RESUMO
Signaling via the pre-T cell antigen receptor (pre-TCR) and the receptor Notch1 induces transient self-renewal (ß-selection) of TCRß(+) CD4(-)CD8(-) double-negative stage 3 (DN3) and DN4 progenitor cells that differentiate into CD4(+)CD8(+) double-positive (DP) thymocytes, which then rearrange the locus encoding the TCR α-chain (Tcra). Interleukin 7 (IL-7) promotes the survival of TCRß(-) DN thymocytes by inducing expression of the pro-survival molecule Bcl-2, but the functions of IL-7 during ß-selection have remained unclear. Here we found that IL-7 signaled TCRß(+) DN3 and DN4 thymocytes to upregulate genes encoding molecules involved in cell growth and repressed the gene encoding the transcriptional repressor Bcl-6. Accordingly, IL-7-deficient DN4 cells lacked trophic receptors and did not proliferate but rearranged Tcra prematurely and differentiated rapidly. Deletion of Bcl6 partially restored the self-renewal of DN4 cells in the absence of IL-7, but overexpression of BCL2 did not. Thus, IL-7 critically acts cooperatively with signaling via the pre-TCR and Notch1 to coordinate proliferation, differentiation and Tcra recombination during ß-selection.
Assuntos
Interleucina-7/genética , Receptor Notch1/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Timócitos/metabolismo , Animais , Antígenos CD4/genética , Antígenos CD4/imunologia , Antígenos CD8/genética , Antígenos CD8/imunologia , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Regulação da Expressão Gênica , Interleucina-7/deficiência , Interleucina-7/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Proteínas Proto-Oncogênicas c-bcl-6/deficiência , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-bcl-6/imunologia , Receptor Notch1/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Recombinação Genética , Transdução de Sinais , Timócitos/citologia , Timócitos/imunologia , Timo/citologia , Timo/imunologia , Timo/metabolismoRESUMO
Despite extensive analysis of pRB phosphorylation in vitro, how this modification influences development and homeostasis in vivo is unclear. Here, we show that homozygous Rb∆K4 and Rb∆K7 knock-in mice, in which either four or all seven phosphorylation sites in the C-terminal region of pRb, respectively, have been abolished by Ser/Thr-to-Ala substitutions, undergo normal embryogenesis and early development, notwithstanding suppressed phosphorylation of additional upstream sites. Whereas Rb∆K4 mice exhibit telomere attrition but no other abnormalities, Rb∆K7 mice are smaller and display additional hallmarks of premature aging including infertility, kyphosis, and diabetes, indicating an accumulative effect of blocking pRb phosphorylation. Diabetes in Rb∆K7 mice is insulin-sensitive and associated with failure of quiescent pancreatic ß-cells to re-enter the cell cycle in response to mitogens, resulting in induction of DNA damage response (DDR), senescence-associated secretory phenotype (SASP), and reduced pancreatic islet mass and circulating insulin level. Pre-treatment with the epigenetic regulator vitamin C reduces DDR, increases cell cycle re-entry, improves islet morphology, and attenuates diabetes. These results have direct implications for cell cycle regulation, CDK-inhibitor therapeutics, diabetes, and longevity.
