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1.
Cell ; 175(4): 998-1013.e20, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30388456

RESUMO

Treatment of cancer has been revolutionized by immune checkpoint blockade therapies. Despite the high rate of response in advanced melanoma, the majority of patients succumb to disease. To identify factors associated with success or failure of checkpoint therapy, we profiled transcriptomes of 16,291 individual immune cells from 48 tumor samples of melanoma patients treated with checkpoint inhibitors. Two distinct states of CD8+ T cells were defined by clustering and associated with patient tumor regression or progression. A single transcription factor, TCF7, was visualized within CD8+ T cells in fixed tumor samples and predicted positive clinical outcome in an independent cohort of checkpoint-treated patients. We delineated the epigenetic landscape and clonality of these T cell states and demonstrated enhanced antitumor immunity by targeting novel combinations of factors in exhausted cells. Our study of immune cell transcriptomes from tumors demonstrates a strategy for identifying predictors, mechanisms, and targets for enhancing checkpoint immunotherapy.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunoterapia/métodos , Melanoma/imunologia , Transcriptoma , Animais , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Antígenos CD/imunologia , Antineoplásicos Imunológicos/imunologia , Antineoplásicos Imunológicos/farmacologia , Apirase/antagonistas & inibidores , Apirase/imunologia , Linhagem Celular Tumoral , Humanos , Antígenos Comuns de Leucócito/antagonistas & inibidores , Antígenos Comuns de Leucócito/imunologia , Melanoma/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fator 1 de Transcrição de Linfócitos T/metabolismo
3.
Nature ; 487(7408): 500-4, 2012 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-22763439

RESUMO

Drug resistance presents a challenge to the treatment of cancer patients. Many studies have focused on cell-autonomous mechanisms of drug resistance. By contrast, we proposed that the tumour micro-environment confers innate resistance to therapy. Here we developed a co-culture system to systematically assay the ability of 23 stromal cell types to influence the innate resistance of 45 cancer cell lines to 35 anticancer drugs. We found that stroma-mediated resistance is common, particularly to targeted agents. We characterized further the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibitors because most patients with this type of cancer show some degree of innate resistance. Proteomic analysis showed that stromal cell secretion of hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-OH kinase (PI(3)K)-AKT signalling pathways, and immediate resistance to RAF inhibition. Immunohistochemistry experiments confirmed stromal cell expression of HGF in patients with BRAF-mutant melanoma and showed a significant correlation between HGF expression by stromal cells and innate resistance to RAF inhibitor treatment. Dual inhibition of RAF and either HGF or MET resulted in reversal of drug resistance, suggesting RAF plus HGF or MET inhibitory combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. More generally, this study indicates that the systematic dissection of interactions between tumours and their micro-environment can uncover important mechanisms underlying drug resistance.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Fator de Crescimento de Hepatócito/metabolismo , Melanoma/metabolismo , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Microambiente Tumoral/fisiologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica , Linhagem Celular Tumoral , Técnicas de Cocultura , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Indóis/farmacologia , Indóis/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Terapia de Alvo Molecular , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteômica , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Vemurafenib
4.
Nucleic Acids Res ; 39(4): 1326-35, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21030442

RESUMO

Promoter hypermethylation and heterochromatinization is a frequent event leading to gene inactivation and tumorigenesis. At the molecular level, inactivation of tumor suppressor genes in cancer has many similarities to the inactive X chromosome in female cells and is defined and maintained by DNA methylation and characteristic histone modifications. In addition, the inactive-X is marked by the histone macroH2A, a variant of H2A with a large non-histone region of unknown function. Studying tumor suppressor genes (TSGs) silenced in cancer cell lines, we find that when active, these promoters are associated with H2A.Z but become enriched for macroH2A1 once silenced. Knockdown of macroH2A1 was not sufficient for reactivation of silenced genes. However, when combined with DNA demethylation, macroH2A1 deficiency significantly enhanced reactivation of the tumor suppressor genes p16, MLH1 and Timp3 and inhibited cell proliferation. Our findings link macroH2A1 to heterochromatin of epigenetically silenced cancer genes and indicate synergism between macroH2A1 and DNA methylation in maintenance of the silenced state.


