RESUMO
Inactivating mutations of the CREBBP and EP300 acetyltransferases are among the most common genetic alterations in diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL). Here, we examined the relationship between these two enzymes in germinal center (GC) B cells, the normal counterpart of FL and DLBCL, and in lymphomagenesis by using conditional GC-directed deletion mouse models targeting Crebbp or Ep300. We found that CREBBP and EP300 modulate common as well as distinct transcriptional programs implicated in separate anatomic and functional GC compartments. Consistently, deletion of Ep300 but not Crebbp impaired the fitness of GC B cells in vivo. Combined loss of Crebbp and Ep300 completely abrogated GC formation, suggesting that these proteins partially compensate for each other through common transcriptional targets. This synthetic lethal interaction was retained in CREBBP-mutant DLBCL cells and could be pharmacologically targeted with selective small molecule inhibitors of CREBBP and EP300 function. These data provide proof-of-principle for the clinical development of EP300-specific inhibitors in FL and DLBCL.
Assuntos
Linfócitos B/fisiologia , Proteína de Ligação a CREB/genética , Proteína p300 Associada a E1A/genética , Epigênese Genética/genética , Centro Germinativo/fisiologia , Linfoma Folicular/etiologia , Linfoma Difuso de Grandes Células B/genética , Acetiltransferases/genética , Animais , Linhagem Celular , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Deleção de Sequência/genética , Transcrição Gênica/genéticaRESUMO
RNA interference is a powerful tool for studying gene function, however, the reproducible generation of RNAi transgenic mice remains a significant limitation. By combining optimized fluorescence-coupled miR30-based shRNAs with high efficiency ES cell targeting, we developed a fast, scalable pipeline for the production of shRNA transgenic mice. Using this system, we generated eight tet-regulated shRNA transgenic lines targeting Firefly and Renilla luciferases, Oct4 and tumor suppressors p53, p16(INK4a), p19(ARF) and APC and demonstrate potent gene silencing and GFP-tracked knockdown in a broad range of tissues in vivo. Further, using an shRNA targeting APC, we illustrate how this approach can identify predicted phenotypes and also unknown functions for a well-studied gene. In addition, through regulated gene silencing we validate APC/Wnt and p19(ARF) as potential therapeutic targets in T cell acute lymphoblastic leukemia/lymphoma and lung adenocarcinoma, respectively. This system provides a cost-effective and scalable platform for the production of RNAi transgenic mice targeting any mammalian gene. PAPERCLIP:
Assuntos
Técnicas de Silenciamento de Genes/métodos , Interferência de RNA , Adenocarcinoma/genética , Adenocarcinoma/terapia , Animais , Células-Tronco Embrionárias/metabolismo , Técnicas de Silenciamento de Genes/economia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Processamento Pós-Transcricional do RNA , RNA Interferente Pequeno/genética , Transdução de Sinais , Proteínas Wnt/metabolismoRESUMO
Mutations disabling the TP53 tumour suppressor gene represent the most frequent events in human cancer and typically occur through a two-hit mechanism involving a missense mutation in one allele and a 'loss of heterozygosity' deletion encompassing the other. While TP53 missense mutations can also contribute gain-of-function activities that impact tumour progression, it remains unclear whether the deletion event, which frequently includes many genes, impacts tumorigenesis beyond TP53 loss alone. Here we show that somatic heterozygous deletion of mouse chromosome 11B3, a 4-megabase region syntenic to human 17p13.1, produces a greater effect on lymphoma and leukaemia development than Trp53 deletion. Mechanistically, the effect of 11B3 loss on tumorigenesis involves co-deleted genes such as Eif5a and Alox15b (also known as Alox8), the suppression of which cooperates with Trp53 loss to produce more aggressive disease. Our results imply that the selective advantage produced by human chromosome 17p deletion reflects the combined impact of TP53 loss and the reduced dosage of linked tumour suppressor genes.
