RESUMO
The forkhead transcription factor FOXP1 is generally regarded as an oncogene in activated B cell-like diffuse large B-cell lymphoma. Previous studies have suggested that a small isoform of FOXP1 rather than full-length FOXP1, may possess this oncogenic activity. Corroborating those studies, we herein show that activated B cell-like diffuse large B-cell lymphoma cell lines and primary activated B cell-like diffuse large B-cell lymphoma cells predominantly express a small FOXP1 isoform, and that the 5'-end of the Foxp1 gene is a common insertion site in murine lymphomas in leukemia virus- and transposon-mediated insertional mutagenesis screens. By combined mass spectrometry, (quantative) reverse transcription polymerase chain reaction/sequencing, and small interfering ribonucleic acid-mediated gene silencing, we determined that the small FOXP1 isoform predominantly expressed in activated B cell-like diffuse large B-cell lymphoma lacks the N-terminal 100 amino acids of full-length FOXP1. Aberrant overexpression of this FOXP1 isoform (ΔN100) in primary human B cells revealed its oncogenic capacity; it repressed apoptosis and plasma cell differentiation. However, no difference in potency was found between this small FOXP1 isoform and full-length FOXP1. Furthermore, overexpression of full-length FOXP1 or this small FOXP1 isoform in primary B cells and diffuse large B-cell lymphoma cell lines resulted in similar gene regulation. Taken together, our data indicate that this small FOXP1 isoform and full-length FOXP1 have comparable oncogenic and transcriptional activity in human B cells, suggesting that aberrant expression or overexpression of FOXP1, irrespective of the specific isoform, contributes to lymphomagenesis. These novel insights further enhance the value of FOXP1 for the diagnostics, prognostics, and treatment of diffuse large B-cell lymphoma patients.
Assuntos
Linfócitos B/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ativação Transcricional , Processamento Alternativo , Sequência de Aminoácidos , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Caspase 3/metabolismo , Caspase 7/metabolismo , Ciclo Celular/genética , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/química , Humanos , Memória Imunológica , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Mutagênese Insercional , Plasmócitos/citologia , Plasmócitos/imunologia , Plasmócitos/metabolismo , Isoformas de Proteínas , Proteínas Repressoras/químicaRESUMO
Many anticancer drugs induce DNA breaks to eliminate tumor cells. The anthracycline topoisomerase II inhibitors additionally cause histone eviction. Here, we performed genome-wide high-resolution mapping of chemotherapeutic effects of various topoisomerase I and II (TopoI and II) inhibitors and integrated this mapping with established maps of genomic or epigenomic features to show their activities in different genomic regions. The TopoI inhibitor topotecan and the TopoII inhibitor etoposide are similar in inducing DNA damage at transcriptionally active genomic regions. The anthracycline daunorubicin induces DNA breaks and evicts histones from active chromatin, thus quenching local DNA damage responses. Another anthracycline, aclarubicin, has a different genomic specificity and evicts histones from H3K27me3-marked heterochromatin, with consequences for diffuse large B-cell lymphoma cells with elevated levels of H3K27me3. Modifying anthracycline structures may yield compounds with selectivity for different genomic regions and activity for different tumor types.
Assuntos
Antineoplásicos/farmacologia , DNA de Neoplasias/química , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Neoplasias/tratamento farmacológico , Inibidores da Topoisomerase/farmacologia , Aclarubicina/química , Aclarubicina/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Cromatina/química , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Dano ao DNA , DNA de Neoplasias/metabolismo , Daunorrubicina/química , Daunorrubicina/farmacologia , Etoposídeo/química , Etoposídeo/farmacologia , Histonas/antagonistas & inibidores , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Terapia de Alvo Molecular , Neoplasias/química , Neoplasias/genética , Neoplasias/patologia , Especificidade de Órgãos , Transporte Proteico/efeitos dos fármacos , Relação Estrutura-Atividade , Inibidores da Topoisomerase/química , Topotecan/química , Topotecan/farmacologiaRESUMO
Genomically distal mutations can contribute to the deregulation of cancer genes by engaging in chromatin interactions. To study this, we overlay viral cancer-causing insertions obtained in a murine retroviral insertional mutagenesis screen with genome-wide chromatin conformation capture data. Here we find that insertions tend to cluster in 3D hotspots within the nucleus. The identified hotspots are significantly enriched for known cancer genes, and bear the expected characteristics of bona fide regulatory interactions, such as enrichment for transcription factor-binding sites. In addition, we observe a striking pattern of mutual exclusive integration. This is an indication that insertions in these loci target the same gene, either in their linear genomic vicinity or in their 3D spatial vicinity. Our findings shed new light on the repertoire of targets obtained from insertional mutagenesis screening and underline the importance of considering the genome as a 3D structure when studying effects of genomic perturbations.
