RESUMO
MicroRNAs and heterogeneous ribonucleoproteins (hnRNPs) are posttranscriptional gene regulators that bind mRNA in a sequence-specific manner. Here, we report that loss of miR-328 occurs in blast crisis chronic myelogenous leukemia (CML-BC) in a BCR/ABL dose- and kinase-dependent manner through the MAPK-hnRNP E2 pathway. Restoration of miR-328 expression rescues differentiation and impairs survival of leukemic blasts by simultaneously interacting with the translational regulator poly(rC)-binding protein hnRNP E2 and with the mRNA encoding the survival factor PIM1, respectively. The interaction with hnRNP E2 is independent of the microRNA's seed sequence and it leads to release of CEBPA mRNA from hnRNP E2-mediated translational inhibition. Altogether, these data reveal the dual ability of a microRNA to control cell fate both through base pairing with mRNA targets and through a decoy activity that interferes with the function of regulatory proteins.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , MicroRNAs/metabolismo , Animais , Crise Blástica , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Complexo de Inativação Induzido por RNA/metabolismoRESUMO
BACKGROUND: Using next-generation sequencing (NGS) to guide cancer therapy has created challenges in analyzing and reporting large volumes of genomic data to patients and caregivers. Specifically, providing current, accurate information on newly approved therapies and open clinical trials requires considerable manual curation performed mainly by human "molecular tumor boards" (MTBs). The purpose of this study was to determine the utility of cognitive computing as performed by Watson for Genomics (WfG) compared with a human MTB. MATERIALS AND METHODS: One thousand eighteen patient cases that previously underwent targeted exon sequencing at the University of North Carolina (UNC) and subsequent analysis by the UNCseq informatics pipeline and the UNC MTB between November 7, 2011, and May 12, 2015, were analyzed with WfG, a cognitive computing technology for genomic analysis. RESULTS: Using a WfG-curated actionable gene list, we identified additional genomic events of potential significance (not discovered by traditional MTB curation) in 323 (32%) patients. The majority of these additional genomic events were considered actionable based upon their ability to qualify patients for biomarker-selected clinical trials. Indeed, the opening of a relevant clinical trial within 1 month prior to WfG analysis provided the rationale for identification of a new actionable event in nearly a quarter of the 323 patients. This automated analysis took <3 minutes per case. CONCLUSION: These results demonstrate that the interpretation and actionability of somatic NGS results are evolving too rapidly to rely solely on human curation. Molecular tumor boards empowered by cognitive computing could potentially improve patient care by providing a rapid, comprehensive approach for data analysis and consideration of up-to-date availability of clinical trials. IMPLICATIONS FOR PRACTICE: The results of this study demonstrate that the interpretation and actionability of somatic next-generation sequencing results are evolving too rapidly to rely solely on human curation. Molecular tumor boards empowered by cognitive computing can significantly improve patient care by providing a fast, cost-effective, and comprehensive approach for data analysis in the delivery of precision medicine. Patients and physicians who are considering enrollment in clinical trials may benefit from the support of such tools applied to genomic data.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias/tratamento farmacológico , Biomarcadores Tumorais , Estudos de Casos e Controles , Terapia Combinada , Seguimentos , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Metástase Linfática , Invasividade Neoplásica , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/patologia , Neoplasias/patologia , Prognóstico , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
Allogeneic stem cell transplantation is the most potent form of effective adoptive immunotherapy. The graft-versus-leukemia (GVL) effect mediated by the allogeneic graft, however, is typically coexpressed with graft-versus-host disease (GVHD), which is the major complication of allogeneic stem cell transplantation. In this study, we used genetic and antibody-based strategies to examine the effect that blockade of interleukin 23 (IL-23) signaling had on GVH and GVL reactivity in murine transplantation recipients. These studies demonstrate that the selective protection of the colon that occurs as a consequence of inhibition of IL-23 signaling reduces GVHD without loss of the GVL effect. The separation of GVH and GVL reactivity was noted in both acute and chronic hematologic malignancy models, indicating that this approach was not restricted by the kinetic profile of the underlying leukemia. Furthermore, a potent GVL response could be mounted in the colon under conditions where tumor cells migrated to this site, indicating that this organ did not serve as a sanctuary site for subsequent systemic relapse in GVHD-protected animals. These studies demonstrate that blockade of IL-23 signaling is an effective strategy for separating GVH and GVL responses and identify IL-23 as a therapeutic target for the regulation of alloresponses in humans.
