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1.
Blood ; 139(7): 1039-1051, 2022 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-34767620

RESUMEN

Human telomere biology disorders (TBD)/short telomere syndromes (STS) are heterogeneous disorders caused by inherited loss-of-function mutations in telomere-associated genes. Here, we identify 3 germline heterozygous missense variants in the RPA1 gene in 4 unrelated probands presenting with short telomeres and varying clinical features of TBD/STS, including bone marrow failure, myelodysplastic syndrome, T- and B-cell lymphopenia, pulmonary fibrosis, or skin manifestations. All variants cluster to DNA-binding domain A of RPA1 protein. RPA1 is a single-strand DNA-binding protein required for DNA replication and repair and involved in telomere maintenance. We showed that RPA1E240K and RPA1V227A proteins exhibit increased binding to single-strand and telomeric DNA, implying a gain in DNA-binding function, whereas RPA1T270A has binding properties similar to wild-type protein. To study the mutational effect in a cellular system, CRISPR/Cas9 was used to knock-in the RPA1E240K mutation into healthy inducible pluripotent stem cells. This resulted in severe telomere shortening and impaired hematopoietic differentiation. Furthermore, in patients with RPA1E240K, we discovered somatic genetic rescue in hematopoietic cells due to an acquired truncating cis RPA1 mutation or a uniparental isodisomy 17p with loss of mutant allele, coinciding with stabilized blood counts. Using single-cell sequencing, the 2 somatic genetic rescue events were proven to be independently acquired in hematopoietic stem cells. In summary, we describe the first human disease caused by germline RPA1 variants in individuals with TBD/STS.


Asunto(s)
Trastornos de Fallo de la Médula Ósea/patología , Mutación con Ganancia de Función , Heterocigoto , Síndromes Mielodisplásicos/patología , Proteína de Replicación A/genética , Acortamiento del Telómero , Telómero/genética , Adolescente , Adulto , Trastornos de Fallo de la Médula Ósea/etiología , Trastornos de Fallo de la Médula Ósea/metabolismo , Diferenciación Celular , Niño , Femenino , Humanos , Recién Nacido , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/etiología , Síndromes Mielodisplásicos/metabolismo , Adulto Joven
2.
Mol Ther ; 26(1): 289-303, 2018 01 03.
Artículo en Inglés | MEDLINE | ID: mdl-29055620

RESUMEN

Existing recombinant adeno-associated virus (rAAV) serotypes for delivering in vivo gene therapy treatments for human liver diseases have not yielded combined high-level human hepatocyte transduction and favorable humoral neutralization properties in diverse patient groups. Yet, these combined properties are important for therapeutic efficacy. To bioengineer capsids that exhibit both unique seroreactivity profiles and functionally transduce human hepatocytes at therapeutically relevant levels, we performed multiplexed sequential directed evolution screens using diverse capsid libraries in both primary human hepatocytes in vivo and with pooled human sera from thousands of patients. AAV libraries were subjected to five rounds of in vivo selection in xenografted mice with human livers to isolate an enriched human-hepatotropic library that was then used as input for a sequential on-bead screen against pooled human immunoglobulins. Evolved variants were vectorized and validated against existing hepatotropic serotypes. Two of the evolved AAV serotypes, NP40 and NP59, exhibited dramatically improved functional human hepatocyte transduction in vivo in xenografted mice with human livers, along with favorable human seroreactivity profiles, compared with existing serotypes. These novel capsids represent enhanced vector delivery systems for future human liver gene therapy applications.


