RESUMEN
Leukemia stem cells (LSCs) are widely believed to reside in well-characterized bone marrow (BM) niches; however, the capacity of the BM niches to accommodate LSCs is insufficient, and a significant proportion of LSCs are instead maintained in regions outside the BM. The molecular basis for this niche-independent behavior of LSCs remains elusive. Here, we show that integrin-α9 overexpression (ITGA9 OE) plays a pivotal role in the extramedullary maintenance of LSCs by molecularly mimicking the niche-interacting status, through the binding with its soluble ligand, osteopontin (OPN). Retroviral insertional mutagenesis conducted on leukemia-prone Runx-deficient mice identified Itga9 OE as a novel leukemogenic event. Itga9 OE activates Akt and p38MAPK signaling pathways. The elevated Myc expression subsequently enhances ribosomal biogenesis to overcome the cell integrity defect caused by the preexisting Runx alteration. The Itga9-Myc axis, originally discovered in mice, was further confirmed in multiple human acute myeloid leukemia (AML) subtypes, other than RUNX leukemias. In addition, ITGA9 was shown to be a functional LSC marker of the best prognostic value among 14 known LSC markers tested. Notably, the binding of ITGA9 with soluble OPN, a known negative regulator against HSC activation, induced LSC dormancy, while the disruption of ITGA9-soluble OPN interaction caused rapid cell propagation. These findings suggest that the ITGA9 OE increases both actively proliferating leukemia cells and dormant LSCs in a well-balanced manner, thereby maintaining LSCs. The ITGA9 OE would serve as a novel therapeutic target in AML.
Asunto(s)
Leucemia Mieloide Aguda , Células Madre Neoplásicas , Animales , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Humanos , Ratones , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Osteopontina/genética , Osteopontina/metabolismo , Nicho de Células Madre , Cadenas alfa de Integrinas/metabolismo , Cadenas alfa de Integrinas/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Regulación Leucémica de la Expresión Génica , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Ratones Endogámicos C57BL , Transducción de SeñalRESUMEN
Intercellular mitochondria transfer is a biological phenomenon implicated in diverse biological processes. However, the physiological role of this phenomenon remains understudied between erythroblasts and their erythroblastic island (EBI) macrophage niche. To gain further insights into the mitochondria transfer functions, we infused EBI macrophages in vivo into mice subjected to different modes of anemic stresses. Interestingly, we observed the occurrence of mitochondria transfer events from the infused EBI macrophages to early stages of erythroblasts coupled with enhanced erythroid recovery. Single-cell RNA-sequencing analysis on erythroblasts receiving exogenous mitochondria revealed a subset of highly proliferative and metabolically active erythroid populations marked by high expression of CD47. Furthermore, CD47 or Sirpα blockade leads to a decline in both the occurrence of mitochondria transfer events and their mediated erythroid recovery. Hence, these data indicate a significant role of mitochondria transfer in the enhancement of erythroid recovery from stress through the alteration of the bioenergetic profiles via CD47-Sirpα interaction in the early stages of erythroblasts.
