BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma

Melanoma is the most lethal form of skin cancer, and the incidence and mortality rates are rapidly rising. Epidemiologically, high numbers of nevi (moles) are associated with higher risk of melanoma . The majority of melanomas exhibit activating mutations in the serine/threonine kinase BRAF . BRAF mutations may be critical for the initiation of melanoma ; however, the direct role of BRAF in nevi and melanoma has not been tested in an animal model. To directly test the role of activated BRAF in nevus and melanoma development, we have generated transgenic zebrafish expressing the most common BRAF mutant form (V600E) under the control of the melanocyte mitfa promoter. Expression of mutant, but not wild-type, BRAF led to dramatic patches of ectopic melanocytes, which we have termed fish (f)-nevi. Remarkably, in p53-deficient fish, activated BRAF induced formation of melanocyte lesions that rapidly developed into invasive melanomas, which resembled human melanomas and could be serially transplanted. These data provide direct evidence that BRAF activation is sufficient for f-nevus formation, that BRAF activation is among the primary events in melanoma development, and that the p53 and BRAF pathways interact genetically to produce 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.     

Co-development of a companion diagnostic for targeted cancer therapy

Oncology drug development is a long and costly process associated with a success rate of 5-10%. The parallel development of companion diagnostic tests that will identify patients most likely to receive benefit has the potential to increase the success rate for oncology drugs and decrease development time and associated costs. Metastatic melanoma is a challenging disease that has been associated with poor survival. Identification of a mutated BRAF kinase gene in many cases of melanoma provided a promising therapeutic target. Here we describe the successful co-development of vemurafenib, a first-in-class selective inhibitor of oncogenic BRAF kinase, and its companion diagnostic, the cobas(?) 4800 BRAF V600 Mutation Test. Key success factors in the development process included early identification of the BRAF V600E biomarker, early development of the diagnostic test, and early and close collaboration between the pharmaceutical and diagnostic development teams. This focused and integrated process resulted in the first personalized medicine for the treatment of metastatic melanoma less than five years after the Investigational New Drug Application, a remarkably short time.     

Detrimental effects of melanocortin‐1 receptor (MC1R) variants on the clinical outcomes of BRAF V600 metastatic melanoma patients treated with BRAF inhibitors

Melanocortin‐1 receptor (MC1R) plays a key role in skin pigmentation, and its variants are linked with a higher melanoma risk. The influence of MC1R variants on the outcomes of patients with metastatic melanoma (MM) treated with BRAF inhibitors (BRAFi) is unknown. We studied the MC1R status in a cohort of 53 consecutive BRAF‐mutated patients with MM treated with BRAFi. We also evaluated the effect of vemurafenib in four ^V600BRAF melanoma cell lines with/without MC1R variants. We found a significant correlation between the presence of MC1R variants and worse outcomes in terms of both overall response rate (ORR; 59% versus 95%, P = 0.011 univariate, P = 0.028 multivariate analysis) and progression‐free survival (PFS) shorter than 6 months (72% versus 33%, P = 0.012 univariate, P = 0.027 multivariate analysis). No difference in overall survival (OS) was reported, probably due to subsequent treatments. Data in vitro showed a significant different phosphorylation of Erk1/2 and p38 MAPK during treatment, associated with a greater increase in vemurafenib IC50 in MC1R variant cell lines.     

Melanocortin-1 receptor variant R151C modifies melanoma risk in Dutch families with melanoma

Germline mutations of the cell-cycle regulator p16 (also called "CDKN2A") in kindreds with melanoma implicate this gene in susceptibility to malignant melanoma. Most families with familial atypical multiple-mole melanoma (FAMMM) who are registered at the Leiden dermatology clinic share the same p16-inactivating deletion (p16-Leiden). Incomplete penetrance and variable clinical expression suggest risk modification by other genetic and/or environmental factors. Variants of the melanocortin-1 receptor (MC1R) gene have been shown to be associated with red hair, fair skin, and melanoma in humans. Carriers of the p16-Leiden deletion in Dutch families with FAMMM show an increased risk of melanoma when they also carry MC1R variant alleles. The R151C variant is overrepresented in patients with melanoma who are from families with the p16-Leiden mutation. Although some of the effect of the R151C variant on melanoma risk may be attributable to its effect on skin type, our analyses indicate that the R151C variant contributes an increased melanoma risk even after statistical correction for its effect on skin type. These findings suggest that the R151C variant may be involved in melanoma tumorigenesis in a dual manner, both as a determinant of fair skin and as a component in an independent additional pathway.     