Assuntos
Genes p53/genética , Neoplasias/genética , Neoplasias/patologia , Deleção de Sequência/genética , Proteína Supressora de Tumor p53/deficiência , Alelos , Animais , Transformação Celular Neoplásica/genética , Cromossomos Humanos Par 17/genética , Cromossomos de Mamíferos/genética , Modelos Animais de Doenças , Progressão da Doença , Feminino , Heterozigoto , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Linfoma/genética , Linfoma/patologia , Masculino , Camundongos , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Sintenia/genética , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
To repurpose compounds for diffuse large B cell lymphoma (DLBCL), we screened a library of drugs and other targeted compounds approved by the US Food and Drug Administration on 9 cell lines and validated the results on a panel of 32 genetically characterized DLBCL cell lines. Dasatinib, a multikinase inhibitor, was effective against 50% of DLBCL cell lines, as well as against in vivo xenografts. Dasatinib was more broadly active than the Bruton kinase inhibitor ibrutinib and overcame ibrutinib resistance. Tumors exhibiting dasatinib resistance were commonly characterized by activation of the PI3K pathway and loss of PTEN expression as a specific biomarker. PI3K suppression by mTORC2 inhibition synergized with dasatinib and abolished resistance in vitro and in vivo. These results provide a proof of concept for the repurposing approach in DLBCL, and point to dasatinib as an attractive strategy for further clinical development in lymphomas.
Assuntos
Dasatinibe/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linfoma Difuso de Grandes Células B , Adenina/análogos & derivados , Animais , Linhagem Celular Tumoral , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Piperidinas , Estudo de Prova de Conceito , Pirazóis/farmacologia , Pirimidinas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PTEN encodes a lipid phosphatase that is underexpressed in many cancers owing to deletions, mutations or gene silencing. PTEN dephosphorylates phosphatidylinositol (3,4,5)-triphosphate, thereby opposing the activity of class I phosphatidylinositol 3-kinases that mediate growth- and survival-factor signalling through phosphatidylinositol 3-kinase effectors such as AKT and mTOR. To determine whether continued PTEN inactivation is required to maintain malignancy, here we generate an RNA interference-based transgenic mouse model that allows tetracycline-dependent regulation of PTEN in a time- and tissue-specific manner. Postnatal Pten knockdown in the haematopoietic compartment produced highly disseminated T-cell acute lymphoblastic leukaemia. Notably, reactivation of PTEN mainly reduced T-cell leukaemia dissemination but had little effect on tumour load in haematopoietic organs. Leukaemia infiltration into the intestine was dependent on CCR9 G-protein-coupled receptor signalling, which was amplified by PTEN loss. Our results suggest that in the absence of PTEN, G-protein-coupled receptors may have an unanticipated role in driving tumour growth and invasion in an unsupportive environment. They further reveal that the role of PTEN loss in tumour maintenance is not invariant and can be influenced by the tissue microenvironment, thereby producing a form of intratumoral heterogeneity that is independent of cancer genotype.
Assuntos
Leucemia/enzimologia , Leucemia/fisiopatologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Microambiente Tumoral/fisiologia , Animais , Quimiocinas/metabolismo , Técnicas de Silenciamento de Genes , Leucemia/genética , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/metabolismo , Interferência de RNA , Receptores Acoplados a Proteínas G/metabolismo , Transdução de SinaisRESUMO
Activating mutations of NOTCH1 (a well-known oncogene in T-cell acute lymphoblastic leukemia) are present in â¼4-13% of chronic lymphocytic leukemia (CLL) cases, where they are associated with disease progression and chemorefractoriness. However, the specific role of NOTCH1 in leukemogenesis remains to be established. Here, we report that the active intracellular portion of NOTCH1 (ICN1) is detectable in â¼50% of peripheral blood CLL cases lacking gene mutations. We identify a "NOTCH1 gene-expression signature" in CLL cells, and show that this signature is significantly enriched in primary CLL cases expressing ICN1, independent of NOTCH1 mutation. NOTCH1 target genes include key regulators of B-cell proliferation, survival, and signal transduction. In particular, we show that NOTCH1 transactivates MYC via binding to B-cell-specific regulatory elements, thus implicating this oncogene in CLL development. These results significantly extend the role of NOTCH1 in CLL pathogenesis, and have direct implications for specific therapeutic targeting.
