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1.
Blood ; 143(13): 1242-1258, 2024 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-38096363

RESUMO

ABSTRACT: To establish a strict p53-dependent gene-expression profile, TP53-/- clones were derived from TP53+/+ and TP53-/mut t(4;14) human myeloma cell lines (HMCLs) using CRISPR/Cas9 technology. From the 17 dysregulated genes shared between the TP53-/- clones from TP53+/+ HMCLs, we established a functional p53 score, involving 13 genes specifically downregulated upon p53 silencing. This functional score segregated clones and myeloma cell lines as well as other cancer cell lines according to their TP53 status. The score efficiently identified samples from patients with myeloma with biallelic TP53 inactivation and was predictive of overall survival in Multiple Myeloma Research Foundation-coMMpass and CASSIOPEA cohorts. At the functional level, we showed that among the 13 genes, p53-regulated BAX expression correlated with and directly affected the MCL1 BH3 mimetic S63845 sensitivity of myeloma cells by decreasing MCL1-BAX complexes. However, resistance to S63845 was overcome by combining MCL1 and BCL2 BH3 mimetics, which displayed synergistic efficacy. The combination of BH3 mimetics was effective in 97% of patient samples with or without del17p. Nevertheless, single-cell RNA sequencing analysis showed that myeloma cells surviving the combination had lower p53 score, showing that myeloma cells with higher p53 score were more sensitive to BH3 mimetics. Taken together, we established a functional p53 score that identifies myeloma cells with biallelic TP53 invalidation, demonstrated that p53-regulated BAX is critical for optimal cell response to BH3 mimetics, and showed that MCL1 and BCL2 BH3 mimetics in combination may be of greater effectiveness for patients with biallelic TP53 invalidation, for whom there is still an unmet medical need.


Assuntos
Antineoplásicos , Mieloma Múltiplo , Pirimidinas , Tiofenos , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Linhagem Celular Tumoral , Apoptose , Antineoplásicos/uso terapêutico
2.
Blood ; 142(18): 1543-1555, 2023 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-37562004

RESUMO

A strategy combining targeted therapies is effective in B-cell lymphomas (BCL), such as mantle cell lymphoma (MCL), but acquired resistances remain a recurrent issue. In this study, we performed integrative longitudinal genomic and single-cell RNA-sequencing analyses of patients with MCL who were treated with targeted therapies against CD20, BCL2, and Bruton tyrosine kinase (OAsIs trial). We revealed the emergence of subclones with a selective advantage against OAsIs combination in vivo and showed that resistant cells were characterized by B-cell receptor (BCR)-independent overexpression of NF-κB1 target genes, especially owing to CARD11 mutations. Functional studies demonstrated that CARD11 gain of function not only resulted in BCR independence but also directly increased the transcription of the antiapoptotic BCL2A1, leading to resistance against venetoclax and OAsIs combination. Based on the transcriptional profile of OAsIs-resistant subclones, we designed a 16-gene resistance signature that was also predictive for patients with MCL who were treated with conventional chemotherapy, underlying a common escape mechanism. Among druggable strategies to inhibit CARD11-dependent NF-κB1 transduction, we evaluated the selective inhibition of its essential partner MALT1. We demonstrated that MALT1 protease inhibition led to a reduction in the expression of genes involved in OAsIs resistance, including BCL2A1. Consequently, MALT1 inhibition induced synergistic cell death in combination with BCL2 inhibition, irrespective of CARD11 mutational status, both in vitro and in vivo. Taken together, our study identified mechanisms of resistance to targeted therapies and provided a novel strategy to overcome resistance in aggressive BCL. The OAsIs trial was registered at www.clinicaltrials.gov #NCT02558816.


