RESUMO
MYB is a master regulator and pioneer factor highly expressed in hematopoietic progenitor cells (HPCs) where it contributes to the reprogramming processes operating during hematopoietic development. MYB plays a complex role being involved in several lineages of the hematopoietic system. At the molecular level, the MYB gene is subject to intricate regulation at many levels through several enhancer and promoter elements, through transcriptional elongation control, as well as post-transcriptional regulation. The protein is modulated by post-translational modifications (PTMs) such as SUMOylation restricting the expression of its downstream targets. Together with a range of interaction partners, cooperating transcription factors (TFs) and epigenetic regulators, MYB orchestrates a fine-tuned symphony of genes expressed during various stages of haematopoiesis. At the same time, the complex MYB system is vulnerable, being a target for unbalanced control and cancer development.
Assuntos
Hematopoese , Células-Tronco Hematopoéticas , Proteínas Proto-Oncogênicas c-myb , Humanos , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Proteínas Proto-Oncogênicas c-myb/genética , Animais , Processamento de Proteína Pós-Traducional , Epigênese Genética , Regulação da Expressão GênicaRESUMO
Myb was identified over four decades ago as the transforming component of acute leukemia viruses in chickens. Since then it has become increasingly apparent that dysregulated MYB activity characterizes many blood cancers, including acute myeloid leukemia, and that it represents the most "addictive" oncoprotein in many, if not all, such diseases. As a consequence of this tumor-specific dependency for MYB, it has become a major focus of efforts to develop specific antileukemia drugs. Much attention is being given to ways to interrupt the interaction between MYB and cooperating factors, in particular EP300/KAT3B and CBP/KAT3A. Aside from candidates identified through screening of small molecules, the most exciting prospect for novel drugs seems to be the design of peptide mimetics that interfere directly at the interface between MYB and its cofactors. Such peptides combine a high degree of target specificity with good efficacy including minimal effects on normal hematopoietic cells.
Assuntos
Leucemia Mieloide Aguda , Proteínas Proto-Oncogênicas c-myb , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-myb/metabolismo , Proteínas Proto-Oncogênicas c-myb/genética , Animais , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia , Terapia de Alvo Molecular , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacosRESUMO
Tuberculosis (TB) is the leading cause of global morbidity and mortality resulting from infectious disease, with over 10.6 million new cases and 1.4 million deaths in 2021. This global emergency is exacerbated by the emergence of multidrug-resistant MDR-TB and extensively drug-resistant XDR-TB; therefore, new drugs and new drug targets are urgently required. From a whole cell phenotypic screen, a series of azetidines derivatives termed BGAz, which elicit potent bactericidal activity with MIC99 values <10 µM against drug-sensitive Mycobacterium tuberculosis and MDR-TB, were identified. These compounds demonstrate no detectable drug resistance. The mode of action and target deconvolution studies suggest that these compounds inhibit mycobacterial growth by interfering with cell envelope biogenesis, specifically late-stage mycolic acid biosynthesis. Transcriptomic analysis demonstrates that the BGAz compounds tested display a mode of action distinct from the existing mycobacterial cell wall inhibitors. In addition, the compounds tested exhibit toxicological and PK/PD profiles that pave the way for their development as antitubercular chemotherapies.
Assuntos
Azetidinas , Tuberculose Extensivamente Resistente a Medicamentos , Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Humanos , Azetidinas/farmacologia , Azetidinas/uso terapêutico , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Extensivamente Resistente a Medicamentos/tratamento farmacológico , Testes de Sensibilidade MicrobianaRESUMO
MYB plays a key role in gene regulation throughout the hematopoietic hierarchy and is critical for the maintenance of normal hematopoietic stem cells (HSC). Acquired genetic dysregulation of MYB is involved in the etiology of a number of leukemias, although inherited noncoding variants of the MYB gene are a susceptibility factor for many hematological conditions, including myeloproliferative neoplasms (MPN). The mechanisms that connect variations in MYB levels to disease predisposition, especially concerning age dependency in disease initiation, are completely unknown. Here, we describe a model of Myb insufficiency in mice that leads to MPN, myelodysplasia, and leukemia in later life, mirroring the age profile of equivalent human diseases. We show that this age dependency is intrinsic to HSC, involving a combination of an initial defective cellular state resulting from small effects on the expression of multiple genes and a progressive accumulation of further subtle changes. Similar to previous studies showing the importance of proteostasis in HSC maintenance, we observed altered proteasomal activity and elevated proliferation indicators, followed by elevated ribosome activity in young Myb-insufficient mice. We propose that these alterations combine to cause an imbalance in proteostasis, potentially creating a cellular milieu favoring disease initiation.
