Mutational analysis of the BRAF gene in human tumor cells

Genes of the RAF family, which mediate cellular responses to growth signals, encode kinases that are regulated by RAS and participate in the RAS, RAF, mitogen/extracellular signal-regulated kinase, extracellular signal-regulated kinase and mitogen-activated protein kinase pathway. As BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation, it may provide possible diagnostic and therapeutic targets in human malignant tumors. We analyzed exon 15 of the BRAF gene for mutations in 58 lung, 12 breast, six kidney, 14 cervical, four endometrial and 10 ovarian carcinoma cell lines by PCR-SSCP and direct sequencing. The T1796A transversion was found in one (2.9%) of 34 small cell lung carcinoma and one (8.3%) of 12 breast carcinoma cell lines, resulting in a valine-to-glutamate substitution at residue 599 (V599E). One (4.2%) of 24 non-small cell lung carcinoma cell line showed the C1786G transversion, leading to a leucine-to-valine substitution at residue 596 (L596V). No BRAF point mutations were found in any of the other cell lines examined. Our present results suggest that BRAF may not be a frequent target of mutations involved in the pathogenesis of human lung, breast, kidney, cervical, endometrial and ovarian carcinomas.     

Mutation of B-Raf in human choroidal melanoma cells mediates cell proliferation and transformation through the MEK/ERK pathway

The BRAF gene, encoding a mitogen-activated protein kinase kinase kinase, is mutated in several human cancers, with the highest incidence occurring in cutaneous melanoma. The activating V599E mutation accounted for 80% of all mutations detected in cutaneous melanoma cell lines. Reconstitution experiments have shown that this mutation increases ectopically expressed B-Raf kinase activity and induces NIH3T3 cell transformation. Here we used tumor-derived cell lines to characterize the activity of endogenous mutated B-Raf protein and assess its specific role in transformation. We show that three cell lines (OCM-1, MKT-BR, and SP-6.5) derived from human choroidal melanoma, the most frequent primary ocular neoplasm in humans, express B-Raf containing the V599E mutation. These melanoma cells showed a 10-fold increase in endogenous B-RafV599E kinase activity and a constitutive activation of the MEK/ERK pathway that is independent of Ras. This, as well as melanoma cell proliferation, was strongly diminished by siRNA-mediated depletion of the mutant B-Raf protein. Moreover, blocking B-RafV599E-induced ERK activation by different experimental approaches significantly reduced cell proliferation and anchorage-independent growth of melanoma cells. Finally, quantitative immunoblot analysis allowed us to identify signaling and cell cycle proteins that are differentially expressed between normal melanocytes and melanoma cells. Although the expression of signaling molecules was not sensitive to U0126 in melanoma cells, the expression of a cluster of cell cycle proteins remained regulated by the B-RafV599E/MEK/ERK pathway. Our results pinpoint this pathway as an important component in choroidal melanoma cell lines.     

Identification of Raf-1 S471 as a novel phosphorylation site critical for Raf-1 and B-Raf kinase activities and for MEK binding

The Ras-Raf-MAPK cascade is a key growth-signaling pathway and its uncontrolled activation results in cell transformation. Although the general features of the signal transmission along the cascade are reasonably defined, the mechanisms underlying Raf activation remain incompletely understood. Here, we show that Raf-1 dephosphorylation, primarily at epidermal growth factor (EGF)-induced sites, abolishes Raf-1 kinase activity. Using mass spectrometry, we identified five novel in vivo Raf-1 phosphorylation sites, one of which, S471, is located in subdomain VIB of Raf-1 kinase domain. Mutational analyses demonstrated that Raf-1 S471 is critical for Raf-1 kinase activity and for its interaction with mitogen-activated protein kinase kinase (MEK). Similarly, mutation of the corresponding B-Raf site, S578, resulted in an inactive kinase, suggesting that the same Raf-1 and B-Raf phosphorylation is needed for Raf kinase activation. Importantly, the naturally occurring, cancer-associated B-Raf activating mutation V599E suppressed the S578A mutation, suggesting that introducing a charged residue at this region eliminates the need for an activating phosphorylation. Our results demonstrate an essential role of specific EGF-induced Raf-1 phosphorylation sites in Raf-1 activation, identify Raf-1 S471 as a novel phosphorylation site critical for Raf-1 and B-Raf kinase activities, and point to the possibility that the V599E mutation activates B-Raf by mimicking a phosphorylation at the S578 site.     