Assuntos
Linfoma Difuso de Grandes Células B , Linfoma de Célula do Manto , Adulto , Humanos , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Linhagem Celular Tumoral , Mutação com Ganho de Função , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma de Célula do Manto/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
3.
Haematologica ; 107(12): 2905-2917, 2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-35263985

RESUMO

Aggressive B-cell malignancies, such as mantle cell lymphoma (MCL), are microenvironment-dependent tumors and a better understanding of the dialogs occurring in lymphoma-protective ecosystems will provide new perspectives to increase treatment efficiency. To identify novel molecular regulations, we performed a transcriptomic analysis based on the comparison of circulating MCL cells (n=77) versus MCL lymph nodes (n=107) together with RNA sequencing of malignant (n=8) versus normal B-cell (n=6) samples. This integrated analysis led to the discovery of microenvironment-dependent and tumor-specific secretion of interleukin-32 beta (IL32ß), whose expression was confirmed in situ within MCL lymph nodes by multiplex immunohistochemistry. Using ex vivo models of primary MCL cells (n=23), we demonstrated that, through the secretion of IL32ß, the tumor was able to polarize monocytes into specific MCL-associated macrophages, which in turn favor tumor survival. We highlighted that while IL32ß-stimulated macrophages secreted several protumoral factors, they supported tumor survival through a soluble dialog, mostly driven by BAFF. Finally, we demonstrated the efficacy of selective NIK/alternative-NFkB inhibition to counteract microenvironment-dependent induction of IL32ß and BAFF-dependent survival of MCL cells. These data uncovered the IL32ß/BAFF axis as a previously undescribed pathway involved in lymphoma-associated macrophage polarization and tumor survival, which could be counteracted through selective NIK inhibition.


Assuntos
Fator Ativador de Células B , Interleucinas , Linfoma de Célula do Manto , Proteínas Serina-Treonina Quinases , Adulto , Humanos , Linhagem Celular Tumoral , Interleucinas/metabolismo , Linfoma de Célula do Manto/patologia , Macrófagos/metabolismo , NF-kappa B/metabolismo , Microambiente Tumoral , Fator Ativador de Células B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Quinase Induzida por NF-kappaB
4.
Int J Mol Sci ; 19(1)2017 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-29295500

RESUMO

(1) Background: TP53 deficiency remains a major adverse event in Multiple Myeloma (MM) despite therapeutic progresses. As it is not possible to target TP53 deficiency with pharmacological agents, we explored the possibility of activating another p53 family member, p73, which has not been well studied in myeloma. (2) Methods: Using human myeloma cell lines (HMCLs) with normal or abnormal TP53 status, we assessed TP73 methylation and expression. (3) Results: Using microarray data, we reported that TP73 is weakly expressed in 47 HMCLs and mostly in TP53 wild type (TP53wt) HMCLs (p = 0.0029). Q-RT-PCR assays showed that TP73 was expressed in 57% of TP53wt HMCLs (4 out of 7) and 11% of TP53 abnormal (TP53abn) HMCLs (2 out of 18) (p = 0.0463). We showed that TP73 is silenced by methylation in TP53abn HMCLs and that decitabine increased its expression, which, however, remained insufficient for significant protein expression. Alkylating drugs increased expression of TP73 only in TP53wt HMCLs but failed to synergize with decitabine in TP53abn HMCLs. (4) Conclusions: Decitabine and melphalan does not appear as a promising combination for inducing p73 and bypassing p53 deficiency in myeloma cells.


Assuntos
Azacitidina/análogos & derivados , Melfalan/farmacologia , Mieloma Múltiplo/metabolismo , Proteína Tumoral p73/metabolismo , Proteína Supressora de Tumor p53/deficiência , Azacitidina/farmacologia , Sequência de Bases , Linhagem Celular Tumoral , Cisplatino/farmacologia , Ilhas de CpG/genética , Metilação de DNA/efeitos dos fármacos , Decitabina , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteína Tumoral p73/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
5.
J Immunol ; 192(4): 1962-71, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24453253

