RESUMEN

Overdiagnosis of early melanoma is a significant problem. Due to subtle unique and overlapping clinical and histological criteria between pigmented lesions and the risk of mortality from melanoma, some benign pigmented lesions are diagnosed as melanoma. Although histopathology is the gold standard to diagnose melanoma, there is a demand to find alternatives that are more accurate and cost-effective. In the current "omics" era, there is gaining interest in biomarkers to help diagnose melanoma early and to further understand the mechanisms driving tumor progression. Genomic investigations have attempted to differentiate malignant melanoma from benign pigmented lesions. However, genetic biomarkers of early melanoma diagnosis have not yet proven their value in the clinical setting. Protein biomarkers may be more promising since they directly influence tissue phenotype, a result of by-products of genomic mutations, posttranslational modifications and environmental factors. Uncovering relevant protein biomarkers could increase confidence in their use as diagnostic signatures. Currently, proteomic investigations of melanoma progression from pigmented lesions are limited. Studies have previously characterised the melanoma proteome from cultured cell lines and clinical samples such as serum and tissue. This has been useful in understanding how melanoma progresses into metastasis and development of resistance to adjuvant therapies. Currently, most studies focus on metastatic melanoma to find potential drug therapy targets, prognostic factors and markers of resistance. This paper reviews recent advancements in the genomics and proteomic fields and reports potential avenues, which could help identify and differentiate melanoma from benign pigmented lesions and prevent the progression of melanoma.

Asunto(s)

Melanoma , Neoplasias Cutáneas , Humanos , Proteómica , Melanoma/metabolismo , Neoplasias Cutáneas/patología , Genómica , Biomarcadores , Diagnóstico Precoz

RESUMEN

In this modern era of precision medicine, molecular signatures identified from advanced omics technologies hold great promise to better guide clinical decisions. However, current approaches are often location-specific due to the inherent differences between platforms and across multiple centres, thus limiting the transferability of molecular signatures. We present Cross-Platform Omics Prediction (CPOP), a penalised regression model that can use omics data to predict patient outcomes in a platform-independent manner and across time and experiments. CPOP improves on the traditional prediction framework of using gene-based features by selecting ratio-based features with similar estimated effect sizes. These components gave CPOP the ability to have a stable performance across datasets of similar biology, minimising the effect of technical noise often generated by omics platforms. We present a comprehensive evaluation using melanoma transcriptomics data to demonstrate its potential to be used as a critical part of a clinical screening framework for precision medicine. Additional assessment of generalisation was demonstrated with ovarian cancer and inflammatory bowel disease studies.

RESUMEN

Purpose: To examine the relationship between immune activity, PD-L1 expression, and tumor cell signaling, in metastatic melanomas prior to and during treatment with targeted MAPK inhibitors.Experimental Design: Thirty-eight tumors from 17 patients treated with BRAF inhibitor (n = 12) or combination BRAF/MEK inhibitors (n = 5) with known PD-L1 expression were analyzed. RNA expression arrays were performed on all pretreatment (PRE, n = 17), early during treatment (EDT, n = 8), and progression (PROG, n = 13) biopsies. HLA-A/HLA-DPB1 expression was assessed by IHC.Results: Gene set enrichment analysis (GSEA) of PRE, EDT, and PROG melanomas revealed that transcriptome signatures indicative of immune cell activation were strongly positively correlated with PD-L1 staining. In contrast, MAPK signaling and canonical Wnt/-ß-catenin activity was negatively associated with PD-L1 melanoma expression. The expression of PD-L1 and immune activation signatures did not simply reflect the degree or type of immune cell infiltration, and was not sufficient for tumor response to MAPK inhibition.Conclusions: PD-L1 expression correlates with immune cells and immune activity signatures in melanoma, but is not sufficient for tumor response to MAPK inhibition, as many PRE and PROG melanomas displayed both PD-L1 positivity and immune activation signatures. This confirms that immune escape is common in MAPK inhibitor-treated tumors. This has important implications for the selection of second-line immunotherapy because analysis of mechanisms of immune escape will likely be required to identify patients likely to respond to such therapies. Clin Cancer Res; 23(20); 6054-61. ©2017 AACR.

