SHOC2 and CRAF Mediate ERK1/2 Reactivation in Mutant NRAS-mediated Resistance to RAF Inhibitor

ERK1/2 signaling is frequently dysregulated in tumors through BRAF mutation. Targeting mutant BRAF with vemurafenib frequently elicits therapeutic responses; however, durable effects are often limited by ERK1/2 pathway reactivation via poorly defined mechanisms. We generated mutant BRAF^V600E melanoma cells that exhibit resistance to PLX4720, the tool compound for vemurafenib, that co-expressed mutant (Q61K) NRAS. In these BRAF^V600E/NRAS^Q61K co-expressing cells, re-activation of the ERK1/2 pathway during PLX4720 treatment was dependent on NRAS. Expression of mutant NRAS in parental BRAF^V600 cells was sufficient to by-pass PLX4720 effects on ERK1/2 signaling, entry into S phase and susceptibility to apoptosis in a manner dependent on the RAF binding site in NRAS. ERK1/2 activation in BRAF^V600E/NRAS^Q61K cells required CRAF only in the presence of PLX4720, indicating a switch in RAF isoform requirement. Both ERK1/2 activation and resistance to apoptosis of BRAF^V600E/NRAS^Q61K cells in the presence of PLX4720 was modulated by SHOC-2/Sur-8 expression, a RAS-RAF scaffold protein. These data show that NRAS mutations confer resistance to RAF inhibitors in mutant BRAF cells and alter RAF isoform and scaffold molecule requirements to re-activate the ERK1/2 pathway.     

Inhibition of mutant BRAF splice variant signaling by next‐generation,selective RAF inhibitors

Vemurafenib and dabrafenib block MEK‐ERK1/2 signaling and cause tumor regression in the majority of advanced‐stage BRAF^V600E melanoma patients; however, acquired resistance and paradoxical signaling have driven efforts for more potent and selective RAF inhibitors. Next‐generation RAF inhibitors, such as PLX7904 (PB04), effectively inhibit RAF signaling in BRAF^V600E melanoma cells without paradoxical effects in wild‐type cells. Furthermore, PLX7904 blocks the growth of vemurafenib‐resistant BRAF^V600E cells that express mutant NRAS. Acquired resistance to vemurafenib and dabrafenib is also frequently driven by expression of mutation BRAF splice variants; thus, we tested the effects of PLX7904 and its clinical analog, PLX8394 (PB03), in BRAF^V600E splice variant‐mediated vemurafenib‐resistant cells. We show that paradox‐breaker RAF inhibitors potently block MEK‐ERK1/2 signaling, G1/S cell cycle events, survival and growth of vemurafenib/PLX4720‐resistant cells harboring distinct BRAF^V600E splice variants. These data support the further investigation of paradox‐breaker RAF inhibitors as a second‐line treatment option for patients failing on vemurafenib or dabrafenib.     

The BRAFV600E inhibitor,PLX4032, increases type I collagen synthesis in melanoma cells

Vertical growth phase (VGP) melanoma is frequently metastatic, a process mediated by changes in gene expression, which are directed by signal transduction pathways in the tumor cells. A prominent signaling pathway is the Ras-Raf-Mek-Erk MAPK pathway, which increases expression of genes that promote melanoma progression. Many melanomas harbor a mutation in this pathway, BRAF^V600E, which constitutively activates MAPK signaling and expression of downstream target genes that facilitate tumor progression. In BRAF^V600E melanoma, the small molecule inhibitor, vemurafenib (PLX4032), has revolutionized therapy for melanoma by inducing rapid tumor regression. This compound down-regulates the expression of many genes. However, in this study, we document that blocking the Ras-Raf-Mek-Erk MAPK pathway, either with an ERK (PLX4032) or a MEK (U1026) signaling inhibitor, in BRAF^V600E human and murine melanoma cell lines increases collagen synthesis in vitro and collagen deposition in vivo. Since TGFß signaling is a major mediator of collagen synthesis, we examined whether blocking TGFß signaling with a small molecule inhibitor would block this increase in collagen. However, there was minimal reduction in collagen synthesis in response to blocking TGFß signaling, suggesting additional mechanism(s), which may include activation of the p38 MAPK pathway. Presently, it is unclear whether this increased collagen synthesis and deposition in melanomas represent a therapeutic benefit or an unwanted “off target” effect of inhibiting the Ras-Raf-Erk-Mek pathway.     

