RESUMO
Mixed-phenotype acute leukemia is a rare subtype of leukemia in which both myeloid and lymphoid markers are co-expressed on the same malignant cells. The pathogenesis is largely unknown, and the treatment is challenging. We previously reported the specific association of the recurrent t(8;12)(q13;p13) chromosomal translocation that creates the ETV6-NCOA2 fusion with T/myeloid leukemias. Here we report that ETV6-NCOA2 initiates T/myeloid leukemia in preclinical models; ectopic expression of ETV6-NCOA2 in mouse bone marrow hematopoietic progenitors induced T/myeloid lymphoma accompanied by spontaneous Notch1-activating mutations. Similarly, cotransduction of human cord blood CD34+ progenitors with ETV6-NCOA2 and a nontransforming NOTCH1 mutant induced T/myeloid leukemia in immunodeficient mice; the immunophenotype and gene expression pattern were similar to those of patient-derived ETV6-NCOA2 leukemias. Mechanistically, we show that ETV6-NCOA2 forms a transcriptional complex with ETV6 and the histone acetyltransferase p300, leading to derepression of ETV6 target genes. The expression of ETV6-NCOA2 in human and mouse nonthymic hematopoietic progenitor cells induces transcriptional dysregulation, which activates a lymphoid program while failing to repress the expression of myeloid genes such as CSF1 and MEF2C. The ETV6-NCOA2 induced arrest at an early immature T-cell developmental stage. The additional acquisition of activating NOTCH1 mutations transforms the early immature ETV6-NCOA2 cells into T/myeloid leukemias. Here, we describe the first preclinical model to depict the initiation of T/myeloid leukemia by a specific somatic genetic aberration.
Assuntos
Regulação Leucêmica da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide/genética , Coativador 2 de Receptor Nuclear/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Repressoras/genética , Animais , Transformação Celular Neoplásica , Células Cultivadas , Feminino , Células-Tronco Hematopoéticas/patologia , Humanos , Leucemia Mieloide/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
Natural killer (NK) cells are important in the immune defense against tumor cells and pathogens, and they regulate other immune cells by cytokine secretion. Although murine NK cell biology has been extensively studied, knowledge about transcriptional circuitries controlling human NK cell development and maturation is limited. By generating ETS1-deficient human embryonic stem cells and by expressing the dominant-negative ETS1 p27 isoform in cord blood hematopoietic progenitor cells, we show that the transcription factor ETS1 is critically required for human NK cell differentiation. Genome-wide transcriptome analysis determined by RNA-sequencing combined with chromatin immunoprecipitation-sequencing analysis reveals that human ETS1 directly induces expression of key transcription factors that control NK cell differentiation (ie, E4BP4, TXNIP, TBET, GATA3, HOBIT, BLIMP1). In addition, ETS1 regulates expression of genes involved in apoptosis and NK cell activation. Our study provides important molecular insights into the role of ETS1 as an important regulator of human NK cell development and terminal differentiation.