Asunto(s)

Antígeno B7-H1/genética , Resistencia a Antineoplásicos/genética , Expresión Génica , Melanoma/genética , Melanoma/inmunología , Inhibidores de Proteínas Quinasas/farmacología , Escape del Tumor/genética , Escape del Tumor/inmunología , Adulto , Anciano , Antígeno B7-H1/metabolismo , Biopsia , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Masculino , Melanoma/tratamiento farmacológico , Melanoma/patología , Persona de Mediana Edad , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Mutación , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Transducción de Señal/efectos de los fármacos , Escape del Tumor/efectos de los fármacos

RESUMEN

Melanoma of the skin is a common cancer only in Europeans, whereas it arises in internal body surfaces (mucosal sites) and on the hands and feet (acral sites) in people throughout the world. Here we report analysis of whole-genome sequences from cutaneous, acral and mucosal subtypes of melanoma. The heavily mutated landscape of coding and non-coding mutations in cutaneous melanoma resolved novel signatures of mutagenesis attributable to ultraviolet radiation. However, acral and mucosal melanomas were dominated by structural changes and mutation signatures of unknown aetiology, not previously identified in melanoma. The number of genes affected by recurrent mutations disrupting non-coding sequences was similar to that affected by recurrent mutations to coding sequences. Significantly mutated genes included BRAF, CDKN2A, NRAS and TP53 in cutaneous melanoma, BRAF, NRAS and NF1 in acral melanoma and SF3B1 in mucosal melanoma. Mutations affecting the TERT promoter were the most frequent of all; however, neither they nor ATRX mutations, which correlate with alternative telomere lengthening, were associated with greater telomere length. Most melanomas had potentially actionable mutations, most in components of the mitogen-activated protein kinase and phosphoinositol kinase pathways. The whole-genome mutation landscape of melanoma reveals diverse carcinogenic processes across its subtypes, some unrelated to sun exposure, and extends potential involvement of the non-coding genome in its pathogenesis.

Asunto(s)

Genoma Humano/genética , Melanoma/genética , Mutación/genética , ADN Helicasas/genética , GTP Fosfohidrolasas/genética , Genes p16 , Humanos , Melanoma/clasificación , Proteínas de la Membrana/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Neurofibromatosis 1/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteínas Proto-Oncogénicas B-raf/genética , Factores de Empalme de ARN/genética , Transducción de Señal/efectos de los fármacos , Telomerasa/genética , Telómero/genética , Proteína p53 Supresora de Tumor/genética , Rayos Ultravioleta/efectos adversos , Proteína Nuclear Ligada al Cromosoma X

RESUMEN

Alternate BRAF splicing is the most common mechanism of acquired resistance to BRAF inhibitor treatment in melanoma. Recently, alternate BRAF exon 4-8 splicing was shown to involve an intronic mutation, located 51 nucleotides upstream of BRAF exon 9 within a predicted splicing branch point. This intronic mutation was identified in a single cell line but has not been examined in vivo. Herein we demonstrate that in three melanomas biopsied from patients with acquired resistance to BRAF inhibitors, alternate BRAF exon 4-8 splicing is not associated with this intronic branch point mutation. We also confirm that melanoma cells expressing BRAF splicing variants retain exquisite sensitivity to existing FDA-approved MEK inhibitors.

