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1.
Sci Rep ; 9(1): 18062, 2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31792264

RESUMO

Recent advances in molecular diagnostics have shown that lesions affecting both copies of the gene for tumor suppressor protein 53 (TP53) count among the most powerful predictors for high-risk disease in multiple myeloma (MM). However, the functional relevance and potential therapeutic implications of single hits to TP53 remain less well understood. Here, we have for the first time approximated the different constellations of mono- and bi-allelic TP53 lesions observed in MM patients within the frame of a single MM cell line model and assessed their potential to disrupt p53 system functionality and to impart drug resistance. Both types of common first hit: point mutation with expression of mutant p53 protein or complete loss of contribution from one of two wildtype alleles strongly impaired p53 system functionality and increased resistance to melphalan. Second hits abolished remaining p53 activity and increased resistance to genotoxic drugs even further. These results fit well with the clinical drive to TP53 single- and double-hit disease in MM patients, provide a rationale for the most commonly observed double-hit constellation (del17p+ TP53 point mutation), and underscore the potential increases in MM cell malignancy associated with any type of initial TP53 lesion.

2.
Front Immunol ; 10: 2024, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31555268

RESUMO

Tumor necrosis factor (TNF) receptor associated factor-2 (TRAF2) knockout (KO) cells were generated to investigate the role of TRAF2 in signaling by TNFR1 and the CD95-type death receptors (DRs) TRAILR1/2 and CD95. To prevent negative selection effects arising from the increased cell death sensitivity of TRAF2-deficient cells, cell lines were used for the generation of the TRAF2 KO variants that were protected from DR-induced apoptosis downstream of caspase-8 activation. As already described in the literature, TRAF2 KO cells displayed enhanced constitutive alternative NFκB signaling and reduced TNFR1-induced activation of the classical NFκB pathway. There was furthermore a significant but only partial reduction in CD95-type DR-induced upregulation of the proinflammatory NFκB-regulated cytokine interleukin-8 (IL8), which could be reversed by reexpression of TRAF2. In contrast, expression of the TRAF2-related TRAF1 protein failed to functionally restore TRAF2 deficiency. TRAF2 deficiency resulted furthermore in enhanced procaspase-8 processing by DRs, but this surprisingly came along with a reduction in net caspase-8 activity. In sum, our data argue for (i) a non-obligate promoting function of TRAF2 in proinflammatory DR signaling and (ii) a yet unrecognized stabilizing effect of TRAF2 on caspase-8 activity.

3.
Cell Death Dis ; 10(8): 611, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31406107

RESUMO

The NEDD8-activating enzyme (NAE) inhibitor MLN4924 inhibits cullin-RING ubiquitin ligase complexes including the SKP1-cullin-F-box E3 ligase ßTrCP. MLN4924 therefore inhibits also the ßTrCP-dependent activation of the classical and the alternative NFĸB pathway. In this work, we found that a subgroup of multiple myeloma cell lines (e.g., RPMI-8226, MM.1S, KMS-12BM) and about half of the primary myeloma samples tested are sensitized to TNF-induced cell death by MLN4924. This correlated with MLN4924-mediated inhibition of TNF-induced activation of the classical NFκB pathway and reduced the efficacy of TNF-induced TNFR1 signaling complex formation. Interestingly, binding studies revealed a straightforward correlation between cell surface TNFR1 expression in multiple myeloma cell lines and their sensitivity for MLN4924/TNF-induced cell death. The cell surface expression levels of TNFR1 in the investigated MM cell lines largely correlated with TNFR1 mRNA expression. This suggests that the variable levels of cell surface expression of TNFR1 in myeloma cell lines are decisive for TNF/MLN4924 sensitivity. Indeed, introduction of TNFR1 into TNFR1-negative TNF/MLN4924-resistant KMS-11BM cells, was sufficient to sensitize this cell line for TNF/MLN4924-induced cell death. Thus, MLN4924 might be especially effective in myeloma patients with TNFR1+ myeloma cells and a TNFhigh tumor microenvironment.