Assuntos
Cromatina/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Genes Neoplásicos/genética , Infecções por Retroviridae/virologia , Integração Viral/genética , Animais , Núcleo Celular/imunologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Camundongos , Mutagênese Insercional/métodos , Infecções por Retroviridae/genéticaRESUMO
EZH2 is frequently overexpressed in glioblastoma (GBM), suggesting an oncogenic function that could be a target for therapeutic intervention. However, reduced EZH2 activity can also promote tumorigenesis, leading to concerns about the use of EZH2 inhibitors. Here, we provide further insight about the effects of prolonged Ezh2 inhibition in glioblastoma using preclinical mouse models and primary tumor-derived human GBM cell lines. Using doxycycline-inducible shRNAs that mimic the effects of a selective EZH2 inhibitor, we demonstrate that prolonged Ezh2 depletion causes a robust switch in cell fate, including significantly enhanced proliferation, DNA damage repair, and activation of part of the pluripotency network, resulting in altered tumor cell identity and tumor progression. Short-term Ezh2 depletion significantly improved survival without the tumor progression observed upon prolonged Ezh2 depletion, suggesting that precise dosing regiments are very important. These results could be of high clinical relevance with regard to how glioblastomas should be treated with epigenetic therapies.
RESUMO
The ability of retroviruses and transposons to insert their genetic material into host DNA makes them widely used tools in molecular biology, cancer research and gene therapy. However, these systems have biases that may strongly affect research outcomes. To address this issue, we generated very large datasets consisting of ~ 120,000 to ~ 180,000 unselected integrations in the mouse genome for the Sleeping Beauty (SB) and piggyBac (PB) transposons, and the Mouse Mammary Tumor Virus (MMTV). We analyzed ~ 80 (epi)genomic features to generate bias maps at both local and genome-wide scales. MMTV showed a remarkably uniform distribution of integrations across the genome. More distinct preferences were observed for the two transposons, with PB showing remarkable resemblance to bias profiles of the Murine Leukemia Virus. Furthermore, we present a model where target site selection is directed at multiple scales. At a large scale, target site selection is similar across systems, and defined by domain-oriented features, namely expression of proximal genes, proximity to CpG islands and to genic features, chromatin compaction and replication timing. Notable differences between the systems are mainly observed at smaller scales, and are directed by a diverse range of features. To study the effect of these biases on integration sites occupied under selective pressure, we turned to insertional mutagenesis (IM) screens. In IM screens, putative cancer genes are identified by finding frequently targeted genomic regions, or Common Integration Sites (CISs). Within three recently completed IM screens, we identified 7%-33% putative false positive CISs, which are likely not the result of the oncogenic selection process. Moreover, results indicate that PB, compared to SB, is more suited to tag oncogenes.
Assuntos
Cromatina/genética , Elementos de DNA Transponíveis/genética , Retroviridae/genética , Animais , Ilhas de CpG/genética , Genoma/genética , Camundongos , Mutagênese Insercional/métodos , Oncogenes/genéticaRESUMO
Retroviruses and DNA transposons are an important part of molecular biologists' toolbox. The applications of these elements range from functional genomics to oncogene discovery and gene therapy. However, these elements do not integrate uniformly across the genome, which is an important limitation to their use. A number of genetic and epigenetic factors have been shown to shape the integration preference of these elements. Insight into integration bias can significantly enhance the analysis and interpretation of results obtained using these elements. For three different applications, we outline how bias can affect results, and can potentially be addressed.
