RESUMO
Human CD34+ hematopoietic stem and progenitor cells (HSPCs) are a standard source of cells for clinical HSC transplantations as well as experimental xenotransplantation to generate "humanized mice". To further extend the range of applications of these humanized mice, we developed a protocol to efficiently edit the genomes of human CD34+ HSPCs before transplantation. In the past, manipulating HSPCs has been complicated by the fact that they are inherently difficult to transduce with lentivectors, and rapidly lose their stemness and engraftment potential during in vitro culture. However, with optimized nucleofection of sgRNA:Cas9 ribonucleoprotein complexes, we are now able to edit a candidate gene in CD34+ HSPCs with almost 100% efficiency, and transplant these modified cells in immunodeficient mice with high engraftment levels and multilineage hematopoietic differentiation. The result is a humanized mouse from which we knocked out a gene of interest from their human immune system.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Humanos , Camundongos , Animais , Antígenos CD34 , Técnicas de Inativação de Genes , Sistema Imunitário , Transplante de Células-Tronco Hematopoéticas/métodos , Camundongos SCIDRESUMO
Background: Adult and pediatric tumors display stark differences in their mutation spectra and chromosome alterations. Here, we attempted to identify common and unique gene dependencies and their associated biomarkers among adult and pediatric tumor isolates using functional genetic lethal screens and computational modeling. Methods: We performed CRISRP-Cas9 lethality screens in two adult glioblastoma (GBM) tumor isolates and five pediatric brain tumor isolates representing atypical teratoid rhabdoid tumors (ATRT), diffuse intrinsic pontine glioma, GBM, and medulloblastoma. We then integrated the screen results with machine learning-based gene-dependency models generated from data from >900 cancer cell lines. Results: We found that >50% of candidate dependencies of 280 identified were shared between adult GBM tumors and individual pediatric tumor isolates. 68% of screen hits were found as nodes in our network models, along with shared and tumor-specific predictors of gene dependencies. We investigated network predictors associated with ADAR, EFR3A, FGFR1 (pediatric-specific), and SMARCC2 (ATRT-specific) gene dependency among our tumor isolates. Conclusions: The results suggest that, despite harboring disparate genomic signatures, adult and pediatric tumor isolates share a preponderance of genetic dependences. Further, combining data from primary brain tumor lethality screens with large cancer cell line datasets produced valuable insights into biomarkers of gene dependency, even for rare cancers. Importance of the Study: Our results demonstrate that large cancer cell lines data sets can be computationally mined to identify known and novel gene dependency relationships in adult and pediatric human brain tumor isolates. Gene dependency networks and lethality screen results represent a key resource for neuro-oncology and cancer research communities. We also highlight some of the challenges and limitations of this approach.
RESUMO
Clinical trials of high-dose androgen (HDA) therapy for prostate cancer (PC) have shown promising efficacy but are limited by lack of criteria to identify likely responders. To elucidate factors that govern the growth-repressive effects of HDAs, we applied an unbiased integrative approach using genetic screens and transcriptional profiling of PC cells with or without demonstrated phenotypic sensitivity to androgen-mediated growth repression. Through this comprehensive analysis, we identified genetic events and related signaling networks that determine the response to both HDA and androgen withdrawal. We applied these findings to develop a gene signature that may serve as an early indicator of treatment response and identify men with tumors that are amenable to HDA therapy.
Assuntos
Androgênios/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Genes p53/genética , Humanos , Masculino , Proteínas de Ligação a Retinoblastoma/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Many of the regulatory features governing erythrocyte specification, maturation, and associated disorders remain enigmatic. To identify new regulators of erythropoiesis, we utilize a functional genomic screen for genes affecting expression of the erythroid marker CD235a/GYPA. Among validating hits are genes coding for the N6-methyladenosine (m6A) mRNA methyltransferase (MTase) complex, including, METTL14, METTL3, and WTAP. We demonstrate that m6A MTase activity promotes erythroid gene expression programs through selective translation of ~300 m6A marked mRNAs, including those coding for SETD histone methyltransferases, ribosomal components, and polyA RNA binding proteins. Remarkably, loss of m6A marks results in dramatic loss of H3K4me3 marks across key erythroid-specific KLF1 transcriptional targets (e.g., Heme biosynthesis genes). Further, each m6A MTase subunit and a subset of their mRNAs targets are required for human erythroid specification in primary bone-marrow derived progenitors. Thus, m6A mRNA marks promote the translation of a network of genes required for human erythropoiesis.
