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1.
Proc Natl Acad Sci U S A ; 117(50): 31945-31953, 2020 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-33268499

RESUMEN

Regulation of quiescence is critical for the maintenance of adult hematopoietic stem cells (HSCs). Disruption of transcription factor gene Prdm16 during mouse embryonic development has been shown to cause a severe loss of fetal liver HSCs; however, the underlying mechanisms and the function of Prdm16 in adult HSCs remain unclear. To investigate the role of Prdm16 in adult HSCs, we generated a novel conditional knockout mouse model and deleted Prdm16 in adult mouse hematopoietic system using the IFN-inducible Mx1-Cre Our results show that Prdm16 deletion in the adult mouse hematopoietic system has a less severe effect on HSCs, causing a gradual decline of adult HSC numbers and a concomitant increase in the multipotent progenitor (MPP) compartment. Prdm16 deletion in the hematopoietic system following transplantation produced the same phenotype, indicating that the defect is intrinsic to adult HSCs. This HSC loss was also exacerbated by stress induced by 5-fluorouracil injections. Annexin V staining showed no difference in apoptosis between wild-type and knockout adult HSCs. In contrast, Bromodeoxyuridine analysis revealed that loss of Prdm16 significantly increased cycling of long-term HSCs (LT-HSCs) with the majority of the cells found in the S to G2/M phase. Consistently, RNA sequencing analysis of mouse LT-HSCs with and without Prdm16 deletion showed that Prdm16 loss induced a significant decrease in the expression of several known cell cycle regulators of HSCs, among which Cdkn1a and Egr1 were identified as direct targets of Prdm16 Our results suggest that Prdm16 preserves the function of adult LT-HSCs by promoting their quiescence.


Asunto(s)
Células Madre Adultas/fisiología , Ciclo Celular/genética , Proteínas de Unión al ADN/metabolismo , Células Madre Hematopoyéticas/fisiología , Factores de Transcripción/metabolismo , Activación Transcripcional , Animales , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Proteínas de Unión al ADN/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Femenino , Trasplante de Células Madre Hematopoyéticas , Ratones , Ratones Noqueados , RNA-Seq , Factores de Transcripción/genética
2.
Haematologica ; 100(8): 1051-7, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26001790

RESUMEN

Next generation sequencing technologies have provided insights into the molecular heterogeneity of various myeloid neoplasms, revealing previously unknown somatic genetic events. In our cohort of 1444 cases analyzed by next generation sequencing, somatic mutations in the gene BRCA1-BRCA2-containing complex 3 (BRCC3) were identified in 28 cases (1.9%). BRCC3 is a member of the JAMM/MPN+ family of zinc metalloproteases capable of cleaving Lys-63 linked polyubiquitin chains, and is implicated in DNA repair. The mutations were located throughout its coding region. The average variant allelic frequency of BRCC3 mutations was 30.1%, and by a serial sample analysis at two different time points a BRCC3 mutation was already identified in the initial stage of a myelodysplastic syndrome. BRCC3 mutations commonly occurred in nonsense (n=12), frameshift (n=4), and splice site (n=5) configurations. Due to the marginal male dominance (odds ratio; 2.00, 0.84-4.73) of BRCC3 mutations, the majority of mutations (n=23; 82%) were hemizygous. Phenotypically, BRCC3 mutations were frequently observed in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms and associated with -Y abnormality (odds ratio; 3.70, 1.25-11.0). Clinically, BRCC3 mutations were also related to higher age (P=0.01), although prognosis was not affected. Knockdown of Brcc3 gene expression in murine bone marrow lineage negative, Sca1 positive, c-kit positive cells resulted in 2-fold more colony formation and modest differentiation defect. Thus, BRCC3 likely plays a role as tumor-associated gene in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms.