Regulation and role of Raf-1/B-Raf heterodimerization

The Ras-Raf-MEK-extracellular signal-regulated kinase (ERK) pathway participates in the control of many fundamental cellular processes including proliferation, survival, and differentiation. The pathway is deregulated in up to 30% of human cancers, often due to mutations in Ras and the B-Raf isoform. Raf-1 and B-Raf can form heterodimers, and this may be important for cellular transformation. Here, we have analyzed the biochemical and biological properties of Raf-1/B-Raf heterodimers. Isolated Raf-1/B-Raf heterodimers possessed a highly increased kinase activity compared to the respective homodimers or monomers. Heterodimers between wild-type Raf-1 and B-Raf mutants with low or no kinase activity still displayed elevated kinase activity, as did heterodimers between wild-type B-Raf and kinase-negative Raf-1. In contrast, heterodimers containing both kinase-negative Raf-1 and kinase-negative B-Raf were completely inactive, suggesting that the kinase activity of the heterodimer specifically originates from Raf and that either kinase-competent Raf isoform is sufficient to confer high catalytic activity to the heterodimer. In cell lines, Raf-1/B-Raf heterodimers were found at low levels. Heterodimerization was enhanced by 14-3-3 proteins and by mitogens independently of ERK. However, ERK-induced phosphorylation of B-Raf on T753 promoted the disassembly of Raf heterodimers, and the mutation of T753 prolonged growth factor-induced heterodimerization. The B-Raf T753A mutant enhanced differentiation of PC12 cells, which was previously shown to be dependent on sustained ERK signaling. Fine mapping of the interaction sites by peptide arrays suggested a complex mode of interaction involving multiple contact sites with a main Raf-1 binding site in B-Raf encompassing T753. In summary, our data suggest that Raf-1/B-Raf heterodimerization occurs as part of the physiological activation process and that the heterodimer has distinct biochemical properties that may be important for the regulation of some biological processes.     

Serine and tyrosine phosphorylations cooperate in Raf-1, but not B-Raf activation

The Raf family of serine/threonine protein kinases couple growth factor receptor stimulation to mitogen activated protein kinase activation, but their own regulation is poorly understood. Using phospho-specific antisera, we show that activated Raf-1 is phosphorylated on S338 and Y341. Expression of Raf-1 with oncogenic Ras gives predominantly S338 phosphorylation, whereas activated Src gives predominantly Y341 phosphorylation. Phosphorylation at both sites is maximal only when both oncogenic Ras and activated Src are present. Raf-1 that cannot interact with Ras-GTP is not phosphorylated, showing that phosphorylation is Ras dependent, presumably occurring at the plasma membrane. Mutations which prevent phosphorylation at either site block Raf-1 activation and maximal activity is seen only when both are phosphorylated. Mutations at S339 or Y340 do not block Raf-1 activation. While B-Raf lacks a tyrosine phosphorylation site equivalent to Y341 of Raf-1, S445 of B-Raf is equivalent to S338 of Raf-1. Phosphorylation of S445 is constitutive and is not stimulated by oncogenic Ras. However, S445 phosphorylation still contributes to B-Raf activation by elevating basal and consequently Ras-stimulated activity. Thus, there are considerable differences between the activation of the Raf proteins; Ras-GTP mediates two phosphorylation events required for Raf-1 activation but does not regulate such events for B-Raf.     

New insight into BRAF mutations in cancer

There has been much recent progress in our understanding of the role played by the RAS-RAF-MEK-ERK cascade in human cancer. RAS is an oncogene and this pathway is known to promote proliferation and malignant transformation. More recently, however, RAF has become the focus of attention, particularly in melanoma, where approximately 70% of cases carry mutations in the BRAF gene. The majority of the mutations in BRAF in cancer are activating, but rare mutants that cannot activate MEK have provided new insight into RAF signalling networks that exist in cancer and normal cells. Surprisingly, germline mutations in BRAF that occur in rare genetic syndromes have also recently been described. The induction of BRAF mutations in melanoma depends on the type of UV exposure that the skin receives, and some studies have suggested the existence of susceptibility loci that make it more likely that some individuals will acquire these mutations. Importantly, genetic profiling and microarray studies have provided insight into the spectrum of melanomas in which BRAF plays a role and also revealed intriguing new data that could be important for the diagnosis and treatment of human cancers.     

