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1.
Sci Rep ; 12(1): 1848, 2022 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-35115608

RESUMO

WDR5 nucleates the assembly of histone-modifying complexes and acts outside this context in a range of chromatin-centric processes. WDR5 is also a prominent target for pharmacological inhibition in cancer. Small-molecule degraders of WDR5 have been described, but most drug discovery efforts center on blocking the WIN site of WDR5, an arginine binding cavity that engages MLL/SET enzymes that deposit histone H3 lysine 4 methylation (H3K4me). Therapeutic application of WIN site inhibitors is complicated by the disparate functions of WDR5, but is generally guided by two assumptions-that WIN site inhibitors disable all functions of WDR5, and that changes in H3K4me drive the transcriptional response of cancer cells to WIN site blockade. Here, we test these assumptions by comparing the impact of WIN site inhibition versus WDR5 degradation on H3K4me and transcriptional processes. We show that WIN site inhibition disables only a specific subset of WDR5 activity, and that H3K4me changes induced by WDR5 depletion do not explain accompanying transcriptional responses. These data recast WIN site inhibitors as selective loss-of-function agents, contradict H3K4me as a relevant mechanism of action for WDR5 inhibitors, and indicate distinct clinical applications of WIN site inhibitors and WDR5 degraders.


Assuntos
Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Linfoma de Células B/tratamento farmacológico , Sítios de Ligação , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Montagem e Desmontagem da Cromatina , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Metilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteólise , Transdução de Sinais , Transcrição Gênica
2.
Elife ; 102021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33416496

RESUMO

The oncoprotein transcription factor MYC is a major driver of malignancy and a highly validated but challenging target for the development of anticancer therapies. Novel strategies to inhibit MYC may come from understanding the co-factors it uses to drive pro-tumorigenic gene expression programs, providing their role in MYC activity is understood. Here we interrogate how one MYC co-factor, host cell factor (HCF)-1, contributes to MYC activity in a human Burkitt lymphoma setting. We identify genes connected to mitochondrial function and ribosome biogenesis as direct MYC/HCF-1 targets and demonstrate how modulation of the MYC-HCF-1 interaction influences cell growth, metabolite profiles, global gene expression patterns, and tumor growth in vivo. This work defines HCF-1 as a critical MYC co-factor, places the MYC-HCF-1 interaction in biological context, and highlights HCF-1 as a focal point for development of novel anti-MYC therapies.


Tumours form when cells lose control of their growth. Usually, cells produce signals that control how much and how often they divide. But if these signals become faulty, cells may grow too quickly or multiply too often. For example, a group of proteins known as MYC proteins activate growth genes in a cell, but too much of these proteins causes cells to grow uncontrollably. With one third of all cancer deaths linked to excess MYC proteins, these molecules could be key targets for anti-cancer drugs. However, current treatments fail to target these proteins. One option for treating cancers linked to MYC proteins could be to target proteins that work alongside MYC proteins, such as the protein HCF-1, which can attach to MYC proteins. To test if HCF-1 could be a potential drug target, Popay et al. first studied how HCF-1 and MYC proteins interacted using specific cancer cells grown in the laboratory. This revealed that when the two proteins connected, they activated genes that trigger rapid cell growth. When these cancer cells were then injected into mice, tumours quickly grew. However, when the MYC and HCF-1 attachments in the cancer cells were disrupted, the tumours shrunk. This suggests that if anti-cancer drugs were able to target HCF-1 proteins, they could potentially reduce or even reverse the growth of tumours. While further research is needed to identify drug candidates, these findings reveal a promising target for treating tumours that stem from over-abundant MYC proteins.