Assuntos
Diferenciação Celular/imunologia , Regulação da Expressão Gênica/imunologia , Células-Tronco Embrionárias Humanas/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Proteína Proto-Oncogênica c-ets-1/imunologia , Apoptose/genética , Apoptose/imunologia , Diferenciação Celular/genética , Linhagem Celular , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células Matadoras Naturais/citologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Proteína Proto-Oncogênica c-ets-1/genéticaRESUMO
T-cell acute lymphoblastic leukemia (T-ALL) and T-cell acute lymphoblastic lymphoma (T-LBL) are aggressive hematological malignancies that are currently treated with high-dose chemotherapy. Over the last several years, the search toward novel and less-toxic therapeutic strategies for T-ALL/T-LBL patients has largely focused on the identification of cell-intrinsic properties of the tumor cell. However, non-cell-autonomous activation of specific oncogenic pathways might also offer opportunities that could be exploited at the therapeutic level. In line with this, we here show that endogenous interleukin 7 (IL7) can increase the expression of the oncogenic kinase proviral integration site for Moloney-murine leukemia 1 (PIM1) in CD127+ T-ALL/T-LBL, thereby rendering these tumor cells sensitive to in vivo PIM inhibition. In addition, using different CD127+ T-ALL/T-LBL xenograft models, we also reveal that residual tumor cells, which remain present after short-term in vivo chemotherapy, display consistent upregulation of PIM1 as compared with bulk nontreated tumor cells. Notably, this effect was transient as increased PIM1 levels were not observed in reestablished disease after abrogation of the initial chemotherapy. Furthermore, we uncover that this phenomenon is, at least in part, mediated by the ability of glucocorticoids to cause transcriptional upregulation of IL7RA in T-ALL/T-LBL patient-derived xenograft (PDX) cells, ultimately resulting in non-cell-autonomous PIM1 upregulation by endogenous IL7. Finally, we confirm in vivo that chemotherapy in combination with a pan-PIM inhibitor can improve leukemia survival in a PDX model of CD127+ T-ALL. Altogether, our work reveals that IL7 and glucocorticoids coordinately drive aberrant activation of PIM1 and suggests that IL7-responsive CD127+ T-ALL and T-LBL patients could benefit from PIM inhibition during induction chemotherapy.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Citocinas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Linfócitos T/imunologia , Animais , Apoptose , Proliferação de Células , Quimioterapia Combinada , Humanos , Camundongos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Linfócitos T/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The ability of natural killer (NK) cells to kill tumor cells without prior sensitization makes them a rising player in immunotherapy. Increased understanding of the development and functioning of NK cells will improve their clinical utilization. As opposed to murine NK cell development, human NK cell development is still less understood. Here, we studied the role of thioredoxin-interacting protein (TXNIP) in human NK cell differentiation by stable TXNIP knockdown or overexpression in cord blood hematopoietic stem cells, followed by in vitro NK cell differentiation. TXNIP overexpression only had marginal effects, indicating that endogenous TXNIP levels are sufficient in this process. TXNIP knockdown, however, reduced proliferation of early differentiation stages and greatly decreased NK cell numbers. Transcriptome analysis and experimental confirmation showed that reduced protein synthesis upon TXNIP knockdown likely caused this low proliferation. Contrary to its profound effects on the early differentiation stages, TXNIP knockdown led to limited alterations in NK cell phenotype, and it had no effect on NK cell cytotoxicity or cytokine production. Thus, TXNIP promotes human NK cell differentiation by affecting protein synthesis and proliferation of early NK cell differentiation stages, but it is redundant for functional NK cell maturation.
Assuntos
Células Matadoras Naturais , Tiorredoxinas , Animais , Proteínas de Transporte/genética , Diferenciação Celular/genética , Citocinas/metabolismo , Expressão Gênica , Humanos , Células Matadoras Naturais/metabolismo , Camundongos , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMO
BACKGROUND: Transgenic zebrafish lines with the expression of a fluorescent reporter under the control of a cell-type specific promoter, enable transcriptome analysis of FACS sorted cell populations. RNA quality and yield are key determinant factors for accurate expression profiling. Limited cell number and FACS induced cellular stress make RNA isolation of sorted zebrafish cells a delicate process. We aimed to optimize a workflow to extract sufficient amounts of high-quality RNA from a limited number of FACS sorted cells from Tg(fli1a:GFP) zebrafish embryos, which can be used for accurate gene expression analysis. RESULTS: We evaluated two suitable RNA isolation kits (the RNAqueous micro and the RNeasy plus micro kit) and determined that sorting cells directly into lysis buffer is a critical step for success. For low cell numbers, this ensures direct cell lysis, protects RNA from degradation and results in a higher RNA quality and yield. We showed that this works well up to 0.5× dilution of the lysis buffer with sorted cells. In our sort settings, this corresponded to 30,000 and 75,000 cells for the RNAqueous micro kit and RNeasy plus micro kit respectively. Sorting more cells dilutes the lysis buffer too much and requires the use of a collection buffer. We also demonstrated that an additional genomic DNA removal step after RNA isolation is required to completely clear the RNA from any contaminating genomic DNA. For cDNA synthesis and library preparation, we combined SmartSeq v4 full length cDNA library amplification, Nextera XT tagmentation and sample barcoding. Using this workflow, we were able to generate highly reproducible RNA sequencing results. CONCLUSIONS: The presented optimized workflow enables to generate high quality RNA and allows accurate transcriptome profiling of small populations of sorted zebrafish cells.
