RESUMEN
Loss-of-function mutations in TET2 occur frequently in patients with clonal hematopoiesis, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML) and are associated with a DNA hypermethylation phenotype. To determine the role of TET2 deficiency in leukemia stem cell maintenance, we generated a reversible transgenic RNAi mouse to model restoration of endogenous Tet2 expression. Tet2 restoration reverses aberrant hematopoietic stem and progenitor cell (HSPC) self-renewal in vitro and in vivo. Treatment with vitamin C, a co-factor of Fe2+ and α-KG-dependent dioxygenases, mimics TET2 restoration by enhancing 5-hydroxymethylcytosine formation in Tet2-deficient mouse HSPCs and suppresses human leukemic colony formation and leukemia progression of primary human leukemia PDXs. Vitamin C also drives DNA hypomethylation and expression of a TET2-dependent gene signature in human leukemia cell lines. Furthermore, TET-mediated DNA oxidation induced by vitamin C treatment in leukemia cells enhances their sensitivity to PARP inhibition and could provide a safe and effective combination strategy to selectively target TET deficiency in cancer. PAPERCLIP.
Asunto(s)
Ácido Ascórbico/farmacología , Proteínas de Unión al ADN/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Síndromes Mielodisplásicos/tratamiento farmacológico , Proteínas Proto-Oncogénicas/metabolismo , Vitaminas/farmacología , Animales , Ácido Ascórbico/administración & dosificación , Muerte Celular , Línea Celular Tumoral , Metilación de ADN , Proteínas de Unión al ADN/genética , Dioxigenasas , Técnicas de Silenciamiento del Gen , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Síndromes Mielodisplásicos/genética , Trasplante de Neoplasias , Poli(ADP-Ribosa) Polimerasa-1/genética , Proteínas Proto-Oncogénicas/genética , Transcripción Genética , Trasplante Heterólogo , Vitaminas/administración & dosificaciónRESUMEN
The response to systemic infection and injury requires the rapid adaptation of hematopoietic stem cells (HSCs), which proliferate and divert their differentiation toward the myeloid lineage. Significant interest has emerged in understanding the signals that trigger the emergency hematopoietic program. However, the mechanisms that halt this response of HSCs, which is critical to restore homeostasis, remain unknown. Here we reveal that the E3 ubiquitin ligase Speckle-type BTB-POZ protein (SPOP) restrains the inflammatory activation of HSCs. In the absence of Spop, systemic inflammation proceeded in an unresolved manner, and the sustained response in the HSCs resulted in a lethal phenotype reminiscent of hyper-inflammatory syndrome or sepsis. Our proteomic studies decipher that SPOP restricted inflammation by ubiquitinating the innate signal transducer myeloid differentiation primary response protein 88 (MYD88). These findings unearth an HSC-intrinsic post-translational mechanism that is essential for reestablishing homeostasis after emergency hematopoiesis.
Asunto(s)
Inflamación/inmunología , Leucocitosis/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Neutrófilos/inmunología , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Animales , Línea Celular , Femenino , Células HEK293 , Hematopoyesis/inmunología , Humanos , Masculino , Ratones , Neutrófilos/citología , Complejos de Ubiquitina-Proteína Ligasa , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Maintenance of stemness is tightly linked to cell cycle regulation through protein phosphorylation by cyclin-dependent kinases (CDKs). However, how this process is reversed during differentiation is unknown. We report here that exit from stemness and differentiation of pluripotent cells along the neural lineage are controlled by CDC14, a CDK-counteracting phosphatase whose function in mammals remains obscure. Lack of the two CDC14 family members, CDC14A and CDC14B, results in deficient development of the neural system in the mouse and impairs neural differentiation from embryonic stem cells (ESCs). Mechanistically, CDC14 directly dephosphorylates specific proline-directed Ser/Thr residues of undifferentiated embryonic transcription Factor 1 (UTF1) during the exit from stemness, triggering its proteasome-dependent degradation. Multiomic single-cell analysis of transcription and chromatin accessibility in differentiating ESCs suggests that increased UTF1 levels in the absence of CDC14 prevent the proper firing of bivalent promoters required for differentiation. CDC14 phosphatases are dispensable for mitotic exit, suggesting that CDC14 phosphatases have evolved to control stemness rather than cell cycle exit and establish the CDK-CDC14 axis as a critical molecular switch for linking cell cycle regulation and self-renewal.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas de Saccharomyces cerevisiae , Animales , Ratones , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Ciclo Celular , Fosforilación/fisiología , Mitosis , Proteínas de Saccharomyces cerevisiae/metabolismo , MamíferosRESUMEN
The bone marrow microenvironment has a key role in regulating haematopoiesis, but its molecular complexity and response to stress are incompletely understood. Here we map the transcriptional landscape of mouse bone marrow vascular, perivascular and osteoblast cell populations at single-cell resolution, both at homeostasis and under conditions of stress-induced haematopoiesis. This analysis revealed previously unappreciated levels of cellular heterogeneity within the bone marrow niche and resolved cellular sources of pro-haematopoietic growth factors, chemokines and membrane-bound ligands. Our studies demonstrate a considerable transcriptional remodelling of niche elements under stress conditions, including an adipocytic skewing of perivascular cells. Among the stress-induced changes, we observed that vascular Notch delta-like ligands (encoded by Dll1 and Dll4) were downregulated. In the absence of vascular Dll4, haematopoietic stem cells prematurely induced a myeloid transcriptional program. These findings refine our understanding of the cellular architecture of the bone marrow niche, reveal a dynamic and heterogeneous molecular landscape that is highly sensitive to stress and illustrate the utility of single-cell transcriptomic data in evaluating the regulation of haematopoiesis by discrete niche populations.
