RESUMO
The treatment of melanoma has been markedly improved by the introduction of targeted therapies and checkpoint blockade immunotherapy. Unfortunately, resistance to these therapies remains a limitation. Novel anticancer therapeutics targeting the MCL1 anti-apoptotic protein have shown impressive responses in haematological cancers but are yet to be evaluated in melanoma. To assess the sensitivity of melanoma to new MCL1 inhibitors, we measured the response of 51 melanoma cell lines to the novel MCL1 inhibitor, S63845. Additionally, we assessed combination of this drug with inhibitors of the bromodomain and extra-terminal (BET) protein family of epigenetic readers, which we postulated would assist MCL1 inhibition by downregulating anti-apoptotic targets regulated by NF-kB such as BCLXL, BCL2A1 and XIAP, and by upregulating pro-apoptotic proteins including BIM and NOXA. Only 14% of melanoma cell lines showed sensitivity to S63845, however, combination of S63845 and I-BET151 induced highly synergistic apoptotic cell death in all melanoma lines tested and in an in vivo xenograft model. Cell death was dependent on caspases and BAX/BAK. Although the combination of drugs increased the BH3-only protein, BIM, and downregulated anti-apoptotic proteins such as BCL2A1, the importance of these proteins in inducing cell death varied between cell lines. ABT-199 or ABT-263 inhibitors against BCL2 or BCL2 and BCLXL, respectively, induced further cell death when combined with S63845 and I-BET151. The combination of MCL1 and BET inhibition appears to be a promising therapeutic approach for metastatic melanoma, and presents opportunities to add further BCL2 family inhibitors to overcome treatment resistance.
Assuntos
Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pirimidinas/farmacologia , Tiofenos/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Mutations in BRAF activate oncogenic MAPK signalling in almost half of cutaneous melanomas. Inhibitors of BRAF (BRAFi) and its target MEK are widely used to treat melanoma patients with BRAF mutations but unfortunately acquired resistance occurs in the majority of patients. Resistance results from mutations or non-genomic changes that either reactivate MAPK signalling or activate other pathways that provide alternate survival and growth signalling. Here, we show the histone deacetylase inhibitor (HDACi) panobinostat overcomes BRAFi resistance in melanoma, but this is dependent on the resistant cells showing a partial response to BRAFi treatment. Using patient- and in vivo-derived melanoma cell lines with acquired BRAFi resistance, we show that combined treatment with the BRAFi encorafenib and HDACi panobinostat in 2D and 3D culture systems synergistically induced caspase-dependent apoptotic cell death. Key changes induced by HDAC inhibition included decreased PI3K pathway activity associated with a reduction in the protein level of a number of receptor tyrosine kinases, and cell line dependent upregulation of pro-apoptotic BIM or NOXA together with reduced expression of anti-apoptotic proteins. Independent of these changes, panobinostat reduced c-Myc and pre-treatment of cells with siRNA against c-Myc reduced BRAFi/HDACi drug-induced cell death. These results suggest that a combination of HDAC and MAPK inhibitors may play a role in treatment of melanoma where the resistance mechanisms are due to activation of MAPK-independent pathways.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Melanoma/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Sinergismo Farmacológico , Xenoenxertos , Inibidores de Histona Desacetilases/administração & dosagem , Humanos , Melanoma/enzimologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Inibidores de Proteínas Quinases/administração & dosagem , Distribuição Aleatória , Transdução de Sinais/efeitos dos fármacosRESUMO
Alzheimer's disease (AD) is an age-related disease, with loss of integrity of the blood-brain barrier (BBB) being an early feature. Cellular senescence is one of the reported nine hallmarks of aging. Here, we show for the first time the presence of senescent cells in the vasculature in AD patients and mouse models of AD. Senescent endothelial cells and pericytes are present in APP/PS1 transgenic mice but not in wild-type littermates at the time of amyloid deposition. In vitro, senescent endothelial cells display altered VE-cadherin expression and loss of cell junction formation and increased permeability. Consistent with this, senescent endothelial cells in APP/PS1 mice are present at areas of vascular leak that have decreased claudin-5 and VE-cadherin expression confirming BBB breakdown. Furthermore, single cell sequencing of endothelial cells from APP/PS1 transgenic mice confirms that adhesion molecule pathways are among the most highly altered pathways in these cells. At the pre-plaque stage, the vasculature shows significant signs of breakdown, with a general loss of VE-cadherin, leakage within the microcirculation, and obvious pericyte perturbation. Although senescent vascular cells were not directly observed at sites of vascular leak, senescent cells were close to the leak area. Thus, we would suggest in AD that there is a progressive induction of senescence in constituents of the neurovascular unit contributing to an increasing loss of vascular integrity. Targeting the vasculature early in AD, either with senolytics or with drugs that improve the integrity of the BBB may be valid therapeutic strategies.
