RESUMEN
Programmed cell death contributes to host defense against pathogens. To investigate the relative importance of pyroptosis, necroptosis, and apoptosis during Salmonella infection, we infected mice and macrophages deficient for diverse combinations of caspases-1, -11, -12, and -8 and receptor interacting serine/threonine kinase 3 (RIPK3). Loss of pyroptosis, caspase-8-driven apoptosis, or necroptosis had minor impact on Salmonella control. However, combined deficiency of these cell death pathways caused loss of bacterial control in mice and their macrophages, demonstrating that host defense can employ varying components of several cell death pathways to limit intracellular infections. This flexible use of distinct cell death pathways involved extensive cross-talk between initiators and effectors of pyroptosis and apoptosis, where initiator caspases-1 and -8 also functioned as executioners when all known effectors of cell death were absent. These findings uncover a highly coordinated and flexible cell death system with in-built fail-safe processes that protect the host from intracellular infections.
Asunto(s)
Apoptosis/inmunología , Macrófagos/inmunología , Necroptosis/inmunología , Piroptosis/inmunología , Infecciones por Salmonella/inmunología , Salmonella/inmunología , Animales , Caspasa 1/deficiencia , Caspasa 1/genética , Caspasa 12/deficiencia , Caspasa 12/genética , Caspasa 8/genética , Caspasas Iniciadoras/deficiencia , Caspasas Iniciadoras/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genéticaRESUMEN
Randomized trials in acute myeloid leukemia (AML) have demonstrated improved survival by the BCL-2 inhibitor venetoclax combined with azacitidine in older patients, and clinical trials are actively exploring the role of venetoclax in combination with intensive chemotherapy in fitter patients with AML. As most patients still develop recurrent disease, improved understanding of relapse mechanisms is needed. We find that 17% of patients relapsing after venetoclax-based therapy for AML have acquired inactivating missense or frameshift/nonsense mutations in the apoptosis effector gene BAX. In contrast, such variants were rare after genotoxic chemotherapy. BAX variants arose within either leukemic or preleukemic compartments, with multiple mutations observed in some patients. In vitro, AML cells with mutated BAX were competitively selected during prolonged exposure to BCL-2 antagonists. In model systems, AML cells rendered deficient for BAX, but not its close relative BAK, displayed resistance to BCL-2 targeting, whereas sensitivity to conventional chemotherapy was variable. Acquired mutations in BAX during venetoclax-based therapy represent a novel mechanism of resistance to BH3-mimetics and a potential barrier to the long-term efficacy of drugs targeting BCL-2 in AML.
Asunto(s)
Leucemia Mieloide Aguda , Proteínas Proto-Oncogénicas c-bcl-2 , Humanos , Anciano , Proteína X Asociada a bcl-2/genética , Línea Celular Tumoral , Proteínas Proto-Oncogénicas c-bcl-2/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Apoptosis , MutaciónRESUMEN
The BCL2 inhibitor venetoclax has established therapeutic roles in chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). As BCL2 is an important determinant of survival of both myeloid progenitor and B cells, we investigated whether clinical and molecular abnormalities arise in the myeloid compartment during long-term continuous venetoclax treatment of CLL in 89 patients (87 with relapsed/refractory CLL). Over a median follow-up of 75 (range 21-98) months, persistent cytopenias (≥1 of neutropenia, thrombocytopenia, anemia) lasting ≥4 months and unrelated to CLL occurred in 25 patients (28%). Of these patients, 20 (80%) displayed clonal hematopoiesis, including 10 with therapy-related myeloid neoplasms (t-MNs). t-MNs occurred exclusively in patients previously exposed to fludarabine-alkylator combination therapy with a cumulative 5-year incidence of 10.4% after venetoclax initiation, consistent with rates reported for patients exposed to fludarabine-alkylator combination therapy without venetoclax. To determine whether the altered myelopoiesis reflected the acquisition of mutations, we analyzed samples from patients with no or minimal bone marrow CLL burden (n = 41). Mutations in the apoptosis effector BAX were identified in 32% (13/41). In cellular assays, C-terminal BAX mutants abrogated outer mitochondrial membrane localization of BAX and engendered resistance to venetoclax killing. BAX-mutated clonal hematopoiesis occurred independently of prior fludarabine-alkylator combination therapy exposure and was not associated with t-MNs. Single-cell sequencing revealed clonal co-occurrence of mutations in BAX with DNMT3A or ASXL1. We also observed simultaneous BCL2 mutations within CLL cells and BAX mutations in the myeloid compartment of the same patients, indicating lineage-specific adaptation to venetoclax therapy.