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1.
Nat Commun ; 12(1): 2482, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33931647

RESUMO

While oncogenes promote tumorigenesis, they also induce deleterious cellular stresses, such as apoptosis, that cancer cells must combat by coopting adaptive responses. Whether tumor suppressor gene haploinsufficiency leads to such phenomena and their mechanistic basis is unclear. Here, we demonstrate that elevated levels of the anti-apoptotic factor, CASP8 and FADD-like apoptosis regulator (CFLAR), promotes apoptosis evasion in acute myeloid leukemia (AML) cells haploinsufficient for the cut-like homeobox 1 (CUX1) transcription factor, whose loss is associated with dismal clinical prognosis. Genome-wide CRISPR/Cas9 screening identifies CFLAR as a selective, acquired vulnerability in CUX1-deficient AML, which can be mimicked therapeutically using inhibitor of apoptosis (IAP) antagonists in murine and human AML cells. Mechanistically, CUX1 deficiency directly alleviates CUX1 repression of the CFLAR promoter to drive CFLAR expression and leukemia survival. These data establish how haploinsufficiency of a tumor suppressor is sufficient to induce advantageous anti-apoptosis cell survival pathways and concurrently nominate CFLAR as potential therapeutic target in these poor-prognosis leukemias.


Assuntos
Apoptose/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Haploinsuficiência , Proteínas de Homeodomínio/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/genética , Imunoprecipitação da Cromatina , Dipeptídeos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Genes Supressores de Tumor , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Indóis/farmacologia , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Leucemia Mielomonocítica Crônica/genética , Leucemia Mielomonocítica Crônica/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Análise Serial de Proteínas , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo
2.
Front Immunol ; 10: 942, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31114583

RESUMO

Staphylococcal and streptococcal superantigens are virulence factors that cause toxic shock by hyperinducing inflammatory cytokines. Effective T-cell activation requires interaction between the principal costimulatory receptor CD28 and its two coligands, B7-1 (CD80) and B7-2 (CD86). To elicit an inflammatory cytokine storm, bacterial superantigens must bind directly into the homodimer interfaces of CD28 and B7-2. Recent evidence revealed that by engaging CD28 and B7-2 directly at their dimer interface, staphylococcal enterotoxin B (SEB) potently enhances intercellular synapse formation mediated by B7-2 and CD28, resulting in T-cell hyperactivation. Here, we addressed the question, whether diverse bacterial superantigens share the property of triggering B7-2/CD28 receptor engagement and if so, whether they are capable of enhancing also the interaction between B7-1 and CD28, which occurs with an order-of-magnitude higher affinity. To this end, we compared the ability of distinct staphylococcal and streptococcal superantigens to enhance intercellular B7-2/CD28 engagement. Each of these diverse superantigens promoted B7-2/CD28 engagement to a comparable extent. Moreover, they were capable of triggering the intercellular B7-1/CD28 interaction, analyzed by flow cytometry of co-cultured cell populations transfected separately to express human CD28 or B7-1. Streptococcal mitogenic exotoxin Z (SMEZ), the most potent superantigen known, was as sensitive as SEB, SEA and toxic shock syndrome toxin-1 (TSST-1) to inhibition of inflammatory cytokine induction by CD28 and B7-2 dimer interface mimetic peptides. Thus, superantigens act not only by mediating unconventional interaction between MHC-II molecule and T-cell receptor but especially, by strongly promoting engagement of CD28 by its B7-2 and B7-1 coligands, a critical immune checkpoint, forcing the principal costimulatory axis to signal excessively. Our results show that the diverse superantigens use a common mechanism to subvert the inflammatory response, strongly enhancing B7-1/CD28 and B7-2/CD28 costimulatory receptor engagement.


