Wild-type Melanoma Cell Lines Research Articles

Abstract 4000: ERBB4 is sufficient and necessary for proliferation of BRAF WT melanoma cell lines

Abstract Approximately 50% of metastatic melanomas possess wild-type (WT) BRAF alleles. These BRAF-WT tumors are a significant clinical problem, despite the fact that these tumors commonly possess mutations in NF1 or RAS genes, as there are no targeted therapeutics for these tumors. Here we demonstrate that ERBB4 is sufficient and necessary for proliferation of BRAF WT melanoma cell lines, suggesting that disrupting ERBB4 signaling may be effective against BRAF WT melanomas. ERBB4 (HER4) is a member of the ERBB family of receptor tyrosine kinases, which includes the epidermal growth factor receptor (EGFR/ERBB1/HER1), ERBB2 (Neu/HER2), and ERBB3 (HER3). EGFR and ERBB2 are validated targets in a variety of solid tumors, whereas the roles that ERBB4 plays in human malignancies are ambiguous. Our previous in silico analyses of BRAF-WT tumor genomes in The Cancer Genome Atlas skin cutaneous melanoma (TCGA-SKCM) dataset suggest that ERBB4 mutation or overexpression cooperates with NF1 or RAS gene mutations to drive melanomas via activation of the PI3 kinase/AKT signaling pathway. These in silico analyses have also enabled us to prioritize the ERBB4 mutants found in BRAF-WT melanomas. We have tested this hypothesis using a panel of human BRAF-WT melanoma cell lines. We have shown that expression of wild-type ERBB4 causes increased clonogenic proliferation, whereas expression of a dominant-negative ERBB4 mutant (K751M) causes decreased clonogenic proliferation. These data indicate that ERBB4 is sufficient and necessary for clonogenic proliferation of BRAF-WT melanoma cell lines. Analgous studies are testing whether ERBB4 regulates anchorage independence in semi-solid medium and proliferation rates and saturation density in culture dishes. The BRAF-WT melanoma cell lines endogenously express ERBB4 ligands, EGFR, and ERBB2. Moreover, ERBB4 homodimers function as tumor suppressors, whereas ERBB4-EGFR and ERBB4-ERBB2 heterodimers are oncogenic. Thus, we are using gene silencing and pharmacologic tools to determine whether endogenous ERBB4 ligands, endogenous EGFR, or endogenous ERBB2 is required for the oncogenic activity of ERBB4. Furthermore, we are determining whether prioritized ERBB4 mutants found in BRAF-WT melanomas exhibit greater signaling and oncogenic activities than WT ERBB4. Consequently, these experiments will establish a functional role for ERBB4 mutations and expression in BRAF-WT melanomas and will identify potential strategies for disrupting oncogenic ERBB4 signaling in these tumors. Citation Format: Lauren M. Lucas, Vipasha Dwivedi, Rania H. Mohamedelhassan, Erin L. Harrell, Connor M. Kelley, Elizabeth L. Knerr, Jessica A. Markham, David J. Riese. ERBB4 is sufficient and necessary for proliferation of BRAF WT melanoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4000.

Mitogen-activated protein kinase dependency in BRAF/RAS wild-type melanoma: A rationale for combination inhibitors.

Inhibitors targeting the mitogen-activated protein kinase (MAPK) pathway and immune checkpoint molecules have dramatically improved the survival of patients with BRAFV600 -mutant melanoma. For BRAF/RAS wild-type (WT) melanoma patients, however, immune checkpoint inhibitors remain the only effective therapeutic option with 40% of patients responding to PD-1 inhibition. In the present study, a large panel of 10 BRAFV600 -mutant and 13 BRAF/RAS WT melanoma cell lines was analyzed to examine MAPK dependency and explore the potential utility of MAPK inhibitors in this melanoma subtype. We now show that the majority of BRAF/RAS WT melanoma cell lines (8/13) display some degree of sensitivity to trametinib treatment and resistance to trametinib in this melanoma subtype is associated with, but not mediated by NF1 suppression. Although knockdown of NF1 stimulates RAS and CRAF activity, the activation of CRAF by NF1 knockdown is limited by ERK-dependent feedback in BRAF-mutant cells, but not in BRAF/RAS WT melanoma cells. Thus, NF1 is not a dominant regulator of MAPK signaling in BRAF/RAS WT melanoma, and co-targeting multiple MAP kinase nodes provides a therapeutic opportunity for this melanoma subtype.