Assuntos
Leucemia , Transtornos Mieloproliferativos , Animais , Camundongos , Humanos , Proteostase , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Proto-Oncogênicas c-myb/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Regulação da Expressão Gênica , Leucemia/metabolismo , Transtornos Mieloproliferativos/metabolismoRESUMO
Liver transplantation is an effective therapy, but increasing demand for donor organs has led to the use of marginal donor organs with increased complication rates. Mesenchymal stromal cells (MSC) pleiotropically modulate aberrant immune-mediated responses and represent a potential therapy to target the inflammation seen post-transplant with marginal donor livers. To avoid the confounding effects of xenotransplantation seen in studies with human MSC, a PDGFRα/Sca-1 (PaS) sorted MSC population was used which was analogous to human MSC populations (LNGFR+Thy-1+VCAM-1Hi). PaS MSC are a well-described population that demonstrate MSC properties without evidence of clonal mutation during expansion. We demonstrate their anti-inflammatory properties herein through their suppression of T-lymphocyte proliferation in vitro and secretion of anti-inflammatory cytokines (IL-10 and OPG) after stimulation (P = .004 and P = .003). The MDR2-/- model of biliary injury and hepatic ischemia-reperfusion (HIR) injury models were used to replicate the non-anastomotic biliary complications seen following liver transplantation. Systemic MSC therapy in MDR2-/- mice led to reduced liver injury with an increase in restorative macrophages (5913 ± 333.9 vs 12 597 ± 665.8, P = .002, n = 7) and a change in lymphocyte ratios (3.55 ± 0.37 vs 2.59 ± 0.139, P = .023, n = 17), whereas subcutaneous administration of MSC showed no beneficial effect. MSC also reduced cell death in the HIR model assessed by Periodic acid-Schiff (PAS) staining (91.7% ± 2.8 vs 80.1% ± 4.6, P = .03). Systemically administered quantum dot-labeled MSC were tracked using single-cell resolution CryoViz imaging which demonstrated their sequestration in the lungs alongside retention/redistribution to injured liver tissue. MSC represent a potential novel therapy in marginal organ transplantation which warrants further study.
Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Traumatismo por Reperfusão , Camundongos , Animais , Humanos , Transplante de Células-Tronco Mesenquimais/métodos , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Modelos Animais de Doenças , Células-Tronco Mesenquimais/metabolismo , Fígado , Traumatismo por Reperfusão/terapiaRESUMO
Transposable elements (TEs) occupy nearly 40% of mammalian genomes and, whilst most are fragmentary and no longer capable of transposition, they can nevertheless contribute to cell function. TEs within genes transcribed by RNA polymerase II can be copied as parts of primary transcripts; however, their full contribution to mature transcript sequences remains unresolved. Here, using long and short read (LR and SR) RNA sequencing data, we show that 26% of coding and 65% of noncoding transcripts in human pluripotent stem cells (hPSCs) contain TE-derived sequences. Different TE families are incorporated into RNAs in unique patterns, with consequences to transcript structure and function. The presence of TE sequences within a transcript is correlated with TE-type specific changes in its subcellular distribution, alterations in steady-state levels and half-life, and differential association with RNA Binding Proteins (RBPs). We identify hPSC-specific incorporation of endogenous retroviruses (ERVs) and LINE:L1 into protein-coding mRNAs, which generate TE sequence-derived peptides. Finally, single cell RNA-seq reveals that hPSCs express ERV-containing transcripts, whilst differentiating subpopulations lack ERVs and express SINE and LINE-containing transcripts. Overall, our comprehensive analysis demonstrates that the incorporation of TE sequences into the RNAs of hPSCs is more widespread and has a greater impact than previously appreciated.