4.
Cell Death Dis ; 10(2): 122, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30741924

RESUMO

We evaluated redundant and receptor-specific activities of TRADD, RIPK1, and FADD in RIPK3-expressing HeLa cells lacking expression of these proteins or any combination of two of these factors. We confirmed the opposing role of FADD in TNF- and TRAIL-induced necroptosis and observed an anti-necroptotic function of TRADD. RIPK1 and TRADD act in a redundant manner in TNF- but not TRAIL-induced apoptosis. Complementary, FADD proved to be sufficient for TRAIL- but not for TNF-induced apoptosis. TRADD and RIPK1, however, redundantly mediated proinflammatory signaling in response to TNF and TRAIL. FADD deficiency sensitized more efficiently for TNFR1-mediated necroptosis than caspase-8 deficiency pointing to a caspase-8 independent inhibitory activity of FADD on TNF-induced necroptosis. Based on these characteristics, we propose a model in which the death receptor-specific activities of TRADD, RIPK1, and FADD are traced back to their hierarchically different position in TNFR1- and TRAIL death receptor signaling.

5.
Cell Death Dis ; 9(11): 1084, 2018 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-30349023

RESUMO

TNF-like weak inducer of apoptosis (TWEAK) and inhibition of protein synthesis with cycloheximide (CHX) sensitize for poly(I:C)-induced cell death. Notably, although CHX preferentially enhanced poly(I:C)-induced apoptosis, TWEAK enhanced primarily poly(I:C)-induced necroptosis. Both sensitizers of poly(I:C)-induced cell death, however, showed no major effect on proinflammatory poly(I:C) signaling. Analysis of a panel of HeLa-RIPK3 variants lacking TRADD, RIPK1, FADD, or caspase-8 expression revealed furthermore similarities and differences in the way how poly(I:C)/TWEAK, TNF, and TRAIL utilize these molecules for signaling. RIPK1 turned out to be essential for poly(I:C)/TWEAK-induced caspase-8-mediated apoptosis but was dispensable for this response in TNF and TRAIL signaling. TRADD-RIPK1-double deficiency differentially affected poly(I:C)-triggered gene induction but abrogated gene induction by TNF completely. FADD deficiency abrogated TRAIL- but not TNF- and poly(I:C)-induced necroptosis, whereas TRADD elicited protective activity against all three death inducers. A general protective activity against poly(I:C)-, TRAIL-, and TNF-induced cell death was also observed in FLIPL and FLIPS transfectrants.

6.
Cell Death Dis ; 9(9): 921, 2018 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-30206205

RESUMO

TNF is not only a major effector molecule of PAMP/DAMP-activated macrophages, but also regulates macrophage function and viability. We recently demonstrated that TNFR2 triggers necroptosis in macrophages with compromised caspase activity by two cooperating mechanisms: induction of endogenous TNF with subsequent stimulation of TNFR1 and depletion of cytosolic TRAF2-cIAP complexes. Here we show that TNFR2 activation in caspase-inhibited macrophages results in the production of endogenous TNF and TNFR1 stimulation followed by upregulation of A20, TRAF1, IL-6, and IL-1ß. Surprisingly, TNFR1-mediated induction of IL-6 and IL-1ß was clearly evident in response to TNFR2 stimulation but occurred not or only weakly in macrophages selectively and directly stimulated via TNFR1. Moreover, TNFR2-induced TNFR1-mediated gene induction was largely inhibited by necrostatin-1, whereas upregulation of A20 and TRAF1 by direct and exclusive stimulation of TNFR1 remained unaffected by this compound. Thus, treatment with TNFR2/ZVAD enables TNFR1 in macrophages to stimulate gene induction via a pathway requiring RIPK1 kinase activity. TNFR2/ZVAD-induced production of IL-6 and IL-1ß was largely blocked in necroptosis-resistant MLKL- and RIPK3-deficient macrophages, whereas induction of A20 and TRAF1 remained unaffected. In sum, our results show that in caspase-inhibited macrophages TNFR2 not only triggers TNF/TNFR1-mediated necroptosis but also TNF/TNFR1-mediated RIPK3/MLKL-dependent and -independent gene induction.

7.
Science ; 361(6404): 810-813, 2018 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-30026316

RESUMO

RIPK1 (receptor-interacting serine/threonine kinase 1) is a master regulator of signaling pathways leading to inflammation and cell death and is of medical interest as a drug target. We report four patients from three unrelated families with complete RIPK1 deficiency caused by rare homozygous mutations. The patients suffered from recurrent infections, early-onset inflammatory bowel disease, and progressive polyarthritis. They had immunodeficiency with lymphopenia and altered production of various cytokines revealed by whole-blood assays. In vitro, RIPK1-deficient cells showed impaired mitogen-activated protein kinase activation and cytokine secretion and were prone to necroptosis. Hematopoietic stem cell transplantation reversed cytokine production defects and resolved clinical symptoms in one patient. Thus, RIPK1 plays a critical role in the human immune system.


