RESUMEN
Over the years, the overall survival of older patients diagnosed with acute myeloid leukemia (AML) has not significantly increased. Although standard cytotoxic therapies that rapidly eliminate dividing myeloblasts are used to induce remission, relapse can occur due to surviving therapy-resistant leukemic stem cells (LSCs). Hence, anti-LSC strategies have become a key target to cure AML. We have recently shown that previously approved cardiac glycosides and glucocorticoids target LSC-enriched CD34+ cells in the primary human AML 8227 model with more efficacy than normal hematopoietic stem cells (HSCs). To translate these in vitro findings into humans, we developed a mathematical model of stem cell dynamics that describes the stochastic evolution of LSCs in AML post-standard-of-care. To this, we integrated population pharmacokinetic-pharmacodynamic (PKPD) models to investigate the clonal reduction potential of several promising candidate drugs in comparison to cytarabine, which is commonly used in high doses for consolidation therapy in AML patients. Our results suggest that cardiac glycosides (proscillaridin A, digoxin and ouabain) and glucocorticoids (budesonide and mometasone) reduce the expansion of LSCs through a decrease in their viability. While our model predicts that effective doses of cardiac glycosides are potentially too toxic to use in patients, simulations show the possibility of mometasone to prevent relapse through the glucocorticoid's ability to drastically reduce LSC population size. This work therefore highlights the prospect of these treatments for anti-LSC strategies and underlines the use of quantitative approaches to preclinical drug translation in AML.
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Leucemia Mieloide Aguda , Células Madre Neoplásicas , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Modelos Teóricos , Citarabina/uso terapéutico , Citarabina/farmacologíaRESUMEN
The leukemia stem cell (LSC) populations of acute myeloid leukemia (AML) exhibit phenotypic, genetic, and functional heterogeneity that contribute to therapy failure and relapse. Progress toward understanding the mechanistic basis for therapy resistance in LSCs has been hampered by difficulties in isolating cell fractions that enrich for the entire heterogeneous population of LSCs within individual AML samples. We previously reported that CD200 gene expression is upregulated in LSC-containing AML fractions. Here, we show that CD200 is present on a greater proportion of CD45dim blasts compared with more differentiated CD45high cells in AML patient samples. In 75% (49 of 65) of AML cases we examined, CD200 was expressed on ≥10% of CD45dim blasts; of these, CD200 identified LSCs within the blast population in 9 of 10 (90%) samples tested in xenotransplantation assays. CD200+ LSCs could be isolated from CD200+ normal HSCs with the use of additional markers. Notably, CD200 expression captured both CD34- and CD34+ LSCs within individual AML samples. Analysis of highly purified CD200+ LSC-containing fractions from NPM1-mutated AMLs, which are commonly CD34-, exhibited an enrichment of primitive gene expression signatures compared with unfractionated cells. Overall, our findings support CD200 as a novel LSC marker that is able to capture the entire LSC compartment from AML patient samples, including those with NPM1 mutation.