BRAF silencing by short hairpin RNA or chemical blockade by PLX4032 leads to different responses in melanoma and thyroid carcinoma cells

BRAF-activating mutations have been reported in several types of cancer, including melanoma ( approximately 70% of cases), thyroid (30-70%), ovarian (15-30%), and colorectal cancer (5-20%). Mutant BRAF has constitutive kinase activity and causes hyperactivation of the mitogen-activated protein kinase pathway. BRAF silencing induces regression of melanoma xenografts, indicating the essential role of BRAF for cell survival. We set up an inducible short hairpin RNA system to compare the role of oncogenic BRAF in thyroid carcinoma versus melanoma cells. Although BRAF knockdown led to apoptosis in the melanoma cell line A375, the anaplastic thyroid carcinoma cell ARO underwent growth arrest upon silencing, with little or no cell death. Reexpression of the thyroid differentiation marker, sodium iodide symporter, was induced after long-term silencing. The different outcome of BRAF down-regulation in the two cell lines was associated with an opposite regulation of p21(CIP1/WAF1) expression levels in response to the block of the BRAF mitogenic signal. These results were confirmed using a specific BRAF small-molecule inhibitor, PLX4032. Restoration of p21(CIP1/WAF1) expression rescued melanoma cells from death. Altogether, our data indicate that oncogenic BRAF inhibition can have a different effect on cell fate depending on the cellular type. Furthermore, we suggest that a BRAF-independent mechanism of cell survival exists in anaplastic thyroid cancer cells.     

Simultaneous knockdown of BRAF and expression of INK4A in melanoma cells leads to potent growth inhibition and apoptosis

Abnormal BRAF and p16INK4A co-exist in 60% of melanomas. BRAF mutation also occurs in 80% of benign nevi where it turns-on p16INK4A resulting in proliferative senescence; loss of p16INK4A removes the inhibitory block leading to melanoma development. Since only melanomas with wild-type BRAF have amplified CDK4 and cyclin D1 genes, p16INK4A-CDK4/6-cyclin D pathway is viewed as linearly downstream of BRAF. Thus, co-occurrence of aberrant BRAF and INK4A may be remnant of changes during melanoma formation without functional significance. To explore this notion, we simultaneously knocked down BRAF (via siRNA) and expressed INK4A cDNA in melanoma cells and observed enhanced growth inhibition. Notably, although each alone had no statistically significant effect on apoptosis, co-expression of BRAF siRNA and INK4A cDNA caused potent apoptosis, which was associated with up-regulation of BIM and down-regulation of BCL2. Our results suggest that aberrant BRAF and INK4A cooperate to promote proliferation and survival of melanoma cells.     

Clinical outcome and pathological features associated with NRAS mutation in cutaneous melanoma

The effect of NRAS mutations on the pathological features and clinical outcomes in patients with cutaneous melanoma was compared with that of tumors containing BRAF(V600E) mutations and tumors wild type for both (WT). Clinical outcome data were obtained from a prospective cohort of 249 patients. Mutations involving NRAS and BRAF(V600E) were detected by PCR and were sequence verified. Cox proportional hazards regression was performed to relate NRAS and BRAF mutations to clinical outcome. Seventy-five percentage of NRAS mutations occurred in tumors >1 mm thick (BRAF(V600E) 40%, WT 34%); 75% of NRAS mutations had >1 mitosis/mm(2) (BRAF(V600E) 40%, WT 55%). When compared to WT, multivariate analysis of melanoma-specific survival (MSS) identified NRAS mutations as an adverse prognostic factor [hazard ratio (HR) 2.96; P = 0.04] but not BRAF(V600E) mutations (HR 1.73; P = 0.23). NRAS mutations were associated with thicker tumors and higher rates of mitosis when compared to BRAF(V600E) and WT melanoma and independently of this, with shorter MSS.     

Selective RAF inhibitor impairs ERK1/2 phosphorylation and growth in mutant NRAS,vemurafenib‐resistant melanoma cells

The RAF inhibitor vemurafenib achieves remarkable clinical responses in mutant BRAF melanoma patients. However, vemurafenib is burdened by acquired drug resistance and by the side effects associated with its paradoxical activation of the ERK1/2 pathway in wild‐type BRAF cells. This paradoxical effect has driven the development of a new class of RAF inhibitors. Here, we tested one of these selective, non‐paradox‐inducing RAF inhibitors termed paradox‐breaker‐04 (PB04) or PLX7904. Consistent with its design, PB04 is able to efficiently inhibit activation of ERK1/2 in mutant BRAF melanoma cells but does not hyperactivate ERK1/2 in mutant RAS‐expressing cells. Importantly, PB04 inhibited ERK1/2 phosphorylation in mutant BRAF melanoma cells with acquired resistance to vemurafenib/PLX4720 that is mediated by a secondary mutation in NRAS. Consistent with ERK1/2 reactivation driving the re‐acquisition of malignant properties, PB04 promoted apoptosis and inhibited entry into S phase and anchorage‐independent growth in mutant N‐RAS‐mediated vemurafenib‐resistant cells. These data indicate that paradox‐breaker RAF inhibitors may be clinically effective as a second‐line option in a cohort of acquired vemurafenib‐resistant patients.     