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Compuestos Bicíclicos Heterocíclicos con Puentes , Neoplasias Hematológicas , Leucemia Linfocítica Crónica de Células B , Mutación , Mielopoyesis/efectos de los fármacos , Trastornos Mieloproliferativos , Neoplasias Primarias Secundarias , Sulfonamidas , Proteína X Asociada a bcl-2 , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Compuestos Bicíclicos Heterocíclicos con Puentes/administración & dosificación , Compuestos Bicíclicos Heterocíclicos con Puentes/efectos adversos , Femenino , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Masculino , Persona de Mediana Edad , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Neoplasias Primarias Secundarias/genética , Neoplasias Primarias Secundarias/metabolismo , Sulfonamidas/administración & dosificación , Sulfonamidas/efectos adversos , Vidarabina/administración & dosificación , Vidarabina/efectos adversos , Vidarabina/análogos & derivados , Proteína X Asociada a bcl-2/antagonistas & inhibidores , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Neutralizing antibodies are important for immunity against SARS-CoV-2 and as therapeutics for the prevention and treatment of COVID-19. Here, we identified high-affinity nanobodies from alpacas immunized with coronavirus spike and receptor-binding domains (RBD) that disrupted RBD engagement with the human receptor angiotensin-converting enzyme 2 (ACE2) and potently neutralized SARS-CoV-2. Epitope mapping, X-ray crystallography, and cryo-electron microscopy revealed two distinct antigenic sites and showed two neutralizing nanobodies from different epitope classes bound simultaneously to the spike trimer. Nanobody-Fc fusions of the four most potent nanobodies blocked ACE2 engagement with RBD variants present in human populations and potently neutralized both wild-type SARS-CoV-2 and the N501Y D614G variant at concentrations as low as 0.1 nM. Prophylactic administration of either single nanobody-Fc or as mixtures reduced viral loads by up to 104-fold in mice infected with the N501Y D614G SARS-CoV-2 virus. These results suggest a role for nanobody-Fc fusions as prophylactic agents against SARS-CoV-2.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , Tratamiento Farmacológico de COVID-19 , COVID-19 , SARS-CoV-2/inmunología , Anticuerpos de Dominio Único , Enzima Convertidora de Angiotensina 2/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/farmacología , COVID-19/inmunología , Camélidos del Nuevo Mundo , Humanos , Ratones , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/farmacologíaRESUMEN
Selective targeting of BCL-2 with the BH3-mimetic venetoclax has been a transformative treatment for patients with various leukemias. TP-53 controls apoptosis upstream of where BCL-2 and its prosurvival relatives, such as MCL-1, act. Therefore, targeting these prosurvival proteins could trigger apoptosis across diverse blood cancers, irrespective of TP53 mutation status. Indeed, targeting BCL-2 has produced clinically relevant responses in blood cancers with aberrant TP-53. However, in our study, TP53-mutated or -deficient myeloid and lymphoid leukemias outcompeted isogenic controls with intact TP-53, unless sufficient concentrations of BH3-mimetics targeting BCL-2 or MCL-1 were applied. Strikingly, tumor cells with TP-53 dysfunction escaped and thrived over time if inhibition of BCL-2 or MCL-1 was sublethal, in part because of an increased threshold for BAX/BAK activation in these cells. Our study revealed the key role of TP-53 in shaping long-term responses to BH3-mimetic drugs and reconciled the disparate pattern of initial clinical response to venetoclax, followed by subsequent treatment failure among patients with TP53-mutant chronic lymphocytic leukemia or acute myeloid leukemia. In contrast to BH3-mimetics targeting just BCL-2 or MCL-1 at doses that are individually sublethal, a combined BH3-mimetic approach targeting both prosurvival proteins enhanced lethality and durably suppressed the leukemia burden, regardless of TP53 mutation status. Our findings highlight the importance of using sufficiently lethal treatment strategies to maximize outcomes of patients with TP53-mutant disease. In addition, our findings caution against use of sublethal BH3-mimetic drug regimens that may enhance the risk of disease progression driven by emergent TP53-mutant clones.