Abstract 3717: New therapies for the treatment of BRAF/NRAS wild type melanoma

Abstract Melanoma represents the common tumor whose incidence has increased the most in the last 30 years and causes more than one death every hour in the US alone. Despite significant advances in targeted and immunotherapies, most patients cannot still be cured. Our aim is to identify new drug combinations that are synergistic in BRAF/NRAS wild type melanoma, a sub-type representing 30% of cases for which targeted therapies are not currently available. We high-throughput screened a collection of 20 BRAF/NRAS wild type melanoma cell lines with 180 drug combinations (60 library drugs used at 5 different concentrations combined with 3 clinically relevant anchor drugs) and generated over 8000 survival curves . We found that 25% of cell lines are highly sensitive to a combination of nilotinib plus trametinib and confirmed this finding with 2 independent assays. We further validated the drug synergy firstly using an independent collection of BRAF/NRAS wild type melanoma cell lines (n=7), then a collection of BRAF/NRAS wild type patient derived xenotransplant cultures (n=3), and finally with a collection of BRAFV600E and NRASQ61 melanoma cell lines (n=12). Further, we generated a gene expression signature of cell lines that display synergy for the nilotinib/trametinib combination, and used it to classify human melanomas from Leeds Melanoma Project (N=171) and TCGA (n=470) cohorts. Tumors classified as “synergistic-like” (27.9 and 36.7%, respectively) are associated to decreased overall and recurrence free survival (P<0.05), suggesting that our combination might be effective in a relevant fraction of aggressive tumors. In order to identify drug resistance mechanisms we deployed a genome-wide CRISPR/Cas9 screen. We found that loss of the tuberous sclerosis complex can confer resistance to nilotinib/trametinib, and validated this mechanism using clonal engineered lines. Since tuberous sclerosis complex genes are mutated in 10% of melanomas, this approach can help to identify patients potentially refractory to the treatment. We also investigated the molecular mechanism of nilotinib/trametinib synergy by analysing the level of several phosphoproteins upon treatment. We discovered that the nilotinib/trametinib combination synergistically reduce the level of P-ERK in synergistic cell lines but not in cell lines resistant to the drug combination, thus pointing out the MAPK pathway dependence of the synergy. This finding provides a putative marker to identify tumors responsive to the treatment. Finally, we tested in vivo the nilotinib/trametinib combination in a patient derived xenotransplant mouse model and showed that the combination is well tolerated and significantly more effective than the 2 drugs alone (P<0.01). These data suggest a strong clinical translation potential for nilotinib/trametinib combination and pave the way to the development of clinical trials for BRAF/NRAS wild type melanoma. Citation Format: Marco Ranzani, Kristel Kemper, Magali Michaut, Oscar Krijgsman, Vivek Iyer, Anneliese Speak, Jeremie Nsengimana, Kim Wong, Vera Grinkevich, Nanne Aben, Martin Del Castillo Velasco-Herrera, Clara Alsinet, Marcela Sjoberg, Mamunur Rashid, Gemma Turner, Fiona Behan, Emmanuelle Supper, Nicola Thompson, Graham Bignell, Ken Dutton-Regester, Antonia Pritchard, Chi Wong, Ultan McDermott, Nicholas K. Hayward, Kosuke Yusa, Julia Newton-Bishop, Lodewyk Wessels, Mathew Garnett, Daniel Peeper, David Adams. New therapies for the treatment of BRAF/NRAS wild type melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3717. doi:10.1158/1538-7445.AM2017-3717

Abstract 4658: Activation mechanisms of Integrated Stress Response by inhibition of oncogenic BRAF in melanoma cell lines