Assuntos
Envelhecimento/fisiologia , Ácido Ascórbico/farmacologia , Diabetes Mellitus Experimental/prevenção & controle , Proteína do Retinoblastoma/metabolismo , Animais , Senescência Celular/efeitos dos fármacos , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Fator de Transcrição E2F1/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Fibroblastos/efeitos dos fármacos , Técnicas de Introdução de Genes , Células Secretoras de Insulina/patologia , Camundongos , Fosforilação , Gravidez , Proteína do Retinoblastoma/genética , Telômero/genéticaRESUMO
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
Designing diagnostic assays to genotype rapidly mutating viruses remains a challenge despite the overall improvements in nucleic acid detection technologies. RT-PCR and next-generation sequencing are unsuitable for genotyping during outbreaks or in point-of-care detection due to their infrastructure requirements and longer turnaround times. We developed a quantum dot barcode multiplexing system to genotype mutated viruses. We designed multiple quantum dot barcodes to target conserved, wildtype, and mutated regions of SARS-CoV-2. We calculated ratios of the signal output from different barcodes that enabled SARS-CoV-2 detection and identified SARS-CoV-2 variant strains from a sample. We detected different sequence types, including conserved genes, nucleotide deletions, and single nucleotide substitutions. Our system detected SARS-CoV-2 patient specimens with 98% sensitivity and 94% specificity across 91 patient samples. Further, we leveraged our barcoding and ratio system to track the emergence of the N501Y SARS-CoV-2 mutation from December 2020 to May 2021 and demonstrated that the more transmissible N501Y mutation started to dominate infections by April 2021. Our barcoding and signal ratio approach can genotype viruses and track the emergence of viral mutations in a single diagnostic test. This technology can be extended to tracking other viruses. Combined with smartphone detection technologies, this assay can be adapted for point-of-care tracking of viral mutations in real time.
Assuntos
COVID-19 , Ácidos Nucleicos , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , Genótipo , Nucleotídeos , MutaçãoRESUMO
In acute myeloid leukaemia, long-term survival is poor as most patients relapse despite achieving remission. Historically, the failure of therapy has been thought to be due to mutations that produce drug resistance, possibly arising as a consequence of the mutagenic properties of chemotherapy drugs. However, other lines of evidence have pointed to the pre-existence of drug-resistant cells. For example, deep sequencing of paired diagnosis and relapse acute myeloid leukaemia samples has provided direct evidence that relapse in some cases is generated from minor genetic subclones present at diagnosis that survive chemotherapy, suggesting that resistant cells are generated by evolutionary processes before treatment and are selected by therapy. Nevertheless, the mechanisms of therapy failure and capacity for leukaemic regeneration remain obscure, as sequence analysis alone does not provide insight into the cell types that are fated to drive relapse. Although leukaemia stem cells have been linked to relapse owing to their dormancy and self-renewal properties, and leukaemia stem cell gene expression signatures are highly predictive of therapy failure, experimental studies have been primarily correlative and a role for leukaemia stem cells in acute myeloid leukaemia relapse has not been directly proved. Here, through combined genetic and functional analysis of purified subpopulations and xenografts from paired diagnosis/relapse samples, we identify therapy-resistant cells already present at diagnosis and two major patterns of relapse. In some cases, relapse originated from rare leukaemia stem cells with a haematopoietic stem/progenitor cell phenotype, while in other instances relapse developed from larger subclones of immunophenotypically committed leukaemia cells that retained strong stemness transcriptional signatures. The identification of distinct patterns of relapse should lead to improved methods for disease management and monitoring in acute myeloid leukaemia. Moreover, the shared functional and transcriptional stemness properties that underlie both cellular origins of relapse emphasize the importance of developing new therapeutic approaches that target stemness to prevent relapse.
Assuntos
Linhagem da Célula , Leucemia Mieloide Aguda/patologia , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/patologia , Animais , Células Clonais/metabolismo , Células Clonais/patologia , Feminino , Humanos , Imunofenotipagem , Leucemia Mieloide Aguda/genética , Camundongos , Mutação , Células Progenitoras Mieloides/metabolismo , Células Progenitoras Mieloides/patologia , Recidiva Local de Neoplasia/genética , Células-Tronco Neoplásicas/metabolismoRESUMO
Through a clustered regularly insterspaced short palindromic repeats (CRISPR) screen to identify mitochondrial genes necessary for the growth of acute myeloid leukemia (AML) cells, we identified the mitochondrial outer membrane protein mitochondrial carrier homolog 2 (MTCH2). In AML, knockdown of MTCH2 decreased growth, reduced engraftment potential of stem cells, and induced differentiation. Inhibiting MTCH2 in AML cells increased nuclear pyruvate and pyruvate dehydrogenase (PDH), which induced histone acetylation and subsequently promoted the differentiation of AML cells. Thus, we have defined a new mechanism by which mitochondria and metabolism regulate AML stem cells and gene expression.
