997; 17: 5598611.
  • 44
    Kerkhoff E, Rapp UR. High-intensity Raf signals convert mitotic cell cycling into cellular growth. Cancer Res 1998; 58: 163640.
  • 45
    Li S, Sedivy JM. Raf-1 protein kinase activates the NF-K B transcription factor by dissociating the cytoplasmic NF-K B-I K B complex. Proc Natl Acad Sci USA 1993; 90: 924751.
  • 46
    Baumann B, Weber CK, Troppmair J, Whiteside S, Israel A, Rapp UR, Wirth T. Raf induces NF-KB by membrane shuttle kinase MEKK1, a signaling pathway critical for transformation. Proc Natl Acad Sci USA 2000; 97: 461520.
  • 47
    Baccarini M. Second nature: biological functions of the Raf-1 “kinase”. FEBS Lett 2005; 579: 32717.
  • 48
    O'Neill E, Rushworth L, Baccarini M, Kolch W. Role of the kinase MST2 in suppression of apoptosis by the proto-oncogene product Raf-1. Science 2004; 306: 226770.
  • 49
    Chen J, Fujii K, Zhang L, Roberts T, Fu H. Raf-1 promotes cell survival by antagonizing apoptosis signal-regulating kinase 1 through a MEK-ERK independent mechanism. Proc Natl Acad Sci USA 2001; 98: 77838.
  • 50
    Ehrenreiter K, Piazzolla D, Velamoor V, Sobczak I, Small JV, Takeda J, Leung T, Baccarini M. Raf-1 regulates Rho signaling and cell migration. J Cell Biol 2005; 168: 95564.
  • 51
    Kolch W. Coordinating ERK/MAPK signalling through scaffolds and inhibitors. Nat Rev Mol Cell Biol 2005; 6: 82737.
  • 52
    Rapp UR, Gotz R, Albert S. BuCy RAFs drive cells into MEK addiction. Cancer Cell 2006; 9: 912.
  • 53
    Zebisch A, Troppmair J. Back to the roots: the remarkable RAF oncogene story. Cell Mol Life Sci 2006; 63: 131430.
  • 54
    Rajalingam K, Wunder C, Brinkmann V, Churin Y, Hekman M, Sievers C, Rapp UR, Rudel T. Prohibitin is required for Ras-induced Raf-MEK-ERK activation and epithelial cell migration. Nat Cell Biol 2005; 7: 83743.
  • 55
    Rodriguez-Viciana P, Oses-Prieto J, Burlingame A, Fried M, McCormick F. A phosphatase holoenzyme comprised of Shoc2/Sur8 and the catalytic subunit of PP1 functions as an M-Ras effector to modulate Raf activity. Mol Cell 2006; 22: 21730.
  • 56
    Douziech M, Sahmi M, Laberge G, Therrien M. A KSR/CNK complex mediated by HYP, a novel SAM domain-containing protein, regulates RAS-dependent RAF activation in Drosophila. Genes Dev 2006; 20: 80719.
  • 57
    Luo Z, Tzivion G, Belshaw PJ, Vavvas D, Marshall M, Avruch J. Oligomerization activates c-Raf-1 through a Ras-dependent mechanism. Nature 1996; 383: 1815.
  • 58
    Farrar MA, Alberol I, Perlmutter RM. Activation of the Raf-1 kinase cascade by coumermycin-induced dimerization. Nature 1996; 383: 17881.
  • 59
    Weber CK, Slupsky JR, Kalmes HA, Rapp UR. Active Ras induces heterodimerization of cRaf and BRaf. Cancer Res 2001; 61: 35958.
  • 60
    Chadee DN, Xu D, Hung G, Andalibi A, Lim DJ, Luo Z, Gutmann DH, Kyriakis JM. Mixed-lineage kinase 3 regulates B-Raf through maintenance of the B-Raf/Raf-1 complex and inhibition by the NF2 tumor suppressor protein. Proc Natl Acad Sci USA 2006; 103: 44638.
  • 61
    Rushworth LK, Hindley AD, O'Neill E, Kolch W. Regulation and role of Raf-1/B-Raf heterodimerization. Mol Cell Biol 2006; 26: 226272.
  • 62
    Wan PT, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, Good VM, Jones CM, Marshall CJ, Springer CJ, Barford D, Marais R. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 2004; 116: 85567.
  • 63
    Garnett MJ, Rana S, Paterson H, Barford D, Marais R. Wild-type and mutant B-RAF activate C-RAF through distinct mechanisms involving heterodimerization. Mol Cell 2005; 20: 9639.
  • 64
    Hirst GL, Balmain A. Forty years of cancer modelling in the mouse. Eur J Cancer 2004; 40: 197480.
  • 65
    Maddison K, Clarke AR. New approaches for modelling cancer mechanisms in the mouse. J Pathol 2005; 205: 18193.
  • 66
    Holland EC. Mouse models of human cancer as tools in drug development. Cancer Cell 2004; 6: 1978.