RESUMEN

The identification of recurrent driver mutations by whole-exome sequencing (WES) of fresh-frozen human cancers and the subsequent development of novel targeted therapies have recently transformed the treatment of many cancers including melanoma. In routine clinical practice, fresh-frozen tissue is rarely available and mutation testing usually needs to be carried out on archival formalin fixed, paraffin embedded (FFPE) tissue, from which DNA is typically fragmented, cross-linked and of lower quality. In this study we aimed to determine whether WES data generated from genomic DNA (gDNA) extracted from FFPE tissues can be produced reliably and of clinically-actionable standard. In this study of ten melanoma patients, we compared WES data produced from analysis of gDNA isolated from FFPE tumour tissue with that isolated from fresh-frozen tumour tissue from the same specimen. FFPE samples were sequenced using both Illumina's Nextera and NimbleGen SeqCap exome capture kits. To examine mutations between the two tissue sources and platforms, somatic mutations in the FFPE exomes were called using the matched fresh tissue sequence as a reference. Of the 10 FFPE DNA samples, seven Nextera and four SeqCap samples passed library preparation. On average, there were 5341 and 2246 variants lost in FFPE compared to matched fresh tissue utilising Nextera and SeqCap kits, respectively. In order to explore the feasibility of future clinical implementation of WES, FFPE variants in 27 genes of important clinical relevance in melanoma were assessed. The average concordance rate was 43.2% over a total of 1299 calls for the chosen genes in the FFPE DNA. For the current clinically most important melanoma mutations, 0/3 BRAF and 6/8 (75%) NRAS FFPE calls were concordant with the fresh tissue result, which was confirmed using a Sequenom OncoCarta Panel. The poor performance of FFPE WES indicates that specialised library construction to account for low quality DNA and further refinements will be necessary before this approach could be used for routine clinical decision making over currently preferred techniques.

Asunto(s)

Toma de Decisiones Clínicas , ADN de Neoplasias/química , Exoma/genética , Melanoma/diagnóstico , Australia , ADN de Neoplasias/genética , Formaldehído , Secciones por Congelación , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Melanoma/genética , Melanoma/patología , Mutación , Adhesión en Parafina , Análisis de Secuencia de ADN

RESUMEN

BACKGROUND: Acquired resistance to BRAF inhibitors (BRAFi) is a near-universal phenomenon caused by numerous genetic and non-genetic alterations. In this study, we evaluated the spectrum, onset, pattern of progression, and subsequent clinical outcomes associated with specific mechanisms of resistance. METHODS: We compiled clinical and genetic data from 100 patients with 132 tissue samples obtained at progression on BRAFi therapy from 3 large, previously published studies of BRAFi resistance. These samples were subjected to whole-exome sequencing and/or polymerase chain reaction-based genetic testing. RESULTS: Among 132 samples, putative resistance mechanisms were identified in 58%, including NRAS or KRAS mutations (20%), BRAF splice variants (16%), BRAF(V600E/K) amplifications (13%), MEK1/2 mutations (7%), and non-mitogen-activated protein kinase pathway alterations (11%). Marked heterogeneity was observed within tumors and patients; 18 of 19 patients (95%) with more than one progression biopsy had distinct/unknown drivers of resistance between samples. NRAS mutations were associated with vemurafenib use (p = 0.045) and intracranial metastases (p = 0.036), and MEK1/2 mutations correlated with hepatic progression (p = 0.011). Progression-free survival and overall survival were similar across resistance mechanisms. The median survival after disease progression was 6.9 months, and responses to subsequent BRAF and MEK inhibition were uncommon (2 of 15; 13%). Post-progression outcomes did not correlate with specific acquired BRAFi-resistance mechanisms. CONCLUSIONS: This is the first study to systematically characterise the clinical implications of particular acquired BRAFi-resistance mechanisms in patients with BRAF-mutant melanoma largest study to compile the landscape of resistance. Despite marked heterogeneity of resistance mechanisms within patients, NRAS mutations correlated with vemurafenib use and intracranial disease involvement.

Asunto(s)

Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/antagonistas & inhibidores , Resistencia a Antineoplásicos , Melanoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Neoplasias Cutáneas/tratamiento farmacológico , Australia , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Análisis Mutacional de ADN , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Resistencia a Antineoplásicos/genética , Europa (Continente) , Femenino , Predisposición Genética a la Enfermedad , Humanos , Estimación de Kaplan-Meier , Masculino , Melanoma/enzimología , Melanoma/genética , Melanoma/mortalidad , Melanoma/patología , Persona de Mediana Edad , Mutación , Fenotipo , Modelos de Riesgos Proporcionales , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Factores de Riesgo , Transducción de Señal/efectos de los fármacos , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/mortalidad , Neoplasias Cutáneas/patología , Factores de Tiempo , Resultado del Tratamiento , Estados Unidos