Assuntos
Citometria de Fluxo , RNA/genética , RNA/isolamento & purificação , Análise de Sequência de RNA , Peixe-Zebra/genética , Animais , Contagem de Células , Poli A/genética , Controle de QualidadeRESUMO
Elevated expression of the Zinc finger E-box binding homeobox transcription factor-2 (ZEB2) is correlated with poor prognosis and patient outcome in a variety of human cancer subtypes. Using a conditional gain-of-function mouse model, we recently demonstrated that ZEB2 is an oncogenic driver of immature T-cell acute lymphoblastic leukemia (T-ALL), a heterogenic subgroup of human leukemia characterized by a high incidence of remission failure or hematological relapse after conventional chemotherapy. Here, we identified the lysine-specific demethylase KDM1A as a novel interaction partner of ZEB2 and demonstrated that mouse and human T-ALLs with increased ZEB2 levels critically depend on KDM1A activity for survival. Therefore, targeting the ZEB2 protein complex through direct disruption of the ZEB2-KDM1A interaction or pharmacological inhibition of the KDM1A demethylase activity itself could serve as a novel therapeutic strategy for this aggressive subtype of human leukemia and possibly other ZEB2-driven malignancies.
Assuntos
Benzoatos/farmacologia , Ciclopropanos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases/metabolismo , Proteínas de Homeodomínio/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Repressoras/metabolismo , Animais , Benzoatos/uso terapêutico , Linhagem Celular Tumoral , Ciclopropanos/uso terapêutico , Regulação Leucêmica da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Repressoras/genética , Regulação para Cima , Homeobox 2 de Ligação a E-box com Dedos de ZincoAssuntos
Rearranjo Gênico , Genes myc , Predisposição Genética para Doença , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Animais , Biomarcadores Tumorais , Variações do Número de Cópias de DNA , Modelos Animais de Doenças , Estudos de Associação Genética , Humanos , Camundongos , Terapia de Alvo Molecular , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Translocação Genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Genetic studies in T-cell acute lymphoblastic leukemia have uncovered a remarkable complexity of oncogenic and loss-of-function mutations. Amongst this plethora of genetic changes, NOTCH1 activating mutations stand out as the most frequently occurring genetic defect, identified in more than 50% of T-cell acute lymphoblastic leukemias, supporting a role as an essential driver for this gene in T-cell acute lymphoblastic leukemia oncogenesis. In this study, we aimed to establish a comprehensive compendium of the long non-coding RNA transcriptome under control of Notch signaling. For this purpose, we measured the transcriptional response of all protein coding genes and long non-coding RNAs upon pharmacological Notch inhibition in the human T-cell acute lymphoblastic leukemia cell line CUTLL1 using RNA-sequencing. Similar Notch dependent profiles were established for normal human CD34(+) thymic T-cell progenitors exposed to Notch signaling activity in vivo. In addition, we generated long non-coding RNA expression profiles (array data) from ex vivo isolated Notch active CD34(+) and Notch inactive CD4(+)CD8(+) thymocytes and from a primary cohort of 15 T-cell acute lymphoblastic leukemia patients with known NOTCH1 mutation status. Integration of these expression datasets with publicly available Notch1 ChIP-sequencing data resulted in the identification of long non-coding RNAs directly regulated by Notch activity in normal and malignant T cells. Given the central role of Notch in T-cell acute lymphoblastic leukemia oncogenesis, these data pave the way for the development of novel therapeutic strategies that target hyperactive Notch signaling in human T-cell acute lymphoblastic leukemia.
