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1.
Blood Adv ; 3(7): 1039-1046, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30940639

RESUMO

Recent studies have identified germline mutations in TP53, PAX5, ETV6, and IKZF1 in kindreds with familial acute lymphoblastic leukemia (ALL), but the genetic basis of ALL in many kindreds is unknown despite mutational analysis of the exome. Here, we report a germline deletion of ETV6 identified by linkage and structural variant analysis of whole-genome sequencing data segregating in a kindred with thrombocytopenia, B-progenitor acute lymphoblastic leukemia, and diffuse large B-cell lymphoma. The 75-nt deletion removed the ETV6 exon 7 splice acceptor, resulting in exon skipping and protein truncation. The ETV6 deletion was also identified by optimal structural variant analysis of exome sequencing data. These findings identify a new mechanism of germline predisposition in ALL and implicate ETV6 germline variation in predisposition to lymphoma. Importantly, these data highlight the importance of germline structural variant analysis in the search for germline variants predisposing to familial leukemia.

2.
Nat Genet ; 51(4): 694-704, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30926971

RESUMO

Acute erythroid leukemia (AEL) is a high-risk leukemia of poorly understood genetic basis, with controversy regarding diagnosis in the spectrum of myelodysplasia and myeloid leukemia. We compared genomic features of 159 childhood and adult AEL cases with non-AEL myeloid disorders and defined five age-related subgroups with distinct transcriptional profiles: adult, TP53 mutated; NPM1 mutated; KMT2A mutated/rearranged; adult, DDX41 mutated; and pediatric, NUP98 rearranged. Genomic features influenced outcome, with NPM1 mutations and HOXB9 overexpression being associated with a favorable prognosis and TP53, FLT3 or RB1 alterations associated with poor survival. Targetable signaling mutations were present in 45% of cases and included recurrent mutations of ALK and NTRK1, the latter of which drives erythroid leukemogenesis sensitive to TRK inhibition. This genomic landscape of AEL provides the framework for accurate diagnosis and risk stratification of this disease, and the rationale for testing targeted therapies in this high-risk leukemia.


Assuntos
Leucemia Eritroblástica Aguda/genética , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Genômica/métodos , Proteínas de Homeodomínio/genética , Humanos , Lactente , Recém-Nascido , Masculino , Mutação/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Nucleares/genética , Prognóstico , Proteína Supressora de Tumor p53/genética , Adulto Jovem , Tirosina Quinase 3 Semelhante a fms/genética
3.
Nat Genet ; 51(2): 296-307, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30643249

RESUMO

Recent genomic studies have identified chromosomal rearrangements defining new subtypes of B-progenitor acute lymphoblastic leukemia (B-ALL), however many cases lack a known initiating genetic alteration. Using integrated genomic analysis of 1,988 childhood and adult cases, we describe a revised taxonomy of B-ALL incorporating 23 subtypes defined by chromosomal rearrangements, sequence mutations or heterogeneous genomic alterations, many of which show marked variation in prevalence according to age. Two subtypes have frequent alterations of the B lymphoid transcription-factor gene PAX5. One, PAX5alt (7.4%), has diverse PAX5 alterations (rearrangements, intragenic amplifications or mutations); a second subtype is defined by PAX5 p.Pro80Arg and biallelic PAX5 alterations. We show that p.Pro80Arg impairs B lymphoid development and promotes the development of B-ALL with biallelic Pax5 alteration in vivo. These results demonstrate the utility of transcriptome sequencing to classify B-ALL and reinforce the central role of PAX5 as a checkpoint in B lymphoid maturation and leukemogenesis.


Assuntos
Fator de Transcrição PAX5/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Doença Aguda , Adolescente , Adulto , Idoso , Animais , Criança , Pré-Escolar , Cromossomos/genética , Feminino , Rearranjo Gênico/genética , Humanos , Lactente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Mutação/genética , Transcriptoma/genética , Adulto Jovem
4.
Cancer Cell ; 35(1): 140-155.e7, 2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-30595505

RESUMO

Diffuse intrinsic pontine gliomas (DIPGs) are incurable childhood brainstem tumors with frequent histone H3 K27M mutations and recurrent alterations in PDGFRA and TP53. We generated genetically engineered inducible mice and showed that H3.3 K27M enhanced neural stem cell self-renewal while preserving regional identity. Neonatal induction of H3.3 K27M cooperated with activating platelet-derived growth factor receptor α (PDGFRα) mutant and Trp53 loss to accelerate development of diffuse brainstem gliomas that recapitulated human DIPG gene expression signatures and showed global changes in H3K27 posttranslational modifications, but relatively restricted gene expression changes. Genes upregulated in H3.3 K27M tumors were enriched for those associated with neural development where H3K27me3 loss released the poised state of apparently bivalent promoters, whereas downregulated genes were enriched for those encoding homeodomain transcription factors.


