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1.
Nat Commun ; 11(1): 896, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-32060274

RESUMO

Predicting the outcome of immunotherapy treatment in melanoma patients is challenging. Alterations in genes involved in antigen presentation and the interferon gamma (IFNγ) pathway play an important role in the immune response to tumors. We describe here that the overexpression of PSMB8 and PSMB9, two major components of the immunoproteasome, is predictive of better survival and improved response to immune-checkpoint inhibitors of melanoma patients. We study the mechanism underlying this connection by analyzing the antigenic peptide repertoire of cells that overexpress these subunits using HLA peptidomics. We find a higher response of patient-matched tumor infiltrating lymphocytes against antigens diferentially presented after immunoproteasome overexpression. Importantly, we find that PSMB8 and PSMB9 expression levels are much stronger predictors of melanoma patients' immune response to checkpoint inhibitors than the tumors' mutational burden. These results suggest that PSMB8 and PSMB9 expression levels can serve as important biomarkers for stratifying melanoma patients for immune-checkpoint treatment.

2.
Pigment Cell Melanoma Res ; 33(2): 334-344, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31549767

RESUMO

NRAS mutations are the most common alterations among RAS isoforms in cutaneous melanoma, with patients harboring these aggressive tumors having a poor prognosis and low survival rate. The main line of treatment for these patients is MAPK pathway-targeted therapies, such as MEK inhibitors, but, unfortunately, the response to these inhibitors is variable due to tumor resistance. Identifying genetic modifiers involved in resistance toward MEK-targeted therapy may assist in the development of new therapeutic strategies, enhancing treatment response and patient survival. Our whole-genome CRISPR-Cas9 knockout screen identified the target Kelch domain-containing F-Box protein 42 (FBXO42) as a factor involved in NRAS-mutant melanoma-acquired resistance to the MEK1/2 inhibitor trametinib. We further show that FBXO42, an E3 ubiquitin ligase, is involved in the TAK1 signaling pathway, possibly prompting an increase in active P38. In addition, we demonstrate that combining trametinib with the TAK1 inhibitor, takinib, is a far more efficient treatment than trametinib alone in NRAS-mutant melanoma cells. Our findings thus show a new pathway involved in NRAS-mutant melanoma resistance and provide new opportunities for novel therapeutic options.

3.
Cell ; 179(1): 219-235.e21, 2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31522890

RESUMO

Although clonal neo-antigen burden is associated with improved response to immune therapy, the functional basis for this remains unclear. Here we study this question in a novel controlled mouse melanoma model that enables us to explore the effects of intra-tumor heterogeneity (ITH) on tumor aggressiveness and immunity independent of tumor mutational burden. Induction of UVB-derived mutations yields highly aggressive tumors with decreased anti-tumor activity. However, single-cell-derived tumors with reduced ITH are swiftly rejected. Their rejection is accompanied by increased T cell reactivity and a less suppressive microenvironment. Using phylogenetic analyses and mixing experiments of single-cell clones, we dissect two characteristics of ITH: the number of clones forming the tumor and their clonal diversity. Our analysis of melanoma patient tumor data recapitulates our results in terms of overall survival and response to immune checkpoint therapy. These findings highlight the importance of clonal mutations in robust immune surveillance and the need to quantify patient ITH to determine the response to checkpoint blockade.