Assuntos
Doenças do Colo/prevenção & controle , Doença Enxerto-Hospedeiro/prevenção & controle , Efeito Enxerto vs Leucemia/imunologia , Subunidade p19 da Interleucina-23 , Leucemia/terapia , Transplante de Células-Tronco , Doença Aguda , Animais , Doença Crônica , Colo/imunologia , Colo/patologia , Doenças do Colo/genética , Doenças do Colo/imunologia , Doenças do Colo/patologia , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Efeito Enxerto vs Leucemia/genética , Humanos , Leucemia/genética , Leucemia/imunologia , Leucemia/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Transplante HomólogoRESUMO
In a previously developed inducible transgenic mouse model of chronic myeloid leukemia, we now demonstrate that the disease is transplantable using BCR-ABL(+) Lin(-)Sca-1(+)c-kit(+) (LSK) cells. Interestingly, the phenotype is more severe when unfractionated bone marrow cells are transplanted, yet neither progenitor cells (Lin(-)Sca-1(-)c-kit(+)), nor mature granulocytes (CD11b(+)Gr-1(+)), nor potential stem cell niche cells (CD45(-)Ter119(-)) are able to transmit the disease or alter the phenotype. The phenotype is largely independent of BCR-ABL priming before transplantation. However, prolonged BCR-ABL expression abrogates the potential of LSK cells to induce full-blown disease in secondary recipients and increases the fraction of multipotent progenitor cells at the expense of long-term hematopoietic stem cells (LT-HSCs) in the bone marrow. BCR-ABL alters the expression of genes involved in proliferation, survival, and hematopoietic development, probably contributing to the reduced LT-HSC frequency within BCR-ABL(+) LSK cells. Reversion of BCR-ABL, or treatment with imatinib, eradicates mature cells, whereas leukemic stem cells persist, giving rise to relapsed chronic myeloid leukemia on reinduction of BCR-ABL, or imatinib withdrawal. Our results suggest that BCR-ABL induces differentiation of LT-HSCs and decreases their self-renewal capacity.
Assuntos
Diferenciação Celular/genética , Transformação Celular Neoplásica/patologia , Proteínas de Fusão bcr-abl/fisiologia , Células-Tronco Hematopoéticas/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Animais , Separação Celular , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Citometria de Fluxo , Genes abl/fisiologia , Transplante de Células-Tronco Hematopoéticas , Camundongos , Camundongos Transgênicos , Estadiamento de Neoplasias , Transplante de Neoplasias , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVES: Describe an augmented intelligence approach to facilitate the update of evidence for associations in knowledge graphs. METHODS: New publications are filtered through multiple machine learning study classifiers, and filtered publications are combined with articles already included as evidence in the knowledge graph. The corpus is then subjected to named entity recognition, semantic dictionary mapping, term vector space modeling, pairwise similarity, and focal entity match to identify highly related publications. Subject matter experts review recommended articles to assess inclusion in the knowledge graph; discrepancies are resolved by consensus. RESULTS: Study classifiers achieved F-scores from 0.88 to 0.94, and similarity thresholds for each study type were determined by experimentation. Our approach reduces human literature review load by 99%, and over the past 12 months, 41% of recommendations were accepted to update the knowledge graph. CONCLUSION: Integrated search and recommendation exploiting current evidence in a knowledge graph is useful for reducing human cognition load.