Assuntos
Metilação de DNA , Inativação Gênica , Genes p16 , Histonas/metabolismo , Alelos , Linhagem Celular , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Técnicas de Silenciamento de Genes , Genes Supressores de Tumor , Histonas/genética , Humanos , Regiões Promotoras Genéticas
5.
Cell Rep Med ; 3(2): 100500, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35243413

RESUMO

Immune checkpoint blockade (CPB) improves melanoma outcomes, but many patients still do not respond. Tumor mutational burden (TMB) and tumor-infiltrating T cells are associated with response, and integrative models improve survival prediction. However, integrating immune/tumor-intrinsic features using data from a single assay (DNA/RNA) remains underexplored. Here, we analyze whole-exome and bulk RNA sequencing of tumors from new and published cohorts of 189 and 178 patients with melanoma receiving CPB, respectively. Using DNA, we calculate T cell and B cell burdens (TCB/BCB) from rearranged TCR/Ig sequences and find that patients with TMBhigh and TCBhigh or BCBhigh have improved outcomes compared to other patients. By combining pairs of immune- and tumor-expressed genes, we identify three gene pairs associated with response and survival, which validate in independent cohorts. The top model includes lymphocyte-expressed MAP4K1 and tumor-expressed TBX3. Overall, RNA or DNA-based models combining immune and tumor measures improve predictions of melanoma CPB outcomes.


Assuntos
Melanoma , Transcriptoma , Humanos , Melanoma/tratamento farmacológico , RNA , Análise de Sequência de RNA , Transcriptoma/genética , Sequenciamento do Exoma
6.
Clin Cancer Res ; 24(19): 4771-4784, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29563139

RESUMO

Purpose: Telomerase promoter mutations are highly prevalent in human tumors including melanoma. A subset of patients with metastatic melanoma often fail multiple therapies, and there is an unmet and urgent need to prolong disease control for those patients.Experimental Design: Numerous preclinical therapy-resistant models of human and mouse melanoma were used to test the efficacy of a telomerase-directed nucleoside, 6-thio-2'-deoxyguanosine (6-thio-dG). Integrated transcriptomics and proteomics approaches were used to identify genes and proteins that were significantly downregulated by 6-thio-dG.Results: We demonstrated the superior efficacy of 6-thio-dG both in vitro and in vivo that results in telomere dysfunction, leading to apoptosis and cell death in various preclinical models of therapy-resistant melanoma cells. 6-thio-dG concomitantly induces telomere dysfunction and inhibits the expression level of AXL.Conclusions: In summary, this study shows that indirectly targeting aberrant telomerase in melanoma cells with 6-thio-dG is a viable therapeutic approach in prolonging disease control and overcoming therapy resistance. Clin Cancer Res; 24(19); 4771-84. ©2018 AACR See related commentary by Teh and Aplin, p. 4629.


Assuntos
Desoxiguanosina/análogos & derivados , Melanoma/tratamento farmacológico , Regiões Promotoras Genéticas/genética , Telomerase/genética , Tionucleosídeos/farmacologia , Animais , Linhagem Celular Tumoral , Desoxiguanosina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Melanoma/genética , Melanoma/patologia , Camundongos , Mutação , Telômero/efeitos dos fármacos , Telômero/genética
7.
Cancer Discov ; 8(2): 196-215, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29101162

