RESUMO
Hematopoietic stem cell (HSC) dormancy is understood as supportive of HSC function and its long-term integrity. Although regulation of stress responses incurred as a result of HSC activation is recognized as important in maintaining stem cell function, little is understood of the preventive machinery present in human HSCs that may serve to resist their activation and promote HSC self-renewal. We demonstrate that the transcription factor PLAG1 is essential for long-term HSC function and, when overexpressed, endows a 15.6-fold enhancement in the frequency of functional HSCs in stimulatory conditions. Genome-wide measures of chromatin occupancy and PLAG1-directed gene expression changes combined with functional measures reveal that PLAG1 dampens protein synthesis, restrains cell growth and division, and enhances survival, with the primitive cell advantages it imparts being attenuated by addition of the potent translation activator, c-MYC. We find PLAG1 capitalizes on multiple regulatory factors to ensure protective diminished protein synthesis including 4EBP1 and translation-targeting miR-127 and does so independently of stress response signaling. Overall, our study identifies PLAG1 as an enforcer of human HSC dormancy and self-renewal through its highly context-specific regulation of protein biosynthesis and classifies PLAG1 among a rare set of bona fide regulators of messenger RNA translation in these cells. Our findings showcase the importance of regulated translation control underlying human HSC physiology, its dysregulation under activating demands, and the potential if its targeting for therapeutic benefit.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células-Tronco Hematopoéticas , Fatores de Transcrição , Diferenciação Celular/fisiologia , Proliferação de Células , Autorrenovação Celular , Células-Tronco Hematopoéticas/metabolismo , Humanos , Fatores de Transcrição/metabolismoRESUMO
Umbilical cord blood-derived haematopoietic stem cells (HSCs) are essential for many life-saving regenerative therapies. However, despite their advantages for transplantation, their clinical use is restricted because HSCs in cord blood are found only in small numbers. Small molecules that enhance haematopoietic stem and progenitor cell (HSPC) expansion in culture have been identified, but in many cases their mechanisms of action or the nature of the pathways they impinge on are poorly understood. A greater understanding of the molecular circuitry that underpins the self-renewal of human HSCs will facilitate the development of targeted strategies that expand HSCs for regenerative therapies. Whereas transcription factor networks have been shown to influence the self-renewal and lineage decisions of human HSCs, the post-transcriptional mechanisms that guide HSC fate have not been closely investigated. Here we show that overexpression of the RNA-binding protein Musashi-2 (MSI2) induces multiple pro-self-renewal phenotypes, including a 17-fold increase in short-term repopulating cells and a net 23-fold ex vivo expansion of long-term repopulating HSCs. By performing a global analysis of MSI2-RNA interactions, we show that MSI2 directly attenuates aryl hydrocarbon receptor (AHR) signalling through post-transcriptional downregulation of canonical AHR pathway components in cord blood HSPCs. Our study gives mechanistic insight into RNA networks controlled by RNA-binding proteins that underlie self-renewal and provides evidence that manipulating such networks ex vivo can enhance the regenerative potential of human HSCs.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Autorrenovação Celular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Contagem de Células , Autorrenovação Celular/genética , Regulação para Baixo/genética , Feminino , Sangue Fetal/citologia , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Receptores de Hidrocarboneto Arílico/genética , Transdução de Sinais/genéticaRESUMO
Copy-number variations (CNVs) are strong risk factors for neurodevelopmental and psychiatric disorders. The 15q13.3 microdeletion syndrome region contains up to ten genes and is associated with numerous conditions, including autism spectrum disorder (ASD), epilepsy, schizophrenia, and intellectual disability; however, the mechanisms underlying the pathogenesis of 15q13.3 microdeletion syndrome remain unknown. We combined whole-genome sequencing, human brain gene expression (proteome and transcriptome), and a mouse model with a syntenic heterozygous deletion (Df(h15q13)/+ mice) and determined that the microdeletion results in abnormal development of cortical dendritic spines and dendrite outgrowth. Analysis of large-scale genomic, transcriptomic, and proteomic data identified OTUD7A as a critical gene for brain function. OTUD7A was found to localize to dendritic and spine compartments in cortical neurons, and its reduced levels in Df(h15q13)/+ cortical neurons contributed to the dendritic spine and dendrite outgrowth deficits. Our results reveal OTUD7A as a major regulatory gene for 15q13.3 microdeletion syndrome phenotypes that contribute to the disease mechanism through abnormal cortical neuron morphological development.
