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
Repeat tumor biopsies to study genomic changes during therapy are difficult, invasive and data are confounded by tumoral heterogeneity. The analysis of circulating tumor DNA (ctDNA) can provide a non-invasive approach to assess prognosis and the genetic evolution of tumors in response to therapy. Mutation-specific droplet digital PCR was used to measure plasma concentrations of oncogenic BRAF and NRAS variants in 48 patients with advanced metastatic melanoma prior to treatment with targeted therapies (vemurafenib, dabrafenib or dabrafenib/trametinib combination) or immunotherapies (ipilimumab, nivolumab or pembrolizumab). Baseline ctDNA levels were evaluated relative to treatment response and progression-free survival (PFS). Tumor-associated ctDNA was detected in the plasma of 35/48 (73%) patients prior to treatment and lower ctDNA levels at this time point were significantly associated with response to treatment and prolonged PFS, irrespective of therapy type. Levels of ctDNA decreased significantly in patients treated with MAPK inhibitors (p < 0.001) in accordance with response to therapy, but this was not apparent in patients receiving immunotherapies. We show that circulating NRAS mutations, known to confer resistance to BRAF inhibitors, were detected in 3 of 7 (43%) patients progressing on kinase inhibitor therapy. Significantly, ctDNA rebound and circulating mutant NRAS preceded radiological detection of progressive disease. Our data demonstrate that ctDNA is a useful biomarker of response to kinase inhibitor therapy and can be used to monitor tumor evolution and detect the early appearance of resistance effectors.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , ADN de Neoplasias/sangre , Melanoma/sangre , Melanoma/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , ADN de Neoplasias/genética , Resistencia a Antineoplásicos/genética , Femenino , GTP Fosfohidrolasas/genética , Humanos , Estimación de Kaplan-Meier , Masculino , Melanoma/genética , Proteínas de la Membrana/genética , Persona de Mediana Edad , Terapia Molecular Dirigida/métodos , Mutación , Metástasis de la Neoplasia , Evaluación de Resultado en la Atención de Salud/métodos , Evaluación de Resultado en la Atención de Salud/estadística & datos numéricos , Pronóstico , Modelos de Riesgos Proporcionales , Proteínas Proto-Oncogénicas B-raf/genética , Adulto JovenRESUMEN
BACKGROUND: MEK1 mutations in melanoma can confer resistance to BRAF inhibitors, although preexisting MEK1(P124) mutations do not preclude clinical responses. We sought to determine whether recurrent, preexisting MEK1(P124) mutations affected clinical outcome in BRAF inhibitor-treated patients with melanoma. METHODS: Data from four published datasets were analyzed to determine whether preexisting MEK1(P124) mutations affect radiologic response or progression-free survival (PFS) in patients with BRAF(V600)-mutant metastatic melanoma treated with vemurafenib or dabrafenib. The effects of MEK1(P124) mutations on MAPK pathway activity and response to BRAF inhibition were also investigated in a series of cell models. RESULTS: In a pooled analysis of 123 patients, the presence of a pretreatment MEK1(P124) mutation (N = 12, 10%) was associated with a poorer RECIST response (33% vs. 72% in MEK1(P124Q/S) vs. MEK1(P124) wild-type, P = 0.018), and a shorter PFS (median 3.1 vs. 4.8 months, P = 0.004). Furthermore, MEK1(P124Q/S) mutations were shown to have independent kinase activity and introduction of these mutations into a BRAF-mutant melanoma cell line diminished inhibition of ERK phosphorylation by dabrafenib and enhanced clonogenic survival in the presence of dabrafenib compared with cells ectopically expressing wild-type MEK1. Consistent with these data, two BRAF-mutant cell lines with endogenous MEK1(P124) mutations showed intermediate sensitivity to dabrafenib, but were highly sensitive to downstream inhibition of MEK or ERK. CONCLUSION: Taken together, our data indicate that preexisting MEK1(P124) mutations are associated with a reduced response to BRAF inhibitor therapy and identify a subset of patients with BRAF-mutant melanoma likely to benefit from combination therapies involving MEK or ERK inhibitors.