B-Raf and Raf-1 are regulated by distinct autoregulatory mechanisms

B-Raf is a key regulator of the ERK pathway and is mutationally activated in two-thirds of human melanomas. In this work, we have investigated the activation mechanism of B-Raf and characterized the roles of Ras and of B-Raf phosphorylation in this regulation. Raf-1 is regulated by an N-terminal autoinhibitory domain whose actions are blocked by interaction with Ras and subsequent phosphorylation of Ser(338). We observed that B-Raf also contains an N-terminal autoinhibitory domain and that the interaction of this domain with the catalytic domain was inhibited by binding to active H-Ras. However, unlike Raf-1, the phosphorylation of B-Raf at Ser(445) was constitutive and was only moderately increased by expression of constitutively active H-Ras or constitutively active PAK1. Ser(445) phosphorylation is important to the B-Raf activation mechanism, however, because mutation of this site to alanine increased the affinity of the regulatory domain for the catalytic domain and increased autoinhibition. Similarly, expression of constitutively active PAK1 also decreased auto-inhibition. B-Raf autoinhibition was negatively regulated by acidic substitutions at phosphorylation sites within the activation loop of B-Raf and by the oncogenic substitution V599E. However, these substitutions did not affect the ability of the regulatory domain to co-immunoprecipitate with the catalytic domain. These data demonstrate that B-Raf activity is autoregulated, that constitutive phosphorylation of Ser(445) primes B-Raf for activation, and that a key feature of phosphorylation within the activation loop or of oncogenic mutations within this region is to block autoinhibition.     

Activation of the Raf/MAP kinase cascade by the Ras-related protein TC21 is required for the TC21-mediated transformation of NIH 3T3 cells

TC21 is a member of the Ras superfamily of small GTP-binding proteins and, like Ras, has been implicated in the regulation of growth-stimulating pathways. Point mutations introduced into TC21 based on equivalent H-Ras oncogenic mutations are transforming in cultured cells, and oncogenic mutations in TC21 have been isolated from several human tumours. The mechanism of TC21 signalling in transformation is poorly understood. While activation of the serine/threonine kinases Raf-1 and B-Raf has been implicated in signalling pathways leading to transformation by H-Ras, it has been argued that TC21 does not activate Raf-1 or B-Raf. Since the Raf-signalling pathway is important in transformation by other Ras proteins, we assessed whether the Raf pathway is important to transformation by TC21. Raf-1 and B-Raf are constitutively active in TC21-transformed cells and the ERK/MAPK cascade is required for the maintenance of the transformed state. We demonstrate that oncogenic V23 TC21, like Ras, interacts with Raf-1 and B-Raf (but not with A-Raf), resulting in the translocation of the Raf proteins to the plasma membrane and in their activation. Furthermore, using point mutations in the effector loop of TC21, we show that the interaction of TC21 with Raf-1 is crucial for transformation.     

Neuronal calcium activates a Rap1 and B-Raf signaling pathway via the cyclic adenosine monophosphate-dependent protein kinase

Activity-dependent regulation of neuronal events such as cell survival and synaptic plasticity is controlled by increases in neuronal calcium levels. These actions often involve stimulation of intracellular kinase signaling pathways. For example, the mitogen-activated protein kinase, or extracellular signal-regulated kinase (ERK), signaling cascade has increasingly been shown to be important for the induction of gene expression and long term potentiation. However, the mechanisms leading to ERK activation by neuronal calcium are still unclear. In the present study, we describe a protein kinase A (PKA)-dependent signaling pathway that may link neuronal calcium influx to ERKs via the small G-protein, Rap1, and the neuronal Raf isoform, B-Raf. Thus, in PC12 cells, depolarization-mediated calcium influx led to the activation of B-Raf, but not Raf-1, via PKA. Furthermore, depolarization also induced the PKA-dependent stimulation of Rap1 and led to the formation of a Rap1/B-Raf signaling complex. In contrast, depolarization did not lead to the association of Ras with B-Raf. The major action of PKA-dependent Rap1/B-Raf signaling in neuronal cells is the activation of ERKs. Thus, we further show that, in both PC12 cells and hippocampal neurons, depolarization-induced calcium influx stimulates ERK activity in a PKA-dependent manner. Given the fact that both Rap1 and B-Raf are highly expressed in the central nervous system, we suggest that this signaling pathway may regulate a number of activity-dependent neuronal functions.     