Asunto(s)
Proteínas de la Cápside/genética , Dependovirus/genética , Ingeniería Genética , Vectores Genéticos/genética , Hígado/metabolismo , Transducción Genética , Animales , Proteínas de la Cápside/química , Femenino , Técnicas de Transferencia de Gen , Hepatocitos/metabolismo , Xenoinjertos , Humanos , Masculino , Ratones , Modelos Moleculares , Conformación Proteica
3.
PLoS Genet ; 11(2): e1004951, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25659124

RESUMEN

Many recurrent chromosome translocations in cancer result in the generation of fusion genes that are directly implicated in the tumorigenic process. Precise modeling of the effects of cancer fusion genes in mice has been inaccurate, as constructs of fusion genes often completely or partially lack the correct regulatory sequences. The reciprocal t(2;13)(q36.1;q14.1) in human alveolar rhabdomyosarcoma (A-RMS) creates a pathognomonic PAX3-FOXO1 fusion gene. In vivo mimicking of this translocation in mice is complicated by the fact that Pax3 and Foxo1 are in opposite orientation on their respective chromosomes, precluding formation of a functional Pax3-Foxo1 fusion via a simple translocation. To circumvent this problem, we irreversibly inverted the orientation of a 4.9 Mb syntenic fragment on chromosome 3, encompassing Foxo1, by using Cre-mediated recombination of two pairs of unrelated oppositely oriented LoxP sites situated at the borders of the syntenic region. We tested if spatial proximity of the Pax3 and Foxo1 loci in myoblasts of mice homozygous for the inversion facilitated Pax3-Foxo1 fusion gene formation upon induction of targeted CRISPR-Cas9 nuclease-induced DNA double strand breaks in Pax3 and Foxo1. Fluorescent in situ hybridization indicated that fore limb myoblasts show a higher frequency of Pax3/Foxo1 co-localization than hind limb myoblasts. Indeed, more fusion genes were generated in fore limb myoblasts via a reciprocal t(1;3), which expressed correctly spliced Pax3-Foxo1 mRNA encoding Pax3-Foxo1 fusion protein. We conclude that locus proximity facilitates chromosome translocation upon induction of DNA double strand breaks. Given that the Pax3-Foxo1 fusion gene will contain all the regulatory sequences necessary for precise regulation of its expression, we propose that CRISPR-Cas9 provides a novel means to faithfully model human diseases caused by chromosome translocation in mice.


Asunto(s)
Proteínas de Fusión Oncogénica/genética , Factores de Transcripción Paired Box/genética , Rabdomiosarcoma Alveolar/genética , Translocación Genética/genética , Animales , Sistemas CRISPR-Cas , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Ratones , Mioblastos/metabolismo , Mioblastos/patología , ARN Mensajero/biosíntesis , Rabdomiosarcoma Alveolar/metabolismo , Rabdomiosarcoma Alveolar/patología
4.
J Neurosci ; 32(31): 10530-40, 2012 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-22855803

RESUMEN

Sox2 plays critical roles in cell fate specification during development and in stem cell formation; however, its role in postmitotic cells is largely unknown. Sox2 is highly expressed in supporting cells (SCs) of the postnatal mammalian auditory sensory epithelium, which unlike non-mammalian vertebrates remains quiescent even after sensory hair cell damage. Here, we induced the ablation of Sox2, specifically in SCs at three different postnatal ages (neonatal, juvenile and adult) in mice. In neonatal mice, Sox2-null inner pillar cells (IPCs, a subtype of SCs) proliferated and generated daughter cells, while other SC subtypes remained quiescent. Furthermore, p27(Kip1), a cell cycle inhibitor, was absent in Sox2-null IPCs. Similarly, upon direct deletion of p27(Kip1), p27(Kip1)-null IPCs also proliferated but retained Sox2 expression. Interestingly, cell cycle control of IPCs by Sox2-mediated expression of p27(Kip1) gradually declined with age. In addition, deletion of Sox2 or p27(Kip1) did not cause a cell fate change. Finally, chromatin immunoprecipitation with Sox2 antibodies and luciferase reporter assays with the p27(Kip1) promoter support that Sox2 directly activates p27(Kip1) transcription in postmitotic IPCs. Hence, in contrast to the well known activity of Sox2 in promoting proliferation and cell fate determination, our data demonstrate that Sox2 plays a novel role as a key upstream regulator of p27(Kip1) to maintain the quiescent state of postmitotic IPCs. Our studies suggest that manipulating Sox2 or p27(Kip1) expression is an effective approach to inducing proliferation of neonatal auditory IPCs, an initial but necessary step toward restoring hearing in mammals.