  • 67
    Morse HC,III, Rapp UR. Tumorigenic activity of artificially activated oncogenes. In: Klein G, ed. Cellular oncogene activation. New York: Marcel Dekker, 1988; 41: 33564.
  • 68
    Jaworski M, Buchmann A, Bauer P, Riess O, Schwarz M. B-raf and Ha-ras mutations in chemically induced mouse liver tumors. Oncogene 2005; 24: 12905.
  • 69
    Collier LS, Carlson CM, Ravimohan S, Dupuy AJ, Largaespada DA. Cancer gene discovery in solid tumours using transposon-based somatic mutagenesis in the mouse. Nature 2005; 436: 2726.
  • 70
    Kerkhoff E, Fedorov LM, Siefken R, Walter AO, Papadopoulos T, Rapp UR. Lung-targeted expression of the c-Raf-1 kinase in transgenic mice exposes a novel oncogenic character of the wild-type protein. Cell Growth Differ 2000; 11: 18590.
  • 71
    Rapp UR, Fensterle J, Albert S, Goetz R. Raf kinases in lung tumor development. Adv Enzyme Regul 2003; 43: 18395.
  • 72
    Fedorov LM, Tyrsin OY, Papadopoulos T, Camarero G, Goetz R, Rapp UR. Bcl-2 determines susceptibility to induction of lung cancer by oncogenic CRaf. Cancer Res 2002; 62: 6297303.
  • 73
    Goetz R, Kramer BW, Camarero G, Rapp UR. BAG-1 haplo-insufficiency impairs lung tumorigenesis. BMC Cancer 2004; 4: 85.
  • 74
    Kramer BW, Goetz R, Rapp UR. Use of mitogenic cascade blockers for treatment of C-Raf induced lung adenoma in vivo: CI-1040 strongly reduces growth and improves lung structure. BMC Cancer 2004; 4: 24.
  • 75
    Goetz R, Wiese S, Takayama S, Camarero GC, Rossoll W, Schweizer U, Troppmair J, Jablonka S, Holtmann B, Reed JC, Rapp UR, Sendtner M. Bag1 is essential for differentiation and survival of hematopoietic and neuronal cells. Nat Neurosci 2005; 8: 116978.
  • 76
    Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 1996; 86: 35364.
  • 77
    Knauf JA, Ma X, Smith EP, Zhang L, Mitsutake N, Liao XH, Refetoff S, Nikiforov YE, Fagin JA. Targeted expression of BRAFV600E in thyroid cells of transgenic mice results in papillary thyroid cancers that undergo dedifferentiation. Cancer Res 2005; 65: 423845.
  • 78
    Mercer K, Giblett S, Green S, Lloyd D, Dias SD, Plumb M, Marais R, Pritchard C. Expression of endogenous oncogenic V600EB-raf induces proliferation and developmental defects in mice and transformation of primary fibroblasts. Cancer Res 2005; 65: 11493500.
  • 79
    Konopleva M, Shi Y, Steelman LS, Shelton JG, Munsell M, Marini F, McQueen T, Contractor R, McCubrey JA, Andreeff M. Development of a conditional in vivo model to evaluate the efficacy of small molecule inhibitors for the treatment of Raf-transformed hematopoietic cells. Cancer Res 2005; 65: 996270.
  • 80
    Hoshino R, Chatani Y, Yamori T, Tsuruo T, Oka H, Yoshida O, Shimada Y, Ari-i S, Wada H, Fujimoto J, Kohno M. Constitutive activation of the 41-/43-kDa mitogen-activated protein kinase signaling pathway in human tumors. Oncogene 1999; 18: 81322.
  • 81
    Malumbres M, Barbacid M. RAS oncogenes: the first 30 years. Nat Rev Cancer 2003; 3: 45965.
  • 82
    McPhillips F, Mullen P, Monia BP, Ritchie AA, Dorr FA, Smyth JF, Langdon SP. Association of c-Raf expression with survival and its targeting with antisense oligonucleotides in ovarian cancer. Br J Cancer 2001; 85: 17538.
  • 83
    Fransen K, Klintenas M, Osterstrom A, Dimberg J, Monstein HJ, Soderkvist P. Mutation analysis of the BRAF, ARAF and RAF-1 genes in human colorectal adenocarcinomas. Carcinogenesis 2004; 25: 52733.
  • 84
    Lee JW, Soung YH, Kim SY, Park WS, Nam SW, Min WS, Kim SH, Lee JY, Yoo NJ, Lee SH. Mutational analysis of the ARAF gene in human cancers. APMIS 2005; 113: 547.
  • 85
    Emuss V, Garnett M, Mason C, Marais R. Mutations of C-RAF are rare in human cancer because C-RAF has a low basal kinase activity compared with B-RAF. Cancer Res 2005; 65: 971926.
  • 86
    Garnett MJ, Marais R. Guilty as charged: B-RAF is a human oncogene. Cancer Cell 2004; 6: 31319.