Assuntos
Artrite/genética , Doenças Inflamatórias Intestinais/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Imunodeficiência Combinada Severa/genética , Alelos , Artrite/imunologia , Citocinas/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Doenças Inflamatórias Intestinais/imunologia , Linfopenia/genética , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Linhagem , Imunodeficiência Combinada Severa/imunologia
8.
Oncogene ; 37(30): 4122-4136, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29706657

RESUMO

Attempts to exploit the cytotoxic activity of death receptors (DR) for treating cancer have thus far been disappointing. DR activation in most malignant cells fails to trigger cell death and may even promote tumor growth by activating cell death-independent DR-associated signaling pathways. Overcoming apoptosis resistance is consequently a prerequisite for successful clinical exploitation of DR stimulation. Here we show that hyperosmotic stress in the tumor microenvironment unleashes the deadly potential of DRs by enforcing BCL-2 addiction of cancer cells. Hypertonicity robustly enhanced cytotoxicity of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and other DR ligands in various cancer entities. Initial events in TRAIL DR signaling remained unaffected, but hypertonic conditions unlocked activation of the mitochondrial death pathway and thus amplified the apoptotic signal. Mechanistically, we demonstrate that hyperosmotic stress imposed a BCL-2-addiction on cancer cells to safeguard the integrity of the outer mitochondrial membrane (OMM), essentially exhausting the protective capacity of BCL-2-like pro-survival proteins. Deprivation of these mitochondrial safeguards licensed DR-generated truncated BH3-interacting domain death agonist (tBID) to activate BCL-2-associated X protein (BAX) and initiated mitochondrial outer membrane permeabilization (MOMP). Our work highlights that hyperosmotic stress in the tumor environment primes mitochondria for death and lowers the threshold for DR-induced apoptosis. Beyond TRAIL-based therapies, our findings could help to strengthen the efficacy of other apoptosis-inducing cancer treatment regimens.

9.
MAbs ; 9(3): 506-520, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28095113

RESUMO

Cell surface antigen-specific antibodies are of substantial diagnostic and therapeutic importance. The binding properties of such antibodies are usually evaluated by cell-free assays, in particular surface plasmon resonance (SPR) analysis, or flow cytometry. SPR analyses allow the detailed quantitative and dynamic evaluation of the binding properties of antibodies, but need purified, typically recombinantly produced antigens. It can, however, be difficult to produce the required antigen. Furthermore, cellular factors influencing the antigen-antibody interaction are not considered by this method. Flow cytometry-based analyses do not have these limitations, but require elaborated calibration controls for absolute quantification of bound molecules. To overcome the limitations of SRP and flow cytometry in the characterization of cell surface antigen-specific antibodies, we developed Fn14-specific antibody 18D1 as an example of an antibody fusion protein format that includes the luciferase of Gaussia princeps (GpL), which enables very simple and highly sensitive cellular binding studies. We found that GpL-tagging of the C-terminus of the antibody light chain does not affect the interaction of 18D1-IgG1 with its antigen and Fc-gamma receptors (FcγRs). In accordance with this, the GpL(LC-CT)-18D1-IgG1 antibody fusion protein showed basically the same FcγR-dependent agonistic properties as the parental 18D1 antibody. Similar results were obtained with isotype switch variants of 18D1 and antibodies specific for CD95, LTßR and CD40. In sum, we demonstrate that antibody GpL fusion proteins are easily manageable and versatile tools for the characterization of cell surface antigen-antibody interactions that have the potential to considerably extend the instrumentarium for the evaluation of antibodies.


Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Superfície/imunologia , Técnicas Imunológicas/métodos , Proteínas Recombinantes de Fusão/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/química , Imunoglobulina G/imunologia , Luciferases , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Receptor de TWEAK/imunologia
10.
FEBS J ; 284(8): 1131-1159, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27865080

RESUMO

Since their identification more than 20 years ago, the death receptors CD95, TRAILR1, and TRAILR2 have been intensively studied with respect to their cell death-inducing activities. These receptors, however, can also trigger a variety of cell death-independent cellular responses reaching from the activation of proinflammatory gene transcription programs over the stimulation of proliferation and differentiation to induction of cell migration. The cell death-inducing signaling mechanisms of CD95 and the TRAIL death receptors are well understood. In contrast, despite the increasing recognition of the biological and pathophysiological relevance of the cell death-independent activities of CD95, TRAILR1, and TRAILR2, the corresponding signaling mechanisms are less understood and give no fully coherent picture. This review is focused on the cell death-independent activities of CD95 and the TRAIL death receptors and addresses mainly three questions: (a) how are these receptors linked to noncell death pathways at the molecular level, (b) which factors determine the balance of cell death and cell death-independent activities of CD95 and the TRAIL death receptors at the cellular level, and (c) what are the consequences of the cell death-independent functions of these receptors for their role in cancer and inflammatory diseases.


Assuntos
Morte Celular/fisiologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Receptor fas/fisiologia , Animais , Apoptose/fisiologia , Caspases/metabolismo , Ativação Enzimática , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais
11.
Cell Death Dis ; 7(9): e2375, 2016 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-27899821

RESUMO

Macrophages express TNFR1 as well as TNFR2 and are also major producers of tumor necrosis factor (TNF), especially upon contact with pathogen-associated molecular patterns. Consequently, TNF not only acts as a macrophage-derived effector molecule but also regulates the activity and viability of macrophages. Here, we investigated the individual contribution of TNFR1 and TNFR2 to TNF-induced cell death in macrophages. Exclusive stimulation of TNFR1 showed no cytotoxic effect whereas selective stimulation of TNFR2 displayed mild cytotoxicity. Intriguingly, the latter was strongly enhanced by the caspase inhibitor zVAD-fmk. The strong cytotoxic activity of TNFR2 in the presence of zVAD-fmk was reversed by necrostatin-1, indicating necroptotic cell death. TNFR1- and TNF-deficient macrophages turned out to be resistant against TNFR2-induced cell death. In addition, the cIAP-depleting SMAC mimetic BV6 also enforced TNF/TNFR1-mediated necroptotic cell death in the presence of zVAD-fmk. In sum, our data suggest a model in which TNFR2 sensitizes macrophages for endogenous TNF-induced TNFR1-mediated necroptosis by the known ability of TNFR2 to interfere with the survival activity of TRAF2-cIAP1/2 complexes.


Assuntos
Apoptose , Macrófagos/citologia , Macrófagos/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Comunicação Autócrina/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Células Progenitoras Mieloides/efeitos dos fármacos , Células Progenitoras Mieloides/metabolismo , Necrose , Oligopeptídeos/farmacologia , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
12.
Blood ; 126(4): 437-44, 2015 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-26012567

RESUMO

Inhibition of the tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible 14 (Fn14) system reduces intestinal cell death and disease development in several models of colitis. In view of the crucial role of TNF and intestinal cell death in graft-versus-host disease (GVHD) and the ability of TWEAK to enhance TNF-induced cell death, we tested here the therapeutic potential of Fn14 blockade on allogeneic hematopoietic cell transplantation (allo-HCT)-induced intestinal GVHD. An Fn14-specific blocking human immunoglobulin G1 antibody variant with compromised antibody-dependent cellular cytotoxicity (ADCC) activity strongly inhibited the severity of murine allo-HCT-induced GVHD. Treatment of the allo-HCT recipients with this monoclonal antibody reduced cell death of gastrointestinal cells but neither affected organ infiltration by donor T cells nor cytokine production. Fn14 blockade also inhibited intestinal cell death in mice challenged with TNF. This suggests that the protective effect of Fn14 blockade in allo-HCT is based on the protection of intestinal cells from TNF-induced apoptosis and not due to immune suppression. Importantly, Fn14 blockade showed no negative effect on graft-versus-leukemia/lymphoma (GVL) activity. Thus, ADCC-defective Fn14-blocking antibodies are not only possible novel GVL effect-sparing therapeutics for the treatment of GVHD but might also be useful for the treatment of other inflammatory bowel diseases where TNF-induced cell death is of relevance.