A twenty year perspective on melanoma therapy

Melanoma had long been considered to be particularly addressable with immunotherapy, but that reputation was built on modestly effective cytokine-based immunotherapy. CTLA-4 antibody therapy reinforced this legacy, but PD-1 antibodies transformed the melanoma treatment landscape and lead the way for immunotherapy to become standard treatment for more than half of the advanced cancer population. BRAF mutations were discovered in 8% of all cancer and nearly 50% of melanomas. Successful development of BRAF inhibitors and BRAF/MEK combination therapy in melanoma preceded regulatory approval across all cancer types. No cancer type saw outcomes improved by the same margin as melanoma in the decade of the 2010s.     

Activities of multiple cancer-related pathways are associated with BRAF mutation and predict the resistance to BRAF/MEK inhibitors in melanoma cells

Drug resistance is a major obstacle in the targeted therapy of melanoma using BRAF/MEK inhibitors. This study was to identify BRAF V600E-associated oncogenic pathways that predict resistance of BRAF-mutated melanoma to BRAF/MEK inhibitors. We took in silico approaches to analyze the activities of 24 cancer-related pathways in melanoma cells and identify those whose activation was associated with BRAF V600E and used the support vector machine (SVM) algorithm to predict the resistance of BRAF-mutated melanoma cells to BRAF/MEK inhibitors. We then experimentally confirmed the in silico findings. In a microarray gene expression dataset of 63 melanoma cell lines, we found that activation of multiple oncogenic pathways preferentially occurred in BRAF-mutated melanoma cells. This finding was reproduced in 5 additional independent melanoma datasets. Further analysis of 46 melanoma cell lines that harbored BRAF mutation showed that 7 pathways, including TNFα, EGFR, IFNα, hypoxia, IFNγ, STAT3, and MYC, were significantly differently expressed in AZD6244-resistant compared with responsive melanoma cells. A SVM classifier built on this 7-pathway activation pattern correctly predicted the response of 10 BRAF-mutated melanoma cell lines to the MEK inhibitor AZD6244 in our experiments. We experimentally showed that TNFα, EGFR, IFNα, and IFNγ pathway activities were also upregulated in melanoma cell A375 compared with its sub-line DRO, while DRO was much more sensitive to AZD6244 than A375. In conclusion, we have identified specific oncogenic pathways preferentially activated in BRAF-mutated melanoma cells and a pathway pattern that predicts resistance of BRAF-mutated melanoma to BRAF/MEK inhibitors, providing novel clinical implications for melanoma therapy.     

Neuroblastoma rat sarcoma mutated melanoma: That’s what we got so far

Neuroblastoma rat sarcoma (NRAS) mutation, occurring in about 20%–30% of cutaneous melanomas, leads to activation of RAS‐RAF‐MAPK cascade and represents a clear distinct clinicopathological entity in melanoma. In contrast with BRAF mutant melanoma, no specific target therapies are available outside the setting of clinical trials. In the field of immunoncology, the predictive role of NRAS mutation with respect to checkpoint inhibitors treatment has not clearly established and deserves further investigation. At present, the standard treatment is the same as for BRAF wild type melanoma. Ongoing trials are exploring novel combination strategies among patients with advanced NRAS mutant melanoma.     

Polymorphisms in the CD28/CTLA4/ICOS genes: role in malignant melanoma susceptibility and prognosis?

The appearance of vitiligo and spontaneous regression of the primary lesion in melanoma patients illustrate a relationship between tumor immunity and autoimmunity. T lymphocytes play a major role both in tumor immunity and autoimmunity. CD28, Cytotoxic T lymphocyte antigen 4 (CTLA4) and inducible costimulator (ICOS) molecules are important secondary signal molecules in the T lymphocyte activation. Single nucleotide polymorphisms (SNPs) in the CD28/CTLA4/ICOS gene region were reported to be associated with several autoimmune diseases including, type-1 diabetes, SLE, autoimmune thyroid diseases and celiac disease. In this study, we investigated the association of SNPs in the CD28, CTLA4 and ICOS genes with the risk of melanoma. We also assessed the prognostic effect of the different polymorphisms in melanoma patients. Twenty-four tagging SNPs across the three genes and four additional SNPs were genotyped in a cohort of 763 German melanoma patients and 734 healthy German controls. Influence on prognosis was determined in 587 melanoma cases belonging to stage I or II of the disease. In general, no differences in genotype or allele frequencies were detected between melanoma patients and controls. However, the variant alleles for two polymorphisms in the CD28 gene were differentially distributed in cases and controls. Similarly no association of any polymorphism with prognosis, except for the rs3181098 polymorphism in the CD28 gene, was observed. In addition, individuals with AA genotype for rs11571323 polymorphism in the ICOS gene showed reduced overall survival. However, keeping in view the correction for multiple hypothesis testing our results suggest that the polymorphisms in the CD28, CTLA4 and ICOS genes at least do not modulate risk of melanoma and nor do those influence the disease prognosis in the investigated population.     