Assuntos
Expressão Gênica , Genes Mitocondriais , Fator C1 de Célula Hospedeira/genética , Biogênese de Organelas , Proteínas Proto-Oncogênicas c-myc/genética , Ribossomos/fisiologia , Animais , Linfoma de Burkitt , Feminino , Fator C1 de Célula Hospedeira/metabolismo , Humanos , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas c-myc/metabolismo
3.
J Med Chem ; 63(8): 4315-4333, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32223236

RESUMO

The frequent deregulation of MYC and its elevated expression via multiple mechanisms drives cells to a tumorigenic state. Indeed, MYC is overexpressed in up to ∼50% of human cancers and is considered a highly validated anticancer target. Recently, we discovered that WD repeat-containing protein 5 (WDR5) binds to MYC and is a critical cofactor required for the recruitment of MYC to its target genes and reported the first small molecule inhibitors of the WDR5-MYC interaction using structure-based design. These compounds display high binding affinity, but have poor physicochemical properties and are hence not suitable for in vivo studies. Herein, we conducted an NMR-based fragment screening to identify additional chemical matter and, using a structure-based approach, we merged a fragment hit with the previously reported sulfonamide series. Compounds in this series can disrupt the WDR5-MYC interaction in cells, and as a consequence, we observed a reduction of MYC localization to chromatin.


Assuntos
Desenho de Fármacos , Descoberta de Drogas/métodos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Sulfonamidas/síntese química , Sulfonamidas/farmacologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-myc/metabolismo , Relação Estrutura-Atividade
4.
Mol Cell Oncol ; 7(2): 1709388, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32158922

RESUMO

The oncoprotein transcription factor MYC is overexpressed in most cancers and is responsible for hundreds of thousands of cancer deaths worldwide every year. MYC is also a highly validated - but currently undruggable - anti-cancer target. We recently showed that breaking the interaction of MYC with its chromatin co-factor WD repeat-containing protein 5 (WDR5) promotes tumor regression in mouse xenografts, laying the foundation for a new strategy to inhibit MYC in the clinic.

5.
J Med Chem ; 63(2): 656-675, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31858797

RESUMO

WD repeat domain 5 (WDR5) is a member of the WD40-repeat protein family that plays a critical role in multiple chromatin-centric processes. Overexpression of WDR5 correlates with a poor clinical outcome in many human cancers, and WDR5 itself has emerged as an attractive target for therapy. Most drug-discovery efforts center on the WIN site of WDR5 that is responsible for the recruitment of WDR5 to chromatin. Here, we describe discovery of a novel WDR5 WIN site antagonists containing a dihydroisoquinolinone bicyclic core using a structure-based design. These compounds exhibit picomolar binding affinity and selective concentration-dependent antiproliferative activities in sensitive MLL-fusion cell lines. Furthermore, these WDR5 WIN site binders inhibit proliferation in MYC-driven cancer cells and reduce MYC recruitment to chromatin at MYC/WDR5 co-bound genes. Thus, these molecules are useful probes to study the implication of WDR5 inhibition in cancers and serve as a potential starting point toward the discovery of anti-WDR5 therapeutics.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/síntese química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Quinolonas/síntese química , Quinolonas/farmacologia , Repetições WD40/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Cromatina/efeitos dos fármacos , Cromatina/genética , Cristalografia por Raios X , Desenho de Fármacos , Descoberta de Drogas , Repressão Epigenética/efeitos dos fármacos , Genes myc/efeitos dos fármacos , Humanos , Relação Estrutura-Atividade
6.
J Med Chem ; 62(24): 11232-11259, 2019 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-31724864

RESUMO

The treatment of tumors driven by overexpression or amplification of MYC oncogenes remains a significant challenge in drug discovery. Here, we present a new strategy toward the inhibition of MYC via the disruption of the protein-protein interaction between MYC and its chromatin cofactor WD Repeat-Containing Protein 5. Blocking the association of these proteins is hypothesized to disrupt the localization of MYC to chromatin, thus disrupting the ability of MYC to sustain tumorigenesis. Utilizing a high-throughput screening campaign and subsequent structure-guided design, we identify small-molecule inhibitors of this interaction with potent in vitro binding affinity and report structurally related negative controls that can be used to study the effect of this disruption. Our work suggests that disruption of this protein-protein interaction may provide a path toward an effective approach for the treatment of multiple tumors and anticipate that the molecules disclosed can be used as starting points for future efforts toward compounds with improved drug-like properties.


