RESUMO
Oncogenic mutations confer on cells the ability to propagate indefinitely, but whether oncogenes alter the cell fate of these cells is unknown. Here, we show that the transcriptional regulator PRDM16s causes oncogenic fate conversion by transforming cells fated to form platelets and erythrocytes into myeloid leukemia stem cells (LSCs). Prdm16s expression in megakaryocyte-erythroid progenitors (MEPs), which normally lack the potential to generate granulomonocytic cells, caused AML by converting MEPs into LSCs. Prdm16s blocked megakaryocytic/erythroid potential by interacting with super enhancers and activating myeloid master regulators, including PU.1. A CRISPR dropout screen confirmed that PU.1 is required for Prdm16s-induced leukemia. Ablating PU.1 attenuated leukemogenesis and reinstated the megakaryocytic/erythroid potential of leukemic MEPs in mouse models and human AML with PRDM16 rearrangement. Thus, oncogenic PRDM16 s expression gives MEPs an LSC fate by activating myeloid gene regulatory networks.
Assuntos
Transformação Celular Neoplásica/patologia , Proteínas de Ligação a DNA/genética , Leucemia Mieloide Aguda/patologia , Células Progenitoras de Megacariócitos e Eritrócitos/patologia , Fatores de Transcrição/genética , Animais , Transformação Celular Neoplásica/genética , Regulação Leucêmica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Leucemia Mieloide Aguda/genética , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas/genética , Transativadores/genética , Translocação GenéticaRESUMO
Altered metabolism fuels 2 hallmark properties of cancer cells: unlimited proliferation and differentiation blockade. Adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of bioenergetics crucial for glucose metabolism in acute myeloid leukemia (AML), and its inhibition delays leukemogenesis, but whether the metabolic function of AMPK alters the AML epigenome remains unknown. Here, we demonstrate that AMPK maintains the epigenome of MLL-rearranged AML by linking acetyl-coenzyme A (CoA) homeostasis to Bromodomain and Extra-Terminal domain (BET) protein recruitment to chromatin. AMPK deletion reduced acetyl-CoA and histone acetylation, displacing BET proteins from chromatin in leukemia-initiating cells. In both mouse and patient-derived xenograft AML models, treating with AMPK and BET inhibitors synergistically suppressed AML. Our results provide a therapeutic rationale to target AMPK and BET for AML therapy.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Acetilcoenzima A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Leucemia Mieloide Aguda/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação Leucêmica da Expressão Gênica , Histonas/metabolismo , Homeostase , Humanos , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/patologia , Camundongos , Gradação de Tumores , Ligação Proteica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Cotton (Gossypium spp.) is the largest producer of natural fibers for textile and is an important crop worldwide. Crop production is comprised primarily of G. hirsutum L., an allotetraploid. However, elite cultivars express very small amounts of variation due to the species monophyletic origin, domestication and further bottlenecks due to selection. Conversely, wild cotton species harbor extensive genetic diversity of prospective utility to improve many beneficial agronomic traits, fiber characteristics, and resistance to disease and drought. Introgression of traits from wild species can provide a natural way to incorporate advantageous traits through breeding to generate higher-producing cotton cultivars and more sustainable production systems. Interspecific introgression efforts by conventional methods are very time-consuming and costly, but can be expedited using marker-assisted selection. RESULTS: Using transcriptome sequencing we have developed the first gene-associated single nucleotide polymorphism (SNP) markers for wild cotton species G. tomentosum, G. mustelinum, G. armourianum and G. longicalyx. Markers were also developed for a secondary cultivated species G. barbadense cv. 3-79. A total of 62,832 non-redundant SNP markers were developed from the five wild species which can be utilized for interspecific germplasm introgression into cultivated G. hirsutum and are directly associated with genes. Over 500 of the G. barbadense markers have been validated by whole-genome radiation hybrid mapping. Overall 1,060 SNPs from the five different species have been screened and shown to produce acceptable genotyping assays. CONCLUSIONS: This large set of 62,832 SNPs relative to cultivated G. hirsutum will allow for the first high-density mapping of genes from five wild species that affect traits of interest, including beneficial agronomic and fiber characteristics. Upon mapping, the markers can be utilized for marker-assisted introgression of new germplasm into cultivated cotton and in subsequent breeding of agronomically adapted types, including cultivar development.