RESUMO

Peptide splicing is a novel mechanism of production of peptides relying on the proteasome and involving the linkage of fragments originally distant in the parental protein. Peptides produced by splicing can be presented on class I molecules of the MHC and recognized by CTLs. In this study, we describe a new antigenic peptide, which is presented by HLA-A3 and comprises two noncontiguous fragments of the melanoma differentiation Ag gp100(PMEL17) spliced together in the reverse order to that in which they appear in the parental protein. Contrary to the previously described spliced peptides, which are produced by the association of fragments of 3-6 aa, the peptide described in this work results from the ultimate association of an 8-aa fragment with a single arginine residue. As described before, peptide splicing takes place in the proteasome by transpeptidation involving an acyl-enzyme intermediate linking one of the peptide fragment to a catalytic subunit of the proteasome. Interestingly, we observe that the peptide causing the nucleophilic attack on the acyl-enzyme intermediate must be at least 3 aa long to give rise to a spliced peptide. The spliced peptide produced from this reaction therefore bears an extended C terminus that needs to be further trimmed to produce the final antigenic peptide. We show that the proteasome is able to perform the final trimming step required to produce the antigenic peptide described in this work.


Assuntos
Melanoma/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína/fisiologia , Antígeno gp100 de Melanoma/genética , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Antígeno HLA-A3/genética , Antígeno HLA-A3/imunologia , Antígeno HLA-A3/metabolismo , Humanos , Melanoma/genética , Melanoma/imunologia , Fragmentos de Peptídeos/genética , Linfócitos T Citotóxicos/imunologia , Antígeno gp100 de Melanoma/imunologia , Antígeno gp100 de Melanoma/metabolismo
6.
Hemasphere ; 8(10): e70016, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39380841

RESUMO

In multiple myeloma, as in B-cell malignancies, mono- and especially bi-allelic TP53 gene inactivation is a high-risk factor for treatment resistance, and there are currently no therapies specifically targeting p53 deficiency. In this study, we evaluated if the loss of cell cycle control in p53-deficient myeloma cells would confer a metabolically actionable vulnerability. We show that CTP synthase 1 (CTPS1), which encodes a CTP synthesis rate-limiting enzyme essential for DNA and RNA synthesis in lymphoid cells, is overexpressed in samples from myeloma patients displaying a high proliferation rate (high MKI67 expression) or a low p53 score (synonymous with TP53 deletion and/or mutation). This overexpression of CTPS1 was associated with reduced survival in two cohorts. Using scRNA-seq analysis in 24 patient samples, we further demonstrate that myeloma cells in the S or G2/M phase display high CTPS1 expression. Pharmacological inhibition of CTPS1 by STP-B induced cell cycle arrest in early S phase in isogenic NCI-H929 or XG7 TP53 +/+, TP53 -/-, and TP53 R175H/R175H cells and in a TP53 -/R123STOP patient sample. The functional annotation of transcriptional changes in 10 STP-B-treated myeloma cell lines revealed a decrease in protein translation and confirmed the blockade of cells into the S phase. The pharmacological inhibition of ATR, which governs the intrinsic S/G2 checkpoint, in STP-B-induced S-phase arrested cells synergistically induced cell death in TP53 +/+, TP53 -/-, and TP53 R175H/R175H isogenic cell lines (Bliss score >15). This combination induced replicative stress and caspase-mediated cell death and was highly effective in resistant/refractory patient samples with TP53 deletion and/or mutation and in TP53 -/- NCI-H929 xenografted NOD-scid IL2Rgamma mice. Our in vitro, ex vivo, and in vivo data provide the rationale for combined CTPS1 and ATR inhibition for the treatment of p53-deficient patients.

8.
J Exp Med ; 202(1): 61-72, 2005 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-15998788

RESUMO

A large array of antigens that are recognized by tumor-specific T cells has been identified and shown to be generated through various processes. We describe a new mechanism underlying T cell recognition of melanoma cells, which involves the generation of a major histocompatibility complex class I-restricted epitope after tumor-mediated uptake and processing of an extracellular protein--a process referred to as cross-presentation-which is believed to be restricted to immune cells. We show that melanoma cells cross-present, in an alpha v beta3-dependent manner, an antigen derived from secreted matrix metalloproteinase-2 (MMP-2) to human leukocyte antigen A*0201-restricted T cells. Because MMP-2 activity is critical for melanoma progression, the MMP-2 peptide should be cross-presented by most progressing melanomas and represents a unique antigen for vaccine therapy of these tumors.