RESUMO

Ex vivo systems that incorporate features of the tumor microenvironment and model the dynamic response to immune checkpoint blockade (ICB) may facilitate efforts in precision immuno-oncology and the development of effective combination therapies. Here, we demonstrate the ability to interrogate ex vivo response to ICB using murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS). MDOTS/PDOTS isolated from mouse and human tumors retain autologous lymphoid and myeloid cell populations and respond to ICB in short-term three-dimensional microfluidic culture. Response and resistance to ICB was recapitulated using MDOTS derived from established immunocompetent mouse tumor models. MDOTS profiling demonstrated that TBK1/IKKε inhibition enhanced response to PD-1 blockade, which effectively predicted tumor response in vivo Systematic profiling of secreted cytokines in PDOTS captured key features associated with response and resistance to PD-1 blockade. Thus, MDOTS/PDOTS profiling represents a novel platform to evaluate ICB using established murine models as well as clinically relevant patient specimens.Significance: Resistance to PD-1 blockade remains a challenge for many patients, and biomarkers to guide treatment are lacking. Here, we demonstrate feasibility of ex vivo profiling of PD-1 blockade to interrogate the tumor immune microenvironment, develop therapeutic combinations, and facilitate precision immuno-oncology efforts. Cancer Discov; 8(2); 196-215. ©2017 AACR.See related commentary by Balko and Sosman, p. 143See related article by Deng et al., p. 216This article is highlighted in the In This Issue feature, p. 127.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Citocinas/metabolismo , Resistencia a Medicamentos Antineoplásicos , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Imunofenotipagem , Camundongos , Técnicas Analíticas Microfluídicas , Receptor de Morte Celular Programada 1/metabolismo , Esferoides Celulares , Imagem com Lapso de Tempo , Células Tumorais Cultivadas
8.
Science ; 357(6356): 1156-1160, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28912244

RESUMO

Growing evidence suggests that microbes can influence the efficacy of cancer therapies. By studying colon cancer models, we found that bacteria can metabolize the chemotherapeutic drug gemcitabine (2',2'-difluorodeoxycytidine) into its inactive form, 2',2'-difluorodeoxyuridine. Metabolism was dependent on the expression of a long isoform of the bacterial enzyme cytidine deaminase (CDDL), seen primarily in Gammaproteobacteria. In a colon cancer mouse model, gemcitabine resistance was induced by intratumor Gammaproteobacteria, dependent on bacterial CDDL expression, and abrogated by cotreatment with the antibiotic ciprofloxacin. Gemcitabine is commonly used to treat pancreatic ductal adenocarcinoma (PDAC), and we hypothesized that intratumor bacteria might contribute to drug resistance of these tumors. Consistent with this possibility, we found that of the 113 human PDACs that were tested, 86 (76%) were positive for bacteria, mainly Gammaproteobacteria.


Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/microbiologia , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/microbiologia , Animais , Neoplasias do Colo/microbiologia , Desoxicitidina/uso terapêutico , Gammaproteobacteria/isolamento & purificação , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mycoplasma hyorhinis/isolamento & purificação , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/microbiologia , Gencitabina , Neoplasias Pancreáticas
9.
Nat Commun ; 8(1): 1136, 2017 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-29070816

RESUMO

Treatment with immune checkpoint blockade (CPB) therapies often leads to prolonged responses in patients with metastatic melanoma, but the common mechanisms of primary and acquired resistance to these agents remain incompletely characterized and have yet to be validated in large cohorts. By analyzing longitudinal tumor biopsies from 17 metastatic melanoma patients treated with CPB therapies, we observed point mutations, deletions or loss of heterozygosity (LOH) in beta-2-microglobulin (B2M), an essential component of MHC class I antigen presentation, in 29.4% of patients with progressing disease. In two independent cohorts of melanoma patients treated with anti-CTLA4 and anti-PD1, respectively, we find that B2M LOH is enriched threefold in non-responders (~30%) compared to responders (~10%) and associated with poorer overall survival. Loss of both copies of B2M is found only in non-responders. B2M loss is likely a common mechanism of resistance to therapies targeting CTLA4 or PD1.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Apresentação de Antígeno/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Melanoma/tratamento farmacológico , Animais , Anticorpos Monoclonais/imunologia , Apresentação de Antígeno/genética , Antígeno CTLA-4/imunologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Perda de Heterozigosidade , Melanoma/genética , Melanoma/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Metástase Neoplásica , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Mutação Puntual , Receptor de Morte Celular Programada 1/imunologia , Microglobulina beta-2/genética
10.
Cell Rep ; 21(7): 1953-1967, 2017 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-29141225