RESUMO
Background: Oncologists increasingly rely on clinical genome sequencing to pursue effective, molecularly targeted therapies. This study assesses the validity and utility of the artificial intelligence Watson for Genomics (WfG) for analyzing clinical sequencing results. Methods: This study identified patients with solid tumors who participated in in-house genome sequencing projects at a single cancer specialty hospital between April 2013 and October 2016. Targeted genome sequencing results of these patients' tumors, previously analyzed by multidisciplinary specialists at the hospital, were reanalyzed by WfG. This study measures the concordance between the two evaluations. Results: In 198 patients, in-house genome sequencing detected 785 gene mutations, 40 amplifications, and 22 fusions after eliminating single nucleotide polymorphisms. Breast cancer (n = 40) was the most frequent diagnosis in this analysis, followed by gastric cancer (n = 31), and lung cancer (n = 30). Frequently detected single nucleotide variants were found in TP53 (n = 107), BRCA2 (n = 24), and NOTCH2 (n = 23). MYC (n = 10) was the most frequently detected gene amplification, followed by ERBB2 (n = 9) and CCND1 (n = 6). Concordant pathogenic classifications (i.e., pathogenic, benign, or variant of unknown significance) between in-house specialists and WfG included 705 mutations (89.8%; 95% CI, 87.5%-91.8%), 39 amplifications (97.5%; 95% CI, 86.8-99.9%), and 17 fusions (77.3%; 95% CI, 54.6-92.2%). After about 12 months, reanalysis using a more recent version of WfG demonstrated a better concordance rate of 94.5% (95% CI, 92.7-96.0%) for gene mutations. Across the 249 gene alterations determined to be pathogenic by both methods, including mutations, amplifications, and fusions, WfG covered 84.6% (88 of 104) of all targeted therapies that experts proposed and offered an additional 225 therapeutic options. Conclusions: WfG was able to scour large volumes of data from scientific studies and databases to analyze in-house clinical genome sequencing results and demonstrated the potential for application to clinical practice; however, we must train WfG in clinical trial settings.
RESUMO
Standard chemotherapy is not curative for many patients with acute myeloid leukemia (AML). New treatment strategies combining demethylating agents, such as decitabine, and drugs that induce myelomonocytic differentiation (i.e. Vitamin D3) may re-establish functional hematopoiesis in these patients. We studied the effects of decitabine alone or in combination with Vitamin D3 (VD3) on U937 cells and AML blasts. Preincubation with decitabine (0.1-1 microM) and subsequent exposure to VD3 (3 nM) synergistically induced monocytic differentiation. To elucidate the mechanisms of decitabine- and VD3-induced monocytic differentiation, we investigated the effects of the two drugs on transcription factors implicated in monocytic differentiation. Northern and Western blotting showed that decitabine induced transcription of c-jun but not PU.1, while VD3 increased PU.1, IRF8, and C/EBPbeta but not c-jun. Using electromobility shift assays, we demonstrated increased DNA binding of nuclear proteins from decitabine- and VD3-induced U937 cells to the CD11b promoter. In addition, we investigated whether the myeloid transcription factor Sp1 played a role in decitabine- and VD3-induced CD14 expression. Indeed, we found that mithramycin A, a specific inhibitor of Sp1, inhibited both VD3- and decitabine-induced upregulation of CD14, which is in line with previous data showing that Sp1 is critical for CD14 promoter activity. Induction of CD11b and/or CD14 by decitabine and/or VD3 was confirmed in primary AML patient samples at the time of diagnosis. In conclusion, decitabine synergizes with Vitamin D3 to induce CD11b and CD14 expression, likely by enhancing PU.1/c-jun and Sp1 transcriptional activity.
