RESUMO
T-cell malignancies comprise a heterogeneous group of cancers resulting from the clonal expansion of T-cells at different developmental stages [...].
RESUMO
Cell differentiation is accompanied by epigenetic changes leading to precise lineage definition and cell identity. Here we present a comprehensive resource of epigenomic data of human T cell precursors along with an integrative analysis of other hematopoietic populations. Although T cell commitment is accompanied by large scale epigenetic changes, we observed that the majority of distal regulatory elements are constitutively unmethylated throughout T cell differentiation, irrespective of their activation status. Among these, the TCRA gene enhancer (Eα) is in an open and unmethylated chromatin structure well before activation. Integrative analyses revealed that the HOXA5-9 transcription factors repress the Eα enhancer at early stages of T cell differentiation, while their decommission is required for TCRA locus activation and enforced αß T lineage differentiation. Remarkably, the HOXA-mediated repression of Eα is paralleled by the ectopic expression of homeodomain-related oncogenes in T cell acute lymphoblastic leukemia. These results highlight an analogous enhancer repression mechanism at play in normal and cancer conditions, but imposing distinct developmental constraints.
Assuntos
Elementos Facilitadores Genéticos , Hematopoese/genética , Receptores de Antígenos de Linfócitos T/genética , Timo/citologia , Animais , Proteínas Reguladoras de Apoptose/genética , Diferenciação Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , Desmetilação do DNA , Metilação de DNA/genética , Epigenoma , Regulação da Expressão Gênica , Rearranjo Gênico do Linfócito T , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Ativação Linfocitária/imunologia , Camundongos , Ligação Proteica , Processamento de Proteína Pós-Traducional , Células-Tronco/citologia , Linfócitos T/citologia , Timócitos/metabolismoRESUMO
Chimeric antigen receptor (CAR) T-cells targeting CD19 demonstrated remarkable efficacy for the treatment of B-cell malignancies. The development of CAR T-cells against T-cell malignancies appears more challenging due to the similarities between the therapeutic, normal and malignant T-cells. The obstacles include CAR T-cell fratricide, T-cell aplasia, and contamination of CAR T-cell products with malignant T-cells. Here, we review these challenges and propose solutions to overcome these limitations.
Assuntos
Linfoma de Células B/imunologia , Linfoma de Células B/terapia , Neoplasias/imunologia , Neoplasias/terapia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Animais , Antígenos CD19/imunologia , Humanos , Imunoterapia Adotiva/métodosRESUMO
Dormant hematopoietic stem cells (dHSCs) are atop the hematopoietic hierarchy. The molecular identity of dHSCs and the mechanisms regulating their maintenance or exit from dormancy remain uncertain. Here, we use single-cell RNA sequencing (RNA-seq) analysis to show that the transition from dormancy toward cell-cycle entry is a continuous developmental path associated with upregulation of biosynthetic processes rather than a stepwise progression. In addition, low Myc levels and high expression of a retinoic acid program are characteristic for dHSCs. To follow the behavior of dHSCs in situ, a Gprc5c-controlled reporter mouse was established. Treatment with all-trans retinoic acid antagonizes stress-induced activation of dHSCs by restricting protein translation and levels of reactive oxygen species (ROS) and Myc. Mice maintained on a vitamin A-free diet lose HSCs and show a disrupted re-entry into dormancy after exposure to inflammatory stress stimuli. Our results highlight the impact of dietary vitamin A on the regulation of cell-cycle-mediated stem cell plasticity. VIDEO ABSTRACT.