Assuntos
Linfócitos B/fisiologia , Leucemia Linfocítica Crônica de Células B/genética , Receptor Notch1/genética , Linfócitos B/patologia , Proliferação de Células/genética , Regulação Leucêmica da Expressão Gênica , Genes myc , Humanos , Mutação , Receptor Notch1/sangueRESUMO
Cellular senescence acts as a potent barrier to tumorigenesis and contributes to the anti-tumor activity of certain chemotherapeutic agents. Senescent cells undergo a stable cell cycle arrest controlled by RB and p53 and, in addition, display a senescence-associated secretory phenotype (SASP) involving the production of factors that reinforce the senescence arrest, alter the microenvironment, and trigger immune surveillance of the senescent cells. Through a proteomics analysis of senescent chromatin, we identified the nuclear factor-κB (NF-κB) subunit p65 as a major transcription factor that accumulates on chromatin of senescent cells. We found that NF-κB acts as a master regulator of the SASP, influencing the expression of more genes than RB and p53 combined. In cultured fibroblasts, NF-κB suppression causes escape from immune recognition by natural killer (NK) cells and cooperates with p53 inactivation to bypass senescence. In a mouse lymphoma model, NF-κB inhibition bypasses treatment-induced senescence, producing drug resistance, early relapse, and reduced survival. Our results demonstrate that NF-κB controls both cell-autonomous and non-cell-autonomous aspects of the senescence program and identify a tumor-suppressive function of NF-κB that contributes to the outcome of cancer therapy.
Assuntos
Senescência Celular/fisiologia , Resistência a Medicamentos/fisiologia , Fenótipo , Fator de Transcrição RelA/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Linfoma/metabolismo , Camundongos , Inibidores da Síntese de Proteínas/farmacologia , RNA Interferente Pequeno/metabolismo , Tetraciclina/farmacologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Tumour suppressor genes encode a broad class of molecules whose mutational attenuation contributes to malignant progression. In the canonical situation, the tumour suppressor is completely inactivated through a two-hit process involving a point mutation in one allele and chromosomal deletion of the other. Here, to identify tumour suppressor genes in lymphoma, we screen a short hairpin RNA library targeting genes deleted in human lymphomas. We functionally identify those genes whose suppression promotes tumorigenesis in a mouse lymphoma model. Of the nine tumour suppressors we identified, eight correspond to genes occurring in three physically linked 'clusters', suggesting that the common occurrence of large chromosomal deletions in human tumours reflects selective pressure to attenuate multiple genes. Among the new tumour suppressors are adenosylmethionine decarboxylase 1 (AMD1) and eukaryotic translation initiation factor 5A (eIF5A), two genes associated with hypusine, a unique amino acid produced as a product of polyamine metabolism through a highly conserved pathway. Through a secondary screen surveying the impact of all polyamine enzymes on tumorigenesis, we establish the polyamine-hypusine axis as a new tumour suppressor network regulating apoptosis. Unexpectedly, heterozygous deletions encompassing AMD1 and eIF5A often occur together in human lymphomas and co-suppression of both genes promotes lymphomagenesis in mice. Thus, some tumour suppressor functions can be disabled through a two-step process targeting different genes acting in the same pathway.
Assuntos
Linfoma de Células B/genética , Lisina/análogos & derivados , Poliaminas/química , Proteínas Supressoras de Tumor/genética , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Deleção de Genes , Redes Reguladoras de Genes , Testes Genéticos , Humanos , Linfoma de Células B/fisiopatologia , Lisina/química , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Reprodutibilidade dos TestesRESUMO
Diffuse Large B-cell lymphoma (DLBCL) includes the Activated B-cell-like (ABC) and Germinal Center B-cell-like (GCB) subtypes, which differ in cell-of-origin, genetics and clinical response. By screening the subtype-specific activity of 211 drugs approved or in active clinical development for other diseases, we identified inhibitors of nicotinamide phosphoribosyl transferase (NAMPTi) as active in a subset of GCB-DLBCLs in vitro and in vivo. We validated three chemically distinct NAMPTi for their on-target activity based on biochemical and genetic rescue approaches, and found the ratio between NAMPT:PARP1 RNA levels was predictive of NAMPTi sensitivity across DLBCL subtypes. Notably, the NAMPT:PARP1 transcript ratio predicts higher anti-tumor activity in BCL2-translocated GCB-DLBCL. Accordingly, pharmacological and genetic inhibition of BCL2 was potently synergistic with NAMPT blockade. These data support the inhibition of NAMPT as a therapeutically relevant strategy for BCL2-translocated DLBCLs.