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Leucemia Mieloide Aguda , Células Madre Neoplásicas , Biomarcadores , Diferenciación Celular , Humanos , Leucemia Mieloide Aguda/genética , NucleofosminaRESUMEN
PURPOSE: Glioblastoma (GBM) is a fatal primary malignant brain tumor. GBM stem cells (GSC) contribute to resistance to the DNA-damaging chemotherapy, temozolomide. The epidermal growth factor receptor (EGFR) displays genomic alterations enabling DNA repair mechanisms in half of GBMs. We aimed to investigate EGFR/DNA combi-targeting in GBM. EXPERIMENTAL DESIGN: ZR2002 is a "combi-molecule" designed to inflict DNA damage through its chlorethyl moiety and induce irreversible EGFR tyrosine kinase inhibition. We assessed its in vitro efficacy in temozolomide-resistant patient-derived GSCs, mesenchymal temozolomide-sensitive and resistant in vivo-derived GSC sublines, and U87/EGFR isogenic cell lines stably expressing EGFR/wild-type or variant III (EGFRvIII). We evaluated its antitumor activity in mice harboring orthotopic EGFRvIII or mesenchymal TMZ-resistant GSC tumors. RESULTS: ZR2002 induced submicromolar antiproliferative effects and inhibited neurosphere formation of all GSCs with marginal effects on normal human astrocytes. ZR2002 inhibited EGF-induced autophosphorylation of EGFR, downstream Erk1/2 phosphorylation, increased DNA strand breaks, and induced activation of wild-type p53; the latter was required for its cytotoxicity through p53-dependent mechanism. ZR2002 induced similar effects on U87/EGFR cell lines and its oral administration significantly increased survival in an orthotopic EGFRvIII mouse model. ZR2002 improved survival of mice harboring intracranial mesenchymal temozolomide-resistant GSC line, decreased EGFR, Erk1/2, and AKT phosphorylation and was detected in tumor brain tissue by MALDI imaging mass spectrometry. CONCLUSIONS: These findings provide the molecular basis of binary EGFR/DNA targeting and uncover the oral bioavailability, blood-brain barrier permeability, and antitumor activity of ZR2002 supporting potential evaluation of this first-in-class drug in recurrent GBM.
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Neoplasias Encefálicas/tratamiento farmacológico , Daño del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Glioblastoma/tratamiento farmacológico , Quinazolinas/farmacología , Temozolomida/farmacología , Animales , Antineoplásicos Alquilantes/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/fisiología , Neoplasias Encefálicas/mortalidad , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Receptores ErbB/antagonistas & inhibidores , Femenino , Glioblastoma/mortalidad , Glioblastoma/patología , Humanos , Ratones , Ratones Desnudos , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Cardiac glycosides are approved for the treatment of heart failure as Na+/K+ pump inhibitors. Their repurposing in oncology is currently investigated in preclinical and clinical studies. However, the identification of a specific cancer type defined by a molecular signature to design targeted clinical trials with cardiac glycosides remains to be characterized. Here, we demonstrate that cardiac glycoside proscillaridin A specifically targets MYC overexpressing leukemia cells and leukemia stem cells by causing MYC degradation, epigenetic reprogramming and leukemia differentiation through loss of lysine acetylation. METHODS: Proscillaridin A anticancer activity was investigated against a panel of human leukemia and solid tumor cell lines with different MYC expression levels, overexpression in vitro systems and leukemia stem cells. RNA-sequencing and differentiation studies were used to characterize transcriptional and phenotypic changes. Drug-induced epigenetic changes were studied by chromatin post-translational modification analysis, expression of chromatin regulators, chromatin immunoprecipitation, and mass-spectrometry. RESULTS: At a clinically relevant dose, proscillaridin A rapidly altered MYC protein half-life causing MYC degradation and growth inhibition. Transcriptomic profile of leukemic cells after treatment showed a downregulation of genes involved in MYC pathways, cell replication and an upregulation of hematopoietic differentiation genes. Functional studies confirmed cell cycle inhibition and the onset of leukemia differentiation even after drug removal. Proscillaridin A induced a significant loss of lysine acetylation in histone H3 (at lysine 9, 14, 18 and 27) and in non-histone proteins such as MYC itself, MYC target proteins, and a series of histone acetylation regulators. Global loss of acetylation correlated with the rapid downregulation of histone acetyltransferases. Importantly, proscillaridin A demonstrated anticancer activity against lymphoid and myeloid stem cell populations characterized by MYC overexpression. CONCLUSION: Overall, these results strongly support the repurposing of proscillaridin A in MYC overexpressing leukemia.