Asunto(s)
Cóclea/citología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Células Ciliadas Auditivas/metabolismo , Células Laberínticas de Soporte/fisiología , Factores de Transcripción SOXB1/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Bromodesoxiuridina/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular Transformada , Proliferación Celular/efectos de los fármacos , Inmunoprecipitación de Cromatina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Desoxiuridina/análogos & derivados , Desoxiuridina/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio/genética , Humanos , Etiquetado Corte-Fin in Situ , Proteínas Luminiscentes/genética , Ratones , Ratones Transgénicos , Cadenas Pesadas de Miosina/metabolismo , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/metabolismo , Factores de Transcripción SOXB1/genética , Tamoxifeno/farmacología , Transfección , Proteínas Supresoras de Tumor/genética
5.
Mol Ther ; 19(11): 1950-60, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21629224

RESUMEN

Intrauterine gene transfer (IUGT) offers ontological advantages including immune naiveté mediating tolerance to the vector and transgenic products, and effecting a cure before development of irreversible pathology. Despite proof-of-principle in rodent models, expression efficacy with a therapeutic transgene has yet to be demonstrated in a preclinical nonhuman primate (NHP) model. We aimed to determine the efficacy of human Factor IX (hFIX) expression after adeno-associated-viral (AAV)-mediated IUGT in NHP. We injected 1.0-1.95 × 10(13) vector genomes (vg)/kg of self-complementary (sc) AAV5 and 8 with a LP1-driven hFIX transgene intravenously in 0.9G late gestation NHP fetuses, leading to widespread transduction with liver tropism. Liver-specific hFIX expression was stably maintained between 8 and 112% of normal activity in injected offspring followed up for 2-22 months. AAV8 induced higher hFIX expression (P = 0.005) and milder immune response than AAV5. Random hepatocellular integration was found with no hotspots. Transplacental spread led to low-level maternal tissue transduction, without evidence of immunotoxicity or germline transduction in maternal oocytes. A single intravenous injection of scAAV-LP1-hFIXco to NHP fetuses in late-gestation produced sustained clinically-relevant levels of hFIX with liver-specific expression and a non-neutralizing immune response. These data are encouraging for conditions where gene transfer has the potential to avert perinatal death and long-term irreversible sequelae.


Asunto(s)
Dependovirus/genética , Factor IX/genética , Regulación de la Expresión Génica , Técnicas de Transferencia de Gen , Terapia Genética , Vectores Genéticos/genética , Hemofilia B/terapia , Animales , Línea Celular , Dependovirus/inmunología , Femenino , Vectores Genéticos/administración & dosificación , Vectores Genéticos/farmacocinética , Células HEK293 , Hemofilia B/genética , Humanos , Inyecciones , Macaca fascicularis , Placenta/metabolismo , Embarazo , Transducción Genética , Integración Viral
6.
Blood Cancer Discov ; 3(3): 194-207, 2022 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-35176137

RESUMEN

The genetics of relapsed pediatric acute myeloid leukemia (AML) has yet to be comprehensively defined. Here, we present the spectrum of genomic alterations in 136 relapsed pediatric AMLs. We identified recurrent exon 13 tandem duplications (TD) in upstream binding transcription factor (UBTF) in 9% of relapsed AML cases. UBTF-TD AMLs commonly have normal karyotype or trisomy 8 with cooccurring WT1 mutations or FLT3-ITD but not other known oncogenic fusions. These UBTF-TD events are stable during disease progression and are present in the founding clone. In addition, we observed that UBTF-TD AMLs account for approximately 4% of all de novo pediatric AMLs, are less common in adults, and are associated with poor outcomes and MRD positivity. Expression of UBTF-TD in primary hematopoietic cells is sufficient to enhance serial clonogenic activity and to drive a similar transcriptional program to UBTF-TD AMLs. Collectively, these clinical, genomic, and functional data establish UBTF-TD as a new recurrent mutation in AML. SIGNIFICANCE: We defined the spectrum of mutations in relapsed pediatric AML and identified UBTF-TDs as a new recurrent genetic alteration. These duplications are more common in children and define a group of AMLs with intermediate-risk cytogenetic abnormalities, FLT3-ITD and WT1 alterations, and are associated with poor outcomes. See related commentary by Hasserjian and Nardi, p. 173. This article is highlighted in the In This Issue feature, p. 171.


