RESUMO
The NOTCH signaling pathway is implicated in a broad range of developmental processes, including cell fate decisions. However, the molecular basis for its role at the different steps of stem cell lineage commitment is unclear. We recently identified the NOTCH signaling pathway as a positive regulator of megakaryocyte lineage specification during hematopoiesis, but the developmental pathways that allow hematopoietic stem cell differentiation into the erythro-megakaryocytic lineages remain controversial. Here, we investigated the role of downstream mediators of NOTCH during megakaryopoiesis and report crosstalk between the NOTCH and PI3K/AKT pathways. We demonstrate the inhibitory role of phosphatase with tensin homolog and Forkhead Box class O factors on megakaryopoiesis in vivo. Finally, our data annotate developmental mechanisms in the hematopoietic system that enable a decision to be made either at the hematopoietic stem cell or the committed progenitor level to commit to the megakaryocyte lineage, supporting the existence of 2 distinct developmental pathways.
Assuntos
Diferenciação Celular , Linhagem da Célula/fisiologia , Megacariócitos/fisiologia , Proteína Oncogênica v-akt/metabolismo , Receptores Notch/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem da Célula/genética , Células Cultivadas , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/fisiologia , Megacariócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Oncogênica v-akt/genética , Proteína Oncogênica v-akt/fisiologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/fisiologia , Receptor Cross-Talk/fisiologia , Receptores Notch/genética , Receptores Notch/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Trombopoese/genéticaRESUMO
The tyrosine kinase JAK3 plays a well-established role during normal lymphocyte development and is constitutively phosphorylated in several lymphoid malignancies. However, its contribution to lymphomagenesis remains elusive. In this study, we used the newly identified activating JAK3A572V mutation to elucidate the effect of constitutive JAK3 signaling on murine lymphopoiesis. In a bone marrow transplantation model, JAK3A572V induces an aggressive, fatal, and transplantable lymphoproliferative disorder characterized by the expansion of CD8(+)TCRalphabeta(+)CD44(+)CD122(+)Ly-6C(+) T cells that closely resemble an effector/memory T-cell subtype. Compared with wild-type counterparts, these cells show increased proliferative capacities in response to polyclonal stimulation, enhanced survival rates with elevated expression of Bcl-2, and increased production of interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha), correlating with enhanced cytotoxic abilities against allogeneic target cells. Of interest, the JAK3A572V disease is epidermotropic and produces intraepidermal microabscesses. Taken together, these clinical features are reminiscent of those observed in an uncommon but aggressive subset of CD8(+) human cutaneous T-cell lymphomas (CTCLs). However, we also observed a CD4(+) CTCL-like phenotype when cells are transplanted in an MHC-I-deficient background. These data demonstrate that constitutive JAK3 activation disrupts T-cell homeostasis and induces lymphoproliferative diseases in mice.
Assuntos
Linfócitos T CD8-Positivos/patologia , Janus Quinase 3/fisiologia , Linfopoese/fisiologia , Transtornos Linfoproliferativos/etiologia , Mutação Puntual , Proteínas Recombinantes de Fusão/fisiologia , Subpopulações de Linfócitos T/patologia , Animais , Antígenos Ly/análise , Transplante de Medula Óssea , Linfócitos T CD4-Positivos/química , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/química , Indução Enzimática , Humanos , Receptores de Hialuronatos/análise , Subunidade beta de Receptor de Interleucina-2/análise , Janus Quinase 3/biossíntese , Janus Quinase 3/genética , Linfoma Cutâneo de Células T/patologia , Linfopoese/genética , Transtornos Linfoproliferativos/enzimologia , Transtornos Linfoproliferativos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Quimera por Radiação , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Pele/patologia , Subpopulações de Linfócitos T/químicaRESUMO