Asunto(s)
Antineoplásicos/efectos adversos , Expresión Génica/efectos de los fármacos , Genes myc , Insuficiencia Cardíaca/etiología , Leucemia/genética , Lisina/metabolismo , Proscilaridina/efectos adversos , Acetilación , Antineoplásicos/uso terapéutico , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cromatina/genética , Cromatina/metabolismo , Relación Dosis-Respuesta a Droga , Epigénesis Genética/efectos de los fármacos , Perfilación de la Expresión Génica , Histonas/metabolismo , Humanos , Leucemia/complicaciones , Leucemia/tratamiento farmacológico , Leucemia/metabolismo , Modelos Biológicos , Proscilaridina/uso terapéutico , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
Our ability to manage acute myeloid leukemia (AML) is limited by our incomplete understanding of the epigenetic disruption central to leukemogenesis, including improper histone methylation. Here we examine 16 histone H3 genes in 434 primary AML samples and identify Q69H, A26P, R2Q, R8H and K27M/I mutations (1.6%), with higher incidence in secondary AML (9%). These mutations occur in pre-leukemic hematopoietic stem cells (HSCs) and exist in the major leukemic clones in patients. They increase the frequency of functional HSCs, alter differentiation, and amplify leukemic aggressiveness. These effects are dependent on the specific mutation. H3K27 mutation increases the expression of genes involved in erythrocyte and myeloid differentiation with altered H3K27 tri-methylation and K27 acetylation. The functional impact of histone mutations is independent of RUNX1 mutation, although they at times co-occur. This study establishes that H3 mutations are drivers of human pre-cancerous stem cell expansion and important early events in leukemogenesis.
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Epigenómica , Regulación Leucémica de la Expresión Génica/fisiología , Histonas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Animales , Animales Modificados Genéticamente , Antineoplásicos/farmacología , Secuencia de Bases , Células de la Médula Ósea , Diferenciación Celular , Transformación Celular Neoplásica , ADN/genética , Drosophila melanogaster/genética , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Mutación , Neoplasias ExperimentalesRESUMEN
Therapy for acute myeloid leukemia (AML) involves intense cytotoxic treatment and yet approximately 70% of AML are refractory to initial therapy or eventually relapse. This is at least partially driven by the chemo-resistant nature of the leukemic stem cells (LSCs) that sustain the disease, and therefore novel anti-LSC therapies could decrease relapses and improve survival. We performed in silico analysis of highly prognostic human AML LSC gene expression signatures using existing datasets of drug-gene interactions to identify compounds predicted to target LSC gene programs. Filtering against compounds that would inhibit a hematopoietic stem cell (HSC) gene signature resulted in a list of 151 anti-LSC candidates. Using a novel in vitro LSC assay, we screened 84 candidate compounds at multiple doses and confirmed 14 drugs that effectively eliminate human AML LSCs. Three drug families presenting with multiple hits, namely antihistamines (astemizole and terfenadine), cardiac glycosides (strophanthidin, digoxin and ouabain) and glucocorticoids (budesonide, halcinonide and mometasone), were validated for their activity against human primary AML samples. Our study demonstrates the efficacy of combining computational analysis of stem cell gene expression signatures with in vitro screening to identify novel compounds that target the therapy-resistant LSC at the root of relapse in AML.
Asunto(s)
Biomarcadores de Tumor , Leucemia Mieloide Aguda/etiología , Leucemia Mieloide Aguda/metabolismo , Células Madre Neoplásicas/metabolismo , Apoptosis/genética , Biomarcadores , Ciclo Celular/genética , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Biología Computacional/métodos , Citarabina/farmacología , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Perfilación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamiento farmacológico , Terapia Molecular Dirigida , Células Madre Neoplásicas/efectos de los fármacos , TranscriptomaRESUMEN
Quiescent and proliferating leukemia cells accumulate highly lethal DNA double-strand breaks that are repaired by 2 major mechanisms: BRCA-dependent homologous recombination and DNA-dependent protein kinase-mediated (DNA-PK-mediated) nonhomologous end-joining, whereas DNA repair pathways mediated by poly(ADP)ribose polymerase 1 (PARP1) serve as backups. Here we have designed a personalized medicine approach called gene expression and mutation analysis (GEMA) to identify BRCA- and DNA-PK-deficient leukemias either directly, using reverse transcription-quantitative PCR, microarrays, and flow cytometry, or indirectly, by the presence of oncogenes such as BCR-ABL1. DNA-PK-deficient quiescent leukemia cells and BRCA/DNA-PK-deficient proliferating leukemia cells were sensitive to PARP1 inhibitors that were administered alone or in combination with current antileukemic drugs. In conclusion, GEMA-guided targeting of PARP1 resulted in dual cellular synthetic lethality in quiescent and proliferating immature leukemia cells, and is thus a potential approach to eradicate leukemia stem and progenitor cells that are responsible for initiation and manifestation of the disease. Further, an analysis of The Cancer Genome Atlas database indicated that this personalized medicine approach could also be applied to treat numerous solid tumors from individual patients.