RESUMEN

Whole genome sequencing (WGS) of cancer patients' tumours offers the most comprehensive method of identifying both novel and known clinically-actionable genomic targets. However, the practicalities of performing WGS on clinical samples are poorly defined.This study was designed to test sample preparation, sequencing specifications and bioinformatic algorithms for their effect on accuracy and cost-efficiency in a large WGS analysis of human melanoma samples.WGS was performed on melanoma cell lines (n = 15) and melanoma fresh frozen tumours (n = 222). The appropriate level of coverage and the optimal mutation detection algorithm for the project pipeline were determined.An incremental increase in sequencing coverage from 36X to 132X in melanoma tissue samples and 30X to 103X for cell lines only resulted in a small increase (1-2%) in the number of mutations detected, and the quality scores of the additional mutations indicated a low probability that the mutations were real. The results suggest that 60X coverage for melanoma tissue and 40X for melanoma cell lines empower the detection of 98-99% of informative single nucleotide variants (SNVs), a sensitivity level at which clinical decision making or landscape research projects can be carried out with a high degree of confidence in the results. Likewise the bioinformatic mutation analysis methodology strongly influenced the number and quality of SNVs detected. Detecting mutations in the blood genomes separate to the tumour genomes generated 41% more SNVs than if the blood and melanoma tissue genomes were analysed simultaneously. Therefore, simultaneous analysis should be employed on matched melanoma tissue and blood genomes to reduce errors in mutation detection.This study provided valuable insights into the accuracy of SNV with WGS at various coverage levels in human clinical cancer specimens. Additionally, we investigated the accuracy of the publicly available mutation detection algorithms to detect cancer specific SNVs which will aid researchers and clinicians in study design and implementation of WGS for the identification of somatic mutations in other cancers.

Asunto(s)

Algoritmos , Análisis Mutacional de ADN/métodos , ADN de Neoplasias/aislamiento & purificación , Melanoma/genética , Manejo de Especímenes/métodos , Análisis Mutacional de ADN/economía , ADN de Neoplasias/análisis , Secuenciación de Nucleótidos de Alto Rendimiento/economía , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Mutación , Análisis de Secuencia de ADN/economía , Análisis de Secuencia de ADN/métodos

RESUMEN

The epigenetic modifier EZH2 is part of the polycomb repressive complex that suppresses gene expression via histone methylation. Activating mutations in EZH2 are found in a subset of melanoma that contributes to disease progression by inactivating tumor suppressor genes. In this study we have targeted EZH2 with a specific inhibitor (GSK126) or depleted EZH2 protein by stable shRNA knockdown. We show that inhibition of EZH2 has potent effects on the growth of both wild-type and EZH2 mutant human melanoma in vitro particularly in cell lines harboring the EZH2Y646 activating mutation. This was associated with cell cycle arrest, reduced proliferative capacity in both 2D and 3D culture systems, and induction of apoptosis. The latter was caspase independent and mediated by the release of apoptosis inducing factor (AIFM1) from mitochondria. Gene expression arrays showed that several well characterized tumor suppressor genes were reactivated by EZH2 inhibition. This included activating transcription factor 3 (ATF3) that was validated as an EZH2 target gene by ChIP-qPCR. These results emphasize a critical role for EZH2 in the proliferation and viability of melanoma and highlight the potential for targeted therapy against EZH2 in treatment of patients with melanoma.

Asunto(s)

Genes Supresores de Tumor , Melanoma/genética , Melanoma/metabolismo , Complejo Represivo Polycomb 2/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Factor de Transcripción Activador 3/genética , Apoptosis , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Progresión de la Enfermedad , Proteína Potenciadora del Homólogo Zeste 2 , Epigenómica , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Genotipo , Histonas/química , Humanos , Indoles/química , Concentración 50 Inhibidora , Mutación , Piridonas/química , Regulación hacia Arriba

RESUMEN

One-third of BRAF-mutant metastatic melanoma patients treated with combined BRAF and MEK inhibition progress within 6 months. Treatment options for these patients remain limited. Here we analyse 20 BRAF(V600)-mutant melanoma metastases derived from 10 patients treated with the combination of dabrafenib and trametinib for resistance mechanisms and genetic correlates of response. Resistance mechanisms are identified in 9/11 progressing tumours and MAPK reactivation occurred in 9/10 tumours, commonly via BRAF amplification and mutations activating NRAS and MEK2. Our data confirming that MEK2(C125S), but not the synonymous MEK1(C121S) protein, confers resistance to combination therapy highlight the functional differences between these kinases and the preponderance of MEK2 mutations in combination therapy-resistant melanomas. Exome sequencing did not identify additional progression-specific resistance candidates. Nevertheless, most melanomas carried additional oncogenic mutations at baseline (for example, RAC1 and AKT3) that activate the MAPK and PI3K pathways and are thus predicted to diminish response to MAPK inhibitors.