Asunto(s)
Proteínas de la Membrana/genética , Mutación , Trastornos Mieloproliferativos/genética , Anciano , Anciano de 80 o más Años , Alelos , Animales , Proteína BRCA1/genética , Aberraciones Cromosómicas , Análisis Mutacional de ADN , Enzimas Desubicuitinizantes , Femenino , Frecuencia de los Genes , Técnicas de Silenciamiento del Gen , Estudios de Asociación Genética , Genotipo , Humanos , Masculino , Ratones , Persona de Mediana Edad , Trastornos Mieloproliferativos/diagnóstico , Fenotipo , ARN Interferente Pequeño/genética
3.
Blood ; 119(25): 6099-108, 2012 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-22566606

RESUMEN

Acquisition of self-renewal capability by myeloid progenitors to become leukemic stem cells during myeloid leukemia development is poorly understood. Here, we show that Setbp1 overexpression efficiently confers self-renewal capability to myeloid progenitors in vitro, causing their immortalization in the presence of stem cell factor and IL-3. Self-renewal after immortalization requires continuous Setbp1 expression. We also found that Hoxa9 and Hoxa10 mRNA are present at dramatically higher levels in Setbp1-immortalized cells compared with other immortalized cells, and are induced shortly after Setbp1 expression in primary myeloid progenitors. Suppression of either gene in Setbp1-immortalized cells drastically reduces their colony-forming capability. Interestingly, Setbp1 protein associates with Hoxa9 and Hoxa10 promoters in chromatin immunoprecipitation assays in these cells, suggesting that both are direct transcriptional targets of Setbp1. Setbp1 also promotes self-renewal of myeloid progenitors in vivo as its coexpression with BCR/ABL transforms primary mouse myeloid progenitors, generating aggressive leukemias in recipient mice resembling chronic myelogenous leukemia (CML) myeloid blast crisis. Increased SETBP1 mRNA levels were also detected in a subset of CML advanced phase/blast crisis patients with high levels of HOXA9 and HOXA10 expression. Thus, Setbp1 activation represents a novel mechanism conferring self-renewal capability to myeloid progenitors in myeloid leukemia development.


Asunto(s)
Proteínas Portadoras/fisiología , Proliferación Celular , Proteínas de Homeodominio/genética , Células Progenitoras Mieloides/fisiología , Proteínas Nucleares/fisiología , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Cultivadas , Proteínas Homeobox A10 , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/fisiología , Humanos , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Células Progenitoras Mieloides/metabolismo , Células 3T3 NIH , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Activación Transcripcional/fisiología , Transfección
4.
iScience ; 25(1): 103679, 2022 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-35036869

RESUMEN

Abnormal activation of SETBP1 due to overexpression or missense mutations occurs frequently in various myeloid neoplasms and associates with poor prognosis. Direct activation of Hoxa9/Hoxa10/Myb transcription by SETBP1 and its missense mutants is essential for their transforming capability; however, the underlying epigenetic mechanisms remain elusive. We found that both SETBP1 and its missense mutant SETBP1(D/N) directly interact with histone methyltransferase MLL1. Using a combination of ChIP-seq and RNA-seq analysis in primary hematopoietic stem and progenitor cells, we uncovered extensive overlap in their genomic occupancy and their cooperation in activating many oncogenic transcription factor genes including Hoxa9/Hoxa10/Myb and a large group of ribosomal protein genes. Genetic ablation of Mll1 as well as treatment with an inhibitor of the MLL1 complex OICR-9429 abrogated Setbp1/Setbp1(D/N)-induced transcriptional activation and transformation. Thus, the MLL1 complex plays a critical role in Setbp1-induced transcriptional activation and transformation and represents a promising target for treating myeloid neoplasms with SETBP1 activation.