Identification of PLX4032‐resistance mechanisms and implications for novel RAF inhibitors

BRAF inhibitors improve melanoma patient survival, but resistance invariably develops. Here we report the discovery of a novel BRAF mutation that confers resistance to PLX4032 employing whole‐exome sequencing of drug‐resistant BRAF^V600K melanoma cells. We further describe a new screening approach, a genome‐wide piggyBac mutagenesis screen that revealed clinically relevant aberrations (N‐terminal BRAF truncations and CRAF overexpression). The novel BRAF mutation, a Leu505 to His substitution (BRAF^L505H), is the first resistance‐conferring second‐site mutation identified in BRAF mutant cells. The mutation replaces a small nonpolar amino acid at the BRAF‐PLX4032 interface with a larger polar residue. Moreover, we show that BRAF^L505H, found in human prostate cancer, is itself a MAPK‐activating, PLX4032‐resistant oncogenic mutation. Lastly, we demonstrate that the PLX4032‐resistant melanoma cells are sensitive to novel, next‐generation BRAF inhibitors, especially the ‘paradox‐blocker’ PLX8394, supporting its use in clinical trials for treatment of melanoma patients with BRAF‐mutations.     

The BRAF mutation confers sensitivity of thyroid cancer cells to the BRAF inhibitor PLX4032 (RG7204)

Aberrant signaling of the Ras-Raf-MEK-ERK (MAP kinase) pathway driven by the mutant kinase BRAF^V600E, as a result of the BRAF^T1799A mutation, plays a fundamental role in thyroid tumorigenesis. This study investigated the therapeutic potential of a BRAF^V600E-selective inhibitor, PLX4032 (RG7204), for thyroid cancer by examining its effects on the MAP kinase signaling and proliferation of 10 thyroid cancer cell lines with wild-type BRAF or BRAF^T1799A mutation. We found that PLX4032 could effectively inhibit the MAP kinase signaling, as reflected by the suppression of ERK phosphorylation, in cells harboring the BRAF^T1799A mutation. PLX4032 also showed a potent and BRAF mutation-selective inhibition of cell proliferation in a concentration-dependent manner. PLX4032 displayed low IC₅₀ values (0.115–1.156 μM) in BRAF^V600E mutant cells, in contrast with wild-type BRAF cells that showed resistance to the inhibitor with high IC₅₀ values (56.674–1349.788 μM). Interestingly, cells with Ras mutations were also sensitive to PLX4032, albeit moderately. Thus, this study has confirmed that the BRAF^T1799A mutation confers cancer cells sensitivity to PLX4032 and demonstrated its specific potential as an effective and BRAF^T1799A mutation-selective therapeutic agent for thyroid cancer.     

PLX4032, a potent inhibitor of the B-Raf V600E oncogene, selectively inhibits V600E-positive melanomas

Targeted intervention of the B-Raf V600E gene product that is prominent in melanoma has been met with modest success. Here, we characterize the pharmacological properties of PLX4032, a next-generation inhibitor with exquisite specificity against the V600E oncogene and striking anti-melanoma activity. PLX4032 induces potent cell cycle arrest, inhibits proliferation, and initiates apoptosis exclusively in V600E-positive cells in a variety of in vitro experimental systems; follow-up xenograft studies demonstrate extreme selectivity and efficacy against melanoma tumors bearing the V600E oncoproduct. The collective data support further exploration of PLX4032 as a candidate drug for patients with metastatic melanoma; accordingly, validation of PLX4032 as a therapeutic tool for patients with melanoma is now underway in advanced human (Phase III) clinical trials.     

Metabolic stress regulates ERK activity by controlling KSR‐RAF heterodimerization

Altered cell metabolism is a hallmark of cancer, and targeting specific metabolic nodes is considered an attractive strategy for cancer therapy. In this study, we evaluate the effects of metabolic stressors on the deregulated ERK pathway in melanoma cells bearing activating mutations of the NRAS or BRAF oncogenes. We report that metabolic stressors promote the dimerization of KSR proteins with CRAF in NRAS‐mutant cells, and with oncogenic BRAF in BRAF^V600E‐mutant cells, thereby enhancing ERK pathway activation. Despite this similarity, the two genomic subtypes react differently when a higher level of metabolic stress is induced. In NRAS‐mutant cells, the ERK pathway is even more stimulated, while it is strongly downregulated in BRAF^V600E‐mutant cells. We demonstrate that this is caused by the dissociation of mutant BRAF from KSR and is mediated by activated AMPK. Both types of ERK regulation nevertheless lead to cell cycle arrest. Besides studying the effects of the metabolic stressors on ERK pathway activity, we also present data suggesting that for efficient therapies of both genomic melanoma subtypes, specific metabolic targeting is necessary.     