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Resistencia a Antineoplásicos/genética , GTP Fosfohidrolasas/genética , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 2/genética , Melanoma/tratamiento farmacológico , Proteínas de la Membrana/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias Cutáneas/tratamiento farmacológico , Humanos , Imidazoles/administración & dosificación , Melanoma/genética , Melanoma/secundario , Oximas/administración & dosificación , Proteínas Proto-Oncogénicas c-akt/genética , Piridonas/administración & dosificación , Pirimidinonas/administración & dosificación , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Proteína de Unión al GTP rac1/genética

RESUMEN

Metabolic heterogeneity is a key factor in cancer pathogenesis. We found that a subset of BRAF- and NRAS-mutant human melanomas resistant to the MEK inhibitor selumetinib displayed increased oxidative phosphorylation (OxPhos) mediated by the transcriptional coactivator PGC1α. Notably, all selumetinib-resistant cells with elevated OxPhos could be resensitized by cotreatment with the mTORC1/2 inhibitor AZD8055, whereas this combination was ineffective in resistant cell lines with low OxPhos. In both BRAF- and NRAS-mutant melanoma cells, MEK inhibition increased MITF expression, which in turn elevated levels of PGC1α. In contrast, mTORC1/2 inhibition triggered cytoplasmic localization of MITF, decreasing PGC1α expression and inhibiting OxPhos. Analysis of tumor biopsies from patients with BRAF-mutant melanoma progressing on BRAF inhibitor ± MEK inhibitor revealed that PGC1α levels were elevated in approximately half of the resistant tumors. Overall, our findings highlight the significance of OxPhos in melanoma and suggest that combined targeting of the MAPK and mTORC pathways may offer an effective therapeutic strategy to treat melanomas with this metabolic phenotype.

Asunto(s)

Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Melanoma/tratamiento farmacológico , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Complejos Multiproteicos/antagonistas & inhibidores , Fosforilación Oxidativa/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Línea Celular Tumoral , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Melanoma/genética , Melanoma/metabolismo , Factor de Transcripción Asociado a Microftalmía/genética , Factor de Transcripción Asociado a Microftalmía/metabolismo , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Outcomes for melanoma patients with stage III disease differ widely even within the same subcategory. Molecular signatures that more accurately predict prognosis are needed to stratify patients according to risk. Proteomic analyses were used to identify differentially abundant proteins in extracts of surgically excised samples from patients with stage IIIc melanoma lymph node metastases. Analysis of samples from patients with poor (n = 14, <1 yr) and good (n = 19, >4 yr) survival outcomes identified 84 proteins that were differentially abundant between prognostic groups. Subsequent selected reaction monitoring analysis verified 21 proteins as potential biomarkers for survival. Poor prognosis patients are characterized by increased levels of proteins involved in protein metabolism, nucleic acid metabolism, angiogenesis, deregulation of cellular energetics and methylation processes, and decreased levels of proteins involved in apoptosis and immune response. These proteins are able to classify stage IIIc patients into prognostic subgroups (P < 0.02). This is the first report of potential prognostic markers from stage III melanoma using proteomic analyses. Validation of these protein markers in larger patient cohorts should define protein signatures that enable better stratification of stage III melanoma patients.