Asunto(s)
Leucemia Mieloide Aguda , Adulto , Niño , Aberraciones Cromosómicas , Exones , Genómica , Humanos , Leucemia Mieloide Aguda/genética , Mutación , Recurrencia
7.
J Neurosci ; 30(17): 5927-36, 2010 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-20427652

RESUMEN

Cochlear hair cells (HCs) are mechanosensory receptors that transduce sound into electrical signals. HC damage in nonmammalian vertebrates induces surrounding supporting cells (SCs) to divide, transdifferentiate and replace lost HCs; however, such spontaneous HC regeneration does not occur in the mammalian cochlea. Here, we acutely ablate the retinoblastoma protein (Rb), a crucial cell cycle regulator, in two subtypes of postmitotic SCs (pillar and Deiters' cells) using an inducible Cre line, Prox1-CreER(T2). Inactivation of Rb in these SCs results in cell cycle reentry of both pillar and Deiters' cells, and completion of cell division with an increase in cell number of pillar cells. Interestingly, nuclei of Rb(-/-) mitotic pillar and Deiters' cells migrate toward the HC layer and divide near the epithelial surface in a manner similar to the SCs in the regenerating avian auditory epithelium. In contrast to postmitotic Rb(-/-) HCs which abort cell division, postmitotic Rb(-/-) pillar cells can proliferate, maintain their SC fate and survive for more than a week. However, no newly formed HCs are detected and SC death followed by HC loss occurs. Our studies accomplish a crucial step toward functional HC regeneration in the mammalian cochlea in vivo, demonstrating the critical role of Rb in maintaining quiescence of postmitotic pillar and Deiters' cells and highlighting the heterogeneity between these two cell types. Therefore, the combination of transient Rb inactivation and further manipulation of transcription factors (i.e., Atoh1 activation) in SCs may represent an effective therapeutic avenue for HC regeneration in the mammalian cochlea.


Asunto(s)
Proliferación Celular , Cóclea/fisiología , Mitosis/fisiología , Proteína de Retinoblastoma/metabolismo , Animales , Animales Recién Nacidos , Muerte Celular/fisiología , Movimiento Celular/fisiología , Núcleo Celular/fisiología , Supervivencia Celular/fisiología , Epitelio/fisiología , Células Ciliadas Auditivas/fisiología , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteína de Retinoblastoma/deficiencia , Proteína de Retinoblastoma/genética , Factores de Tiempo
8.
Proc Natl Acad Sci U S A ; 105(2): 781-5, 2008 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-18178626

RESUMEN

Unlike lower vertebrates, mammals are unable to replace damaged mechanosensory hair cells (HCs) in the cochlea. Recently, ablation of the retinoblastoma protein (Rb) in undifferentiated mouse HC precursors was shown to cause cochlear HC proliferation and the generation of new HCs, raising the hope that inactivation of Rb in postmitotic HCs could trigger cell division and regenerate functional HCs postnatally. Here, we acutely inactivated Rb in nearly all cochlear HCs of newborn mice, using a newly developed HC-specific inducible Cre mouse line. Beginning 48 h after Rb deletion, approximately 40% of HCs were in the S and M phases of the cell cycle, demonstrating an overriding role for Rb in maintaining the quiescent state of postnatal HCs. Unlike Rb-null HC precursors, such HCs failed to undergo cell division and died rapidly. HC clusters were restricted to the less differentiated cochlear regions, consistent with differentiation-dependent roles of Rb. Moreover, outer HCs expressed the maturation marker prestin, suggesting an embryonic time window for Rb-dependent HC specification. We conclude that Rb plays essential and age-dependent roles during HC proliferation and differentiation, and, in contrast to previous hypotheses, cell death after forced cell-cycle reentry presents a major challenge for mammalian HC regeneration from residual postnatal HCs.