Assuntos
Cruzamento , Mapeamento Cromossômico , Genes de Plantas , Gossypium/genética , Polimorfismo de Nucleotídeo Único , Cromossomos de Plantas , Biologia Computacional , Cruzamentos Genéticos , Marcadores Genéticos , Genoma de Planta , Técnicas de Genotipagem , Reprodutibilidade dos Testes , Deleção de Sequência , TranscriptomaRESUMO
Hematopoietic stem cells (HSCs) are capable of regenerating the blood system, but the instructive cues that direct HSCs to regenerate particular lineages lost to the injury remain elusive. Here, we show that iron is increasingly taken up by HSCs during anemia and induces erythroid gene expression and regeneration in a Tet2-dependent manner. Lineage tracing of HSCs reveals that HSCs respond to hemolytic anemia by increasing erythroid output. The number of HSCs in the spleen, but not bone marrow, increases upon anemia and these HSCs exhibit enhanced proliferation, erythroid differentiation, iron uptake, and TET2 protein expression. Increased iron in HSCs promotes DNA demethylation and expression of erythroid genes. Suppressing iron uptake or TET2 expression impairs erythroid genes expression and erythroid differentiation of HSCs; iron supplementation, however, augments these processes. These results establish that the physiological level of iron taken up by HSCs has an instructive role in promoting erythroid-biased differentiation of HSCs.
Assuntos
Anemia , Dioxigenases , Humanos , Baço , Células-Tronco Hematopoéticas/metabolismo , Diferenciação Celular , Ferro/metabolismo , Anemia/metabolismo , Células Eritroides , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/metabolismoRESUMO
Metabolic dysregulation underlies malignant phenotypes attributed to cancer stem cells, such as unlimited proliferation and differentiation blockade. Here, we demonstrate that NAD+ metabolism enables acute myeloid leukemia (AML) to evade apoptosis, another hallmark of cancer stem cells. We integrated whole-genome CRISPR screening and pan-cancer genetic dependency mapping to identify NAMPT and NMNAT1 as AML dependencies governing NAD+ biosynthesis. While both NAMPT and NMNAT1 were required for AML, the presence of NAD+ precursors bypassed the dependence of AML on NAMPT but not NMNAT1, pointing to NMNAT1 as a gatekeeper of NAD+ biosynthesis. Deletion of NMNAT1 reduced nuclear NAD+, activated p53, and increased venetoclax sensitivity. Conversely, increased NAD+ biosynthesis promoted venetoclax resistance. Unlike leukemia stem cells (LSCs) in both murine and human AML xenograft models, NMNAT1 was dispensable for hematopoietic stem cells and hematopoiesis. Our findings identify NMNAT1 as a previously unidentified therapeutic target that maintains NAD+ for AML progression and chemoresistance.
Assuntos
Leucemia Mieloide Aguda , Nicotinamida-Nucleotídeo Adenililtransferase , Animais , Apoptose/genética , Homeostase , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Camundongos , NAD/metabolismo , Células-Tronco Neoplásicas/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismoRESUMO
Histone variants contribute to the complexity of the chromatin landscape and play an integral role in defining DNA domains and regulating gene expression. The histone H3 variant H3.3 is incorporated into genic elements independent of DNA replication by its chaperone HIRA. Here we demonstrate that Hira is required for the self-renewal of adult hematopoietic stem cells (HSCs) and to restrain erythroid differentiation. Deletion of Hira led to rapid depletion of HSCs while differentiated hematopoietic cells remained largely unaffected. Depletion of HSCs after Hira deletion was accompanied by increased expression of bivalent and erythroid genes, which was exacerbated upon cell division and paralleled increased erythroid differentiation. Assessing H3.3 occupancy identified a subset of polycomb-repressed chromatin in HSCs that depends on HIRA to maintain the inaccessible, H3.3-occupied state for gene repression. HIRA-dependent H3.3 incorporation thus defines distinct repressive chromatin that represses erythroid differentiation of HSCs.
