RESUMEN
Leukemia stem cells (LSCs) have the capacity to self-renew and propagate disease upon serial transplantation in animal models, and elimination of this cell population is required for curative therapies. Here, we describe a series of pooled, in vivo RNAi screens to identify essential transcription factors (TFs) in a murine model of acute myeloid leukemia (AML) with genetically and phenotypically defined LSCs. These screens reveal the heterodimeric, circadian rhythm TFs Clock and Bmal1 as genes required for the growth of AML cells in vitro and in vivo. Disruption of canonical circadian pathway components produces anti-leukemic effects, including impaired proliferation, enhanced myeloid differentiation, and depletion of LSCs. We find that both normal and malignant hematopoietic cells harbor an intact clock with robust circadian oscillations, and genetic knockout models reveal a leukemia-specific dependence on the pathway. Our findings establish a role for the core circadian clock genes in AML.
Asunto(s)
Factores de Transcripción ARNTL/genética , Proteínas CLOCK/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/patología , Animales , Ritmo Circadiano , Modelos Animales de Enfermedad , Técnicas de Inactivación de Genes , Hematopoyesis , Humanos , Leucemia Mieloide Aguda/metabolismo , Ratones , Ratones Endogámicos C57BL , Células Madre Neoplásicas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
Acute myeloid leukemia (AML) is a heterogeneous disease with complex molecular pathophysiology. To systematically characterize AML's genetic dependencies, we conducted genome-scale short hairpin RNA screens in 17 AML cell lines and analyzed dependencies relative to parallel screens in 199 cell lines of other cancer types. We identified 353 genes specifically required for AML cell proliferation. To validate the in vivo relevance of genetic dependencies observed in human cell lines, we performed a secondary screen in a syngeneic murine AML model driven by the MLL-AF9 oncogenic fusion protein. Integrating the results of these interference RNA screens and additional gene expression data, we identified the transcription factor ZEB2 as a novel AML dependency. ZEB2 depletion impaired the proliferation of both human and mouse AML cells and resulted in aberrant differentiation of human AML cells. Mechanistically, we showed that ZEB2 transcriptionally represses genes that regulate myeloid differentiation, including genes involved in cell adhesion and migration. In addition, we found that epigenetic silencing of the miR-200 family microRNAs affects ZEB2 expression. Our results extend the role of ZEB2 beyond regulating epithelial-mesenchymal transition (EMT) and establish ZEB2 as a novel regulator of AML proliferation and differentiation.
Asunto(s)
Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Proteínas de Homeodominio/genética , Leucemia Mieloide Aguda/genética , MicroARNs/genética , Proteínas Represoras/genética , Animales , Adhesión Celular , Diferenciación Celular , Línea Celular Tumoral , Movimiento Celular , Epigénesis Genética , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , MicroARNs/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/metabolismo , Transducción de Señal , Transcripción Genética , Caja Homeótica 2 de Unión a E-Box con Dedos de ZincRESUMEN
The developmental switch from human fetal (gamma) to adult (beta) hemoglobin represents a clinically important example of developmental gene regulation. The transcription factor BCL11A is a central mediator of gamma-globin silencing and hemoglobin switching. Here we determine chromatin occupancy of BCL11A at the human beta-globin locus and other genomic regions in vivo by high-resolution chromatin immunoprecipitation (ChIP)-chip analysis. BCL11A binds the upstream locus control region (LCR), epsilon-globin, and the intergenic regions between gamma-globin and delta-globin genes. A chromosome conformation capture (3C) assay shows that BCL11A reconfigures the beta-globin cluster by modulating chromosomal loop formation. We also show that BCL11A and the HMG-box-containing transcription factor SOX6 interact physically and functionally during erythroid maturation. BCL11A and SOX6 co-occupy the human beta-globin cluster along with GATA1, and cooperate in silencing gamma-globin transcription in adult human erythroid progenitors. These findings collectively demonstrate that transcriptional silencing of gamma-globin genes by BCL11A involves long-range interactions and cooperation with SOX6. Our findings provide insight into the mechanism of BCL11A action and new clues for the developmental gene regulatory programs that function at the beta-globin locus.