Abstract The eIF2α-ATF4 signaling pathway termed “Integrated stress response (ISR)” plays an important role in protecting cells against various cellular stresses such as deprivation of glucose and amino acids, virus infection, and deficiency of heme. Under such stress conditions, eIF2α kinases, including PERK, GCN2, PKR and HRI, phosphorylate eIF2α, resulting in reduction of general protein synthesis while facilitating selective translation of ATF4, a central transcription factor that upregulates expression of stress-induced genes for cellular adaptation. Recently, it was reported that BRAF inhibitor vemurafenib triggers activation of the eIF2α-ATF4 signaling in BRAF-mutant melanoma cell lines; however, activation mechanisms of vemurafenib-induced ISR signaling not fully understood. In this study, we first compared vemurafenib-induced ISR activation in BRAF-mutant and -wild type melanoma cell lines. Consistent with previous studies, vemurafenib activated ISR signaling preferentially in BRAF-mutant cell lines, as determined by phosphorylation of eIF2α and ATF4 expression. We also found that the ISR activation by vemurafenib was impaired by knockdown of BRAF in BRAF-mutant cell lines. These results indicated that existence of mutated BRAF is important for vemurafenib to trigger activation of ISR signaling pathway. We next examined which eIF2α kinase is involved in the ISR signaling activation by vemurafenib. We found that knockdown of GCN2, an eIF2α kinase sensing depletion of amino acids, impaired phosphorylation of eIF2α and ATF4 induction by vemurafenib. In addition, we confirmed that vemurafenib induced phosphorylation of GCN2 on Thr899 that is known to occur in response to amino acids starvation. In agreement with these results, gene expression profiling with ontology analysis revealed that vemurafenib upregulated genes that are involved in amino acids biosynthesis via ISR signaling. Collectively, our results suggested that vemurafenib activated ISR signaling pathway possibly through mimicking amino acid deprivation in BRAF-mutant melanoma cell lines. Citation Format: Ikuko Nagasawa, Satomi Tsukahara, Kazuhiro Kunimasa, Akihiro Tomida. Activation mechanisms of Integrated Stress Response by inhibition of oncogenic BRAF in melanoma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4658.

Abstract 4783: Identification of new therapies for the treatment of BRAF/NRAS wild-type melanomas by functional screening approaches

Abstract Melanoma causes 9000 deaths each year in the USA alone, more than one every hour. It represents the common tumor whose incidence has increased the most in the last 30 years. Despite promising advances, including immunotherapies, the therapeutic regimens are curative in only a fraction of patients. Implementation and combination of different therapeutic modalities is therefore required to improve patient survival. Given the clinical efficacy of drug combinations for BRAF mutant melanomas, we performed a high-throughput drug screening to identify new drug combinations for the treatment of BRAF/NRAS wild type melanomas. Effective targeted therapies are not currently available for this sub-type of the disease, which represents up to 30% of melanomas. By viability assays we characterized the sensitivity to 240 combinations of clinically relevant drugs in a collection of 21 BRAF/NRAS wild type melanoma cell lines. We analysed 8360 survival curves and found that 16 (73%) and 5 (23%) cell lines are highly sensitive to temozolomide plus olaparib and to nilotinib plus trametinib combinations, respectively. Two independent experimental approaches validated these drug synergies. By -omics technologies we deeply characterized the cell line profiles of mutations, copy number changes, DNA methylation, and gene and microRNA expression. We generated gene expression signatures of synergistic and resistant cell lines for the 2 drug combinations and used them to classify 374 human melanomas. Tumors significantly associated to nilotinib plus trametinib synergism signature were significantly enriched for high immune response and proliferative Jonsson's expression classes, while tumors associated to the signature of drug resistance are enriched for pigmentation class. Melanomas associated to temozolomide plus olaparib resistance signature displayed enrichment for normal class. We are currently confirming the representation of these signatures in an independent cohort of melanomas and looking for other clinical correlates. We are also validating the predictivity of these gene expression signatures in independent cohorts of melanoma cell lines. Prospectively, this may represent an approach to identify patients that could have the maximal benefit from these drug combinations. Additionally, we have recently performed a genome-wide CRISPR/Cas9 screen to identify drug resistance genes for these 2 drug combinations. Preliminary results indicate a prominent role of tuberous sclerosis complex in the regulation of the sensitivity to nilotinib plus trametinib combination. These results may pave the way to the development of novel patient-tailored targeted therapies for the efficient eradication of BRAF/NRAS wild type melanomas. Citation Format: Marco Ranzani, Magali Michaut, Clara Alsinet, Vera Grinkevich, Kim Wong, Vivek Iyer, Nanne Aben, Martin Del Castillo Velasco-Herrera, Marcela Sjoberg, Mamunur Rashid, Graham Bignell, Ken Dutton-Regester, Antonia Pritchard, Daniel Vis, Gemma Turner, Ultan McDermott, Nicholas K. Hayward, Kosuke Yusa, Lodewyk Wessels, Mathew Garnett, David Adams. Identification of new therapies for the treatment of BRAF/NRAS wild-type melanomas by functional screening approaches. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4783.