Loss of PI(4,5)P2 5-Phosphatase A Contributes to Resistance of Human Melanoma Cells to RAF/MEK Inhibitors

Past studies have shown that the inositol polyphosphate 5-phosphatase, phosphatidylinositol 4,5-bisphosphate 5-phosphatase (PIB5PA), is commonly downregulated or lost in melanomas, which contributes to elevated activation of phosphatidylinositol 3-kinase (PI3K)/Akt in melanoma cells. In this report, we provide evidence that PIB5PA deficiency plays a role in resistance of melanoma cells to RAF/mitogen-activated protein kinase kinase (MEK) inhibitors. Ectopic expression of PIB5PA enhanced apoptosis induced by the RAF inhibitor PLX4720 in BRAF^V600E and by the MEK inhibitor U0126 in both BRAF^V600E and wild-type BRAF melanoma cells. This was due to inhibition of PI3K/Akt, as co-introduction of an active form of Akt (myr-Akt) abolished the effect of overexpression of PIB5PA on apoptosis induced by PLX4720 or U0126. While overexpression of PIB5PA triggered activation of Bad and down-regulation of Mcl-1, knockdown of Bad or overexpression of Mcl-1 recapitulated, at least in part, the effect of myr-Akt, suggesting that regulation of Bad and Mcl-1 is involved in PIB5PA-mediated sensitization of melanoma cells to the inhibitors. The role of PIB5PA deficiency in BRAF inhibitor resistance was confirmed by knockdown of PIB5PA, which led to increased growth of BRAF^V600E melanoma cells selected for resistance to PLX4720. Consistent with its role in vitro, overexpression of PIB5PA and the MEK inhibitor selumetinib cooperatively inhibited melanoma tumor growth in a xenograft model. Taken together, these results identify loss of PIB5PA as a novel resistance mechanism of melanoma to RAF/MEK inhibitors and suggest that restoration of PIB5PA may be a useful strategy to improve the therapeutic efficacy of the inhibitors in the treatment of melanoma.     

NRAS and BRAF Mutations in Melanoma-Associated Nevi and Uninvolved Nevi

According to the prevailing multistep model of melanoma development, oncogenic BRAF or NRAS mutations are crucial initial events in melanoma development. It is not known whether melanocytic nevi that are found in association with a melanoma are more likely to carry BRAF or NRAS mutations than uninvolved nevi. By laser microdissection we were able to selectively dissect and genotype cells either from the nevus or from the melanoma part of 46 melanomas that developed in association with a nevus. In 25 cases we also genotyped a control nevus of the same patients. Available tissue was also immunostained using the BRAF^V600E-mutation specific antibody VE1. The BRAF^V600E mutation was found in 63.0% of melanomas, 65.2% of associated nevi and 50.0% of control nevi. No significant differences in the distribution of BRAF or NRAS mutations could be found between melanoma and associated nevi or between melanoma associated nevi and control nevi. In concordant cases immunohistochemistry showed a higher expression (intensity of immunohistochemistry) of the mutated BRAF^V600E-protein in melanomas compared to their associated nevi. In this series the presence of a BRAF- or NRAS mutation in a nevus was not associated with the risk of malignant transformation. Our findings do not support the current traditional model of stepwise tumor progression.     

Tumor Homogeneity between Primary and Metastatic Sites for BRAF Status in Metastatic Melanoma Determined by Immunohistochemical and Molecular Testing

BRAF inhibitors have demonstrated improvement of overall survival in patients with metastatic melanoma and BRAF^V600 mutations. In order to evaluate BRAF tumor heterogeneity between primary and metastatic site, we have evaluated the performance of immunohistochemistry (IHC) with an anti-BRAF^V600E antibody in both localization by comparison with high resolution melting analysis followed by Sanger sequencing in a parallel blinded study. A total of 230 samples distributed as primary melanoma (n = 88) and different types of metastatic samples (n = 142) were studied in 99 patients with advanced or metastatic melanoma (stage III or IV). The prevalence of each BRAF mutation was c.1799T>A, BRAF^V600E (45.2%), c.1799_1800TG>AA, BRAF^V600E2 (3.0%), c.1798_1799GT>AA, BRAF^V600K (3.0%), c.1801 A>G, BRAF^K601E (1.3%), c.1789_1790CT>TC, BRAF^L597S (0.4%), c.1780G>A, BRAF^D594N (0.9%) respectively. IHC was positive in 109/112 samples harboring BRAF^V600E/E2 mutations and negative in other cases. The cytoplasmic staining was either strongly positive in tumor cells of BRAF^V600E mutated cases. It appeared strong brown, different from the vesicular grey cytoplasmic pigmentation of melanophages. Concordance between the two techniques was 96.4%. Sensitivity of IHC for detecting the BRAF^V600E/E2 mutations was 97.3%, while specificity was 100%. Both our IHC and molecular study demonstrated homogeneity between primary and metastatic sites for BRAF status in melanoma. This study also provides evidence that IHC may be a cost-effective first-line method for BRAF^V600E detection. Thereafter, molecular techniques should be used in negative, ambiguous or non-contributive cases.     