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Indolizinas/farmacología , Isoquinolinas/farmacología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Mieloide Aguda/tratamiento farmacológico , Morfolinas/farmacología , Proteínas de Neoplasias/fisiología , Fragmentos de Péptidos/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Sulfonamidas/farmacología , Proteína p53 Supresora de Tumor/fisiología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/uso terapéutico , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis/fisiología , Compuestos Bicíclicos Heterocíclicos con Puentes/administración & dosificación , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Sistemas CRISPR-Cas , Línea Celular Tumoral , Daño del ADN , Genes p53 , Humanos , Indolizinas/uso terapéutico , Subunidad alfa del Receptor de Interleucina-2/deficiencia , Isoquinolinas/uso terapéutico , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/terapia , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Morfolinas/uso terapéutico , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Proteínas de Neoplasias/antagonistas & inhibidores , Fosforilación Oxidativa/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Sulfonamidas/administración & dosificación , Sulfonamidas/uso terapéutico , Proteína p53 Supresora de Tumor/deficiencia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BAK and BAX are the essential effectors of apoptosis because without them a cell is resistant to most apoptotic stimuli. BAK and BAX undergo conformation changes to homooligomerize then permeabilize the mitochondrial outer membrane during apoptosis. How BCL-2 homology 3 (BH3)-only proteins bind to activate BAK and BAX is unclear. We report that BH3-only proteins bind inactive full-length BAK at mitochondria and then dissociate following exposure of the BAK BH3 and BH4 domains before BAK homodimerization. Using a functional obstructive labeling approach, we show that activation of BAK involves important interactions of BH3-only proteins with both the canonical hydrophobic binding groove (α2-5) and α6 at the rear of BAK, with interaction at α6 promoting an open groove to receive a BH3-only protein. Once activated, how BAK homodimers multimerize to form the putative apoptotic pore is unknown. Obstructive labeling of BAK beyond the BH3 domain and hydrophobic groove did not inhibit multimerization and mitochondrial damage, indicating that critical protein-protein interfaces in BAK self-association are limited to the α2-5 homodimerization domain.
Asunto(s)
Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/química , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Animales , Apoptosis , Sitios de Unión , Línea Celular , Citocromos c/metabolismo , Disulfuros/química , Epítopos/química , Fibroblastos/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Unión Proteica , Dominios Proteicos , Mapeo de Interacción de Proteínas , Multimerización de Proteína , Proteína X Asociada a bcl-2/metabolismoRESUMEN
AIMS: The purpose of this clinical trial was to investigate whether cardiovascular magnetic resonance imaging (CMR) using ferumoxytol (Feraheme™, FH), an ultrasmall superparamagnetic iron oxide nanoparticle (USPIO), allows more detailed characterization of infarct pathology compared with conventional gadolinium-based necrosis/fibrosis imaging in patients with acute myocardial infarction. METHODS AND RESULTS: Fourteen patients who had experienced an acute ST-elevation myocardial infarction were included in this study. Following coronary angiography, a first baseline study (pre-FH) was performed followed by subsequent CMR studies (post-FH) 48 h after intravenous ferumoxytol administration. The CMR studies comprised cine-CMR, T(2)-weighted short tau inversion recovery spin echo imaging, T(2)-mapping, and T(1)-weighted late gadolinium enhancement (LGE) imaging. The median extent of short-axis in-plane LGE was 30% [inter-quartile range (IQR) 26-40%]. The median in-plane extent of T(2)-weighted 'hypoenhancement' in the region of myocardial infarction, which was not present prior to ferumoxytol administration in any patient, was 19% (IQR 14-22%; P < 0.001 compared with the extent of LGE). The median in-plane extent of areas showing signal void in T(2)-mapping images post-FH in the region of myocardial infarction was 16% (IQR 12-18%; P < 0.001 compared with the extent of LGE; P = 0.34 compared with the extent of T(2)-weighted hypoenhancement). A substantial drop in absolute T(2)-values was observed not only in the infarct core and peri-infarct zone, but also in the remote 'healthy' myocardium, although there was only a minor change in the skeletal muscle. Substantial ferumoxytol uptake was detected only in cultured macrophages, but not in peripheral blood monocytes from study patients. CONCLUSION: We could demonstrate in humans that USPIO-based contrast agents enable a more detailed characterization of myocardial infarct pathology mainly by detecting infiltrating macrophages. Considering the multi-functionality of USPIO-based particles and their superior safety profile compared with gadolinium-based compounds, these observations open up new vistas for the clinical application of USPIO.