Assuntos
Retrovirus Endógenos/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Células-Tronco Pluripotentes/metabolismo , Transcriptoma , Linhagem Celular , Humanos , RNA não Traduzido/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Mutations at the N- or C-terminus of C/EBPα are frequent in acute myeloid leukaemia (AML) with normal karyotype. Here, we investigate the role of the transcription factor Myb in AMLs driven by different combinations of CEBPA mutations. Using knockdown of Myb in murine cell lines modelling the spectrum of CEBPA mutations, we show that the effect of reduced Myb depends on the mutational status of the two Cebpa alleles. Importantly, Myb knockdown fails to override the block in myeloid differentiation in cells with biallelic N-terminal C/EBPα mutations, demonstrating for the first time that the dependency on Myb is much lower in AML with this mutational profile. By comparing gene expression following Myb knockdown and chromatin immunoprecipitation sequencing data for the binding of C/EBPα isoforms, we provide evidence for a functional cooperation between C/EBPα and Myb in the maintenance of AML. This co-dependency breaks down when both alleles of CEBPA harbour N-terminal mutations, as a subset of C/EBPα-regulated genes only bind the short p30 C/EBPα isoform and, unlike other C/EBPα-regulated genes, do so without a requirement for Myb.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Mutação/genética , Proteínas Proto-Oncogênicas c-myb/genética , Alelos , Animais , Apoptose/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Camundongos , Fenótipo , Isoformas de Proteínas/genética , RNA Interferente Pequeno/genética , TransfecçãoRESUMO
The gene encoding the transcription factor C/EBPα is mutated in 10-15% of acute myeloid leukemia (AML) patients. N-terminal CEBPA mutations cause ablation of full-length C/EBPα without affecting the expression of a shorter oncogenic isoform, termed p30. The mechanistic basis of p30-induced leukemogenesis is incompletely understood. Here, we demonstrate that the MLL1 histone-methyltransferase complex represents a critical actionable vulnerability in CEBPA-mutated AML. Oncogenic C/EBPα p30 and MLL1 show global co-localization on chromatin and p30 exhibits robust physical interaction with the MLL1 complex. CRISPR/Cas9-mediated mutagenesis of MLL1 results in proliferation arrest and myeloid differentiation in C/EBPα p30-expressing cells. In line, CEBPA-mutated hematopoietic progenitor cells are hypersensitive to pharmacological targeting of the MLL1 complex. Inhibitor treatment impairs proliferation and restores myeloid differentiation potential in mouse and human AML cells with CEBPA mutations. Finally, we identify the transcription factor GATA2 as a direct critical target of the p30-MLL1 interaction. Altogether, we show that C/EBPα p30 requires the MLL1 complex to regulate oncogenic gene expression and that CEBPA-mutated AML is hypersensitive to perturbation of the MLL1 complex. These findings identify the MLL1 complex as a potential therapeutic target in AML with CEBPA mutations.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/genética , Sistemas CRISPR-Cas , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Leucemia Mieloide Aguda/patologia , Mutação , Proteína de Leucina Linfoide-Mieloide/antagonistas & inibidores , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Diferenciação Celular , Proliferação de Células , Fator de Transcrição GATA2 , Hematopoese , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Células Tumorais CultivadasRESUMO
During somatic reprogramming, Yamanaka's pioneer factors regulate a complex sequence of molecular events leading to the activation of a network of pluripotency factors, ultimately resulting in the acquisition and maintenance of a pluripotent state. Here, we show that, contrary to the pluripotency factors studied so far, overexpression of Mybl2 inhibits somatic reprogramming. Our results demonstrate that Mybl2 levels are crucial to the dynamics of the reprogramming process. Mybl2 overexpression changes chromatin conformation, affecting the accessibility of pioneer factors to the chromatin and promoting accessibility for early immediate response genes known to be reprogramming blockers. These changes in the chromatin landscape ultimately lead to a deregulation of key genes that are important for the mesenchymal-to-epithelial transition. This work defines Mybl2 level as a gatekeeper for the initiation of reprogramming, providing further insights into the tight regulation and required coordination of molecular events that are necessary for changes in cell fate identity during the reprogramming process.