Assuntos
Neoplasias do Tronco Encefálico/genética , Perfilação da Expressão Gênica/métodos , Glioma/genética , Histonas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Proteína Supressora de Tumor p53/genética , Animais , Autorrenovação Celular , Células Cultivadas , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Camundongos , Mutação , Células-Tronco Neurais/citologia , Rombencéfalo/patologia , Análise de Sequência de RNA/métodos
5.
Cancer Chemother Pharmacol ; 82(2): 251-263, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29855693

RESUMO

PURPOSE: The anaplastic lymphoma kinase (ALK) has been demonstrated to be a valid clinical target in diseases such as anaplastic large cell lymphoma and non-small cell lung cancer. Recent studies have indicated that ALK is overexpressed in pediatric rhabdomyosarcoma (RMS) and hence we hypothesized that this kinase may be a suitable candidate for therapeutic intervention in this tumor. METHODS: We evaluated the expression of ALK in a panel of pediatric RMS cell lines and patient-derived xenografts (PDX), and sensitivity to ALK inhibitors was assessed both in vitro and in vivo. RESULTS: Essentially, all RMS lines were sensitive to crizotinib, NVP-TAE684 or LDK-378 in vitro, and molecular analyses demonstrated inhibition of RMS cell proliferation following siRNA-mediated reduction of ALK expression. However, in vivo PDX studies using ALK kinase inhibitors demonstrated no antitumor activity when used as single agents or when combined with standard of care therapy (vincristine, actinomycin D and cyclophosphamide). More alarmingly, however, crizotinib actually accelerated the growth of these tumors in vivo. CONCLUSIONS: While ALK appears to be a relevant target in RMS in vitro, targeting this kinase in vivo yields no therapeutic efficacy, warranting extreme caution when considering the use of these agents in pediatric RMS patients.


Assuntos
Quinase do Linfoma Anaplásico/antagonistas & inibidores , Quinase do Linfoma Anaplásico/biossíntese , Rabdomiossarcoma/tratamento farmacológico , Rabdomiossarcoma/enzimologia , Quinase do Linfoma Anaplásico/genética , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Crizotinibe/administração & dosagem , Crizotinibe/farmacologia , Ciclofosfamida/administração & dosagem , Dactinomicina/administração & dosagem , Interações Medicamentosas , Humanos , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/biossíntese , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-met/biossíntese , Proteínas Proto-Oncogênicas c-met/genética , Pirimidinas/farmacologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Rabdomiossarcoma/genética , Transfecção , Vincristina/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Cancer Cell ; 33(5): 937-948.e8, 2018 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-29681510

RESUMO

Somatic genetic alterations of IKZF1, which encodes the lymphoid transcription factor IKAROS, are common in high-risk B-progenitor acute lymphoblastic leukemia (ALL) and are associated with poor prognosis. Such alterations result in the acquisition of stem cell-like features, overexpression of adhesion molecules causing aberrant cell-cell and cell-stroma interaction, and decreased sensitivity to tyrosine kinase inhibitors. Here we report coding germline IKZF1 variation in familial childhood ALL and 0.9% of presumed sporadic B-ALL, identifying 28 unique variants in 45 children. The majority of variants adversely affected IKZF1 function and drug responsiveness of leukemic cells. These results identify IKZF1 as a leukemia predisposition gene, and emphasize the importance of germline genetic variation in the development of both familial and sporadic ALL.