Assuntos
Apresentação de Antígeno , Antígenos de Neoplasias/metabolismo , Integrina alfaVbeta3/metabolismo , Metaloproteinase 2 da Matriz/imunologia , Melanoma/enzimologia , Melanoma/imunologia , Animais , Sequência de Bases , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Vesículas Revestidas por Clatrina/enzimologia , Vesículas Revestidas por Clatrina/imunologia , DNA Complementar/genética , Epitopos/metabolismo , Antígenos HLA-A/metabolismo , Antígeno HLA-A2 , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Linfócitos T Citotóxicos/imunologia
9.
Cancer Immunol Immunother ; 59(3): 431-9, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19730858

RESUMO

We characterized a new melanoma antigen derived from one of the multiple open reading frames (ORFs) of the meloe transcript. The meloe gene is overexpressed in melanomas as compared to other cancer cell lines and normal tissues. The corresponding transcript is rather unusual, in that it does not contain a long unique ORF but multiple short ORFs. We recently characterized a tumor epitope derived from a polypeptide (MELOE-1) encoded by the ORF(1230-1370) and involved in relapse prevention of melanoma patients treated with autologous tumor infiltrating lymphocytes (TIL). Here we show that the ORF(285-404) encodes a polypeptide called MELOE-2 that also generated a HLA-A2 epitope recognized by a melanoma-specific T cell clone derived from the same TIL population from which we derived the MELOE-1-specific T cell clone. We also showed that HLA-A2 melanoma cells were spontaneously recognized by the MELOE-2-specific T cell clone, and we detected the presence of MELOE-2 reactive T cells in another TIL population infused to a patient who remained relapse-free after TIL treatment. These results demonstrate that translation of meloe transcript in melanoma cells can produce at least two immunogenic polypeptides, MELOE-1 and MELOE-2, from two distinct ORFs that could be relevant target for melanoma immunotherapy.


Assuntos
Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/fisiologia , Antígeno HLA-A2/imunologia , Melanoma/genética , Melanoma/imunologia , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/fisiologia , Fases de Leitura Aberta/imunologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Linfócitos T/imunologia , Antígenos de Neoplasias/genética , Sequência de Bases , Ensaios Clínicos como Assunto , DNA Complementar/química , DNA Complementar/imunologia , Humanos , Linfócitos do Interstício Tumoral/imunologia , Dados de Sequência Molecular , Proteínas de Neoplasias/genética
11.
Nat Commun ; 11(1): 259, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937780

RESUMO

A fascinating but uncharacterized action of antimitotic chemotherapy is to collectively prime cancer cells to apoptotic mitochondrial outer membrane permeabilization (MOMP), while impacting only on cycling cell subsets. Here, we show that a proapoptotic secretory phenotype is induced by activation of cGAS/STING in cancer cells that are hit by antimitotic treatment, accumulate micronuclei and maintain mitochondrial integrity despite intrinsic apoptotic pressure. Organotypic cultures of primary human breast tumors and patient-derived xenografts sensitive to paclitaxel exhibit gene expression signatures typical of type I IFN and TNFα exposure. These cytokines induced by cGAS/STING activation trigger NOXA expression in neighboring cells and render them acutely sensitive to BCL-xL inhibition. cGAS/STING-dependent apoptotic effects are required for paclitaxel response in vivo, and they are amplified by sequential, but not synchronous, administration of BH3 mimetics. Thus anti-mitotic agents propagate apoptotic priming across heterogeneously sensitive cancer cells through cytosolic DNA sensing pathway-dependent extracellular signals, exploitable by delayed MOMP targeting.