Assuntos
Descoberta de Drogas , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Ácido Salicílico/química , Bibliotecas de Moléculas Pequenas/farmacologia , Sulfonamidas/farmacologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Repetições WD40
7.
Proc Natl Acad Sci U S A ; 116(50): 25260-25268, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31767764

RESUMO

The oncoprotein transcription factor MYC is overexpressed in the majority of cancers. Key to its oncogenic activity is the ability of MYC to regulate gene expression patterns that drive and maintain the malignant state. MYC is also considered a validated anticancer target, but efforts to pharmacologically inhibit MYC have failed. The dependence of MYC on cofactors creates opportunities for therapeutic intervention, but for any cofactor this requires structural understanding of how the cofactor interacts with MYC, knowledge of the role it plays in MYC function, and demonstration that disrupting the cofactor interaction will cause existing cancers to regress. One cofactor for which structural information is available is WDR5, which interacts with MYC to facilitate its recruitment to chromatin. To explore whether disruption of the MYC-WDR5 interaction could potentially become a viable anticancer strategy, we developed a Burkitt's lymphoma system that allows replacement of wild-type MYC for mutants that are defective for WDR5 binding or all known nuclear MYC functions. Using this system, we show that WDR5 recruits MYC to chromatin to control the expression of genes linked to biomass accumulation. We further show that disrupting the MYC-WDR5 interaction within the context of an existing cancer promotes rapid and comprehensive tumor regression in vivo. These observations connect WDR5 to a core tumorigenic function of MYC and establish that, if a therapeutic window can be established, MYC-WDR5 inhibitors could be developed as anticancer agents.


Assuntos
Linfoma de Burkitt/metabolismo , Cromatina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Linfoma de Burkitt/genética , Carcinogênese , Linhagem Celular Tumoral , Cromatina/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Nus , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética
8.
Cell Rep ; 26(11): 2916-2928.e13, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30865883

RESUMO

The chromatin-associated protein WDR5 is a promising target for pharmacological inhibition in cancer. Drug discovery efforts center on the blockade of the "WIN site" of WDR5, a well-defined pocket that is amenable to small molecule inhibition. Various cancer contexts have been proposed to be targets for WIN site inhibitors, but a lack of understanding of WDR5 target genes and of the primary effects of WIN site inhibitors hampers their utility. Here, by the discovery of potent WIN site inhibitors, we demonstrate that the WIN site links WDR5 to chromatin at a small cohort of loci, including a specific subset of ribosome protein genes. WIN site inhibitors rapidly displace WDR5 from chromatin and decrease the expression of associated genes, causing translational inhibition, nucleolar stress, and p53 induction. Our studies define a mode by which WDR5 engages chromatin and forecast that WIN site blockade could have utility against multiple cancer types.


Assuntos
Cromatina/metabolismo , Inibidores Enzimáticos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Inibidores Enzimáticos/síntese química , Feminino , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/química , Masculino , Ligação Proteica/efeitos dos fármacos
9.
BMC Cancer ; 17(1): 241, 2017 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-28376728