Assuntos
Mutação em Linhagem Germinativa , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Animais , Criança , Feminino , Mutação da Fase de Leitura , Predisposição Genética para Doença , Humanos , Masculino , Camundongos , Transplante de Neoplasias , Linhagem , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Análise de Sequência de DNA
7.
Cancer Res ; 77(17): 4626-4638, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28684529

RESUMO

Histone lysine demethylases facilitate the activity of oncogenic transcription factors, including possibly MYC. Here we show that multiple histone demethylases influence the viability and poor prognosis of neuroblastoma cells, where MYC is often overexpressed. We also identified the approved small-molecule antifungal agent ciclopirox as a novel pan-histone demethylase inhibitor. Ciclopirox targeted several histone demethylases, including KDM4B implicated in MYC function. Accordingly, ciclopirox inhibited Myc signaling in parallel with mitochondrial oxidative phosphorylation, resulting in suppression of neuroblastoma cell viability and inhibition of tumor growth associated with an induction of differentiation. Our findings provide new insights into epigenetic regulation of MYC function and suggest a novel pharmacologic basis to target histone demethylases as an indirect MYC-targeting approach for cancer therapy. Cancer Res; 77(17); 4626-38. ©2017 AACR.


Assuntos
Antifúngicos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Histona Desmetilases/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-myc/metabolismo , Piridonas/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclopirox , Epigênese Genética , Histonas/metabolismo , Humanos , Camundongos , Camundongos SCID , Neuroblastoma/enzimologia , Neuroblastoma/patologia , Fosforilação Oxidativa/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/genética , RNA Interferente Pequeno/genética , Transcrição Genética/efeitos dos fármacos , Células Tumorais Cultivadas
8.
Cancer Res ; 77(1): 123-133, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-27815386

RESUMO

Loss of the tumor suppressor gene PTEN exerts diverse outcomes on cancer in different developmental contexts. To gain insight into the effect of its loss on outcomes in the brain, we conditionally inactivated the murine Pten gene in neonatal neural stem/progenitor cells. Pten inactivation created an abnormal perivascular proliferative niche in the cerebellum that persisted in adult animals but did not progress to malignancy. Proliferating cells showed undifferentiated morphology and expressed the progenitor marker Nestin but not Math1, a marker of committed granule neuron progenitors. Codeletion of Pten and Trp53 resulted in fully penetrant medulloblastoma originating from the perivascular niche, which exhibited abnormal blood vessel networks and advanced neuronal differentiation of tumor cells. EdU pulse-chase experiments demonstrated a perivascular cancer stem cell population in Pten/Trp53 double mutant medulloblastomas. Genetic analyses revealed recurrent somatic inactivations of the tumor suppressor gene Ptch1 and a recapitulation of the sonic hedgehog subgroup of human medulloblastomas. Overall, our results showed that PTEN acts to prevent the proliferation of a progenitor niche in postnatal cerebellum predisposed to oncogenic induction of medulloblastoma. Cancer Res; 77(1); 123-33. ©2016 AACR.


Assuntos
Neoplasias Cerebelares/genética , Meduloblastoma/genética , Células-Tronco Neoplásicas/patologia , Células-Tronco Neurais/patologia , PTEN Fosfo-Hidrolase/genética , Animais , Neoplasias Cerebelares/patologia , Hibridização Genômica Comparativa , Modelos Animais de Doenças , Imunofluorescência , Imuno-Histoquímica , Meduloblastoma/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Transdução de Sinais/fisiologia , Nicho de Células-Tronco/fisiologia , Proteína Supressora de Tumor p53/genética
9.
Nat Genet ; 48(12): 1551-1556, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27798625

RESUMO

Acute myeloid leukemia (AML) comprises a heterogeneous group of leukemias frequently defined by recurrent cytogenetic abnormalities, including rearrangements involving the core-binding factor (CBF) transcriptional complex. To better understand the genomic landscape of CBF-AMLs, we analyzed both pediatric (n = 87) and adult (n = 78) samples, including cases with RUNX1-RUNX1T1 (n = 85) or CBFB-MYH11 (n = 80) rearrangements, by whole-genome or whole-exome sequencing. In addition to known mutations in the Ras pathway, we identified recurrent stabilizing mutations in CCND2, suggesting a previously unappreciated cooperating pathway in CBF-AML. Outside of signaling alterations, RUNX1-RUNX1T1 and CBFB-MYH11 AMLs demonstrated remarkably different spectra of cooperating mutations, as RUNX1-RUNX1T1 cases harbored recurrent mutations in DHX15 and ZBTB7A, as well as an enrichment of mutations in epigenetic regulators, including ASXL2 and the cohesin complex. This detailed analysis provides insights into the pathogenesis and development of CBF-AML, while highlighting dramatic differences in the landscapes of cooperating mutations for these related AML subtypes.


