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1.
Blood ; 2024 Aug 16.
Artículo en Inglés | MEDLINE | ID: mdl-39158067

RESUMEN

Menin inhibitors that disrupt Menin-MLL interaction hold promise for treating specific acute myeloid leukemia subtypes, including KMT2A rearrangements (KMT2A-r), yet resistance remains a challenge. Here, through systematic chromatin-focused CRISPR screens, along with genetic, epigenetic, and pharmacologic studies in a variety of human and mouse KMT2A-r AML models, we uncover a potential resistance mechanism independent of canonical Menin-MLL targets. We show that a group of non-canonical Menin targets, which are bivalently co-occupied by active Menin and repressive H2AK119ub marks, are typically downregulated following Menin inhibition. The loss of Polycomb Repressive Complex 1.1 (PRC1.1) subunits, such as PCGF1 or BCOR, leads to Menin inhibitor resistance by epigenetic reactivation of these non-canonical targets, including MYC. Genetic and pharmacological inhibition of MYC can resensitize PRC1.1-deficent leukemia cells to Menin inhibition. Moreover, we demonstrate that leukemia cells with the loss of PRC1.1 subunits exhibit reduced monocytic gene signatures and are susceptible to the BCL2 inhibition, and combinational treatment of venetoclax overcomes the resistance to Menin inhibition in PRC1.1-deficient leukemia cells. These findings highlight the important roles of PRC1.1 and its regulated non-canonical Menin targets in modulating Menin inhibitor response and provide potential strategies to treat leukemias with compromised PRC1.1 function.

2.
Blood ; 140(9): 992-1008, 2022 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-35639948

RESUMEN

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.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Células Madre Hematopoyéticas , Factores de Transcripción , Diferenciación Celular/fisiología , Proliferación Celular , Autorrenovación de las Células , Células Madre Hematopoyéticas/metabolismo , Humanos , Factores de Transcripción/metabolismo
3.
Blood ; 137(25): 3518-3532, 2021 06 24.
Artículo en Inglés | MEDLINE | ID: mdl-33720355

RESUMEN

Acute myeloid leukemia (AML) cells have an atypical metabolic phenotype characterized by increased mitochondrial mass, as well as a greater reliance on oxidative phosphorylation and fatty acid oxidation (FAO) for survival. To exploit this altered metabolism, we assessed publicly available databases to identify FAO enzyme overexpression. Very long chain acyl-CoA dehydrogenase (VLCAD; ACADVL) was found to be overexpressed and critical to leukemia cell mitochondrial metabolism. Genetic attenuation or pharmacological inhibition of VLCAD hindered mitochondrial respiration and FAO contribution to the tricarboxylic acid cycle, resulting in decreased viability, proliferation, clonogenic growth, and AML cell engraftment. Suppression of FAO at VLCAD triggered an increase in pyruvate dehydrogenase activity that was insufficient to increase glycolysis but resulted in adenosine triphosphate depletion and AML cell death, with no effect on normal hematopoietic cells. Together, these results demonstrate the importance of VLCAD in AML cell biology and highlight a novel metabolic vulnerability for this devastating disease.


Asunto(s)
Ácidos Grasos/metabolismo , Leucemia Mieloide Aguda/metabolismo , Acil-CoA Deshidrogenasa de Cadena Larga/genética , Acil-CoA Deshidrogenasa de Cadena Larga/metabolismo , Línea Celular Tumoral , Ciclo del Ácido Cítrico , Ácidos Grasos/genética , Glucólisis , Humanos , Cetona Oxidorreductasas/metabolismo , Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo
4.
Nature ; 532(7600): 508-511, 2016 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-27121842

RESUMEN

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.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Autorrenovación de las Células , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Transducción de Señal , Animales , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Recuento de Células , Autorrenovación de las Células/genética , Regulación hacia Abajo/genética , Femenino , Sangre Fetal/citología , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Receptores de Hidrocarburo de Aril/genética , Transducción de Señal/genética
5.
Am J Hum Genet ; 102(2): 278-295, 2018 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-29395074

RESUMEN

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.


Asunto(s)
Trastornos de los Cromosomas/enzimología , Trastornos de los Cromosomas/genética , Enzimas Desubicuitinizantes/fisiología , Endopeptidasas/genética , Discapacidad Intelectual/enzimología , Discapacidad Intelectual/genética , Trastornos del Neurodesarrollo/enzimología , Trastornos del Neurodesarrollo/genética , Convulsiones/enzimología , Convulsiones/genética , Animales , Trastorno del Espectro Autista/genética , Deleción Cromosómica , Cromosomas Humanos Par 15/enzimología , Cromosomas Humanos Par 15/genética , Espinas Dendríticas/metabolismo , Enzimas Desubicuitinizantes/genética , Endopeptidasas/metabolismo , Femenino , Eliminación de Gen , Estudios de Asociación Genética , Humanos , Masculino , Ratones , Fenotipo , Prosencéfalo/patología
6.
Mol Ther ; 27(6): 1074-1086, 2019 06 05.
Artículo en Inglés | MEDLINE | ID: mdl-31023523

RESUMEN

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.