Assuntos
Adenosina/análogos & derivados , Eritropoese/genética , Biossíntese de Proteínas , Adenosina/genética , Antígenos CD34/genética , Antígenos CD34/metabolismo , Células da Medula Óssea/fisiologia , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Leucemia Eritroblástica Aguda/genética , Metiltransferases/genética , Regiões Promotoras Genéticas , Fatores de Processamento de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RegulonRESUMO
The miR-106a~363 cluster encodes 6 miRNAs on the X-chromosome which are abundant in blood cells and overexpressed in a variety of malignancies. The constituent miRNA of miR-106a~363 have functional activities in vitro that are predicted to be both oncogenic and tumor suppressive, yet little is known about their physiological functions in vivo. Mature miR-106a~363 (Mirc2) miRNAs are processed from an intragenic, non-protein encoding gene referred to as Xpcl1 (or Kis2), situated at an X-chromosomal locus frequently targeted by retroviruses in murine lymphomas. The oncogenic potential of miR-106a~363 Xpcl1 has not been proven, nor its potential role in T cell development. We show that miR106a~363 levels normally drop at the CD4+/CD8+ double positive (DP) stage of thymocyte development. Forced expression of Xpcl1 at this stage impairs thymocyte maturation and induces T-cell lymphomas. Surprisingly, miR-106a~363 Xpcl1 also induces p27 transcription via Foxo3/4 transcription factors. As a haploinsufficient tumor suppressor, elevated p27 is expected to inhibit lymphomagenesis. Consistent with this, concurrent p27 Kip1 deletion dramatically accelerated lymphomagenesis, indicating that p27 is rate limiting for tumor development by Xpcl1. Whereas down-regulation of miR-106a~363 is important for normal T cell differentiation and for the prevention of lymphomas, eliminating p27 reveals Xpcl1's full oncogenic potential.
RESUMO
Mice lacking the p27(Kip1) Cdk inhibitor (Cdkn1b) exhibit increased susceptibility to lymphomas from the Maloney murine leukemia virus (M-MuLV), and exhibit a high frequency of viral integrations at Xpcl1 (Kis2), a locus on the X-chromosome. Xpcl1 encodes miR-106a~363, a cluster of microRNAs that are expressed in response to adjacent retroviral integrations. We report the first large-scale profile of microRNA expression in MuLV-induced lymphomas, in combination with microarray gene expression analysis. The source material was T-cell lymphomas induced by M-MuLV in p27(Kip1) knockout mice and normal thymus. Surprisingly, the overall levels of miRNA expression were equivalent in lymphomas and normal thymus. Nonetheless, the expression of specific microRNAs was altered in tumors. The miR-106a~363 miRNA were over-expressed in lymphomas, particularly those with viral integrations at the Xpcl1 locus. In contrast, p27(Kip1) deletion itself was associated with a different pattern of microRNA expression. Gene expression was dramatically altered in lymphomas, yet paralleled data from T-cell lymphomas induced by other mechanisms. Genes with altered expression in association with the p27(Kip1) null genotype were of similar functional classes to those associated with Xpcl1 integration, but with the opposite pattern of expression. Thus, the effect of p27(Kip1) deletion may be to oppose an anti-oncogenic effect of Xpcl1 rather than enhancing its oncogenic functions. A subset of miR-106a~363 target genes was consistently reduced in lymphomas with Xpcl1 integrations, particularly genes with cell cycle and immune functions. We identify four predicted target genes of miR-106a~363 miRNA, including N-Myc (Mycn), and the TGF-beta receptor (Tgfbr2) using 3'UTR reporter assays. Still, bioinformatic miRNA target predictions were poor predictors of altered gene expression in lymphomas with Xpcl1 integration. Confirmation of miR-106a~363 gene targeting relevant to the tumor phenotype requires in vivo validation, because only a subset of predicted targets are consistently reduced in tumors that overexpress miR-106a~363.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/deficiência , Regulação Neoplásica da Expressão Gênica , Linfoma/genética , MicroRNAs/genética , Animais , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Perfilação da Expressão Gênica , Genótipo , Camundongos , MicroRNAs/metabolismo , Vírus da Leucemia Murina de Moloney/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Timo/metabolismo , Integração Viral/genéticaRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) establishes a predominantly latent infection in the infected host. Importantly, during latency, only a small number of viral encoded genes are expressed. This viral gene expression pattern contributes to the establishment of long-term infection as well as the ability of the virus to evade the immune system. Previous studies have been shown that the replication and transcription activator (RTA) encoded by ORF50 activates it downstream genes and initiates viral lytic reactivation through functional interaction with RBP-Jkappa, the major downstream effector of the Notch signaling pathway. This indicates that RTA can usurp the conserved Notch signaling pathway and mimic the activities of intracellular Notch1 to modulate gene expression. In this report, we show that the activated intracellular domain of Notch1 (ICN) is aberrantly accumulated in KSHV latently infected pleural effusion lymphoma (PEL) cells. ICN activated the RTA promoter in a dose-dependent manner, and forced expression of ICN in latently infected KSHV-positive cells initiated full blown lytic replication with the production of infectious viral progeny. However, latency-associated nuclear antigen (LANA) which is predominantly expressed during latency can specifically down-modulate ICN-mediated transactivation of RTA and so control KSHV for lytic reactivation. These results demonstrate that LANA can inhibit viral lytic replication by antagonizing ICN function and suggest that LANA is a critical component of the regulatory control mechanism for switching between viral latent and lytic replication by directly interacting with effectors of the conserved cellular Notch1 pathway.