To study the impact of oncogenic K-Ras on T-cell leukemia/lymphoma development and progression, we made use of a conditional K-Ras(G12D) murine knockin model, in which oncogenic K-Ras is expressed from its endogenous promoter. Transplantation of whole bone marrow cells that express oncogenic K-Ras into wild-type recipient mice resulted in a highly penetrant, aggressive T-cell leukemia/lymphoma. The lymphoblasts were composed of a CD4/CD8 double-positive population that aberrantly expressed CD44. Thymi of primary donor mice showed reduced cellularity, and immunophenotypic analysis demonstrated a block in differentiation at the double-negative 1 stage. With progression of disease, approximately 50% of mice acquired Notch1 mutations within the PEST domain. Of note, primary lymphoblasts were hypersensitive to gamma-secretase inhibitor treatment, which is known to impair Notch signaling. This inhibition was Notch-specific as assessed by down-regulation of Notch1 target genes and intracellular cleaved Notch. We also observed that the oncogenic K-Ras-induced T-cell disease was responsive to rapamycin and inhibitors of the RAS/MAPK pathway. These data indicate that patients with T-cell leukemia with K-Ras mutations may benefit from therapies that target the NOTCH pathway alone or in combination with inhibition of the PI3K/AKT/MTOR and RAS/MAPK pathways.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Regulação da Expressão Gênica , Genes ras , Leucemia de Células T/genética , Linfoma de Células T/genética , Receptor Notch1/genética , Proteínas ras/fisiologia , Animais , Células da Medula Óssea/citologia , Diferenciação Celular , Humanos , Receptores de Hialuronatos/biossíntese , Camundongos , Camundongos Transgênicos , MutaçãoRESUMO
JAK3-activating mutations are commonly seen in chronic or acute hematologic malignancies affecting the myeloid, megakaryocytic, lymphoid, and natural killer (NK) cell compartment. Overexpression models of mutant JAK3 or pharmacologic inhibition of its kinase activity have highlighted the role that these constitutively activated mutants play in the T-cell, NK cell, and megakaryocytic lineages, but to date, the functional impact of JAK3 mutations at an endogenous level remains unknown. Here, we report a JAK3A572V knockin mouse model and demonstrate that activated JAK3 leads to a progressive and dose-dependent expansion of CD8+ T cells in the periphery before colonization of the bone marrow. This phenotype is dependent on the γc chain of cytokine receptors and presents several features of the human leukemic form of cutaneous T-cell lymphoma (L-CTCL), including skin involvements. We also showed that the JAK3A572V-positive malignant cells are transplantable and phenotypically heterogeneous in bone marrow transplantation assays. Interestingly, we revealed that activated JAK3 functionally cooperates with partial trisomy 21 in vivo to enhance the L-CTCL phenotype, ultimately leading to a lethal and fully penetrant disorder. Finally, we assessed the efficacy of JAK3 inhibition and showed that CTCL JAK3A572V-positive T cells are sensitive to tofacitinib, which provides additional preclinical insights into the use of JAK3 inhibitors in these disorders. Altogether, this JAK3A572V knockin model is a relevant new tool for testing the efficacy of JAK inhibitors in JAK3-related hematopoietic malignancies.
Assuntos
Cromossomos de Mamíferos/metabolismo , Neoplasias Hematológicas/metabolismo , Janus Quinase 3/metabolismo , Linfoma Cutâneo de Células T/metabolismo , Mutação de Sentido Incorreto , Neoplasias Experimentais/metabolismo , Trissomia , Substituição de Aminoácidos , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Cromossomos de Mamíferos/genética , Técnicas de Introdução de Genes , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Janus Quinase 3/genética , Linfoma Cutâneo de Células T/tratamento farmacológico , Linfoma Cutâneo de Células T/genética , Linfoma Cutâneo de Células T/patologia , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologiaRESUMO
JAK3 is a non-receptor tyrosine kinase, predominantly expressed in hematopoietic cells and that has been implicated in the signal transduction of the common gamma chain subfamily of cytokine receptors. As a result, JAK3 plays an essential role in hematopoieisis during T cell development. JAK3 inactivating mutations result in immunodeficiency syndromes (SCID) in both humans and mice. Recent data indicate that abnormal activation of JAK3 due to activating mutations is also found in human hematological malignancies, including acute megakaryoblastic leukemia (AMKL) and cutaneous T cell lymphoma (CTCL). After a brief summary of the JAK3 structure and function, we will review the evidence on the emerging role of JAK3 activation in hematological malignancies that warrant further studies to test the relevance of specific inhibition of JAK3 as a therapeutic approach to these challenging clinical entities.