RAF Inhibition Overcomes Resistance to TRAIL-Induced Apoptosis in Melanoma Cells

Mutated BRAF represents a critical oncogene in melanoma, and selective inhibitors have been approved for melanoma therapy. However, the molecular consequences of RAF inhibition in melanoma cells remained largely elusive. Here, we investigated the effects of the pan-RAF inhibitor L-779,450, which inhibited cell proliferation both in BRAF-mutated and wild-type melanoma cell lines. It furthermore enhanced apoptosis in combination with the death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and overcame TRAIL resistance in melanoma cells. Enhanced apoptosis coincided with activation of mitochondrial pathways, seen by loss of mitochondrial membrane potential and release of cytochrome c, Smac (second mitochondria-derived activator of caspases), and apoptosis-inducing factor (AIF). Subsequently, caspase-9 and -3 were activated. Apoptosis induction by L-779,450/TRAIL was prevented by Bcl-2 overexpression and was dependent on Bax. Thus, activation of Bax by L-779,450 alone was demonstrated by Bax conformational changes, whereas Bak was not activated. Furthermore, the BH3-only protein Bim was upregulated in response to L-779,450. The significant roles of Smac, Bax, and Bim in this setting were proven by small interfering RNA (siRNA)-mediated knockdown experiments. L-779,450 also resulted in morphological changes indicating autophagy confirmed by the autophagy marker light chain 3-II (LC3-II). The pro-apoptotic effects of L-779,450 may explain the antitumor effects of RAF inhibition and may be considered when evaluating RAF inhibitors for melanoma therapy.

Abstract C138: Loss of NF1 in melanoma cell lines is associated with active Ras and dependence on MEK even in the absence of BRAF or NRAS mutation.

Abstract Though two-thirds of cutaneous melanomas harbor activating mutations in the BRAF and NRAS genes, the alterations that drive tumor progression in the roughly 30% of melanoma tumors wild-type for BRAF and NRAS remain largely uncharacterized. The selective RAF inhibitors vemurafenib and dabrafenib inhibit MAPK pathway activity only in BRAF mutant cells, thus their clinical utility is restricted to patients with BRAF mutant tumors. MEK inhibitors, which have broader antitumor activity and have shown promising activity in NRAS mutant melanoma, may be effective in a broader range of MAPK pathway-dependent tumors. We performed a functional and genomic analysis of melanoma cell lines wild-type for BRAF and NRAS to determine whether occult mutations in RAS signaling were present. Elevated RAS-GTP was common in BRAF/NRAS wild-type melanoma cell lines, a subset of which exhibited total loss of NF1 protein expression. The proliferation of NF1-null melanoma cells was dependent upon MEK, though the cellular potency of several allosteric MEK inhibitors that are currently in clinical testing (PD0325901, AZD6244, MEK162 and trametinib) varied widely in NF1-null melanoma cells. The greatest antitumor activity was noted with trametinib, a MEK inhibitor that also blocks RAF mediated phosphorylation of MEK. Notably, alterations in NF1 also co-occurred with RAS and BRAF mutations in both cell lines and human melanomas. In the setting of BRAF co-mutation, loss of NF1 abrogated upstream negative feedback on RAS activation resulting in constitutive expression of RAS-GTP and resistance to RAF but not MEK inhibition. In summary, NF1 loss is a common event in cutaneous melanoma that is associated with RAS activation, MEK dependence and RAF inhibitor resistance. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C138. Citation Format: Moriah H. Nissan, Christine Pratilas, Alexis Jones, Helen Won, Li Kong, Zhan Yao, Taha Merghoub, Antoni Ribas, Paul Chapman, Rona Yaeger, Barry Taylor, Nikolaus Shultz, Michael F. Berger, Neal Rosen, David B. Solit. Loss of NF1 in melanoma cell lines is associated with active Ras and dependence on MEK even in the absence of BRAF or NRAS mutation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C138.