4.
Cancer Res ; 79(10): 2634-2648, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30914429

RESUMO

The invasive phenotype of metastatic cancer cells is accompanied by the formation of actin-rich invadopodia, which adhere to the extracellular matrix and degrade it. In this study, we explored the role of the tyrosine kinome in the formation of invadopodia in metastatic melanoma cells. Using a microscopy-based siRNA screen, we identified a series of regulators, the knockdown of which either suppresses (e.g., TYK2, IGFR1, ERBB3, TYRO3, FES, ALK, PTK7) or enhances (e.g., ABL2, AXL, CSK) invadopodia formation and function. Notably, the receptor tyrosine kinase AXL displayed a dual regulatory function, where both depletion or overexpression enhanced invadopodia formation and activity. This apparent contradiction was attributed to the capacity of AXL to directly stimulate invadopodia, yet its suppression upregulates the ERBB3 signaling pathway, which can also activate core invadopodia regulators and enhance invadopodia function. Bioinformatic analysis of multiple melanoma cell lines points to an inverse expression pattern of AXL and ERBB3. High expression of AXL in melanoma cells is associated with high expression of invadopodia components and an invasive phenotype. These results provide new insights into the complexity of metastasis-promoting mechanisms and suggest that targeting of multiple invadopodia signaling networks may serve as a potential anti-invasion therapy in melanoma. SIGNIFICANCE: These findings uncover a unique interplay between AXL and ERBB3 in invadopodia regulation that points to the need for combined therapy in order to prevent invadopodia-mediated metastasis in melanoma.


Assuntos
Melanoma/metabolismo , Melanoma/patologia , Podossomos/metabolismo , Podossomos/patologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptor ErbB-3/metabolismo , Actinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Células HEK293 , Humanos , Invasividade Neoplásica/patologia , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologia
5.
J Invest Dermatol ; 139(9): 1985-1992.e10, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30905807

RESUMO

ABCB5 is an ABC transporter that was shown to confer low-level multidrug resistance in cancer. In this study, we show that ABCB5 was mutated in 13.75% of the 640 melanoma samples analyzed. Besides nonsense mutations, two mutation hotspots were found in the ABCB5 protein, in the drug-binding pocket and the nucleotide-binding domains. Four mutations, which are representative of the mutation pattern, were selected. ATPase assays showed that these mutations resulted in a decrease in basal ATP hydrolysis by ABCB5. To select informative melanoma cell lines, mutational profiles of the clinical samples were further analyzed. This study showed mutations in the tumor suppressor CDKN2A gene and the NRAS oncogene in 62.5% and 75%, respectively of the samples that had mutations in the ABCB5 gene. No mutation was found in the tumor suppressor PTEN gene, whereas the activating V600E mutation in the BRAF oncogene was found in 25% of the samples with a mutated ABCB5 gene. Studies in four melanoma cell lines with various genetic backgrounds showed an increase in the proliferation and migration capacity of mutant ABCB5-expressing cells, suggesting that ABCB5 plays a role in the development of melanoma as a tumor suppressor gene.

6.
Sci Rep ; 9(1): 4672, 2019 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-30858388

RESUMO

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

9.
Semin Cancer Biol ; 59: 36-49, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30742905

RESUMO

Almost thirty years ago, PI3K was discovered as a lipid kinase associated with certain oncoproteins. The first decade of research on PI3K saw the identification, purification and cloning of PI3Kα. The second decade of research was noted for the identification of some of PI3K's activators and effectors. This was accompanied by the discovery that PI3K acts as a retroviral oncogene. The third decade was known for the establishment of the direct involvement of PI3K in cancer, demonstrated by the identification of cancer-specific mutations. Efforts to target PI3K were on the rise from that moment on, accompanied by the first clinical trials for PI3K inhibitor therapies. In the fourth decade of research, PI3K-based cancer drugs will continue to emerge, as will new knowledge regarding other uncovered functions of this protein and pathway.

10.
Methods Mol Biol ; 1884: 203-214, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30465205

RESUMO

Neo-antigens expressed on tumors are targets for development of cancer immunotherapy strategies. Use of prediction algorithms to identify neo-antigens yields a significant number of peptides that must be validated in laborious and time-consuming methods; many prove to be false-positive identifications. The use of HLA peptidomics allows the isolation of the HLA-peptide complexes directly from cells and can be done on fresh tumor, patient-derived xerographs, or cell lines when the tissue sample is limited. This method can be used to identify both HLA class I and HLA class II or any different MHC from different species. Here we describe the steps to create the immune-affinity columns used from the process, the immunoprecipitation procedure, and also the isolation of the peptides that will be analyzed by mass spectrometry.