Assuntos
Neoplasias Hematológicas/imunologia , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Janus Quinase 3/metabolismo , Receptores de Citocinas/metabolismo , Animais , Regulação Neoplásica da Expressão Gênica , Neoplasias Hematológicas/tratamento farmacológico , Humanos , Janus Quinase 3/genética , Janus Quinase 3/imunologia , Linfócitos/imunologia , Camundongos , Piperidinas , Pirimidinas/uso terapêutico , Pirróis/uso terapêutico , Transdução de SinaisRESUMO
Acute megakaryoblastic leukemia (AMKL) is a form of acute myeloid leukemia (AML) associated with a poor prognosis. The genetics and pathophysiology of AMKL are not well understood. We generated a knockin mouse model of the one twenty-two-megakaryocytic acute leukemia (OTT-MAL) fusion oncogene that results from the t(1;22)(p13;q13) translocation specifically associated with a subtype of pediatric AMKL. We report here that OTT-MAL expression deregulated transcriptional activity of the canonical Notch signaling pathway transcription factor recombination signal binding protein for immunoglobulin kappa J region (RBPJ) and caused abnormal fetal megakaryopoiesis. Furthermore, cooperation between OTT-MAL and an activating mutation of the thrombopoietin receptor myeloproliferative leukemia virus oncogene (MPL) efficiently induced a short-latency AMKL that recapitulated all the features of human AMKL, including megakaryoblast hyperproliferation and maturation block, thrombocytopenia, organomegaly, and extensive fibrosis. Our results establish that concomitant activation of RBPJ (Notch signaling) and MPL (cytokine signaling) transforms cells of the megakaryocytic lineage and suggest that specific targeting of these pathways could be of therapeutic value for human AMKL.
Assuntos
Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Leucemia Megacarioblástica Aguda/genética , Fusão Oncogênica , Proteínas de Fusão Oncogênica/genética , Animais , Modelos Animais de Doenças , Hematopoese , Humanos , Leucemia Megacarioblástica Aguda/etiologia , Leucemia Megacarioblástica Aguda/metabolismo , Leucemia Megacarioblástica Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Receptores Notch/metabolismo , Receptores de Trombopoetina/genética , Transdução de Sinais , Transcrição GênicaRESUMO
We report the unexpected finding that loss of Hh signaling through conditional deletion of Smoothened (Smo) in the adult hematopoietic compartment has no apparent effect on adult hematopoiesis, including peripheral blood count, number or cell-cycle status of stem or progenitor cells, hematopoietic colony-forming potential, long-term repopulating activity in competitive repopulation assays, or stress response to serial 5-fluorouracil treatment. Furthermore, pharmacologic inhibition of Hh signaling with a potent and selective small molecule antagonist has no substantive effect on hematopoiesis in the mouse. In addition, Hh signaling is not required for the development of MLL-AF9-mediated acute myeloid leukemia (AML). Taken together, these data demonstrate that Hh signaling is dispensable for normal hematopoietic development and hematopoietic stem cell function, indicating that targeting of Hh signaling in solid tumors is not likely to result in hematopoietic toxicity. Furthermore, the Hh pathway may not be a compelling target in certain hematopoietic malignancies.
Assuntos
Proteínas Hedgehog/fisiologia , Hematopoese , Células-Tronco Hematopoéticas/fisiologia , Transdução de Sinais/fisiologia , Células-Tronco Adultas/fisiologia , Animais , Leucemia Mieloide Aguda/etiologia , Camundongos , Neoplasias Experimentais/etiologia , Receptores Acoplados a Proteínas G/deficiência , Receptor SmoothenedRESUMO
In the hematopoietic system, Notch signaling specifies T cell lineage fate, in part through negative regulation of B cell and myeloid lineage development. However, we unexpectedly observed the development of megakaryocytes when using heterotypic cocultures of hematopoietic stem cells with OP9 cells expressing Delta-like1, but not with parental OP9 cells. This effect was abrogated by inhibition of Notch signaling either with gamma-secretase inhibitors or by expression of the dominant-negative Mastermind-like1. The importance of Notch signaling for megakaryopoietic development in vivo was confirmed by using mutant alleles that either activate or inhibit Notch signaling. These findings indicate that Notch is a positive regulator of megakaryopoiesis and plays a more complex role in cell-fate decisions among myeloid progenitors than previously appreciated.