Assuntos
Células-Tronco Hematopoéticas/citologia , Transdução de Sinais , Tretinoína/farmacologia , Vitamina A/administração & dosagem , Animais , Vias Biossintéticas , Técnicas de Cultura de Células , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular , Dieta , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/efeitos dos fármacos , Camundongos , Poli I-C/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Análise de Célula Única , Estresse Fisiológico , Vitamina A/farmacologia , Vitaminas/administração & dosagem , Vitaminas/farmacologiaRESUMO
UNLABELLED: Cancer onset and progression involves the accumulation of multiple oncogenic hits, which are thought to dominate or bypass the physiologic regulatory mechanisms in tissue development and homeostasis. We demonstrate in T-cell acute lymphoblastic leukemia (T-ALL) that, irrespective of the complex oncogenic abnormalities underlying tumor progression, experimentally induced, persistent T-cell receptor (TCR) signaling has antileukemic properties and enforces a molecular program resembling thymic negative selection, a major developmental event in normal T-cell development. Using mouse models of T-ALL, we show that induction of TCR signaling by high-affinity self-peptide/MHC or treatment with monoclonal antibodies to the CD3ε chain (anti-CD3) causes massive leukemic cell death. Importantly, anti-CD3 treatment hampered leukemogenesis in mice transplanted with either mouse- or patient-derived T-ALLs. These data provide a strong rationale for targeted therapy based on anti-CD3 treatment of patients with TCR-expressing T-ALL and demonstrate that endogenous developmental checkpoint pathways are amenable to therapeutic intervention in cancer cells. SIGNIFICANCE: T-ALLs are aggressive malignant lymphoid proliferations of T-cell precursors characterized by high relapse rates and poor prognosis, calling for the search for novel therapeutic options. Here, we report that the lineage-specific TCR/CD3 developmental checkpoint controlling cell death in normal T-cell progenitors remains switchable to induce massive tumor cell apoptosis in T-ALL and is amenable to preclinical therapeutic intervention. Cancer Discov; 6(9); 972-85. ©2016 AACR.See related commentary by Lemonnier and Mak, p. 946This article is highlighted in the In This Issue feature, p. 932.
Assuntos
Leucemia de Células T/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Complexo CD3/imunologia , Complexo CD3/metabolismo , Seleção Clonal Mediada por Antígeno , Modelos Animais de Doenças , Feminino , Humanos , Imunofenotipagem , Leucemia de Células T/tratamento farmacológico , Leucemia de Células T/genética , Leucemia de Células T/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
The status of long-term quiescence and dormancy guarantees the integrity of hematopoietic stem cells (HSCs) during adult homeostasis. However the molecular mechanisms regulating HSC dormancy remain poorly understood. Here we show that cylindromatosis (CYLD), a tumor suppressor gene and negative regulator of NF-κB signaling with deubiquitinase activity, is highly expressed in label-retaining dormant HSCs (dHSCs). Moreover, Cre-mediated conditional elimination of the catalytic domain of CYLD induced dHSCs to exit quiescence and abrogated their repopulation and self-renewal potential. This phenotype is dependent on the interactions between CYLD and its substrate TRAF2 (tumor necrosis factor-associated factor 2). HSCs expressing a mutant CYLD with an intact catalytic domain, but unable to bind TRAF2, showed the same HSC phenotype. Unexpectedly, the robust cycling of HSCs lacking functional CYLD-TRAF2 interactions was not elicited by increased NF-κB signaling, but instead by increased activation of the p38MAPK pathway. Pharmacological inhibition of p38MAPK rescued the phenotype of CYLD loss, identifying the CYLD-TRAF2-p38MAPK pathway as a novel important regulator of HSC function restricting HSC cycling and promoting dormancy.