Assuntos
Proteínas de Ciclo Celular/genética , Transativadores/genética , Reprogramação Celular , Transição Epitelial-Mesenquimal , Humanos , TransfecçãoRESUMO
We investigated the adhesive behavior of mesenchymal stem cells (MSC) in blood, which might influence their fate when infused as therapy. Isolated human bone marrow MSC (BMMSC) or umbilical cord MSC (UCMSC) adhered efficiently from flow to the matrix proteins, collagen, or fibronectin, but did not adhere to endothelial selectins. However, when suspended in blood, BMMSC no longer adhered to collagen, while UCMSC adhered along with many aggregated platelets. Neither MSC adhered to fibronectin from flowing blood, although the fibronectin surface did become coated with a platelet monolayer. UCMSC induced platelet aggregation in platelet rich plasma, and caused a marked drop in platelet count when mixed with whole human or mouse blood in vitro, or when infused into mice. In contrast, BMMSC did not activate platelets or induce changes in platelet count. Interestingly, isolated UCMSC and BMMSC both adhered to predeposited platelets. The differences in behavior in blood were attributable to expression of podoplanin (an activating ligand for the platelet receptor CLEC-2), which was detected on UCMSC, but not BMMSC. Thus, platelets were activated when bound to UCMSC, but not BMMSC. Platelet aggregation by UCMSC was inhibited by recombinant soluble CLEC-2, and UCMSC did not cause a reduction in platelet count when mixed with blood from mice deficient in CLEC-2. We predict that both MSC would carry platelets in the blood, but their interaction with vascular endothelium would depend on podoplanin-induced activation of the bound platelets. Such interactions with platelets might target MSC to damaged tissue, but could also be thrombotic. Stem Cells 2018;36:1062-1074.
Assuntos
Plaquetas/metabolismo , Adesão Celular/genética , Células-Tronco Mesenquimais/metabolismo , Animais , Humanos , CamundongosRESUMO
Cumulatively, breast, cervical, ovarian, and uterine cancer account for more than 70% of cancers in women in India. Distinct differences in the clinical presentation of women with cancer suggest underlying differences in cancer biology and genetics. The peak age of onset of breast and ovarian cancer appears to be a decade earlier in India (age 45-50 years) than in high-income countries (age >60 years). Understanding these differences through research to develop diagnosis, screening, prevention, and treatment frameworks that ar e specific to the Indian population are critical and essential to improving women's health in India. Since the sequencing of the human genome in 2001, applications of advanced technologies, such as massively parallel sequencing, have transformed the understanding of the genetic and environmental drivers of cancer. How can advanced technologies be harnessed to provide health-care solutions at a scale and to a budget suitable for a country of 1·2 billion people? What research programmes are necessary to answer questions specific to India, and to build capacity for innovative solutions using these technologies? In order to answer these questions, we convened a workshop with key stakeholders to address these issues. In this Series paper, we highlight challenges in tackling the growing cancer burden in India, discuss ongoing genomics research and developments in infrastructure, and suggest key priorities for future research in cancer in India.