  • 87
    Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, Woffendin H, Garnett MJ, Bottomley W, Davis N, Dicks E, et al. Mutations of the BRAF gene in human cancer. Nature 2002; 417: 94954.
  • 88
    Satyamoorthy K, Li G, Gerrero MR, Brose MS, Volpe P, Weber BL, Van Belle P, Elder DE, Herlyn M. Constitutive mitogen-activated protein kinase activation in melanoma is mediated by both BRAF mutations and autocrine growth factor stimulation. Cancer Res 2003; 63: 7569.
  • 89
    Ikenoue T, Hikiba Y, Kanai F, Tanaka Y, Imamura J, Imamura T, Ohta M, Ijichi H, Tateishi K, Kawakami T, Aragaki J, Matsumura M, et al. Functional analysis of mutations within the kinase activation segment of B-Raf in human colorectal tumors. Cancer Res 2003; 63: 81327.
  • 90
    Wellbrock C, Ogilvie L, Hedley D, Karasarides M, Martin J, Niculescu-Duvaz D, Springer CJ, Marais R. V599EB-RAF is an oncogene in melanocytes. Cancer Res 2004; 64: 233842.
  • 91
  • 92
    Dibb NJ, Dilworth SM, Mol CD. Switching on kinases: oncogenic activation of BRAF and the PDGFR family. Nat Rev Cancer 2004; 4: 71827.
  • 93
    Solit DB, Garraway LA, Pratilas CA, Sawai A, Getz G, Basso A, Ye Q, Lobo JM, She Y, Osman I, Golub TR, Sebolt-Leopold J, et al. BRAF mutation predicts sensitivity to MEK inhibition. Nature 2006; 439: 35862.
  • 94
    Ciampi R, Knauf JA, Kerler R, Gandhi M, Zhu Z, Nikiforova MN, Rabes HM, Fagin JA, Nikiforov YE. Oncogenic AKAP9-BRAF fusion is a novel mechanism of MAPK pathway activation in thyroid cancer. J Clin Invest 2005; 115: 94101.
  • 95
    Ciampi R, Zhu Z, Nikiforov YE. BRAF copy number gains in thyroid tumors detected by fluorescence in situ hybridization. Endocr Pathol 2005; 16: 99105.
  • 96
    Zebisch A, Staber PB, Delavar A, Bodner C, Hiden K, Fischereder K, Janakiraman M, Linkesch W, Auner HW, Emberger W, Windpassinger C, Schimek MG, et al. Two transforming C-RAF germ-line mutations identified in patients with therapy-related acute myeloid leukemia. Cancer Res 2006; 66: 34018.
  • 97
    Niihori T, Aoki Y, Narumi Y, Neri G, Cave H, Verloes A, Okamoto N, Hennekam RC, Gillessen-Kaesbach G, Wieczorek D, Kavamura MI, Kurosawa K, et al. Germline KRAS and BRAF mutations in cardio-facio-cutaneous syndrome. Nat Genet 2006; 38: 2946.
  • 98
    Rodriguez-Viciana P, Tetsu O, Tidyman WE, Estep AL, Conger BA, Cruz MS, McCormick F, Rauen KA. Germline mutations in genes within the MAPK pathway cause cardio-facio-cutaneous syndrome. Science 2006; 311: 128790.
  • 99
    Houben R, Becker JC, Kappel A, Terheyden P, Brocker EB, Goetz R, Rapp UR. Constitutive activation of the Ras-Raf signaling pathway in metastatic melanoma is associated with poor prognosis. J Carcinog 2004; 3: 6.
  • 100
    Dong J, Phelps RG, Qiao R, Yao S, Benard O, Ronai Z, Aaronson SA. BRAF oncogenic mutations correlate with progression rather than initiation of human melanoma. Cancer Res 2003; 63: 38835.
  • 101
    Samowitz WS, Sweeney C, Herrick J, Albertsen H, Levin TR, Murtaugh MA, Wolff RK, Slattery ML. Poor survival associated with the BRAF V600E mutation in microsatellite-stable colon cancers. Cancer Res 2005; 65: 60639.
  • 102
    Xing M, Westra WH, Tufano RP, Cohen Y, Rosenbaum E, Rhoden KJ, Carson KA, Vasko V, Larin A, Tallini G, Tolaney S, Holt EH et al. BRAF Mutation predicts a poorer clinical prognosis for papillary thyroid cancer. J Clin Endocrinol Metab 2005; 90: 63739.
  • 103
    Weir B, Zhao X, Meyerson M. Somatic alterations in the human cancer genome. Cancer Cell 2004; 6: 4338.
  • 104
    Gray-Schopfer VC, da Rocha Dias S, Marais R. The role of B-RAF in melanoma. Cancer Metastasis Rev 2005; 24: 16583.
  • 105
    Wong CW, Fan YS, Chan TL, Chan AS, Ho LC, Ma TK, Yuen ST, Leung SY. BRAF and NRAS mutations are uncommon in melanomas arising in diverse internal organs. J Clin Pathol 2005; 58: 6404.