Asunto(s)
Médula Ósea/irrigación sanguínea , Microambiente Celular , Hematopoyesis , Células Madre Hematopoyéticas , Análisis de la Célula Individual , Nicho de Células Madre , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adipocitos/citología , Adipocitos/metabolismo , Animales , Proteínas de Unión al Calcio/metabolismo , Diferenciación Celular , Linaje de la Célula , Endotelio Vascular/citología , Femenino , Regulación de la Expresión Génica , Hematopoyesis/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Masculino , Ratones , Células Mieloides/citología , Células Mieloides/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , RNA-Seq , Receptores Notch/metabolismo , Nicho de Células Madre/genética , Estrés Fisiológico/genética , Transcriptoma/genéticaRESUMEN
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Tissue homeostasis depends largely on the ability to replenish impaired or aged cells. Thus, tissue-resident stem cells need to provide functional progeny throughout the lifetime of an organism. Significant work in the past years has characterized how stem cells integrate signals from their environment to shape regulatory transcriptional networks and chromatin-regulating factors that control stem cell differentiation or maintenance. There is increasing interest in how post-translational modifications, and specifically ubiquitylation, control these crucial decisions. Ubiquitylation modulates the stability and function of important factors that regulate key processes in stem cell behavior. In this review, we analyze the role of ubiquitylation in embryonic stem cells and different adult multipotent stem cell systems and discuss the underlying mechanisms that control the balance between quiescence, self-renewal, and differentiation. We also discuss deregulated processes of ubiquitin-mediated protein degradation that lead to the development of tumor-initiating cells.
Asunto(s)
Células Madre/fisiología , Ubiquitinación , Animales , Diferenciación Celular , División Celular , Cromatina/fisiología , Epigénesis Genética , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Células-Madre Neurales , Proteolisis , Ubiquitina-Proteína Ligasas/fisiologíaRESUMEN
Inflammation has enduring impacts on organismal immunity. However, the precise mechanisms by which tissue-restricted inflammation conditions systemic responses are poorly understood. Here, we leveraged a highly compartmentalized model of skin inflammation and identified a surprising type I interferon (IFN)- mediated activation of hematopoietic stem/progenitor cells (HSPCs) that results in profound changes to systemic host responses. Post-inflamed mice were protected from atherosclerosis and had worse outcomes following influenza virus infection. This IFN-mediated HSPC modulation was dependent on IFNAR signaling and could be recapitulated with the administration of recombinant IFNα. Importantly, the transfer of post-inflamed HSPCs was sufficient to transmit the immune suppression phenotype. IFN modulation of HSPCs was rooted both in long-term changes in chromatin accessibility and the emergence of an IFN- responsive functional state from multiple progenitor populations. Collectively, our data reveal the profound and enduring effect of transient inflammation and more specifically type I IFN signaling and set the stage for a more nuanced understanding of HSPC functional modulation by peripheral immune signals.