Assuntos
Leucemia Mieloide Aguda/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Proteínas de Neoplasias/fisiologia , Acetilação , Animais , Sistemas CRISPR-Cas , Diferenciação Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Sangue Fetal/citologia , Regulação Leucêmica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Histonas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína de Leucina Linfoide-Mieloide/fisiologia , Proteínas de Fusão Oncogênica/fisiologia , Processamento de Proteína Pós-Traducional , Ácido Pirúvico/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologiaRESUMO
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
Stress hormones bind and activate the glucocorticoid receptor (GR) in many tissues including the brain. We identified arginine and glutamate rich 1 (ARGLU1) in a screen for new modulators of glucocorticoid signaling in the CNS. Biochemical studies show that the glutamate rich C-terminus of ARGLU1 coactivates multiple nuclear receptors including the glucocorticoid receptor (GR) and the arginine rich N-terminus interacts with splicing factors and binds to RNA. RNA-seq of neural cells depleted of ARGLU1 revealed significant changes in the expression and alternative splicing of distinct genes involved in neurogenesis. Loss of ARGLU1 is embryonic lethal in mice, and knockdown in zebrafish causes neurodevelopmental and heart defects. Treatment with dexamethasone, a GR activator, also induces changes in the pattern of alternatively spliced genes, many of which were lost when ARGLU1 was absent. Importantly, the genes found to be alternatively spliced in response to glucocorticoid treatment were distinct from those under transcriptional control by GR, suggesting an additional mechanism of glucocorticoid action is present in neural cells. Our results thus show that ARGLU1 is a novel factor for embryonic development that modulates basal transcription and alternative splicing in neural cells with consequences for glucocorticoid signaling.
Assuntos
Desenvolvimento Embrionário , Glucocorticoides/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Splicing de RNA/genética , Ativação Transcricional/genética , Processamento Alternativo/efeitos dos fármacos , Processamento Alternativo/genética , Animais , Animais Geneticamente Modificados , Células Cultivadas , Embrião não Mamífero , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Glucocorticoides/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neurogênese/efeitos dos fármacos , Neurogênese/genética , Splicing de RNA/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Transativadores/fisiologia , Ativação Transcricional/efeitos dos fármacos , Peixe-ZebraRESUMO
Mitochondrial DNA (mtDNA) biosynthesis requires replication factors and adequate nucleotide pools from the mitochondria and cytoplasm. We performed gene expression profiling analysis of 542 human acute myeloid leukemia (AML) samples and identified 55% with upregulated mtDNA biosynthesis pathway expression compared with normal hematopoietic cells. Genes that support mitochondrial nucleotide pools, including mitochondrial nucleotide transporters and a subset of cytoplasmic nucleoside kinases, were also increased in AML compared with normal hematopoietic samples. Knockdown of cytoplasmic nucleoside kinases reduced mtDNA levels in AML cells, demonstrating their contribution in maintaining mtDNA. To assess cytoplasmic nucleoside kinase pathway activity, we used a nucleoside analog 2'3'-dideoxycytidine (ddC), which is phosphorylated to the activated antimetabolite, 2'3'-dideoxycytidine triphosphate by cytoplasmic nucleoside kinases. ddC is a selective inhibitor of the mitochondrial DNA polymerase γ. ddC was preferentially activated in AML cells compared with normal hematopoietic progenitor cells. ddC treatment inhibited mtDNA replication, oxidative phosphorylation, and induced cytotoxicity in a panel of AML cell lines. Furthermore, ddC preferentially inhibited mtDNA replication in a subset of primary human leukemia cells and selectively targeted leukemia cells while sparing normal progenitor cells. In animal models of human AML, treatment with ddC decreased mtDNA, electron transport chain proteins, and induced tumor regression without toxicity. ddC also targeted leukemic stem cells in secondary AML xenotransplantation assays. Thus, AML cells have increased cytidine nucleoside kinase activity that regulates mtDNA biogenesis and can be leveraged to selectively target oxidative phosphorylation in AML.