Phosphatidylinositol-3-OH kinase or RAS pathway mutations in human breast cancer cell lines

Constitutive activation of the phosphatidylinositol-3-OH kinase (PI3K) and RAS signaling pathways are important events in tumor formation. This is illustrated by the frequent genetic alteration of several key players from these pathways in a wide variety of human cancers. Here, we report a detailed sequence analysis of the PTEN, PIK3CA, KRAS, HRAS, NRAS, and BRAF genes in a collection of 40 human breast cancer cell lines. We identified a surprisingly large proportion of cell lines with mutations in the PI3K or RAS pathways (54% and 25%, respectively), with mutants for each of the six genes. The PIK3CA, KRAS, and BRAF mutation spectra of the breast cancer cell lines were similar to those of colorectal cancers. Unlike in colorectal cancers, however, mutational activation of the PI3K pathway was mutually exclusive with mutational activation of the RAS pathway in all but 1 of 30 mutant breast cancer cell lines (P = 0.001). These results suggest that there is a fine distinction between the signaling activators and downstream effectors of the oncogenic PI3K and RAS pathways in breast epithelium and those in other tissues.     

Application of a novel [3+2] cycloaddition reaction to prepare substituted imidazoles and their use in the design of potent DFG-out allosteric B-Raf inhibitors

B-Raf protein kinase, which is a key signaling molecule in the RAS–RAF–MEK–ERK signaling pathway, plays an important role in many cancers. The B-Raf V600E mutation represents the most frequent oncogenic kinase mutation known and is responsible for increased kinase activity in approximately 7% of all human cancers, establishing B-Raf as an important therapeutic target for inhibition. Through the use of an iterative program that utilized a chemocentric approach and a rational structure based design, we have developed novel, potent, and specific DFG-out allosteric inhibitors of B-Raf kinase. Here, we present efficient and versatile chemistry that utilizes a key one pot, [3+2] cycloaddition reaction to obtain highly substituted imidazoles and their application in the design of allosteric B-Raf inhibitors. Inhibitors based on this scaffold display subnanomolar potency and a favorable kinase profile.     

A selective cellular screening assay for B-Raf and c-Raf kinases

The Ras/Raf signaling pathway has been recognized as an important process in cancer biology. Recently, activating mutations in the BRAF gene were reported to be present in approximately 66% of malignant melanomas as well as other malignancies such as colon cancer. Here, the authors report the development of a B-Raf-specific cellular assay to profile cell-active B-Raf inhibitors. Expression of the active B-Raf mutant (V600E) and the kinase-inactive form of its substrate, MEK1, was regulated by mifepristone, and the catalytic activity of B-Raf was monitored by following MEK1 phosphorylation. Target specificity was ensured because the phosphorylation of MEK1 was significantly inhibited when kinase-inactive B-Raf was used in place of the active kinase. A cellular c-Raf assay was similarly established to monitor the selectivity between B-Raf and c-Raf. Z' factor values were consistently above 0.50 with either kinase, indicating that assay performance was sufficiently robust for use as cellular profiling assays. The authors used this system to demonstrate that the selectivity profile of compounds targeted against B-Raf and c-Raf kinases could be quantitatively determined. This platform provides a quantitative cellular readout for a spectrum of specific inhibitors of B-Raf and c-Raf kinases that is particularly suitable for use in drug discovery.     

Prevalence of class I–III BRAF mutations among 114,662 cancer patients in a large genomic database

BRAF mutations are relatively common in many cancers, particularly melanoma, colorectal cancer, and thyroid cancer and to a lesser extent in lung cancer. These mutations can be targeted by BRAF and MEK inhibitors, which exhibit good clinical activity. There are conflicting reports of the various relative rates of BRAF Class I mutations (V600 locus), defined as those that exhibit extremely strong kinase activity by stimulating monomeric activation of BRAF, Class II, define as non-V600 mutations that activate BRAF to signal as a RAS-independent dimer, and Class III mutations, defined as “kinase-dead” with low kinase activity as compared to wild type BRAF. Prospective studies have largely focused on patients with tumors harboring Class I BRAF mutations (limited to the V600 locus) where response rates up to 70% with BRAF plus MEK inhibition have been demonstrated. We report on the relative prevalence of various types of BRAF mutations across human cancers in a cohort of 114,662 patients that received comprehensive genomic profiling using next-generation sequencing. Of these patients, 4517 (3.9%) a pathogenic or presumed pathogenic BRAF mutation (3.9%). Of these, 1271 were seen in melanoma, representing 39.7% of all melanomas sequenced, representing the highest rate in all tumors. Class I (V600) mutations were seen overall in 2841 patients (62.1% of BRAF mutations, 2.4% of total cancers). Class II mutations were seen in 746 tumors (16.5% of BRAF mutant, 0.7% of total), and Class III mutations were seen in 801 tumors (17.7% of BRAF, 0.7% of total). Knowledge of the relative prevalence of these types of mutations can aid in the development of agents that might better address non-V600 mutations in cancer.Impact statementThese data represent the largest aggregation of BRAF mutations within a single clinical database to our knowledge. The relative proportions of both BRAF V600 mutations and non-V600 mutations are informative in all cancers and by malignancy, and can serve as a definitive gold-standard for BRAF mutation cancer incidence by malignancy. The rate of BRAF mutation in human cancer in a real-world large database is lower than previously reported likely representing testing more broadly across tumor types. The relative percentages of Class II and Class III BRAF mutations are higher than previously reported, representing almost 35% of BRAF mutations in cancer. These findings provide support for the development of effective treatments for non-V600 BRAF mutations in cancer.     