RESUMO

BACKGROUND: Triple negative breast cancer (TNBC) is a heterogeneous disease that lacks unifying molecular alterations that can guide therapy decisions. We previously identified distinct molecular subtypes of TNBC (TNBCtype) using gene expression data generated on a microarray platform using frozen tumor specimens. Tumors and cell lines representing the identified subtypes have distinct enrichment in biologically relevant transcripts with differing sensitivity to standard chemotherapies and targeted agents. Since our initial discoveries, RNA-sequencing (RNA-seq) has evolved as a sensitive and quantitative tool to measure transcript abundance. METHODS: To demonstrate that TNBC subtypes were similar between platforms, we compared gene expression from matched specimens profiled by both microarray and RNA-seq from The Cancer Genome Atlas (TCGA). In the clinical care of patients with TNBC, tumor specimens collected for diagnostic purposes are processed by formalin fixation and paraffin-embedding (FFPE). Thus, for TNBCtype to eventually have broad and practical clinical utility we performed RNA-seq gene expression and molecular classification comparison between fresh-frozen (FF) and FFPE tumor specimens. RESULTS: Analysis of TCGA showed consistent subtype calls between 91% of evaluable samples demonstrating conservation of TNBC subtypes across microarray and RNA-seq platforms. We compared RNA-seq performed on 21-paired FF and FFPE TNBC specimens and evaluated genome alignment, transcript coverage, differential transcript enrichment and concordance of TNBC molecular subtype calls. We demonstrate that subtype accuracy between matched FF and FFPE samples increases with sequencing depth and correlation strength to an individual TNBC subtype. CONCLUSIONS: TNBC subtypes were reliably identified from FFPE samples, with highest accuracy if the samples were less than 4 years old and reproducible subtyping increased with sequencing depth. To reproducibly subtype tumors using gene expression, it is critical to select genes that do not vary due to platform type, tissue processing or RNA isolation method. The majority of differentially expressed transcripts between matched FF and FFPE samples could be attributed to transcripts selected for by RNA enrichment method. While differentially expressed transcripts did not impact TNBC subtyping, they will provide guidance on determining which transcripts to avoid when implementing a gene set size reduction strategy. TRIAL REGISTRATION: NCT00930930 07/01/2009.


Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Neoplasias/genética , Neoplasias de Mama Triplo Negativas/genética , Feminino , Formaldeído , Humanos , Inclusão em Parafina , RNA/genética , Fixação de Tecidos/métodos , Neoplasias de Mama Triplo Negativas/classificação , Neoplasias de Mama Triplo Negativas/patologia
10.
Cancer Res ; 75(19): 4012-5, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26383167

RESUMO

The MYC oncogenes encode a family of transcription factors that feature prominently in cancer. MYC proteins are overexpressed or deregulated in a majority of malignancies and drive tumorigenesis by inducing widespread transcriptional reprogramming that promotes cell proliferation, metabolism, and genomic instability. The ability of MYC to regulate transcription depends on its dimerization with MAX, which creates a DNA-binding domain that recognizes specific sequences in the regulatory elements of MYC target genes. Recently, we discovered that recognition of target genes by MYC also depends on its interaction with WDR5, a WD40-repeat protein that exists as part of several chromatin-regulatory complexes. Here, we discuss how interaction of MYC with WDR5 could create an avidity-based chromatin recognition mechanism that allows MYC to select its target genes in response to both genetic and epigenetic determinants. We rationalize how the MYC-WDR5 interaction provides plasticity in target gene selection by MYC and speculate on the biochemical and genomic contexts in which this interaction occurs. Finally, we discuss how properties of the MYC-WDR5 interface make it an attractive point for discovery of small-molecule inhibitors of MYC function in cancer cells.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/fisiologia , Montagem e Desmontagem da Cromatina/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Histona-Lisina N-Metiltransferase/fisiologia , Proteínas de Neoplasias/fisiologia , Neoplasias/genética , Proteínas Proto-Oncogênicas c-myc/fisiologia , Proteínas Repressoras/fisiologia , Transdução de Sinais/fisiologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Montagem e Desmontagem da Cromatina/genética , DNA/genética , DNA/metabolismo , Metilação de DNA , Descoberta de Drogas , Epigênese Genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes myc , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Genéticos , Terapia de Alvo Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Neoplasias/tratamento farmacológico , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos
11.
Mol Cell ; 58(3): 440-52, 2015 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-25818646