RESUMO
The most common risk factor for developing hepatocellular carcinoma (HCC) is chronic infection with hepatitis B virus (HBV). To better understand the evolutionary forces driving HCC, we performed a near-saturating transposon mutagenesis screen in a mouse HBV model of HCC. This screen identified 21 candidate early stage drivers and a very large number (2,860) of candidate later stage drivers that were enriched for genes that are mutated, deregulated or functioning in signaling pathways important for human HCC, with a striking 1,199 genes being linked to cellular metabolic processes. Our study provides a comprehensive overview of the genetic landscape of HCC.
Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virologia , Hepatite B Crônica/complicações , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virologia , Mutagênese , Animais , Carcinoma Hepatocelular/metabolismo , Elementos de DNA Transponíveis , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/metabolismo , Redes e Vias Metabólicas/genética , Metabolômica/métodos , Camundongos , Camundongos Transgênicos , Mutagênese Insercional , Ácido Pirúvico/metabolismoRESUMO
Histone deacetylases (HDACs) are epigenetic erasers of lysine-acetyl marks. Inhibition of HDACs using small molecule inhibitors (HDACi) is a potential strategy in the treatment of various diseases and is approved for treating hematological malignancies. Harnessing the therapeutic potential of HDACi requires knowledge of HDAC-function in vivo. Here, we generated a thymocyte-specific gradient of HDAC-activity using compound conditional knockout mice for Hdac1 and Hdac2. Unexpectedly, gradual loss of HDAC-activity engendered a dosage-dependent accumulation of immature thymocytes and correlated with the incidence and latency of monoclonal lymphoblastic thymic lymphomas. Strikingly, complete ablation of Hdac1 and Hdac2 abrogated lymphomagenesis due to a block in early thymic development. Genomic, biochemical and functional analyses of pre-leukemic thymocytes and tumors revealed a critical role for Hdac1/Hdac2-governed HDAC-activity in regulating a p53-dependent barrier to constrain Myc-overexpressing thymocytes from progressing into lymphomas by regulating Myc-collaborating genes. One Myc-collaborating and p53-suppressing gene, Jdp2, was derepressed in an Hdac1/2-dependent manner and critical for the survival of Jdp2-overexpressing lymphoma cells. Although reduced HDAC-activity facilitates oncogenic transformation in normal cells, resulting tumor cells remain highly dependent on HDAC-activity, indicating that a critical level of Hdac1 and Hdac2 governed HDAC-activity is required for tumor maintenance.
Assuntos
Dosagem de Genes/fisiologia , Genes Supressores de Tumor/fisiologia , Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Proteínas Proto-Oncogênicas c-myc/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Células Cultivadas , Epistasia Genética/fisiologia , Dosagem de Genes/genética , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Genes/fisiologia , Histona Desacetilase 1/metabolismo , Histona Desacetilase 1/fisiologia , Histona Desacetilase 2/metabolismo , Histona Desacetilase 2/fisiologia , Linfoma/genética , Linfoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Insertional mutagenesis is a potent forward genetic screening technique used to identify candidate cancer genes in mouse model systems. An important, yet unresolved issue in the analysis of these screens, is the identification of the genes affected by the insertions. To address this, we developed Kernel Convolved Rule Based Mapping (KC-RBM). KC-RBM exploits distance, orientation and insertion density across tumors to automatically map integration sites to target genes. We perform the first genome-wide evaluation of the association of insertion occurrences with aberrant gene expression of the predicted targets in both retroviral and transposon data sets. We demonstrate the efficiency of KC-RBM by showing its superior performance over existing approaches in recovering true positives from a list of independently, manually curated cancer genes. The results of this work will significantly enhance the accuracy and speed of cancer gene discovery in forward genetic screens. KC-RBM is available as R-package.