ROCK1 is a potential combinatorial drug target for BRAF mutant melanoma

Treatment of BRAF mutant melanomas with specific BRAF inhibitors leads to tumor remission. However, most patients eventually relapse due to drug resistance. Therefore, we designed an integrated strategy using (phospho)proteomic and functional genomic platforms to identify drug targets whose inhibition sensitizes melanoma cells to BRAF inhibition. We found many proteins to be induced upon PLX4720 (BRAF inhibitor) treatment that are known to be involved in BRAF inhibitor resistance, including FOXD3 and ErbB3. Several proteins were down‐regulated, including Rnd3, a negative regulator of ROCK1 kinase. For our genomic approach, we performed two parallel shRNA screens using a kinome library to identify genes whose inhibition sensitizes to BRAF or ERK inhibitor treatment. By integrating our functional genomic and (phospho)proteomic data, we identified ROCK1 as a potential drug target for BRAF mutant melanoma. ROCK1 silencing increased melanoma cell elimination when combined with BRAF or ERK inhibitor treatment. Translating this to a preclinical setting, a ROCK inhibitor showed augmented melanoma cell death upon BRAF or ERK inhibition in vitro. These data merit exploration of ROCK1 as a target in combination with current BRAF mutant melanoma therapies.     

Prenylation Inhibition-Induced Cell Death in Melanoma: Reduced Sensitivity in BRAF Mutant/PTEN Wild-Type Melanoma Cells

While targeted therapy brought a new era in the treatment of BRAF mutant melanoma, therapeutic options for non-BRAF mutant cases are still limited. In order to explore the antitumor activity of prenylation inhibition we investigated the response to zoledronic acid treatment in thirteen human melanoma cell lines with known BRAF, NRAS and PTEN mutational status. Effect of zoledronic acid on proliferation, clonogenic potential, apoptosis and migration of melanoma cells as well as the activation of downstream elements of the RAS/RAF pathway were investigated in vitro with SRB, TUNEL and PARP cleavage assays and videomicroscopy and immunoblot measurements, respectively. Subcutaneous and spleen-to-liver colonization xenograft mouse models were used to evaluate the influence of zoledronic acid treatment on primary and disseminated tumor growth of melanoma cells in vivo. Zoledronic acid more efficiently decreased short-term in vitro viability in NRAS mutant cells when compared to BRAF mutant and BRAF/NRAS wild-type cells. In line with this finding, following treatment decreased activation of ribosomal protein S6 was found in NRAS mutant cells. Zoledronic acid demonstrated no significant synergism in cell viability inhibition or apoptosis induction with cisplatin or DTIC treatment in vitro. Importantly, zoledronic acid could inhibit clonogenic growth in the majority of melanoma cell lines except in the three BRAF mutant but PTEN wild-type melanoma lines. A similar pattern was observed in apoptosis induction experiments. In vivo zoledronic acid did not inhibit the subcutaneous growth or spleen-to-liver colonization of melanoma cells. Altogether our data demonstrates that prenylation inhibition may be a novel therapeutic approach in NRAS mutant melanoma. Nevertheless, we also demonstrated that therapeutic sensitivity might be influenced by the PTEN status of BRAF mutant melanoma cells. However, further investigations are needed to identify drugs that have appropriate pharmacological properties to efficiently target prenylation in melanoma cells.     

The broad‐spectrum receptor tyrosine kinase inhibitor dovitinib suppresses growth of BRAF‐mutant melanoma cells in combination with other signaling pathway inhibitors

BRAF inhibitors have revolutionized treatment of mutant BRAF metastatic melanomas. However, resistance develops rapidly following BRAF inhibitor treatment. We have found that BRAF‐mutant melanoma cell lines are more sensitive than wild‐type BRAF cells to the small molecule tyrosine kinase inhibitor dovitinib. Sensitivity is associated with inhibition of a series of known dovitinib targets. Dovitinib in combination with several agents inhibits growth more effectively than either agent alone. These combinations inhibit BRAF‐mutant melanoma and colorectal carcinoma cell lines, including cell lines with intrinsic or selected BRAF inhibitor resistance. Hence, combinations of dovitinib with second agents are potentially effective therapies for BRAF‐mutant melanomas, regardless of their sensitivity to BRAF inhibitors.