Asunto(s)
Terapia Genética/métodos , Vectores Genéticos/uso terapéutico , Hematopoyesis/genética , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/virología , Lentivirus/genética , Transducción Genética , Animales , Antígenos CD34/metabolismo , Células Clonales , Terapia Genética/efectos adversos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Sustancias Luminiscentes/metabolismo , Macaca mulatta , Mutagénesis Insercional/genética , Regiones Promotoras Genéticas , Empalme de Proteína/genética , Secuencias Repetidas Terminales/genética , Trasplante Autólogo
7.
Biochem Cell Biol ; 97(1): 10-20, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-29898370

RESUMEN

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.


Asunto(s)
Regulación de la Expresión Génica , Hematopoyesis , Células Madre Hematopoyéticas/metabolismo , Procesamiento Postranscripcional del ARN , Proteínas de Unión al ARN/metabolismo , Animales , Diferenciación Celular , Células Madre Hematopoyéticas/citología , Humanos
9.
Transfusion ; 56(5): 1066-74, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-26756864

RESUMEN

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.


Asunto(s)
Megacariocitos/citología , Células Madre de Sangre Periférica/citología , Trombopoyesis/efectos de los fármacos , Antígenos CD34/análisis , Técnicas de Cultivo de Célula , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Humanos , Complejo GPIb-IX de Glicoproteína Plaquetaria/análisis , Trombopoyetina/farmacología , Factores de Tiempo
10.
BMC Health Serv Res ; 16(1): 637, 2016 11 08.
Artículo en Inglés | MEDLINE | ID: mdl-27825345

RESUMEN

BACKGROUND: Over the past decade the healthcare workforce has diversified in several directions with formalised roles for health care assistants, specialised roles for nurses and technicians, advanced roles for physician associates and nurse practitioners and new professions for new services, such as case managers. Hence the composition of health care teams has become increasingly diverse. The exact extent of this diversity is unknown across the different countries of Europe, as are the drivers of this change. The research questions guiding this study were: What extended professional roles are emerging on health care teams? How are extended professional roles created? What main drivers explain the observed differences, if any, in extended roles in and between countries? METHODS: We performed a case-based comparison of the extended roles in care pathways for breast cancer, heart disease and type 2 diabetes. We conducted 16 case studies in eight European countries, including in total 160 interviews with physicians, nurses and other health care professionals in new roles and 600+ hours of observation in health care clinics. RESULTS: The results show a relatively diverse composition of roles in the three care pathways. We identified specialised roles for physicians, extended roles for nurses and technicians, and independent roles for advanced nurse practitioners and physician associates. The development of extended roles depends upon the willingness of physicians to delegate tasks, developments in medical technology and service (re)design. Academic training and setting a formal scope of practice for new roles have less impact upon the development of new roles. While specialised roles focus particularly on a well-specified technical or clinical domain, the generic roles concentrate on organising and integrating care and cure. CONCLUSION: There are considerable differences in the number and kind of extended roles between both countries and care pathways. The main drivers for new roles reside in the technological development of medical treatment and the need for more generic competencies. Extended roles develop in two directions: 1) specialised roles and 2) generic roles.


Asunto(s)
Atención a la Salud , Personal de Salud , Grupo de Atención al Paciente , Rol Profesional , Neoplasias de la Mama/terapia , Atención a la Salud/organización & administración , Diabetes Mellitus Tipo 2/terapia , Europa (Continente) , Cardiopatías/terapia , Humanos , Recursos Humanos
11.
Blood ; 122(9): 1545-55, 2013 Aug 29.
Artículo en Inglés | MEDLINE | ID: mdl-23777767

RESUMEN

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.


Asunto(s)
Proteínas de Unión al ADN/fisiología , Hematopoyesis/genética , Células Madre Hematopoyéticas/fisiología , Ensayos Analíticos de Alto Rendimiento/métodos , Histona Demetilasas con Dominio de Jumonji/fisiología , Interferencia de ARN/fisiología , Animales , Células Cultivadas , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Histona Demetilasas/genética , Histona Demetilasas/fisiología , Histona Demetilasas con Dominio de Jumonji/genética , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Estudios de Validación como Asunto
12.
Nat Methods ; 8(4 Suppl): S36-40, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21451511

RESUMEN

Individual stem cells are functionally defined by their self-renewal and differentiation potential. Methods for clonal analysis are essential for understanding stem cells, particularly given the increasing evidence for stem-cell heterogeneity. Stem cells reside within complex microenvironments, making single-cell analysis particularly challenging. Furthermore, simultaneous molecular and functional characterization of single stem cells is not trivial. Here we explore clonal assays applied to stem cell biology and their use in understanding the cellular and molecular basis of stem-cell identity.