Assuntos
Antimitóticos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Proteínas de Membrana/metabolismo , Comunicação Parácrina/efeitos dos fármacos , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular , Feminino , Técnicas de Inativação de Genes , Humanos , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Proteínas de Membrana/genética , Camundongos , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Paclitaxel/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/metabolismo
12.
J Hematol Oncol ; 11(1): 137, 2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30545397

RESUMO

BACKGROUND: Human myeloma cell lines (HMCLs) are widely used for their representation of primary myeloma cells because they cover patient diversity, although not fully. Their genetic background is mostly undiscovered, and no comprehensive study has ever been conducted in order to reveal those details. METHODS: We performed whole-exon sequencing of 33 HMCLs, which were established over the last 50 years in 12 laboratories. Gene expression profiling and drug testing for the 33 HMCLs are also provided and correlated to exon-sequencing findings. RESULTS: Missense mutations were the most frequent hits in genes (92%). HMCLs harbored between 307 and 916 mutations per sample, with TP53 being the most mutated gene (67%). Recurrent bi-allelic losses were found in genes involved in cell cycle regulation (RB1, CDKN2C), the NFκB pathway (TRAF3, BIRC2), and the p53 pathway (TP53, CDKN2A). Frequency of mutations/deletions in HMCLs were either similar to that of patients (e.g., DIS3, PRDM1, KRAS) or highly increased (e.g., TP53, CDKN2C, NRAS, PRKD2). MAPK was the most altered pathway (82% of HMCLs), mainly by RAS mutants. Surprisingly, HMCLs displayed alterations in epigenetic (73%) and Fanconi anemia (54%) and few alterations in apoptotic machinery. We further identified mutually exclusive and associated mutations/deletions in genes involved in the MAPK and p53 pathways as well as in chromatin regulator/modifier genes. Finally, by combining the gene expression profile, gene mutation, gene deletion, and drug response, we demonstrated that several targeted drugs overcome or bypass some mutations. CONCLUSIONS: With this work, we retrieved genomic alterations of HMCLs, highlighting that they display numerous and unprecedented abnormalities, especially in DNA regulation and repair pathways. Furthermore, we demonstrate that HMCLs are a reliable model for drug screening for refractory patients at diagnosis or at relapse.


Assuntos
Reparo do DNA/genética , DNA/genética , Mieloma Múltiplo/genética , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular , Éxons , Humanos , Mieloma Múltiplo/metabolismo , Mutação
13.
Blood Adv ; 2(23): 3492-3505, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30530776

RESUMO

In this study, we assessed the sensitivity of myeloma cells to the oncolytic measles virus (MV) in relation to p53 using 37 cell lines and 23 primary samples. We showed that infection and cell death were correlated with CD46 expression, which was associated with TP53 status; TP53 abn cell lines highly expressed CD46 and were preferentially infected by MV when compared with the TP53 wt cell lines (P = .046 and P = .045, respectively). Infection of myeloma cells was fully dependent on CD46 expression in both cell lines and primary cells. In the TP53 wt cell lines, but not the TP53 abn cell lines, activation of the p53 pathway with nutlin3a inhibited both CD46 expression and MV infection, while TP53 silencing reciprocally increased CD46 expression and MV infection. We showed using a p53 chromatin immunoprecipitation assay and microRNA assessment that CD46 gene expression was directly and indirectly regulated by p53. Primary myeloma cells overexpressed CD46 as compared with normal cells and were highly infected and killed by MV. CD46 expression and MV infection were inhibited by nutlin3a in primary p53-competent myeloma cells, but not in p53-deficient myeloma cells, and the latter were highly sensitive to MV infection. In summary, myeloma cells were highly sensitive to MV and infection inhibition by the p53 pathway was abrogated in p53-deficient myeloma cells. These results argue for an MV-based clinical trial for patients with p53 deficiency.