RESUMO

MYC is an oncoprotein transcription factor that is overexpressed in the majority of malignancies. The oncogenic potential of MYC stems from its ability to bind regulatory sequences in thousands of target genes, which depends on interaction of MYC with its obligate partner, MAX. Here, we show that broad association of MYC with chromatin also depends on interaction with the WD40-repeat protein WDR5. MYC binds WDR5 via an evolutionarily conserved "MYC box IIIb" motif that engages a shallow, hydrophobic cleft on the surface of WDR5. Structure-guided mutations in MYC that disrupt interaction with WDR5 attenuate binding of MYC at ∼80% of its chromosomal locations and disable its ability to promote induced pluripotent stem cell formation and drive tumorigenesis. Our data reveal WDR5 as a key determinant for MYC recruitment to chromatin and uncover a tractable target for the discovery of anticancer therapies against MYC-driven tumors.


Assuntos
Carcinogênese/metabolismo , Cromatina/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Anisotropia , Sítios de Ligação/genética , Carcinogênese/genética , Cromatina/química , Cromatina/genética , Polarização de Fluorescência , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Nus , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Células NIH 3T3 , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/química , Proteínas/genética , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/genética , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-Híbrido
12.
Adv Cancer Res ; 110: 77-106, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21704229

RESUMO

The c-Myc oncogene encodes a multifunctional transcription factor that directs the expression of genes required for cell growth and proliferation. Consistent with its potent growth-promoting properties, cells have evolved numerous mechanisms that limit the expression and activity of Myc. One of the most prominent of these mechanisms is proteolysis, which destroys Myc within minutes of its synthesis. The rapid and controlled destruction of Myc keeps its levels low and precisely tied to processes that regulate Myc production. In this review, we discuss how Myc protein stability is regulated and the influence of Myc proteolysis on its function. We describe what is known about how Myc is destroyed by ubiquitin (Ub)-mediated proteolysis, attempt to rationalize the role of different Ub-protein ligases and deubiquitylating enzymes (dUbs) in the regulation of Myc stability, and detail how these processes go awry in cancer. Finally, we discuss how our understanding of Myc regulation by the ubiquitin-proteasome system (UPS) can expose strategies for therapeutic intervention in human malignancies.


Assuntos
Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
13.
Adv Exp Med Biol ; 650: 103-15, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19731805

RESUMO

A hallmark feature of adaptive immunity is the production of lymphocytes bearing an enormous repertoire of receptors for foreign antigens. This repertoire is generated early in B and T-cell development by the process of V(D)J recombination, which randomly assembles functional immunoglobulin (Ig) and T-cell receptor (TCR) genes from large arrays of DNA segments. Precursor lymphocytes must target then retarget a single V(D)J recombinase enzyme to distinct regions within antigen receptor loci to guide lymphocyte development and to ensure that each mature B and T-cell expresses only a single antigen receptor specificity. Proper targeting of V(D)J recombinase is also essential to avoid chromosomal aberrations that result in lymphoid malignancies. Early studies suggested that changes in the specificity of V(D)J recombination are achieved by differentially opening or closing chromatin associated with Ig and TCR gene segments at the proper developmental time point. This accessibility model has been extended significantly in recent years and it has become clear that control mechanisms governing antigen receptor gene assembly are multifaceted and vary from locus to locus. In this chapter we review how genetic and epigenetic mechanisms as well as widespread changes in chromosomal conformation synergize to orchestrate the diversification of genes encoding B and T-cell antigen receptors.


Assuntos
Rearranjo Gênico do Linfócito B , Rearranjo Gênico do Linfócito T , Genes de Imunoglobulinas , Genes Codificadores dos Receptores de Linfócitos T , Recombinação Genética , Alelos , Animais , Cromatina/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , VDJ Recombinases/genética
14.
J Immunol ; 181(1): 485-93, 2008 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-18566414