Assuntos
Biomarcadores Tumorais/genética , Fatores de Ligação ao Core/genética , Genômica/métodos , Leucemia Mieloide Aguda/genética , Mutação/genética , Proteínas de Fusão Oncogênica/genética , Adulto , Criança , Humanos
10.
Nat Genet ; 48(12): 1481-1489, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27776115

RESUMO

Chromosomal rearrangements deregulating hematopoietic transcription factors are common in acute lymphoblastic leukemia (ALL). Here we show that deregulation of the homeobox transcription factor gene DUX4 and the ETS transcription factor gene ERG is a hallmark of a subtype of B-progenitor ALL that comprises up to 7% of B-ALL. DUX4 rearrangement and overexpression was present in all cases and was accompanied by transcriptional deregulation of ERG, expression of a novel ERG isoform, ERGalt, and frequent ERG deletion. ERGalt uses a non-canonical first exon whose transcription was initiated by DUX4 binding. ERGalt retains the DNA-binding and transactivation domains of ERG, but it inhibits wild-type ERG transcriptional activity and is transforming. These results illustrate a unique paradigm of transcription factor deregulation in leukemia in which DUX4 deregulation results in loss of function of ERG, either by deletion or induced expression of an isoform that is a dominant-negative inhibitor of wild-type ERG function.


Assuntos
Transformação Celular Neoplásica/genética , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Rearranjo Gênico , Proteínas de Homeodomínio/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adolescente , Adulto , Transformação Celular Neoplásica/patologia , Perfilação da Expressão Gênica , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Isoformas de Proteínas , Regulador Transcricional ERG/genética , Adulto Jovem
11.
PLoS Genet ; 12(9): e1006281, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27622269

RESUMO

Drosophila melanogaster Piwi functions within the germline stem cells (GSCs) and the somatic niche to regulate GSC self-renewal and differentiation. How Piwi influences GSCs is largely unknown. We uncovered a genetic interaction between Piwi and c-Fos in the somatic niche that influences GSCs. c-Fos is a proto-oncogene that influences many cell and developmental processes. In wild-type ovarian cells, c-Fos is post-transcriptionally repressed by Piwi, which destabilized the c-Fos mRNA by promoting the processing of its 3' untranslated region (UTR) into Piwi-interacting RNAs (piRNAs). The c-Fos 3' UTR was sufficient to trigger Piwi-dependent destabilization of a GFP reporter. Piwi represses c-Fos in the somatic niche to regulate GSC maintenance and differentiation and in the somatic follicle cells to affect somatic cell disorganization, tissue dysmorphogenesis, oocyte maturation arrest, and infertility.


Assuntos
Proteínas Argonauta/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Oogônios/metabolismo , Ovário/crescimento & desenvolvimento , Proteínas Proto-Oncogênicas c-fos/genética , Regiões 3' não Traduzidas , Animais , Proteínas Argonauta/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Feminino , Oogênese , Oogônios/citologia , Ovário/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Nicho de Células-Tronco
12.
Mol Cell ; 62(2): 207-221, 2016 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-27105116

RESUMO

Nucleosome remodeling and deacetylation (NuRD) complexes are co-transcriptional regulators implicated in differentiation, development, and diseases. Methyl-CpG binding domain (MBD) proteins play an essential role in recruitment of NuRD complexes to their target sites in chromatin. The related SHREC complex in fission yeast drives transcriptional gene silencing in heterochromatin through cooperation with HP1 proteins. How remodeler and histone deacetylase (HDAC) cooperate within NuRD complexes remains unresolved. We determined that in SHREC the two modules occupy distant sites on the scaffold protein Clr1 and that repressive activity of SHREC can be modulated by the expression level of the HDAC-associated Clr1 domain alone. Moreover, the crystal structure of Clr2 reveals an MBD-like domain mediating recruitment of the HDAC module to heterochromatin. Thus, SHREC bi-functionality is organized in two separate modules with separate recruitment mechanisms, which work together to elicit transcriptional silencing at heterochromatic loci.