Assuntos
DNA Mitocondrial/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Fosforilação Oxidativa , Fosfotransferases/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Replicação do DNA , Humanos , Camundongos SCID , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Núcleosídeo-Fosfato Quinase/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Zalcitabina/metabolismoRESUMO
Human Epidermal Growth Factor Receptor 2-positive (HER2(+)) breast cancer (BC) is a highly aggressive disease commonly treated with chemotherapy and anti-HER2 drugs, including trastuzumab. There is currently no way to predict which HER2(+) BC patients will benefit from these treatments. Previous prognostic signatures for HER2(+) BC were developed irrespective of the subtype or the hierarchical organization of cancer in which only a fraction of cells, tumor-initiating cells (TICs), can sustain tumor growth. Here, we used serial dilution and single-cell transplantation assays to identify MMTV-Her2/Neu mouse mammary TICs as CD24(+):JAG1(-) at a frequency of 2-4.5%. A 17-gene Her2-TIC-enriched signature (HTICS), generated on the basis of differentially expressed genes in TIC versus non-TIC fractions and trained on one HER2(+) BC cohort, predicted clinical outcome on multiple independent HER2(+) cohorts. HTICS included up-regulated genes involved in S/G2/M transition and down-regulated genes involved in immune response. Its prognostic power was independent of other predictors, stratified lymph node(+) HER2(+) BC into low and high-risk subgroups, and was specific for HER2(+):estrogen receptor alpha-negative (ERα(-)) patients (10-y overall survival of 83.6% for HTICS(-) and 24.0% for HTICS(+) tumors; hazard ratio = 5.57; P = 0.002). Whereas HTICS was specific to HER2(+):ERα(-) tumors, a previously reported stroma-derived signature was predictive for HER2(+):ERα(+) BC. Retrospective analyses revealed that patients with HTICS(+) HER2(+):ERα(-) tumors resisted chemotherapy but responded to chemotherapy plus trastuzumab. HTICS is, therefore, a powerful prognostic signature for HER2(+):ERα(-) BC that can be used to identify high risk patients that would benefit from anti-HER2 therapy.
Assuntos
Neoplasias da Mama/genética , Receptor alfa de Estrogênio/metabolismo , Perfilação da Expressão Gênica , Genes Neoplásicos/genética , Células-Tronco Neoplásicas/patologia , Receptor ErbB-2/metabolismo , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Antígeno CD24/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-1 , Proteínas de Membrana/metabolismo , Camundongos , Terapia Neoadjuvante , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Prognóstico , Proteínas Serrate-Jagged , Transdução de Sinais/efeitos dos fármacos , Trastuzumab , Resultado do TratamentoRESUMO
Clonal hematopoiesis (CH) arises when a hematopoietic stem cell (HSC) acquires a mutation that confers a competitive advantage over wild-type (WT) HSCs, resulting in its clonal expansion. Individuals with CH are at an increased risk of developing hematologic neoplasms and a range of age-related inflammatory illnesses1-3. Therapeutic interventions that suppress the expansion of mutant HSCs have the potential to prevent these CH-related illnesses; however, such interventions have not yet been identified. The most common CH driver mutations are in the DNA methyltransferase 3 alpha (DNMT3A) gene with arginine 882 (R882) being a mutation hotspot. Here we show that murine hematopoietic stem and progenitor cells (HSPCs) carrying the Dnmt3aR878H/+ mutation, which is equivalent to human DNMT3AR882H/+, have increased mitochondrial respiration compared with WT cells and are dependent on this metabolic reprogramming for their competitive advantage. Importantly, treatment with metformin, an oral anti-diabetic drug with inhibitory activity against complex I in the electron transport chain (ETC), reduced the fitness of Dnmt3aR878H/+ HSCs. Through a multi-omics approach, we discovered that metformin acts by enhancing the methylation potential in Dnmt3aR878H/+ HSPCs and reversing their aberrant DNA CpG methylation and histone H3K27 trimethylation (H3K27me3) profiles. Metformin also reduced the fitness of human DNMT3AR882H HSPCs generated by prime editing. Our findings provide preclinical rationale for investigating metformin as a preventive intervention against illnesses associated with DNMT3AR882 mutation-driven CH in humans.