Dual MEK/AKT inhibition with trametinib and GSK2141795 does not yield clinical benefit in metastatic NRAS‐mutant and wild‐type melanoma

Aberrant MAPK and PI3K pathway signaling may drive the malignant phenotype in NRAS‐mutant and BRAF^WT NRAS^WT metastatic melanoma. To target these pathways, NRAS‐mutant and BRAF^WT NRAS^WT patients received oral trametinib at 1.5 mg daily and GSK2141795 at 50 mg daily in a two‐cohort Simon two‐stage design. Participants had adequate end‐organ function and no more than two prior treatment regimens. Imaging assessments were performed at 8‐week intervals. A total of 10 NRAS‐mutant and 10 BRAF^WT NRAS^WT patients were enrolled. No objective responses were noted in either cohort. The median PFS and OS were 2.3 and 4.0 months in the NRAS‐mutant cohort and 2.8 and 3.5 months in the wild‐type cohort. Grade 3 and grade 4 adverse events, primarily rash, were observed in 25% of patients. We conclude that the combination of trametinib and GSK2141795 does not have significant clinical activity in NRAS‐mutant or BRAF^WT NRAS^WT melanoma.     

BH3-only protein silencing contributes to acquired resistance to PLX4720 in human melanoma

B-RAF is mutated to a constitutively active form in 8% of human cancers including 50% of melanomas. In clinical trials, the RAF inhibitor, PLX4032 (vemurafenib), caused partial or complete responses in 48–81% of mutant B-RAF harboring melanoma patients. However, the average duration of response was 6–7 months before tumor regrowth, indicating the acquisition of resistance to PLX4032. To understand the mechanisms of resistance, we developed mutant B-RAF melanoma cells that displayed resistance to RAF inhibition through continuous culture with PLX4720 (the tool compound for PLX4032). Resistance was associated with a partial reactivation of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling, recovery of G1/S cell-cycle events, and suppression of the pro-apoptotic B-cell leukemia/lymphoma 2 (Bcl-2) homology domain 3 (BH3)-only proteins, Bcl-2-interacting mediator of cell death-extra large (Bim-EL) and Bcl-2 modifying factor (Bmf). Preventing ERK1/2 reactivation with MEK (mitogen-activated protein/extracellular signal-regulated kinase kinase) inhibitors blocked G1-S cell-cycle progression but failed to induce apoptosis or upregulate Bim-EL and Bmf. Treatment with the histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid, led to de-repression of Bim-EL and enhanced cell death in the presence of PLX4720 or AZD6244 in resistant cells. These data indicate that acquired resistance to PLX4032/4720 likely involves ERK1/2 pathway reactivation as well as ERK1/2-independent silencing of BH3-only proteins. Furthermore, combined treatment of HDAC inhibitors and MEK inhibitors may contribute to overcoming PLX4032 resistance.     

Regulating the response to targeted MEK inhibition in melanoma: Enhancing apoptosis in NRAS- and BRAF-mutant melanoma cells with Wnt/β-catenin activation