RESUMO
Activating transcription factor 3 (ATF3) is an activation transcription factor/cyclic adenosine monophosphate (cAMP) responsive element-binding (CREB) protein family member. It is recognized as an important regulator of cancer progression by repressing expression of key inflammatory factors such as interferon-γ and chemokine (C-C motif) ligand 4 (CCL4). Here, we describe a novel library screening approach that probes individual leucine zipper components before combining them to search exponentially larger sequence spaces not normally accessible to intracellular screening. To do so, we employ two individual semirational library design approaches and screen using a protein-fragment complementation assay (PCA). First, a 248,832-member library explored 12 amino acid positions at all five a positions to identify those that provided improved binding, with all e/g positions fixed as Q, placing selection pressure onto the library options provided. Next, a 59,049-member library probed all ten e/g positions with 3 options. Similarly, during e/g library screening, a positions were locked into a generically bindable sequence pattern (AIAIA), weakly favoring leucine zipper formation, while placing selection pressure onto e/g options provided. The combined a/e/g library represents â¼14.7 billion members, with the resulting peptide, ATF3W_aeg, binding ATF3 with high affinity (Tm = 60 °C; Kd = 151 nM) while strongly disfavoring homodimerization. Moreover, ATF3W_aeg is notably improved over component PCA hits, with target specificity found to be driven predominantly by electrostatic interactions. The combined a/e/g exponential library screening approach provides a robust, accelerated platform for exploring larger peptide libraries, toward derivation of potent yet selective antagonists that avoid homoassociation to provide new insight into rational peptide design.
Assuntos
Fator 3 Ativador da Transcrição , Biblioteca de Peptídeos , Fator 3 Ativador da Transcrição/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/química , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Peptídeos/metabolismoRESUMO
MYC oncoprotein deregulation is a common catastrophic event in human cancer and limiting its activity restrains tumor development and maintenance, as clearly shown via Omomyc, an MYC-interfering 90 amino acid mini-protein. MYC is a multifunctional transcription factor that regulates many aspects of transcription by RNA polymerase II (RNAPII), such as transcription activation, pause release, and elongation. MYC directly associates with Protein Arginine Methyltransferase 5 (PRMT5), a protein that methylates a variety of targets, including RNAPII at the arginine residue R1810 (R1810me2s), crucial for proper transcription termination and splicing of transcripts. Therefore, we asked whether MYC controls termination as well, by affecting R1810me2S. We show that MYC overexpression strongly increases R1810me2s, while Omomyc, an MYC shRNA, or a PRMT5 inhibitor and siRNA counteract this phenomenon. Omomyc also impairs Serine 2 phosphorylation in the RNAPII carboxyterminal domain, a modification that sustains transcription elongation. ChIP-seq experiments show that Omomyc replaces MYC and reshapes RNAPII distribution, increasing occupancy at promoter and termination sites. It is unclear how this may affect gene expression. Transcriptomic analysis shows that transcripts pivotal to key signaling pathways are both up- or down-regulated by Omomyc, whereas genes directly controlled by MYC and belonging to a specific signature are strongly down-regulated. Overall, our data point to an MYC/PRMT5/RNAPII axis that controls termination via RNAPII symmetrical dimethylation and contributes to rewiring the expression of genes altered by MYC overexpression in cancer cells. It remains to be clarified which role this may have in tumor development.
RESUMO
Interplay between chromatin-associated complexes and modifications critically contribute to the partitioning of epigenome into stable and functionally distinct domains. Yet there is a lack of systematic identification of chromatin crosstalk mechanisms, limiting our understanding of the dynamic transition between chromatin states during development and disease. Here we perform co-dependency mapping of genes using CRISPR-Cas9-mediated fitness screens in pan-cancer cell lines to quantify gene-gene functional relationships. We identify 145 co-dependency modules and further define the molecular context underlying the essentiality of these modules by incorporating mutational, epigenome, gene expression and drug sensitivity profiles of cell lines. These analyses assign new protein complex composition and function, and predict new functional interactions, including an unexpected co-dependency between two transcriptionally counteracting chromatin complexes - polycomb repressive complex 2 (PRC2) and MLL-MEN1 complex. We show that PRC2-mediated H3K27 tri-methylation regulates the genome-wide distribution of MLL1 and MEN1. In lymphoma cells with EZH2 gain-of-function mutations, the re-localization of MLL-MEN1 complex drives oncogenic gene expression and results in a hypersensitivity to pharmacologic inhibition of MEN1. Together, our findings provide a resource for discovery of trans-regulatory interactions as mechanisms of chromatin regulation and potential targets of synthetic lethality.