RESUMO

Lymphocyte development is controlled by dynamic repression and activation of gene expression. These developmental programs include the ordered, tissue-specific assembly of Ag receptor genes by V(D)J recombination. Changes in gene expression and the targeting of V(D)J recombination are largely controlled by patterns of epigenetic modifications imprinted on histones and DNA, which alter chromatin accessibility to nuclear factors. An important component of this epigenetic code is methylation of histone H3 at lysine 9 (H3K9me), which is catalyzed by histone methyltransferases and generally leads to gene repression. However, the function and genetic targets of H3K9 methyltransferases during lymphocyte development remain unknown. To elucidate the in vivo function of H3K9me, we generated mice lacking G9a, a major H3K9 histone methyltransferase, in lymphocytes. Surprisingly, lymphocyte development is unperturbed in G9a-deficient mice despite a significant loss of H3K9me2 in precursor B cells. G9a deficiency is manifest as modest defects in the proliferative capacity of mature B cells and their differentiation into plasma cells following stimulation with LPS and IL-4. Precursor lymphocytes from the mutant mice retain tissue- and stage-specific control over V(D)J recombination. However, G9a deficiency results in reduced usage of Iglambda L chains and a corresponding inhibition of Iglambda gene assembly in bone marrow precursors. These findings indicate that the H3K9me2 epigenetic mark affects a highly restricted set of processes during lymphocyte development and activation.


Assuntos
Linfócitos B/enzimologia , Histona-Lisina N-Metiltransferase/metabolismo , Linfócitos T/enzimologia , Animais , Formação de Anticorpos/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Proliferação de Células , Células Cultivadas , Ativação Enzimática , Histona-Lisina N-Metiltransferase/deficiência , Histona-Lisina N-Metiltransferase/genética , Cadeias lambda de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/imunologia , Cadeias lambda de Imunoglobulina/metabolismo , Camundongos , Camundongos Knockout , Especificidade de Órgãos , Linfócitos T/citologia , Linfócitos T/imunologia , VDJ Recombinases/metabolismo
15.
J Biol Chem ; 282(38): 28189-94, 2007 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-17666396

RESUMO

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a cytokine that preferentially induces apoptosis in tumor cells compared with normal cells through two receptors (DR4 and DR5). Somatic mutations in these receptors have been found in different kinds of cancer; however, it is poorly understood how the mutations affect signaling. We found that point mutations (L334F, E326K, E338K, and K386N) that were identified in human tumors result in the DR5 receptor losing its ability to form a functional death-inducing signaling complex and induce apoptosis. The mutant receptors also have a "dominant negative" effect whereby they inhibit the ability of TRAIL to induce apoptosis through functional DR4 receptors. This dominant negative mechanism is achieved through competition for TRAIL binding as shown by experiments where the ability of the mutant DR5 receptor to bind with the ligand was abolished, thus restoring TRAIL signaling through DR4. The inhibitory effect on signaling through the wild-type DR4 protein can be overcome if the inhibitory mechanism is bypassed by using a DR4-agonistic antibody that is not subject to this competition. This study provides a molecular basis for the use of specific therapeutic agonists of TRAIL receptors in people whose tumors harbor somatic DR5 mutations.


Assuntos
Regulação Neoplásica da Expressão Gênica , Mutação , Neoplasias/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligação Competitiva , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Eletroporação , Humanos , Ligantes , Modelos Biológicos , Ligação Proteica , Transdução de Sinais , Frações Subcelulares/metabolismo
16.
Nat Immunol ; 7(12): 1309-16, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17057722