Asunto(s)
Análisis de la Célula Individual/métodos , Células Madre/citología , Células Madre/metabolismo , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Linaje de la Célula , Células Clonales/citología , Células Clonales/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo
13.
Blood ; 119(11): 2510-22, 2012 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-22174158

RESUMEN

The stem cell-intrinsic model of self-renewal via asymmetric cell division (ACD) posits that fate determinants be partitioned unequally between daughter cells to either activate or suppress the stemness state. ACD is a purported mechanism by which hematopoietic stem cells (HSCs) self-renew, but definitive evidence for this cellular process remains open to conjecture. To address this issue, we chose 73 candidate genes that function within the cell polarity network to identify potential determinants that may concomitantly alter HSC fate while also exhibiting asymmetric segregation at cell division. Initial gene-expression profiles of polarity candidates showed high and differential expression in both HSCs and leukemia stem cells. Altered HSC fate was assessed by our established in vitro to in vivo screen on a subcohort of candidate polarity genes, which revealed 6 novel positive regulators of HSC function: Ap2a2, Gpsm2, Tmod1, Kif3a, Racgap1, and Ccnb1. Interestingly, live-cell videomicroscopy of the endocytic protein AP2A2 shows instances of asymmetric segregation during HSC/progenitor cell cytokinesis. These results contribute further evidence that ACD is functional in HSC self-renewal, suggest a role for Ap2a2 in HSC activity, and provide a unique opportunity to prospectively analyze progeny from HSC asymmetric divisions.


Asunto(s)
Complejo 2 de Proteína Adaptadora/metabolismo , Subunidades alfa de Complejo de Proteína Adaptadora/metabolismo , División Celular Asimétrica/fisiología , Polaridad Celular/genética , Endocitosis/genética , Células Madre Hematopoyéticas/citología , Células Madre Neoplásicas/patología , Células Madre/citología , Complejo 2 de Proteína Adaptadora/antagonistas & inhibidores , Complejo 2 de Proteína Adaptadora/genética , Subunidades alfa de Complejo de Proteína Adaptadora/antagonistas & inhibidores , Subunidades alfa de Complejo de Proteína Adaptadora/genética , Animales , Biomarcadores/metabolismo , Western Blotting , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Citometría de Flujo , Perfilación de la Expresión Génica , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/fisiología , Leucemia/metabolismo , Leucemia/patología , Ratones , Células Madre Neoplásicas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/fisiología
14.
Hemasphere ; 8(8): e116, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-39175825

RESUMEN

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.

15.
Blood ; 117(2): e27-38, 2011 Jan 13.
Artículo en Inglés | MEDLINE | ID: mdl-20980679

RESUMEN

The molecular mechanisms regulating self-renewal of leukemia stem cells remain poorly understood. Here we report the generation of 2 closely related leukemias created through the retroviral overexpression of Meis1 and Hoxa9. Despite their apparent common origin, these clonal leukemias exhibit enormous differences in stem cell frequency (from 1 in 1.4, FLA2; to 1 in 347, FLB1), suggesting that one of these leukemias undergoes nearly unlimited self-renewal divisions. Using next-generation RNA-sequencing, we characterized the transcriptomes of these phenotypically similar, but biologically distinct, leukemias, identifying hundreds of differentially expressed genes and a large number of structural differences (eg, alternative splicing and promoter usage). Focusing on ligand-receptor pairs, we observed high expression levels of Sdf1-Cxcr4; Jagged2-Notch2/1; Osm-Gp130; Scf-cKit; and Bmp15-Tgfb1/2. Interestingly, the integrin beta 2-like gene (Itgb2l) is both highly expressed and differentially expressed between our 2 leukemias (∼ 14-fold higher in FLA2 than FLB1). In addition, gene ontology analysis indicated G-protein-coupled receptor had a much higher proportion of differential expression (22%) compared with other classes (∼ 5%), suggesting a potential role regulating subtle changes in cellular behavior. These results provide the first comprehensive transcriptome analysis of a leukemia stem cell and document an unexpected level of transcriptome variation between phenotypically similar leukemic cells.


