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1.
EMBO J ; 40(6): e104296, 2021 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-33459422

RESUMEN

The IκB kinase (IKK)-NF-κB pathway is activated as part of the DNA damage response and controls both inflammation and resistance to apoptosis. How these distinct functions are achieved remained unknown. We demonstrate here that DNA double-strand breaks elicit two subsequent phases of NF-κB activation in vivo and in vitro, which are mechanistically and functionally distinct. RNA-sequencing reveals that the first-phase controls anti-apoptotic gene expression, while the second drives expression of senescence-associated secretory phenotype (SASP) genes. The rapidly activated first phase is driven by the ATM-PARP1-TRAF6-IKK cascade, which triggers proteasomal destruction of inhibitory IκBα, and is terminated through IκBα re-expression from the NFKBIA gene. The second phase, which is activated days later in senescent cells, is on the other hand independent of IKK and the proteasome. An altered phosphorylation status of NF-κB family member p65/RelA, in part mediated by GSK3ß, results in transcriptional silencing of NFKBIA and IKK-independent, constitutive activation of NF-κB in senescence. Collectively, our study reveals a novel physiological mechanism of NF-κB activation with important implications for genotoxic cancer treatment.


Asunto(s)
Senescencia Celular/fisiología , Quinasa I-kappa B/metabolismo , Inhibidor NF-kappaB alfa/biosíntesis , Factor de Transcripción ReIA/metabolismo , Transcripción Genética/genética , Animales , Apoptosis/genética , Línea Celular , Proliferación Celular/genética , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Femenino , Silenciador del Gen/fisiología , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Inhibidor NF-kappaB alfa/genética , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo
2.
Blood ; 137(20): 2785-2799, 2021 05 20.
Artículo en Inglés | MEDLINE | ID: mdl-33232972

RESUMEN

Aberrant B-cell receptor/NF-κB signaling is a hallmark feature of B-cell non-Hodgkin lymphomas, especially in diffuse large B-cell lymphoma (DLBCL). Recurrent mutations in this cascade, for example, in CD79B, CARD11, or NFKBIZ, and also in the Toll-like receptor pathway transducer MyD88, all deregulate NF-κB, but their differential impact on lymphoma development and biology remains to be determined. Here, we functionally investigate primary mouse lymphomas that formed in recipient mice of Eµ-myc transgenic hematopoietic stem cells stably transduced with naturally occurring NF-κB mutants. Although most mutants supported Myc-driven lymphoma formation through repressed apoptosis, CARD11- or MyD88-mutant lymphoma cells selectively presented with a macrophage-activating secretion profile, which, in turn, strongly enforced transforming growth factor ß (TGF-ß)-mediated senescence in the lymphoma cell compartment. However, MyD88- or CARD11-mutant Eµ-myc lymphomas exhibited high-level expression of the immune-checkpoint mediator programmed cell death ligand 1 (PD-L1), thus preventing their efficient clearance by adaptive host immunity. Conversely, these mutant-specific dependencies were therapeutically exploitable by anti-programmed cell death 1 checkpoint blockade, leading to direct T-cell-mediated lysis of predominantly but not exclusively senescent lymphoma cells. Importantly, mouse-based mutant MyD88- and CARD11-derived signatures marked DLBCL subgroups exhibiting mirroring phenotypes with respect to the triad of senescence induction, macrophage attraction, and evasion of cytotoxic T-cell immunity. Complementing genomic subclassification approaches, our functional, cross-species investigation unveils pathogenic principles and therapeutic vulnerabilities applicable to and testable in human DLBCL subsets that may inform future personalized treatment strategies.


Asunto(s)
Inmunidad Adaptativa , Proteínas Adaptadoras de Señalización CARD/genética , Senescencia Celular/fisiología , Guanilato Ciclasa/genética , Linfoma de Células B Grandes Difuso/inmunología , Factor 88 de Diferenciación Mieloide/genética , Proteínas de Neoplasias/genética , Linfocitos T Citotóxicos/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Antígeno B7-H1/antagonistas & inhibidores , Antígenos CD79/genética , Línea Celular Tumoral , Quimiotaxis , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Genes Reporteros , Genes myc , Humanos , Inhibidores de Puntos de Control Inmunológico , Linfoma de Células B Grandes Difuso/patología , Linfoma de Células B Grandes Difuso/terapia , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación Missense , FN-kappa B/genética , FN-kappa B/metabolismo , Mutación Puntual , Proteína 2 Ligando de Muerte Celular Programada 1/antagonistas & inhibidores , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Transcriptoma
3.
Mol Cell ; 47(2): 306-19, 2012 Jul 27.
Artículo en Inglés | MEDLINE | ID: mdl-22683265

RESUMEN

The CD95 (Fas/APO-1) death-inducing signaling complex (DISC) is essential for the initiation of CD95-mediated apoptotic and nonapoptotic responses. The CD95 DISC comprises CD95, FADD, procaspase-8, procaspase-10, and c-FLIP proteins. Procaspase-8 and procaspase-10 are activated at the DISC, leading to the formation of active caspases and apoptosis initiation. In this study we analyzed the stoichiometry of the CD95 DISC. Using quantitative western blots, mass spectrometry, and mathematical modeling, we reveal that the amount of DED proteins procaspase-8/procaspase-10 and c-FLIP at the DISC exceeds that of FADD by several-fold. Furthermore, our findings imply that procaspase-8, procaspase-10, and c-FLIP could form DED chains at the DISC, enabling the formation of dimers and efficient activation of caspase-8. Taken together, our findings provide an enhanced understanding of caspase-8 activation and initiation of apoptosis at the DISC.