Assuntos
Linfoma , Neoplasias , Humanos , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Histonas/genética , Histonas/metabolismo , CromatinaRESUMO
Multiple myeloma (MM) shows constitutive activation of canonical and noncanonical nuclear factor κB (NF-κB) signaling via genetic mutations or tumor microenvironment (TME) stimulations. A subset of MM cell lines showed dependency for cell growth and survival on the canonical NF-κB transcription factor RELA alone, suggesting a critical role for a RELA-mediated biological program in MM pathogenesis. Here, we determined the RELA-dependent transcriptional program in MM cell lines and found the expression of the cell surface molecules interleukin-27 receptor-α (IL-27Rα) and the adhesion molecule JAM2 to be responsive to RELA at the messenger RNA and protein levels. IL-27Rα and JAM2 were expressed on primary MM cells at higher levels than on healthy long-lived plasma cells (PCs) in the bone marrow. IL-27 activated STAT1, and to a lesser extent STAT3, in MM cell lines and in PCs generated from memory B cells in an IL-21-dependent in vitro PC differentiation assay. Concomitant activity of IL-21 and IL-27 enhanced differentiation into PCs and increased the cell-surface expression of the known STAT target gene CD38. In accordance, a subset of MM cell lines and primary MM cells cultured with IL-27 upregulated CD38 cell-surface expression, a finding with potential implications for enhancing the efficacy of CD38-directed monoclonal antibody therapies by increasing CD38 expression on tumor cells. The elevated expression of IL-27Rα and JAM2 on MM cells compared with that on healthy PCs may be exploited for the development of targeted therapeutic strategies that modulate the interaction of MM cells with the TME.
Assuntos
Interleucina-27 , Mieloma Múltiplo , Humanos , Interleucina-27/metabolismo , Mieloma Múltiplo/genética , NF-kappa B/metabolismo , Receptores de Citocinas/metabolismo , Microambiente Tumoral , Regulação para CimaRESUMO
PURPOSE: Met, the tyrosine kinase receptor for hepatocyte growth factor, is frequently deregulated in human cancer. Recent evidence indicates that Met amplification may confer resistance to treatments directed toward other receptor tyrosine kinases. Thus, there is a need to develop Met inhibitors into therapeutic tools, to be used alone or in combination with other molecularly targeted drugs. Preclinical validation of Met inhibitors has thus far been done in nude mice bearing cancer cells xenografts. A far superior model would be a transgenic line developing spontaneous Met-driven tumors with high penetrance and short latency. EXPERIMENTAL DESIGN: To this end, we introduced into the mouse genome TPR-MET, the oncogenic form of MET. The Tpr-Met protein ensures deregulation of Met signaling because dimerization motifs in the Tpr moiety cause ligand-independent activation of the Met kinase. RESULTS: Here, we describe a TPR-MET transgenic line that develops thymic T-cell lymphoma with full penetrance and very short latency. In the tumors, Tpr-Met and its effectors were phosphorylated. Treatment of tumor-derived T lymphocytes with the selective Met inhibitor PHA-665752 at nanomolar concentrations abolished phosphorylation of Met and downstream effectors and led to caspase-mediated apoptosis. I.v. administration of PHA-665752 to transgenic mice bearing lymphomas in exponential growth phase led to a significant decrease in tumor growth and, in some cases, to tumor regression. CONCLUSIONS: Our transgenic line, which within 2 months reliably develops Tpr-Met-driven T-cell lymphoma, represents a valuable tool to explore the efficacy and therapeutic potential of Met kinase inhibitors as anticancer drugs.