Assuntos
Transtornos Cromossômicos/enzimologia , Transtornos Cromossômicos/genética , Enzimas Desubiquitinantes/fisiologia , Endopeptidases/genética , Deficiência Intelectual/enzimologia , Deficiência Intelectual/genética , Transtornos do Neurodesenvolvimento/enzimologia , Transtornos do Neurodesenvolvimento/genética , Convulsões/enzimologia , Convulsões/genética , Animais , Transtorno do Espectro Autista/genética , Deleção Cromossômica , Cromossomos Humanos Par 15/enzimologia , Cromossomos Humanos Par 15/genética , Espinhas Dendríticas/metabolismo , Enzimas Desubiquitinantes/genética , Endopeptidases/metabolismo , Feminino , Deleção de Genes , Estudos de Associação Genética , Humanos , Masculino , Camundongos , Fenótipo , Prosencéfalo/patologiaRESUMO
Lentiviral vectors (LVs) are used for delivery of genes into hematopoietic stem and progenitor cells (HSPCs) in clinical trials worldwide. LVs, in contrast to retroviral vectors, are not associated with insertion site-associated malignant clonal expansions and, thus, are considered safer. Here, however, we present a case of markedly abnormal dysplastic clonal hematopoiesis affecting the erythroid, myeloid, and megakaryocytic lineages in a rhesus macaque transplanted with HSPCs that were transduced with a LV containing a strong retroviral murine stem cell virus (MSCV) constitutive promoter-enhancer in the LTR. Nine insertions were mapped in the abnormal clone, resulting in overexpression and aberrant splicing of several genes of interest, including the cytokine stem cell factor and the transcription factor PLAG1. This case represents the first clear link between lentiviral insertion-induced clonal expansion and a clinically abnormal transformed phenotype following transduction of normal primate or human HSPCs, which is concerning, and suggests that strong constitutive promoters should not be included in LVs.
Assuntos
Terapia Genética/métodos , Vetores Genéticos/uso terapêutico , Hematopoese/genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/virologia , Lentivirus/genética , Transdução Genética , Animais , Antígenos CD34/metabolismo , Células Clonais , Terapia Genética/efeitos adversos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Substâncias Luminescentes/metabolismo , Macaca mulatta , Mutagênese Insercional/genética , Regiões Promotoras Genéticas , Processamento de Proteína/genética , Sequências Repetidas Terminais/genética , Transplante AutólogoRESUMO
Normal hematopoiesis is sustained through a carefully orchestrated balance between hematopoietic stem cell (HSC) self-renewal and differentiation. The functional importance of this axis is underscored by the severity of disease phenotypes initiated by abnormal HSC function, including myelodysplastic syndromes and hematopoietic malignancies. Major advances in the understanding of transcriptional regulation of primitive hematopoietic cells have been achieved; however, the post-transcriptional regulatory layer that may impinge on their behavior remains underexplored by comparison. Key players at this level include RNA-binding proteins (RBPs), which execute precise and highly coordinated control of gene expression through modulation of RNA properties that include its splicing, polyadenylation, localization, degradation, or translation. With the recent identification of RBPs having essential roles in regulating proliferation and cell fate decisions in other systems, there has been an increasing appreciation of the importance of post-transcriptional control at the stem cell level. Here we discuss our current understanding of RBP-driven post-transcriptional regulation in HSCs, its implications for normal, perturbed, and malignant hematopoiesis, and the most recent technological innovations aimed at RBP-RNA network characterization at the systems level. Emerging evidence highlights RBP-driven control as an underappreciated feature of primitive hematopoiesis, the greater understanding of which has important clinical implications.