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Antineoplásicos/farmacología , Proliferación Celular , Leucemia/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica , Cricetinae , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Genes BRCA1 , Genes BRCA2 , Genes Letales , Genes abl , Humanos , Leucemia/tratamiento farmacológico , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Células Madre Embrionarias de Ratones/fisiología , Ftalazinas/farmacología , Piperazinas/farmacología , Transcriptoma , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Extracellular vesicles (EVs) enable the exit of regulatory, mutant and oncogenic macromolecules (proteins, RNA and DNA) from their parental tumor cells and uptake of this material by unrelated cellular populations. Among the resulting biological effects of interest is the notion that cancer-derived EVs may mediate horizontal transformation of normal cells through transfer of mutant genes, including mutant ras. Here, we report that H-ras-mediated transformation of intestinal epithelial cells (IEC-18) results in the emission of exosome-like EVs containing genomic DNA, HRAS oncoprotein and transcript. However, EV-mediated horizontal transformation of non-transformed cells (epithelial, astrocytic, fibroblastic and endothelial) is transient, limited or absent due to barrier mechanisms that curtail the uptake, retention and function of oncogenic H-ras in recipient cells. Thus, epithelial cells and astrocytes are resistant to EV uptake, unless they undergo malignant transformation. In contrast, primary and immortalized fibroblasts are susceptible to the EV uptake, retention of H-ras DNA and phenotypic transformation, but these effects are transient and fail to produce a permanent tumorigenic conversion of these cells in vitro and in vivo, even after several months of observation. Increased exposure to EVs isolated from H-ras-transformed cancer cells, but not to those from their indolent counterparts, triggers demise of recipient fibroblasts. Uptake of H-ras-containing EVs stimulates but fails to transform primary endothelial cells. Thus, we suggest that intercellular transfer of oncogenes exerts regulatory rather than transforming influence on recipient cells, while cancer cells may often act as preferential EV recipients.
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Comunicación Celular/fisiología , Transformación Celular Neoplásica/genética , Vesículas Extracelulares/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica/patología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Vesículas Extracelulares/genética , Vesículas Extracelulares/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Xenoinjertos , Humanos , Ratones , Ratones SCID , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
The involvement of the complement pathway in cancer is supported by a growing body of evidence, and yet its role in acute myeloid leukemia (AML) has not been extensively studied. We examined the expression of 87 genes in the complement, coagulation, and fibrinolysis-proteolytic pathways in 374 cytogenetically normal AML samples and observed that these samples can be divided into subgroups on the basis of complement gene expression. Three complement regulatory genes were linked to poor outcome as individual factors in a multivariate analysis (CFH, CFD, and SERPING1) in multiple cohorts. The combined expression of these genes was significantly associated with poorer overall survival in two cohorts of patients <60 years of age, independent of other factors (p ≤ 0.0004). For patients with an intermediate molecular risk, this three-gene risk marker enabled stratification of patients into prognostic subgroups with survival ranging from 17.4% to 44.1%. Thus, the expression of complement pathway genes is linked to outcome in AML, and a three-gene risk marker may improve the risk assessment of patients.