Assuntos
Modelos Animais de Doenças , Linfoma/tratamento farmacológico , Linfoma/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/genética , Animais , Western Blotting , Técnicas de Transferência de Genes , Humanos , Imuno-Histoquímica , Indóis/farmacologia , Linfoma/patologia , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-met/efeitos dos fármacos , Sulfonas/farmacologiaRESUMO
In this issue of Cancer Cell, Parvin and colleagues identify the expression of LMO2 as a biomarker for DNA repair defects in lymphomas. Using isogenic cell lines and xenografts, the authors show that expression of LMO2 predicts sensitivity to PARP inhibition, especially in combination with genotoxic therapy.
Assuntos
Proteínas com Domínio LIM , Inibidores de Poli(ADP-Ribose) Polimerases , Proteínas Adaptadoras de Transdução de Sinal , Dano ao DNA , Proteínas Proto-Oncogênicas/genética , Mutações Sintéticas LetaisRESUMO
Rhabdomyosarcoma (RMS) is a highly malignant soft-tissue tumor of childhood deriving from skeletal muscle cells. RMS can be classified in two major histologic subtypes: embryonal (ERMS) and alveolar (ARMS), the latter being characterized by the PAX3/7-FKHR translocation. Here we first investigated whether the Met receptor, a transcriptional target of PAX3 and PAX7, has a role in PAX3-FKHR-mediated transformation. Following PAX3-FKHR transduction, Met was up-regulated in mouse embryonal fibroblasts (MEF), NIH 3T3 and C2C12 cells, and they all acquired anchorage independence. This property was lost in low serum but addition of hepatocyte growth factor/scatter factor (HGF/SF) rescued soft-agar growth. Genetic proof that Met is necessary for this PAX3-FKHR-mediated effect was obtained by transducing with PAX3-FKHR MEFs derived from Met mutant (Met(D/D)) and wild-type (Met(+/+)) embryos. Only Met(+/+) MEFs acquired anchorage-independent growth whereas PAX3-FKHR-transduced Met(D/D) cells were unable to form colonies in soft agar. To verify if Met had a role in RMS maintenance, we silenced the receptor by transducing ERMS and ARMS cell lines with an inducible lentivirus expressing an anti-Met short hairpin RNA (shRNA). Met down-regulation significantly affected RMS cells proliferation, survival, invasiveness, and anchorage-independent growth. Finally, induction of the Met-directed shRNA promoted a dramatic reduction of tumor mass in a xenograft model of RMS. Our data show that both ARMS- and ERMS-derived cell lines, in spite of the genetic drift which may have occurred in years of culture, seem to have retained an "addiction" to the Met oncogene and suggest that Met may represent a target of choice to develop novel therapeutic strategies for ARMS.
Assuntos
Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/fisiologia , Receptores de Fatores de Crescimento/antagonistas & inibidores , Receptores de Fatores de Crescimento/fisiologia , Rabdomiossarcoma Alveolar/terapia , Rabdomiossarcoma Embrionário/terapia , Animais , Apoptose/genética , Processos de Crescimento Celular/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Feminino , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Inativação Gênica , Células HeLa , Fator de Crescimento de Hepatócito , Humanos , Camundongos , Camundongos Nus , Células NIH 3T3 , Invasividade Neoplásica , Proteínas de Fusão Oncogênica/genética , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-met , Interferência de RNA , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/genética , Receptores de Fatores de Crescimento/genética , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Alveolar/patologia , Rabdomiossarcoma Embrionário/genética , Rabdomiossarcoma Embrionário/metabolismo , Rabdomiossarcoma Embrionário/patologia , Transdução Genética , Regulação para CimaRESUMO
Hydrocephalus is a common and variegated pathology often emerging in newborn children after genotoxic insults during pregnancy (Hicks and D'Amato, 1980). Cre recombinase is known to have possible toxic effects that can compromise normal cell cycle and survival. Here we show, by using three independent nestin Cre transgenic lines, that high levels of Cre recombinase expression into the nucleus of neuronal progenitors can compromise normal brain development. The transgenics analyzed are the nestin Cre Balancer (Bal1) line, expressing the Cre recombinase with a nuclear localization signal, and two nestin CreER(T2) (Cre recombinase fused with a truncated estrogen receptor) mice lines with different levels of expression of a hybrid CreER(T2) recombinase that translocates into the nucleus after tamoxifen treatment. All homozygous Bal1 nestin Cre embryos displayed reduced neuronal proliferation, increased aneuploidy and cell death, as well as defects in ependymal lining and lamination of the cortex, leading to microencephaly and to a form of communicating hydrocephalus. An essentially overlapping phenotype was observed in the two nestin CreER(T2) transgenic lines after tamoxifen mediated-CreER(T2) translocation into the nucleus. Neither tamoxifen-treated wild-type nor nestin CreER(T2) oil-treated control mice displayed these defects. These results indicate that some forms of hydrocephalus may derive from a defect in neuronal precursors proliferation. Furthermore, they underscore the potential risks for developmental studies of high levels of nuclear Cre in neurogenic cells.