The limitations of revolutionary new mutation-specific inhibitors of BRAF^V600E include the universal recurrence seen in melanoma patients treated with this novel class of drugs. Recently, our lab showed that simultaneous activation of the Wnt/β-catenin signaling pathway and targeted inhibition of BRAF^V600E by PLX4720 synergistically induces apoptosis across a spectrum of BRAF^V600E melanoma cell lines. As a follow-up to that study, treatment of BRAF-mutant and NRAS-mutant melanoma lines with WNT3A and the MEK inhibitor AZD6244 also induces apoptosis. The susceptibility of BRAF-mutant lines and NRAS-mutant lines to apoptosis correlates with negative regulation of Wnt/β-catenin signaling by ERK/MAPK signaling and dynamic decreases in abundance of the downstream scaffolding protein, AXIN1. Apoptosis-resistant NRAS-mutant lines can sensitize to AZD6244 by pretreatment with AXIN1 siRNA, similar to what we previously reported in BRAF-mutant cell lines. Taken together, these findings indicate that NRAS-mutant melanoma share with BRAF-mutant melanoma the potential to regulate apoptosis upon MEK inhibition through WNT3A and dynamic regulation of cellular AXIN1. Understanding the cellular context that makes melanoma cells susceptible to this combination treatment will contribute to the study and development of novel therapeutic combinations that may lead to more durable responses.     

Regulating the response to targeted MEK inhibition in melanoma

The limitations of revolutionary new mutation-specific inhibitors of BRAF^V600E include the universal recurrence seen in melanoma patients treated with this novel class of drugs. Recently, our lab showed that simultaneous activation of the Wnt/β-catenin signaling pathway and targeted inhibition of BRAF^V600E by PLX4720 synergistically induces apoptosis across a spectrum of BRAF^V600E melanoma cell lines. As a follow-up to that study, treatment of BRAF-mutant and NRAS-mutant melanoma lines with WNT3A and the MEK inhibitor AZD6244 also induces apoptosis. The susceptibility of BRAF-mutant lines and NRAS-mutant lines to apoptosis correlates with negative regulation of Wnt/β-catenin signaling by ERK/MAPK signaling and dynamic decreases in abundance of the downstream scaffolding protein, AXIN1. Apoptosis-resistant NRAS-mutant lines can sensitize to AZD6244 by pretreatment with AXIN1 siRNA, similar to what we previously reported in BRAF-mutant cell lines. Taken together, these findings indicate that NRAS-mutant melanoma share with BRAF-mutant melanoma the potential to regulate apoptosis upon MEK inhibition through WNT3A and dynamic regulation of cellular AXIN1. Understanding the cellular context that makes melanoma cells susceptible to this combination treatment will contribute to the study and development of novel therapeutic combinations that may lead to more durable responses.     

The BRAFT1799A mutation confers sensitivity of thyroid cancer cells to the BRAFV600E inhibitor PLX4032 (RG7204)

Aberrant signaling of the Ras-Raf-MEK-ERK (MAP kinase) pathway driven by the mutant kinase BRAF(V600E), as a result of the BRAF(T1799A) mutation, plays a fundamental role in thyroid tumorigenesis. This study investigated the therapeutic potential of a BRAF(V600E)-selective inhibitor, PLX4032 (RG7204), for thyroid cancer by examining its effects on the MAP kinase signaling and proliferation of 10 thyroid cancer cell lines with wild-type BRAF or BRAF(T1799A) mutation. We found that PLX4032 could effectively inhibit the MAP kinase signaling, as reflected by the suppression of ERK phosphorylation, in cells harboring the BRAF(T1799A) mutation. PLX4032 also showed a potent and BRAF mutation-selective inhibition of cell proliferation in a concentration-dependent manner. PLX4032 displayed low IC(50) values (0.115-1.156μM) in BRAF(V600E) mutant cells, in contrast with wild-type BRAF cells that showed resistance to the inhibitor with high IC(50) values (56.674-1349.788μM). Interestingly, cells with Ras mutations were also sensitive to PLX4032, albeit moderately. Thus, this study has confirmed that the BRAF(T1799A) mutation confers cancer cells sensitivity to PLX4032 and demonstrated its specific potential as an effective and BRAF(T1799A) mutation-selective therapeutic agent for thyroid cancer.     