Assuntos
Regulação da Expressão Gênica , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Animais , Diferenciação Celular , Células-Tronco Hematopoéticas/citologia , HumanosRESUMO
BACKGROUND: Cultured megakaryocytes could prove useful in the study of human diseases, but it is difficult to produce sufficient numbers for study. We describe and evaluate the use of an expansion process to develop mature megakaryocytes from peripheral blood-derived human hematopoietic stem and progenitor cells (HSPCs). STUDY DESIGN AND METHODS: HSPCs (CD34+) were isolated from peripheral blood by positive selection and expanded using an optimal CD34+ expansion supplement. We evaluated megakaryocyte growth, maturation, and morphology in response to thrombopoietin (TPO) stimulation using flow cytometry and electron microscopy. TPO demonstrated a dose-dependent stimulatory effect on both megakaryocyte number and maturation. RESULTS: From 90 to 120 mL of unmanipulated peripheral blood, we isolated a mean of 1.5 × 10(5) HSPCs (1.5 × 10(3) cells/mL of whole blood). HSPCs expanded nine-fold after a 4-day culture using an expansion supplement. Expanded cells were cultured for an additional 8 days with TPO (20 ng/mL), which resulted in a 2.9-fold increase in megakaryocytic cells where 83% of live cells expressed CD41a+, a marker of megakaryocyte commitment, and 50% expressed CD42b+, a marker for megakaryocyte maturation. The expanded HSPCs responded to TPO stimulation to yield more than 1.0 × 10(6) megakaryocytes. This cell number was sufficient for morphologic studies that demonstrated these expanded HSPCs produced mature polyploid megakaryocytes capable of forming proplatelet extensions. CONCLUSIONS: Peripheral blood HSPCs can be expanded and differentiated into functional, mature megakaryocytes, a finding that supports the use of this process to study inherent platelet (PLT) production disorders as well as study factors that impair normal PLT production.
Assuntos
Megacariócitos/citologia , Células-Tronco de Sangue Periférico/citologia , Trombopoese/efeitos dos fármacos , Antígenos CD34/análise , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Humanos , Complexo Glicoproteico GPIb-IX de Plaquetas/análise , Trombopoetina/farmacologia , Fatores de TempoRESUMO
Histone methylation is a dynamic and reversible process proposed to directly impact on stem cell fate. The Jumonji (JmjC) domain-containing family of demethylases comprises 27 members that target mono-, di-, and trimethylated lysine residues of histone (or nonhistone) proteins. To evaluate their role in regulation of hematopoietic stem cell (HSC) behavior, we performed an in vivo RNAi-based functional screen and demonstrated that Jarid1b and Jhdm1f play opposing roles in regulation of HSC activity. Decrease in Jarid1b levels correlated with an in vitro expansion of HSCs with preserved long-term in vivo lymphomyeloid differentiation potential. Through RNA sequencing analysis, Jarid1b knockdown was associated with increased expression levels of several HSC regulators (Hoxa7, Hoxa9, Hoxa10, Hes1, Gata2) and reduced levels of differentiation-associated genes. shRNA against Jhdmlf, in contrast, impaired hematopoietic reconstitution of bone marrow cells. Together, our studies identified Jarid1b as a negative regulator of HSC activity and Jhdmlf as a positive regulator of HSC activity.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Hematopoese/genética , Células-Tronco Hematopoéticas/fisiologia , Ensaios de Triagem em Larga Escala/métodos , Histona Desmetilases com o Domínio Jumonji/fisiologia , Interferência de RNA/fisiologia , Animais , Células Cultivadas , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Histona Desmetilases/genética , Histona Desmetilases/fisiologia , Histona Desmetilases com o Domínio Jumonji/genética , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Estudos de Validação como AssuntoRESUMO