Assuntos
Encéfalo/anormalidades , Hidrocefalia/enzimologia , Integrases/metabolismo , Microcefalia/enzimologia , Malformações do Sistema Nervoso/enzimologia , Células-Tronco/enzimologia , Aneuploidia , Animais , Biomarcadores/metabolismo , Encéfalo/enzimologia , Encéfalo/fisiopatologia , Morte Celular/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células , Epêndima/anormalidades , Epêndima/metabolismo , Epêndima/patologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hidrocefalia/genética , Hidrocefalia/fisiopatologia , Integrases/genética , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Camundongos , Camundongos Transgênicos , Microcefalia/genética , Microcefalia/fisiopatologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/fisiopatologia , Nestina , Neurônios/enzimologia , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Tamoxifeno/farmacologiaRESUMO
An enormous amount of tumor sequencing data has been generated through large scale sequencing efforts. The functional consequences of the majority of mutations identified by such projects remain an open, unexplored question. This problem is particularly complicated in the case of rare mutations where frequency of occurrence alone or prediction of functional consequences are insufficient to distinguish driver from passenger or bystander mutations. We combine genome editing technology with a powerful mouse cancer model to uncover previously unsuspected rare oncogenic mutations in Burkitt's lymphoma. We identify two candidate tumor suppressors whose loss cooperate with MYC over-expression to accelerate lymphomagenesis. Our results highlight the utility of in vivo CRISPR/Cas9 screens combined with powerful mouse models to identify and validate rare oncogenic modifier events from tumor mutational data.
Assuntos
Linfoma de Burkitt/genética , Sistemas CRISPR-Cas , Transformação Celular Neoplásica/genética , Genes Supressores de Tumor , Animais , Feminino , CamundongosRESUMO
Tpr-Met, the oncogenic counterpart of the Met receptor, has been detected in gastric cancers, as well as in precursor lesions and in the adjacent normal gastric mucosa. This has prompted the suggestion that Tpr-Met may predispose to the development of gastric tumors. Given the sequence specificity of RNA interference, oncogenes activated by point mutation or rearrangements can be targeted while spearing the product of the wild-type allele. In this work, we report specific suppression of Tpr-Met expression and inhibition of Tpr-Met-mediated transformation and tumorigenesis by means of a short interfering RNA (siRNA) directed toward the Tpr-Met junction (anti-TM2). When delivered by a lentiviral vector, anti-TM2 siRNA was effective also in mouse embryonal fibroblasts or epithelial cells expressing high levels of Tpr-Met. Our results suggest that lentiviral-mediated delivery of anti-TM2 siRNA may be developed into a powerful tool to treat Tpr-Met-positive cancers.
Assuntos
Vetores Genéticos/genética , Lentivirus/genética , Neoplasias Experimentais/terapia , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Interferência de RNA , Animais , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Regulação da Expressão Gênica , Terapia Genética , Humanos , Camundongos , Neoplasias Experimentais/etiologia , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , RNA Interferente Pequeno/genética , Transdução GenéticaRESUMO
Despite the pressing need for novel cancer treatments, our improved understanding of tumor biology is not being successfully translated into better therapies. Here we present a lentiviral vector that enables in vivo validation of cancer therapeutic targets when combined with existing cancer animal models that faithfully reproduce the natural history of human disease. Unlike the conventional genetic approaches with targeted alleles, the outlined experimental strategy could be used to assess the preclinical efficacy of a growing number of putative therapeutic hits in a rapid and cost-effective manner.