Assuntos
Pesquisa Biomédica/estatística & dados numéricos , Detecção Precoce de Câncer/estatística & dados numéricos , Neoplasias dos Genitais Femininos/epidemiologia , Neoplasias dos Genitais Femininos/genética , Genômica , Adulto , Idade de Início , Idoso , Pesquisa Biomédica/economia , Atenção à Saúde/economia , Atenção à Saúde/métodos , Feminino , Neoplasias dos Genitais Femininos/diagnóstico , Neoplasias dos Genitais Femininos/terapia , Prioridades em Saúde , Humanos , Índia/epidemiologia , Programas de Rastreamento/organização & administração , Pessoa de Meia-Idade , Avaliação das Necessidades , Prevalência , Medição de RiscoRESUMO
Growth Factor Independence 1 (GFI1) is a transcriptional repressor that plays a critical role during both myeloid and lymphoid haematopoietic lineage commitment. Several studies have demonstrated the involvement of GFI1 in haematological malignancies and have suggested that low expression of GFI1 is a negative indicator of disease progression for both myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). In this study, we have stratified AML patients into those defined as having a normal karyotype (CN-AML). Unlike the overall pattern in AML, those patients with CN-AML have a poorer survival rate when GFI1 expression is high. In this group, high GFI1 expression is paralleled by higher FLT3 expression, and, even when the FLT3 gene is not mutated, exhibit a FLT3-ITD signature of gene expression. Knock-down of GFI1 expression in the human AML Fujioka cell line led to a decrease in the level of FLT3 RNA and protein and to the down regulation of FLT3-ITD signature genes, thus linking two major prognostic indicators for AML.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação Leucêmica da Expressão Gênica , Cariótipo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Sequências de Repetição em Tandem , Fatores de Transcrição/genética , Tirosina Quinase 3 Semelhante a fms/genética , Biomarcadores Tumorais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/patologia , Avaliação de Resultados da Assistência ao Paciente , Prognóstico , Fatores de Transcrição/metabolismo , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
BACKGROUND & AIMS: There is growing interest in the use of bone marrow cells to treat liver fibrosis, however, little is known about their antifibrotic efficacy or the identity of their effector cell(s). Sphingosine-1-phosphate (S1P) mediates egress of immune cells from the lymphoid organs into the lymphatic vessels; we investigated its role in the response of hematopoietic stem cells (HSCs) to liver fibrosis in mice. METHODS: Purified (c-kit+/sca1+/lin-) HSCs were infused repeatedly into mice undergoing fibrotic liver injury. Chronic liver injury was induced in BoyJ mice by injection of carbon tetrachloride (CCl4) or placement on a methionine-choline-deficient diet. Some mice were irradiated and given transplants of bone marrow cells from C57BL6 mice, with or without the S1P antagonist FTY720; we then studied HSC mobilization and localization. Migration of HSC lines was quantified in Transwell assays. Levels of S1P in liver, bone marrow, and lymph fluid were measured using an enzyme-linked immunosorbent assay. Liver tissues were collected and analyzed by immunohistochemical quantitative polymerase chain reaction and sphingosine kinase activity assays. We performed quantitative polymerase chain reaction analyses of the expression of sphingosine kinase 1 and 2, sphingosine-1-phosphate lyase 1, and sphingosine-1-phosphate phosphatase 1 in normal human liver and cirrhotic liver from patients with alcohol-related liver disease (n = 6). RESULTS: Infusions of HSCs into mice with liver injury reduced liver scarring based on picrosirius red staining (49.7% reduction in mice given HSCs vs control mice; P < .001), and hepatic hydroxyproline content (328 mg/g in mice given HSCs vs 428 mg/g in control mice; P < .01). HSC infusion also reduced hepatic expression of α-smooth muscle actin (0.19 ± 0.007-fold compared with controls; P < .0001) and collagen type I α 1 chain (0.29 ± 0.17-fold compared with controls; P < .0001). These antifibrotic effects were maintained with infusion of lymphoid progenitors that lack myeloid potential and were associated with increased numbers of recipient neutrophils and macrophages in liver. In studies of HSC cell lines, we found HSCs to recruit monocytes, and this process to require C-C motif chemokine receptor 2. In fibrotic liver tissue from mice and patients, hepatic S1P levels increased owing to increased hepatic sphingosine kinase-1 expression, which contributed to a reduced liver:lymph S1P gradient and limited HSC egress from the liver. Mice given the S1P antagonist (FTY720) with HSCs had increased hepatic retention of HSCs (1697 ± 247 cells in mice given FTY720 vs 982 ± 110 cells in controls; P < .05), and further reductions in fibrosis. CONCLUSIONS: In studies of mice with chronic liver injury, we showed the antifibrotic effects of repeated infusions of purified HSCs. We found that HSCs promote recruitment of endogenous macrophages and neutrophils. Strategies to reduce SIP signaling and increase retention of HSCs in the liver could increase their antifibrotic activities and be developed for treatment of patients with liver fibrosis.