Assuntos
Montagem e Desmontagem da Cromatina , Inativação Gênica , Heterocromatina/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Acetilação , Sítios de Ligação , Ilhas de CpG , DNA Fúngico/metabolismo , Regulação Fúngica da Expressão Gênica , Heterocromatina/química , Heterocromatina/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/química , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Modelos Moleculares , Nucleossomos/enzimologia , Nucleossomos/genética , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Fúngico/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Relação Estrutura-Atividade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Genética
13.
Proc Natl Acad Sci U S A ; 112(49): 15172-7, 2015 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-26598706

RESUMO

A majority of breast cancers are driven by estrogen via estrogen receptor-α (ERα). Our previous studies indicate that hypoxia-inducible factor 1α (HIF-1α) cooperates with ERα in breast cancer cells. However, whether ERα is implicated in the direct regulation of HIF-1α and the role of HIF-1α in endocrine therapy response are unknown. In this study we found that a subpopulation of HIF-1α targets, many of them bearing both hypoxia response elements and estrogen response elements, are regulated by ERα in normoxia and hypoxia. Interestingly, the HIF-1α gene itself also bears an estrogen response element, and its expression is directly regulated by ERα. Clinical data revealed that expression of the HIF-1α gene or a hypoxia metagene signature is associated with a poor outcome to endocrine treatment in ERα(+) breast cancer. HIF-1α was able to confer endocrine therapy resistance to ERα(+) breast cancer cells. Our findings define, for the first time to our knowledge, a direct regulatory pathway between ERα and HIF-1α, which might modulate hormone response in treatment.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Moduladores de Receptor Estrogênico/uso terapêutico , Receptor alfa de Estrogênio/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Transdução de Sinais , Tamoxifeno/uso terapêutico , Transcrição Genética/fisiologia
14.
Cancer Cell ; 28(3): 343-56, 2015 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-26321221

RESUMO

Alterations of IKZF1, encoding the lymphoid transcription factor IKAROS, are a hallmark of high-risk acute lymphoblastic leukemia (ALL), however the role of IKZF1 alterations in ALL pathogenesis is poorly understood. Here, we show that in mouse models of BCR-ABL1 leukemia, Ikzf1 and Arf alterations synergistically promote the development of an aggressive lymphoid leukemia. Ikzf1 alterations result in acquisition of stem cell-like features, including self-renewal and increased bone marrow stromal adhesion. Retinoid receptor agonists reversed this phenotype, partly by inducing expression of IKZF1, resulting in abrogation of adhesion and self-renewal, cell cycle arrest, and attenuation of proliferation without direct cytotoxicity. Retinoids potentiated the activity of dasatinib in mouse and human BCR-ABL1 ALL, providing an additional therapeutic option in IKZF1-mutated ALL.


Assuntos
Proteínas de Fusão bcr-abl/genética , Fator de Transcrição Ikaros/genética , Mutação/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Retinoides/metabolismo , Animais , Pontos de Checagem do Ciclo Celular/genética , Humanos , Camundongos , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Receptores do Ácido Retinoico/metabolismo
15.
Cell Stem Cell ; 17(1): 101-15, 2015 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-26140606

RESUMO

Cell-based therapies to treat retinal degeneration are now being tested in clinical trials. However, it is not known whether the source of stem cells is important for the production of differentiated cells suitable for transplantation. To test this, we generated induced pluripotent stem cells (iPSCs) from murine rod photoreceptors (r-iPSCs) and scored their ability to make retinae by using a standardized quantitative protocol called STEM-RET. We discovered that r-iPSCs more efficiently produced differentiated retinae than did embryonic stem cells (ESCs) or fibroblast-derived iPSCs (f-iPSCs). Retinae derived from f-iPSCs had fewer amacrine cells and other inner nuclear layer cells. Integrated epigenetic analysis showed that DNA methylation contributes to the defects in f-iPSC retinogenesis and that rod-specific CTCF insulator protein-binding sites may promote r-iPSC retinogenesis. Together, our data suggest that the source of stem cells is important for producing retinal neurons in three-dimensional (3D) organ cultures.