Assuntos
Azacitidina/análogos & derivados , Colecalciferol/biossíntese , Monócitos/citologia , Transcrição Gênica , Azacitidina/farmacocinética , Antígeno CD11b/biossíntese , Diferenciação Celular , Decitabina , Humanos , Receptores de Lipopolissacarídeos/biossíntese , Modelos Biológicos , Monócitos/metabolismo , Plicamicina/farmacologia , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismo , Células U937RESUMO
The transcription factor, CCAAT/enhancer binding protein alpha (C/EBPalpha) is important in the terminal differentiation of granulocytes, hepatocytes, and adipocytes, and recurrent mutations of C/EBPalpha were described in acute myeloid leukemia. In the lung, C/EBPalpha is expressed in bronchial cells and type II pneumocytes. Abnormal proliferation of the latter cell type was reported in C/EBPalpha knockout mice. We determined the expression of C/EBPalpha by Northern blot analysis in 30 lung cancer cell lines and found significant down-regulation in 24 cell lines. Immunohistochemical study of primary tumor specimens showed undetectable or low expression of C/EBPalpha in 23 of 53 specimens. Its expression was more frequently down-regulated in adenocarcinoma and poorly differentiated cancer specimens than in squamous cell cancers. A higher frequency of reduced expression was found in more advanced stages. To investigate the consequences of C/EBPalpha expression in lung cancer cells, we stably transfected two cell lines that do not express the gene (Calu1 and H358) with a plasmid allowing for induction of C/EBPalpha protein expression. Induction of C/EBPalpha led to significant growth reduction attributable to proliferation arrest, morphological changes characteristic of differentiation, and apoptosis. These results suggest that C/EBPalpha is down-regulated in a large proportion of lung cancers and that it has growth-inhibitory properties in airway epithelial cells. Genetic analysis of the C/EBPalpha gene is in progress to fully evaluate its role as a novel tumor suppressor in lung cancer.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/fisiologia , Neoplasias Pulmonares/patologia , Apoptose/genética , Proteína alfa Estimuladora de Ligação a CCAAT/biossíntese , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Diferenciação Celular/genética , Divisão Celular/genética , Regulação para Baixo , Genes Supressores de Tumor , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transfecção , Células Tumorais Cultivadas , Sulfato de Zinco/farmacologiaRESUMO
We showed previously that CCAAT/enhancer binding protein alpha (C/EBP alpha), a tissue-specific transcription factor, is a candidate tumor suppressor in lung cancer. In the present study, we have performed a transcriptional profiling study of C/EBP alpha target genes using an inducible cell line system. This study led to the identification of hepatocyte nuclear factor 3beta (HNF3 beta), a transcription factor known to play a role in airway differentiation, as a downstream target of C/EBP alpha. We found down-regulation of HNF3 beta expression in a large proportion of lung cancer cell lines examined and identified two novel mutants of HNF3 beta, as well as hypermethylation of the HNF3 beta promoter. We also developed a tetracycline-inducible cell line model to study the cellular consequences of HNF3 beta expression. Conditional expression of HNF3 beta led to significant growth reduction, proliferation arrest, apoptosis, and loss of clonogenic ability, suggesting additionally that HNF3 beta is a novel tumor suppressor in lung cancer. This is the first study to show genetic abnormalities of lung-specific differentiation pathways in the development of lung cancer.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas de Ligação a DNA/genética , Neoplasias Pulmonares/genética , Proteínas Nucleares/genética , Fatores de Transcrição , Apoptose/genética , Divisão Celular/genética , Metilação de DNA , Proteínas de Ligação a DNA/biossíntese , Regulação para Baixo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Fator 3-beta Nuclear de Hepatócito , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Mutação , Proteínas Nucleares/biossíntese , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
The success of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) depends on the requirement for BCR-ABL1 kinase activity in CML progenitors. However, CML quiescent HSCs are TKI resistant and represent a BCR-ABL1 kinase-independent disease reservoir. Here we have shown that persistence of leukemic HSCs in BM requires inhibition of the tumor suppressor protein phosphatase 2A (PP2A) and expression--but not activity--of the BCR-ABL1 oncogene. Examination of HSCs from CML patients and healthy individuals revealed that PP2A activity was suppressed in CML compared with normal HSCs. TKI-resistant CML quiescent HSCs showed increased levels of BCR-ABL1, but very low kinase activity. BCR-ABL1 expression, but not kinase function, was required for recruitment of JAK2, activation of a JAK2/ß-catenin survival/self-renewal pathway, and inhibition of PP2A. PP2A-activating drugs (PADs) markedly reduced survival and self-renewal of CML quiescent HSCs, but not normal quiescent HSCs, through BCR-ABL1 kinase-independent and PP2A-mediated inhibition of JAK2 and ß-catenin. This led to suppression of human leukemic, but not normal, HSC/progenitor survival in BM xenografts and interference with long-term maintenance of BCR-ABL1-positive HSCs in serial transplantation assays. Targeting the JAK2/PP2A/ß-catenin network in quiescent HSCs with PADs (e.g., FTY720) has the potential to treat TKI-refractory CML and relieve lifelong patient dependence on TKIs.