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The Graffi murine leukemia virus induces a large spectrum of leukemias in mice and thus provides a good model to compare the transcriptome of all types of leukemias. We analyzed the gene expression profiles of both T and B leukemias induced by the virus with DNA microarrays. Given that we considered that a 4-fold change in expression level was significant, 388 probe sets were associated to B, to T, or common to both leukemias. Several of them were not yet associated with lymphoid leukemia. We confirmed specific deregulation of Fmn2, Arntl2, Bfsp2, Gfra2, Gpm6a, and Gpm6b in B leukemia, of Nln, Fbln1, and Bmp7 in T leukemias, and of Etv5 in both leukemias. More importantly, we show that the mouse Fmn2 induced an anchorage-independent growth, a drastic modification in cell shape with a concomitant disruption of the actin cytoskeleton. Interestingly, we found that human FMN2 is overexpressed in approximately 95% of pre-B acute lymphoblastic leukemia with the highest expression levels in patients with a TEL/AML1 rearrangement. These results, surely related to the role of FMN2 in meiotic spindle maintenance, suggest its important role in leukemogenesis. Finally, we propose a new panel of genes potentially involved in T and/or B leukemias.
Assuntos
Vírus da Leucemia Murina/patogenicidade , Leucemia Experimental/genética , Proteínas dos Microfilamentos/genética , Proteínas Nucleares/genética , Oncogenes , Infecções por Retroviridae/genética , Infecções Tumorais por Vírus/genética , Adulto , Animais , Biomarcadores Tumorais/genética , Criança , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Forminas , Perfilação da Expressão Gênica , Marcadores Genéticos , Humanos , Leucemia Experimental/metabolismo , Leucemia de Células T/genética , Camundongos , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Infecções por Retroviridae/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infecções Tumorais por Vírus/metabolismoRESUMO
Many long-lived species of animals require the function of adult stem cells throughout their lives. However, the transcriptomes of stem cells in invertebrates and vertebrates have not been compared, and consequently, ancestral regulatory circuits that control stem cell populations remain poorly defined. In this study, we have used data from high-throughput RNA sequencing to compare the transcriptomes of pluripotent adult stem cells from planarians with the transcriptomes of human and mouse pluripotent embryonic stem cells. From a stringently defined set of 4,432 orthologs shared between planarians, mice and humans, we identified 123 conserved genes that are ≥5-fold differentially expressed in stem cells from all three species. Guided by this gene set, we used RNAi screening in adult planarians to discover novel stem cell regulators, which we found to affect the stem cell-associated functions of tissue homeostasis, regeneration, and stem cell maintenance. Examples of genes that disrupted these processes included the orthologs of TBL3, PSD12, TTC27, and RACK1. From these analyses, we concluded that by comparing stem cell transcriptomes from diverse species, it is possible to uncover conserved factors that function in stem cell biology. These results provide insights into which genes comprised the ancestral circuitry underlying the control of stem cell self-renewal and pluripotency.
Assuntos
Células-Tronco Pluripotentes/fisiologia , Animais , Diferenciação Celular/genética , Perfilação da Expressão Gênica , Humanos , Mamíferos , Camundongos , Planárias , Células-Tronco Pluripotentes/citologiaRESUMO
Transition between activation and quiescence programs in hematopoietic stem and progenitor cells (HSC/HSPCs) is perceived to be governed intrinsically and by microenvironmental co-adaptation. However, HSC programs dictating both transition and adaptability, remain poorly defined. Single cell multiome analysis divulging differential transcriptional activity between distinct HSPC states, indicated for the exclusive absence of Fli-1 motif from quiescent HSCs. We reveal that Fli-1 activity is essential for HSCs during regenerative hematopoiesis. Fli-1 directs activation programs while manipulating cellular sensory and output machineries, enabling HSPCs co-adoptability with a stimulated vascular niche. During regenerative conditions, Fli-1 presets and enables propagation of niche-derived Notch1 signaling. Constitutively induced Notch1 signaling is sufficient to recuperate functional HSC impairments in the absence of Fli-1. Applying FLI-1 modified-mRNA transduction into lethargic adult human mobilized HSPCs, enables their vigorous niche-mediated expansion along with superior engraftment capacities. Thus, decryption of stem cell activation programs offers valuable insights for immune regenerative medicine.