CXCR3 Signaling in BRAFWT Melanoma Increases IL-8 Expression and Tumorigenicity

Patients with early stage, radial growth phase (RGP) melanoma have a 97% survival rate; however, when the melanoma progresses to the invasive vertical growth phase (VGP), survival rates decrease to 15%. The targets of many clinical trials are the known genetic and molecular mechanisms involved in melanoma progression, with the most common oncogenic mutation being the BRAF^V600E. However, less than half of melanomas harbor this mutation, and consequently, do not respond to the current BRAF targeted treatments. It is therefore critical to elucidate alternative mechanisms regulating melanoma progression. Increased expression of the chemokine receptor, CXCR3, on melanoma cells is correlated with increased metastasis and poor patient outcomes, suggesting a role for CXCR3 in the RGP to VGP transition. We found that endogenous CXCR3 can be induced in two RGP cell lines, BOWES (BRAF^WT) and WM35 (BRAF^V600E), with in vitro environmental stress and nutrient deprivation. Signaling via induced endogenous CXCR3 is linked with IL-8 expression in BOWES cells. Ectopic overexpression of CXCR3 in BOWES cells leads to increased ligand-mediated phERK, cellular migration, and IL-8 expression in vitro, and to increased tumorigenesis and lymph node metastasis in vivo. Our results demonstrate that, in BRAF^WT melanomas, CXCR3 signaling mediates significant increases in IL-8 expression, suggesting that CXCR3 expression and signaling may represent a transformative event that drives the progression of BRAF^WT melanomas. Implications: Expression of CXCR3 on BRAF^WT melanoma cells may be a mediator of melanoma progression.     

AEBP1 upregulation confers acquired resistance to BRAF (V600E) inhibition in melanoma

An activating BRAF (V600E) kinase mutation occurs in approximately half of melanomas. Recent clinical studies have demonstrated that vemurafenib (PLX4032) and dabrafenib, potent and selective inhibitors of mutant v-raf murine sarcoma viral oncogene homolog B1 (BRAF), exhibit remarkable activities in patients with V600 BRAF mutant melanomas. However, acquired drug resistance invariably develops after the initial treatment. Identification of acquired resistance mechanisms may inform the development of new therapies that elicit long-term responses of melanomas to BRAF inhibitors. Here we report that increased expression of AEBP1 (adipocyte enhancer-binding protein 1) confers acquired resistance to BRAF inhibition in melanoma. AEBP1 is shown to be highly upregulated in PLX4032-resistant melanoma cells because of the hyperactivation of the PI3K/Akt-cAMP response element-binding protein (CREB) signaling pathway. This upregulates AEBP1 expression and thus leads to the activation of NF-κB via accelerating IκBa degradation. In addition, inhibition of the PI3K/Akt-CREB-AEBP1-NF-κB pathway greatly reverses the PLX4032-resistant phenotype of melanoma cells. Furthermore, increased expression of AEBP1 is validated in post-treatment tumors in patients with acquired resistance to BRAF inhibitor. Therefore, these results reveal a novel PI3K/Akt-CREB-AEBP1-NF-κB pathway whose activation contributes to acquired resistance to BRAF inhibition, and suggest that this pathway, particularly AEBP1, may represent a novel therapeutic target for treating BRAF inhibitor-resistant melanoma.     

Reactivation of Mitogen-activated Protein Kinase (MAPK) Pathway by FGF Receptor 3 (FGFR3)/Ras Mediates Resistance to Vemurafenib in Human B-RAF V600E Mutant Melanoma

Oncogenic B-RAF V600E mutation is found in 50% of melanomas and drives MEK/ERK pathway and cancer progression. Recently, a selective B-RAF inhibitor, vemurafenib (PLX4032), received clinical approval for treatment of melanoma with B-RAF V600E mutation. However, patients on vemurafenib eventually develop resistance to the drug and demonstrate tumor progression within an average of 7 months. Recent reports indicated that multiple complex and context-dependent mechanisms may confer resistance to B-RAF inhibition. In the study described herein, we generated B-RAF V600E melanoma cell lines of acquired-resistance to vemurafenib, and investigated the underlying mechanism(s) of resistance. Biochemical analysis revealed that MEK/ERK reactivation through Ras is the key resistance mechanism in these cells. Further analysis of total gene expression by microarray confirmed a significant increase of Ras and RTK gene signatures in the vemurafenib-resistant cells. Mechanistically, we found that the enhanced activation of fibroblast growth factor receptor 3 (FGFR3) is linked to Ras and MAPK activation, therefore conferring vemurafenib resistance. Pharmacological or genetic inhibition of the FGFR3/Ras axis restored the sensitivity of vemurafenib-resistant cells to vemurafenib. Additionally, activation of FGFR3 sufficiently reactivated Ras/MAPK signaling and conferred resistance to vemurafenib in the parental B-RAF V600E melanoma cells. Finally, we demonstrated that vemurafenib-resistant cells maintain their addiction to the MAPK pathway, and inhibition of MEK or pan-RAF activities is an effective therapeutic strategy to overcome acquired-resistance to vemurafenib. Together, we describe a novel FGFR3/Ras mediated mechanism for acquired-resistance to B-RAF inhibition. Our results have implications for the development of new therapeutic strategies to improve the outcome of patients with B-RAF V600E melanoma.