RESUMEN
Clonal hematopoiesis (CH) is an aging-associated condition characterized by the clonal outgrowth of mutated preleukemic cells. Individuals with CH are at an increased risk of developing hematopoietic malignancies. Here, we describe a novel animal model carrying a recurrent TET2 missense mutation frequently found in patients with CH and leukemia. In a fashion similar to CH, animals show signs of disease late in life when they develop a wide range of myeloid neoplasms, including acute myeloid leukemia (AML). Using single-cell transcriptomic profiling of the bone marrow, we show that disease progression in aged animals correlates with an enhanced inflammatory response and the emergence of an aberrant inflammatory monocytic cell population. The gene signature characteristic of this inflammatory population is associated with poor prognosis in patients with AML. Our study illustrates an example of collaboration between a genetic lesion found in CH and inflammation, leading to transformation and the establishment of blood neoplasms. SIGNIFICANCE: Progression from a preleukemic state to transformation, in the presence of TET2 mutations, is coupled with the emergence of inflammation and a novel population of inflammatory monocytes. Genes characteristic of this inflammatory population are associated with the worst prognosis in patients with AML. These studies connect inflammation to progression to leukemia. See related commentary by Pietras and DeGregori, p. 2234 . This article is highlighted in the In This Issue feature, p. 2221.
Asunto(s)
Leucemia Mieloide Aguda , Trastornos Mieloproliferativos , Animales , Hematopoyesis/genética , Inflamación/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Mutación , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/patologíaRESUMEN
Clonal hematopoiesis (CH) is an aging-associated condition characterized by the clonal outgrowth of pre-leukemic cells that acquire specific mutations. Although individuals with CH are healthy, they are at an increased risk of developing myeloid malignancies, suggesting that additional alterations are needed for the transition from a pre-leukemia stage to frank leukemia. To identify signaling states that cooperate with pre-leukemic cells, we used an in vivo RNAi screening approach. One of the most prominent genes identified was the ubiquitin ligase TRAF6. Loss of TRAF6 in pre-leukemic cells results in overt myeloid leukemia and is associated with MYC-dependent stem cell signatures. TRAF6 is repressed in a subset of patients with myeloid malignancies, suggesting that subversion of TRAF6 signaling can lead to acute leukemia. Mechanistically, TRAF6 ubiquitinates MYC, an event that does not affect its protein stability but rather represses its functional activity by antagonizing an acetylation modification.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia Mieloide Aguda , Trastornos Mieloproliferativos , Hematopoyesis , Humanos , Leucemia Mieloide Aguda/patología , Mutación , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/metabolismoRESUMEN
Aberrant DNA methylation is a characteristic feature of cancer including blood malignancies. Mutations in the DNA methylation regulators DNMT3A, TET1/2 and IDH1/2 are recurrent in leukemia and lymphoma. Specific and distinct DNA methylation patterns characterize subtypes of AML and lymphoma. Regulatory regions such as promoter CpG islands, CpG shores and enhancers show changes in methylation during transformation. However, the reported poor correlation between changes in methylation and gene expression in many mouse models and human studies reflects the complexity in the precise molecular mechanism for why aberrant DNA methylation promotes malignancies. This review will summarize current concepts regarding the mechanisms behind aberrant DNA methylation in hematopoietic malignancy and discuss its importance in cancer prognosis, tumor heterogeneity and relapse.
RESUMEN
Hematopoietic-specific transcription factors require coactivators to communicate with the general transcription machinery and establish transcriptional programs that maintain hematopoietic stem cell (HSC) self-renewal, promote differentiation, and prevent malignant transformation. Mediator is a large coactivator complex that bridges enhancer-localized transcription factors with promoters, but little is known about Mediator function in adult stem cell self-renewal and differentiation. We show that MED12, a member of the Mediator kinase module, is an essential regulator of HSC homeostasis, as in vivo deletion of Med12 causes rapid bone marrow aplasia leading to acute lethality. Deleting other members of the Mediator kinase module does not affect HSC function, suggesting kinase-independent roles of MED12. MED12 deletion destabilizes P300 binding at lineage-specific enhancers, resulting in H3K27Ac depletion, enhancer de-activation, and consequent loss of HSC stemness signatures. As MED12 mutations have been described recently in blood malignancies, alterations in MED12-dependent enhancer regulation may control both physiological and malignant hematopoiesis.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Hematopoyesis , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Complejo Mediador/metabolismo , Animales , Apoptosis/genética , Médula Ósea/patología , Supervivencia Celular/genética , Cromatina/metabolismo , Eliminación de Gen , Perfilación de la Expresión Génica , Ratones , Unión Proteica , Factores de Transcripción/metabolismo , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Little is known on post-transcriptional regulation of adult and embryonic stem cell maintenance and differentiation. Here we characterize the role of Ddb1, a component of the CUL4-DDB1 ubiquitin ligase complex. Ddb1 is highly expressed in multipotent hematopoietic progenitors and its deletion leads to abrogation of both adult and fetal hematopoiesis, targeting specifically transiently amplifying progenitor subsets. However, Ddb1 deletion in non-dividing lymphocytes has no discernible phenotypes. Ddb1 silencing activates Trp53 pathway and leads to significant effects on cell cycle progression and rapid apoptosis. The abrogation of hematopoietic progenitor cells can be partially rescued by simultaneous deletion of Trp53. Conversely, depletion of DDB1 in embryonic stem cell (ESC) leads to differentiation albeit negative effects on cell cycle and apoptosis. Mass spectrometry reveals differing protein interactions between DDB1 and distinct DCAFs, the substrate recognizing components of the E3 complex, between cell types. Our studies identify CUL4-DDB1 complex as a novel post-translational regulator of stem and progenitor maintenance and differentiation.