  • 106
    Ciampi R, Knauf JA, Rabes HM, Fagin JA, Nikiforov YE. BRAF kinase activation via chromosomal rearrangement in radiation-induced and sporadic thyroid cancer. Cell Cycle 2005; 4: 5478.
  • 107
    Xing M. BRAF mutation in thyroid cancer. Endocr Relat Cancer 2005; 12: 24562.
  • 108
    Bell DA. Origins and molecular pathology of ovarian cancer. Mod Pathol 2005; 18 ( Suppl 2): S19S32.
  • 109
    Dunn GP, Old LJ, Schreiber RD. The immunobiology of cancer immunosurveillance and immunoediting. Immunity 2004; 21: 13748.
  • 110
    Andersen MH, Fensterle J, Ugurel S, Reker S, Houben R, Guldberg P, Berger TG, Schadendorf D, Trefzer U, Brocker EB, Straten P, Rapp UR et al. Immunogenicity of constitutively active V599EBRaf. Cancer Res 2004; 64: 545660.
  • 111
    Somasundaram R, Swoboda R, Caputo L, Otvos L, Weber B, Volpe P, van Belle P, Hotz S, Elder DE, Marincola FM, Schuchter L, Guerry D, et al. Human leukocyte antigen-A2-restricted CTL responses to mutated BRAF peptides in melanoma patients. Cancer Res 2006; 66: 328793.
  • 112
    Sharkey MS, Lizee G, Gonzales MI, Patel S, Topalian SL. CD4+ T-cell recognition of mutated B-RAF in melanoma patients harboring the V599E mutation. Cancer Res 2004; 64: 15959.
  • 113
    Fensterle J, Becker JC, Potapenko T, Heimbach V, Vetter CS, Brocker EB, Rapp UR. B-Raf specific antibody responses in melanoma patients. BMC Cancer 2004; 4: 62.
  • 114
    Strumberg D, Seeber S. Raf kinase inhibitors in oncology. Onkologie 2005; 28: 1017.
  • 115
    Sridhar SS, Hedley D, Siu LL. Raf kinase as a target for anticancer therapeutics. Mol Cancer Ther 2005; 4: 67785.
  • 116
    Thompson N, Lyons J. Recent progress in targeting the Raf/MEK/ERK pathway with inhibitors in cancer drug discovery. Curr Opin Pharmacol 2005; 5: 3506.
  • 117
    von Eschenbach AC. A vision for the national cancer program in the United States. Nat Rev Cancer 2004; 4: 8208.
  • 118
    Dumas J, Smith RA, Lowinger TB. Recent developments in the discovery of protein kinase inhibitors from the urea class. Curr Opin Drug Discov Devel 2004; 7: 60016.
  • 119
    Lowinger TB, Riedl B, Dumas J, Smith RA. Design and discovery of small molecules targeting raf-1 kinase. Curr Pharm Des 2002; 8: 226978.
  • 120
    Lee JT, McCubrey JA. BAY-43–9006 Bayer/Onyx. Curr Opin Investig Drugs 2003; 4: 75763.
  • 121
    Lyons JF, Wilhelm S, Hibner B, Bollag G. Discovery of a novel Raf kinase inhibitor. Endocr Relat Cancer 2001; 8: 21925.
  • 122
    Wilhelm S, Chien DS. BAY 43–9006: preclinical data. Curr Pharm Des 2002; 8: 22557.
  • 123
    Wilhelm SM, Carter C, Tang L, Wilkie D, McNabola A, Rong H, Chen C, Zhang X, Vincent P, McHugh M, Cao Y, Shujath J, et al. BAY 43–9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res 2004; 64: 7099109.
  • 124
    Carlomagno F, Anaganti S, Guida T, Salvatore G, Troncone G, Wilhelm SM, Santoro M. BAY 43–9006 inhibition of oncogenic RET mutants. J Natl Cancer Inst 2006; 98: 32634.
  • 125
    Salvatore G, De Falco V, Salerno P, Nappi TC, Pepe S, Troncone G, Carlomagno F, Melillo RM, Wilhelm SM, Santoro M. BRAF is a therapeutic target in aggressive thyroid carcinoma. Clin Cancer Res 2006; 12: 16239.
  • 126
    Lierman E, Folens C, Stover EH, Mentens N, Van Miegroet H, Scheers W, Boogaerts M, Vandenberghe P, Marynen P, Cools J. Sorafenib (BAY43–9006) is a potent inhibitor of FIP1L1-PDGFRα and the imatinib resistant FIP1L1-PDGFRα T674I mutant. Blood [Epub ahead of print].
  • 127
    Yu C, Bruzek LM, Meng XW, Gores GJ, Carter CA, Kaufmann SH, Adjei AA. The role of Mcl-1 downregulation in the proapoptotic activity of the multikinase inhibitor BAY 43–9006. Oncogene 2005; 24: 68619.
