RESUMO
RUNX1 is a member of RUNX transcription factors and plays important roles in hematopoiesis. Disruption of RUNX1 activity has been implicated in the development of hematopoietic neoplasms. Chromosomal translocations involving the RUNX1 gene are associated with several types of leukemia, including acute myeloid leukemia driven by a leukemogenic fusion protein RUNX1-RUNX1T1. Previous studies have shown that RUNX1 is an unstable protein and is subjected to proteolytic degradation mediated by the ubiquitin-proteasome pathway. However, the precise mechanisms of RUNX1 ubiquitination have not been fully understood. Furthermore, much less is known about the mechanisms to regulate the stability of RUNX1-RUNX1T1. In this study, we identified several RUNX1-interacting E3 ubiquitin ligases using a novel high-throughput binding assay. Among them, we found that STUB1 bound to RUNX1 and induced its ubiquitination and degradation mainly in the nucleus. Immunofluorescence analyses revealed that the STUB1-induced ubiquitination also promoted nuclear export of RUNX1, which probably contributes to the reduced transcriptional activity of RUNX1 in STUB1-overexpressing cells. STUB1 also induced ubiquitination of RUNX1-RUNX1T1 and down-regulated its expression. Importantly, STUB1 overexpression showed a substantial growth-inhibitory effect in myeloid leukemia cells that harbor RUNX1-RUNX1T1, whereas it showed only a marginal effect in other non-RUNX1-RUNX1T1 leukemia cells and normal human cord blood cells. Taken together, these data suggest that the E3 ubiquitin ligase STUB1 is a negative regulator of both RUNX1 and RUNX1-RUNX1T1. Activation of STUB1 could be a promising therapeutic strategy for RUNX1-RUNX1T1 leukemia.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Humanos , Estabilidade Proteica , Proteína 1 Parceira de Translocação de RUNX1RESUMO
RUNX1 is a member of RUNX transcription factors and plays important roles in hematopoiesis. RUNX1 function is under the tight control through posttranslational modifications, including phosphorylation and ubiquitination. We previously developed a luminescence-based binding assay (AlphaScreen) to systematically detect RUNX1-interacting E3 ubiquitin ligases. In this study, we showed that a nuclear ubiquitin ligase RNF38 induced ubiquitination of RUNX1. RNF38-induced RUNX1 ubiquitination did not promote RUNX1 degradation, but rather stabilized RUNX1 protein. We also found that RNF38 enhanced RUNX1-mediated transcriptional repression of the erythroid master regulator KLF1 in K562â¯cells. Consequently, RNF38 cooperated with RUNX1 to inhibit erythroid differentiation of K562â¯cells. Thus, our study identified RNF38 as a novel E3 ligase that modifies RUNX1 function without inducing its degradation.
Assuntos
Proteínas de Transporte/farmacologia , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Ubiquitinação/efeitos dos fármacos , Diferenciação Celular , Subunidade alfa 2 de Fator de Ligação ao Core/efeitos dos fármacos , Células Eritroides/citologia , Células Eritroides/efeitos dos fármacos , Humanos , Células K562 , Fatores de Transcrição Kruppel-Like , Estabilidade Proteica/efeitos dos fármacos , Ubiquitina-Proteína Ligases/farmacologiaRESUMO
Enhancement of intestinal IgA responses is a primary strategy in the development of oral vaccine. Dietary fatty acids are known to regulate host immune responses. In this study, we show that dietary palmitic acid (PA) and its metabolites enhance intestinal IgA responses. Intestinal IgA production was increased in mice maintained on a PA-enriched diet. These mice also showed increased intestinal IgA responses against orally immunized Ag, without any effect on serum Ab responses. We found that PA directly stimulates plasma cells to produce Ab. In addition, mice receiving a PA-enriched diet had increased numbers of IgA-producing plasma cells in the large intestine; this effect was abolished when serine palmitoyltransferase was inhibited. These findings suggest that dietary PA regulates intestinal IgA responses and has the potential to be a diet-derived mucosal adjuvant.
Assuntos
Gorduras na Dieta/metabolismo , Imunoglobulina A/imunologia , Mucosa Intestinal/imunologia , Ácido Palmítico/imunologia , Adjuvantes Imunológicos/metabolismo , Animais , Células Cultivadas , Toxina da Cólera/imunologia , Óleo de Coco , Suplementos Nutricionais , Ácidos Graxos Monoinsaturados , Feminino , Imunidade nas Mucosas , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Ovalbumina/imunologia , Óleo de Palmeira , Ácido Palmítico/administração & dosagem , Óleos de Plantas/administração & dosagem , Óleos de Plantas/metabolismo , Plasmócitos/imunologia , Óleo de Brassica napus , Serina C-Palmitoiltransferase/antagonistas & inibidores , Óleo de Soja/administração & dosagemRESUMO
Immunotherapy has attracted considerable attention as a therapeutic strategy for cancers including acute myeloid leukemia (AML). In this study, we found that the development of several aggressive subtypes of AML is slower in Rag2-/- mice despite the lack of B and T lymphocytes, even compared to the immunologically normal C57BL/6 mice. Furthermore, an orally active p53-activating drug shows stronger antileukemia effect on AML in Rag2-/- mice than C57BL/6 mice. Intriguingly, Natural Killer (NK) cells in Rag2-/- mice are increased in number, highly express activation markers, and show increased cytotoxicity to leukemia cells in a coculture assay. B2m depletion that triggers missing-self recognition of NK cells impairs the growth of AML cells in vivo. In contrast, NK cell depletion accelerates AML progression in Rag2-/- mice. Interestingly, immunogenicity of AML keeps changing during tumor evolution, showing a trend that the aggressive AMLs generate through serial transplantations are susceptible to NK cell-mediated tumor suppression in Rag2-/- mice. Thus, we show the critical role of NK cells in suppressing the development of certain subtypes of AML using Rag2-/- mice, which lack functional lymphocytes but have hyperactive NK cells.