Strict control over hematopoietic stem cell decision making is essential for healthy life-long blood production and underpins the origins of hematopoietic diseases. Acute myeloid leukemia (AML) in particular is a devastating hematopoietic malignancy that arises from the clonal evolution of disease-initiating primitive cells which acquire compounding genetic changes over time and culminate in the generation of leukemic stem cells (LSCs). Understanding the molecular underpinnings of these driver cells throughout their development will be instrumental in the interception of leukemia, the enabling of effective treatment of pre-leukemic conditions, as well as the development of strategies to target frank AML disease. To this point, a number of precancerous myeloid disorders and age-related alterations are proving as instructive models to gain insights into the initiation of LSCs. Here, we explore this myeloid dysregulation at the level of post-transcriptional control, where RNA-binding proteins (RBPs) function as core effectors. Through regulating the interplay of a myriad of RNA metabolic processes, RBPs orchestrate transcript fates to govern gene expression in health and disease. We describe the expanding appreciation of the role of RBPs and their post-transcriptional networks in sustaining healthy hematopoiesis and their dysregulation in the pathogenesis of clonal myeloid disorders and AML, with a particular emphasis on findings described in human stem cells. Lastly, we discuss key breakthroughs that highlight RBPs and post-transcriptional control as actionable targets for precision therapy of AML.
RESUMO
The long-term survival of patients with acute myeloid leukemia (AML) is dismally poor. A permanent cure of AML requires elimination of leukemic stem cells (LSCs), the only cell type capable of initiating and maintaining the leukemic clonal hierarchy. We report a therapeutic approach using an activating monoclonal antibody directed to the adhesion molecule CD44. In vivo administration of this antibody to nonobese diabetic-severe combined immune-deficient mice transplanted with human AML markedly reduced leukemic repopulation. Absence of leukemia in serially transplanted mice demonstrated that AML LSCs are directly targeted. Mechanisms underlying this eradication included interference with transport to stem cell-supportive microenvironmental niches and alteration of AML-LSC fate, identifying CD44 as a key regulator of AML LSCs. The finding that AML LSCs require interaction with a niche to maintain their stem cell properties provides a therapeutic strategy to eliminate quiescent AML LSCs and may be applicable to other types of cancer stem cells.
Assuntos
Receptores de Hialuronatos/biossíntese , Imunoterapia/métodos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/terapia , Células-Tronco/citologia , ADP-Ribosil Ciclase 1/biossíntese , Animais , Anticorpos Monoclonais/química , Antígenos CD34/biossíntese , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Acute myeloid leukemia (AML) is fueled by leukemic stem cells (LSC) whose determinants are challenging to discern from hematopoietic stem cells (HSC) or uncover by approaches focused on general cell properties. We have identified a set of RNA-binding proteins (RBP) selectively enriched in human AML LSCs. Using an in vivo two-step CRISPR-Cas9 screen to assay stem cell functionality, we found 32 RBPs essential for LSCs in MLL-AF9;NrasG12D AML. Loss-of-function approaches targeting key hit RBP ELAVL1 compromised LSC-driven in vivo leukemic reconstitution, and selectively depleted primitive malignant versus healthy cells. Integrative multiomics revealed differentiation, splicing, and mitochondrial metabolism as key features defining the leukemic ELAVL1-mRNA interactome with mitochondrial import protein, TOMM34, being a direct ELAVL1-stabilized target whose repression impairs AML propagation. Altogether, using a stem cell-adapted in vivo CRISPR screen, this work demonstrates pervasive reliance on RBPs as regulators of LSCs and highlights their potential as therapeutic targets in AML. SIGNIFICANCE: LSC-targeted therapies remain a significant unmet need in AML. We developed a stem-cell-adapted in vivo CRISPR screen to identify key LSC drivers. We uncover widespread RNA-binding protein dependencies in LSCs, including ELAVL1, which we identify as a novel therapeutic vulnerability through its regulation of mitochondrial metabolism. This article is highlighted in the In This Issue feature, p. 171.