  • 128
    Panka DJ, Wang W, Atkins MB, Mier JW. The Raf inhibitor BAY 43–9006 (Sorafenib) induces caspase-independent apoptosis in melanoma cells. Cancer Res 2006; 66: 161119.
  • 129
    Wright JJ, Zerivitz K, Gravell A. Clinical trials referral resource. Current clinical trials of BAY 43–9006, Part 1. Oncology (Huntingt) 2005; 19: 499502.
  • 130
    Wright JJ, Zerivitz K, Gravell A. Clinical trials referral resource. Current clinical trials of BAY 43–9006, Part 2. Oncology (Williston Park) 2005; 19: 7228.
  • 131
    Rini BI. Sorafenib. Expert Opin Pharmacother 2006; 7: 45361.
  • 132
    Kolch W, Heidecker G, Lloyd P, Rapp UR. Raf-1 protein kinase is required for growth of induced NIH/3T3 cells. Nature 1991; 349: 4268.
  • 133
    Monia BP, Sasmor H, Johnston JF, Freier SM, Lesnik EA, Muller M, Geiger T, Altmann KH, Moser H, Fabbro D. Sequence-specific antitumor activity of a phosphorothioate oligodeoxyribonucleotide targeted to human C-raf kinase supports an antisense mechanism of action in vivo. Proc Natl Acad Sci USA 1996; 93: 154814.
  • 134
    Monia BP, Johnston JF, Geiger T, Muller M, Fabbro D. Antitumor activity of a phosphorothioate antisense oligodeoxynucleotide targeted against C-raf kinase. Nat Med 1996; 2: 66875.
  • 135
    Stevenson JP, Yao KS, Gallagher M, Friedland D, Mitchell EP, Cassella A, Monia B, Kwoh TJ, Yu R, Holmlund J, Dorr FA, O'Dwyer PJ. Phase I clinical/pharmacokinetic and pharmacodynamic trial of the c-raf-1 antisense oligonucleotide ISIS 5132 (CGP 69846A). J Clin Oncol 1999; 17: 222736.
  • 136
    Cunningham CC, Holmlund JT, Schiller JH, Geary RS, Kwoh TJ, Dorr A, Nemunaitis J. A phase I trial of c-Raf kinase antisense oligonucleotide ISIS 5132 administered as a continuous intravenous infusion in patients with advanced cancer. Clin Cancer Res 2000; 6: 162631.
  • 137
    Rudin CM, Holmlund J, Fleming GF, Mani S, Stadler WM, Schumm P, Monia BP, Johnston JF, Geary R, Yu RZ, Kwoh TJ, Dorr FA, et al. Phase I trial of ISIS 5132, an antisense oligonucleotide inhibitor of c-raf-1, administered by 24-hour weekly infusion to patients with advanced cancer. Clin Cancer Res 2001; 7: 121420.
  • 138
    Coudert B, Anthoney A, Fiedler W, Droz JP, Dieras V, Borner M, Smyth JF, Morant R, de Vries MJ, Roelvink M, Fumoleau P. Phase II trial with ISIS 5132 in patients with small-cell (SCLC) and non-small cell (NSCLC) lung cancer. A European Organization for Research and Treatment of Cancer (EORTC) early clinical studies group report. Eur J Cancer 2001; 37: 21948.
  • 139
    Cripps MC, Figueredo AT, Oza AM, Taylor MJ, Fields AL, Holmlund JT, McIntosh LW, Geary RS, Eisenhauer EA. Phase II randomized study of ISIS 3521 and ISIS 5132 in patients with locally advanced or metastatic colorectal cancer: a National Cancer Institute of Canada clinical trials group study. Clin Cancer Res 2002; 8: 218892.
  • 140
    Tolcher AW, Reyno L, Venner PM, Ernst SD, Moore M, Geary RS, Chi K, Hall S, Walsh W, Dorr A, Eisenhauer E. A randomized phase II and pharmacokinetic study of the antisense oligonucleotides ISIS 3521 and ISIS 5132 in patients with hormone-refractory prostate cancer. Clin Cancer Res 2002; 8: 25305.
  • 141
    Oza AM, Elit L, Swenerton K, Faught W, Ghatage P, Carey M, McIntosh L, Dorr A, Holmlund JT, Eisenhauer E. Phase II study of CGP 69846A (ISIS 5132) in recurrent epithelial ovarian cancer: an NCIC clinical trials group study (NCIC IND. 116). Gynecol Oncol 2003; 89: 12933.
  • 142
    Mullen P, McPhillips F, Monia BP, Smyth JF, Langdon SP. Comparison of strategies targeting Raf-1 mRNA in ovarian cancer. Int J Cancer 2006; 118: 156571.
  • 143
    Gokhale PC, Zhang C, Newsome JT, Pei J, Ahmad I, Rahman A, Dritschilo A, Kasid UN. Pharmacokinetics, toxicity, and efficacy of ends-modified raf antisense oligodeoxyribonucleotide encapsulated in a novel cationic liposome. Clin Cancer Res 2002; 8: 361121.