Asunto(s)
Antígeno CD47 , Eritropoyesis , Mitocondrias , Animales , Antígeno CD47/metabolismo , Metabolismo Energético , Eritroblastos/metabolismo , Eritropoyesis/fisiología , Ratones , Mitocondrias/metabolismo , ARN/metabolismo , Receptores de Eritropoyetina/metabolismo , Análisis de la Célula Individual , Estrés FisiológicoRESUMEN
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive hematologic malignancy with poor clinical outcomes. Dysregulated MYC expression, which is associated with protein arginine methyltransferase 5 (PRMT5) dependency, is a recurrent feature of BPDCN. Although recent studies have reported a PRMT5 gene signature in BPDCN patient samples, the role of PRMT5 in BPDCN remains unexplored. Here, we demonstrate that BPDCN is highly sensitive to PRMT5 inhibition. Consistent with the upregulation of PRMT5 in BPDCN, we show that pharmacological inhibition (GSK3326595) of PRMT5 inhibits the growth of the patient-derived BPDCN cell line CAL-1 in vitro and mitigated tumor progression in our mouse xenograft model. Interestingly, RNA-sequencing (RNA-seq) analysis revealed that PRMT5 inhibition increases intron retention in several key RNA methylation genes, including METTL3, which was accompanied by a dose-dependent decrease in METTL3 expression. Notably, the function of cellular m6A RNA modification of METTL3 was also affected by PRMT5 inhibition in CAL-1 cells. Intriguingly, METTL3 depletion in CAL-1 caused a significant increase in interferon (IFN) signaling, which was further elevated upon PRMT5 inhibition. Importantly, we discovered that this increase in IFN signaling attenuated the sensitivity of METTL3-depleted CAL-1 cells to PRMT5 inhibition. Correspondingly, stimulation of IFN signaling via TLR7 agonists weakened CAL-1 cell sensitivity to PRMT5 inhibition. Overall, our findings implicate PRMT5 as a therapeutic target in BPDCN and provide insight into the involvement of METTL3 and the IFN pathway in regulating the response to PRMT5 inhibition.
Asunto(s)
Neoplasias Hematológicas , Trastornos Mieloproliferativos , Neoplasias Cutáneas , Animales , Células Dendríticas/metabolismo , Neoplasias Hematológicas/genética , Humanos , Interferones/metabolismo , Metiltransferasas/metabolismo , Ratones , Trastornos Mieloproliferativos/patología , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , ARN/metabolismo , Neoplasias Cutáneas/genética , Receptor Toll-Like 7/metabolismoRESUMEN
Hematopoietic stem cells (HSC) rarely divide, rest in quiescence, and proliferate only upon stress hematopoiesis. The cytokine thrombopoietin (Thpo) has been perplexingly described to induce quiescence and promote self-renewal divisions in HSCs. To clarify the contradictory effect of Thpo, we conducted a detailed analysis on conventional (Thpo-/-) and liver-specific (Thpofl/fl;AlbCre+/-) Thpo-deletion models. Thpo-/- HSCs exhibited profound loss of quiescence, impaired cell cycle progression, and increased apoptosis. Thpo-/- HSCs also exhibited diminished mitochondrial mass and impaired mitochondrial bioenergetics. Abnormal HSC phenotypes in Thpo-/- mice were reversible after HSC transplantation into wild-type recipients. Moreover, Thpo-/- HSCs acquired quiescence with extended administration of a Thpo receptor agonist, romiplostim, and were prone to subsequent stem cell exhaustion during competitive bone marrow transplantation. Thpofl/fl;AlbCre+/- HSCs exhibited similar stem cell phenotypes but to a lesser degree compared with Thpo-/- HSCs. HSCs that survive Thpo deficiency acquire quiescence in a dose-dependent manner through the modification of their metabolic state.