Assuntos
Anticorpos Monoclonais Murinos/uso terapêutico , Apoptose , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Intestinos/patologia , Receptores do Fator de Necrose Tumoral/antagonistas & inibidores , Fatores de Necrose Tumoral/antagonistas & inibidores , Animais , Citotoxicidade Celular Dependente de Anticorpos , Western Blotting , Células Cultivadas , Citocina TWEAK , Modelos Animais de Doenças , Feminino , Imunofluorescência , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/metabolismo , Doença Enxerto-Hospedeiro/patologia , Humanos , Imunoglobulina G/administração & dosagem , Imunoglobulina G/imunologia , Mucosa Intestinal/metabolismo , Intestinos/imunologia , Medições Luminescentes , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/imunologia , Receptores do Fator de Necrose Tumoral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rituximab , Receptor de TWEAK , Fator de Necrose Tumoral alfa/farmacologia , Fatores de Necrose Tumoral/imunologia , Fatores de Necrose Tumoral/metabolismo
13.
Br J Pharmacol ; 172(5): 1222-36, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25363690

RESUMO

BACKGROUND AND PURPOSE: MLN4924 prevents the formation of active cullin-RING ubiquitin ligase complexes and thus inhibits NF-κB signalling. Here, we evaluated the effects of this compound on monocytes and dendritic cells (DCs). EXPERIMENTAL APPROACH: Monocytes and DCs were challenged with TNF or LPS in the presence and absence of MLN4924. The effects of MLN4924 on cellular viability, pro-inflammatory gene induction and DC maturation were investigated using the MTT assay, elisa and FACS analysis. Mechanisms of cell death induction were evaluated by using inhibitors of caspases, RIPK1 and MLKL. KEY RESULTS: MLN4924 inhibited NF-κB activation and sensitized monocytes and immature DCs (iDCs) for TNFR1-induced cell death. Neither the caspase inhibitor zVAD-fmk, the RIPK1 inhibitor necrostatin-1 (nec-1) nor the MLKL inhibitor necrosulfonamide (NSA) alone prevented TNF-induced cell death. A combination of zVAD-fmk and nec-1 or NSA, however, rescued monocytes and iDCs from MLN4924/TNF-induced cell death indicating that MLN4924 affects anti-apoptotic and anti-necrotic activities in TNFR1 signalling. MLN4924 also converted the response of iDCs to LPS from maturation to cell death. LPS-induced cell death in MLN4924-treated iDCs was again only effectively blocked by cotreatment with zVAD-fmk and nec-1 or NSA. Noteworthy, MLN4924/LPS-induced cell death was almost completely independent of endogenous TNF. MLN4924 also strongly inhibited maturation and activation of iDCs that were rescued from cell death by zVAD-fmk and nec-1. CONCLUSIONS AND IMPLICATIONS: Our data reveal a strong dual suppressive effect of MLN4924 on DC activity. The targeting of NAE by MLN4924 could be a new way to treat inflammatory diseases.


Assuntos
Apoptose/efeitos dos fármacos , Ciclopentanos/farmacologia , Células Dendríticas/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Necrose/tratamento farmacológico , Pirimidinas/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Ciclopentanos/química , Relação Dose-Resposta a Droga , Humanos , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Pirimidinas/química , Relação Estrutura-Atividade
14.
Mol Cancer ; 13: 85, 2014 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-24741998

RESUMO

BACKGROUND: Stimulation of CD40 can augment anti-cancer T cell immune responses by triggering effective activation and maturation of antigen-presenting cells (APCs). Although CD40 agonists have clinical activity in humans, the associated systemic activation of the immune system triggers dose-limiting side-effects. METHODS: To increase the tumor selectivity of CD40 agonist-based therapies, we developed an approach in which soluble trimeric CD40L (sCD40L) is genetically fused to tumor targeting antibody fragments, yielding scFv:CD40L fusion proteins. We hypothesized that scFv:CD40L fusion proteins would have reduced CD40 agonist activity similar to sCD40L but will be converted to a highly agonistic membrane CD40L-like form of CD40L upon anchoring to cell surface exposed antigen via the scFv domain. RESULTS: Targeted delivery of CD40L to the carcinoma marker EpCAM on carcinoma cells induced dose-dependent paracrine maturation of DCs ~20-fold more effective than a non-targeted control scFv:CD40L fusion protein. Similarly, targeted delivery of CD40L to the B cell leukemia marker CD20 induced effective paracrine maturation of DCs. Of note, the CD20-selective delivery of CD40L also triggered loss of cell viability in certain B cell leukemic cell lines as a result of CD20-induced apoptosis. CONCLUSIONS: Targeted delivery of CD40L to cancer cells is a promising strategy that may help to trigger cancer-localized activation of CD40 and can be modified to exert additional anti-cancer activity via the targeting domain.