Assuntos
Antígeno B7-1/imunologia , Antígeno B7-2/imunologia , Toxinas Bacterianas/toxicidade , Antígenos CD28/imunologia , Staphylococcus aureus/imunologia , Streptococcus pyogenes/imunologia , Superantígenos/toxicidade , Linfócitos T/imunologia , Toxinas Bacterianas/imunologia , Células HEK293 , Humanos , Inflamação/induzido quimicamente , Inflamação/imunologia , Inflamação/patologia , Ativação Linfocitária/efeitos dos fármacos , Superantígenos/imunologia , Linfócitos T/patologia
3.
Proc Natl Acad Sci U S A ; 113(42): E6437-E6446, 2016 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-27708164

RESUMO

Full T-cell activation requires interaction between the costimulatory receptors B7-2 and CD28. By binding CD28, bacterial superantigens elicit harmful inflammatory cytokine overexpression through an unknown mechanism. We show that, by engaging not only CD28 but also its coligand B7-2 directly, superantigens potently enhance the avidity between B7-2 and CD28, inducing thereby T-cell hyperactivation. Using the same 12-aa ß-strand-hinge-α-helix domain, superantigens engage both B7-2 and CD28 at their homodimer interfaces, areas remote from where these coreceptors interact, implying that inflammatory signaling can be controlled through the receptor homodimer interfaces. Short B7-2 dimer interface mimetic peptides bind diverse superantigens, prevent superantigen binding to cell-surface B7-2 or CD28, attenuate inflammatory cytokine overexpression, and protect mice from lethal superantigen challenge. Thus, superantigens induce a cytokine storm not only by mediating the interaction between MHC-II molecule and T-cell receptor but also, critically, by promoting B7-2/CD28 coreceptor engagement, forcing the principal costimulatory axis to signal excessively. Our results reveal a role for B7-2 as obligatory receptor for superantigens. B7-2 homodimer interface mimotopes prevent superantigen lethality by blocking the superantigen-host costimulatory receptor interaction.


Assuntos
Antígeno B7-2/metabolismo , Antígenos CD28/metabolismo , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Superantígenos/imunologia , Sequência de Aminoácidos , Animais , Antígeno B7-2/química , Antígeno B7-2/genética , Linhagem Celular Tumoral , Citocinas/genética , Enterotoxinas/química , Enterotoxinas/imunologia , Feminino , Humanos , Camundongos , Modelos Moleculares , Mimetismo Molecular , Peptídeos/química , Peptídeos/imunologia , Peptídeos/metabolismo , Ligação Proteica/imunologia , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes de Fusão , Transdução de Sinais , Superantígenos/química , Superantígenos/metabolismo
4.
Genome Biol ; 16: 213, 2015 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-26415775

RESUMO

BACKGROUND: Pluripotent embryonic stem cells (ESCs) have the unique ability to differentiate into every cell type and to self-renew. These characteristics correlate with a distinct nuclear architecture, epigenetic signatures enriched for active chromatin marks and hyperdynamic binding of structural chromatin proteins. Recently, several chromatin-related proteins have been shown to regulate ESC pluripotency and/or differentiation, yet the role of the major heterochromatin proteins in pluripotency is unknown. RESULTS: Here we identify Heterochromatin Protein 1ß (HP1ß) as an essential protein for proper differentiation, and, unexpectedly, for the maintenance of pluripotency in ESCs. In pluripotent and differentiated cells HP1ß is differentially localized and differentially associated with chromatin. Deletion of HP1ß, but not HP1α, in ESCs provokes a loss of the morphological and proliferative characteristics of embryonic pluripotent cells, reduces expression of pluripotency factors and causes aberrant differentiation. However, in differentiated cells, loss of HP1ß has the opposite effect, perturbing maintenance of the differentiation state and facilitating reprogramming to an induced pluripotent state. Microscopy, biochemical fractionation and chromatin immunoprecipitation reveal a diffuse nucleoplasmic distribution, weak association with chromatin and high expression levels for HP1ß in ESCs. The minor fraction of HP1ß that is chromatin-bound in ESCs is enriched within exons, unlike the situation in differentiated cells, where it binds heterochromatic satellite repeats and chromocenters. CONCLUSIONS: We demonstrate an unexpected duality in the role of HP1ß: it is essential in ESCs for maintaining pluripotency, while it is required for proper differentiation in differentiated cells. Thus, HP1ß function both depends on, and regulates, the pluripotent state.