Assuntos
Células Matadoras Naturais , Leucemia Mieloide Aguda , Animais , Camundongos , Camundongos Knockout , Camundongos Endogâmicos C57BL , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Linfócitos T , Proteínas de Ligação a DNA/genéticaRESUMO
Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in de novo guanine nucleotide synthesis pathway. Although IMPDH inhibitors are widely used as effective immunosuppressants, their antitumor effects have not been proven in the clinical setting. Here, we found that acute myeloid leukemias (AMLs) with MLL-fusions are susceptible to IMPDH inhibitors in vitro. We also showed that alternate-day administration of IMPDH inhibitors suppressed the development of MLL-AF9-driven AML in vivo without having a devastating effect on immune function. Mechanistically, IMPDH inhibition induced overactivation of Toll-like receptor (TLR)-TRAF6-NF-κB signaling and upregulation of an adhesion molecule VCAM1, which contribute to the antileukemia effect of IMPDH inhibitors. Consequently, combined treatment with IMPDH inhibitors and the TLR1/2 agonist effectively inhibited the development of MLL-fusion AML. These findings provide a rational basis for clinical testing of IMPDH inhibitors against MLL-fusion AMLs and potentially other aggressive tumors with active TLR signaling.
Assuntos
Leucemia Mieloide Aguda , Proteína de Leucina Linfoide-Mieloide , Humanos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Inibidores Enzimáticos/farmacologia , NF-kappa B , Imunossupressores/uso terapêuticoRESUMO
Somatic mutations of ASXL1 are frequently detected in age-related clonal hematopoiesis (CH). However, how ASXL1 mutations drive CH remains elusive. Using knockin (KI) mice expressing a C-terminally truncated form of ASXL1-mutant (ASXL1-MT), we examined the influence of ASXL1-MT on physiological aging in hematopoietic stem cells (HSCs). HSCs expressing ASXL1-MT display competitive disadvantage after transplantation. Nevertheless, in genetic mosaic mouse model, they acquire clonal advantage during aging, recapitulating CH in humans. Mechanistically, ASXL1-MT cooperates with BAP1 to deubiquitinate and activate AKT. Overactive Akt/mTOR signaling induced by ASXL1-MT results in aberrant proliferation and dysfunction of HSCs associated with age-related accumulation of DNA damage. Treatment with an mTOR inhibitor rapamycin ameliorates aberrant expansion of the HSC compartment as well as dysregulated hematopoiesis in aged ASXL1-MT KI mice. Our findings suggest that ASXL1-MT provokes dysfunction of HSCs, whereas it confers clonal advantage on HSCs over time, leading to the development of CH.
Assuntos
Envelhecimento/genética , Hematopoiese Clonal/genética , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Proteínas Repressoras/genética , Serina-Treonina Quinases TOR/metabolismo , Idoso , Envelhecimento/metabolismo , Envelhecimento/fisiologia , Animais , Apoptose/genética , Ciclo Celular/genética , Proliferação de Células/genética , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Técnicas de Introdução de Genes , Hematopoese/fisiologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/fisiologia , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/genética , Camundongos , Camundongos Transgênicos , Mutação , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA-Seq , Espécies Reativas de Oxigênio/farmacologia , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sirolimo/farmacologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genéticaRESUMO
ASXL1 mutations occur frequently in myeloid neoplasms and are associated with poor prognosis. However, the mechanisms by which mutant ASXL1 induces leukaemogenesis remain unclear. In this study, we report mutually reinforcing effects between a C-terminally truncated form of mutant ASXL1 (ASXL1-MT) and BAP1 in promoting myeloid leukaemogenesis. BAP1 expression results in increased monoubiquitination of ASXL1-MT, which in turn increases the catalytic function of BAP1. This hyperactive ASXL1-MT/BAP1 complex promotes aberrant myeloid differentiation of haematopoietic progenitor cells and accelerates RUNX1-ETO-driven leukaemogenesis. Mechanistically, this complex induces upregulation of posterior HOXA genes and IRF8 through removal of H2AK119 ubiquitination. Importantly, BAP1 depletion inhibits posterior HOXA gene expression and leukaemogenicity of ASXL1-MT-expressing myeloid leukemia cells. Furthermore, BAP1 is also required for the growth of MLL-fusion leukemia cells with posterior HOXA gene dysregulation. These data indicate that BAP1, which has long been considered a tumor suppressor, in fact plays tumor-promoting roles in myeloid neoplasms.