Assuntos
Doença de Alzheimer , Barreira Hematoencefálica , Humanos , Camundongos , Animais , Barreira Hematoencefálica/metabolismo , Doença de Alzheimer/metabolismo , Células Endoteliais , Camundongos Transgênicos , EnvelhecimentoRESUMO
The development of resistance to treatments of melanoma is commonly associated with an upregulation of the MAPK pathway and the development of an undifferentiated state. Previous studies have suggested that melanoma with these resistance characteristics may be susceptible to innate death mechanisms such as pyroptosis triggered by the activation of inflammasomes. In this study, we have taken cell lines from patients before and after the development of resistance to BRAF V600 inhibitors and exposed the resistant melanoma to temozolomide (a commonly used chemotherapy) with and without chloroquine to inhibit autophagy. It was found that melanoma with an inflammatory undifferentiated state appeared susceptible to this combination when tested in vitro and in vivo against xenografts in nonobese diabetic scid gamma mice. Translation of the latter results into patients would promise durable responses in patients treated by the combination. The inflammasome and death mechanism involved appeared to vary between melanoma and involved either AIM2 or NLRP3 inflammasomes and gasdermin D or E. These preliminary studies have raised questions as to the selectivity for different inflammasomes in different melanoma and their selective targeting by chemotherapy. They also question whether the inflammatory state of melanoma may be used as biomarkers to select patients for inflammasome-targeted therapy.
Assuntos
Inflamassomos , Melanoma , Animais , Humanos , Inflamassomos/metabolismo , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética , PiroptoseRESUMO
Dysregulation of epigenetic modifiers is a frequent event in melanoma and underlies many aspects of melanoma biology, including resistance to targeted therapy and immunotherapies. Here, we report that dual targeting of BET and CDK9 proteins have synergistic effects against melanoma cells in vitro and in vivo. The BET inhibitor (IBET151) and CDK9 inhibitor (CDKI73) synergistically killed melanoma cells in vitro independent of their BRAF or NRAS mutation status. The combination of drugs markedly inhibited the growth of human melanoma C002M cells in vitro in three-dimensional spheroids and in vivo in NOD-SCID gamma mice compared with vehicle control and the individual drugs (P < 0.05). Cell death was associated with mitochondrial depolarization, caspase-dependent apoptosis with cleavage of PARP1, and downregulation of anti-apoptotic proteins BCL2, BCLXL, and MCL1. Gene set enrichment analysis revealed downregulation of hallmark gene sets associated with E2F, G2M checkpoint, and c-MYC. Survival analysis showed worse prognosis with high G2M, E2F, or c-MYC gene signatures, suggesting biomarkers of response of BET and CDK9 inhibitors in melanoma. This combination of epigenetic inhibitors targets multiple downstream genes, leading to cell death of melanoma cells in vitro and in vivo, and warrants further investigation for treatment of melanoma in patients not responding to current therapies.
Assuntos
Antineoplásicos/uso terapêutico , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Melanoma/tratamento farmacológico , Proteínas do Tecido Nervoso/genética , Pirimidinas/uso terapêutico , Receptores de Superfície Celular/genética , Neoplasias Cutâneas/tratamento farmacológico , Sulfonamidas/uso terapêutico , Animais , Apoptose , Biomarcadores Farmacológicos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Quimioterapia Combinada , Epigenômica , Humanos , Camundongos , Camundongos SCID , Metástase Neoplásica , Estadiamento de Neoplasias , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The histone methylase EZH2 is frequently dysregulated in melanoma and is associated with DNA methylation and silencing of genes involved in tumor suppression. In this study, we used chromatin immunoprecipitation and sequencing to identify key suppressor genes that are silenced by histone methylation in constitutively active EZH2(Y641) mutant melanoma and assessed whether these regions were also sites of DNA methylation. The genes identified were validated by their re-expression after treatment with EZH2 and DNA methyltransferase inhibitors. The expression of putative EZH2 target genes was shown to be highly relevant to the survival of patients with melanoma in clinical datasets. To determine correlates of response to EZH2 inhibitors, we screened a panel of 53 melanoma cell lines for drug sensitivity. We compared RNA sequencing profiles of sensitive to resistant melanoma cells and performed pathway analysis. Sensitivity was associated with strong downregulation of IFN-γ and IFN-α gene signatures that were reversed by treatment with EZH2 inhibitors. This is consistent with EZH2-driven dedifferentiated invasive states associated with treatment resistance and defects in antigen presentation. These results suggest that EZH2 inhibitors may be most effectively targeted to immunologically cold melanoma to both induce direct cytotoxicity and increase immune responses in the context of checkpoint inhibitor immunotherapy.