Assuntos
Proteínas Cromossômicas não Histona/genética , Células-Tronco Embrionárias , Heterocromatina/genética , Células-Tronco Pluripotentes Induzidas , Animais , Diferenciação Celular/genética , Reprogramação Celular/genética , Cromatina/genética , Proteínas Cromossômicas não Histona/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Camundongos , Camundongos Knockout
6.
PLoS One ; 10(7): e0134026, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26218064

RESUMO

Expression of the Bcr-Abl fusion gene in hematopoietic progenitor cells (HPCs) results in the development of chronic myelogenous leukemia (CML), for which hematopoietic microenvironment plays an important role. We investigated the specific effects of an HPC line transduced with Bcr-Abl, KOBA, on BM-derived OP9 stroma cells. DNA microarray analysis revealed that OP9 cells co-cultured with KOBA cells (OP9/L) show diverse changes in the gene expression. OP9/L cells showed significant down-regulation of Cdkn genes and up-regulation of Icam1, leading to the increased proliferation capacity of OP9 cells and enhanced transmigration of leukemia cells through them. The effects were attributed to direct Notch activation of OP9 cells by KOBA cells. OP9/L cells also showed a markedly altered cytokine gene expression pattern, including a robust increase in a variety of proinflammatory genes and a decrease in hematopoietic cytokines such as Cxcl12, Scf, and Angpt1. Consequently, OP9/L cells promoted the proliferation of KOBA cells more efficiently than parental OP9 cells, whereas the activity supporting normal myelopoiesis was attenuated. In mice bearing KOBA leukemia, the characteristic genetic changes observed in OP9/L cells were reflected differentially in the endothelial cells (ECs) and mesenchymal stroma cells (MCs) of the BM. The ECs were markedly increased with Notch-target gene activation and decreased Cdkn expression, whereas the MCs showed a marked increase in proinflammatory gene expression and a profound decrease in hematopoietic genes. Human CML cell lines also induced essentially similar genetic changes in OP9 cells. Our results suggest that CML cells remodel the hematopoietic microenvironment by changing the gene expression patterns differentially in ECs and MCs of BM.


Assuntos
Proliferação de Células , Proteínas de Fusão bcr-abl/genética , Proteínas Ativadoras de GTPase/fisiologia , Regulação Leucêmica da Expressão Gênica , Células-Tronco Hematopoéticas/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas Nucleares/fisiologia , Células Estromais/patologia , Animais , Apoptose , Western Blotting , Adesão Celular , Movimento Celular , Células-Tronco Hematopoéticas/metabolismo , Humanos , Técnicas Imunoenzimáticas , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/metabolismo , Células Tumorais Cultivadas
7.
PLoS Biol ; 9(9): e1001149, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21931534

RESUMO

Bacterial superantigens, a diverse family of toxins, induce an inflammatory cytokine storm that can lead to lethal shock. CD28 is a homodimer expressed on T cells that functions as the principal costimulatory ligand in the immune response through an interaction with its B7 coligands, yet we show here that to elicit inflammatory cytokine gene expression and toxicity, superantigens must bind directly into the dimer interface of CD28. Preventing access of the superantigen to CD28 suffices to block its lethality. Mice were protected from lethal superantigen challenge by short peptide mimetics of the CD28 dimer interface and by peptides selected to compete with the superantigen for its binding site in CD28. Superantigens use a conserved ß-strand/hinge/α-helix domain of hitherto unknown function to engage CD28. Mutation of this superantigen domain abolished inflammatory cytokine gene induction and lethality. Structural analysis showed that when a superantigen binds to the T cell receptor on the T cell and major histocompatibility class II molecule on the antigen-presenting cell, CD28 can be accommodated readily as third superantigen receptor in the quaternary complex, with the CD28 dimer interface oriented towards the ß-strand/hinge/α-helix domain in the superantigen. Our findings identify the CD28 homodimer interface as a critical receptor target for superantigens. The novel role of CD28 as receptor for a class of microbial pathogens, the superantigen toxins, broadens the scope of pathogen recognition mechanisms.


Assuntos
Antígenos CD28/imunologia , Citocinas/genética , Choque Séptico/imunologia , Superantígenos/imunologia , Sequência de Aminoácidos , Animais , Toxinas Bacterianas/imunologia , Antígenos CD28/genética , Linhagem Celular Tumoral , Citocinas/imunologia , Enterotoxinas/imunologia , Mapeamento de Epitopos , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Regulação da Expressão Gênica , Vetores Genéticos , Humanos , Imunidade Celular , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Ligação Proteica , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Choque Séptico/genética , Staphylococcus aureus/genética , Staphylococcus aureus/imunologia , Superantígenos/administração & dosagem , Ressonância de Plasmônio de Superfície
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