Abstract 917: High antitumor activity of the ERK inhibitor SCH722984 against BRAF-mutant, NRAS-mutant and wild-type melanoma cell lines.

Abstract MAPK pathway dysregulation via mutations in proteins such as BRAFV600E and NRASQ61 is a key feature in melanomas, leading to constitutive ERK signaling. Currently, there are no FDA-approved targeted therapies effective in vemurafenib-resistant BRAFV600E mutant, NRAS mutant, or wild-type melanoma. The effect of SCH722984, an allosteric inhibitor of ERK 1/2, was determined in a panel of 53 melanoma cell lines, including wild-type, NRAS-mutant and vemurafenib-sensitive and resistant BRAF-mutant melanoma. 54% (13 of 24) of BRAF mutants, including 4 with innate vemurafenib resistance, were sensitive to SCH722984 with an IC50 <1μM. 100% (3 of 3) of BRAF/NRAS double mutants, 71% (10 of 14) of NRAS mutants and 71% (5 of 7) of wild-type melanomas were sensitive. 13% (5 of 24) of BRAF mutants, 14% (2 of 14) of NRAS mutants and 29% (2 of 7) of wild-type melanomas were intermediately sensitive (IC50 1-1.5μM). Among BRAFV600E mutants with in vitro acquired resistance to vemurafenib, those with MAPK pathway reactivation as the mechanism of resistance were sensitive to SCH722984. Combining vemurafenib and SCH722984 in BRAF mutant melanoma was synergistic in a majority of cell lines, resulting in up to 10-fold decrease in the 50% inhibitory concentration (IC50) and with combination indices (CI) below 1. SCH722984 inhibited phosphorylation of RSK, an ERK downstream target. Finally, long term cultures of two BRAFV600E-mutant cell lines sensitive to SCH722984 showed that treatment with SCH722984 alone or in combination with vemurafenib was more potent than vemurafenib alone and resulted in a significant delay in developing acquired resistance. SCH722984 may therefore be clinically applicable as a treatment for non-BRAF mutant melanoma or in BRAF-mutant melanoma with innate or acquired resistance, alone or in combination with BRAF inhibitors. Citation Format: Deborah J. Wong, Lidia Robert, Mohammad S. Atefi, David Foulad, Earl V. Avramis, Begonya Comin-Anduix, Ahmed A. Samatar, Antoni Ribas. High antitumor activity of the ERK inhibitor SCH722984 against BRAF-mutant, NRAS-mutant and wild-type melanoma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 917. doi:10.1158/1538-7445.AM2013-917

Abstract 1914: Potent anticancer activity against both BRAF-mutant and BRAF wild-type melanoma cell lines using a novel CRM1 nuclear export inhibitor