Asunto(s)
Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Transducción de Señal , Receptor fas/química , Apoptosis , Caspasa 10/metabolismo , Caspasa 8/metabolismo , Dimerización , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Células HeLa , Humanos , Espectrometría de Masas/métodos , Microscopía Fluorescente/métodos , Modelos Biológicos , Modelos Teóricos , Receptor fas/metabolismo
4.
Blood ; 129(1): 71-81, 2017 01 05.
Artículo en Inglés | MEDLINE | ID: mdl-27733358

RESUMEN

Classical Hodgkin lymphoma (cHL), although originating from B cells, is characterized by the virtual lack of gene products whose expression constitutes the B-cell phenotype. Epigenetic repression of B-cell-specific genes via promoter hypermethylation and histone deacetylation as well as compromised expression of B-cell-committed transcription factors were previously reported to contribute to the lost B-cell phenotype in cHL. Restoring the B-cell phenotype may not only correct a central malignant property, but it may also render cHL susceptible to clinically established antibody therapies targeting B-cell surface receptors or small compounds interfering with B-cell receptor signaling. We conducted a high-throughput pharmacological screening based on >28 000 compounds in cHL cell lines carrying a CD19 reporter to identify drugs that promote reexpression of the B-cell phenotype. Three chemicals were retrieved that robustly enhanced CD19 transcription. Subsequent chromatin immunoprecipitation-based analyses indicated that action of 2 of these compounds was associated with lowered levels of the transcriptionally repressive lysine 9-trimethylated histone H3 mark at the CD19 promoter. Moreover, the antileukemia agents all-trans retinoic acid and arsenic trioxide (ATO) were found to reconstitute the silenced B-cell transcriptional program and reduce viability of cHL cell lines. When applied in combination with a screening-identified chemical, ATO evoked reexpression of the CD20 antigen, which could be further therapeutically exploited by enabling CD20 antibody-mediated apoptosis of cHL cells. Furthermore, restoration of the B-cell phenotype also rendered cHL cells susceptible to the B-cell non-Hodgkin lymphoma-tailored small-compound inhibitors ibrutinib and idelalisib. In essence, we report here a conceptually novel, redifferentiation-based treatment strategy for cHL.


Asunto(s)
Antineoplásicos/farmacología , Linfocitos B/inmunología , Diferenciación Celular/efectos de los fármacos , Enfermedad de Hodgkin/inmunología , Transcriptoma/efectos de los fármacos , Antígenos CD19/inmunología , Antígenos CD20/inmunología , Linfocitos B/efectos de los fármacos , Inmunoprecipitación de Cromatina , Citometría de Flujo , Ensayos Analíticos de Alto Rendimiento , Humanos , Fenotipo , Reacción en Cadena de la Polimerasa , Células Tumorales Cultivadas
5.
Cell Commun Signal ; 11(1): 44, 2013 Jun 26.
Artículo en Inglés | MEDLINE | ID: mdl-23803157

RESUMEN

Apoptosis is a form of programmed cell death, which is fundamental to all multicellular organisms. Deregulation of apoptosis leads to a number of severe diseases including cancer. Apoptosis is initiated either by extrinsic signals via stimulation of receptors at the cellular surface or intrinsic signals, such as DNA damage or growth factor withdrawal. Apoptosis has been extensively studied using systems biology which substantially contributed to the understanding of this death signaling network. This review gives an overview of mathematical models of apoptosis and the potential of systems biology to contribute to the development of novel therapies for cancer or other apoptosis-related diseases.

6.
Exp Cell Res ; 318(11): 1324-31, 2012 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-22309778

RESUMEN

c-FLIP proteins (isoforms: c-FLIP(L), c-FLIP(S), and c-FLIP(R)) play an essential role in the regulation of death receptor (DR)-induced apoptosis and NF-κB activation. Here, we discuss multiple mechanisms by which c-FLIPs control NF-κB activation and the life/death decision made in cancer and immune cells. We focus on the role of c-FLIP in cellular signaling. We concentrate on c-FLIP protein modifications as well as on the regulation of c-FLIP expression levels. Furthermore, we discuss in detail how the exact quantity and dynamics of different c-FLIP isoforms in the cell influence the induction of pro- versus anti-apoptotic pathways.