RESUMO
Sequencing of bulk tumor populations has improved genetic classification and risk assessment of B-ALL, but does not directly examine intratumor heterogeneity or infer leukemia cellular origins. We profiled 89 B-ALL samples by single-cell RNA-seq (scRNA-seq) and compared them to a reference map of normal human B-cell development established using both functional and molecular assays. Intra-sample heterogeneity was driven by cell cycle, metabolism, differentiation, and inflammation transcriptional programs. By inference of B lineage developmental state composition, nearly all samples possessed a high abundance of pro-B cells, with variation between samples mainly driven by sub-populations. However, ZNF384- r and DUX4- r B-ALL showed composition enrichment of hematopoietic stem cells, BCR::ABL1 and KMT2A -r ALL of Early Lymphoid progenitors, MEF2D -r and TCF3::PBX1 of Pre-B cells. Enrichment of Early Lymphoid progenitors correlated with high-risk clinical features. Understanding variation in transcriptional programs and developmental states of B-ALL by scRNA-seq refines existing clinical and genomic classifications and improves prediction of treatment outcome.
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Central nervous system (CNS) dissemination of B-precursor acute lymphoblastic leukemia (B-ALL) has poor prognosis and remains a therapeutic challenge. Here we performed targeted DNA sequencing as well as transcriptional and proteomic profiling of paired leukemia-infiltrating cells in the bone marrow (BM) and CNS of xenografts. Genes governing mRNA translation were upregulated in CNS leukemia, and subclonal genetic profiling confirmed this in both BM-concordant and BM-discordant CNS mutational populations. CNS leukemia cells were exquisitely sensitive to the translation inhibitor omacetaxine mepesuccinate, which reduced xenograft leptomeningeal disease burden. Proteomics demonstrated greater abundance of secreted proteins in CNS-infiltrating cells, including complement component 3 (C3), and drug targeting of C3 influenced CNS disease in xenografts. CNS-infiltrating cells also exhibited selection for stemness traits and metabolic reprogramming. Overall, our study identifies targeting of mRNA translation as a potential therapeutic approach for B-ALL leptomeningeal disease. SIGNIFICANCE: Cancer metastases are often driven by distinct subclones with unique biological properties. Here we show that in B-ALL CNS disease, the leptomeningeal environment selects for cells with unique functional dependencies. Pharmacologic inhibition of mRNA translation signaling treats CNS disease and offers a new therapeutic approach for this condition.This article is highlighted in the In This Issue feature, p. 1.
Assuntos
Doenças do Sistema Nervoso Central , Neoplasias do Sistema Nervoso Central , Neoplasias Meníngeas , Leucemia-Linfoma Linfoblástico de Células Precursoras , Sistema Nervoso Central/metabolismo , Doenças do Sistema Nervoso Central/patologia , Neoplasias do Sistema Nervoso Central/tratamento farmacológico , Humanos , Neoplasias Meníngeas/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Biossíntese de Proteínas/genética , ProteômicaRESUMO
The treatment landscape of acute myeloid leukemia (AML) is evolving, with promising therapies entering clinical translation, yet patient responses remain heterogeneous, and biomarkers for tailoring treatment are lacking. To understand how disease heterogeneity links with therapy response, we determined the leukemia cell hierarchy makeup from bulk transcriptomes of more than 1,000 patients through deconvolution using single-cell reference profiles of leukemia stem, progenitor and mature cell types. Leukemia hierarchy composition was associated with functional, genomic and clinical properties and converged into four overall classes, spanning Primitive, Mature, GMP and Intermediate. Critically, variation in hierarchy composition along the Primitive versus GMP or Primitive versus Mature axes were associated with response to chemotherapy or drug sensitivity profiles of targeted therapies, respectively. A seven-gene biomarker derived from the Primitive versus Mature axis was associated with response to 105 investigational drugs. Cellular hierarchy composition constitutes a novel framework for understanding disease biology and advancing precision medicine in AML.