  • 144
    Rudin CM, Marshall JL, Huang CH, Kindler HL, Zhang C, Kumar D, Gokhale PC, Steinberg J, Wanaski S, Kasid UN, Ratain MJ. Delivery of a liposomal c-raf-1 antisense oligonucleotide by weekly bolus dosing in patients with advanced solid tumors: a phase I study. Clin Cancer Res 2004; 10: 724451.
  • 145
    Dritschilo A, Huang CH, Rudin CM, Marshall J, Collins B, Dul JL, Zhang C, Kumar D, Gokhale PC, Ahmad A, Ahmad I, Sherman JW et al. Phase I study of liposome-encapsulated c-raf antisense oligodeoxyribonucleotide infusion in combination with radiation therapy in patients with advanced malignancies. Clin Cancer Res 2006; 12: 12519.
  • 146
    Hingorani SR, Jacobetz MA, Robertson GP, Herlyn M, Tuveson DA. Suppression of BRAF(V599E) in human melanoma abrogates transformation. Cancer Res 2003; 63: 5198202.
  • 147
    Sumimoto H, Miyagishi M, Miyoshi H, Yamagata S, Shimizu A, Taira K, Kawakami Y. Inhibition of growth and invasive ability of melanoma by inactivation of mutated BRAF with lentivirus-mediated RNA interference. Oncogene 2004; 23: 60319.
  • 148
    Karasarides M, Chiloeches A, Hayward R, Niculescu-Duvaz D, Scanlon I, Friedlos F, Ogilvie L, Hedley D, Martin J, Marshall CJ, Springer CJ, et al. B-RAF is a therapeutic target in melanoma. Oncogene 2004; 23: 62928.
  • 149
    Sharma A, Trivedi NR, Zimmerman MA, Tuveson DA, Smith CD, Robertson GP. Mutant V599EB-Raf regulates growth and vascular development of malignant melanoma tumors. Cancer Res 2005; 65: 241221.
  • 150
    Pal A, Ahmad A, Khan S, Sakabe I, Zhang C, Kasid UN, Ahmad I. Systemic delivery of RafsiRNA using cationic cardiolipin liposomes silences Raf-1 expression and inhibits tumor growth in xenograft model of human prostate cancer. Int J Oncol 2005; 26: 108791.
  • 151
    Leng Q, Mixson AJ. Small interfering RNA targeting Raf-1 inhibits tumor growth in vitro and in vivo. Cancer Gene Ther 2005; 12: 68290.
  • 152
    Miyata Y. Hsp90 inhibitor geldanamycin and its derivatives as novel cancer chemotherapeutic agents. Curr Pharm Des 2005; 11: 11318.
  • 153
    Workman P. Combinatorial attack on multistep oncogenesis by inhibiting the Hsp90 molecular chaperone. Cancer Lett 2004; 206: 14957.
  • 154
    Kamal A, Thao L, Sensintaffar J, Zhang L, Boehm MF, Fritz LC, Burrows FJ. A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors. Nature 2003; 425: 40710.
  • 155
    Supko JG, Hickman RL, Grever MR, Malspeis L. Preclinical pharmacologic evaluation of geldanamycin as an antitumor agent. Cancer Chemother Pharmacol 1995; 36: 30515.
  • 156
    Budillon A, Bruzzese F, Di Gennaro E, Caraglia M. Multiple-target drugs: inhibitors of heat shock protein 90 and of histone deacetylase. Curr Drug Targets 2005; 6: 33751.
  • 157
    da Rocha Dias S, Friedlos F, Light Y, Springer C, Workman P, Marais R. Activated B-RAF is an Hsp90 client protein that is targeted by the anticancer drug 17-allylamino-17-demethoxygeldanamycin. Cancer Res 2005; 65: 1068691.
  • 158
    Grbovic OM, Basso AD, Sawai A, Ye Q, Friedlander P, Solit D, Rosen N. V600E B-Raf requires the Hsp90 chaperone for stability and is degraded in response to Hsp90 inhibitors. Proc Natl Acad Sci USA 2006; 103: 5762.
  • 159
    Smith V, Sausville EA, Camalier RF, Fiebig HH, Burger AM. Comparison of 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17DMAG) and 17-allylamino-17-demethoxygeldanamycin (17AAG) in vitro: effects on Hsp90 and client proteins in melanoma models. Cancer Chemother Pharmacol 2005; 56: 12637.
  • 160
    Hollingshead M, Alley M, Burger AM, Borgel S, Pacula-Cox C, Fiebig HH, Sausville EA. In vivo antitumor efficacy of 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride), a water-soluble geldanamycin derivative. Cancer Chemother Pharmacol 2005; 56: 11525.
  • 161
    Sittler A, Lurz R, Lueder G, Priller J, Lehrach H, Hayer-Hartl MK, Hartl FU, Wanker EE. Geldanamycin activates a heat shock response and inhibits huntingtin aggregation in a cell culture model of Huntington's disease. Hum Mol Genet 2001; 10: 130715.