RESUMO

Therapy of advanced melanoma is changing dramatically. Following mutational and biological subclassification of this heterogeneous cancer, several targeted and immune therapies were approved and increased survival significantly. To facilitate further advancements through pre-clinical in vivo modeling, we have established 459 patient-derived xenografts (PDX) and live tissue samples from 384 patients representing the full spectrum of clinical, therapeutic, mutational, and biological heterogeneity of melanoma. PDX have been characterized using targeted sequencing and protein arrays and are clinically annotated. This exhaustive live tissue resource includes PDX from 57 samples resistant to targeted therapy, 61 samples from responders and non-responders to immune checkpoint blockade, and 31 samples from brain metastasis. Uveal, mucosal, and acral subtypes are represented as well. We show examples of pre-clinical trials that highlight how the PDX collection can be used to develop and optimize precision therapies, biomarkers of response, and the targeting of rare genetic subgroups.


Assuntos
Xenoenxertos/patologia , Melanoma/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Células Cultivadas , Xenoenxertos/metabolismo , Humanos , Melanoma/classificação , Melanoma/genética , Camundongos
11.
Cancer Discov ; 4(7): 816-27, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24771846

RESUMO

UNLABELLED: Most melanomas harbor oncogenic BRAF(V600) mutations, which constitutively activate the MAPK pathway. Although MAPK pathway inhibitors show clinical benefit in BRAF(V600)-mutant melanoma, it remains incompletely understood why 10% to 20% of patients fail to respond. Here, we show that RAF inhibitor-sensitive and inhibitor-resistant BRAF(V600)-mutant melanomas display distinct transcriptional profiles. Whereas most drug-sensitive cell lines and patient biopsies showed high expression and activity of the melanocytic lineage transcription factor MITF, intrinsically resistant cell lines and biopsies displayed low MITF expression but higher levels of NF-κB signaling and the receptor tyrosine kinase AXL. In vitro, these MITF-low/NF-κB-high melanomas were resistant to inhibition of RAF and MEK, singly or in combination, and ERK. Moreover, in cell lines, NF-κB activation antagonized MITF expression and induced both resistance marker genes and drug resistance. Thus, distinct cell states characterized by MITF or NF-κB activity may influence intrinsic resistance to MAPK pathway inhibitors in BRAF(V600)-mutant melanoma. SIGNIFICANCE: Although most BRAF(V600)-mutant melanomas are sensitive to RAF and/or MEK inhibitors, a subset fails to respond to such treatment. This study characterizes a transcriptional cell state distinction linked to MITF and NF-κB that may modulate intrinsic sensitivity of melanomas to MAPK pathway inhibitors.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/genética , Melanoma/patologia , Subunidade p50 de NF-kappa B/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Anilidas/farmacologia , Benzimidazóis/farmacologia , Benzocicloeptenos/farmacologia , Linhagem Celular Tumoral , Células Cultivadas , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Indóis/farmacologia , Melanócitos/citologia , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Melanoma/tratamento farmacológico , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Piridinas/farmacologia , Quinolinas/farmacologia , Sulfonamidas/farmacologia , Triazóis/farmacologia
12.
PLoS One ; 6(2): e17262, 2011 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-21364955

RESUMO

MacroH2A1 is a histone H2A variant which contains a large non-histone C-terminal region of largely unknown function. Within this region is a macro domain which can bind ADP-ribose and related molecules. Most studies of macroH2A1 focus on the involvement of this variant in transcriptional repression. Studies in mouse embryos and in embryonic stem cells suggested that during early development macroH2A can be found at the centrosome. Centrosomal localization of macroH2A was later reported in somatic cells. Here we provide data showing that macroH2A1 does not localize to the centrosome and that the centrosomal signal observed with antibodies directed against the macroH2A1 non-histone region may be the result of antibody cross-reactivity.


Assuntos
Centrossomo/metabolismo , Histonas/genética , Histonas/metabolismo , Animais , Células Cultivadas , Cromatina/metabolismo , Técnicas de Silenciamento de Genes , Variação Genética/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Histonas/antagonistas & inibidores , Humanos , Camundongos , Proteínas Mutantes/metabolismo , Transporte Proteico/genética , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Distribuição Tecidual , Transfecção
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