Asunto(s)
Medios de Contraste , Dextranos , Nanopartículas de Magnetita , Infarto del Miocardio/diagnóstico , Células Cultivadas , Medios de Contraste/farmacocinética , Dextranos/farmacocinética , Óxido Ferrosoférrico/farmacocinética , Humanos , Leucocitos Mononucleares/metabolismo , Angiografía por Resonancia Magnética/métodos , Persona de Mediana Edad , Estudios Prospectivos , Factores de TiempoRESUMEN
Ferroptosis is a recently defined form of regulated cell death, which is biochemically and morphologically distinct from traditional forms of programmed cell death such as apoptosis or necrosis. It is driven by iron, reactive oxygen species, and phospholipids that are oxidatively damaged, ultimately resulting in mitochondrial damage and breakdown of membrane integrity. Numerous cellular signaling pathways and molecules are involved in the regulation of ferroptosis, including enzymes that control the cellular redox status. Alterations in the ferroptosis-regulating network can contribute to the development of various diseases, including cancer. Evidence suggests that ferroptosis is commonly suppressed in cancer cells, allowing them to survive and progress. However, cancer cells which are resistant to common chemotherapeutic drugs seem to be highly susceptible to ferroptosis inducers, highlighting the great potential of pharmacologic modulation of ferroptosis for cancer treatment. Non-coding RNAs (ncRNAs) are considered master regulators of various cellular processes, particularly in cancer where they have been implicated in all hallmarks of cancer. Recent work also demonstrated their involvement in the molecular control of ferroptosis. Hence, ncRNA-based therapeutics represent an exciting alternative to modulate ferroptosis for cancer therapy. This review summarizes the ncRNAs implicated in the regulation of ferroptosis in cancer and highlights their underlying molecular mechanisms in the light of potential therapeutic applications.
Asunto(s)
Ferroptosis , Neoplasias , Apoptosis/genética , Muerte Celular/fisiología , Ferroptosis/genética , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismoRESUMEN
A variety of cancer entities are driven by KRAS mutations, which remain difficult to target clinically. Survival pathways, such as resistance to cell death, may represent a promising treatment approach in KRAS mutated cancers. Based on the frequently observed genomic deletions of BCL-2-related ovarian killer (BOK) in cancer patients, we explored the function of BOK in a mutant KrasG12D-driven murine model of lung cancer. Using KrasG12D/+ Bok-/- mice, we observed an overall tumor-promoting function of BOK in vivo. Specifically, loss of BOK reduced proliferation both in cell lines in vitro as well as in KrasG12D-driven tumor lesions in vivo. During tumor development in vivo, loss of BOK resulted in a lower tumor burden, with fewer, smaller, and less advanced tumors. Using KrasG12D/+ Tp53Δ/Δ Bok-/- mice, we identified that this phenotype was entirely dependent on the presence of functional p53. Furthermore, analysis of a human dataset of untreated early-stage lung tumors did not identify any common deletion of the BOK locus, independently of the TP53 status or the histopathological classification. Taken together our data indicate that BOK supports tumor progression in Kras-driven lung cancer.
Asunto(s)
Proteína p53 Supresora de TumorRESUMEN
The conformational changes converting BAX from an inert cytosolic monomer into the homo-oligomers that permeabilize the mitochondrial outer membrane (MOM) are crucial steps toward apoptosis. Here, we have explored the potential role of the BAX α1-α2 loop in this process by three mutagenic approaches: replacing loop segments with cognate loop regions from closely related proteins, alanine scanning and analysis of BAX α1-α2 loop missense mutations observed in tumours. Responsiveness to a death signal, such as tBID, was reduced by mutations in the N-terminal but not C-terminal half of the loop. N-terminal loop variants, which were enriched in tumours, impaired MOM integration by allosterically reducing exposure of the BAX α9 transmembrane anchor. Most C-terminal loop variants reduced BAX stability, leading to increased BAX apoptotic function in some variants. Thus, our systematic mutagenesis suggests that the two halves of the α1-α2 loop have distinct functions. We show that the N-terminal half of the loop (its first nine residues) comprises an important allosteric regulator of BAX activation by setting the proportion of MOM-integrated BAX following a death signal. The enrichment of N-terminal loop mutations in tumours indicates that they may promote tumour cell survival and underscore the loop as a target for therapeutic manipulation of BAX function.
