RESUMEN
The identity of the RNA-binding proteins (RBPs) that govern cancer stem cells remains poorly characterized. The MSI2 RBP is a central regulator of translation of cancer stem cell programs. Through proteomic analysis of the MSI2-interacting RBP network and functional shRNA screening, we identified 24 genes required for in vivo leukemia. Syncrip was the most differentially required gene between normal and myeloid leukemia cells. SYNCRIP depletion increased apoptosis and differentiation while delaying leukemogenesis. Gene expression profiling of SYNCRIP-depleted cells demonstrated a loss of the MLL and HOXA9 leukemia stem cell program. SYNCRIP and MSI2 interact indirectly though shared mRNA targets. SYNCRIP maintains HOXA9 translation, and MSI2 or HOXA9 overexpression rescued the effects of SYNCRIP depletion. Altogether, our data identify SYNCRIP as a new RBP that controls the myeloid leukemia stem cell program. We propose that targeting these RBP complexes might provide a novel therapeutic strategy in leukemia.
Asunto(s)
Regulación Leucémica de la Expresión Génica , Ribonucleoproteínas Nucleares Heterogéneas/genética , Leucemia Mieloide/genética , Proteínas de Unión al ARN/metabolismo , Animales , Supervivencia Celular , Femenino , Hematopoyesis/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Proteínas de Homeodominio/genética , Humanos , Leucemia Bifenotípica Aguda/genética , Leucemia Bifenotípica Aguda/patología , Leucemia Mieloide/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Células Progenitoras Mieloides/metabolismo , Células Progenitoras Mieloides/patología , ARN Interferente Pequeño , Proteínas de Unión al ARN/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Impaired erythropoiesis in the deletion 5q (del(5q)) subtype of myelodysplastic syndrome (MDS) has been linked to heterozygous deletion of RPS14, which encodes the ribosomal protein small subunit 14. We generated mice with conditional inactivation of Rps14 and demonstrated an erythroid differentiation defect that is dependent on the tumor suppressor protein p53 (encoded by Trp53 in mice) and is characterized by apoptosis at the transition from polychromatic to orthochromatic erythroblasts. This defect resulted in age-dependent progressive anemia, megakaryocyte dysplasia and loss of hematopoietic stem cell (HSC) quiescence. As assessed by quantitative proteomics, mutant erythroblasts expressed higher levels of proteins involved in innate immune signaling, notably the heterodimeric S100 calcium-binding proteins S100a8 and S100a9. S100a8--whose expression was increased in mutant erythroblasts, monocytes and macrophages--is functionally involved in the erythroid defect caused by the Rps14 deletion, as addition of recombinant S100a8 was sufficient to induce a differentiation defect in wild-type erythroid cells, and genetic inactivation of S100a8 expression rescued the erythroid differentiation defect of Rps14-haploinsufficient HSCs. Our data link Rps14 haploinsufficiency in del(5q) MDS to activation of the innate immune system and induction of S100A8-S100A9 expression, leading to a p53-dependent erythroid differentiation defect.
Asunto(s)
Anemia/genética , Calgranulina A/genética , Calgranulina B/genética , Eritropoyesis/genética , Haploinsuficiencia/genética , Síndromes Mielodisplásicos/genética , Proteínas Ribosómicas/genética , Anemia/inmunología , Animales , Western Blotting , Médula Ósea/patología , Calgranulina A/metabolismo , Citocinas/inmunología , Modelos Animales de Enfermedad , Células Precursoras Eritroides/metabolismo , Eritropoyesis/inmunología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Células Madre Hematopoyéticas , Humanos , Inmunidad Innata/genética , Inmunidad Innata/inmunología , Inmunohistoquímica , Hibridación Fluorescente in Situ , Técnicas In Vitro , Espectrometría de Masas , Megacariocitos , Ratones , Ratones Noqueados , Microscopía Confocal , Síndromes Mielodisplásicos/inmunología , Síndromes Mielodisplásicos/patología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The casein kinase 1A1 gene (CSNK1A1) is a putative tumor suppressor gene located in the common deleted region for del(5q) myelodysplastic syndrome (MDS). We generated a murine model with conditional inactivation of Csnk1a1 and found that Csnk1a1 haploinsufficiency induces hematopoietic stem cell expansion and a competitive repopulation advantage, whereas homozygous deletion induces hematopoietic stem cell failure. Based on this finding, we found that heterozygous inactivation of Csnk1a1 sensitizes cells to a CSNK1 inhibitor relative to cells with two intact alleles. In addition, we identified recurrent somatic mutations in CSNK1A1 on the nondeleted allele of patients with del(5q) MDS. These studies demonstrate that CSNK1A1 plays a central role in the biology of del(5q) MDS and is a promising therapeutic target.
Asunto(s)
Quinasa de la Caseína I/metabolismo , Deleción Cromosómica , Cromosomas Humanos Par 5 , Síndromes Mielodisplásicos/genética , Anciano , Animales , Secuencia de Bases , Quinasa de la Caseína I/genética , Cartilla de ADN , Femenino , Citometría de Flujo , Haploinsuficiencia , Humanos , Masculino , Ratones , Mutación , Reacción en Cadena de la Polimerasa , Adulto JovenRESUMEN
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
Efforts to develop more effective therapies for acute leukemia may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including leukemia stem cells (LSCs) and supportive interactions with the bone marrow microenvironment. The therapeutic targeting of LSCs is challenging because LSCs are highly similar to normal hematopoietic stem and progenitor cells (HSPCs) and are protected by stromal cells in vivo. We screened 14,718 compounds in a leukemia-stroma co-culture system for inhibition of cobblestone formation, a cellular behavior associated with stem-cell function. Among those compounds that inhibited malignant cells but spared HSPCs was the cholesterol-lowering drug lovastatin. Lovastatin showed anti-LSC activity in vitro and in an in vivo bone marrow transplantation model. Mechanistic studies demonstrated that the effect was on target, via inhibition of HMG-CoA reductase. These results illustrate the power of merging physiologically relevant models with high-throughput screening.
Asunto(s)
Antineoplásicos/farmacología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Leucemia , Células Madre Neoplásicas/efectos de los fármacos , Línea Celular Tumoral , Células Madre Hematopoyéticas , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Lovastatina/farmacología , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/fisiologíaRESUMEN
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.