Distinct requirement for an intact dimer interface in wild-type, V600E and kinase-dead B-Raf signalling

The dimerisation of Raf kinases involves a central cluster within the kinase domain, the dimer interface (DIF). Yet, the importance of the DIF for the signalling potential of wild-type B-Raf (B-Raf(wt)) and its oncogenic counterparts remains unknown. Here, we show that the DIF plays a pivotal role for the activity of B-Raf(wt) and several of its gain-of-function (g-o-f) mutants. In contrast, the B-Raf(V600E), B-Raf(insT) and B-Raf(G469A) oncoproteins are remarkably resistant to mutations in the DIF. However, compared with B-Raf(wt), B-Raf(V600E) displays extended protomer contacts, increased homodimerisation and incorporation into larger protein complexes. In contrast, B-Raf(wt) and Raf-1(wt) mediated signalling triggered by oncogenic Ras as well as the paradoxical activation of Raf-1 by kinase-inactivated B-Raf require an intact DIF. Surprisingly, the B-Raf DIF is not required for dimerisation between Raf-1 and B-Raf, which was inactivated by the D594A mutation, sorafenib or PLX4720. This suggests that paradoxical MEK/ERK activation represents a two-step mechanism consisting of dimerisation and DIF-dependent transactivation. Our data further implicate the Raf DIF as a potential target against Ras-driven Raf-mediated (paradoxical) ERK activation.     

Involvement of B-Raf in Ras-induced Raf-1 activation.

The mechanism of Ras-induced Raf-1 activation is not fully understood. Previously, we identified a 400-kDa protein complex as a Ras-dependent Raf-1 activator. In this study, we identified B-Raf as a component of this complex. B-Raf was concentrated during the purification of the activator. Immunodepletion of B-Raf abolished the effect of the activator on Raf-1. Furthermore, B-Raf and Ras-activated Raf-1 co-operatively, when co-transfected into human embryonic kidney 293 cells. On the other hand, Ras-dependent extracellular signal-regulated kinase/mitogen-activated protein kinase kinase stimulator (a complex of B-Raf and 14-3-3) failed to activate Raf-1 in our cell-free system. These results suggest that B-Raf is an essential component of the Ras-dependent Raf-1 activator.     

The requirement of specific membrane domains for Raf-1 phosphorylation and activation

Activation of Raf-1 by Ras requires recruitment to the membrane as well as additional phosphorylations, including phosphorylation at serine 338 (Ser-338) and tyrosine 341 (Tyr-341). In this study we show that Tyr-341 participates in the recruitment of Raf-1 to specialized membrane domains called "rafts," which are required for Raf-1 to be phosphorylated on Ser-338. Raf-1 is also thought to be recruited to the small G protein Rap1 upon GTP loading of Rap1. However, this does not result in Raf-1 activation. We propose that this is because Raf-1 is not phosphorylated on Tyr-341 upon recruitment to Rap1. Redirecting Rap1 to Ras-containing membranes or mimicking Tyr-341 phosphorylation of Raf-1 by mutation converts Rap1 into an activator of Raf-1. In contrast to Raf-1, B-Raf is activated by Rap1. We suggest that this is because B-Raf activation is independent of tyrosine phosphorylation. Moreover, mutants that render B-Raf dependent on tyrosine phosphorylation are no longer activated by Rap1.     

beta 2-adrenergic receptor activates extracellular signal-regulated kinases (ERKs) via the small G protein rap1 and the serine/threonine kinase B-Raf