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Regulação Neoplásica da Expressão Gênica/imunologia , Melanoma/imunologia , Neoplasias Cutâneas/imunologia , Proteínas Supressoras de Tumor/genética , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Sequenciamento de Cromatina por Imunoprecipitação , Metilação de DNA/imunologia , Conjuntos de Dados como Assunto , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/genética , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Feminino , Inativação Gênica/imunologia , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Interferon-alfa/genética , Interferon gama/genética , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/mortalidade , Camundongos , Mutação , Invasividade Neoplásica/genética , Invasividade Neoplásica/imunologia , RNA-Seq , Pele/imunologia , Pele/patologia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/mortalidade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Monoclonal antibodies against immune checkpoint blockade have proven to be a major success in the treatment of melanoma. The programmed death receptor-1 ligand-1 (PD-L1) expression on melanoma cells is believed to have an inhibitory effect on T cell responses and to be an important escape mechanism from immune attack. Previous studies have shown that PD-L1 can be expressed constitutively or can be induced by IFN-γ secreted by infiltrating lymphocytes. In the present study we have investigated the mechanism underlying these two modes of PD-L1 expression in melanoma cells including cells that had acquired resistance to the BRAF inhibitor vemurafenib. PD-L1 expression was examined by flow cytometry and immunoblotting. Specific inhibitors and siRNA knockdown approaches were used to examine the roles of the RAF/ MEK, PI3K, NF-κB, STAT3 and AP1/ c-Jun pathways. IFN-γ inducible expression of PD-L1 was dependent on NF-κB as shown by inhibition with BMS-345541, an inhibitor of IκB and the BET protein inhibitor I-BET151, as well as by siRNA knockdown of NF-κB subunits. We were unable to implicate the BRAF/MEK pathway as major regulators in PD-L1 expression on vemurafenib resistant cells. Similarly the PI3K/AKT pathway and the transcription factors STAT3 and c-Jun had only minor roles in IFN-γ induced expression of PD-L1. The mechanism underlying constitutive expression remains unresolved. We suggest these results have significance in selection of treatments that can be used in combination with monoclonal antibodies against PD1, to enhance their effectiveness and to reduce inhibitory effects melanoma cells have against cytotoxic T cell activity.
Assuntos
Antígeno B7-H1/metabolismo , Indóis/farmacologia , Interferon gama/farmacologia , Melanoma/metabolismo , NF-kappa B/metabolismo , Sulfonamidas/farmacologia , Antígeno B7-H1/genética , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Quinoxalinas/farmacologia , Fatores de Tempo , VemurafenibRESUMO
Histone acetylation marks have an important role in controlling gene expression and are removed by histone deacetylases (HDACs). These marks are read by bromodomain and extra-terminal (BET) proteins and novel inhibitiors of these proteins are currently in clinical development. Inhibitors of HDAC and BET proteins have individually been shown to cause apoptosis and reduce growth of melanoma cells. Here we show that combining the HDAC inhibitor LBH589 and BET inhibitor I-BET151 synergistically induce apoptosis of melanoma cells but not of melanocytes. Induction of apoptosis proceeded through the mitochondrial pathway, was caspase dependent and involved upregulation of the BH3 pro-apoptotic protein BIM. Analysis of signal pathways in melanoma cell lines resistant to BRAF inhibitors revealed that treatment with the combination strongly downregulated anti-apoptotic proteins and proteins in the AKT and Hippo/YAP signaling pathways. Xenograft studies showed that the combination of inhibitors was more effective than single drug treatment and confirmed upregulation of BIM and downregulation of XIAP as seen in vitro. These results support the combination of these two classes of epigenetic regulators in treatment of melanoma including those resistant to BRAF inhibitors.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Inibidores de Histona Desacetilases/administração & dosagem , Melanoma/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Epigênese Genética , Feminino , Compostos Heterocíclicos de 4 ou mais Anéis/química , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/química , Imuno-Histoquímica , Indóis/química , Melanócitos/metabolismo , Melanoma/tratamento farmacológico , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mitocôndrias/metabolismo , Transplante de Neoplasias , Panobinostat , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Neoplasias Cutâneas/tratamento farmacológico , Fatores de Transcrição , Proteínas de Sinalização YAPRESUMO
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.
Assuntos
Genes Supressores de Tumor , Melanoma/genética , Melanoma/metabolismo , Complexo Repressor Polycomb 2/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Fator 3 Ativador da Transcrição/genética , Apoptose , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Progressão da Doença , Proteína Potenciadora do Homólogo 2 de Zeste , Epigenômica , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Genótipo , Histonas/química , Humanos , Indóis/química , Concentração Inibidora 50 , Mutação , Piridonas/química , Regulação para CimaRESUMO
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.
Assuntos
Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Melanoma/metabolismo , NF-kappa B/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Comunicação Autócrina/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimiocinas/biossíntese , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Melanoma/genética , Melanoma/patologia , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Fatores de TranscriçãoRESUMO
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.