Assuntos
Linfócitos B/efeitos dos fármacos , Ligante de CD40/genética , Células Dendríticas/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Anticorpos de Cadeia Única/genética , Anticorpos Monoclonais Murinos/farmacologia , Antígenos CD20/genética , Antígenos CD20/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Ligante de CD40/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Molécula de Adesão da Célula Epitelial , Expressão Gênica , Células HEK293 , Humanos , Terapia de Alvo Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Rituximab , Anticorpos de Cadeia Única/metabolismo
15.
Front Immunol ; 5: 63, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24600451

RESUMO

Soluble tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), in contrast to membrane TWEAK and TNF, is only a weak activator of the classical NFκB pathway. We observed that soluble TWEAK was regularly more potent than TNF with respect to the induction of TNF receptor-associated factor 1 (TRAF1), a NFκB-controlled signaling protein involved in the regulation of inflammatory signaling pathways. TNF-induced TRAF1 expression was efficiently blocked by inhibition of the classical NFκB pathway using the IKK2 inhibitor, TPCA1. In contrast, in some cell lines, TWEAK-induced TRAF1 production was only partly inhibited by TPCA1. The NEDD8-activating enzyme inhibitor MLN4924, however, which inhibits classical and alternative NFκB signaling, blocked TNF- and TWEAK-induced TRAF1 expression. This suggests that TRAF1 induction by soluble TWEAK is based on the cooperative activity of the two NFκB signaling pathways. We have previously shown that oligomerization of soluble TWEAK results in ligand complexes with membrane TWEAK-like activity. Oligomerization of soluble TWEAK showed no effect on the dose response of TRAF1 induction, but potentiated the ability of soluble TWEAK to trigger production of the classical NFκB-regulated cytokine IL8. Transfectants expressing soluble TWEAK and membrane TWEAK showed similar induction of TRAF1 while only the membrane TWEAK expressing cells robustly stimulated IL8 production. These data indicate that soluble TWEAK may efficiently induce a distinct subset of the membrane TWEAK-targeted genes and argue again for a crucial role of classical NFκB pathway-independent signaling in TWEAK-induced TRAF1 expression. Other TWEAK targets, which can be equally well induced by soluble and membrane TWEAK, remain to be identified and the relevance of the ability of soluble TWEAK to induce such a distinct subset of membrane TWEAK-targeted genes for TWEAK biology will have to be clarified in future studies.

16.
MAbs ; 6(1): 297-308, 2014 Jan-Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24135629

RESUMO

Expression of fibroblast growth factor (FGF)-inducible 14 (Fn14), a member of the tumor necrosis factor receptor superfamily, is typically low in healthy adult organisms, but strong Fn14 expression is induced in tissue injury and tissue remodeling. High Fn14 expression is also observed in solid tumors, which is why this receptor is under consideration as a therapeutic target in oncology. Here, we describe various novel mouse-human cross-reactive llama-derived recombinant Fn14-specific antibodies (5B6, 18D1, 4G5) harboring the human IgG1 Fc domain. In contrast to recombinant variants of the established Fn14-specific antibodies PDL192 and P4A8, all three llama-derived antibodies efficiently bound to the W42A and R56P mutants of human Fn14. 18D1 and 4G5, but not 5B6, efficiently blocked TNF-like weak inducer of apoptosis(TWEA K) binding at low concentrations (0.2­2 µg/ml). Oligomerization and Fcγ receptor (FcγR) binding converted all antibodies into strong Fn14 agonists. Variants of 18D1 with enhanced and reduced antibody-dependent cell-mediated cytotoxicity (ADCC) activity were further analyzed in vivo with respect to their effect on metastasis. In a xenogeneic model using human colon carcinoma cancer cells, both antibody variants were effective in reducing metastasis to the liver. In contrast, only the 18D1 variant with enhanced ADCC activity, but not its ADCC-defective counterpart, suppressed lung metastasis in the RE NCA model. In sum, this suggests that Fn14 targeting might primarily act by triggering of antibody effector functions, but also by blockade of TWEA K-Fn14 interaction in some cases


Assuntos
Anticorpos Antineoplásicos/farmacologia , Camelídeos Americanos/imunologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Proteínas de Neoplasias/imunologia , Receptores do Fator de Necrose Tumoral/imunologia , Animais , Anticorpos Antineoplásicos/imunologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Células HEK293 , Xenoenxertos , Humanos , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Receptor de TWEAK , Ensaios Antitumorais Modelo de Xenoenxerto
17.
J Immunol ; 191(5): 2308-18, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-23918987