Asunto(s)
Genes de Retinoblastoma , Células Ciliadas Auditivas/fisiología , Proteína de Retinoblastoma/metabolismo , Animales , Animales Recién Nacidos , Ciclo Celular , Proliferación Celular , Cromatina/química , Células Ciliadas Auditivas/metabolismo , Humanos , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Mitosis , Proteínas Motoras Moleculares/biosíntesis , Recombinación Genética , Regeneración
9.
Cancer Discov ; 11(11): 2846-2867, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34103329

RESUMEN

Lineage-ambiguous leukemias are high-risk malignancies of poorly understood genetic basis. Here, we describe a distinct subgroup of acute leukemia with expression of myeloid, T lymphoid, and stem cell markers driven by aberrant allele-specific deregulation of BCL11B, a master transcription factor responsible for thymic T-lineage commitment and specification. Mechanistically, this deregulation was driven by chromosomal rearrangements that juxtapose BCL11B to superenhancers active in hematopoietic progenitors, or focal amplifications that generate a superenhancer from a noncoding element distal to BCL11B. Chromatin conformation analyses demonstrated long-range interactions of rearranged enhancers with the expressed BCL11B allele and association of BCL11B with activated hematopoietic progenitor cell cis-regulatory elements, suggesting BCL11B is aberrantly co-opted into a gene regulatory network that drives transformation by maintaining a progenitor state. These data support a role for ectopic BCL11B expression in primitive hematopoietic cells mediated by enhancer hijacking as an oncogenic driver of human lineage-ambiguous leukemia. SIGNIFICANCE: Lineage-ambiguous leukemias pose significant diagnostic and therapeutic challenges due to a poorly understood molecular and cellular basis. We identify oncogenic deregulation of BCL11B driven by diverse structural alterations, including de novo superenhancer generation, as the driving feature of a subset of lineage-ambiguous leukemias that transcend current diagnostic boundaries.This article is highlighted in the In This Issue feature, p. 2659.


Asunto(s)
Elementos de Facilitación Genéticos , Leucemia Mieloide Aguda , Proteínas Represoras , Proteínas Supresoras de Tumor , Redes Reguladoras de Genes , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/genética
10.
J Clin Oncol ; 22(18): 3790-7, 2004 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-15365076

RESUMEN

PURPOSE: Osteosarcoma is distinct from most cancers in that the majority of osteosarcomas lack telomerase expression and use the alternative lengthening of telomeres (ALT) mechanism to maintain telomeres. Laboratory studies suggest that compared with ALT, telomerase expression is associated with increased tumor aggressiveness. We evaluated the clinical significance of telomerase expression in human osteosarcoma. PATIENTS AND METHODS: Fifty-six osteosarcomas from 51 patients treated at St Jude Children's Research Hospital between 1982 and 2003 were evaluated for telomerase enzyme activity, mRNA expression of the catalytic component of telomerase (TERT), and presence of the ALT pathway. RESULTS: Outcome analysis was based on TERT mRNA expression in the primary tumor samples from 44 patients. Fourteen primary tumors expressed TERT mRNA (32%; eight TERT only, six TERT and ALT) and 30 did not express TERT mRNA (68%; 29 ALT, one no ALT). Progression-free survival (PFS) was inferior in the TERT-positive group compared with the TERT-negative group (3-year estimates, 21.4% +/- 9.5% v 63.7% +/- 11.1%; P =.014). Likewise, overall survival was inferior in the TERT-positive group compared with the TERT-negative group (3-year estimates, 42.9% +/- 12.2% v 70.0% +/- 9.9%; P =.031). Among 31 patients with nonmetastatic disease at diagnosis, PFS was lower in the TERT-positive group compared with the TERT-negative group (3-year estimates, 33.3% +/- 13.6% v 72.0% +/- 11.5%; P =.092). CONCLUSION: Telomerase expression in primary tumor samples is associated with decreased PFS and OS in patients with osteosarcoma. Additional studies are warranted to better define the clinical utility of this molecular marker.