Asunto(s)
Sitio Alostérico/genética , Mitocondrias/metabolismo , Proteína X Asociada a bcl-2/genética , Animales , Humanos , Ratones , Modelos Moleculares , TransfecciónRESUMEN
BAK and BAX, which drive commitment to apoptosis, are activated principally by certain BH3-only proteins that bind them and trigger major rearrangements. One crucial conformation change is exposure of their BH3 domain which allows BAK or BAX to form homodimers, and potentially to autoactivate other BAK and BAX molecules to ensure robust pore formation and cell death. Here, we test whether full-length BAK or mitochondrial BAX that are specifically activated by antibodies can then activate other BAK or BAX molecules. We found that antibody-activated BAK efficiently activated BAK as well as mitochondrial or cytosolic BAX, but antibody-activated BAX unexpectedly proved a poor activator. Notably, autoactivation by BAK involved transient interactions, as BAK and BAX molecules it activated could dissociate and homodimerize. The results suggest that BAK-driven autoactivation may play a substantial role in apoptosis, including recruitment of BAX to the mitochondria. Hence, directly targeting BAK rather than BAX may prove particularly effective in inhibiting unwanted apoptosis, or alternatively, inducing apoptosis in cancer cells.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , Apoptosis , Humanos , Ratones , Pliegue de ProteínaRESUMEN
Apoptosis-regulating BCL-2 family members, which can promote malignant transformation and resistance to therapy, have become prime therapeutic targets, as illustrated by the striking efficacy in certain lymphoid malignancies of the BCL-2-specific inhibitor venetoclax. In other lymphoid malignancies, however, such as the aggressive mantle cell lymphoma (MCL), cell survival might rely instead or also on BCL-2 relative MCL-1. We have explored MCL-1 as a target for killing MCL cells by both genetic and pharmacologic approaches. In several MCL cell lines, MCL-1 knockout with an inducible CRISPR/Cas9 system triggered spontaneous apoptosis. Accordingly, most MCL cell lines proved sensitive to the specific MCL-1 inhibitor S63845, and MCL-1 inhibition also proved efficacious in an MCL xenograft model. Furthermore, its killing efficacy rose on combination with venetoclax, the BCL-XL-specific inhibitor A-1331852, or Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, which reduced pro-survival signals. We also tested the MCL-1 inhibitor in primary samples from 13 MCL patients, using CD40L-expressing feeder cells to model their microenvironmental support. Notably, all unstimulated primary MCL samples were very sensitive to S63845, but the CD40L stimulation attenuated their sensitivity. Mass cytometric analysis revealed that the stimulation likely conveyed protection by elevating BCL-XL and MCL-1. Accordingly, sensitivity of the CD40L-stimulated cells to S63845 was substantially restored by co-treatment with venetoclax, the BCL-XL-specific inhibitor or ibrutinib. Overall, our findings indicate that MCL-1 is very important for survival of MCL cells and that the MCL-1 inhibitor, both alone and together with ibrutinib, venetoclax or a BCL-XL inhibitor, offers promise for novel improved MCL therapies.
Asunto(s)
Linfoma de Células del Manto/tratamiento farmacológico , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Pirimidinas/farmacología , Tiofenos/farmacología , Proteína bcl-X/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Apoptosis , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Proliferación Celular , Humanos , Linfoma de Células del Manto/metabolismo , Linfoma de Células del Manto/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Sulfonamidas/farmacología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
To elicit apoptosis, BAX metamorphoses from an inert cytosolic monomer into homo-oligomers that permeabilize the mitochondrial outer membrane (MOM). A long-standing puzzle is that BH3 domains apparently activate BAX by not only its canonical groove but also a proposed site involving helices α1 and α6. Our mutagenesis studies reveal that late steps like oligomerization require activation through the groove but probably not earlier steps like MOM association. Conversely, α1 or α6 obstruction and alanine mutagenesis scanning implicate these helices early in BAX activation. The α1 and α6 mutations lowered BH3 binding, altered the BAX conformation, and reduced its MOM translocation and integration; their exposure of the BAX α1-α2 loop allosterically sequestered its α9 membrane anchor in the groove. The crystal structure of an α6 mutant revealed additional allosteric effects. The results suggest that the α1 and α6 region drives MOM association and integration, whereas groove binding favors subsequent steps toward oligomerization.