Assuntos
Cisteína Endopeptidases/imunologia , Células-Tronco Hematopoéticas/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Fator 2 Associado a Receptor de TNF/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Animais , Cisteína Endopeptidases/genética , Enzima Desubiquitinante CYLD , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Células-Tronco Hematopoéticas/citologia , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Knockout , Mutação , NF-kappa B/genética , NF-kappa B/imunologia , Fator 2 Associado a Receptor de TNF/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
The phosphatase and tumor suppressor PTEN inhibits the phosphoinositol-3-kinase (PI3K) signaling pathway and plays a key role in cell growth, proliferation, survival, and migration. Pten conditional deletion using MxCre or Scl-CreER(T) leads to splenomegaly and leukemia formation, which occurs after the relocation of normal hematopoietic stem cells (HSCs) from the bone marrow to the spleen. Unexpectedly, dormant HSCs in the bone marrow do not enter the cell cycle upon Pten loss, they do not lose self-renewal activity, and they are not exhausted. Instead, Pten deficiency causes an up-regulation of the PI3K pathway in myeloid cells, but not in HSCs. Strikingly, myeloid cells secrete high levels of G-CSF upon Pten loss, leading to the mobilization of HSCs from the bone marrow and accumulation in the spleen. After deletion of Pten in mice lacking G-CSF, the splenomegaly, myeloproliferative disease, and splenic HSC accumulation are rescued. Our data show that although PTEN has little if any role in normal HSCs, it is essential to prevent overt G-CSF production by myeloid and stromal cells which otherwise causes HSCs to relocate to the spleen followed by lethal leukemia initiation.
Assuntos
Medula Óssea/enzimologia , Fator Estimulador de Colônias de Granulócitos/metabolismo , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/enzimologia , PTEN Fosfo-Hidrolase/deficiência , Alelos , Animais , Citocinas/metabolismo , Deleção de Genes , Fator Estimulador de Colônias de Granulócitos/deficiência , Integrases/metabolismo , Camundongos , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/patologia , PTEN Fosfo-Hidrolase/metabolismo , Fenótipo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/enzimologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Baço/metabolismo , Baço/patologiaRESUMO
The cell cycle regulators involved in maintaining the quiescence, and thereby the self-renewal capacity, of somatic stem cells have long been elusive. Two new Cell Stem Cell articles in this issue (Matsumoto et al., 2011; Zou et al., 2011) now show that the CDK inhibitor p57 is a crucial brake for cycling HSCs, and links self-renewal activity to cell cycle quiescence.
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Células-Tronco Adultas/metabolismo , Sobrevivência Celular , Senescência Celular , Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Células-Tronco Hematopoéticas/metabolismo , AnimaisRESUMO
Steady-state egress of hematopoietic progenitor cells can be rapidly amplified by mobilizing agents such as AMD3100, the mechanism, however, is poorly understood. We report that AMD3100 increased the homeostatic release of the chemokine stromal cell derived factor-1 (SDF-1) to the circulation in mice and non-human primates. Neutralizing antibodies against CXCR4 or SDF-1 inhibited both steady state and AMD3100-induced SDF-1 release and reduced egress of murine progenitor cells over mature leukocytes. Intra-bone injection of biotinylated SDF-1 also enhanced release of this chemokine and murine progenitor cell mobilization. AMD3100 directly induced SDF-1 release from CXCR4(+) human bone marrow osteoblasts and endothelial cells and activated uPA in a CXCR4/JNK-dependent manner. Additionally, ROS inhibition reduced AMD3100-induced SDF-1 release, activation of circulating uPA and mobilization of progenitor cells. Norepinephrine treatment, mimicking acute stress, rapidly increased SDF-1 release and progenitor cell mobilization, whereas ß2-adrenergic antagonist inhibited both steady state and AMD3100-induced SDF-1 release and progenitor cell mobilization in mice. In conclusion, this study reveals that SDF-1 release from bone marrow stromal cells to the circulation emerges as a pivotal mechanism essential for steady-state egress and rapid mobilization of hematopoietic progenitor cells, but not mature leukocytes.