Asunto(s)

Estimación de Kaplan-Meier , Melanoma/metabolismo , Melanoma/patología , Proteínas de Neoplasias/metabolismo , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Análisis por Conglomerados , Electroforesis en Gel Bidimensional , Redes Reguladoras de Genes , Humanos , Marcaje Isotópico , Estadificación de Neoplasias , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem

RESUMEN

The transcription factor NF-kappaB (NF-kB) is a key regulator of cytokine and chemokine production in melanoma and is responsible for symptoms such as anorexia, fatigue, and weight loss. In addition, NF-kB is believed to contribute to progression of the disease by upregulation of cell cycle and anti-apoptotic genes and to contribute to resistance against targeted therapies and immunotherapy. In this study, we have examined the ability of the bromodomain and extra-terminal (BET) protein inhibitor I-BET151 to inhibit NF-kB in melanoma cells. We show that I-BET151 is a potent, selective inhibitor of a number of NF-kB target genes involved in induction of inflammation and cell cycle regulation and downregulates production of cytokines such as IL-6 and IL-8. SiRNA studies indicate that BRD2 is the main BET protein involved in regulation of NF-kB and that I-BET151 caused transcriptional downregulation of the NF-kB subunit p105/p50. These results suggest that BET inhibitors may have an important role in treatment of melanoma where activation of NF-kB may have a key pathogenic role.

Asunto(s)

Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Melanoma/metabolismo , FN-kappa B/metabolismo , Neoplasias Cutáneas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Comunicación Autocrina/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quimiocinas/biosíntesis , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Melanoma/genética , Melanoma/patología , Ratones , Proteínas Serina-Treonina Quinasas/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Factores de Transcripción

RESUMEN

Epigenetic changes are widespread in melanoma and contribute to the pathogenic biology of this disease. In the present study, we show that I-BET151, which belongs to a new class of drugs that target the BET family of epigenetic "reader" proteins, inhibits melanoma growth in vivo and induced variable degrees of apoptosis in a panel of melanoma cells. Apoptosis was caspase dependent and associated with G1 cell cycle arrest. All melanoma cells tested had increased levels of the BH3 proapoptotic protein BIM, which appeared to be regulated by the BRD2 BET protein and to some extent by BRD3. In contrast, knockdown experiments indicated that inhibition of BRD4 was associated with decreased levels of BIM. Apoptosis was dependent on BIM in some but not all cell lines, indicating that other factors were determinants of apoptosis, such as downregulation of antiapoptotic proteins revealed in gene expression arrays. G1 cell cycle arrest appeared to be mediated by p21 and resulted from inhibition of the BRD4 protein. The activity of BET protein inhibitors appears independent of the BRAF and NRAS mutational status of melanoma, and further studies to assess their therapeutic role in melanoma are warranted.

Asunto(s)

Proteínas Reguladoras de la Apoptosis/metabolismo , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Compuestos Heterocíclicos de 4 o más Anillos/química , Melanoma/genética , Melanoma/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Apoptosis , Proteína 11 Similar a Bcl2 , Caspasas/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular , Línea Celular , Línea Celular Tumoral , Supervivencia Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Análisis Mutacional de ADN , Femenino , GTP Fosfohidrolasas/metabolismo , Humanos , Melanoma/tratamiento farmacológico , Ratones , Ratones Endogámicos NOD , Proteínas de Unión al GTP Monoméricas/metabolismo , Trasplante de Neoplasias , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Factores de Transcripción/metabolismo , Transcriptoma

RESUMEN

Acquired resistance to BRAF inhibitors often involves MAPK re-activation, yet the MEK inhibitor trametinib showed minimal clinical activity in melanoma patients that had progressed on BRAF-inhibitor therapy. Selective ERK inhibitors have been proposed as alternative salvage therapies. We show that ERK inhibition is more potent than MEK inhibition at suppressing MAPK activity and inhibiting the proliferation of multiple BRAF inhibitor resistant melanoma cell models. Nevertheless, melanoma cells often failed to undergo apoptosis in response to ERK inhibition, because the relief of ERK-dependent negative feedback activated RAS and PI3K signalling. Consequently, the combination of ERK and PI3K/mTOR inhibition was effective at promoting cell death in all resistant melanoma cell models, and was substantially more potent than the MEK/PI3K/mTOR inhibitor combination. Our data indicate that a broader targeting strategy concurrently inhibiting ERK, rather than MEK, and PI3K/mTOR may circumvent BRAF inhibitor resistance, and should be considered during the clinical development of ERK inhibitors.