Assuntos
Diferenciação Celular , Células-Tronco Hematopoéticas/citologia , Megacariócitos/citologia , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Proteínas de Ligação ao Cálcio , Células-Tronco Hematopoéticas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Megacariócitos/metabolismo , Camundongos , Células Estromais/metabolismo , Transcrição GênicaRESUMO
Activated tyrosine kinases have been frequently implicated in the pathogenesis of cancer, including acute myeloid leukemia (AML), and are validated targets for therapeutic intervention with small-molecule kinase inhibitors. To identify novel activated tyrosine kinases in AML, we used a discovery platform consisting of immunoaffinity profiling coupled to mass spectrometry that identifies large numbers of tyrosine-phosphorylated proteins, including active kinases. This method revealed the presence of an activated colony-stimulating factor 1 receptor (CSF1R) kinase in the acute megakaryoblastic leukemia (AMKL) cell line MKPL-1. Further studies using siRNA and a small-molecule inhibitor showed that CSF1R is essential for the growth and survival of MKPL-1 cells. DNA sequence analysis of cDNA generated by 5'RACE from CSF1R coding sequences identified a novel fusion of the RNA binding motif 6 (RBM6) gene to CSF1R gene generated presumably by a t(3;5)(p21;q33) translocation. Expression of the RBM6-CSF1R fusion protein conferred interleukin-3 (IL-3)-independent growth in BaF3 cells, and induces a myeloid proliferative disease (MPD) with features of megakaryoblastic leukemia in a murine transplant model. These findings identify a novel potential therapeutic target in leukemogenesis, and demonstrate the utility of phosphoproteomic strategies for discovery of tyrosine kinase alleles.
Assuntos
Leucemia Megacarioblástica Aguda/genética , Proteínas de Fusão Oncogênica/fisiologia , Proteínas de Ligação a RNA/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Cromossomos Humanos Par 3 , Cromossomos Humanos Par 5 , Humanos , Leucemia Megacarioblástica Aguda/etiologia , Camundongos , Transplante de Neoplasias , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/isolamento & purificação , Proteínas Tirosina Quinases/isolamento & purificação , Receptor de Fator Estimulador de Colônias de Macrófagos/isolamento & purificação , Análise de Sequência de DNA , Translocação Genética , Transplante HeterólogoRESUMO
Fas-induced apoptosis is a critical process for normal immune system development and function. Although many molecular components in the Fas signaling pathway have been identified, a systematic understanding of how they work together to determine network dynamics and apoptosis itself has remained elusive. To address this, we generated a computational model for interpreting and predicting effects of pathway component properties. The model integrates current information concerning the signaling network downstream of Fas activation, through both type I and type II pathways, until activation of caspase-3. Unknown parameter values in the model were estimated using experimental data obtained from human Jurkat T cells. To elucidate critical signaling network properties, we examined the effects of altering the level of Bcl-2 on the kinetics of caspase-3 activation, using both overexpression and knockdown in the model and experimentally. Overexpression was used to distinguish among alternative hypotheses for inhibitory binding interactions of Bcl-2 with various components in the mitochondrial pathway. In comparing model simulations with experimental results, we find the best agreement when Bcl-2 blocks the release of cytochrome c by binding to both Bax and truncated Bid instead of Bax, truncated Bid, or Bid alone. Moreover, although Bcl-2 overexpression strongly reduces caspase-3 activation, Bcl-2 knockdown has a negligible effect, demonstrating a general model finding that varying the expression levels of signal molecules frequently has asymmetric effects on the outcome. Finally, we demonstrate that the relative dominance of type I vs type II pathways can be switched by varying particular signaling component levels without changing network structure.