5.
Oncotarget ; 8(58): 98853-98864, 2017 Nov 17.
Artículo en Inglés | MEDLINE | ID: mdl-29228732

RESUMEN

Mechanisms underlying the progression of Chronic Myeloid Leukemia (CML) from chronic phase to myeloid blast crisis are poorly understood. Our previous studies have suggested that overexpression of SETBP1 can drive this progression by conferring unlimited self-renewal capability to granulocyte macrophage progenitors (GMPs). Here we show that overexpression of Hoxa9 or Hoxa10, both transcriptional targets of Setbp1, is also sufficient to induce self-renewal of primary myeloid progenitors, causing their immortalization in culture. More importantly, both are able to cooperate with BCR/ABL to consistently induce transformation of mouse GMPs and development of aggressive leukemias resembling CML myeloid blast crisis, suggesting that either gene can drive CML progression by promoting the self-renewal of GMPs. We further identify Myb as a common critical target for Hoxa9 and Hoxa10 in inducing self-renewal of myeloid progenitors as Myb knockdown significantly reduced colony-forming potential of myeloid progenitors immortalized by the expression of either gene. Interestingly, Myb is also capable of immortalizing primary myeloid progenitors in culture and cooperating with BCR/ABL to induce leukemic transformation of mouse GMPs. Significantly increased levels of MYB transcript also were detected in all human CML blast crisis samples examined over chronic phase samples, further suggesting the possibility that MYB overexpression may play a prevalent role in driving human CML myeloid blast crisis development. In summary, our results identify overexpression of HOXA9, HOXA10, and MYB as critical drivers of CML progression, and suggest MYB as a key therapeutic target for inhibiting the self-renewal of leukemia-initiating cells in CML myeloid blast crisis patients.

6.
Cancer Chemother Pharmacol ; 57(1): 7-14, 2006 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-16001179

RESUMEN

BACKGROUND: Anaplastic thyroid cancer (ATC) is one of the most aggressive and highly lethal human cancers. Median survival after diagnosis is 4-6 months despite available radiotherapy and chemotherapy. Additional treatments are needed for ATC. Vascular endothelial growth factor (VEGF) is a potent angiogenic stimulus, which is expressed by ATC. Previously, anti-VEGF antibody was used to block VEGF-dependent angiogenesis in ATC xenografts. This treatment induced partial (56%) but not complete tumor regression. Aplidin (APLD) is a marine derived antitumor agent currently in phase II clinical studies. Multiple activities of this compound have been described which likely contribute to its antiproliferative effect. Notably, APLD has been shown to have antiangiogenic properties which include: inhibition of VEGF secretion, reduction in the synthesis of the VEGF receptor (FLT-1), and blockade of matrix metalloproteinase production by endothelial cells. We hypothesized that Aplidin, with its broad spectrum of action and antiangiogenic properties, would be a potentially effective drug against ATC. METHODS: Thirty BALB/c nu/nu mice were injected with ATC cells (ARO-81, 1 x 10(6)) and allowed to implant for 3 weeks. Animals were randomized to receive daily intraperitoneal injections of vehicle, low dose (0.5 mg/kg/day), or high dose (1.0 mg/kg/day) APLD. After 3 days, the animals were killed and the tumors were removed, weighed, and divided for RNA and protein analyses. RESULTS: APLD significantly reduced ATC xenograft growth (low dose, 20% reduction, P = 0.01; high dose, 40% reduction, P < 0.001). This was associated with increased levels of apoptosis related proteins polyadenosylribose polymerase 85 (PARP-85, 75% increase, P = 0.024) and caspase 8 (greater than fivefold increase, P = 0.03). APLD treatment was further associated with lost or reduced expression of several genes that support angiogenesis to include: VEGF, hypoxia inducible factor 1(HIF-1), transforming growth factor-beta (TGFbeta), TGFbeta receptor 2 (TGFbetaR2), melanoma growth stimulating factor 1 (GRO1), cadherin, and vasostatin. CONCLUSIONS: This data supports the hypothesis that APLD may be an effective adjunctive therapy against ATC. The demonstrated molecular impact against angiogenic related genes specifically supports future strategies combining APLD with VEGF interacting agents.


Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Antineoplásicos/uso terapéutico , Carcinoma/tratamiento farmacológico , Depsipéptidos/uso terapéutico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias de la Tiroides/tratamiento farmacológico , Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/farmacología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Western Blotting , Carcinoma/irrigación sanguínea , Carcinoma/genética , Depsipéptidos/administración & dosificación , Depsipéptidos/farmacología , Relación Dosis-Respuesta a Droga , Ratones , Ratones Endogámicos BALB C , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/genética , Péptidos Cíclicos , Neoplasias de la Tiroides/irrigación sanguínea , Neoplasias de la Tiroides/genética , Ensayos Antitumor por Modelo de Xenoinjerto
7.
Oncotarget ; 7(52): 86300-86312, 2016 Dec 27.
Artículo en Inglés | MEDLINE | ID: mdl-27863435

RESUMEN

SETBP1 missense mutations have been frequently identified in multiple myeloid neoplasms; however, their oncogenic potential remains unclear. Here we show that expression of Setbp1 mutants carrying two such mutations in mouse bone marrow progenitors efficiently induced development of acute myeloid leukemias (AMLs) in irradiated recipient mice with significantly shorter latencies and greater penetrance than expression of wild-type Setbp1, suggesting that these mutations are highly oncogenic. The increased oncogenicity of Setbp1 missense mutants could be due in part to their capability to drive significantly higher target gene transcription. We further identify Myb as a critical mediator of Setbp1-induced self-renewal as its knockdown caused efficient differentiation of myeloid progenitors immortalized by wild-type Setbp1 and Setbp1 missense mutants. Interestingly, Myb is also a direct transcriptional target of Setbp1 and Setbp1 missense mutants as they directly bind to the Myb locus in immortalized cells and dramatically activate a critical enhancer/promoter region of Myb in luciferase reporter assays. Furthermore, Myb knockdown in Setbp1 and Setbp1 missense mutations-induced AML cells also efficiently induced their differentiation in culture and significantly prolonged the survival of their secondary recipient mice, suggesting that targeting MYB pathway could be a promising strategy for treating human myeloid neoplasms with SETBP1 activation.


Asunto(s)
Proteínas Portadoras/fisiología , Leucemia Mieloide Aguda/etiología , Proteínas Nucleares/fisiología , Proteínas Proto-Oncogénicas c-myb/fisiología , Animales , Proteínas Portadoras/genética , Femenino , Proteínas Homeobox A10 , Proteínas de Homeodominio/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos C57BL , Mutación Missense , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas c-myb/antagonistas & inhibidores
8.
Thyroid ; 15(2): 105-13, 2005 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-15753667

RESUMEN

The immune response might suppress thyroid cancer recurrence. Although the factors that control this are unknown, CD-40 and CD-40 ligand might be important. To test this, we stained 36 papillary (PTC) and four follicular (FTC) thyroid carcinomas for CD-40 (n = 37) and CD-40 ligand (n = 36) and graded staining from absent (grade 0) to intense (grade 3). Follicular cells of the majority of thyroid tumors expressed CD-40 (30/37, 81%) and CD-40 ligand (15/24, 69%). Cancers from young patients (< or =21 years of age) that expressed CD-40 contained more numerous lymphocytes/high-power field (36 +/- 11) than cancers that failed to express CD-40 (4 +/- 3, p = 0.01), but there was no correlation with clinical outcome. Among young patients, CD-40 ligand expression was more intense in multifocal (1.1 +/- 0.2 vs. 0.45 +/- 0.2, p = 0.037), aggressive (1.14 +/- 0.14 vs. 0.65 +/- 0.2, p = 0.05) and recurrent tumors (1.2 +/- 0.2 vs. 0.65 +/- 0.2, p = 0.05) and associated with reduced disease-free survival (p = 0.03). We conclude that the majority of thyroid cancers express CD-40 and CD-40 ligand. In patients < or =21 years of age, tumors with intense expression of CD-40 ligand are more often multifocal, aggressive, and recurrent.