Assuntos
Células-Tronco Adultas/metabolismo , Proteínas de Ciclo Celular/genética , Diferenciação Celular/genética , Células Eritroides/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Chaperonas de Histonas/genética , Fatores de Transcrição/genética , Fatores Etários , Animais , Animais Recém-Nascidos , Proteínas de Ciclo Celular/metabolismo , Autorrenovação Celular/genética , Perfilação da Expressão Gênica/métodos , Ontologia Genética , Hematopoese/genética , Chaperonas de Histonas/metabolismo , Histonas/genética , Histonas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , RNA-Seq/métodos , Fatores de Transcrição/metabolismoRESUMO
Bone marrow stromal cells (BMSCs) that express high levels of stem cell factor (SCF) and CXC chemokine ligand 12 (CXCL12) are one crucial component of the hematopoietic stem cell (HSC) niche. While the secreted factors produced by BMSCs to support HSCs have been well described, little is known regarding the transcriptional regulators controlling the cell fate of BMSCs and thus indirectly maintaining HSCs. BMI1 is a polycomb group protein that regulates HSCs both cell intrinsically and extrinsically, but it is unknown in which cell type and how BMI1 functions to maintain HSCs extrinsically. Here we show that Bmi1 maintains HSCs by preventing adipogenic differentiation of BMSCs. Bmi1 is highly expressed in BMSCs but becomes downregulated upon adipogenic differentiation and during aging. Deleting Bmi1 from BMSCs increased marrow adipocytes, induced HSC quiescence and depletion, and impaired hematopoiesis. We found that BMI1 repressed multiple developmental programs in BMSCs by safeguarding the repressive epigenetic marks histone H2A ubiquitylation and H3 lysine 27 trimethylation. We identified a novel adipogenic program governed by Pax3, which BMI1 repressed in BMSCs. Our results establish Bmi1 as a critical regulator of BMSC cell fate that suppresses marrow adipogenesis to create a supportive niche for HSCs.
Assuntos
Adipócitos/metabolismo , Células-Tronco Mesenquimais/citologia , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Adipócitos/citologia , Adipogenia , Animais , Diferenciação Celular , Senescência Celular , Feminino , Hematopoese , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Histonas/metabolismo , Masculino , Células-Tronco Mesenquimais/metabolismo , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Fator de Transcrição PAX3/genética , Fator de Transcrição PAX3/metabolismo , Complexo Repressor Polycomb 1/genética , Proteínas Proto-Oncogênicas/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Nicho de Células-TroncoRESUMO
Recent advances in next-generation sequencing have identified novel mutations and revealed complex genetic architectures in human hematological malignancies. Moving forward, new methods to quickly generate animal models that recapitulate the complex genetics of human hematological disorders are needed to transform the genetic information to new therapies. Here, we used a ribonucleoprotein-based CRISPR/Cas9 system to model human clonal hematopoiesis of indeterminate potential and acute myeloid leukemia (AML). We edited multiple genes recurrently mutated in hematological disorders, including those encoding epigenetic regulators, transcriptional regulators, and signaling components in murine hematopoietic stem/progenitor cells. Tracking the clonal dynamics by sequencing the indels induced by CRISPR/Cas9 revealed clonal expansion in some recipient mice that progressed to AML initiated by leukemia-initiating cells. Our results establish that the CRISPR/Cas9-mediated multiplex mutagenesis can be used to engineer a variety of murine models of hematological malignancies with complex genetic architectures seen in human disease.
Assuntos
Sistemas CRISPR-Cas , Células Clonais/patologia , Edição de Genes/métodos , Células-Tronco Hematopoéticas/patologia , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/patologia , Animais , Transplante de Medula Óssea , Proteína 9 Associada à CRISPR , Modelos Animais de Doenças , Progressão da Doença , Feminino , Genes Neoplásicos , Humanos , Mutação INDEL , Leucemia Mieloide Aguda/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Pré-Leucemia/genética , Pré-Leucemia/patologia , Ribonucleoproteínas/genética , Organismos Livres de Patógenos EspecíficosRESUMO
Characterization of hematopoietic stem cells (HSCs) has advanced largely owing to transplantation assays, in which the developmental potential of HSCs is assessed generally in nonhomeostatic conditions. These studies established that adult HSCs extensively contribute to multilineage hematopoietic regeneration upon transplantation. On the contrary, recent studies performing lineage tracing of HSCs under homeostatic conditions have shown that adult HSCs may contribute far less to steady-state hematopoiesis than would be anticipated based on transplantation assays. Here, we used 2 independent HSC-lineage-tracing models to examine the contribution of adult HSCs to steady-state hematopoiesis. We show that adult HSCs contribute robustly to steady-state hematopoiesis, exhibiting faster efflux toward the myeloid lineages compared with lymphoid lineages. Platelets were robustly labeled by HSCs, reaching the same level of labeling as HSCs by 1 year of chase. Our results support the view that adult HSCs contribute to the continuous influx of blood cells during steady-state hematopoiesis.