RESUMO

We found recently that TNF-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor-inducible-14 (Fn14) by virtue of their strong capability to reduce the freely available cytoplasmic pool of TNFR-associated factor (TRAF)2 and cellular inhibitors of apoptosis (cIAPs) antagonize the functions of these molecules in TNFR1 signaling, resulting in sensitization for apoptosis and inhibition of classical NF-κB signaling. In this study, we demonstrate that priming of cells with TWEAK also interferes with activation of the classical NF-κB pathway by CD40. Likewise, there was strong inhibition of CD40 ligand (CD40L)-induced activation of MAPKs in TWEAK-primed cells. FACS analysis and CD40L binding studies revealed unchanged CD40 expression and normal CD40L-CD40 interaction in TWEAK-primed cells. CD40L immunoprecipitates, however, showed severely reduced amounts of CD40 and CD40-associated proteins, indicating impaired formation or reduced stability of CD40L-CD40 signaling complexes. The previously described inhibitory effect of TWEAK on TNFR1 signaling has been traced back to reduced activity of the TNFR1-associated TRAF2-cIAP1/2 ubiquitinase complex and did not affect the stability of the immunoprecipitable TNFR1 receptor complex. Thus, the inhibitory effect of TWEAK on CD40 signaling must be based at least partly on other mechanisms. In line with this, signaling by the CD40-related TRAF2-interacting receptor TNFR2 was also attenuated but still immunoprecipitable in TWEAK-primed cells. Collectively, we show that Fn14 activation by soluble TWEAK impairs CD40L-CD40 signaling complex formation and inhibits CD40 signaling and thus identify the Fn14-TWEAK system as a potential novel regulator of CD40-related cellular functions.


Assuntos
Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/fisiologia , Fator 2 Associado a Receptor de TNF/metabolismo , Fatores de Necrose Tumoral/metabolismo , Western Blotting , Antígenos CD40/imunologia , Ligante de CD40/imunologia , Linhagem Celular , Citocina TWEAK , Citometria de Fluxo , Humanos , Imunoprecipitação , Microscopia Confocal , Receptores do Fator de Necrose Tumoral/imunologia , Fator 2 Associado a Receptor de TNF/imunologia , Receptor de TWEAK , Fatores de Necrose Tumoral/imunologia
18.
PLoS One ; 8(3): e59292, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23527154

RESUMO

Multiple myeloma (MM) displays an NFκB activity-related gene expression signature and about 20% of primary MM samples harbor genetic alterations conducive to intrinsic NFκB signaling activation. The relevance of blocking the classical versus the alternative NFκB signaling pathway and the molecular execution mechanisms involved, however, are still poorly understood. Here, we comparatively tested NFκB activity abrogation through TPCA-1 (an IKK2 inhibitor), BAY 11-7082 (an IKK inhibitor poorly selective for IKK1 and IKK2), and MLN4924 (an NEDD8 activating enzyme (NAE)-inhibitor), and analyzed their anti-MM activity. Whereas TPCA-1 interfered selectively with activation of the classical NFκB pathway, the other two compounds inhibited classical and alternative NFκB signaling without significant discrimination. Noteworthy, whereas TPCA-1 and MLN4924 elicited rather mild anti-MM effects with slight to moderate cell death induction after 1 day BAY 11-7082 was uniformly highly toxic to MM cell lines and primary MM cells. Treatment with BAY 11-7082 induced rapid cell swelling and its initial effects were blocked by necrostatin-1 or the ROS scavenger BHA, but a lasting protective effect was not achieved even with additional blockade of caspases. Because MLN4924 inhibits the alternative NFκB pathway downstream of IKK1 at the level of p100 processing, the quite discordant effects between MLN4924 and BAY 11-7082 must thus be due to blockade of IKK1-mediated NFκB-independent necrosis-inhibitory functions or represent an off-target effect of BAY 11-7082. In accordance with the latter, we further observed that concomitant knockdown of IKK1 and IKK2 did not have any major short-term adverse effect on the viability of MM cells.