Asunto(s)
Resistencia a Antineoplásicos/genética , MAP Quinasa Quinasa 1/genética , Melanoma/genética , Proteínas Proto-Oncogénicas B-raf/genética , Supervivencia sin Enfermedad , Humanos , Imidazoles/administración & dosificación , Indoles/administración & dosificación , Melanoma/tratamiento farmacológico , Melanoma/patología , Mutación , Oximas/administración & dosificación , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Sulfonamidas/administración & dosificación , VemurafenibRESUMEN
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 , VemurafenibRESUMEN
Cancer cells commonly undergo chronic endoplasmic reticulum (ER) stress, to which the cells have to adapt for survival and proliferation. We report here that in melanoma cells intrinsic activation of the ER stress response/unfolded protein response (UPR) is, at least in part, caused by increased outputs of protein synthesis driven by oncogenic activation of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) and promotes proliferation and protects against apoptosis induced by acute ER stress. Inhibition of oncogenic BRAF(V600E) or MEK-attenuated activation of inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6) signaling of the UPR in melanoma cells. This was associated with decreased phosphorylation of eukaryotic initiation factor 4E (eIF4E) and nascent protein synthesis and was recapitulated by knockdown of eIF4E. In line with this, introduction of BRAF(V600E) into melanocytes led to increases in eIF4E phosphorylation and protein production and triggered activation of the UPR. Similar to knockdown of glucose-regulated protein 78 (GRP78), inhibition of XBP1 decelerated melanoma cell proliferation and enhanced apoptosis induced by the pharmacological ER stress inducers tunicamycin and thapasigargin. Collectively, these results reveal that potentiation of adaptation to chronic ER stress is another mechanism by which oncogenic activation of the MEK/ERK pathway promotes the pathogenesis of melanoma.
Asunto(s)
Adaptación Fisiológica/fisiología , Estrés del Retículo Endoplásmico/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Melanoma/metabolismo , Neoplasias Cutáneas/metabolismo , Factor de Transcripción Activador 6/metabolismo , Apoptosis/fisiología , Línea Celular Tumoral , Supervivencia Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Chaperón BiP del Retículo Endoplásmico , Endorribonucleasas/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Indoles/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Melanocitos/metabolismo , Melanocitos/patología , Melanoma/patología , Proteínas de la Membrana/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/metabolismo , Factores de Transcripción del Factor Regulador X , Neoplasias Cutáneas/patología , Sulfonamidas/farmacología , Factores de Transcripción/metabolismo , Proteína 1 de Unión a la X-BoxRESUMEN
Approximately 50% of melanomas require oncogenic B-RAF(V600E) signaling for proliferation, survival, and metastasis, and the use of highly selective B-RAF inhibitors has yielded remarkable, although short-term, clinical responses. Reactivation of signaling downstream of B-RAF is frequently associated with acquired resistance to B-RAF inhibitors, and the identification of B-RAF targets may therefore provide new strategies for managing melanoma. In this report, we applied whole-genome expression analyses to reveal that oncogenic B-RAF(V600E) regulates genes associated with epithelial-mesenchymal transition in normal cutaneous human melanocytes. Most prominent was the B-RAF-mediated transcriptional repression of E-cadherin, a keratinocyte-melanoma adhesion molecule whose loss is intimately associated with melanoma invasion and metastasis. Here we identify a link between oncogenic B-RAF, the transcriptional repressor Tbx3, and E-cadherin. We show that B-RAF(V600E) induces the expression of Tbx3, which potently represses E-cadherin expression in melanocytes and melanoma cells. Tbx3 expression is normally restricted to developmental embryonic tissues and promoting cell motility, but it is also aberrantly increased in various cancers and has been linked to tumor cell invasion and metastasis. We propose that this B-RAF/Tbx3/E-cadherin pathway has a critical role in promoting the metastasis of B-RAF-mutant melanomas.