Asunto(s)
Células Madre Hematopoyéticas/citología , Trombopoyetina/deficiencia , Animales , Apoptosis , Ciclo Celular , Autorrenovación de las Células , Metabolismo Energético/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Ratones , Ratones Noqueados , Ratones Transgénicos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Estrés Oxidativo/efectos de los fármacos , Receptores Fc , Receptores de Trombopoyetina/agonistas , Proteínas Recombinantes de Fusión/farmacología , Transducción de Señal , Trombopoyetina/genética , Trombopoyetina/farmacología , TranscriptomaRESUMEN
Multiple myeloma (MM) is an intractable hematological malignancy characterized by abnormal plasma cells in the bone marrow (BM) and increased osteolytic lesions. Within the BM niche, mesenchymal stem cells (MSCs) have been proposed to contribute to functionally important MM-MSC interactions. However, despite various studies on MM pathology, the impact of MM on MSCs during the early stages of malignancy has not been adequately addressed. We previously identified tissue inhibitor of matrix metalloproteinase 1 (TIMP-1) as a cytokine that is modulated in vivo within the MM BM niche, and highlighted its potential relevance in MM. Given the role of TIMP-1 in preventing migration of breast cancer cells, this study aimed to investigate the relationship between MSC-secreted TIMP-1 and MM progression. Here, we examined the effect of MSC-derived TIMP-1 on MM cell migration, and found that TIMP-1 secreted by human MSCs play a role in preventing migration of MM cells by reducing the levels of MM cell-derived MMP-9. We also investigated how MM cells regulate expression of TIMP-1 in MSCs. Using a knockdown approach in MSCs, we implicated TGF-B activated kinase 1 binding protein 1 (TAB1) as an upstream effector of TIMP-1 that was downregulated in the presence of MM cells, which resulted in reduced TIMP-1 secretion. Overall, our findings uncover how MSCs in the MM BM niche are modulated to promote MM progression, and unravel a previously unreported role of the TAB1-TIMP-1 axis in the context of the MM BM niche.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Madre Mesenquimatosas/patología , Mieloma Múltiple/patología , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Línea Celular , Línea Celular Tumoral , Movimiento Celular , Técnicas de Cocultivo , Regulación Neoplásica de la Expresión Génica , Humanos , Metaloproteinasa 9 de la Matriz/metabolismo , Células Madre Mesenquimatosas/metabolismo , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/genéticaRESUMEN
RUNX1 is among the most frequently mutated genes in human leukemia, and the loss or dominant-negative suppression of RUNX1 function is found in myelodysplastic syndrome and acute myeloid leukemia (AML). How posttranslational modifications (PTMs) of RUNX1 affect its in vivo function, however, and whether PTM dysregulation of RUNX1 can cause leukemia are largely unknown. We performed targeted deep sequencing on a family with 3 occurrences of AML and identified a novel RUNX1 mutation, R237K. The mutated R237 residue is a methylation site by protein arginine methyltransferase 1, and loss of methylation reportedly impairs the transcriptional activity of RUNX1 in vitro. To explore the biologic significance of RUNX1 methylation in vivo, we used RUNX1 R233K/R237K double-mutant mice, in which 2 arginine-to-lysine mutations precluded RUNX1 methylation. Genetic ablation of RUNX1 methylation led to loss of quiescence and expansion of hematopoietic stem cells (HSCs), and it changed the genomic and epigenomic signatures of phenotypic HSCs to a poised progenitor state. Furthermore, loss of RUNX1 R233/R237 methylation suppressed endoplasmic reticulum stress-induced unfolded protein response genes, including Atf4, Ddit3, and Gadd34; the radiation-induced p53 downstream genes Bbc3, Pmaip1, and Cdkn1a; and subsequent apoptosis in HSCs. Mechanistically, activating transcription factor 4 was identified as a direct transcriptional target of RUNX1. Collectively, defects in RUNX1 methylation in HSCs confer resistance to apoptosis and survival advantage under stress conditions, a hallmark of a preleukemic clone that may predispose affected individuals to leukemia. Our study will lead to a better understanding of how dysregulation of PTMs can contribute to leukemogenesis.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Células Madre Hematopoyéticas/fisiología , Leucemia/genética , Metiltransferasas/metabolismo , Procesamiento Proteico-Postraduccional/genética , Animales , Apoptosis/genética , Supervivencia Celular/genética , Familia , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia/metabolismo , Leucemia/patología , Leucemia Mieloide Aguda/sangre , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Masculino , Metilación , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Mutación Missense , Síndromes Mielodisplásicos/sangre , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/metabolismo , LinajeRESUMEN
Protein arginine methyltransferase 5 (PRMT5) is essential for hematopoiesis, while PRMT5 inhibition remains a promising therapeutic strategy against various cancers. Here, we demonstrate that hematopoietic stem cell (HSC) quiescence and viability are severely perturbed upon PRMT5 depletion, which also increases HSC size, PI3K/AKT/mechanistic target of rapamycin (mTOR) pathway activity, and protein synthesis rate. We uncover a critical role for PRMT5 in maintaining HSC genomic integrity by modulating splicing of genes involved in DNA repair. We found that reducing PRMT5 activity upregulates exon skipping and intron retention events that impair gene expression. Genes across multiple DNA repair pathways are affected, several of which mediate interstrand crosslink repair and homologous recombination. Consequently, loss of PRMT5 activity leads to endogenous DNA damage that triggers p53 activation, induces apoptosis, and culminates in rapid HSC exhaustion, which is significantly delayed by p53 depletion. Collectively, these findings establish the importance of cell-intrinsic PRMT5 activity in HSCs.