RESUMO

The products of recombination-activating gene 1 (Rag1) and Rag2 are required for T cell receptor gene assembly and thymocyte maturation, yet their transcriptional control mechanisms remain unclear. A congenic strain (called 'ZORI' here) with defects in Rag1 and Rag2 expression, thymocyte maturation and peripheral T cell homeostasis has been developed. Here, we mapped the mutation in this strain to a chromosome 18 locus containing a single known gene encoding the zinc finger protein Zfp608. This gene (Zfp608) was highly expressed in neonatal thymus but was extinguished thereafter. In contrast to wild-type mice, ZORI mice had sustained thymocyte expression of Zfp608 throughout life. The ZORI mutation produced a thymocyte-intrinsic developmental defect. Overexpression of Zfp608 in BALB/c thymocytes substantially impaired Rag1 and Rag2 expression, indicating the underlying mechanism for the defect in ZORI thymocyte development. Thus, the normal function of Zfp608 may be to prevent Rag1 and Rag2 expression in utero.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Genes RAG-1/fisiologia , Linfócitos/citologia , Timo/embriologia , Dedos de Zinco/imunologia , Animais , Proteínas de Ligação a DNA/genética , Citometria de Fluxo , Expressão Gênica/imunologia , Immunoblotting , Hibridização In Situ , Linfócitos/metabolismo , Camundongos , Camundongos Congênicos , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa
17.
Biochim Biophys Acta ; 1736(3): 228-36, 2005 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-16154383

RESUMO

MDA-MB-231, MCF7, and SKOV3 cancer cells, but not HEK-293 cells, expressed mRNA for the leukocyte G protein-coupled 5-oxo-eicosatetraenoate (ETE) OXE receptor. 5-Oxo-ETE, 5-oxo-15-OH-ETE, and 5-HETE stimulated the cancer cell lines but not HEK-293 cells to mount pertussis toxin-sensitive proliferation responses. Their potencies in eliciting this response were similar to their known potencies in activating leukocytes and OXE receptor-transfected cells. However, high concentrations of 5-oxo-ETE and 5-oxo-15-OH-ETE, but not 5-HETE, arrested growth and caused apoptosis in all four cell lines; these responses were pertussis toxin-resistant. The same high concentrations of the oxo-ETEs but again not 5-HETE also activated peroxisome proliferator-activated receptor (PPAR)-gamma. Pharmacological studies indicated that this activation did not mediate their effects on proliferation. These results are the first to implicate the OXE receptor in malignant cell growth and to show that 5-oxo-ETEs activate cell death programs as well as PPARgamma independently of this receptor.


Assuntos
Ácidos Araquidônicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Receptores Eicosanoides/fisiologia , Anilidas/farmacologia , Apoptose/efeitos dos fármacos , Ácidos Araquidônicos/metabolismo , Sítios de Ligação/genética , Caspase 3 , Caspases/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Expressão Gênica/genética , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Ácidos Hidroxieicosatetraenoicos/farmacologia , Mitose/efeitos dos fármacos , PPAR gama/antagonistas & inibidores , PPAR gama/genética , PPAR gama/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/genética , Toxina Pertussis/farmacologia , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Prostaglandina D2/análogos & derivados , Prostaglandina D2/metabolismo , Prostaglandina D2/farmacologia , Ligação Proteica , Receptores Eicosanoides/genética , Transfecção
18.
Mol Biol Cell ; 16(3): 1189-99, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15635090

RESUMO

Although evasion of apoptosis is thought to be required for the development of cancer, it is unclear which cell death pathways are evaded. We previously identified a novel epithelial cell death pathway that works in normal cells but is inactivated in tumor cells, implying that it may be targeted during tumor development. The pathway can be activated by the Fas-associated death domain (FADD) of the adaptor protein but is distinct from the known mechanism of FADD-induced apoptosis through caspase-8. Here, we show that a physiological signal (tumor necrosis factor-related apoptosis-inducing ligand) can kill normal epithelial cells through the endogenous FADD protein by using the novel FADD death domain pathway, which activates both apoptosis and autophagy. We also show that selective resistance to this pathway occurs when primary epithelial cells are immortalized and that this occurs through a mechanism that is independent of known events (telomerase activity, and loss of function of p53, Rb, INK4a, and ARF) that are associated with immortalization. These data identify a novel cell death pathway that combines apoptosis and autophagy and that is selectively inactivated at the earliest stages of epithelial cancer development.