Assuntos
Biomarcadores Tumorais/genética , Mutação/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , RNA Longo não Codificante/genética , Receptor Notch1/metabolismo , Timócitos/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Biomarcadores Tumorais/metabolismo , Western Blotting , Estudos de Casos e Controles , Transformação Celular Neoplásica/patologia , Células Cultivadas , Imunoprecipitação da Cromatina , Estudos de Coortes , Inibidores Enzimáticos/farmacologia , Seguimentos , Perfilação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Timócitos/citologia , Timócitos/efeitos dos fármacosRESUMO
Studies defining normal and disrupted human neural crest cell development have been challenging given its early timing and intricacy of development. Consequently, insight into the early disruptive events causing a neural crest related disease such as pediatric cancer neuroblastoma is limited. To overcome this problem, we developed an in vitro differentiation model to recapitulate the normal in vivo developmental process of the sympathoadrenal lineage which gives rise to neuroblastoma. We used human in vitro pluripotent stem cells and single-cell RNA sequencing to recapitulate the molecular events during sympathoadrenal development. We provide a detailed map of dynamically regulated transcriptomes during sympathoblast formation and illustrate the power of this model to study early events of the development of human neuroblastoma, identifying a distinct subpopulation of cell marked by SOX2 expression in developing sympathoblast obtained from patient derived iPSC cells harboring a germline activating mutation in the anaplastic lymphoma kinase (ALK) gene.
RESUMO
T-lineage acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that accounts for 10%-15% of pediatric and 25% of adult ALL cases. Although the prognosis of T-ALL has improved over time, the outcome of T-ALL patients with primary resistant or relapsed leukemia remains poor. Therefore, further progress in the treatment of T-ALL requires a better understanding of its biology and the development of more effective precision oncologic therapies. The proto-oncogene MYB is highly expressed in diverse hematologic malignancies, including T-ALLs with genomic aberrations that further potentiate its expression and activity. Previous studies have associated MYB with a malignant role in the pathogenesis of several cancers. However, its role in the induction and maintenance of T-ALL remains relatively poorly understood. In this study, we found that an increased copy number of MYB is associated with higher MYB expression levels, and might be associated with inferior event-free survival of pediatric T-ALL patients. Using our previously described conditional Myb overexpression mice, we generated two distinct MYB-driven T-ALL mouse models. We demonstrated that the overexpression of Myb synergizes with Pten deletion but not with the overexpression of Lmo2 to accelerate the development of T-cell lymphoblastic leukemias. We also showed that MYB is a dependency factor in T-ALL since RNA interference of Myb blocked cell cycle progression and induced apoptosis in both human and murine T-ALL cell lines. Finally, we provide preclinical evidence that targeting the transcriptional activity of MYB can be a useful therapeutic strategy for the treatment of T-ALL.
Assuntos
Proteínas de Homeodomínio/genética , Poli A/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Proto-Oncogênicas/genética , RNA Longo não Codificante/genética , Análise de Sequência de RNA/métodos , Azepinas/farmacologia , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Interferência de RNA , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Triazóis/farmacologiaRESUMO
T-cell lymphoblastic lymphoma (T-LBL) is a rare and aggressive lymphatic cancer, often diagnosed at a young age. Patients are treated with intensive chemotherapy, potentially followed by a hematopoietic stem cell transplantation. Although prognosis of T-LBL has improved with intensified treatment protocols, they are associated with side effects and 10-20% of patients still die from relapsed or refractory disease. Given this, the search toward less toxic anti-lymphoma therapies is ongoing. Here, we targeted the recently described DNA hypermethylated profile in T-LBL with the DNA hypomethylating agent decitabine. We evaluated the anti-lymphoma properties and downstream effects of decitabine, using patient derived xenograft (PDX) models. Decitabine treatment resulted in prolonged lymphoma-free survival in all T-LBL PDX models, which was associated with downregulation of the oncogenic MYC pathway. However, some PDX models showed more benefit of decitabine treatment compared to others. In more sensitive models, differentially methylated CpG regions resulted in more differentially expressed genes in open chromatin regions. This resulted in stronger downregulation of cell cycle genes and upregulation of immune response activating transcripts. Finally, we suggest a gene signature for high decitabine sensitivity in T-LBL. Altogether, we here delivered pre-clinical proof of the potential use of decitabine as a new therapeutic agent in T-LBL.
