RESUMO
SHARPIN regulates signaling from the tumor necrosis factor (TNF) superfamily and pattern-recognition receptors. An inactivating Sharpin mutation in mice causes TNF-mediated dermatitis. Blocking cell death prevents the phenotype, implicating TNFR1-induced cell death in causing the skin disease. However, the source of TNF that drives dermatitis is unknown. Immune cells are a potent source of TNF in vivo and feature prominently in the skin pathology; however, T cells, B cells, and eosinophils are dispensable for the skin phenotype. We use targeted in vivo cell ablation, immune profiling, and extensive imaging to identify immune populations driving dermatitis. We find that systemic depletion of Langerin+ cells significantly reduces disease severity. This is enhanced in mice that lack Langerhans cells (LCs) from soon after birth. Reconstitution of LC-depleted Sharpin mutant mice with TNF-deficient LCs prevents dermatitis, implicating LCs as a potential cellular source of pathogenic TNF and highlighting a T cell-independent role in driving skin inflammation.
Assuntos
Dermatite , Células de Langerhans , Animais , Dermatite/patologia , Inflamação/patologia , Camundongos , Camundongos Knockout , Pele/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Tumor necrosis factor (TNF) is a key component of the innate immune response. Upon binding to its receptor, TNFR1, it promotes production of other cytokines via a membrane-bound complex 1 or induces cell death via a cytosolic complex 2. To understand how TNF-induced cell death is regulated, we performed mass spectrometry of complex 2 and identified tankyrase-1 as a native component that, upon a death stimulus, mediates complex 2 poly-ADP-ribosylation (PARylation). PARylation promotes recruitment of the E3 ligase RNF146, resulting in proteasomal degradation of complex 2, thereby limiting cell death. Expression of the ADP-ribose-binding/hydrolyzing severe acute respiratory syndrome coronavirus 2 macrodomain sensitizes cells to TNF-induced death via abolishing complex 2 PARylation. This suggests that disruption of ADP-ribosylation during an infection can prime a cell to retaliate with an inflammatory cell death.
RESUMO
The specific targeting of inhibitor of apoptosis (IAP) proteins by Smac-mimetic (SM) drugs, such as birinapant, has been tested in clinical trials of acute myeloid leukemia (AML) and certain solid cancers. Despite their promising safety profile, SMs have had variable and limited success. Using a library of more than 5700 bioactive compounds, we screened for approaches that could sensitize AML cells to birinapant and identified multidrug resistance protein 1 inhibitors (MDR1i) as a class of clinically approved drugs that can enhance the efficacy of SM therapy. Genetic or pharmacological inhibition of MDR1 increased intracellular levels of birinapant and sensitized AML cells from leukemia murine models, human leukemia cell lines, and primary AML samples to killing by birinapant. The combination of clinical MDR1 and IAP inhibitors was well tolerated in vivo and more effective against leukemic cells, compared with normal hematopoietic progenitors. Importantly, birinapant combined with third-generation MDR1i effectively killed murine leukemic stem cells (LSCs) and prolonged survival of AML-burdened mice, suggesting a therapeutic opportunity for AML. This study identified a drug combination strategy that, by efficiently killing LSCs, may have the potential to improve outcomes in patients with AML.
Assuntos
Leucemia Mieloide Aguda , Animais , Disponibilidade Biológica , Dipeptídeos , Humanos , Indóis , Proteínas Inibidoras de Apoptose/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , CamundongosRESUMO
Chronic hepatitis B virus (HBV) infection remains a global health threat and affects hundreds of millions worldwide. Small molecule compounds that mimic natural antagonists of inhibitor of apoptosis (IAP) proteins, known as Smac-mimetics (second mitochondria-derived activator of caspases-mimetics), can promote the death of HBV-replicating liver cells and promote clearance of infection in preclinical models of HBV infection. The Smac-mimetic birinapant is a substrate of the multidrug resistance protein 1 (MDR1) efflux pump, and therefore inhibitors of MDR1 increase intracellular concentration of birinapant in MDR1 expressing cells. Liver cells are known to express MDR1 and other drug pump proteins. In this study, we investigated whether combining the clinical drugs, birinapant and the MDR1 inhibitor zosuquidar, increases the efficacy of birinapant in killing HBV expressing liver cells. We showed that this combination treatment is well tolerated and, compared to birinapant single agent, was more efficient at inducing death of HBV-positive liver cells and improving HBV-DNA and HBV surface antigen (HBsAg) control kinetics in an immunocompetent mouse model of HBV infection. Thus, this study identifies a novel and safe combinatorial treatment strategy to potentiate substantial reduction of HBV replication using an IAP antagonist.
Assuntos
Antivirais/uso terapêutico , Dibenzocicloeptenos/uso terapêutico , Dipeptídeos/uso terapêutico , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/tratamento farmacológico , Indóis/uso terapêutico , Quinolinas/uso terapêutico , Replicação Viral/efeitos dos fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Modelos Animais de Doenças , Quimioterapia Combinada , Células Hep G2 , Vírus da Hepatite B/fisiologia , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
MLKL is the essential effector of necroptosis, a form of programmed lytic cell death. We have isolated a mouse strain with a single missense mutation, MlklD139V, that alters the two-helix 'brace' that connects the killer four-helix bundle and regulatory pseudokinase domains. This confers constitutive, RIPK3 independent killing activity to MLKL. Homozygous mutant mice develop lethal postnatal inflammation of the salivary glands and mediastinum. The normal embryonic development of MlklD139V homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO).