Asunto(s)
Mitocondrias Hepáticas/metabolismo , Membranas Mitocondriales/metabolismo , Mutación , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Fragmentos de Péptidos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Alineación de SecuenciaRESUMEN
Apoptotic cell death removes unwanted cells and is regulated by interactions between pro-survival and pro-apoptotic members of the BCL-2 protein family. The regulation of apoptosis is thought to be crucial for normal embryonic development. Accordingly, complete loss of pro-survival MCL-1 or BCL-XL (BCL2L1) causes embryonic lethality. However, it is not known whether minor reductions in pro-survival proteins could cause developmental abnormalities. We explored the rate-limiting roles of MCL-1 and BCL-XL in development and show that combined loss of single alleles of Mcl-1 and Bcl-x causes neonatal lethality. Mcl-1+/-;Bcl-x+/- mice display craniofacial anomalies, but additional loss of a single allele of pro-apoptotic Bim (Bcl2l11) restores normal development. These findings demonstrate that the control of cell survival during embryogenesis is finely balanced and suggest that some human craniofacial defects, for which causes are currently unknown, may be due to subtle imbalances between pro-survival and pro-apoptotic BCL-2 family members.
Asunto(s)
Proteína 11 Similar a Bcl2/genética , Anomalías Craneofaciales/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína bcl-X/genética , Animales , Apoptosis , Proteína 11 Similar a Bcl2/metabolismo , Células Cultivadas , Femenino , Heterocigoto , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteína bcl-X/metabolismoRESUMEN
Treatment response of follicular lymphomas (FL) is highly variable. We, therefore, investigated the role of FL cancer-associated fibroblasts (CAFs) on tumor cell viability, in particular in response to treatment with cytotoxic drugs. Stromal cells outgrown from FL patients were characterized and pure CAF populations were co-cultivated with FL cells. To analyze fibroblast-mediated effects, cells in co-culture were treated with ABT-737 and Bortezomib. The adherent cell population was positive for all fibroblastic markers tested and showed increased mRNA-expression of the activation marker FAP. No effect on FL cell viability was noted when co-cultivating them with CAFs. However, stromal cells protected tumor cells from apoptosis in response to cytotoxic treatment. This might be explained by mRNA-induction of ABCC1 and ABCG2 and up-regulation of BCL2L1 in FL cells. Our finding of protective mechanisms mediated by CAFs is of pivotal impact for further studies of cytotoxic agents in FL.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Fibroblastos Asociados al Cáncer/efectos de los fármacos , Fibroblastos Asociados al Cáncer/metabolismo , Supervivencia Celular/efectos de los fármacos , Linfoma Folicular/metabolismo , Linfoma Folicular/patología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Antígenos CD19/genética , Antígenos CD19/metabolismo , Antígenos CD20/genética , Antígenos CD20/metabolismo , Biomarcadores , Compuestos de Bifenilo/farmacología , Línea Celular Tumoral , Técnicas de Cocultivo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Linfoma Folicular/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Clasificación del Tumor , Neprilisina/genética , Neprilisina/metabolismo , Nitrofenoles/farmacología , Piperazinas/farmacología , Sulfonamidas/farmacología , Proteína bcl-X/genética , Proteína bcl-X/metabolismoAsunto(s)
Anticuerpos Monoclonales/análisis , Antígenos de Histocompatibilidad Menor/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Antígenos de Histocompatibilidad Menor/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Testicular germ cell tumors (TGCT) are considered a paradigm of chemosensitive tumors. Embryonal carcinoma cells represent the pluripotent entity of TGCTs and are characterized by expression of Oct-4, a key regulator of pluripotency and a determinant of their inherent hypersensitivity to cisplatin. However, the mechanisms underlying this Oct-4-mediated sensitivity are poorly understood. We previously showed that p53 is a major player in cisplatin hypersensitivity and therefore investigated whether Oct-4 may directly affect p53 activity. Despite a significant decrease in sensitivity, depletion of Oct-4 neither did alter cisplatin-induced transactivation of p53 target genes nor its subcellular localization. These data indicate that, rather than directly modulating p53 activity, Oct-4 provides a cellular context that augments the proapoptotic activity of p53. As mitochondrial priming by the Bcl-2 family is a known determinant of chemosensitivity, we compared the constitutive levels of these proteins in Oct-4-positive and -depleted cells. We identified Noxa as the only Bcl-2 family protein to be highly correlated with Oct-4 status and cisplatin sensitivity. Compared with differentiated cells, constitutive Noxa levels were significantly higher in Oct-4-positive cell lines and cancer patient samples. Furthermore, RNA interference-mediated knockdown of Oct-4 resulted in reduced Noxa transcript, in an almost complete loss of constitutive Noxa protein and decreased cisplatin hypersensitivity to a similar extent as did Noxa depletion. In conclusion, our study indicates that Noxa is a central determinant of hypersensitivity to cisplatin. Oct-4-dependent high constitutive levels of this BH3-only protein prime embryonal carcinoma cells to undergo rapid and massive apoptosis in response to p53 activation.