Assuntos
Antígenos de Neoplasias/isolamento & purificação , Exoma/imunologia , Neoplasias/imunologia , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Algoritmos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Exoma/genética , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/isolamento & purificação , Humanos , Hibridomas , Imunoprecipitação/instrumentação , Imunoprecipitação/métodos , Neoplasias/patologia , Proteômica/instrumentação , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/instrumentação
12.
Cancer Discov ; 8(11): 1366-1375, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30209080

RESUMO

The quest for tumor-associated antigens (TAA) and neoantigens is a major focus of cancer immunotherapy. Here, we combine a neoantigen prediction pipeline and human leukocyte antigen (HLA) peptidomics to identify TAAs and neoantigens in 16 tumors derived from seven patients with melanoma and characterize their interactions with their tumor-infiltrating lymphocytes (TIL). Our investigation of the antigenic and T-cell landscapes encompassing the TAA and neoantigen signatures, their immune reactivity, and their corresponding T-cell identities provides the first comprehensive analysis of cancer cell T-cell cosignatures, allowing us to discover remarkable antigenic and TIL similarities between metastases from the same patient. Furthermore, we reveal that two neoantigen-specific clonotypes killed 90% of autologous melanoma cells, both in vitro and in vivo, showing that a limited set of neoantigen-specific T cells may play a central role in melanoma tumor rejection. Our findings indicate that combining HLA peptidomics with neoantigen predictions allows robust identification of targetable neoantigens, which could successfully guide personalized cancer immunotherapies.Significance: As neoantigen targeting is becoming more established as a powerful therapeutic approach, investigating these molecules has taken center stage. Here, we show that a limited set of neoantigen-specific T cells mediates tumor rejection, suggesting that identifying just a few antigens and their corresponding T-cell clones could guide personalized immunotherapy. Cancer Discov; 8(11); 1366-75. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1333.


Assuntos
Apresentação do Antígeno/imunologia , Antígenos de Neoplasias/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Linfócitos do Interstício Tumoral/imunologia , Melanoma/imunologia , Linfócitos T/imunologia , Animais , Antígenos de Neoplasias/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Linfócitos T/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Cell ; 174(6): 1559-1570.e22, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-30100185

RESUMO

The urea cycle (UC) is the main pathway by which mammals dispose of waste nitrogen. We find that specific alterations in the expression of most UC enzymes occur in many tumors, leading to a general metabolic hallmark termed "UC dysregulation" (UCD). UCD elicits nitrogen diversion toward carbamoyl-phosphate synthetase2, aspartate transcarbamylase, and dihydrooratase (CAD) activation and enhances pyrimidine synthesis, resulting in detectable changes in nitrogen metabolites in both patient tumors and their bio-fluids. The accompanying excess of pyrimidine versus purine nucleotides results in a genomic signature consisting of transversion mutations at the DNA, RNA, and protein levels. This mutational bias is associated with increased numbers of hydrophobic tumor antigens and a better response to immune checkpoint inhibitors independent of mutational load. Taken together, our findings demonstrate that UCD is a common feature of tumors that profoundly affects carcinogenesis, mutagenesis, and immunotherapy response.


Assuntos
Genômica , Metabolômica , Neoplasias/patologia , Ureia/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Animais , Aspartato Carbamoiltransferase/genética , Aspartato Carbamoiltransferase/metabolismo , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Linhagem Celular Tumoral , Di-Hidro-Orotase/genética , Di-Hidro-Orotase/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Neoplasias/metabolismo , Ornitina Carbamoiltransferase/antagonistas & inibidores , Ornitina Carbamoiltransferase/genética , Ornitina Carbamoiltransferase/metabolismo , Fosforilação/efeitos dos fármacos , Pirimidinas/biossíntese , Pirimidinas/química , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo
14.
Oncotarget ; 9(58): 31264-31277, 2018 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-30131853