Assuntos
Vírus do Sarampo/fisiologia , Proteína Cofatora de Membrana/metabolismo , Mieloma Múltiplo/patologia , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Humanos , Proteína Cofatora de Membrana/antagonistas & inibidores , Proteína Cofatora de Membrana/genética , MicroRNAs/metabolismo , Mieloma Múltiplo/metabolismo , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/química , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética
14.
Melanoma Res ; 25(4): 279-83, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25968572

RESUMO

We recently characterized two melanoma antigens MELOE-1 and MELOE-2 derived from a polycistronic RNA overexpressed in the melanocytic lineage. This transcription profile was because of hypomethylation of the meloe proximal promoter in melanomas and melanocytes. Here, we investigate whether this demethylation was restricted to the meloe promoter or was linked to a general lack of methylation at the meloe locus in the melanocytic lineage. We establish the methylation pattern of the locus spanning more than 40 kbp, focusing on CpG islands, using DNA bisulfite conversion and pyrosequencing. The study was carried out on cultured cell lines (melanoma, melanocyte, colon cancer, and mesothelioma cell lines), healthy tissues (skin and colon), and melanoma tumors. Demethylation, specifically observed in the melanocytic lineage, involves a large promoter area and not the entire meloe locus. This enables updating a tight regulation of meloe transcription in this lineage, suggesting tissue-specific epigenetic mechanisms. Associated with the previously described translational mechanisms, leading to the specific expression of MELOE-1 and MELOE-2 in melanomas, this makes MELOE-derived antigens a relevant candidate for immunotherapy of melanoma.


Assuntos
Antígenos de Neoplasias/genética , Metilação de DNA/genética , Antígenos Específicos de Melanoma/genética , Melanoma/genética , Proteínas de Neoplasias/genética , Sequência de Bases , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoterapia , Melanócitos/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Análise de Sequência de DNA , Transcrição Gênica/genética
15.
PLoS One ; 8(9): e75421, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24086527

RESUMO

The melanoma antigens MELOE-1 and MELOE-2 are encoded by a messenger, called meloe, overexpressed in melanomas compared with other tumour cell types and healthy tissues. They are both able to elicit melanoma-specific T cell responses in melanoma patients, and MELOE-1-specific CD8 T cells have been involved in melanoma immunosurveillance. With the aim to develop immunotherapies targeting this antigen, we investigated the transcriptional mechanisms leading to the preferential expression of meloe messenger in the melanocytic lineage. We defined the minimal promoter region of meloe gene and identified binding motifs for a set of transcription factors. Using mutagenesis, co-transfection experiments and chromatin immunoprecipitation, we showed that transcription factors involved in meloe promoter activity in melanomas were the melanocytic specific SOX9 and SOX10 proteins together with the activated P-CREB protein. Furthermore, we showed that meloe promoter was hypomethylated in melanomas and melanocytes, and hypermethylated in colon cancer cell lines and mesotheliomas, thus explaining the absence of P-CREB binding in these cell lines. This was a second key to explain the overerexpression of meloe messenger in the melanocytic lineage. To our knowledge, such a dual transcriptional control conferring tissue-specificity has never been described for the expression of tumour antigens.


Assuntos
Antígenos de Neoplasias/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/metabolismo , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOXE/metabolismo , Western Blotting , Imunoprecipitação da Cromatina , Metilação de DNA , Primers do DNA/genética , Humanos , Imunoterapia/métodos , Luciferases , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/imunologia , Mutagênese , Reação em Cadeia da Polimerase
16.
PLoS One ; 8(9): e75233, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24086473

RESUMO

Our previous studies on melanoma antigens identified two new polypeptides, named MELOE-1 and MELOE-2, that are involved in immunosurveillance. Intriguingly, these antigens are coded by distinct open reading frames (ORF) of the meloe mRNA which is significantly expressed only in the melanocytic lineage. In addition, MELOE-1 and -2 specific T cell clones recognized melanoma cells but very poorly normal melanocytes suggesting differential translation of meloe in normal vs tumor cells. This prompted us to elucidate the mechanisms of translation of these antigens in melanoma cells. We first demonstrated that no splicing event or cryptic promoter could generate shorter meloe transcripts containing only one of the two ORFs. Triggering meloe RNA degradation with a siRNA close to the ORF coding for MELOE-2 abrogated expression of both MELOE-1 and MELOE-2, thus confirming that the two ORFs are always associated. Next we showed, in a bicistronic reporter system, that IRES activities could be detected upstream of MELOE-1 and MELOE-2 and finally confirmed their translation from full length meloe cDNA in melanoma cells with eGFP constructs. In conclusion, meloe is a polycistronic mRNA that generates both MELOE-1 and MELOE-2 antigens through IRES-dependent translation in melanoma cells and that may explain their tumor specificity.