  • 162
    Whitesell L, Lindquist SL. HSP90 and the chaperoning of cancer. Nat Rev Cancer 2005; 5: 76172.
  • 163
    Guo F, Rocha K, Bali P, Pranpat M, Fiskus W, Boyapalle S, Kumaraswamy S, Balasis M, Greedy B, Armitage ES, Lawrence N, Bhalla K. Abrogation of heat shock protein 70 induction as a strategy to increase antileukemia activity of heat shock protein 90 inhibitor 17-allylamino-demethoxy geldanamycin. Cancer Res 2005; 65: 1053644.
  • 164
    Calderwood SK, Theriault JR, Gong J. Message in a bottle: role of the 70-kDa heat shock protein family in anti-tumor immunity. Eur J Immunol 2005; 35: 251827.
  • 165
    Drummond DC, Noble CO, Kirpotin DB, Guo Z, Scott GK, Benz CC. Clinical development of histone deacetylase inhibitors as anticancer agents. Annu Rev Pharmacol Toxicol 2005; 45: 495528.
  • 166
    Acharya MR, Sparreboom A, Venitz J, Figg WD. Rational development of histone deacetylase inhibitors as anti-cancer agents: a review. Mol Pharmacol 2005; 68: 91732.
  • 167
    Villar-Garea A, Esteller M. Histone deacetylase inhibitors: understanding a new wave of anticancer agents. Int J Cancer 2004; 112: 1718.
  • 168
    Mitsiades CS, Mitsiades NS, McMullan CJ, Poulaki V, Shringarpure R, Hideshima T, Akiyama M, Chauhan D, Munshi N, Gu X, Bailey C, Joseph M, et al. Transcriptional signature of histone deacetylase inhibition in multiple myeloma: biological and clinical implications. Proc Natl Acad Sci USA 2004; 101: 5405.
  • 169
    Fuino L, Bali P, Wittmann S, Donapaty S, Guo F, Yamaguchi H, Wang HG, Atadja P, Bhalla K. Histone deacetylase inhibitor LAQ824 down-regulates Her-2 and sensitizes human breast cancer cells to trastuzumab, taxotere, gemcitabine, and epothilone B. Mol Cancer Ther 2003; 2: 97184.
  • 170
    Yu C, Rahmani M, Almenara J, Subler M, Krystal G, Conrad D, Varticovski L, Dent P, Grant S. Histone deacetylase inhibitors promote STI571-mediated apoptosis in STI571-sensitive and -resistant Bcr/Abl+ human myeloid leukemia cells. Cancer Res 2003; 63: 211826.
  • 171
    Bali P, Pranpat M, Bradner J, Balasis M, Fiskus W, Guo F, Rocha K, Kumaraswamy S, Boyapalle S, Atadja P, Seto E, Bhalla K. Inhibition of histone deacetylase 6 acetylates and disrupts the chaperone function of heat shock protein 90: a novel basis of antileukemia activity of histone deacetylase inhibitors. J Biol Chem 2005; 280: 2672934.
  • 172
    Blattman JN, Greenberg PD. Cancer immunotherapy: a treatment for the masses. Science 2004; 305: 2005.
  • 173
    Mahnke YD, Speiser D, Luescher IF, Cerottini JC, Romero P. Recent advances in tumour antigen-specific therapy: in vivo veritas. Int J Cancer 2005; 113: 1738.
  • 174
    Robubi A, Mueller T, Fueller J, Hekman M, Rapp UR, Dandekar T. B-Raf and C-Raf signaling investigated in a simplified model of the mitogenic kinase cascade. Biol Chem 2005; 386: 116571.
  • 175
    Orton RJ, Sturm OE, Vyshemirsky V, Calder M, Gilbert DR, Kolch W. Computational modelling of the receptor-tyrosine-kinase-activated MAPK pathway. Biochem J 2005; 392: 24961.
  • 176
    Strumberg D. Preclinical and clinical development of the oral multikinase inhibitor sorafenib in cancer treatment. Drugs Today (Barc) 2005; 41: 77384.
  • 177
    Tsai J, Zhang J, Bremer R, Artis R, Hirth P, Bollag G. Development of a novel inhibitor of oncogenic B-Raf. In the 97th AACR annual meeting, Washington DC, 2006. Abstract No 2412.
  • 178
    Amiri P, Aikawa ME, Dove J, Stuart DD, Poon D, Pick T, Ramurthy S, Subramanian S, Levine B, Costales A, Harris A, Paul R. CHIR-265 is a potent selective inhibitor of c-Raf/B-Raf/mutB-Raf that effectively inhibits proliferation and survival of cancer cell lines with Ras/Raf pathway mutations. In the 97th AACR annual meeting, Washington DC, 2006. Abstract No 4855.
  • 179
    Stuart DS, Aardalen KM, Lorenzana EG, Salangsang FD, Venetsanakos E, Tan N, Zhang W, Garrett E, Jallal B, Mendel DB. Characterization of a novel Raf kinase inhibitor that causes target dependent tumor regression in human melanoma xenografts expressing mutant B-Raf. In the 97th AACR annual meeting, Washington DC, 2006. Abstract No 4856.