RESUMO
The pediatric extra-cranial tumor neuroblastoma displays a low mutational burden while recurrent copy number alterations are present in most high-risk cases. Here, we identify SOX11 as a dependency transcription factor in adrenergic neuroblastoma based on recurrent chromosome 2p focal gains and amplifications, specific expression in the normal sympatho-adrenal lineage and adrenergic neuroblastoma, regulation by multiple adrenergic specific (super-)enhancers and strong dependency on high SOX11 expression in adrenergic neuroblastomas. SOX11 regulated direct targets include genes implicated in epigenetic control, cytoskeleton and neurodevelopment. Most notably, SOX11 controls chromatin regulatory complexes, including 10 SWI/SNF core components among which SMARCC1, SMARCA4/BRG1 and ARID1A. Additionally, the histone deacetylase HDAC2, PRC1 complex component CBX2, chromatin-modifying enzyme KDM1A/LSD1 and pioneer factor c-MYB are regulated by SOX11. Finally, SOX11 is identified as a core transcription factor of the core regulatory circuitry (CRC) in adrenergic high-risk neuroblastoma with a potential role as epigenetic master regulator upstream of the CRC.
Assuntos
Neuroblastoma , Humanos , Criança , Neuroblastoma/genética , Fatores de Transcrição/genética , Cromatina , Núcleo Celular , Aberrações Cromossômicas , Adrenérgicos , DNA Helicases , Proteínas Nucleares/genética , Fatores de Transcrição SOXC/genética , Histona DesmetilasesRESUMO
Natural killer (NK) cells are cytotoxic and cytokine-producing lymphocytes that play an important role in the first line of defense against malignant or virus-infected cells. A better understanding of the transcriptional regulation of human NK cell differentiation is crucial to improve the efficacy of NK cell-mediated immunotherapy for cancer treatment. Here, we studied the role of the transcription factor interferon regulatory factor (IRF) 2 in human NK cell differentiation by stable knockdown or overexpression in cord blood hematopoietic stem cells and investigated its effect on development and function of the NK cell progeny. IRF2 overexpression had limited effects in these processes, indicating that endogenous IRF2 expression levels are sufficient. However, IRF2 knockdown greatly reduced the cell numbers of all early differentiation stages, resulting in decimated NK cell numbers. This was not caused by increased apoptosis, but by decreased proliferation. Expression of IRF2 is also required for functional maturation of NK cells, as the remaining NK cells after silencing of IRF2 had a less mature phenotype and showed decreased cytotoxic potential, as well as a greatly reduced cytokine secretion. Thus, IRF2 plays an important role during development and functional maturation of human NK cells.
Assuntos
Células Matadoras Naturais , Fatores de Transcrição , Humanos , Células Matadoras Naturais/metabolismo , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica , Diferenciação Celular/genética , Citocinas/metabolismo , Fator Regulador 2 de Interferon/genética , Fator Regulador 2 de Interferon/metabolismoRESUMO
Natural killer (NK) cells are innate lymphocytes that eliminate virus-infected and cancer cells by cytotoxicity and cytokine secretion. In addition to circulating NK cells, distinct tissue-resident NK subsets have been identified in various organs. Although transcription factors regulating NK cell development and function have been extensively studied in mice, the role of RUNX2 in these processes has not been investigated, neither in mice nor in human. Here, by manipulating RUNX2 expression with either knockdown or overexpression in human haematopoietic stem cell-based NK cell differentiation cultures, combined with transcriptomic and ChIP-sequencing analyses, we established that RUNX2 drives the generation of NK cells, possibly through induction of IL-2Rß expression in NK progenitor cells. Importantly, RUNX2 promotes tissue residency in human NK cells. Our findings have the potential to improve existing NK cell-based cancer therapies and can impact research fields beyond NK cell biology, since tissue-resident subsets have also been described in other lymphocyte subpopulations.