Assuntos
Células-Tronco Adultas/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Adultas/citologia , Animais , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos TransgênicosRESUMO
Activation of the unfolded protein response (UPR) sustains protein homeostasis (proteostasis) and plays a fundamental role in tissue maintenance and longevity of organisms. Long-range control of UPR activation has been demonstrated in invertebrates, but such mechanisms in mammals remain elusive. Here, we show that the female sex hormone estrogen regulates the UPR in hematopoietic stem cells (HSCs). Estrogen treatment increases the capacity of HSCs to regenerate the hematopoietic system upon transplantation and accelerates regeneration after irradiation. We found that estrogen signals through estrogen receptor α (ERα) expressed in hematopoietic cells to activate the protective Ire1α-Xbp1 branch of the UPR. Further, ERα-mediated activation of the Ire1α-Xbp1 pathway confers HSCs with resistance against proteotoxic stress and promotes regeneration. Our findings reveal a systemic mechanism through which HSC function is augmented for hematopoietic regeneration.
Assuntos
Endorribonucleases/metabolismo , Receptor alfa de Estrogênio/metabolismo , Estrogênios/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Resposta a Proteínas não Dobradas , Animais , Células Cultivadas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Camundongos , Transdução de SinaisRESUMO
Our understanding of the mechanisms that regulate hematopoietic stem/progenitor cells (HSPCs) has been advanced by the ability to genetically manipulate mice; however, germline modification is time consuming and expensive. Here, we describe fast, efficient, and cost-effective methods to directly modify the genomes of mouse and human HSPCs using the CRISPR/Cas9 system. Using plasmid and virus-free delivery of guide RNAs alone into Cas9-expressing HSPCs or Cas9-guide RNA ribonucleoprotein (RNP) complexes into wild-type cells, we have achieved extremely efficient gene disruption in primary HSPCs from mouse (>60%) and human (â¼75%). These techniques enabled rapid evaluation of the functional effects of gene loss of Eed, Suz12, and DNMT3A. We also achieved homology-directed repair in primary human HSPCs (>20%). These methods will significantly expand applications for CRISPR/Cas9 technologies for studying normal and malignant hematopoiesis.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Células-Tronco Hematopoéticas/metabolismo , Animais , Reparo do DNA , Deleção de Genes , Técnicas de Inativação de Genes , Humanos , Antígenos Comuns de Leucócito/metabolismo , CamundongosRESUMO
Extensive use of targeted PCR-based genotyping is precluded for many plant research laboratories by the cost and time required for DNA extraction. Using cotton (Gossypium hirsutum) as a model for plants with medium-sized seeds, we report here manual procedures for inexpensive non-destructive high-throughput extraction of DNA suitable for PCR-based genotyping of large numbers of individual seeds and seedlings. By sampling only small amounts of cotyledon tissue of ungerminated seed or young seedlings, damage is minimized, and viability is not discernibly affected. The yield of DNA from each seed or seedling is typically sufficient for 1000 or 500 PCR reactions, respectively. For seeds, the tissue sampling procedure relies on a modified 96-well plate that is used subsequently for seed storage. For seeds and seedlings, the DNA is extracted in a strongly basic DNA buffer that is later neutralized and diluted. Extracts can be used directly for high-throughput PCR-based genotyping. Any laboratory can thus extract DNA from thousands of individual seeds/seedlings per person-day at a very modest cost for consumables (~$0.05 per sample). Being non-destructive, our approach enables a wide variety of time- and resource-saving applications, such as marker-assisted selection (MAS), before planting, transplanting, and flowering.