Asunto(s)

Resistencia a Antineoplásicos , Melanoma/tratamiento farmacológico , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Humanos , Melanoma/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas B-raf/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Células Tumorales Cultivadas

RESUMEN

UNLABELLED: Deregulated glucose metabolism fulfills the energetic and biosynthetic requirements for tumor growth driven by oncogenes. Because inhibition of oncogenic BRAF causes profound reductions in glucose uptake and a strong clinical benefit in BRAF-mutant melanoma, we examined the role of energy metabolism in responses to BRAF inhibition. We observed pronounced and consistent decreases in glycolytic activity in BRAF-mutant melanoma cells. Moreover, we identified a network of BRAF-regulated transcription factors that control glycolysis in melanoma cells. Remarkably, this network of transcription factors, including hypoxia-inducible factor-1α, MYC, and MONDOA (MLXIP), drives glycolysis downstream of BRAF(V600), is critical for responses to BRAF inhibition, and is modulated by BRAF inhibition in clinical melanoma specimens. Furthermore, we show that concurrent inhibition of BRAF and glycolysis induces cell death in BRAF inhibitor (BRAFi)-resistant melanoma cells. Thus, we provide a proof-of-principle for treatment of melanoma with combinations of BRAFis and glycolysis inhibitors. SIGNIFICANCE: BRAF is suppress glycolysis and provide strong clinical benefi t in BRAF V600 melanoma. We show that BRAF inhibition suppresses glycolysis via a network of transcription factors that are critical for complete BRAFi responses. Furthermore, we provide evidence for the clinical potential of therapies that combine BRAFis with glycolysis inhibitors.

Asunto(s)

Glucólisis/efectos de los fármacos , Melanoma/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Células HEK293 , Humanos , Indoles/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Melanoma/patología , Piperazinas/farmacología , Piridinas/farmacología , Sulfonamidas/farmacología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Vemurafenib

RESUMEN

PURPOSE: Multiple BRAF inhibitor resistance mechanisms have been described, however, their relative frequency, clinical correlates, and effect on subsequent therapy have not been assessed in patients with metastatic melanoma. EXPERIMENTAL DESIGN: Fifty-nine BRAF(V600)-mutant melanoma metastases from patients treated with dabrafenib or vemurafenib were analyzed. The genetic profile of resistance mechanisms and tumor signaling pathway activity was correlated with clinicopathologic features and therapeutic outcomes. RESULTS: Resistance mechanisms were identified in 58% progressing tumors and BRAF alterations were common. Gene expression analysis revealed that mitogen-activated protein kinase (MAPK) activity remained inhibited in 21% of resistant tumors, and the outcomes of patients with these tumors were poor. Resistance mechanisms also occurred in pretreatment biopsies and heterogeneity of resistance mechanisms occurred within patients and within tumors. There were no responses to subsequent targeted therapy, even when a progressing tumor had a resistance mechanism predicted to be responsive. CONCLUSIONS: Selecting sequential drugs based on the molecular characteristics of a single progressing biopsy is unlikely to provide improved responses, and first-line therapies targeting multiple pathways will be required.

Asunto(s)

Resistencia a Antineoplásicos/genética , Melanoma/tratamiento farmacológico , Quinasas de Proteína Quinasa Activadas por Mitógenos/biosíntesis , Metástasis de la Neoplasia/tratamiento farmacológico , Proteínas Proto-Oncogénicas B-raf/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Imidazoles/administración & dosificación , Indoles/administración & dosificación , Masculino , Melanoma/genética , Melanoma/patología , Persona de Mediana Edad , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Mutación , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Oximas/administración & dosificación , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Sulfonamidas/administración & dosificación , Vemurafenib

RESUMEN

Melanoma of unknown primary (MUP) is an uncommon phenomenon whereby patients present with metastatic disease without an evident primary site. To determine their likely site of origin, we combined exome sequencing from 33 MUPs to assess the total rate of somatic mutations and degree of UV mutagenesis. An independent cohort of 91 archival MUPs was also screened for 46 hot spot mutations highly prevalent in melanoma including BRAF, NRAS, KIT, GNAQ, and GNA11. Results showed that the majority of MUPs exhibited high somatic mutation rates, high ratios of C>T/G>A transitions, and a high rate of BRAF (45 of 101, 45%) and NRAS (32 of 101, 32%) mutations, collectively indicating a mutation profile consistent with cutaneous sun-exposed melanomas. These data suggest that a significant proportion of MUPs arise from regressed or unrecognized primary cutaneous melanomas or arise de novo in lymph nodes from nevus cells that have migrated from the skin.