Assuntos
Células da Medula Óssea/metabolismo , Quimiocina CXCL12/fisiologia , Mobilização de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Compostos Heterocíclicos/farmacologia , Norepinefrina/farmacologia , Receptores CXCR4/fisiologia , Animais , Benzilaminas , Células Cultivadas , Ciclamos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células Estromais/metabolismoRESUMO
Mechanisms governing stress-induced hematopoietic progenitor cell mobilization are not fully deciphered. We report that during granulocyte colony-stimulating factor-induced mobilization c-Met expression and signaling are up-regulated on immature bone marrow progenitors. Interestingly, stromal cell-derived factor 1/CXC chemokine receptor-4 signaling induced hepatocyte growth factor production and c-Met activation. We found that c-Met inhibition reduced mobilization of both immature progenitors and the more primitive Sca-1(+)/c-Kit(+)/Lin(-) cells and interfered with their enhanced chemotactic migration to stromal cell-derived factor 1. c-Met activation resulted in cellular accumulation of reactive oxygen species by mammalian target of rapamycin inhibition of Forkhead Box, subclass O3a. Blockage of mammalian target of rapamycin inhibition or reactive oxygen species signaling impaired c-Met-mediated mobilization. Our data show dynamic c-Met expression and function in the bone marrow and show that enhanced c-Met signaling is crucial to facilitate stress-induced mobilization of progenitor cells as part of host defense and repair mechanisms.
Assuntos
Movimento Celular/fisiologia , Fator Estimulador de Colônias de Granulócitos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Animais , Quimiocina CXCL12/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Fator de Crescimento de Hepatócito/metabolismo , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The signals regulating the survival of mature splenic B cells have become a major focus in recent studies of B cell immunology. Durable B cell persistence in the periphery is dependent on survival signals that are transduced by cell surface receptors. In this study, we describe a novel biological mechanism involved in mature B cell homeostasis, the hepatocyte growth factor/scatter factor (HGF)/c-Met pathway. We demonstrate that c-Met activation by HGF leads to a survival cascade, whereas its blockade results in induction of mature B cell death. Our results emphasize a unique and critical function for c-Met signaling in the previously described macrophage migration inhibitory factor/CD74-induced survival pathway. Macrophage migration inhibitory factor recruits c-Met to the CD74/CD44 complex and thereby enables the induction of a signaling cascade within the cell. This signal results in HGF secretion, which stimulates the survival of the mature B cell population in an autocrine manner. Thus, the CD74-HGF/c-Met axis defines a novel physiologic survival pathway in mature B cells, resulting in the control of the humoral immune response.
Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Linfócitos B/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Antígenos de Diferenciação de Linfócitos B/genética , Apoptose/efeitos dos fármacos , Linfócitos B/citologia , Western Blotting , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citometria de Fluxo , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/farmacologia , Antígenos de Histocompatibilidade Classe II/genética , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Fatores Inibidores da Migração de Macrófagos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Proteínas Proto-Oncogênicas c-met/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacosRESUMO
The mechanisms governing hematopoietic progenitor cell mobilization are not fully understood. We report higher membrane type 1-MMP (MT1-MMP) and lower expression of the MT1-MMP inhibitor, reversion-inducing cysteine-rich protein with Kazal motifs (RECK), on isolated circulating human CD34+ progenitor cells compared with immature BM cells. The expression of MT1-MMP correlated with clinical mobilization of CD34+ cells in healthy donors and patients with lymphoid malignancies. Treatment with G-CSF further increased MT1-MMP and decreased RECK expression in human and murine hematopoietic cells in a PI3K/Akt-dependent manner, resulting in elevated MT1-MMP activity. Blocking MT1-MMP function by Abs or siRNAs impaired chemotaxis and homing of G-CSF-mobilized human CD34+ progenitors. The mobilization of immature and maturing human progenitors in chimeric NOD/SCID mice by G-CSF was inhibited by anti-MT1-MMP treatment, while RECK neutralization promoted motility and egress of BM CD34+ cells. BM c-kit+ cells from MT1-MMP-deficient mice also exhibited inferior chemotaxis, reduced homing and engraftment capacities, and impaired G-CSF-induced mobilization in murine chimeras. Membranal CD44 cleavage by MT1-MMP was enhanced following G-CSF treatment, reducing CD34+ cell adhesion. Accordingly, CD44-deficient mice had a higher frequency of circulating progenitors. Our results reveal that the motility, adhesion, homing, and mobilization of human hematopoietic progenitor cells are regulated in a cell-autonomous manner by dynamic and opposite changes in MT1-MMP and RECK expression.