Assuntos
Fatores de Transcrição , Humanos , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica , Células Matadoras Naturais/metabolismo , Fatores de Transcrição/metabolismoRESUMO
High-risk neuroblastoma, a pediatric tumor originating from the sympathetic nervous system, has a low mutation load but highly recurrent somatic DNA copy number variants. Previously, segmental gains and/or amplifications allowed identification of drivers for neuroblastoma development. Using this approach, combined with gene dosage impact on expression and survival, we identified ribonucleotide reductase subunit M2 (RRM2) as a candidate dependency factor further supported by growth inhibition upon in vitro knockdown and accelerated tumor formation in a neuroblastoma zebrafish model coexpressing human RRM2 with MYCN. Forced RRM2 induction alleviates excessive replicative stress induced by CHK1 inhibition, while high RRM2 expression in human neuroblastomas correlates with high CHK1 activity. MYCN-driven zebrafish tumors with RRM2 co-overexpression exhibit differentially expressed DNA repair genes in keeping with enhanced ATR-CHK1 signaling activity. In vitro, RRM2 inhibition enhances intrinsic replication stress checkpoint addiction. Last, combinatorial RRM2-CHK1 inhibition acts synergistic in high-risk neuroblastoma cell lines and patient-derived xenograft models, illustrating the therapeutic potential.
RESUMO
T-bet and Eomes are transcription factors that are known to be important in maturation and function of murine natural killer (NK) cells. Reduced T-BET and EOMES expression results in dysfunctional NK cells and failure to control tumor growth. In contrast to mice, the current knowledge on the role of T-BET and EOMES in human NK cells is rudimentary. Here, we ectopically expressed either T-BET or EOMES in human hematopoietic progenitor cells. Combined transcriptome, chromatin accessibility and protein expression analyses revealed that T-BET or EOMES epigenetically represses hematopoietic stem cell quiescence and non-NK lineage differentiation genes, while activating an NK cell-specific transcriptome and thereby drastically accelerating NK cell differentiation. In this model, the effects of T-BET and EOMES are largely overlapping, yet EOMES shows a superior role in early NK cell maturation and induces faster NK receptor and enhanced CD16 expression. T-BET particularly controls transcription of terminal maturation markers and epigenetically controls strong induction of KIR expression. Finally, NK cells generated upon T-BET or EOMES overexpression display improved functionality, including increased IFN-γ production and killing, and especially EOMES overexpression NK cells have enhanced antibody-dependent cellular cytotoxicity. Our findings reveal novel insights on the regulatory role of T-BET and EOMES in human NK cell maturation and function, which is essential to further understand human NK cell biology and to optimize adoptive NK cell therapies.
Assuntos
Diferenciação Celular , Células-Tronco Hematopoéticas/metabolismo , Células Matadoras Naturais/metabolismo , Proteínas com Domínio T/metabolismo , Animais , Citotoxicidade Celular Dependente de Anticorpos , Linhagem da Célula , Montagem e Desmontagem da Cromatina , Técnicas de Cocultura , Epigênese Genética , Sangue Fetal/citologia , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Células-Tronco Hematopoéticas/imunologia , Humanos , Interferon gama/metabolismo , Células K562 , Células Matadoras Naturais/imunologia , Camundongos , Fenótipo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Receptores KIR/genética , Receptores KIR/metabolismo , Proteínas com Domínio T/genética , TranscriptomaRESUMO
Juvenile myelomonocytic leukemia (JMML) treatment primarily relies on hematopoietic stem cell transplantation and results in long-term overall survival of 50-60%, demonstrating a need to develop novel treatments. Dysregulation of the non-coding RNA transcriptome has been demonstrated before in this rare and unique disorder of early childhood. In this study, we investigated the therapeutic potential of targeting overexpressed long non-coding RNAs (lncRNAs) in JMML. Total RNA sequencing of bone marrow and peripheral blood mononuclear cell preparations from 19 untreated JMML patients and three healthy children revealed 185 differentially expressed lncRNA genes (131 up- and 54 downregulated). LNA GapmeRs were designed for 10 overexpressed and validated lncRNAs. Molecular knockdown (≥ 70% compared to mock control) after 24 h of incubation was observed with two or more independent GapmeRs in 6 of them. For three lncRNAs (lnc-THADA-4, lnc-ACOT9-1 and NRIR) knockdown resulted in a significant decrease of cell viability after 72 h of incubation in primary cultures of JMML mononuclear cells, respectively. Importantly, the extent of cellular damage correlated with the expression level of the lncRNA of interest. In conclusion, we demonstrated in primary JMML cell cultures that knockdown of overexpressed lncRNAs such as lnc-THADA-4, lnc-ACOT9-1 and NRIR may be a feasible therapeutic strategy.