Asunto(s)
Cisplatino/farmacología , Neoplasias de Células Germinales y Embrionarias/tratamiento farmacológico , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Neoplasias Testiculares/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Neoplasias de Células Germinales y Embrionarias/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Neoplasias Testiculares/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Consistent with the excellent clinical results in testicular germ cell tumors (TGCT), most cell lines derived from this cancer show an exquisite sensitivity to Cisplatin. It is well accepted that the high susceptibility of TGCT cells to apoptosis plays a central role in this hypersensitive phenotype. The role of the tumor suppressor p53 in this response, however, remains controversial. Here we show that siRNA-mediated silencing of p53 is sufficient to completely abrogate hypersensitivity not only to Cisplatin but also to non-genotoxic inducers of p53 such as the Mdm2 antagonist Nutlin-3 and the proteasome inhibitor Bortezomib. The close relationship between p53 protein levels and induction of apoptosis is lost upon short-term differentiation, indicating that this predominant pro-apoptotic function of p53 is unique in pluripotent embryonal carcinoma (EC) cells. RNA interference experiments as well as microarray analysis demonstrated a central role of the pro-apoptotic p53 target gene NOXA in the p53-dependent apoptotic response of these cells. In conclusion, our data indicate that the hypersensitivity of TGCT cells is a result of their unique sensitivity to p53 activation. Furthermore, in the very specific cellular context of germ cell-derived pluripotent EC cells, p53 function appears to be limited to induction of apoptosis.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , Neoplasias de Células Germinales y Embrionarias/patología , Neoplasias Testiculares/patología , Proteína p53 Supresora de Tumor/fisiología , Secuencia de Bases , Línea Celular Tumoral , Cartilla de ADN , Humanos , Masculino , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/genéticaRESUMEN
In response to deregulated oncogene activation, mammalian cells activate disposal programs such as programmed cell death. To investigate the mechanisms behind this oncogenic stress response we used Bcr-Abl over-expressing cells cultivated in presence of imatinib. Imatinib deprivation led to rapid induction of Bcr-Abl activity and over-stimulation of PI3K/Akt-, Ras/MAPK-, and JAK/STAT pathways. This resulted in a delayed necrosis-like cell death starting not before 48 hours after imatinib withdrawal. Cell death was preceded by enhanced glycolysis, glutaminolysis, and amino acid metabolism leading to elevated ATP and protein levels. This enhanced metabolism could be linked to induction of cell death as inhibition of glycolysis or glutaminolysis was sufficient to sustain cell viability. Therefore, these data provide first evidence that metabolic changes induced by Bcr-Abl hyper-activation are important mediators of oncogenic stress-induced cell death.During the first 30 hours after imatinib deprivation, Bcr-Abl hyper-activation did not affect proliferation but resulted in cellular swelling, vacuolization, and induction of eIF2α phosphorylation, CHOP expression, as well as alternative splicing of XPB, indicating endoplasmic reticulum stress response. Cell death was dependent on p38 and RIP1 signaling, whereas classical death effectors of ER stress, namely CHOP-BIM were antagonized by concomitant up-regulation of Bcl-xL.Screening of 1,120 compounds for their potential effects on oncogenic stress-induced cell death uncovered that corticosteroids antagonize cell death upon Bcr-Abl hyper-activation by normalizing cellular metabolism. This protective effect is further demonstrated by the finding that corticosteroids rendered lymphocytes permissive to the transforming activity of Bcr-Abl. As corticosteroids are used together with imatinib for treatment of Bcr-Abl positive acute lymphoblastic leukemia these data could have important implications for the design of combination therapy protocols.In conclusion, excessive induction of Warburg type metabolic alterations can cause cell death. Our data indicate that these metabolic changes are major mediators of oncogenic stress induced by Bcr-Abl.