Assuntos
Epigênese Genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Retina/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Bastonetes/citologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Linhagem Celular , Reprogramação Celular , Metilação de DNA , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Retina/citologia , Retina/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Degeneração Retiniana/terapia
16.
J Natl Cancer Inst ; 107(6): djv080, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25925418

RESUMO

BACKGROUND: Epigenetic alterations, such as histone methylation, modulate Myc signaling, a pathway central to oncogenesis. We investigated the role of the histone demethylase KDM4B in N-Myc-mediated neuroblastoma pathogenesis. METHODS: Spearman correlation was performed to correlate MYCN and KDM4B expression. RNA interference, microarray analysis, gene set enrichment analysis, and real-time polymerase chain reaction were used to define the functions of KDM4B. Immunoprecipitation and immunofluorescence were used to assess protein-protein interactions between N-Myc and KDM4B. Chromatin immunoprecipitation was used to assess the binding of Myc targets. Constitutive and inducible lentiviral-mediated KDM4B knockdown with shRNA was used to assess the effects on tumor growth. Kaplan-Meier survival analysis was used to assess the prognostic value of KDM4B expression. All statistical tests were two-sided. RESULTS: KDM4B and MYCN expression were found to be statistically significantly correlated in a variety of cancers, including neuroblastoma (R = 0.396, P < .001). Functional studies demonstrated that KDM4B regulates the Myc pathway. N-Myc was found to physically interact with and recruit KDM4B. KDM4B was found to regulate neuroblastoma cell proliferation and differentiation in vitro and xenograft growth in vivo (5 mice/group, two-tailed t test, P ≤ 0.001). Finally, together with MYCN amplification, KDM4B was found to stratify a subgroup of poor-prognosis patients (122 case patients, P < .001). CONCLUSIONS: Our findings provide insight into the epigenetic regulation of Myc via histone demethylation and proof-of-concept for inhibition of histone demethylases to target Myc signaling in cancers such as neuroblastoma.


Assuntos
Histona Desmetilases com o Domínio Jumonji/metabolismo , Neuroblastoma/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Imunoprecipitação da Cromatina , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Estimativa de Kaplan-Meier , Camundongos , Neuroblastoma/genética , Prognóstico , Análise Serial de Proteínas , Reação em Cadeia da Polimerase em Tempo Real
17.
Blood ; 126(1): 69-75, 2015 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-25987655

RESUMO

Asparaginase is used to treat acute lymphoblastic leukemia (ALL); however, hypersensitivity reactions can lead to suboptimal asparaginase exposure. Our objective was to use a genome-wide approach to identify loci associated with asparaginase hypersensitivity in children with ALL enrolled on St. Jude Children's Research Hospital (SJCRH) protocols Total XIIIA (n = 154), Total XV (n = 498), and Total XVI (n = 271), or Children's Oncology Group protocols POG 9906 (n = 222) and AALL0232 (n = 2163). Germline DNA was genotyped using the Affymetrix 500K, Affymetrix 6.0, or the Illumina Exome BeadChip array. In multivariate logistic regression, the intronic rs6021191 variant in nuclear factor of activated T cells 2 (NFATC2) had the strongest association with hypersensitivity (P = 4.1 × 10(-8); odds ratio [OR] = 3.11). RNA-seq data available from 65 SJCRH ALL tumor samples and 52 Yoruba HapMap samples showed that samples carrying the rs6021191 variant had higher NFATC2 expression compared with noncarriers (P = 1.1 × 10(-3) and 0.03, respectively). The top ranked nonsynonymous polymorphism was rs17885382 in HLA-DRB1 (P = 3.2 × 10(-6); OR = 1.63), which is in near complete linkage disequilibrium with the HLA-DRB1*07:01 allele we previously observed in a candidate gene study. The strongest risk factors for asparaginase allergy are variants within genes regulating the immune response.


Assuntos
Asparaginase/uso terapêutico , Hipersensibilidade a Drogas/genética , Fatores de Transcrição NFATC/genética , Adolescente , Adulto , Criança , Pré-Escolar , Hipersensibilidade a Drogas/epidemiologia , Terapia de Reposição de Enzimas/efeitos adversos , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Cadeias HLA-DRB1/genética , Humanos , Lactente , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/epidemiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adulto Jovem
18.
Blood ; 125(23): 3609-17, 2015 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-25855603