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Expresión Génica , Redes Reguladoras de Genes , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidad , Adolescente , Adulto , Análisis por Conglomerados , Estudios de Cohortes , Bases de Datos de Ácidos Nucleicos , Femenino , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Cariotipificación , Leucemia Mieloide Aguda/diagnóstico , Masculino , Persona de Mediana Edad , Pronóstico , Transcriptoma , Adulto JovenRESUMEN
Acute myeloid leukemia (AML) is a genetically heterogeneous hematologic malignancy, which is initiated and driven by a rare fraction of leukemia stem cells (LSCs). Despite the difficulties of identifying a common LSC phenotype, there is increasing evidence that high expression of stem cell gene signatures is associated with poor clinical outcome. Identification of functionally distinct subpopulations in this disease is therefore crucial to dissecting the molecular machinery underlying LSC self-renewal. Here, we combined next-generation sequencing technology with in vivo assessment of LSC frequencies and identified the adhesion G protein-coupled receptor 56 (GPR56) as a novel and stable marker for human LSCs for the majority of AML samples. High GPR56 expression was significantly associated with high-risk genetic subgroups and poor outcome. Analysis of GPR56 in combination with CD34 expression revealed engraftment potential of GPR56(+)cells in both the CD34(-)and CD34(+)fractions, thus defining a novel LSC compartment independent of the CD34(+)CD38(-)LSC phenotype.
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Biomarcadores de Tumor/metabolismo , Proliferación Celular , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/patología , Receptores Acoplados a Proteínas G/metabolismo , Adulto , Animales , Separación Celular , Células Cultivadas , Células HEK293 , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidad , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Células Madre Neoplásicas/fisiología , Receptores Acoplados a Proteínas G/fisiología , Análisis de SupervivenciaRESUMEN
To investigate miRNA function in human acute myeloid leukemia (AML) stem cells (LSC), we generated a prognostic LSC-associated miRNA signature derived from functionally validated subpopulations of AML samples. For one signature miRNA, miR-126, high bioactivity aggregated all in vivo patient sample LSC activity into a single sorted population, tightly coupling miR-126 expression to LSC function. Through functional studies, miR-126 was found to restrain cell cycle progression, prevent differentiation, and increase self-renewal of primary LSC in vivo. Compared with prior results showing miR-126 regulation of normal hematopoietic stem cell (HSC) cycling, these functional stem effects are opposite between LSC and HSC. Combined transcriptome and proteome analysis demonstrates that miR-126 targets the PI3K/AKT/MTOR signaling pathway, preserving LSC quiescence and promoting chemotherapy resistance.
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Células Madre Hematopoyéticas/patología , Leucemia Mieloide Aguda/patología , MicroARNs/fisiología , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Ratones SCID , MicroARNs/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Pronóstico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The blood system is sustained by a pool of haematopoietic stem cells (HSCs) that are long-lived due to their capacity for self-renewal. A consequence of longevity is exposure to stress stimuli including reactive oxygen species (ROS), nutrient fluctuation and DNA damage. Damage that occurs within stressed HSCs must be tightly controlled to prevent either loss of function or the clonal persistence of oncogenic mutations that increase the risk of leukaemogenesis. Despite the importance of maintaining cell integrity throughout life, how the HSC pool achieves this and how individual HSCs respond to stress remain poorly understood. Many sources of stress cause misfolded protein accumulation in the endoplasmic reticulum (ER), and subsequent activation of the unfolded protein response (UPR) enables the cell to either resolve stress or initiate apoptosis. Here we show that human HSCs are predisposed to apoptosis through strong activation of the PERK branch of the UPR after ER stress, whereas closely related progenitors exhibit an adaptive response leading to their survival. Enhanced ER protein folding by overexpression of the co-chaperone ERDJ4 (also called DNAJB9) increases HSC repopulation capacity in xenograft assays, linking the UPR to HSC function. Because the UPR is a focal point where different sources of stress converge, our study provides a framework for understanding how stress signalling is coordinated within tissue hierarchies and integrated with stemness. Broadly, these findings reveal that the HSC pool maintains clonal integrity by clearance of individual HSCs after stress to prevent propagation of damaged stem cells.