Asunto(s)
Antígenos CD40/metabolismo , Ligando de CD40/metabolismo , Carcinoma Papilar/metabolismo , Recurrencia Local de Neoplasia/metabolismo , Neoplasias de la Tiroides/metabolismo , Adenocarcinoma Folicular/epidemiología , Adenocarcinoma Folicular/inmunología , Adenocarcinoma Folicular/metabolismo , Adolescente , Adulto , Distribución por Edad , Antígenos CD40/genética , Ligando de CD40/genética , Carcinoma Papilar/epidemiología , Carcinoma Papilar/inmunología , Niño , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/inmunología , Humanos , Inmunohistoquímica , Masculino , Recurrencia Local de Neoplasia/epidemiología , Recurrencia Local de Neoplasia/inmunología , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Riesgo , Neoplasias de la Tiroides/epidemiología , Neoplasias de la Tiroides/inmunología
9.
Thyroid ; 15(4): 320-5, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15876153

RESUMEN

Mortality is low for young patients (younger than 21 years) with papillary thyroid cancer (PTC), and different mutations might contribute to this. Previous studies detected ret/PTC rearrangements more frequently in PTC from children than adults, and recent reports describe a high incidence of BRAF T1796A transversion in adult PTC. However, BRAF mutations have not been adequately studied in PTC from young patients. We amplified and sequenced segments of the BRAF gene spanning the T1796A transversion site in 14 PTC from patients 10-21 years of age (mean, 17.5 +/- 3.5 years). The PTC (7 = class 1; 5 = class 2; 1 = class 3) ranged from 0.7-2.9 cm in diameter (mean, 1.4 +/- 0.75 cm). None of them (0/14) contained BRAF T1796A and none recurred (mean follow-up, 66 +/- 40 months). This incidence of BRAF T1796A is significantly less than that reported for adult PTC (270/699, 38.6%, p = 0.0015) in several series. None of our PTC (0/10) contained ras mutations, but 7/12 (58%) contained ret/PTC rearrangements. We conclude that BRAF mutations are less common in PTC from young patients, and ret/PTC rearrangements were the most common mutation found in these childhood PTC.


Asunto(s)
Carcinoma Papilar/genética , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias de la Tiroides/genética , Adolescente , Adulto , Niño , Femenino , Reordenamiento Génico , Humanos , Masculino , Proteínas de Fusión Oncogénica/genética , Proteínas Tirosina Quinasas/genética
10.
Cancer Cell ; 27(5): 658-70, 2015 May 11.
Artículo en Inglés | MEDLINE | ID: mdl-25920683

RESUMEN

Most cases of adult myeloid neoplasms are routinely assumed to be sporadic. Here, we describe an adult familial acute myeloid leukemia (AML) syndrome caused by germline mutations in the DEAD/H-box helicase gene DDX41. DDX41 was also found to be affected by somatic mutations in sporadic cases of myeloid neoplasms as well as in a biallelic fashion in 50% of patients with germline DDX41 mutations. Moreover, corresponding deletions on 5q35.3 present in 6% of cases led to haploinsufficient DDX41 expression. DDX41 lesions caused altered pre-mRNA splicing and RNA processing. DDX41 is exemplary of other RNA helicase genes also affected by somatic mutations, suggesting that they constitute a family of tumor suppressor genes.


Asunto(s)
ARN Helicasas DEAD-box/genética , Mutación de Línea Germinal , Leucemia Mieloide Aguda/genética , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Animales , ARN Helicasas DEAD-box/química , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Linaje , Empalme del ARN , Homología de Secuencia de Aminoácido
11.
Methods Mol Biol ; 1194: 313-25, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25064111

RESUMEN

Long-term hematopoietic stem cells (LT-HSCs) have the ability to self-renew and differentiate into all blood cell lineages. Understanding the genetic networks that regulate LT-HSC function in the adult bone marrow requires inducible gene targeting and bone marrow transplantations. In this chapter we describe the use of the inducible Mx1-Cre mouse model to delete genes in LT-HSCs and methodologies for examining the function of LT-HSCs following deletion.


Asunto(s)
Células Madre Adultas/metabolismo , Marcación de Gen/métodos , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Integrasas/metabolismo , Interferones/farmacología , Proteínas de Resistencia a Mixovirus/genética , Animales , Trasplante de Médula Ósea , Citometría de Flujo , Técnicas de Genotipaje , Células Madre Hematopoyéticas/citología , Inyecciones , Ratones , Mutagénesis Sitio-Dirigida/métodos , Poli C/farmacología
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