Asunto(s)

Melanoma/genética , Melanoma/patología , Mutación/genética , Neoplasias Primarias Desconocidas/genética , Neoplasias Primarias Desconocidas/patología , Neoplasias Cutáneas/genética , Luz Solar , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Estudios de Cohortes , Análisis Mutacional de ADN , Exoma/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Cutáneas/patología , Melanoma Cutáneo Maligno

RESUMEN

Inhibitors of the mitogen-activated protein kinases (MAPK), BRAF, and MAP-ERK kinase (MEK) induce tumor regression in the majority of patients with BRAF-mutant metastatic melanoma. The clinical benefit of MAPK inhibitors is restricted by the development of acquired resistance with half of those who benefit having progressed by 6 to 7 months and long-term responders uncommon. There remains no agreed treatment strategy on disease progression in these patients. Without published evidence, fears of accelerated disease progression on inhibitor withdrawal have led to the continuation of drugs beyond formal disease progression. We now show that treatment with MAPK inhibitors beyond disease progression can provide significant clinical benefit, and the withdrawal of these inhibitors led to a marked increase in the rate of disease progression in two patients. We also show that MAPK inhibitors retain partial activity in acquired resistant melanoma by examining drug-resistant clones generated to dabrafenib, trametinib, or the combination of these drugs. All resistant sublines displayed a markedly slower rate of proliferation when exposed to MAPK inhibitors, and this coincided with a reduction in MAPK signaling, decrease in bromodeoxyuridine incorporation, and S-phase inhibition. This cytostatic effect was also associated with diminished levels of cyclin D1 and p-pRb. Two short-term melanoma cultures generated from resistant tumor biopsies also responded to MAPK inhibition, with comparable inhibitory changes in proliferation and MAPK signaling. These data provide a rationale for the continuation of BRAF and MEK inhibitors after disease progression and support the development of clinical trials to examine this strategy.

Asunto(s)

Imidazoles/uso terapéutico , Melanoma/tratamiento farmacológico , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Oximas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Piridonas/uso terapéutico , Pirimidinonas/uso terapéutico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos , Expresión Génica , Humanos , Imidazoles/efectos adversos , Masculino , Melanoma/patología , Persona de Mediana Edad , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Oximas/efectos adversos , Proteínas Proto-Oncogénicas B-raf/metabolismo , Piridonas/efectos adversos , Pirimidinonas/efectos adversos

RESUMEN

Activated oncogenes restrict cell proliferation and transformation by triggering a DNA damage-dependent senescence checkpoint in response to DNA hyper-replication. Here, we show that loss of the p16(INK) (4a) cyclin-dependent kinase inhibitor and melanoma tumour suppressor facilitates a DNA damage response after a hyper-replicative phase in human melanocytes. Unlike cells expressing activated oncogenes, however, melanocytes depleted for p16(INK) (4a) display enhanced proliferation and an extended replicative lifespan in the presence of replication-associated DNA damage. Analysis of human benign naevi confirmed that DNA damage and loss of p16(INK) (4a) expression co-segregate closely. Thus, we propose that loss of p16(INK) (4a) facilitates tumourigenesis by promoting the proliferation of genetically unstable cells.

Asunto(s)

Inhibidor p16 de la Quinasa Dependiente de Ciclina/deficiencia , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Replicación del ADN , Inestabilidad Genómica , Melanocitos/metabolismo , Melanocitos/patología , Puntos de Control del Ciclo Celular/genética , Proliferación Celular , Quinasas Ciclina-Dependientes/metabolismo , Daño del ADN/genética , Células HEK293 , Histonas/metabolismo , Humanos , Melanocitos/enzimología , Nevo/metabolismo , Nevo/patología , Proteína de Retinoblastoma/metabolismo , Proteína p53 Supresora de Tumor/metabolismo