Assuntos
Antígenos de Neoplasias/genética , Melanoma/imunologia , Proteínas de Neoplasias/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Melanoma/metabolismo , RNA Interferente Pequeno
17.
PLoS One ; 5(1): e8437, 2010 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-20052413

RESUMO

BACKGROUND: Double positive (DP) CD4CD8 Talphabeta cells have been reported in normal individuals as well as in different pathological conditions including inflammatory diseases, viral infections and cancer, but their function remains to be elucidated. We recently reported the increased frequency of DP Talphabeta cells in human breast pleural effusions. This manuscript addresses the question of the existence and above all the role of this non-conventional DP sub-population among tumor associated lymphocytes in melanomas. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the intratumoral cell infiltrate in solid metastasis (n = 6) and tumor invaded lymph nodes (n = 26) samples from melanomas patients by multiparametric cytometry. Here we documented for the first time significant increased frequency of DP T cells in about 60% of melanoma tumors compared to blood samples. Interestingly, a high proportion of these cells produced TNF-alpha in response to autologous melanoma cell lines. Besides, they are characterized by a unique cytokine profile corresponding to higher secretion of IL-13, IL-4 and IL-5 than simple positive T cells. In deep analysis, we derived a representative tumor-reactive DP T cell clone from a melanoma patient's invaded lymph node. This clone was restricted by HLA-A*2402 and recognized both autologous and allogeneic tumor cells of various origins as well as normal cells, suggesting that the target antigen was a ubiquitous self antigen. However, this DP T cell clone failed to kill HLA-A*2402 EBV-transformed B cells, probably due to the constitutive expression of immunoproteasome by these cells. CONCLUSIONS/SIGNIFICANCE: In conclusion, we can postulate that, according to their broad tumor reactivity and to their original cytokine profile, the tumor associated DP T cells could participate in immune responses to tumors in vivo. Therefore, the presence of these cells and their role will be crucial to address in cancer patients, especially in the context of immunotherapies.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Melanoma/imunologia , Células Clonais , Citometria de Fluxo , Humanos , Melanoma/patologia , Metástase Neoplásica
18.
J Exp Med ; 205(11): 2673-82, 2008 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-18936238

RESUMO

A cytotoxic T lymphocyte (CTL) clone was derived from a tumor-infiltrating lymphocyte (TIL) population infused to a melanoma patient who remained relapse free for 10 yr after this adoptive transfer. This clone recognized all melanoma cell lines tested and, to a lower extent, melanocytes, in the context of human histocompatibility leukocyte antigen A2 (HLA-A2), but it did not recognize other tumor cell types. The gene coding for the antigen recognized by this clone was identified by the screening of a melanoma complementary DNA expression library. This antigen is overexpressed in melanomas, compared with other cancer cell lines and healthy tissues, and was thus called melanoma-overexpressed antigen (meloe). Remarkably, the structure of meloe was unusual, with multiple short open reading frames (ORFs). The peptide recognized by the CTL clone was encoded by one of these ORFs, called MELOE-1. Using a specific HLA-A2/peptide tetramer, we showed a correlation between the infusion of TILs containing MELOE-1-specific T cells and relapse prevention in HLA-A2 patients. Indeed, 5 out of 9 patients who did not relapse were infused with TILs that contained MELOE-1-specific T cells, whereas 0 out of the 21 patients who relapsed was infused with such TIL-containing lymphocytes. Overall, our results suggest that this new antigen is involved in immunosurveillance and, thus, represents an attractive target for immunotherapy protocols of melanoma.