Asunto(s)
Diferenciación Celular , Proteínas Cullin/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/fisiología , Animales , Proteínas de Unión al ADN/genética , Eliminación de Gen , Regulación de la Expresión Génica , Silenciador del Gen , Homeostasis , Ratones , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Cdc14 is a dual-specific phosphatase with relevant functions during mitotic exit in yeast. The relevance of vertebrate Cdc14 phosphatases is not well understood due to the presence of two paralogs, Cdc14A and Cdc14B, and their dispensability for cell cycle progression. Here, we report that overexpression of mammalian Cdc14B, but not Cdc14A, leads to dramatic changes in morphology and malignant transformation of normal murine fibroblasts. Cdc14B disrupts the cytoskeletal F-actin organization with loss of actin stress fibers and vinculin adhesions in a phosphatase-dependent manner. These morphological changes are associated to cellular transformation, as Cdc14B-overexpressing cells display anchorage-independent growth and are able to form tumors in vivo. These alterations are similar to those induced by Ras oncogenes ,and both Cdc14B and H-RasV12 lead to similar changes in the transcriptional profile of transformed cells. Pharmacologic inhibition of the Ras-Mek pathway rescues these defects. These data suggest that Cdc14B, but not Cdc14A, is one of the few phosphatases that display oncogenic activity in mammals and point to the Ras-MAP kinase pathway as a major effector pathway during oncogenic transformation by Cdc14B.
Asunto(s)
Fosfatasas de Especificidad Dual/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas ras/metabolismo , Actinas/metabolismo , Animales , Fosfatasas de Especificidad Dual/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Sistema de Señalización de MAP Quinasas , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Vinculina/metabolismo , Proteínas ras/antagonistas & inhibidoresRESUMEN
Cdc14 is an essential phosphatase in yeast but its role in the mammalian cell cycle remains obscure. We report here that Cdc14b-knockout cells display unscheduled induction of multiple cell cycle regulators resulting in early entry into DNA replication and mitosis from quiescence. Cdc14b dephosphorylates Ser5 at the C-terminal domain (CTD) of RNA polymerase II, a major substrate of cyclin-dependent kinases. Lack of Cdc14b results in increased CTD-Ser5 phosphorylation, epigenetic modifications that mark active chromatin, and transcriptional induction of cell cycle regulators. These data suggest a function for mammalian Cdc14 phosphatases in the control of transcription during the cell cycle.
Asunto(s)
Ciclo Celular , Fosfatasas de Especificidad Dual/metabolismo , ARN Polimerasa II/metabolismo , Transcripción Genética , Alelos , Animales , Sitios de Unión , Células Cultivadas , Epigénesis Genética , Exones , Fibroblastos/citología , Regulación Enzimológica de la Expresión Génica , Ratones , Mitosis , Fosfatos/química , Fosforilación , Activación TranscripcionalRESUMEN
Targeting mitotic exit has been recently proposed as a relevant therapeutic approach against cancer. By using genetically engineered mice, we show that the APC/C cofactor Cdc20 is essential for anaphase onset in vivo in embryonic or adult cells, including progenitor/stem cells. Ablation of Cdc20 results in efficient regression of aggressive tumors, whereas current mitotic drugs display limited effects. Yet, Cdc20 null cells can exit from mitosis upon inactivation of Cdk1 and the kinase Mastl (Greatwall). This mitotic exit depends on the activity of PP2A phosphatase complexes containing B55α or B55δ regulatory subunits. These data illustrate the relevance of critical players of mitotic exit in mammals and their implications in the balance between cell death and mitotic exit in tumor cells.