Asunto(s)
Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/genética , Células Madre Neoplásicas , Animales , Células Clonales , Citometría de Flujo , Vectores Genéticos , Proteínas de Homeodominio/genética , Ratones , Ratones Endogámicos C57BL , Análisis por Micromatrices , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide , Proteínas de Neoplasias/genética , Retroviridae , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ARN
16.
Nat Med ; 12(10): 1167-74, 2006 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16998484

RESUMEN

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.


Asunto(s)
Receptores de Hialuranos/biosíntesis , Inmunoterapia/métodos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/terapia , Células Madre/citología , ADP-Ribosil Ciclasa 1/biosíntesis , Animales , Anticuerpos Monoclonales/química , Antígenos CD34/biosíntesis , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Humanos , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID
17.
Blood Cancer Discov ; 4(3): 180-207, 2023 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-36763002

RESUMEN

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.


Asunto(s)
Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Diferenciación Celular , Células Madre Hematopoyéticas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/uso terapéutico , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteína 1 Similar a ELAV/genética , Proteína 1 Similar a ELAV/metabolismo
18.
Curr Opin Hematol ; 18(4): 203-7, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21577104

RESUMEN

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.


Asunto(s)
Células Madre Hematopoyéticas/fisiología , Proteínas de Homeodominio/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Diferenciación Celular/fisiología , Proteínas de Homeodominio/genética , Humanos , Proteínas de Unión al ARN/genética , Proteínas Supresoras de Tumor/genética
19.
Mol Cancer Res ; 20(11): 1659-1673, 2022 11 03.
Artículo en Inglés | MEDLINE | ID: mdl-35994381

RESUMEN

Acute myeloid leukemia (AML) is a hematologic malignancy metabolically dependent on oxidative phosphorylation and mitochondrial electron transport chain (ETC) activity. AML cells are distinct from their normal hematopoietic counterparts by this metabolic reprogramming, which presents targets for new selective therapies. Here, metabolic changes in AML cells after ETC impairment are investigated. Genetic knockdown of the ETC complex II (CII) chaperone protein SDHAF1 (succinate dehydrogenase assembly factor 1) suppressed CII activity and delayed AML cell growth in vitro and in vivo. As a result, a novel small molecule that directly binds to the ubiquinone binding site of CII and inhibits its activity was identified. Pharmacologic inhibition of CII induced selective death of AML cells while sparing normal hematopoietic progenitors. Through stable isotope tracing, results show that genetic or pharmacologic inhibition of CII truncates the tricarboxylic acid cycle (TCA) and leads to anaplerotic glutamine metabolism to reestablish the truncated cycle. The inhibition of CII showed divergent fates, as AML cells lacked the metabolic plasticity to adequately utilize glutamine metabolism, resulting in preferential depletion of key TCA metabolites and death; normal cells were unaffected. These findings provide insight into the metabolic mechanisms that underlie AML's selective inhibition of CII. IMPLICATIONS: This work highlights the effects of direct CII inhibition in mediating selective AML cell death and provides insights into glutamine anaplerosis as a metabolic adaptation that can be therapeutically targeted.


Asunto(s)
Glutamina , Leucemia Mieloide Aguda , Humanos , Glutamina/genética , Succinato Deshidrogenasa/metabolismo , Succinato Deshidrogenasa/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Fosforilación Oxidativa
20.
Cell Rep ; 39(11): 110947, 2022 06 14.
Artículo en Inglés | MEDLINE | ID: mdl-35705031

RESUMEN

A recurrent chromosomal translocation found in acute myeloid leukemia leads to an in-frame fusion of the transcription repressor ZMYND11 to MBTD1, a subunit of the NuA4/TIP60 histone acetyltransferase complex. To understand the abnormal molecular events that ZMYND11-MBTD1 expression can create, we perform a biochemical and functional characterization comparison to each individual fusion partner. ZMYND11-MBTD1 is stably incorporated into the endogenous NuA4/TIP60 complex, leading to its mislocalization on the body of genes normally bound by ZMYND11. This can be correlated to increased chromatin acetylation and altered gene transcription, most notably on the MYC oncogene, and alternative splicing. Importantly, ZMYND11-MBTD1 expression favors Myc-driven pluripotency during embryonic stem cell differentiation and self-renewal of hematopoietic stem/progenitor cells. Altogether, these results indicate that the ZMYND11-MBTD1 fusion functions primarily by mistargeting the NuA4/TIP60 complex to the body of genes, altering normal transcription of specific genes, likely driving oncogenesis in part through the Myc regulatory network.


Asunto(s)
Cromatina , Histona Acetiltransferasas , Proteínas de Fusión Oncogénica , Sistemas de Lectura Abierta , Acetilación , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Co-Represoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Humanos , Lisina Acetiltransferasa 5/genética , Lisina Acetiltransferasa 5/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Sistemas de Lectura Abierta/genética , Translocación Genética
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