Assuntos
Apoptose , Células Epiteliais/citologia , Fatores de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Adenoviridae/genética , Proteínas Reguladoras de Apoptose , Autofagia , Mama/metabolismo , Carcinoma/patologia , Caspase 8 , Caspases/metabolismo , Morte Celular , Linhagem Celular , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Células Epiteliais/metabolismo , Proteína de Domínio de Morte Associada a Fas , Proteínas de Fluorescência Verde/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Microscopia Eletrônica , Fagocitose , Próstata/patologia , Estrutura Terciária de Proteína , Proteína do Retinoblastoma/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismo , Proteína Supressora de Tumor p53/metabolismo
19.
J Biol Chem ; 279(50): 52479-86, 2004 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-15452120

RESUMO

Members of the tumor necrosis factor (TNF) superfamily of receptors such as Fas/CD95 and the TNF-related apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5 induce apoptosis by recruiting adaptor molecules and caspases. The central adaptor molecule for these receptors is a death domain-containing protein, FADD, which binds to the activated receptor via death domain-death domain interactions. Here, we show that in addition to the death domain, the C-terminal tails of DR4 and DR5 positively regulate FADD binding, caspase activation and apoptosis. In contrast, the corresponding region in the Fas receptor has the opposite effect and inhibits binding to the receptor death domain. Replacement of wild-type or mutant DR5 molecules into DR5-deficient BJAB cells indicates that some agonistic antibodies display an absolute requirement for the C-terminal tail for FADD binding and signaling while other antibodies can function in the absence of this mechanism. These data demonstrate that regions outside the death domains of DR4 and DR5 have opposite effects to that of Fas in regulating FADD recruitment and show that different death receptor agonists can use distinct molecular mechanisms to activate signaling from the same receptor.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/metabolismo , Receptor fas/química , Receptor fas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/agonistas , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose , Proteínas Reguladoras de Apoptose , Sítios de Ligação , Caspases/metabolismo , Linhagem Celular , Proteína de Domínio de Morte Associada a Fas , Teste de Complementação Genética , Células HeLa , Humanos , Técnicas In Vitro , Células Jurkat , Glicoproteínas de Membrana/genética , Camundongos , Modelos Biológicos , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/genética , Receptor fas/genética
20.
J Biol Chem ; 279(31): 32780-5, 2004 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-15173180

RESUMO

Members of the tumor necrosis factor superfamily of receptors induce apoptosis by recruiting adaptor molecules through death domain interactions. The central adaptor molecule for these receptors is the death domain-containing protein Fas-associated death domain (FADD). FADD binds a death domain on a receptor or additional adaptor and recruits caspases to the activated receptor. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signals apoptosis through two receptors, DR4 and DR5. Although there is much interest in TRAIL, the mechanism by which FADD is recruited to the TRAIL receptors is not clear. Using a reverse two-hybrid system we previously identified mutations in the death effector domain of FADD that prevented binding to Fas/CD95. Here we show that these mutations also prevent binding to DR5. FADD-deficient Jurkat cells stably expressing these FADD mutations did not transduce TRAIL or Fas/CD95 signaling. Second site compensating mutations that restore binding to and signaling through Fas/CD95 and DR5 were also in the death effector domain. We conclude that in contrast to current models where the death domain of FADD functions independently of the death effector domain, the death effector domain of FADD comes into direct contact with both TRAIL and Fas/CD95 receptors.


Assuntos
Proteínas de Transporte/química , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Nucleares/química , Receptores do Fator de Necrose Tumoral/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Apoptose , Proteínas de Transporte/metabolismo , Caspases/metabolismo , Linhagem Celular , Clonagem Molecular , Proteínas Correpressoras , Ativação Enzimática , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Células Jurkat , Ligantes , Proteínas Luminescentes/metabolismo , Camundongos , Modelos Moleculares , Chaperonas Moleculares , Mutação , Proteínas Nucleares/metabolismo , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/química , Transdução de Sinais , Termodinâmica , Técnicas do Sistema de Duplo-Híbrido , Valina/química , Receptor fas/química , Receptor fas/metabolismo
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