Assuntos
Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Diferenciação Celular , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/uso terapêutico , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismoRESUMO
PURPOSE OF REVIEW: The MSI2 and PROX1 proteins are increasingly recognized for their critical roles in the biology of primitive hematopoietic cells and for their potential contributions to leukemic pathogenesis. Here we summarize the studies that have shed light on the hematopoietic-specific roles of MSI2 and PROX1 and give an overview of the molecular mechanisms underlying their function. RECENT FINDINGS: In addition to a likely role in cell cycle restraint, the hematopoietic stem cell agonist MSI2 is essential for the maintenance of primitive cell fate through ensuring appropriate balance between self-renewal and differentiation. Overexpression of Msi2 can contribute to the progression of murine myeloid leukemia and in the human setting is associated with poor prognosis. Regulatory control imposed by MSI2 may be achieved partly through regulation of the Notch signaling pathway. Prox1 behaves in an opposing manner to Msi2, resulting in elevated stem cell numbers when depleted. It has a potential role in cell cycle control and may act at the level of primitive hematopoietic stem and progenitor cells as it does in other systems by directly promoting commitment and differentiation. PROX1 functions as a tumor suppressor in numerous tissue types and has been found mutated in hematopoietic cell lines and primary blood malignancies. SUMMARY: Deciphering the molecular mechanisms through which MSI2 and PROX1 affect primitive hematopoietic cell fate will provide insight into the regulation of normal hematopoiesis and facilitate better understanding of the leukemic transformation process. This will be directly applicable to the development of effective regenerative therapies and targeted leukemia treatments.
Assuntos
Células-Tronco Hematopoéticas/fisiologia , Proteínas de Homeodomínio/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Diferenciação Celular/fisiologia , Proteínas de Homeodomínio/genética , Humanos , Proteínas de Ligação a RNA/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Chemo-resistance in acute myeloid leukemia (AML) patients is driven by leukemic stem cells (LSCs) resulting in high rates of relapse and low overall survival. Here, we demonstrate that upregulation of the splicing factor, RBM17 preferentially marks and sustains LSCs and directly correlates with shorten patient survival. RBM17 knockdown in primary AML cells leads to myeloid differentiation and impaired colony formation and in vivo engraftment. Integrative multi-omics analyses show that RBM17 repression leads to inclusion of poison exons and production of nonsense-mediated decay (NMD)-sensitive transcripts for pro-leukemic factors and the translation initiation factor, EIF4A2. We show that EIF4A2 is enriched in LSCs and its inhibition impairs primary AML progenitor activity. Proteomic analysis of EIF4A2-depleted AML cells shows recapitulation of the RBM17 knockdown biological effects, including pronounced suppression of proteins involved in ribosome biogenesis. Overall, these results provide a rationale to target RBM17 and/or its downstream NMD-sensitive splicing substrates for AML treatment.
Assuntos
Leucemia Mieloide Aguda , Células-Tronco Neoplásicas , Fatores de Processamento de RNA , Hematopoese , Humanos , Leucemia Mieloide Aguda/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteômica , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismoRESUMO
How hematopoietic stem cells (HSCs) coordinate their divisional axis and whether this orientation is important for stem cell-driven hematopoiesis is poorly understood. Single-cell RNA sequencing data from patients with Shwachman-Diamond syndrome (SDS), an inherited bone marrow failure syndrome, show that ARHGEF2, a RhoA-specific guanine nucleotide exchange factor and determinant of mitotic spindle orientation, is specifically downregulated in SDS hematopoietic stem and progenitor cells (HSPCs). We demonstrate that transplanted Arhgef2-/- fetal liver and bone marrow cells yield impaired hematopoietic recovery and a production deficit from long-term HSCs, phenotypes that are not the result of differences in numbers of transplanted HSCs, their cell cycle status, level of apoptosis, progenitor output, or homing ability. Notably, these defects are functionally restored in vivo by overexpression of ARHGEF2 or its downstream activated RHOA GTPase. By using live imaging of dividing HSPCs, we show an increased frequency of misoriented divisions in the absence of Arhgef2. ARHGEF2 knockdown in human HSCs also impairs their ability to regenerate hematopoiesis, culminating in significantly smaller xenografts. Together, these data demonstrate a conserved role for Arhgef2 in orienting HSPC division and suggest that HSCs may divide in certain orientations to establish hematopoiesis, the loss of which could contribute to HSC dysfunction in bone marrow failure.