Asunto(s)
Proteínas Portadoras/metabolismo , Células Eritroides/metabolismo , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Proteínas Nucleares/metabolismo , Factores de Transcripción SOXD/metabolismo , gamma-Globinas/metabolismo , Adulto , Animales , Línea Celular , Células Eritroides/citología , Eritropoyesis/fisiología , Factor de Transcripción GATA1/metabolismo , Histonas/metabolismo , Humanos , Ratones , Unión Proteica , Transporte de Proteínas , Proteínas RepresorasAsunto(s)
Antígenos de Plaqueta Humana/inmunología , Autoanticuerpos/inmunología , Eptifibatida/efectos adversos , Inhibidores de Agregación Plaquetaria/efectos adversos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Púrpura Trombocitopénica Idiopática/etiología , Infarto del Miocardio con Elevación del ST/sangre , Trombosis/etiología , Arginina/análogos & derivados , Arginina/uso terapéutico , Aspirina/uso terapéutico , Terapia Combinada , Trombosis Coronaria/etiología , Trombosis Coronaria/cirugía , Sustitución de Medicamentos , Quimioterapia Combinada , Eptifibatida/inmunología , Eptifibatida/uso terapéutico , Humanos , Masculino , Persona de Mediana Edad , Intervención Coronaria Percutánea , Ácidos Pipecólicos/uso terapéutico , Inhibidores de Agregación Plaquetaria/inmunología , Inhibidores de Agregación Plaquetaria/uso terapéutico , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/inmunología , Transfusión de Plaquetas , Púrpura Trombocitopénica Idiopática/inmunología , Púrpura Trombocitopénica Idiopática/terapia , Infarto del Miocardio con Elevación del ST/tratamiento farmacológico , Infarto del Miocardio con Elevación del ST/terapia , Choque Cardiogénico/etiología , Stents/efectos adversos , Sulfonamidas/uso terapéutico , Trombectomía , Terapia Trombolítica , Trombosis/tratamiento farmacológico , Ticagrelor/uso terapéutico , Warfarina/uso terapéuticoRESUMEN
Genome sequencing studies have shown that human malignancies often bear mutations in four or more driver genes, but it is difficult to recapitulate this degree of genetic complexity in mouse models using conventional breeding. Here we use the CRISPR-Cas9 system of genome editing to overcome this limitation. By delivering combinations of small guide RNAs (sgRNAs) and Cas9 with a lentiviral vector, we modified up to five genes in a single mouse hematopoietic stem cell (HSC), leading to clonal outgrowth and myeloid malignancy. We thereby generated models of acute myeloid leukemia (AML) with cooperating mutations in genes encoding epigenetic modifiers, transcription factors and mediators of cytokine signaling, recapitulating the combinations of mutations observed in patients. Our results suggest that lentivirus-delivered sgRNA:Cas9 genome editing should be useful to engineer a broad array of in vivo cancer models that better reflect the complexity of human disease.
Asunto(s)
Neoplasias de la Médula Ósea/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Animales , Modelos Animales de Enfermedad , RatonesRESUMEN
Despite extensive insights into the underlying genetics and biology of acute myeloid leukemia (AML), overall survival remains poor and new therapies are needed. We found that casein kinase 1 α (Csnk1a1), a serine-threonine kinase, is essential for AML cell survival in vivo. Normal hematopoietic stem and progenitor cells (HSPCs) were relatively less affected by shRNA-mediated knockdown of Csnk1a1. To identify downstream mediators of Csnk1a1 critical for leukemia cells, we performed an in vivo pooled shRNA screen and gene expression profiling. We found that Csnk1a1 knockdown results in decreased Rps6 phosphorylation, increased p53 activity, and myeloid differentiation. Consistent with these observations, p53-null leukemias were insensitive to Csnk1a1 knockdown. We further evaluated whether D4476, a casein kinase 1 inhibitor, would exhibit selective antileukemic effects. Treatment of leukemia stem cells (LSCs) with D4476 showed highly selective killing of LSCs over normal HSPCs. In summary, these findings demonstrate that Csnk1a1 inhibition causes reduced Rps6 phosphorylation and activation of p53, resulting in selective elimination of leukemia cells, revealing Csnk1a1 as a potential therapeutic target for the treatment of AML.
Asunto(s)
Caseína Quinasa Ialfa/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/enzimología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Caseína Quinasa Ialfa/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Silenciador del Gen/efectos de los fármacos , Humanos , Ratones , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteína S6 Ribosómica/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Resultado del TratamientoRESUMEN
We used an in vivo small hairpin RNA (shRNA) screening approach to identify genes that are essential for MLL-AF9 acute myeloid leukemia (AML). We found that Integrin Beta 3 (Itgb3) is essential for murine leukemia cells in vivo and for human leukemia cells in xenotransplantation studies. In leukemia cells, Itgb3 knockdown impaired homing, downregulated LSC transcriptional programs, and induced differentiation via the intracellular kinase Syk. In contrast, loss of Itgb3 in normal hematopoietic stem and progenitor cells did not affect engraftment, reconstitution, or differentiation. Finally, using an Itgb3 knockout mouse model, we confirmed that Itgb3 is dispensable for normal hematopoiesis but is required for leukemogenesis. Our results establish the significance of the Itgb3 signaling pathway as a potential therapeutic target in AML.