Assuntos
Herpesvirus Humano 8/fisiologia , Receptor Notch1/metabolismo , Ativação Viral/fisiologia , Latência Viral/fisiologia , Linfócitos B , Linhagem Celular , Regulação Viral da Expressão Gênica , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/virologia , Ativação TranscricionalRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus expressing latent antigens critical for pathogenesis. The mechanism by which KSHV mediates oncogenesis has not been fully elucidated. Notch signaling is an evolutionarily conserved pathway controlling diverse events related to development, proliferation, and tissue homeostasis. Deregulation of Notch signaling has also been shown to be highly correlated with oncogenesis. Here we show that the activated intracellular domain of Notch1 (ICN) is aberrantly accumulated in latently KSHV-infected pleural effusion lymphoma cells and results in increased proliferation. Specifically, growth of the infected cells was dramatically inhibited at the G(1) phase by treatment with a gamma-secretase inhibitor which specifically blocks the production of ICN. Increased ICN also up-regulated the cyclin D1 cell cycle regulator. Taken together, these studies define an important mechanism directly linking latent KSHV infection to induction of oncogenesis through dysregulation of the conserved Notch signaling pathway.
Assuntos
Transformação Celular Viral , Fase G1 , Infecções por Herpesviridae/metabolismo , Herpesvirus Humano 8/metabolismo , Linfoma de Células B/metabolismo , Neoplasias Pleurais/metabolismo , Receptor Notch1/metabolismo , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Linhagem Celular Tumoral , Transformação Celular Viral/efeitos dos fármacos , Ciclina D1/metabolismo , Endopeptidases/metabolismo , Inibidores Enzimáticos/farmacologia , Fase G1/efeitos dos fármacos , Infecções por Herpesviridae/patologia , Humanos , Linfoma de Células B/patologia , Linfoma de Células B/virologia , Neoplasias Pleurais/patologia , Neoplasias Pleurais/virologia , Estrutura Terciária de Proteína , Transdução de Sinais/efeitos dos fármacosRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) is the major biological cofactor contributing to development of Kaposi's sarcoma. KSHV establishes a latent infection in human B cells expressing the latency-associated nuclear antigen (LANA), a critical factor in the regulation of viral latency. LANA controls KSHV latent infection through repression of RTA, an activator of many lytic promoters. RTA activates the expression of several lytic viral genes by interacting with recombination signal sequence-binding protein Jkappa (RBP-Jkappa), a transcriptional repressor and the target of the Notch signaling pathway. The recognition that a number of KSHV lytic gene promoters, including RTA, contain RBP-Jkappa binding sites raised the possibility that RBP-Jkappa-mediated repression may be central to the establishment of latency. Here, we tested this hypothesis by examining the regulation of RTA by LANA through binding to RBP-Jkappa. This study demonstrates that LANA physically associates with RBP-Jkappa in vitro and in KSHV-infected cells, with the complex formed capable of binding to RBP-Jkappa cognate sequences. RBP-Jkappa binding sites within the RTA promoter have been found to be critical for LANA-mediated repression. Our study describes a novel mechanism through which LANA maintains KSHV latency by targeting a major downstream effector of the Notch signaling pathway.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/fisiologia , Proteínas Imediatamente Precoces/biossíntese , Proteínas Nucleares/metabolismo , Transativadores/biossíntese , Proteínas Virais/biossíntese , Ativação Viral , Antígenos Virais , Linhagem Celular , Ensaio de Desvio de Mobilidade Eletroforética , Herpesvirus Humano 8/genética , Humanos , Proteínas Imediatamente Precoces/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina , Imunoprecipitação , Ligação Proteica , RNA Mensageiro/análise , RNA Viral/análise , Transdução de Sinais , Transativadores/genética , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Epstein-Barr virus latent protein EBNA3C has been shown to bind Nm23-H1, a known suppresser of cell migration and metastasis and a regulator of the guanine exchange factor Tiam-1. This interaction results in cellular translocation of Nm23-H1 to the nucleus and suppression of the antimigratory effect in vitro. Furthermore, these proteins can synergistically increase transcription of a basal promoter when targeted to DNA by fusion to a Gal4 DNA binding domain. In this report, we show that EBNA3C and Nm23-H1 can cooperate to upregulate expression of MMP-9, known to be expressed in aggressive forms of lymphomas. This upregulation resulted in increased levels of MMP-9 mRNA, as well as a detectable increase in MMP-9 gelatinolytic activity. Specific mutations in the MMP-9 promoter showed that the Ap1 and NFkappaB binding sites are important for upregulation by the proteins. Additionally, it was shown for the first time that EBNA3C and Nm23-H1 can bind subunits of these transcription factors. This suggests that the ability of EBNA3C to reverse the antimigratory effects of Nm23-H1 is likely to be in part through the synergistic upregulation of MMP-9, mediated through interactions with the AP1 and NFkappaB transcription factors.