Abstract INTRODUCTION: BRAF and NRAS oncogenic mutations occur in over half of all human melanomas. BRAF-mutant tumors are highly sensitive to small molecule inhibitors directed against the oncogenic BRAF protein. However, in tumors that lack the specific mutation, patients have few effective therapeutic options. To address the need for new therapies for patients with BRAF-wild-type (wt) melanoma, we hypothesized that the inhibition of the major nuclear export receptor CRM1 would result in nuclear accumulation of nuclear proteins, increase apoptosis and induce cell cycle arrest independent of BRAF mutational status. To test our hypothesis, we examined the effects of a novel CRM1 inhibitor on melanoma cell lines with various BRAF and NRAS mutational backgrounds. METHODS: We used human malignant melanoma cell lines with BRAF mutation (Sk-Mel-28, A375, Mel-624, UACC903, MalMe-3M), BRAF-wt (MeWo) and BRAF-wt and NRAS mutation (Hs940T) to assess the effects of CRM1 inhibition, BRAF inhibition (PLX4032) or MEK inhibition (U0126) using both mono- or combination therapy. MTT assays were used to evaluate the effects of treatment on cell proliferation. FACS analysis with annexin/PI staining was performed to assess effects on cell cycle and cell death. The Chou-Talalay method was used to assess synergistic drug combination effects. Treatment effects on cleaved caspase-3, PARP cleavage, P53, and FOXO3 were analyzed by western blot analysis. RESULTS: As mono therapy, CRM1 inhibition led to a significant decrease in cell proliferation, cell cycle arrest and an increase in apoptosis in both BRAF wt and mutant cell lines. The degree of inhibition and cell death achieved by CRM1 inhibition across all melanoma cell lines is comparable to the results achieved by BRAF inhibitor PLX4032 in BRAF mutant cell lines. Furthermore, the combination of CRM1 inhibition and BRAF inhibition results in a strongly synergistic combination against BRAF mutant cell lines with a statistically significant decrease in cell proliferation and increased apoptotic cell death. CONCLUSION: We found that relative to other cancer cell types, both BRAF-mutant and BRAF-wt melanoma cells are exquisitely sensitive to CRM1 inhibition. In addition, CRM1 inhibition significantly enhanced the effect of PLX4032 in BRAF-mutant cell lines, suggesting that the combination of these two drugs may address the emergence of acquired resistance to BRAF monotherapy. Based on these results, CRM1 inhibition warrants further investigation to determine its potential benefit in treating melanoma patients independent of BRAF mutation status. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1914. doi:1538-7445.AM2012-1914

High-level expression of wild-type p53 in melanoma cells is frequently associated with inactivity in p53 reporter gene assays.

BackgroundInactivation of the p53 pathway that controls cell cycle progression, apoptosis and senescence, has been proposed to occur in virtually all human tumors and p53 is the protein most frequently mutated in human cancer. However, the mutational status of p53 in melanoma is still controversial; to clarify this notion we analysed the largest series of melanoma samples reported to date.Methodology/Principal FindingsImmunohistochemical analysis of more than 180 melanoma specimens demonstrated that high levels of p53 are expressed in the vast majority of cases. Subsequent sequencing of the p53 exons 5–8, however, revealed only in one case the presence of a mutation. Nevertheless, by means of two different p53 reporter constructs we demonstrate transcriptional inactivity of wild type p53 in 6 out of 10 melanoma cell lines; the 4 other p53 wild type melanoma cell lines exhibit p53 reporter gene activity, which can be blocked by shRNA knock down of p53.Conclusions/SignificanceIn melanomas expressing high levels of wild type p53 this tumor suppressor is frequently inactivated at transcriptional level.

Growth-Inhibitory and Antiangiogenic Activity of the MEK Inhibitor PD0325901 in Malignant Melanoma with or without BRAF Mutations

The Raf/MEK/ERK pathway is an importantmediator of tumor cell proliferation and angiogenesis. Here, weinvestigated the growth-inhibitory and antiangiogenic properties of PD0325901, a novel MEK inhibitor, in human melanoma cells. PD0325901 effects were determined in a panel of melanoma cell lines with different genetic aberrations. PD0325901 markedly inhibited ERK phosphorylation and growth of both BRAF mutant and wild-type melanoma cell lines, with IC50 in the nanomolar range even in the least responsive models. Growth inhibition was observed both in vitro and in vivo in xenograft models, regardless of BRAF mutation status, and was due to G1-phase cell cycle arrest and subsequent induction of apoptosis. Cell cycle (cyclin D1, c-Myc, and p27KIP1) and apoptosis (Bcl-2 and survivin) regulators were modulated by PD0325901 at the protein level. Gene expression profiling revealed profound modulation of several genes involved in the negative control of MAPK signaling and melanoma cell differentiation, suggesting alternative, potentially relevant mechanisms of action. Finally, PD0325901 inhibited the production of the proangiogenic factors vascular endothelial growth factor and interleukin 8 at a transcriptional level. In conclusion, PD0325901 exerts potent growth-inhibitory, proapoptotic, and antiangiogenic activity in melanoma lines, regardless of their BRAF mutation status. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective treatment strategies for patients experiencing malignant melanoma.