Assuntos
Antineoplásicos/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteína Fosfatase 2/metabolismo , Animais , Apoptose , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Ativadores de Enzimas/farmacologia , Cloridrato de Fingolimode , Proteínas de Fusão bcr-abl/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/enzimologia , Humanos , Janus Quinase 2/metabolismo , Células K562 , Camundongos , Camundongos Transgênicos , Células-Tronco Neoplásicas/enzimologia , Propilenoglicóis/farmacologia , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Via de Sinalização Wnt , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/metabolismoRESUMO
Activated protein C (aPC) therapy reduces mortality in adult patients with severe sepsis. In mouse endotoxemia and sepsis models, mortality reduction requires the cell signaling function of aPC, mediated through protease-activated receptor-1 (PAR1) and endothelial protein C receptor (EPCR; also known as Procr). Candidate cellular targets of aPC include vascular endothelial cells and leukocytes. Here, we show that expression of EPCR and PAR1 on hematopoietic cells is required in mice for an aPC variant that mediates full cell signaling activity but only minimal anticoagulant function (5A-aPC) to reduce the mortality of endotoxemia. Expression of EPCR in mature murine immune cells was limited to a subset of CD8+ conventional dendritic cells. Adoptive transfer of splenic CD11chiPDCA-1- dendritic cells from wild-type mice into animals with hematopoietic EPCR deficiency restored the therapeutic efficacy of aPC, whereas transfer of EPCR-deficient CD11chi dendritic cells or wild-type CD11chi dendritic cells depleted of EPCR+ cells did not. In addition, 5A-aPC inhibited the inflammatory response of conventional dendritic cells independent of EPCR and suppressed IFN-gamma production by natural killer-like dendritic cells. These data reveal an essential role for EPCR and PAR1 on hematopoietic cells, identify EPCR-expressing dendritic immune cells as a critical target of aPC therapy, and document EPCR-independent antiinflammatory effects of aPC on innate immune cells.
Assuntos
Células Dendríticas/efeitos dos fármacos , Endotoxemia/metabolismo , Proteína C/metabolismo , Proteína C/fisiologia , Animais , Anticoagulantes/metabolismo , Anticoagulantes/farmacologia , Linfócitos T CD8-Positivos/metabolismo , Células Dendríticas/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotoxemia/mortalidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína C/farmacologia , Sepse/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Imatinib mesylate (IM) induces remission in chronic myelogenous leukemia (CML) patients but does not eliminate leukemia stem cells (LSCs), which remain a potential source of relapse. Here we investigated the ability of HDAC inhibitors (HDACis) to target CML stem cells. Treatment with HDACis combined with IM effectively induced apoptosis in quiescent CML progenitors resistant to elimination by IM alone, and eliminated CML stem cells capable of engrafting immunodeficient mice. In vivo administration of HDACis with IM markedly diminished LSCs in a transgenic mouse model of CML. The interaction of IM and HDACis inhibited genes regulating hematopoietic stem cell maintenance and survival. HDACi treatment represents an effective strategy to target LSCs in CML patients receiving tyrosine kinase inhibitors.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Piperazinas/farmacologia , Pirimidinas/farmacologia , Animais , Antineoplásicos/uso terapêutico , Apoptose , Benzamidas , Proliferação de Células , Proteínas de Fusão bcr-abl/metabolismo , Inibidores de Histona Desacetilases/uso terapêutico , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Camundongos Transgênicos , Piperazinas/uso terapêutico , Pirimidinas/uso terapêuticoRESUMO
The kinase inhibitors imatinib mesylate and dasatinib are the preferred treatment for Philadelphia chromosome-positive (Ph+) leukemias, and they are highly successful in the chronic phase of chronic myeloid leukemia (CML). However, they are not efficient in Ph+ B-cell acute lymphoblastic leukemia (B-ALL). Ph+ leukemia cells are highly resistant to apoptosis, and evidence from cell lines and primary cells suggest Bcl-xL as a critical mediator of resistance to apoptosis: however, this concept has never been rigorously tested in an animal model. To clarify the role of Bcl-xL in Ph+ B-ALL, we generated 2 mouse models. In the first model, Ph+ B-ALL and loss of Bcl-xL expression are coinduced; in the second model, leukemia is induced with expression of Bcl-xL protein well above the levels found in wild-type lymphoblasts. Deletion of Bcl-xL did not inhibit leukemogenesis or affect apoptosis, but increased cellular proliferation. Consistent with this result, overexpression of Bcl-xL led to decreased cellular proliferation. These models reveal an unexpected role for Bcl-xL in cell-cycle entry and the proliferation of tumor cells.