Assuntos
Antineoplásicos/farmacologia , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Mielomonocítica Juvenil/genética , RNA Longo não Codificante/metabolismo , Adolescente , Antineoplásicos/uso terapêutico , Medula Óssea/patologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Técnicas de Silenciamento de Genes , Voluntários Saudáveis , Humanos , Lactente , Leucemia Mielomonocítica Juvenil/sangue , Leucemia Mielomonocítica Juvenil/tratamento farmacológico , Leucemia Mielomonocítica Juvenil/patologia , Leucócitos Mononucleares , Masculino , Cultura Primária de Células , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , RNA-Seq , Células Tumorais CultivadasRESUMO
Mantle cell lymphoma (MCL) is an aggressive B cell lymphoma with poor long-term overall survival. Currently, MCL research and development of potential cures is hampered by the lack of good in vivo models. MCL is characterized by recurrent translocations of CCND1 or CCND2, resulting in overexpression of the cell cycle regulators cyclin D1 or D2, respectively. Here, we show, for the first time, that hematopoiesis-specific activation of cyclin D2 is sufficient to drive murine MCL-like lymphoma development. Furthermore, we demonstrate that cyclin D2 overexpression can synergize with loss of p53 to form aggressive and transplantable MCL-like lymphomas. Strikingly, cyclin D2-driven lymphomas display transcriptional, immunophenotypic, and functional similarities with B1a B cells. These MCL-like lymphomas have B1a-specific B cell receptors (BCRs), show elevated BCR and NF-κB pathway activation, and display increased MALT1 protease activity. Finally, we provide preclinical evidence that inhibition of MALT1 protease activity, which is essential for the development of early life-derived B1a cells, can be an effective therapeutic strategy to treat MCL.
Assuntos
Ciclina D2/genética , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/patologia , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/antagonistas & inibidores , Aloenxertos , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Ciclina D2/metabolismo , Regulação Neoplásica da Expressão Gênica , Linfoma de Célula do Manto/tratamento farmacológico , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Células Neoplásicas Circulantes , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Roughly half of all high-risk neuroblastoma patients present with MYCN amplification. The molecular consequences of MYCN overexpression in this aggressive pediatric tumor have been studied for decades, but thus far, our understanding of the early initiating steps of MYCN-driven tumor formation is still enigmatic. We performed a detailed transcriptome landscaping during murine TH-MYCN-driven neuroblastoma tumor formation at different time points. The neuroblastoma dependency factor MEIS2, together with ASCL1, was identified as a candidate tumor-initiating factor and shown to be a novel core regulatory circuit member in adrenergic neuroblastomas. Of further interest, we found a KEOPS complex member (gm6890), implicated in homologous double-strand break repair and telomere maintenance, to be strongly upregulated during tumor formation, as well as the checkpoint adaptor Claspin (CLSPN) and three chromosome 17q loci CBX2, GJC1 and LIMD2. Finally, cross-species master regulator analysis identified FOXM1, together with additional hubs controlling transcriptome profiles of MYCN-driven neuroblastoma. In conclusion, time-resolved transcriptome analysis of early hyperplastic lesions and full-blown MYCN-driven neuroblastomas yielded novel components implicated in both tumor initiation and maintenance, providing putative novel drug targets for MYCN-driven neuroblastoma.