Asunto(s)
Neoplasias Óseas/genética , Neoplasias Óseas/patología , Osteosarcoma/genética , Osteosarcoma/patología , Telomerasa/biosíntesis , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Masculino , Valor Predictivo de las Pruebas , ARN Mensajero/biosíntesis , Estudios Retrospectivos , Análisis de Supervivencia , Telomerasa/análisis
11.
PLoS One ; 8(12): e82513, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24349301

RESUMEN

Neuroblastoma is an embryonal tumor of childhood with a heterogenous clinical presentation that reflects differences in activation of complex biological signaling pathways. Protein phosphorylation is a key component of cellular signal transduction and plays a critical role in processes that control cancer cell growth and survival. We used shotgun LC/MS to compare phosphorylation between a human MYCN amplified neuroblastoma cell line (NB10), modeling a resistant tumor, and a human neural precursor cell line (NPC), modeling a normal baseline neural crest cell. 2181 unique phosphorylation sites representing 1171 proteins and 2598 phosphopeptides were found. Protein kinases accounted for 6% of the proteome, with a predominance of tyrosine kinases, supporting their prominent role in oncogenic signaling pathways. Highly abundant receptor tyrosine kinase (RTK) phosphopeptides in the NB10 cell line relative to the NPC cell line included RET, insulin-like growth factor 1 receptor/insulin receptor (IGF-1R/IR), and fibroblast growth factor receptor 1 (FGFR1). Multiple phosphorylated peptides from downstream mediators of the PI3K/AKT/mTOR and RAS pathways were also highly abundant in NB10 relative to NPC. Our analysis highlights the importance of RET, IGF-1R/IR and FGFR1 as RTKs in neuroblastoma and suggests a methodology that can be used to identify potential novel biological therapeutic targets. Furthermore, application of this previously unexploited technology in the clinic opens the possibility of providing a new wide-scale molecular signature to assess disease progression and prognosis.


Asunto(s)
Neuroblastoma/metabolismo , Fosfoproteínas/metabolismo , Proteómica , Proteínas Proto-Oncogénicas c-ret/genética , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Transducción de Señal , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas , Anotación de Secuencia Molecular , Células-Madre Neurales/metabolismo , Neuroblastoma/genética , Fosfatidilinositol 3-Quinasas , Fosfoproteínas/genética , Mapas de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , Proteoma , Proteómica/métodos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-ret/antagonistas & inhibidores , Receptor IGF Tipo 1/antagonistas & inhibidores , Receptor de Insulina/antagonistas & inhibidores , Quinasas raf/metabolismo
12.
Cancer Res ; 73(13): 4086-97, 2013 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-23536557

RESUMEN

Neuroblastoma, the most common extracranial pediatric solid tumor, is responsible for 15% of all childhood cancer deaths. Patients frequently present at diagnosis with metastatic disease, particularly to the bone marrow. Advances in therapy and understanding of the metastatic process have been limited due, in part, to the lack of animal models harboring bone marrow disease. The widely used transgenic model, the Th-MYCN mouse, exhibits limited metastasis to this site. Here, we establish the first genetic immunocompetent mouse model for metastatic neuroblastoma with enhanced secondary tumors in the bone marrow. This model recapitulates 2 frequent alterations in metastatic neuroblastoma, overexpression of MYCN and loss of caspase-8 expression. Mouse caspase-8 gene was deleted in neural crest lineage cells by crossing a Th-Cre transgenic mouse with a caspase-8 conditional knockout mouse. This mouse was then crossed with the neuroblastoma prone Th-MYCN mouse. Although overexpression of MYCN by itself rarely caused bone marrow metastasis, combining MYCN overexpression and caspase-8 deletion significantly enhanced bone marrow metastasis (37% incidence). Microarray expression studies of the primary tumors mRNAs and microRNAs revealed extracellular matrix structural changes, increased expression of genes involved in epithelial to mesenchymal transition, inflammation, and downregulation of miR-7a and miR-29b. These molecular changes have been shown to be associated with tumor progression and activation of the cytokine TGF-ß pathway in various tumor models. Cytokine TGF-ß can preferentially promote single cell motility and blood-borne metastasis and therefore activation of this pathway may explain the enhanced bone marrow metastasis observed in this animal model.