Assuntos
Movimento Celular/efeitos dos fármacos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Cirrose Hepática/prevenção & controle , Lisofosfolipídeos/antagonistas & inibidores , Lisofosfolipídeos/metabolismo , Esfingosina/análogos & derivados , Actinas/metabolismo , Aldeído Liases/genética , Animais , Linhagem Celular , Doença Hepática Crônica Induzida por Substâncias e Drogas/complicações , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Feminino , Cloridrato de Fingolimode/uso terapêutico , Expressão Gênica , Humanos , Imunossupressores/uso terapêutico , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/etiologia , Cirrose Hepática/genética , Cirrose Hepática/patologia , Linfa/metabolismo , Macrófagos , Masculino , Proteínas de Membrana/genética , Camundongos , Monócitos , Neutrófilos , Monoéster Fosfórico Hidrolases/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingosina/antagonistas & inibidores , Esfingosina/metabolismoRESUMO
Hereditary tyrosinemia type 1 (HT1) is a severe human autosomal recessive disorder caused by the deficiency of fumarylacetoacetate hydroxylase (FAH), an enzyme catalyzing the last step in the tyrosine degradation pathway. Lack of FAH causes accumulation of toxic metabolites (fumarylacetoacetate and succinylacetone) in blood and tissues, ultimately resulting in severe liver and kidney damage with onset that ranges from infancy to adolescence. This tissue damage is lethal but can be controlled by administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), which inhibits tyrosine catabolism upstream of the generation of fumarylacetoacetate and succinylacetone. Notably, in animals lacking FAH, transient withdrawal of NTBC can be used to induce liver damage and a concomitant regenerative response that stimulates the growth of healthy hepatocytes. Among other things, this model has raised tremendous interest for the in vivo expansion of human primary hepatocytes inside these animals and for exploring experimental gene therapy and cell-based therapies. Here, we report the generation of FAH knock-out rabbits via pronuclear stage embryo microinjection of transcription activator-like effector nucleases. FAH-/- rabbits exhibit phenotypic features of HT1 including liver and kidney abnormalities but additionally develop frequent ocular manifestations likely caused by local accumulation of tyrosine upon NTBC administration. We also show that allogeneic transplantation of wild-type rabbit primary hepatocytes into FAH-/- rabbits enables highly efficient liver repopulation and prevents liver insufficiency and death. Because of significant advantages over rodents and their ease of breeding, maintenance, and manipulation compared with larger animals including pigs, FAH-/- rabbits are an attractive alternative for modeling the consequences of HT1.
Assuntos
Hidrolases/genética , Tirosinemias/genética , Animais , Modelos Animais de Doenças , Feminino , Técnicas de Inativação de Genes , Hepatócitos/transplante , Humanos , Hidrolases/metabolismo , Rim/metabolismo , Rim/patologia , Fígado/metabolismo , Fígado/patologia , Falência Hepática/etiologia , Falência Hepática/metabolismo , Falência Hepática/patologia , Falência Hepática/terapia , Masculino , Coelhos , Tirosinemias/complicações , Tirosinemias/metabolismo , Tirosinemias/patologiaRESUMO
The transcription factor Myb is a key regulator of hematopoietic cell proliferation, differentiation, and survival and has been implicated in the development of leukemia and several other human cancers. Pharmacological inhibition of Myb is therefore emerging as a potential therapeutic strategy. Recently, the first low-molecular-weight compounds that show Myb inhibitory activity have been identified. Characterization of these compounds suggests disruption of the protein-protein-interaction of Myb and the coactivator p300 as a suitable strategy to inhibit Myb.