Assuntos
Leucemia Mieloide Aguda , Biomarcadores , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismoRESUMO
AML cells are arranged in a hierarchy with stem/progenitor cells giving rise to more differentiated bulk cells. Despite the importance of stem/progenitors in the pathogenesis of AML, the determinants of the AML stem/progenitor state are not fully understood. Through a comparison of genes that are significant for growth and viability of AML cells by way of a CRISPR screen, with genes that are differentially expressed in leukemia stem cells (LSC), we identified importin 11 (IPO11) as a novel target in AML. Importin 11 (IPO11) is a member of the importin ß family of proteins that mediate transport of proteins across the nuclear membrane. In AML, knockdown of IPO11 decreased growth, reduced engraftment potential of LSC, and induced differentiation. Mechanistically, we identified the transcription factors BZW1 and BZW2 as novel cargo of IPO11. We further show that BZW1/2 mediate a transcriptional signature that promotes stemness and survival of LSC. Thus, we demonstrate for the first time how specific cytoplasmic-nuclear regulation supports stem-like transcriptional signature in relapsed AML.
Assuntos
Leucemia Mieloide Aguda , beta Carioferinas , Transporte Ativo do Núcleo Celular , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/patologia , Células-Tronco/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMO
Mitochondrial metabolites regulate leukaemic and normal stem cells by affecting epigenetic marks. How mitochondrial enzymes localize to the nucleus to control stem cell function is less understood. We discovered that the mitochondrial metabolic enzyme hexokinase 2 (HK2) localizes to the nucleus in leukaemic and normal haematopoietic stem cells. Overexpression of nuclear HK2 increases leukaemic stem cell properties and decreases differentiation, whereas selective nuclear HK2 knockdown promotes differentiation and decreases stem cell function. Nuclear HK2 localization is phosphorylation-dependent, requires active import and export, and regulates differentiation independently of its enzymatic activity. HK2 interacts with nuclear proteins regulating chromatin openness, increasing chromatin accessibilities at leukaemic stem cell-positive signature and DNA-repair sites. Nuclear HK2 overexpression decreases double-strand breaks and confers chemoresistance, which may contribute to the mechanism by which leukaemic stem cells resist DNA-damaging agents. Thus, we describe a non-canonical mechanism by which mitochondrial enzymes influence stem cell function independently of their metabolic function.
Assuntos
Hexoquinase , Leucemia Mieloide Aguda , Cromatina/metabolismo , DNA/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Hexoquinase/genética , Hexoquinase/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismoRESUMO
Gene expression profiling and proteome analysis of normal and malignant hematopoietic stem cells (HSCs) point to shared core stemness properties. However, discordance between mRNA and protein signatures highlights an important role for post-transcriptional regulation by microRNAs (miRNAs) in governing this critical nexus. Here, we identify miR-130a as a regulator of HSC self-renewal and differentiation. Enforced expression of miR-130a impairs B lymphoid differentiation and expands long-term HSCs. Integration of protein mass spectrometry and chimeric AGO2 crosslinking and immunoprecipitation (CLIP) identifies TBL1XR1 as a primary miR-130a target, whose loss of function phenocopies miR-130a overexpression. Moreover, we report that miR-130a is highly expressed in t(8;21) acute myeloid leukemia (AML), where it is critical for maintaining the oncogenic molecular program mediated by the AML1-ETO complex. Our study establishes that identification of the comprehensive miRNA targetome within primary cells enables discovery of genes and molecular networks underpinning stemness properties of normal and leukemic cells.