  • 180
    Venetsanakos E, Stuart D, Tan N, Ye H, Salangsang F, Aardalen K, Faure M, Heise C, Mendel D, Jallal B. CHIR-265, a novel inhibitor that targets B-Raf and VEGFR, shows efficacy in a broad range of preclinical models. In the 97th AACR annual meeting, Washington DC, 2006. Abstract No 4854.
  • 181
    Niculescu-Duvaz I, Roman E, Whittaker SR, Friedlos F, Kirk R, Scanlon IJ, Davies LC, Niculescu-Duvaz D, Marais R, Springer CJ. Novel inhibitors of B-RAF based on a disubstituted pyrazine scaffold. Generation of a nanomolar lead. J Med Chem 2006; 49: 40716.
  • 182
    Newbatt Y, Burns S, Hayward R, Whittaker S, Kirk R, Marshall C, Springer C, McDonald E, Marais R, Workman P, Aherne W. Identification of inhibitors of the kinase activity of oncogenic V600E BRAF in an enzyme cascade high-throughput screen. J Biomol Screen 2006; 11: 14554.
  • 183
    Takle AK, Brown MJ, Davies S, Dean DK, Francis G, Gaiba A, Hird AW, King FD, Lovell PJ, Naylor A, Reith AD, Steadman JG, et al. The identification of potent and selective imidazole-based inhibitors of B-Raf kinase. Bioorg Med Chem Lett 2006; 16: 37881.
  • 184
    Ouyang B, Knauf JA, Smith EP, Zhang L, Ramsey T, Yusuff N, Batt D, Fagin JA. Inhibitors of Raf kinase activity block growth of thyroid cancer cells with RET/PTC or BRAF mutations in vitro and in vivo. Clin Cancer Res 2006; 12: 178593.
  • 185
    Khire UR, Bankston D, Barbosa J, Brittelli DR, Caringal Y, Carlson R, Dumas J, Gane T, Heald SL, Hibner B, Johnson JS, Katz ME, et al. Omega-carboxypyridyl substituted ureas as Raf kinase inhibitors: SAR of the amide substituent. Bioorg Med Chem Lett 2004; 14: 7836.
  • 186
    Lackey K, Cory M, Davis R, Frye SV, Harris PA, Hunter RN, Jung DK, McDonald OB, McNutt RW, Peel MR, Rutkowske RD, Veal JM, et al. The discovery of potent cRaf1 kinase inhibitors. Bioorg Med Chem Lett 2000; 10: 2236.
  • 187
    Hall-Jackson CA, Eyers PA, Cohen P, Goedert M, Boyle FT, Hewitt N, Plant H, Hedge P. Paradoxical activation of Raf by a novel Raf inhibitor. Chem Biol 1999; 6: 55968.
  • 188
    Heimbrook DC, Huber HE, Stirdivant SM, Claremon D, Liverton N, Patrick DR, Selnick H, Ahern J, Conroy R, Drakas R, Falconi N, Hancock P, et al. Identification of potent, selective kinase inhibitors of Raf. Am Assoc Cancer Res 1998; 39: 558. [Abstract No 3793].
  • 189
    Peterson JR, Bickford LC, Morgan D, Kim AS, Ouerfelli O, Kirschner MW, Rosen MK. Chemical inhibition of N-WASP by stabilization of a native autoinhibited conformation. Nat Struct Mol Biol 2004; 11: 74755.
  • 190
    Loregian A, Palu G. Disruption of protein-protein interactions: towards new targets for chemotherapy. J Cell Physiol 2005; 204: 75062.
  • 191
  • 192
    Giordano TJ, Kuick R, Thomas DG, Misek DE, Vinco M, Sanders D, Zhu Z, Ciampi R, Roh M, Shedden K, Gauger P, Doherty G, et al. Molecular classification of papillary thyroid carcinoma: distinct BRAF, RAS, and RET/PTC mutation-specific gene expression profiles discovered by DNA microarray analysis. Oncogene 2005; 24: 664656.
  • 193
    Kim IJ, Kang HC, Jang SG, Kim K, Ahn SA, Yoon HJ, Yoon SN, Park JG. Oligonucleotide microarray analysis of distinct gene expression patterns in colorectal cancer tissues harboring BRAF and K-ras mutations. Carcinogenesis 2006; 27: 392404.
  • 194
    Park IK, Morrison SJ, Clarke MF. Bmi1, stem cells, and senescence regulation. J Clin Invest 2004; 113: 1759.
  • 195
    Voncken JW, Niessen H, Neufeld B, Rennefahrt U, Dahlmans V, Kubben N, Holzer B, Ludwig S, Rapp UR. MAPKAP kinase 3pK phosphorylates and regulates chromatin association of the polycomb group protein Bmi1. J Biol Chem 2005; 280: 517887.