Assuntos
Hematopoese , Células-Tronco Hematopoéticas , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Apoptose , Células da Medula Óssea , Humanos , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fuso AcromáticoRESUMO
Lineage tracing and single-cell sequencing methods have been used independently to profile fate outcomes or molecular phenotypes, respectively. Weinreb et al. (2020) and Pei et al. (2020) (the latter in this issue of Cell Stem Cell) advance the shared principle that a simultaneous accounting of clonal and transcriptional trajectories provides critical new insights into organization and decision-making in hematopoiesis.
Assuntos
Hematopoese , Análise de Célula Única , Diferenciação Celular , Linhagem da CélulaRESUMO
Despite a surge in the preclinical development of immunotherapies, current models are unable to predict putative toxicity, particularly the "on-target, off-tumor" effects of these therapeutics. To address this gap, we used a humanized mouse model of hematopoiesis to examine the toxicity profile of CAR-Ts targeting brain tumor-antigens also expressed in the hematopoietic system. In assessing the safety of cell-based therapies, we aim to develop and integrate a preclinical evaluation protocol as a necessary step in the clinical development pathway. For complete details on the use and execution of this protocol, please refer to Vora et al. (2020).
Assuntos
Neoplasias Encefálicas/terapia , Hematopoese/imunologia , Imunoterapia Adotiva/métodos , Animais , Neoplasias Encefálicas/imunologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Imunoterapia/métodos , Camundongos , Modelos Animais , Receptores de Antígenos Quiméricos/imunologiaRESUMO
Eliminating leukemic stem cells (LSC) is a sought after therapeutic paradigm for the treatment of acute myeloid leukemia (AML). While repression of aryl hydrocarbon receptor (AHR) signaling has been shown to promote short-term maintenance of primitive AML cells in culture, no work to date has examined whether altered AHR signaling plays a pathologic role in human AML or whether it contributes at all to endogenous LSC function. Here, we show AHR signaling is repressed in human AML blasts and preferentially downregulated in LSC-enriched populations within leukemias. A core set of AHR targets are uniquely repressed in LSCs across diverse genetic AML subtypes. In vitro and in vivo administration of the specific AHR agonist FICZ significantly impaired leukemic growth, promoted differentiation, and repressed self-renewal. Furthermore, LSCs suppressed a set of FICZ-responsive AHR target genes that function as tumor suppressors and promoters of differentiation. FICZ stimulation did not impair normal hematopoietic stem and progenitor (HSPC) function, and failed to upregulate a prominent LSC-specific AHR target in HSPCs, suggesting that differential mechanisms govern FICZ-induced AHR signaling manifestations in HSCs versus LSCs. Altogether, this work highlights AHR signaling suppression as a key LSC-regulating control mechanism and provides proof of concept in a preclinical model that FICZ-mediated AHR pathway activation enacts unique transcriptional programs in AML that identify it as a novel chemotherapeutic approach to selectively target human LSCs. SIGNIFICANCE: The AHR pathway is suppressed in leukemic stem cells (LSC), therefore activating AHR signaling is a potential therapeutic option to target LSCs and to treat acute myeloid leukemia.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Leucemia Mieloide Aguda/genética , Células-Tronco Neoplásicas/patologia , Receptores de Hidrocarboneto Arílico/genética , Transdução de Sinais/genética , Animais , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Células-Tronco Hematopoéticas/patologia , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Regiões Promotoras Genéticas/genéticaRESUMO