Assuntos
Antígenos CD34/análise , Fator Estimulador de Colônias de Granulócitos/farmacologia , Células-Tronco Hematopoéticas/fisiologia , Metaloproteinase 14 da Matriz/genética , Glicoproteínas de Membrana/genética , Animais , Antígenos CD/análise , Células da Medula Óssea/fisiologia , Movimento Celular/fisiologia , Quimiotaxia , Quimera/genética , Proteínas Ligadas por GPI , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/imunologia , Humanos , Metaloproteinase 14 da Matriz/deficiência , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Metaloproteinase 14 da Matriz/metabolismo , Glicoproteínas de Membrana/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , RNA Mensageiro/genética , RNA Interferente Pequeno/genéticaRESUMO
The CD45 phosphatase is uniquely expressed by all leukocytes, but its role in regulating hematopoietic progenitors is poorly understood. We show that enhanced CD45 expression on bone marrow (BM) leukocytes correlates with increased cell motility in response to stress signals. Moreover, immature CD45 knockout (KO) cells showed defective motility, including reduced homing (both steady state and in response to stromal-derived factor 1) and reduced granulocyte colony-stimulating factor mobilization. These defects were associated with increased cell adhesion mediated by reduced matrix metalloproteinase 9 secretion and imbalanced Src kinase activity. Poor mobilization of CD45KO progenitors by the receptor activator of nuclear factor kappaB ligand, and impaired modulation of the endosteal components osteopontin and stem cell factor, suggested defective osteoclast function. Indeed, CD45KO osteoclasts exhibited impaired bone remodeling and abnormal morphology, which we attributed to defective cell fusion and Src function. This led to irregular distribution of metaphyseal bone trabecules, a region enriched with stem cell niches. Consequently, CD45KO mice had less primitive cells in the BM and increased numbers of these cells in the spleen, yet with reduced homing and repopulation potential. Uncoupling environmental and intrinsic defects in chimeric mice, we demonstrated that CD45 regulates progenitor movement and retention by influencing both the hematopoietic and nonhematopoietic compartments.
Assuntos
Remodelação Óssea , Osso e Ossos , Movimento Celular/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Antígenos Comuns de Leucócito/metabolismo , Osteoclastos/fisiologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Osso e Ossos/anatomia & histologia , Osso e Ossos/fisiologia , Adesão Celular/fisiologia , Quimiocina CXCL12/metabolismo , Ativação Enzimática , Fator Estimulador de Colônias de Granulócitos/metabolismo , Células-Tronco Hematopoéticas/citologia , Antígenos Comuns de Leucócito/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Osteoclastos/citologia , Ligante RANK/metabolismo , Transdução de Sinais/fisiologia , Quinases da Família src/metabolismoRESUMO
As mobilized peripheral blood (MPB) represents an attractive cell source for gene therapy, we investigated the ability of third-generation lentiviral vectors (LVs) to transfer the enhanced green fluorescent protein gene into MPB CD34(+) cells in culture conditions allowing expansion of transplantable human hematopoietic stem cells. To date, few studies have reported transduction of MPB cells with vesicular stomatitis virus G pseudotyped LVs. The critical issue remains whether primitive, hematopoietic repopulating cells have, indeed, been transduced. In vitro (5 weeks' culture in FLT3 ligand + thrombopoietin + stem cell factor + interleukin 6) and in vivo (serial transplantation in NOD/SCID mice) experiments show that MPB CD34(+) cells can be effectively long-term transduced by LV and maintain their proliferation, self-renewal, and multilineage differentiation potentials. We show that expansion following transduction improves the engraftment of transduced MPB CD34(+) (4.6-fold expansion of SCID repopulating cells by limiting dilution studies). We propose ex vivo expansion after transduction as an effective tool to improve gene therapy protocols with MPB. Disclosure of potential conflicts of interest is found at the end of this article.