Assuntos
Antineoplásicos/farmacologia , Terapia de Alvo Molecular , Proteínas Proto-Oncogênicas c-myb/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia/tratamento farmacológico , Leucemia/etiologia , Leucemia/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-myb/química , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Proto-Oncogênicas c-myb/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
The transcription factor Myb plays a key role in the hematopoietic system and has been implicated in the development of leukemia and other human cancers. Inhibition of Myb is therefore emerging as a potential therapeutic strategy for these diseases. However, because of a lack of suitable inhibitors, the feasibility of therapeutic approaches based on Myb inhibition has not been explored. We have identified the triterpenoid Celastrol as a potent low-molecular-weight inhibitor of the interaction of Myb with its cooperation partner p300. We demonstrate that Celastrol suppresses the proliferative potential of acute myeloid leukemia (AML) cells while not affecting normal hematopoietic progenitor cells. Furthermore, Celastrol prolongs the survival of mice in a model of an aggressive AML. Overall, our work demonstrates the therapeutic potential of a small molecule inhibitor of the Myb/p300 interaction for the treatment of AML and provides a starting point for the further development of Myb-inhibitory compounds for the treatment of leukemia and, possibly, other tumors driven by deregulated Myb.
Assuntos
Proteína p300 Associada a E1A/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Terapia de Alvo Molecular , Proteínas Proto-Oncogênicas c-myb/metabolismo , Bibliotecas de Moléculas Pequenas/uso terapêutico , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Galinhas , Modelos Animais de Doenças , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos Endogâmicos C57BL , Células Mieloides/efeitos dos fármacos , Células Mieloides/patologia , Triterpenos Pentacíclicos , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Bibliotecas de Moléculas Pequenas/farmacologia , Triterpenos/farmacologia , Triterpenos/uso terapêuticoRESUMO
The glycoprotein VI (GPVI)-Fc receptor γ (FcRγ) chain is the major platelet signaling receptor for collagen. Paradoxically, in a FeCl3 injury model, occlusion, but not initiation of thrombus formation, is delayed in GPVI-deficient and GPVI-depleted mice. In this study, we demonstrate that GPVI is a receptor for fibrin and speculate that this contributes to development of an occlusive thrombus. We observed a marked increase in tyrosine phosphorylation, including the FcRγ chain and Syk, in human and mouse platelets induced by thrombin in the presence of fibrinogen and the αIIbß3 blocker eptifibatide. This was not seen in platelets stimulated by a protease activated receptor (PAR)-4 peptide, which is unable to generate fibrin from fibrinogen. The pattern of tyrosine phosphorylation was similar to that induced by activation of GPVI. Consistent with this, thrombin did not induce tyrosine phosphorylation of Syk and the FcRγ chain in GPVI-deficient mouse platelets. Mouse platelets underwent full spreading on fibrin but not fibrinogen, which was blocked in the presence of a Src kinase inhibitor or in the absence of GPVI. Spreading on fibrin was associated with phosphatidylserine exposure (procoagulant activity), and this too was blocked in GPVI-deficient platelets. The ectodomain of GPVI was shown to bind to immobilized monomeric and polymerized fibrin. A marked increase in embolization was seen following FeCl3 injury in GPVI-deficient mice, likely contributing to the delay in occlusion in this model. These results demonstrate that GPVI is a receptor for fibrin and provide evidence that this interaction contributes to thrombus growth and stability.
Assuntos
Coagulação Sanguínea , Plaquetas/metabolismo , Fibrina/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Trombose/metabolismo , Animais , Plaquetas/citologia , Humanos , Camundongos , FosforilaçãoRESUMO
Mesenchymal stem cells (MSCs) have shown therapeutic promise in many experimental and clinical models of inflammation. However, a commonly reported feature of MSC transplantation is poor homing to injured tissues. Previously, we have shown that pretreatment with cytokines/chemical factors enhances hematopoietic SC adhesion within intestinal microvasculature following ischemia-reperfusion (IR) injury. Using intravital microscopy, the ability of similar pretreatment strategies to enhance the recruitment of murine MSCs to murine intestinal microvasculature following IR injury was investigated. Primary MSCs were isolated from bone marrow and selected on the basis of platelet-derived growth factor receptor-α and SC antigen-1 positivity (PDGFRα(+) /Sca-1(+) ). MSC recruitment was similar in IR injured gut mucosa when compared with sham operated controls, with limited cell adhesion observed. MSCs appeared contorted in microvessels, suggesting physical entrapment. Although not recruited specifically by injury, MSC administration significantly reduced neutrophil recruitment and improved tissue perfusion in the severely injured jejunum. Vasculoprotective effects were not demonstrated in the lesser injured ileum. Pretreatment of MSCs with tumor necrosis factor (TNF)-α, CXCL12, interferon (IFN)-γ, or hydrogen peroxide did not enhance their intestinal recruitment. In fact, TNFα and IFNγ removed the previous therapeutic ability of transplanted MSCs to reduce neutrophil infiltration and improve perfusion in the jejunum. We provide direct evidence that MSCs can rapidly limit leukocyte recruitment and improve tissue perfusion following intestinal IR injury. However, this study also highlights complexities associated with strategies to improve MSC therapeutic efficacy. Future studies using cytokine/chemical pretreatments to enhance MSC recruitment/function require careful consideration and validation to ensure therapeutic function is not impeded.