Asunto(s)
Neoplasias de la Médula Ósea/enzimología , Caspasa 8/genética , Ganglioneuroblastoma/enzimología , Neoplasias del Sistema Nervioso Periférico/enzimología , Proteínas Proto-Oncogénicas/genética , Animales , Neoplasias de la Médula Ósea/genética , Neoplasias de la Médula Ósea/secundario , Caspasa 8/metabolismo , Modelos Animales de Enfermedad , Ganglioneuroblastoma/genética , Ganglioneuroblastoma/secundario , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , MicroARNs/genética , MicroARNs/metabolismo , Proteína Proto-Oncogénica N-Myc , Neoplasias del Sistema Nervioso Periférico/genética , Neoplasias del Sistema Nervioso Periférico/patología , Transcriptoma
13.
Cancer Cell ; 24(6): 710-24, 2013 Dec 09.
Artículo en Inglés | MEDLINE | ID: mdl-24332040

RESUMEN

Rhabdomyosarcoma is a soft-tissue sarcoma with molecular and cellular features of developing skeletal muscle. Rhabdomyosarcoma has two major histologic subtypes, embryonal and alveolar, each with distinct clinical, molecular, and genetic features. Genomic analysis shows that embryonal tumors have more structural and copy number variations than alveolar tumors. Mutations in the RAS/NF1 pathway are significantly associated with intermediate- and high-risk embryonal rhabdomyosarcomas (ERMS). In contrast, alveolar rhabdomyosarcomas (ARMS) have fewer genetic lesions overall and no known recurrently mutated cancer consensus genes. To identify therapeutics for ERMS, we developed and characterized orthotopic xenografts of tumors that were sequenced in our study. High-throughput screening of primary cultures derived from those xenografts identified oxidative stress as a pathway of therapeutic relevance for ERMS.


Asunto(s)
Estrés Oxidativo , Rabdomiosarcoma Embrionario/genética , Animales , Evolución Clonal , Dosificación de Gen , Homeostasis , Humanos , Pérdida de Heterocigocidad , Ratones , Mutación , Rabdomiosarcoma Embrionario/tratamiento farmacológico , Rabdomiosarcoma Embrionario/metabolismo
15.
Cell Cycle ; 6(13): 1646-54, 2007 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-17611414

RESUMEN

Human DDX11 and DDX12 are closely related genes encoding the helicases ChlR1 and ChlR2, which belong to the CHL1 DNA helicase family. Recently, it was shown that human ChlR1 interacts with components of the cohesin complex and is required for proper centromeric cohesion. To establish the function of ChlR1 in development we made a mutant mouse lacking Ddx11, the single mouse ChlR gene. The absence of Ddx11 resulted in embryonic lethality at E10.5. The mutant embryos were smaller in size, malformed and exhibited sparse cellularity in comparison to normal or heterozygous litter mates. Importantly, loss of Ddx11 resulted in the inability to form a proper placenta, indicating that ChlR1 is essential for placental formation. Detailed analysis of cells isolated from Ddx11-/- embryos revealed a G2/M cell cycle delay, an increased frequency of chromosome missegregation, decreased chromosome cohesion, and increased aneuploidy. To examine whether ChlR proteins are required for arm cohesion and for loading of the cohesin complex, further studies were preformed in ChlR1 siRNA treated cells. These studies revealed that ChlR1 is required for proper sister chromatid arm cohesion and that cohesin complexes bind more loosely to chromatin in the absence of ChlR1. Taken together, these studies provide the first data indicating that the ChlR1 helicase is essential for proper binding of the cohesin complex to both the centromere and the chromosome arms, and indicate that ChlR1 is essential for embryonic development and the prevention of aneuploidy in mammals.


Asunto(s)
Aneuploidia , ARN Helicasas DEAD-box/genética , Desarrollo Embrionario/genética , Placenta/anomalías , Preñez , Intercambio de Cromátides Hermanas/genética , Animales , Apoptosis/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/genética , Femenino , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitosis/genética , Proteínas Nucleares/metabolismo , Embarazo , Preñez/genética , Cohesinas
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