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Estrés del Retículo Endoplásmico , Células Madre Hematopoyéticas/citología , Respuesta de Proteína Desplegada/fisiología , Factor de Transcripción Activador 4/metabolismo , Animales , Apoptosis/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Factor 2 Eucariótico de Iniciación/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Células Madre Hematopoyéticas/efectos de los fármacos , Xenoinjertos , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Chaperonas Moleculares/metabolismo , Pliegue de Proteína , Proteína Fosfatasa 1/metabolismo , Transducción de Señal , Factor de Transcripción CHOP/metabolismo , Tunicamicina/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacos , eIF-2 Quinasa/metabolismoRESUMEN
The hematopoietic system sustains regeneration throughout life by balancing self-renewal and differentiation. To stay poised for mature blood production, hematopoietic stem cells (HSCs) maintain low-level expression of lineage-associated genes, a process termed lineage priming. Here, we modulated expression levels of Inhibitor of DNA binding (ID) proteins to ask whether lineage priming affects self-renewal of human HSCs. We found that lentiviral overexpression of ID proteins in cord blood HSCs biases myeloerythroid commitment at the expense of lymphoid differentiation. Conversely, reducing ID2 expression levels increases lymphoid potential. Mechanistically, ID2 inhibits the transcription factor E47 to attenuate B-lymphoid priming in HSCs and progenitors. Strikingly, ID2 overexpression also results in a 10-fold expansion of HSCs in serial limiting dilution assays, indicating that early lymphoid transcription factors antagonize human HSC self-renewal. The relationship between lineage priming and self-renewal can be exploited to increase expansion of transplantable human HSCs and points to broader implications for other stem cell populations.
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Diferenciación Celular , Linaje de la Célula , Células Madre Hematopoyéticas/citología , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Linfocitos/citología , Animales , Biomarcadores/metabolismo , Western Blotting , Proliferación Celular , Células Cultivadas , Sangre Fetal/citología , Sangre Fetal/metabolismo , Perfilación de la Expresión Génica , Humanos , Proteína 2 Inhibidora de la Diferenciación/genética , Linfocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Mieloides/citología , Células Mieloides/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 , Factor de Transcripción 3/genética , Factor de Transcripción 3/metabolismo , Trasplante HeterólogoRESUMEN
Despite efforts to understand and treat acute myeloid leukemia (AML), there remains a need for more comprehensive therapies to prevent AML-associated relapses. To identify new therapeutic strategies for AML, we screened a library of on- and off-patent drugs and identified the antimalarial agent mefloquine as a compound that selectively kills AML cells and AML stem cells in a panel of leukemia cell lines and in mice. Using a yeast genome-wide functional screen for mefloquine sensitizers, we identified genes associated with the yeast vacuole, the homolog of the mammalian lysosome. Consistent with this, we determined that mefloquine disrupts lysosomes, directly permeabilizes the lysosome membrane, and releases cathepsins into the cytosol. Knockdown of the lysosomal membrane proteins LAMP1 and LAMP2 resulted in decreased cell viability, as did treatment of AML cells with known lysosome disrupters. Highlighting a potential therapeutic rationale for this strategy, leukemic cells had significantly larger lysosomes compared with normal cells, and leukemia-initiating cells overexpressed lysosomal biogenesis genes. These results demonstrate that lysosomal disruption preferentially targets AML cells and AML progenitor cells, providing a rationale for testing lysosomal disruption as a novel therapeutic strategy for AML.