G protein-coupled receptors can induce cellular proliferation by stimulating the mitogen-activated protein (MAP) kinase cascade. Heterotrimeric G proteins are composed of both alpha and betagamma subunits that can signal independently to diverse intracellular signaling pathways including those that activate MAP kinases. In this study, we examined the ability of isoproterenol, an agonist of the beta(2)-adrenergic receptor (beta(2)AR), to stimulate extracellular signal-regulated kinases (ERKs). Using HEK293 cells, which express endogenous beta(2)AR, we show that isoproterenol stimulates ERKs via beta(2)AR. This action of isoproterenol requires cAMP-dependent protein kinase and is insensitive to pertussis toxin, suggesting that Galpha(s) activation of cAMP-dependent protein kinase is required. Interestingly, beta(2)AR activates both the small G proteins Rap1 and Ras, but only Rap1 is capable of coupling to Raf isoforms. beta(2)AR inhibits the Ras-dependent activation of both Raf isoforms Raf-1 and B-Raf, whereas Rap1 activation by isoproterenol recruits and activates B-Raf. beta(2)AR activation of ERKs is not blocked by expression of RasN17, an interfering mutant of Ras, but is blocked by expression of either RapN17 or Rap1GAP1, both of which interfere with Rap1 signaling. We propose that isoproterenol can activate ERKs via Rap1 and B-Raf in these cells.     

Nerve growth factor-stimulated B-Raf catalytic activity is refractory to inhibition by cAMP-dependent protein kinase

The cAMP-dependent protein kinase (PKA) exhibits both inhibitory and stimulatory effects upon growth factor signaling mediated by the mitogen-activated protein kinase signaling pathway. PKA has been demonstrated to inhibit Raf-1-mediated cellular proliferation. PKA can both prevent Ras-dependent Raf-1 activation and directly inhibit Raf-1 catalytic activity. In contrast to the inhibitory effect of PKA on Raf-1-dependent processes, PKA potentiates nerve growth factor-stimulated PC12 cell differentiation, a B-Raf mediated process. This potentiation, rather than inhibition, of PC12 cell differentiation is curious in light of the ability of PKA to inhibit Raf-1 catalytic activity. The kinase domains of Raf-1 and B-Raf are highly conserved, and it has been predicted that B-Raf catalytic activity would also be inhibited by PKA. In this study we examined the ability of PKA to regulate the kinase activity of the B-raf proto-oncogene. We report that nerve growth factor-stimulated B-Raf activity is not inhibited by PKA. By contrast, an N-terminally truncated, constitutively active form of B-Raf is inhibited by PKA both in vitro and in transfected PC12 cells. These results suggest that the N-terminal regulatory domain interferes with the ability of PKA to modulate B-Raf catalytic activity and provide an explanation for the observed resistance of B-Raf-dependent processes to PKA inhibition.     

The role of autophagy in cytotoxicity induced by new oncogenic B-Raf inhibitor UI-152 in v-Ha-ras transformed fibroblasts

In human cancers, B-Raf is the most frequently mutated protein kinase in the MAPK signaling cascade, making it an important therapeutic target. We recently discovered a potent and selective B-Raf inhibitor, UI-152, by using a structure-based drug design strategy. In this study, we examined whether B-Raf inhibition by UI-152 may be an effective therapeutic strategy for eliminating cancer cells transformed with v-Ha-ras (Ras-NIH 3T3). UI-152 displayed selective cytotoxicity toward Ras-NIH 3T3 cells while having little to no effect on non-transformed NIH 3T3 cells. We found that treatment with UI-152 markedly increased autophagy and, to a lesser extent, apoptosis. However, inhibition of autophagy by addition of 3-MA failed to reverse the cytotoxic effects of UI-152 on Ras-NIH 3T3 cells, demonstrating that apoptosis and autophagy can act as cooperative partners to induce growth inhibition in Ras-NIH 3T3 cells treated with UI-152. Most interestingly, cell responses to UI-152 appear to be paradoxical. Here, we showed that although UI-152 inhibited ERK, it induced B-Raf binding to Raf-1 as well as Raf-1 activation. This paradoxical activation of Raf-1 by UI-152 is likely to be coupled with the inhibition of the mTOR pathway, an intracellular signaling pathway involved in autophagy. We also showed for the first time that, in multi-drug resistant cells, the combination of UI-152 with verapamil significantly decreased cell proliferation and increased autophagy. Thus, our findings suggest that the inhibition of autophagy, in combination with UI-152, offers a more effective therapeutic strategy for v-Ha-ras-transformed cells harboring wild-type B-Raf.