Asunto(s)
Apoptosis , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/fisiología , FN-kappa B/metabolismo , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Humanos , Isoformas de Proteínas , Transducción de Señal
7.
Nat Commun ; 11(1): 3651, 2020 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-32686676

RESUMEN

Lesion-based targeting strategies underlie cancer precision medicine. However, biological principles - such as cellular senescence - remain difficult to implement in molecularly informed treatment decisions. Functional analyses in syngeneic mouse models and cross-species validation in patient datasets might uncover clinically relevant genetics of biological response programs. Here, we show that chemotherapy-exposed primary Eµ-myc transgenic lymphomas - with and without defined genetic lesions - recapitulate molecular signatures of patients with diffuse large B-cell lymphoma (DLBCL). Importantly, we interrogate the murine lymphoma capacity to senesce and its epigenetic control via the histone H3 lysine 9 (H3K9)-methyltransferase Suv(ar)39h1 and H3K9me3-active demethylases by loss- and gain-of-function genetics, and an unbiased clinical trial-like approach. A mouse-derived senescence-indicating gene signature, termed "SUVARness", as well as high-level H3K9me3 lymphoma expression, predict favorable DLBCL patient outcome. Our data support the use of functional genetics in transgenic mouse models to incorporate basic biology knowledge into cancer precision medicine in the clinic.


Asunto(s)
Senescencia Celular , Histona Metiltransferasas , Linfoma de Células B Grandes Difuso , Células 3T3 , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Epigénesis Genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Histona Metiltransferasas/genética , Histona Metiltransferasas/metabolismo , Humanos , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Ratones , Ratones Transgénicos , Pronóstico
8.
Cancer Cell ; 33(2): 322-336.e8, 2018 02 12.
Artículo en Inglés | MEDLINE | ID: mdl-29438700

RESUMEN

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


Asunto(s)
Histona Demetilasas/genética , Histona Demetilasas con Dominio de Jumonji/genética , Melanoma/genética , Animales , Histonas/metabolismo , Humanos , Lisina/genética , Lisina/metabolismo , Metilación , Ratones Desnudos , Regiones Promotoras Genéticas/genética
9.
Mol Biosyst ; 12(5): 1486-95, 2016 04 26.
Artículo en Inglés | MEDLINE | ID: mdl-27004466

RESUMEN

A number of mathematical models of apoptosis generated recently allowed us to understand intrinsic mechanisms of life/death decisions in a cell. Nevertheless, the parameters for the mathematical models are often experimentally difficult to obtain and there is an emerging need for the development of efficient approaches for parameter estimation. In this study we suggest a new method for parameter estimation, which is based on stochastic simulations and can be used when the number of molecules in the system is small. Our approach comprised the following steps: we start from the selection of parameters that lead to a good ordinary differential equation (ODE) fit. We continued by carrying out stochastic simulations for each of these parameters. Comparing the correlation structure of these simulations with the data, we finally could identify the best parameter set. The method was applied for a model of CD95-induced apoptosis, the new best identified parameters fit well to the experimental data. The best parameter set allowed us to get new insights into CD95 apoptosis regulation and can be applied for the comprehensive analysis of other signaling networks. The modeling approach allowed us to get new insights into network regulation, in particular, to identify robustness in CD95 apoptotic response. Taken together, this new method provides valuable predictions and can be applied for the analysis of other signaling networks.


Asunto(s)
Apoptosis , Modelos Biológicos , Receptor fas/química , Receptor fas/metabolismo , Algoritmos , Proteínas Portadoras/metabolismo , Simulación por Computador , Unión Proteica , Transducción de Señal
10.
Cells ; 2(3): 476-95, 2013 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-24709794

RESUMEN

Contemporary quantitative mass spectrometry provides fascinating opportunities in defining the stoichiometry of high-molecular weight complexes or multiprotein platforms. The composition stoichiometry of multiprotein platforms is a key to understand the regulation of complex signaling pathways and provides a basis for constructing models in systems biology. Here we present an improved AQUA technique workflow that we adapted for the quantitative mass spectrometry analysis of the stoichiometry of the CD95 (Fas/APO-1) death inducing signaling complex (DISC). The DISC is a high-molecular weight platform essential for the initiation of CD95-mediated apoptotic and non-apoptotic responses. For protein quantification, CD95 DISCs were immunoprecipitated and proteins in the immunoprecipitations were separated by one-dimensional gel electrophoresis, followed by protein quantification using the AQUA technique. We will discuss in detail AQUA analysis of the CD95 DISC focusing on the key issues of this methodology, i.e., selection and validation of AQUA peptides. The application of this powerful method allowed getting new insights into mechanisms of procaspase-8 activation at the DISC and apoptosis initiation [1]. Here we discuss the AQUA methodology adapted by us for the analysis of the CD95 DISC in more detail. This approach paves the way for the successful quantification of multiprotein complexes and thereby delineating the intrinsic details of molecular interactions.

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