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Sistema Hematopoético/patologia , Necroptose/genética , Proteínas Quinases/genética , Animais , Animais Recém-Nascidos , Doenças Hereditárias Autoinflamatórias , Humanos , Inflamação/genética , Camundongos , Mutação de Sentido Incorreto , Osteomielite/genética , Proteínas Quinases/metabolismoRESUMO
Resistance to chemotherapy is a major problem in cancer treatment, and it is frequently associated with failure of tumor cells to undergo apoptosis. Birinapant, a clinical SMAC mimetic, had been designed to mimic the interaction between inhibitor of apoptosis proteins (IAPs) and SMAC/Diablo, thereby relieving IAP-mediated caspase inhibition and promoting apoptosis of cancer cells. We show that acute myeloid leukemia (AML) cells are sensitive to birinapant-induced death and that the clinical caspase inhibitor emricasan/IDN-6556 augments, rather than prevents, killing by birinapant. Deletion of caspase-8 sensitized AML to birinapant, whereas combined loss of caspase-8 and the necroptosis effector MLKL (mixed lineage kinase domain-like) prevented birinapant/IDN-6556-induced death, showing that inhibition of caspase-8 sensitizes AML cells to birinapant-induced necroptosis. However, loss of MLKL alone did not prevent a caspase-dependent birinapant/IDN-6556-induced death, implying that AML will be less likely to acquire resistance to this drug combination. A therapeutic breakthrough in AML has eluded researchers for decades. Demonstrated antileukemic efficacy and safety of the birinapant/emricasan combination in vivo suggest that induction of necroptosis warrants clinical investigation as a therapeutic opportunity in AML.
Assuntos
Caspase 8/metabolismo , Inibidores de Caspase/farmacologia , Dipeptídeos/farmacologia , Indóis/farmacologia , Ácidos Pentanoicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Leucemia Mieloide Aguda/metabolismo , Necrose/metabolismo , Células Tumorais CultivadasRESUMO
Deregulated expression of Hox genes such as HoxA9 is associated with development of myeloproliferative disorders and leukemia and indicates a poor prognosis. To investigate the molecular mechanisms by which HoxA9 promotes immortalization of hematopoietic cells, we generated growth factor dependent myeloid cells in which HoxA9 expression is regulated by administration of 4-hydroxy-tamoxifen. Maintenance of HoxA9 overexpression is required for continued cell survival and proliferation, even in the presence of growth factors. We show for the first time that maintenance of Bcl-2 expression is critical for HoxA9-dependent immortalization and influences the latency of HoxA9-dependent leukemia. Hematopoietic cells lacking Bcl-2 were not immortalized by HoxA9 in vitro. Furthermore, deletion of Bcl-2 delayed the onset and reduced the severity of HoxA9/Meis1 and MLL-AF9 leukemias. This is the first description of a molecular link between HoxA9 and the regulation of Bcl-2 family members in acute myeloid leukemia.
Assuntos
Proteínas de Homeodomínio/metabolismo , Leucemia Mieloide Aguda/metabolismo , Células Progenitoras Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Animais , Apoptose/fisiologia , Processos de Crescimento Celular/fisiologia , Sobrevivência Celular/fisiologia , Regulação Leucêmica da Expressão Gênica , Genes bcl-2 , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Interleucina-3/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Transgênicos , Células Mieloides/metabolismo , Células Mieloides/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologiaRESUMO
Loss of p53-dependent apoptosis contributes to the development of hematologic malignancies and failure to respond to treatment. Proapoptotic Bcl-2 family member Puma is essential for apoptosis in HoxB8-immortalized interleukin-3 (IL-3)-dependent myeloid cell lines (FDM cells) provoked by IL-3 deprivation. p53 and FoxO3a can transcriptionally regulate Puma. To investigate which transcriptional regulator is responsible for IL-3 deprivation-induced Puma expression and apoptosis, we generated wild-type (WT), p53(-/-), and FoxO3a(-/-) FDM cells and found that p53(-/-) but not FoxO3a(-/-) cells were protected against IL-3 withdrawal. Loss of p21(cip/waf), which is critical for p53-mediated cell-cycle arrest, afforded no protection against IL-3 deprivation. A survival advantage was also observed in untransformed p53(-/-) hematopoietic progenitor cells cultured in the presence or absence of cytokines. In response to IL-3 deprivation, increased Puma protein levels in p53(-/-) cells were substantially delayed compared with WT cells. Increased p53 transcriptional activity was detected after cytokine deprivation. This was substantially less than that induced by DNA damage and associated not with increased p53 protein levels but with loss of the p53 regulator, MDM2. Thus, we conclude that p53 protein is activated after IL-3 deprivation by loss of MDM2. Activated p53 transcriptionally up-regulates Puma, which initiates apoptosis.