RESUMO

Alterations of genes encoding transcriptional regulators of lymphoid development are a hallmark of B-progenitor acute lymphoblastic leukemia (B-ALL) and most commonly involve PAX5, encoding the DNA-binding transcription factor paired-box 5. The majority of PAX5 alterations in ALL are heterozygous, and key PAX5 target genes are expressed in leukemic cells, suggesting that PAX5 may be a haploinsufficient tumor suppressor. To examine the role of PAX5 alterations in leukemogenesis, we performed mutagenesis screens of mice heterozygous for a loss-of-function Pax5 allele. Both chemical and retroviral mutagenesis resulted in a significantly increased penetrance and reduced latency of leukemia, with a shift to B-lymphoid lineage. Genomic profiling identified a high frequency of secondary genomic mutations, deletions, and retroviral insertions targeting B-lymphoid development, including Pax5, and additional genes and pathways mutated in ALL, including tumor suppressors, Ras, and Janus kinase-signal transducer and activator of transcription signaling. These results show that in contrast to simple Pax5 haploinsufficiency, multiple sequential alterations targeting lymphoid development are central to leukemogenesis and contribute to the arrest in lymphoid maturation characteristic of ALL. This cross-species analysis also validates the importance of concomitant alterations of multiple cellular growth, signaling, and tumor suppression pathways in the pathogenesis of B-ALL.


Assuntos
Deleção de Genes , Neoplasias Experimentais/metabolismo , Fator de Transcrição PAX5/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Camundongos , Camundongos Mutantes , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Fator de Transcrição PAX5/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Supressoras de Tumor/genética
19.
Dev Biol ; 407(2): 344-55, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-25863122

RESUMO

Neuroblastoma is a pediatric cancer of the developing sympathoadrenal lineage. The tumors are known to develop from the adrenal gland or paraspinal ganglia and have molecular and cellular features of sympathetic neurons such as dense core vesicles and catecholamine production. Here we present the detailed molecular, cellular, genetic and epigenetic characterization of an orthotopic xenograft derived from a high-risk stage 4 neuroblastoma patient. Overall, the xenografted tumor retained the high risk features of the primary tumor and showed aggressive growth and metastasis in the mouse. Also, the genome was preserved with no additional copy number variations, structural variations or aneuploidy. There were 13 missense mutations identified in the xenograft that were not present in the patient's primary tumor and there were no new nonsense mutations. None of the missense mutations acquired in the xenograft were in known cancer genes. We also demonstrate the feasibility of using the orthotopic neuroblastoma xenograft to test standard of care chemotherapy and molecular targeted therapeutics. Finally, we optimized a new approach to produce primary cultures of the neuroblastoma xenografts for high-throughput drug screening which can be used to test new combinations of therapeutic agents for neuroblastoma.


Assuntos
Neuroblastoma/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto , Animais , Linhagem Celular Tumoral , Ensaios de Triagem em Larga Escala , Humanos , Imuno-Histoquímica , Camundongos , Neuroblastoma/ultraestrutura , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
20.
Nucleic Acids Res ; 42(18): 11363-82, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25249627

RESUMO

Genome-wide distribution of histone H3K18 and H3K27 acetyltransferases, CBP (CREBBP) and p300 (EP300), is used to map enhancers and promoters, but whether these elements functionally require CBP/p300 remains largely uncertain. Here we compared global CBP recruitment with gene expression in wild-type and CBP/p300 double-knockout (dKO) fibroblasts. ChIP-seq using CBP-null cells as a control revealed nearby CBP recruitment for 20% of constitutively-expressed genes, but surprisingly, three-quarters of these genes were unaffected or slightly activated in dKO cells. Computationally defined enhancer-promoter-units (EPUs) having a CBP peak near the enhancer-like element were more predictive, with CBP/p300 deletion attenuating expression of 40% of such constitutively-expressed genes. Examining signal-responsive (Hypoxia Inducible Factor) genes showed that 97% were within 50 kilobases of an inducible CBP peak, and 70% of these required CBP/p300 for full induction. Unexpectedly, most inducible CBP peaks occurred near signal-nonresponsive genes. Finally, single-cell expression analysis revealed additional context dependence where some signal-responsive genes were not uniformly dependent on CBP/p300 in individual cells. While CBP/p300 was needed for full induction of some genes in single-cells, for other genes CBP/p300 increased the probability of maximal expression. Thus, target gene context influences the transcriptional requirement for CBP/p300, possibly by multiple mechanisms.


Assuntos
Proteína de Ligação a CREB/metabolismo , Ativação Transcricional , 2,2'-Dipiridil/farmacologia , Animais , Proteína de Ligação a CREB/genética , Células Cultivadas , Imunoprecipitação da Cromatina , Elementos Facilitadores Genéticos , Genoma , Camundongos , Regiões Promotoras Genéticas , Elementos de Resposta , Análise de Célula Única , Sítio de Iniciação de Transcrição , Transcrição Genética , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismo
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