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Membranas Intracelulares/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Lisosomas/metabolismo , Células Madre Neoplásicas/metabolismo , Animales , Antimaláricos/farmacocinética , Antimaláricos/farmacología , Supervivencia Celular/efectos de los fármacos , Femenino , Técnicas de Silenciamiento del Gen , Estudio de Asociación del Genoma Completo , Humanos , Membranas Intracelulares/patología , Células K562 , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Proteína 2 de la Membrana Asociada a los Lisosomas , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/genética , Lisosomas/fisiología , Masculino , Mefloquina/farmacocinética , Mefloquina/farmacología , Ratones , Células Madre Neoplásicas/patología , Permeabilidad/efectos de los fármacos , Saccharomyces cerevisiae/genéticaRESUMEN
Early T-cell precursor acute lymphoblastic leukaemia (ETP ALL) is an aggressive malignancy of unknown genetic basis. We performed whole-genome sequencing of 12 ETP ALL cases and assessed the frequency of the identified somatic mutations in 94 T-cell acute lymphoblastic leukaemia cases. ETP ALL was characterized by activating mutations in genes regulating cytokine receptor and RAS signalling (67% of cases; NRAS, KRAS, FLT3, IL7R, JAK3, JAK1, SH2B3 and BRAF), inactivating lesions disrupting haematopoietic development (58%; GATA3, ETV6, RUNX1, IKZF1 and EP300) and histone-modifying genes (48%; EZH2, EED, SUZ12, SETD2 and EP300). We also identified new targets of recurrent mutation including DNM2, ECT2L and RELN. The mutational spectrum is similar to myeloid tumours, and moreover, the global transcriptional profile of ETP ALL was similar to that of normal and myeloid leukaemia haematopoietic stem cells. These findings suggest that addition of myeloid-directed therapies might improve the poor outcome of ETP ALL.
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Predisposición Genética a la Enfermedad/genética , Mutación/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Edad de Inicio , Niño , Variaciones en el Número de Copia de ADN/genética , Genes ras/genética , Genoma Humano/genética , Genómica , Hematopoyesis/genética , Histonas/metabolismo , Humanos , Quinasas Janus/genética , Quinasas Janus/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Datos de Secuencia Molecular , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Receptores de Interleucina-7/genética , Proteína Reelina , Análisis de Secuencia de ADN , Transducción de Señal/genética , Células Madre/metabolismo , Células Madre/patología , Linfocitos T/metabolismo , Linfocitos T/patología , Translocación Genética/genéticaRESUMEN
Gene regulatory networks that govern hematopoietic stem cells (HSCs) and leukemia-initiating cells (L-ICs) are deeply entangled. Thus, the discovery of compounds that target L-ICs while sparing HSC is an attractive but difficult endeavor. Presently, most screening approaches fail to counter-screen compounds against normal hematopoietic stem/progenitor cells (HSPCs). Here, we present a multistep in vitro and in vivo approach to identify compounds that can target L-ICs in acute myeloid leukemia (AML). A high-throughput screen of 4000 compounds on novel leukemia cell lines derived from human experimental leukemogenesis models yielded 80 hits, of which 10 were less toxic to HSPC. We characterized a single compound, kinetin riboside (KR), on AML L-ICs and HSPCs. KR demonstrated comparable efficacy to standard therapies against blast cells in 63 primary leukemias. In vitro, KR targeted the L-IC-enriched CD34(+)CD38(-) AML fraction, while sparing HSPC-enriched fractions, although these effects were mitigated on HSC assayed in vivo. KR eliminated L-ICs in 2 of 4 primary AML samples when assayed in vivo and highlights the importance of in vivo L-IC and HSC assays to measure function. Overall, we provide a novel approach to screen large drug libraries for the discovery of anti-L-IC compounds for human leukemias.