Assuntos
Movimento Celular/fisiologia , Íleo/irrigação sanguínea , Íleo/lesões , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/fisiologia , Traumatismo por Reperfusão/prevenção & controle , Animais , Movimento Celular/efeitos dos fármacos , Citocinas/metabolismo , Citocinas/farmacologia , Íleo/metabolismo , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão/metabolismoRESUMO
Mice with a constitutive or platelet-specific deletion of the C-type-lectin-like receptor (CLEC-2) exhibit hemorrhaging in the brain at mid-gestation. We sought to investigate the basis of this defect, hypothesizing that it is mediated by the loss of CLEC-2 activation by its endogenous ligand, podoplanin, which is expressed on the developing neural tube. To induce deletion of podoplanin at the 2-cell stage, we generated a podoplanin(fl/fl) mouse crossed to a PGK-Cre mouse. Using 3-dimensional light-sheet microscopy, we observed cerebral vessels were tortuous and aberrantly patterned at embryonic (E) day 10.5 in podoplanin- and CLEC-2-deficient mice, preceding the formation of large hemorrhages throughout the fore-, mid-, and hindbrain by E11.5. Immunofluorescence and electron microscopy revealed defective pericyte recruitment and misconnections between the endothelium of developing blood vessels and surrounding pericytes and neuro-epithelial cells. Nestin-Cre-driven deletion of podoplanin on neural progenitors also caused widespread cerebral hemorrhaging. Hemorrhaging was also seen in the ventricles of embryos deficient in the platelet integrin subunit glycoprotein IIb or in embryos in which platelet α-granule and dense granule secretion is abolished. We propose a novel role for podoplanin on the neuro-epithelium, which interacts with CLEC-2 on platelets, mediating platelet adhesion, aggregation, and secretion to guide the maturation and integrity of the developing vasculature and prevent hemorrhage.
Assuntos
Encéfalo/irrigação sanguínea , Encéfalo/embriologia , Circulação Cerebrovascular , Lectinas Tipo C/genética , Glicoproteínas de Membrana/genética , Animais , Plaquetas/metabolismo , Padronização Corporal , Encéfalo/metabolismo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Hemorragias Intracranianas/genética , Hemorragias Intracranianas/metabolismo , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Ativação Plaquetária , Agregação Plaquetária , Glicoproteína IIb da Membrana de Plaquetas/genética , Glicoproteína IIb da Membrana de Plaquetas/metabolismoRESUMO
Recent studies have boosted our understanding of long noncoding RNAs (lncRNAs) in numerous biological processes, but few have examined their roles in somatic cell reprogramming. Through expression profiling and functional screening, we have identified that the large intergenic noncoding RNA p21 (lincRNA-p21) impairs reprogramming. Notably, lincRNA-p21 is induced by p53 but does not promote apoptosis or cell senescence in reprogramming. Instead, lincRNA-p21 associates with the H3K9 methyltransferase SETDB1 and the maintenance DNA methyltransferase DNMT1, which is facilitated by the RNA-binding protein HNRNPK. Consequently, lincRNA-p21 prevents reprogramming by sustaining H3K9me3 and/or CpG methylation at pluripotency gene promoters. Our results provide insight into the role of lncRNAs in reprogramming and establish a novel link between p53 and heterochromatin regulation.