Asunto(s)
Células Madre Hematopoyéticas/enzimología , Proteína-Arginina N-Metiltransferasas/fisiología , Proteostasis , Empalme del ARN , Animales , Apoptosis , Línea Celular , Daño del ADN , Reparación del ADN , Genoma , Células Madre Hematopoyéticas/metabolismo , Ratones , Biosíntesis de Proteínas , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Erythropoiesis is a highly coordinated stepwise process involving the progressive clearance of mitochondria via mitophagy. Based on the expression of several macroautophagy and mitophagy specific genes, we identified a sequential change in the transcriptional pattern during terminal erythroid differentiation. Because erythroid cells are a major source of serum sphingosine-1-phosphate, we analyzed the role of sphingolipid signaling in erythropoiesis and demonstrate that sphingosine kinase activity promotes terminal erythroid differentiation by regulating the expression of key mitophagy genes Pink1 and Bnip3l/Nix. Sphingosine kinase 1 (Sphk1) inhibition also disrupted Pink1-p62 mediated mitochondria clearance in late erythroblasts. Notably, we show that supplementing sphingosine-1-phosphate in vitro can promote erythroid differentiation. Our study clarifies the role of sphingolipid signaling in regulating mitophagy during terminal erythroid differentiation and highlights the potential utility of modulating sphingolipid signaling to facilitate the large-scale production of transfusable red blood cells.
Asunto(s)
Diferenciación Celular/fisiología , Eritropoyesis/fisiología , Lisofosfolípidos/metabolismo , Mitofagia/fisiología , Transducción de Señal/fisiología , Esfingosina/análogos & derivados , Animales , Lisofosfolípidos/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Esfingosina/genética , Esfingosina/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Tumor-associated macrophages (TAMs) are known to regulate tumor response to many anti-cancer therapies, including oncolytic virotherapy. Oncolytic virotherapy employing oncolytic paramyxoviruses, such as attenuated measles (MeV) and mumps (MuV) viruses, has demonstrated therapeutic potential against various malignancies. However, the response of TAMs to oncolytic paramyxoviruses and the consequent effect on virotherapeutic efficacy remains to be characterized. Here, we demonstrate that the presence of human monocyte-derived macrophages (MDMs), irrespective of initial polarization state, enhances the virotherapeutic effect of MeV and MuV on breast cancer cells. Notably, our finding contrasts those of several studies involving other oncolytic viruses, which suggest that TAMs negatively impact virotherapeutic efficacy by impeding virus replication and dissemination. We found that the enhanced virotherapeutic effect in the presence of MDMs was due to slightly delayed proliferation and significantly elevated cell death that was not a result of increased virus replication. Instead, we found that the enhanced virotherapeutic effect involved several macrophage-associated anti-tumor mediators, and was associated with the modulation of MDMs towards an anti-tumor phenotype. Our findings present an alternative view on the role of TAMs in oncolytic virotherapy, and highlight the immunotherapeutic potential of oncolytic paramyxoviruses; possibly contributing towards the overall efficacy of oncolytic virotherapy.