Assuntos
Antígenos de Neoplasias/imunologia , Regulação Neoplásica da Expressão Gênica/imunologia , Linfócitos do Interstício Tumoral/imunologia , Melanoma/imunologia , Proteínas de Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Transferência Adotiva , Animais , Antígenos de Neoplasias/genética , Células COS , Chlorocebus aethiops , Primers do DNA/genética , DNA Complementar/genética , Humanos , Separação Imunomagnética , Linfócitos do Interstício Tumoral/metabolismo , Melanoma/metabolismo , Antígenos Específicos de Melanoma , Proteínas de Neoplasias/genética , Oligonucleotídeos , Recidiva , Linfócitos T Citotóxicos/metabolismo
19.
J Immunol ; 177(5): 3100-7, 2006 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-16920947

RESUMO

HLA-E are nonclassical MHC molecules with poorly characterized tissue distribution and functions. Because of their capacity to bind the inhibitory receptor, CD94/NKG2A, expressed by NK cells and CTL, HLA-E molecules might play an important role in immunomodulation. In particular, expression of HLA-E might favor tumor cell escape from CTL and NK immunosurveillance. To address the potential role of HLA-E in melanoma immunobiology, we assessed the expression of these molecules ex vivo in human melanoma biopsies and in melanoma and melanocyte cell lines. Melanoma cell lines expressed no or low surface, but significant intracellular levels of HLA-E. We also report for the first time that some of them produced a soluble form of this molecule. IFN-gamma significantly increased the surface expression of HLA-E and the shedding of soluble HLA-E by these cells, in a metalloproteinase-dependent fashion. In contrast, melanocyte cell lines constitutively expressed HLA-E molecules that were detectable both at the cell surface and in the soluble form, at levels that were poorly affected by IFN-gamma treatment. On tumor sections, a majority of tumor cells of primary, but a low proportion of metastatic melanomas (30-70 and 10-20%, respectively), expressed HLA-E. Finally, HLA-E expression at the cell surface of melanoma cells decreased their susceptibility to CTL lysis. These data demonstrate that HLA-E expression and shedding are normal features of melanocytes, which are conserved in melanoma cells of primary tumors, but become dependent on IFN-gamma induction after metastasis. The biological significance of these findings warrants further investigation.


Assuntos
Antígenos HLA/metabolismo , Melanócitos/metabolismo , Melanoma/metabolismo , Linfócitos T Citotóxicos/metabolismo , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Suscetibilidade a Doenças , Humanos , Imuno-Histoquímica , Interferon gama/farmacologia , Melanócitos/efeitos dos fármacos , Melanoma/patologia , Solubilidade
20.
Int Immunol ; 17(9): 1193-200, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16027137

RESUMO

We investigated the generation of myeloma-specific CTLs from normal donors HLA mismatched with the myeloma cell line SBN. The T-cell line obtained was cloned and each CTL was assessed against SBN and SBN-EBV (a B-EBV cell line obtained by EBV infection of B cells from SBN patient) simultaneously. Among >270 clones evaluated, 2 CTLs (Vbeta13.1 and Vbeta17) killed SBN but spared SBN-EBV cells. Antibodies against HLA-I, but not HLA-A2, molecules abrogated their recognition of SBN. Moreover, SBN recognition was abrogated by anti-HLA-Cw6 antiserum. Both clones recognized two other HLA-Cw*0602 myeloma cell lines. Neither of them recognized HLA-Cw*0602 B-EBV cell lines, the PBMCs of HLA-Cw*0602-unrelated donors or HLA-Cw*0602 melanoma cell lines. We showed that HLA-Cw6 molecules were more expressed at the cell surface of B-EBV cells as compared with myeloma cells, suggesting that the lack of reactivity against B-EBV cells was not related to a low level of HLA expression. Since CTL clones did not express any KIR or NKG2D, we excluded the fact that NK cell receptors could be involved in myeloma-specific recognition through KIR-HLA-I or NKG2D-MICA,B interactions. Cold target competition and acid elution experiments confirmed that myeloma cell recognition was peptide dependent.


Assuntos
Antígenos de Neoplasias/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Mieloma Múltiplo/imunologia , Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Linhagem Celular Tumoral , Humanos
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