RESUMO

Neurofibromin 1 (NF1), a tumor suppressor that negatively regulates RAS through its GTPase activity, is highly mutated in various types of sporadic human cancers, including melanoma. However, the binding partners of NF1 and the pathways in which it is involved in melanoma have not been characterized in an in depth manner. Utilizing a mass spectrometry analysis of NF1 binding partners, we revealed Calpain1 (CAPN1), a calcium-dependent neutral cysteine protease, as a novel NF1 binding partner that regulates NF1 degradation in melanoma cells. ShRNA-mediated knockdown of CAPN1 or treatment with a CAPN1 inhibitor stabilizes NF1 protein levels, downregulates AKT signaling and melanoma cell growth. Combination treatment of Calpain inhibitor I with MEKi Trametinib in different melanoma cells is more effective in reducing melanoma cell growth compared to treatment with Trametinib alone, suggesting that this combination may have a therapeutic potential in melanoma. This novel mechanism for regulating NF1 in melanoma provides a molecular basis for targeting CAPN1 in order to stabilize NF1 levels and, in doing so, suppressing Ras activation; this mechanism can be exploited therapeutically in melanoma and other cancers.

15.
Nat Commun ; 9(1): 2546, 2018 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-29959327

RESUMO

While synthetic lethality (SL) holds promise in developing effective cancer therapies, SL candidates found via experimental screens often have limited translational value. Here we present a data-driven approach, ISLE (identification of clinically relevant synthetic lethality), that mines TCGA cohort to identify the most likely clinically relevant SL interactions (cSLi) from a given candidate set of lab-screened SLi. We first validate ISLE via a benchmark of large-scale drug response screens and by predicting drug efficacy in mouse xenograft models. We then experimentally test a select set of predicted cSLi via new screening experiments, validating their predicted context-specific sensitivity in hypoxic vs normoxic conditions and demonstrating cSLi's utility in predicting synergistic drug combinations. We show that cSLi can successfully predict patients' drug treatment response and provide patient stratification signatures. ISLE thus complements existing actionable mutation-based methods for precision cancer therapy, offering an opportunity to expand its scope to the whole genome.


Assuntos
Antineoplásicos/uso terapêutico , Ensaios de Triagem em Larga Escala , Neoplasias/tratamento farmacológico , Medicina de Precisão/métodos , Mutações Sintéticas Letais/efeitos dos fármacos , Animais , Biomarcadores Farmacológicos , Hipóxia Celular , Linhagem Celular Tumoral , Combinação de Medicamentos , Sinergismo Farmacológico , Humanos , Camundongos , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/mortalidade , Seleção de Pacientes , Medicina de Precisão/estatística & dados numéricos , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Nat Commun ; 9(1): 2372, 2018 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-29985391

RESUMO

Dysregulation of the Hippo signaling pathway and the consequent YAP1 activation is a frequent event in human malignancies, yet the underlying molecular mechanisms are still poorly understood. A pancancer analysis of core Hippo kinases and their candidate regulating molecules revealed few alterations in the canonical Hippo pathway, but very frequent genetic alterations in the FAT family of atypical cadherins. By focusing on head and neck squamous cell carcinoma (HNSCC), which displays frequent FAT1 alterations (29.8%), we provide evidence that FAT1 functional loss results in YAP1 activation. Mechanistically, we found that FAT1 assembles a multimeric Hippo signaling complex (signalome), resulting in activation of core Hippo kinases by TAOKs and consequent YAP1 inactivation. We also show that unrestrained YAP1 acts as an oncogenic driver in HNSCC, and that targeting YAP1 may represent an attractive precision therapeutic option for cancers harboring genomic alterations in the FAT1 tumor suppressor genes.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Caderinas/fisiologia , Neoplasias de Cabeça e Pescoço/genética , Fosfoproteínas/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Caderinas/genética , Caderinas/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Fatores de Transcrição
17.
Pigment Cell Melanoma Res ; 31(5): 641-648, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29665313

RESUMO

The NRAS oncoprotein is highly mutated in melanoma. However, to date, no comprehensive proteomic study has been reported for NRAS. Here, we utilized the endogenous epitope tagging (EET) approach for the identification of novel NRAS binding partners. Using EET, an epitope tag is added to the endogenously expressed protein, via modification of its genomic coding sequence. Existing EET systems are not robust, suffer from high background, and are labor-intensive. To this end, we present a polyadenylation signal-trap construct for N'-tagging that generates a polycistronic mRNA with the gene of interest. This system requires the integration of the tagging cassette in frame with the target gene to be expressed. Using this design, we demonstrate, for the first time, endogenous tagging of NRAS in melanoma cells allowing the identification of the E3 ubiquitin ligase c-CBL as a novel NRAS binding partner. Thus, our developed EET technology allows the characterization of new RAS effectors, which could be beneficial for the design of future drugs that inhibit constitutive signaling of RAS oncogenic mutants.