Assuntos
Carcinogênese/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide/genética , Proteínas Repressoras/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Animais , Medula Óssea/metabolismo , Medula Óssea/patologia , Transplante de Medula Óssea , Sistemas CRISPR-Cas , Carcinogênese/metabolismo , Carcinogênese/patologia , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Feminino , Edição de Genes , Células HEK293 , Células HeLa , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Leucemia Mieloide/metabolismo , Leucemia Mieloide/mortalidade , Leucemia Mieloide/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteína 1 Parceira de Translocação de RUNX1/genética , Proteína 1 Parceira de Translocação de RUNX1/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Análise de Sobrevida , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação , Irradiação Corporal TotalRESUMO
Bioenergetic metabolism varies during cell differentiation, but details of B cell metabolism remain unclear. Here, we show the metabolic changes during B cell differentiation in the intestine, where B cells differentiate into IgA(+) plasma cells (PCs). Naive B cells in the Peyer's patches (PPs) and IgA(+) PCs in the intestinal lamina propria (iLP) both used the tricarboxylic acid (TCA) cycle, but only IgA(+) PCs underwent glycolysis. These metabolic differences reflected their dependencies on vitamin B1, an essential cofactor for the TCA cycle. Indeed, the diminished activity of the TCA cycle after dietary vitamin B1 depletion decreased the number of naive B cells in PPs without affecting IgA(+) PCs in the iLP. The maintenance of naive B cells by dietary vitamin B1 was required to induce-but not maintain-intestinal IgA responses against oral antigens. These findings reveal the diet-mediated maintenance of B cell immunometabolism in organized and diffuse intestinal tissues.
Assuntos
Linfócitos B/metabolismo , Imunidade nas Mucosas , Mucosa Intestinal/metabolismo , Plasmócitos/metabolismo , Tiamina/metabolismo , Deficiência de Vitaminas do Complexo B/metabolismo , Animais , Anticorpos/metabolismo , Linfócitos B/citologia , Linfócitos B/imunologia , Diferenciação Celular , Linhagem da Célula/imunologia , Ciclo do Ácido Cítrico/imunologia , Feminino , Glicólise/imunologia , Imunidade Humoral , Imunoglobulina A/biossíntese , Imunoglobulina M/biossíntese , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Linfonodos/citologia , Linfonodos/imunologia , Linfonodos/metabolismo , Ativação Linfocitária , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos BALB C , Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/metabolismo , Plasmócitos/citologia , Plasmócitos/imunologia , Tiamina/imunologia , Deficiência de Vitaminas do Complexo B/imunologia , Deficiência de Vitaminas do Complexo B/patologiaRESUMO
ω3 polyunsaturated fatty acids (PUFAs) have anti-allergic and anti-inflammatory properties, but the immune-metabolic progression from dietary oil remains to be investigated. Here we identified 17,18-epoxyeicostetraenoic acid (17,18-EpETE) as an anti-allergic metabolite generated in the gut from dietary ω3 α-linolenic acid (ALA). Biochemical and imaging mass spectrometry analyses revealed increased ALA and its metabolites, especially eicosapentaenoic acid (EPA), in the intestines of mice receiving ALA-rich linseed oil (Lin-mice). In murine food allergy model, the decreased incidence of allergic diarrhea in Lin-mice was due to impairment of mast cell degranulation without affecting allergen-specific serum IgE. Liquid chromatography-tandem mass spectrometry-based mediator lipidomics identified 17,18-EpETE as a major ω3 EPA-derived metabolite generated from dietary ALA in the gut, and 17,18-EpETE exhibits anti-allergic function when administered in vivo. These findings suggest that metabolizing dietary ω3 PUFAs generates 17,18-EpETE, which is an endogenous anti-allergic metabolite and potentially is a therapeutic target to control intestinal allergies.
Assuntos
Ácidos Graxos Ômega-3 , Hipersensibilidade Alimentar/tratamento farmacológico , Mucosa Intestinal/metabolismo , Óleo de Semente do Linho/farmacologia , Animais , Modelos Animais de Doenças , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-3/farmacologia , Feminino , Hipersensibilidade Alimentar/metabolismo , Hipersensibilidade Alimentar/patologia , Intestinos/patologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Intestinal plasma cells predominantly produce immunoglobulin (Ig) A, however, their functional diversity remains poorly characterized. Here we show that murine intestinal IgA plasma cells can be newly classified into two populations on the basis of CD11b expression, which cannot be discriminated by currently known criteria such as general plasma cell markers, B cell origin and T cell dependence. CD11b(+) IgA(+) plasma cells require the lymphoid structure of Peyer's patches, produce more IgA than CD11b(-) IgA(+) plasma cells, proliferate vigorously, and require microbial stimulation and IL-10 for their development and maintenance. These features allow CD11b(+) IgA(+) plasma cells to mediate early-phase antigen-specific intestinal IgA responses induced by oral immunization with protein antigen. These findings reveal the functional diversity of IgA(+) plasma cells in the murine intestine.