Asunto(s)
Adenosina/uso terapéutico , Ensayos Analíticos de Alto Rendimiento/métodos , Cinetina/uso terapéutico , Leucemia/tratamiento farmacológico , Leucemia/patología , Células Madre Neoplásicas/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/análisis , Adenosina/análisis , Adenosina/aislamiento & purificación , Adenosina/farmacología , Animales , Antineoplásicos/análisis , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Humanos , Cinetina/análisis , Cinetina/aislamiento & purificación , Cinetina/farmacología , Ratones , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Células Madre Neoplásicas/patología , Resultado del Tratamiento , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Xenograft studies indicate that some solid tumors and leukemias are organized as cellular hierarchies sustained by cancer stem cells (CSCs). Despite the promise of the CSC model, its relevance in humans remains uncertain. Here we show that acute myeloid leukemia (AML) follows a CSC model on the basis of sorting multiple populations from each of 16 primary human AML samples and identifying which contain leukemia stem cells (LSCs) using a sensitive xenograft assay. Analysis of gene expression from all functionally validated populations yielded an LSC-specific signature. Similarly, a hematopoietic stem cell (HSC) gene signature was established. Bioinformatic analysis identified a core transcriptional program shared by LSCs and HSCs, revealing the molecular machinery underlying 'stemness' properties. Both stem cell programs were highly significant independent predictors of patient survival and were found in existing prognostic signatures. Thus, determinants of stemness influence the clinical outcome of AML, establishing that LSCs are clinically relevant and not artifacts of xenotransplantation.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Leucemia Mieloide Aguda/fisiopatología , Modelos Biológicos , Células Madre Neoplásicas/metabolismo , Animales , Ensayo de Unidades Formadoras de Colonias , Biología Computacional , Citometría de Flujo , Humanos , Leucemia Mieloide Aguda/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Highly regenerative tissues such as blood must possess effective DNA damage responses (DDR) that balance long-term regeneration with protection from leukemogenesis. Hematopoietic stem cells (HSCs) sustain life-long blood production, yet their response to DNA damage remains largely unexplored. We report that human HSCs exhibit delayed DNA double-strand break rejoining, persistent gammaH2AX foci, and enhanced p53- and ASPP1-dependent apoptosis after gamma-radiation compared to progenitors. p53 inactivation or Bcl-2 overexpression reduced radiation-induced apoptosis and preserved in vivo repopulating HSC function. Despite similar protection from irradiation-induced apoptosis, only Bcl-2-overexpressing HSCs showed higher self-renewal capacity, establishing that intact p53 positively regulates self-renewal independently from apoptosis. The reduced self-renewal of HSCs with inactivated p53 was associated with increased spontaneous gammaH2AX foci in secondary transplants of HSCs. Our data reveal distinct physiological roles of p53 that together ensure optimal HSC function: apoptosis regulation and prevention of gammaH2AX foci accumulation upon HSC self-renewal.
Asunto(s)
Apoptosis , Daño del ADN , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Apoptosis/efectos de la radiación , Proteínas Reguladoras de la Apoptosis/metabolismo , Linaje de la Célula/efectos de la radiación , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Citoprotección/efectos de la radiación , Roturas del ADN de Doble Cadena/efectos de la radiación , Células HeLa , Células Madre Hematopoyéticas/efectos de la radiación , Humanos , Ratones , Mielopoyesis/efectos de la radiación , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Radiación Ionizante , Recombinación Genética/efectos de la radiaciónRESUMEN
The classical model of hematopoiesis posits the segregation of lymphoid and myeloid lineages as the earliest fate decision. The validity of this model in the mouse has been questioned; however, little is known about the lineage potential of human progenitors. Here we provide a comprehensive analysis of the human hematopoietic hierarchy by clonally mapping the developmental potential of seven progenitor classes from neonatal cord blood and adult bone marrow. Human multilymphoid progenitors, identified as a distinct population of Thy-1(neg-lo)CD45RA(+) cells in the CD34(+)CD38(-) stem cell compartment, gave rise to all lymphoid cell types, as well as monocytes, macrophages and dendritic cells, which indicated that these myeloid lineages arise in early lymphoid lineage specification. Thus, as in the mouse, human hematopoiesis does not follow a rigid model of myeloid-lymphoid segregation.