Assuntos
Apoptose/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Quinase I-kappa B/antagonistas & inibidores , Mieloma Múltiplo/metabolismo , NF-kappa B/metabolismo , Nitrilos/farmacologia , Transdução de Sinais/genética , Sulfonas/farmacologia , Amidas/farmacologia , Western Blotting , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ciclopentanos/farmacologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Microscopia de Fluorescência , Pirimidinas/farmacologia , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Tiofenos/farmacologia
19.
J Biol Chem ; 288(19): 13455-66, 2013 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-23532848

RESUMO

BACKGROUND: Fn14 is a therapeutic target in various diseases. RESULTS: Anti-Fn14 antibodies activate the alternative NFκB pathway but not other Fn14-related activities induced by soluble or membrane-bound TWEAK. FcγR-bound anti-Fn14 antibodies, however, activate the full spectrum of Fn14-associated activities. CONCLUSION: Anti-Fn14 antibodies elicit agonistic activities differing from those of the natural Fn14 ligand TWEAK. SIGNIFICANCE: These findings influence the rationale of designing Fn14-targeted therapies. The Fn14-specific monoclonal antibodies PDL192 and P4A8, which are under consideration in clinical trials, showed no agonistic activity with respect to IL8 production and cell death induction. However, oligomerization with protein G or binding to Fcγ receptors converted both anti-Fn14 antibodies into potent agonists. TNF-like weak inducer of apoptosis (TWEAK), the ligand of Fn14, occurs naturally in two forms with partly different signaling capabilities, as a membrane-bound ligand and as a soluble trimeric molecule. Although membrane TWEAK strongly triggers all Fn14-associated pathways, soluble TWEAK predominately triggers the alternative nuclear factor κB (NFκB) pathway and enhances TNF-induced cell death but has only a poor effect on the classical NFκB pathway and chemokine production. Thus, the oligomerized and FcγR-bound anti-Fn14 mAbs mimicked the activity of membrane TWEAK. Notably, both anti-Fn14 antibodies significantly triggered p100 processing, the hallmark of the alternative NFκB pathway, and therefore resembled soluble TWEAK. In contrast to the latter, however, the anti-Fn14s showed no effect on TNF receptor 1-induced cell death and P4A8 even blocked the corresponding TWEAK response. Thus, we showed that Fn14 antibodies display an alternative NFκB pathway-specific agonistic activity but fail to phenocopy other activities of soluble TWEAK, whereas oligomerized or FcγR-bound Fn14 antibodies fully mimic the activity of membrane TWEAK. In view of the trivalent nature of the TWEAK-Fn14 interaction, this suggests that the alternative NFκB pathway is uniquely responsive already to Fn14 dimerization enabling antibodies to elicit an unnatural response pattern distinct from that of the naturally occurring Fn14 ligands.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais/farmacologia , Receptores do Fator de Necrose Tumoral/imunologia , Transdução de Sinais/efeitos dos fármacos , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais Humanizados/química , Proteínas de Bactérias/química , Linhagem Celular Tumoral , Citocina TWEAK , Células HEK293 , Humanos , Interleucina-8/biossíntese , Macaca fascicularis , Camundongos , Mutagênese Sítio-Dirigida , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Ligação Proteica , Multimerização Proteica , Receptores de IgG/agonistas , Receptores de IgG/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Receptor de TWEAK , Fator de Necrose Tumoral alfa/fisiologia , Fatores de Necrose Tumoral/antagonistas & inibidores , Fatores de Necrose Tumoral/fisiologia
20.
Leuk Res ; 36(9): 1165-71, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22658851

RESUMO

We investigated the utility of integrin-linked kinase (ILK) as a target for therapeutic intervention in multiple myeloma (MM). ILK (over-)expression was assessed in primary samples and MM cell lines, and the molecular and physiological consequences of siRNA-mediated ILK ablation were compared to treatment with the small molecule inhibitor QLT0267. Whereas ILK expression was ubiquitous, overexpression was only rarely observed in patient biopsies. ILK knockdown had no effect on the viability or survival pathway activity pattern of MM cells. Conversely, QLT0267 induced cell death in MM cell lines and most primary tumor samples via the intrinsic apoptotic pathway. Although this effect was largely tumor cell-specific it is unlikely to have been mediated via ILK. We conclude that ILK does not play a prominent role in the promotion or sustenance of established MM.


Assuntos
Mieloma Múltiplo/patologia , Proteínas Serina-Treonina Quinases/fisiologia , Compostos Azo/farmacologia , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Mieloma Múltiplo/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Pirazóis/farmacologia , RNA Interferente Pequeno/farmacologia
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