Assuntos
Antígenos de Neoplasias/fisiologia , Antígenos Nucleares do Vírus Epstein-Barr/fisiologia , Herpesvirus Humano 4/fisiologia , Metaloproteinase 9 da Matriz/metabolismo , Núcleosídeo-Difosfato Quinase/fisiologia , Complexo 1 de Proteínas Adaptadoras , Linfócitos B , Sítios de Ligação/fisiologia , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Gelatina , Humanos , Metaloproteinase 9 da Matriz/genética , NF-kappa B/metabolismo , Nucleosídeo NM23 Difosfato Quinases , Núcleosídeo-Difosfato Quinase/metabolismo , RNA Mensageiro , Regulação para CimaRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent contributing to development of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman desease. Following primary infection, latency is typically established. However, the mechanism by which KSHV establishes latency is not understood. We have reported that the latency-associated nuclear antigen (LANA) can repress RTA (for replication and transcription activator) expression by down-regulating its promoter. In this study, we show that RTA is associated with the virion particle. We also show that RTA can activate the LANA promoter and induce LANA expression in transient reporter assays. Additionally, the transcription of RTA correlates with LANA expression in the early stages of de novo infection of KSHV, and induction of LANA transcription is responsive to induction of RTA with an inducible system. This induction in LANA transcription was dependent on recombination signal sequence binding protein Jkappa (RBP-Jkappa), as a RBP-Jkappa-deficient cell line was significantly delayed and inefficient in LANA transcription with expression of RTA. These studies suggest that RTA contributes to establishment of KSHV latency by activating LANA expression in the early stages of infection by utilizing the major effector of the Notch signaling pathway RBP-Jkappa. This describes a feedback mechanism by which LANA and RTA can regulate each other and is likely to be a key event in the establishment of KSHV latency.
Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Proteínas Virais/metabolismo , Latência Viral , Antígenos Virais , Linhagem Celular , Herpesvirus Humano 8/genética , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Transativadores/genética , Ativação Transcricional , Transfecção , Proteínas Virais/genética , Vírion/metabolismo , Replicação ViralRESUMO
Like other herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV, also designated human herpesvirus 8) can establish a latent infection in the infected host. During latency a small number of genes are expressed. One of those genes encodes latency-associated nuclear antigen (LANA), which is constitutively expressed in cells during latent as well as lytic infection. LANA has previously been shown to be important for the establishment of latent episome maintenance through tethering of the viral genome to the host chromosomes. Under specific conditions, KSHV can undergo lytic replication, with the production of viral progeny. The immediate-early Rta, encoded by open reading frame 50 of KSHV, has been shown to play a critical role in switching from viral latent replication to lytic replication. Overexpression of Rta from a heterologous promoter is sufficient for driving KSHV lytic replication and the production of viral progeny. In the present study, we show that LANA down-modulates Rta's promoter activity in transient reporter assays, thus repressing Rta-mediated transactivation. This results in a decrease in the production of KSHV progeny virions. We also found that LANA interacts physically with Rta both in vivo and in vitro. Taken together, our results demonstrate that LANA can inhibit viral lytic replication by inhibiting expression as well as antagonizing the function of Rta. This suggests that LANA may play a critical role in maintaining latency by controlling the switch between viral latency and lytic replication.