Assuntos
Mapeamento de Epitopos/métodos , Epitopos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Melanoma/metabolismo , Proteínas de Membrana/metabolismo , Proteômica/métodos , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Epitopos/genética , GTP Fosfo-Hidrolases/genética , Humanos , Melanoma/genética , Melanoma/patologia , Proteínas de Membrana/genética , Mutação , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas c-cbl/genética , Células Tumorais Cultivadas
18.
J Invest Dermatol ; 138(10): 2216-2223, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29679610

RESUMO

Melanoma, a melanocyte origin neoplasm, is the most lethal type of skin cancer, and incidence is increasing. Several familial and somatic mutations have been identified in the gene encoding the melanocyte lineage master regulator, MITF; however, the neoplastic mechanisms of these mutant MITF variants are mostly unknown. Here, by performing unbiased analysis of the transcriptomes in cells expressing mutant MITF, we identified calcium-binding protein S100A4 as a downstream target of MITF-E87R. By using wild-type and mutant MITF melanoma lines, we found that both endogenous wild-type and MITF-E87R variants occupy the S100A4 promoter. Remarkably, whereas wild-type MITF represses S100A4 expression, MITF-E87R activates its transcription. The opposite effects of wild-type and mutant MITF result in opposing cellular phenotypes, because MITF-E87R via S100A4 enhanced invasion and reduced adhesion in contrast to wild-type MITF activity. Finally, we found that melanoma patients with altered S100A4 expression have poor prognosis. These data show that a change in MITF transcriptional activity from repression to activation of S100A4 that results from a point mutation in MITF alters melanoma invasive ability. These data suggest new opportunities for diagnosis and treatment of metastatic melanoma.


Assuntos
DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Fator de Transcrição Associado à Microftalmia/genética , Mutação , Proteína A4 de Ligação a Cálcio da Família S100/genética , Neoplasias Cutâneas/genética , Análise Mutacional de DNA , Progressão da Doença , Humanos , Immunoblotting , Melanoma/metabolismo , Melanoma/patologia , Fator de Transcrição Associado à Microftalmia/metabolismo , Proteína A4 de Ligação a Cálcio da Família S100/biossíntese , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Células Tumorais Cultivadas
20.
Cancer Cell ; 33(2): 322-336.e8, 2018 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-29438700

RESUMO

Oncogene-induced senescence, e.g., in melanocytic nevi, terminates the expansion of pre-malignant cells via transcriptional silencing of proliferation-related genes due to decoration of their promoters with repressive trimethylated histone H3 lysine 9 (H3K9) marks. We show here that structurally distinct H3K9-active demethylases-the lysine-specific demethylase-1 (LSD1) and several Jumonji C domain-containing moieties (such as JMJD2C)-disable senescence and permit Ras/Braf-evoked transformation. In mouse and zebrafish models, enforced LSD1 or JMJD2C expression promoted Braf-V600E-driven melanomagenesis. A large subset of established melanoma cell lines and primary human melanoma samples presented with a collective upregulation of related and unrelated H3K9 demethylase activities, whose targeted inhibition restored senescence, even in Braf inhibitor-resistant melanomas, evoked secondary immune effects and controlled tumor growth in vivo.


Assuntos
Histona Desmetilases/genética , Histona Desmetilases com o Domínio Jumonji/genética , Melanoma/genética , Animais , Histonas/metabolismo , Humanos , Lisina/genética , Lisina/metabolismo , Metilação , Camundongos Nus , Regiões Promotoras Genéticas/genética
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