MSI2, which is expressed predominantly in hematopoietic stem and progenitor cells (HSPCs), enforces HSPC expansion when overexpressed and is upregulated in myeloid leukemias, indicating its regulated transcription is critical to balanced self-renewal and leukemia restraint. Despite this, little is understood of the factors that enforce appropriate physiological levels of MSI2 in the blood system. Here, we define a promoter region that reports on endogenous expression of MSI2 and identify USF2 and PLAG1 as transcription factors whose promoter binding drives reporter activity. We show that these factors co-regulate, and are required for, efficient transactivation of endogenous MSI2. Coincident overexpression of USF2 and PLAG1 in primitive cord blood cells enhanced MSI2 transcription and yielded cellular phenotypes, including expansion of CD34+ cells in vitro, consistent with that achieved by direct MSI2 overexpression. Global chromatin immunoprecipitation sequencing analyses confirm a preferential co-binding of PLAG1 and USF2 at the promoter of MSI2, as well as regulatory regions corresponding to genes with roles in HSPC homeostasis. PLAG1 and USF2 cooperation is thus an important contributor to stem cell-specific expression of MSI2 and HSPC-specific transcriptional circuitry.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Ligação a RNA/genética , Fatores Estimuladores Upstream/metabolismo , Sequência de Bases , Sítios de Ligação , Genoma Humano , Humanos , Células K562 , Regiões Promotoras Genéticas/genética , Ligação Proteica , Transcrição Gênica , Ativação Transcricional/genéticaRESUMO
The development of neural connectivity is essential for brain function, and disruption of this process is associated with autism spectrum disorders (ASDs). DIX domain containing 1 (DIXDC1) has previously been implicated in neurodevelopmental disorders, but its role in postnatal brain function remains unknown. Using a knockout mouse model, we determined that DIXDC1 is a regulator of excitatory neuron dendrite development and synapse function in the cortex. We discovered that MARK1, previously linked to ASDs, phosphorylates DIXDC1 to regulate dendrite and spine development through modulation of the cytoskeletal network in an isoform-specific manner. Finally, rare missense variants in DIXDC1 were identified in ASD patient cohorts via genetic sequencing. Interestingly, the variants inhibit DIXDC1 isoform 1 phosphorylation, causing impairment to dendrite and spine growth. These data reveal that DIXDC1 is a regulator of cortical dendrite and synaptic development and provide mechanistic insight into morphological defects associated with neurodevelopmental disorders.
Assuntos
Dendritos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutação/genética , Animais , Transtorno Autístico/metabolismo , Transtorno Autístico/patologia , Encéfalo/metabolismo , Espinhas Dendríticas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microtúbulos/metabolismo , Mutação de Sentido Incorreto/genética , Fosforilação , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sinapses/metabolismoRESUMO
Two fundamental problems in cancer research are identification of the normal cell within which cancer initiates and identification of the cell type capable of sustaining the growth of the neoplastic clone. There is overwhelming evidence that virtually all cancers are clonal and represent the progeny of a single cell. What is less clear for most cancers is which cells within the tumor clone possess tumorigenic or 'cancer stem cell' (CSC) properties and are capable of maintaining tumor growth. The concept that only a subpopulation of rare CSC is responsible for maintenance of the neoplasm emerged nearly 50 years ago. Testing of this hypothesis is most advanced for the hematopoietic system due to the establishment of functional in vitro and in vivo assays for stem and progenitor cells at all stages of development. This body of work led to conclusive proof for CSC with the identification and purification of leukemic stem cells capable of repopulating NOD/SCID mice. This review will focus on the historical development of the CSC hypothesis, the mechanisms necessary to subvert normal developmental programs, and the identification of the cell in which these leukemogenic events first occur.