Assuntos
Ciclo Celular , Leucemia de Células B/metabolismo , Cromossomo Filadélfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteína bcl-X/biossíntese , Animais , Ciclo Celular/genética , Modelos Animais de Doenças , Regulação Leucêmica da Expressão Gênica/genética , Humanos , Leucemia de Células B/genética , Leucemia de Células B/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Camundongos Knockout , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteína bcl-X/genéticaRESUMO
Genes that are strongly repressed after B-cell activation are candidates for being inactivated, mutated, or repressed in B-cell malignancies. Krüppel-like factor 4 (Klf4), a gene down-regulated in activated murine B cells, is expressed at low levels in several types of human B-cell lineage lymphomas and leukemias. The human KLF4 gene has been identified as a tumor suppressor gene in colon and gastric cancer; in concordance with this, overexpression of KLF4 can suppress proliferation in several epithelial cell types. Here we investigate the effects of KLF4 on pro/pre-B-cell transformation by v-Abl and BCR-ABL, oncogenes that cause leukemia in mice and humans. We show that overexpression of KLF4 induces arrest and apoptosis in the G1 phase of the cell cycle. KLF4-mediated death, but not cell-cycle arrest, can be rescued by Bcl-XL overexpression. Transformed pro/pre-B cells expressing KLF4 display increased expression of p21CIP and decreased expression of c-Myc and cyclin D2. Tetracycline-inducible expression of KLF4 in B-cell progenitors of transgenic mice blocks transformation by BCR-ABL and depletes leukemic pre-B cells in vivo. Collectively, our work identifies KLF4 as a putative tumor suppressor in B-cell malignancies.
Assuntos
Linfócitos B/metabolismo , Transformação Celular Neoplásica/metabolismo , Proteínas de Fusão bcr-abl/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Oncogênicas v-abl/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Linfócitos B/citologia , Ciclo Celular , Morte Celular , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fusão bcr-abl/genética , Fase G1 , Regulação Leucêmica da Expressão Gênica/genética , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Transgênicos , Proteínas Oncogênicas v-abl/genética , alfa-Amilases Salivares , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
To develop murine models of leukemogenesis, a series of transgenic mice expressing BCR-ABL in different hematopoietic cell subsets was generated. Here we describe targeted expression of P210 BCR-ABL in stem and progenitor cells of murine bone marrow using the tet-off system. The transactivator protein tTA was placed under the control of the murine stem cell leukemia (SCL) gene 3' enhancer. Induction of BCR-ABL resulted in neutrophilia and leukocytosis, and the mice became moribund within 29 to 122 days. Autopsy of sick mice demonstrated splenomegaly, myeloid bone marrow hyperplasia, and extramedullary myeloid cell infiltration of multiple organs. BCR-ABL mRNA and protein were detectable in the affected organs. Fluorescence-activated cell sorter (FACS) analysis demonstrated a significant increase in mature and immature myeloid cells in bone marrow and spleen, together with increased bilineal B220+/Mac-1+ cells in the bone marrow. tTA mRNA was expressed in FACS-sorted hematopoietic stem cells expanded 26-fold after BCR-ABL induction. Thirty-one percent of the animals demonstrated a biphasic phenotype, consisting of neutrophilia and subsequent B-cell lymphoblastic disease, reminiscent of blast crisis. In summary, this mouse model recapitulates many characteristics of human chronic myeloid leukemia (CML) and may help elucidate basic leukemogenic mechanisms in CML stem cells during disease initiation and progression.
Assuntos
Modelos Animais de Doenças , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Células-Tronco Hematopoéticas/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Animais , Medula Óssea/metabolismo , Medula Óssea/patologia , Transformação Celular Neoplásica/genética , Progressão da Doença , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Leucocitose/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Células Mieloides/metabolismo , Células Mieloides/patologia , Invasividade Neoplásica , Neutrófilos/metabolismo , Neutrófilos/patologia , Fenótipo , Baço/metabolismo , Baço/patologia , Transplante de Células-Tronco , Taxa de Sobrevida , Ativação Transcricional/genéticaRESUMO
The PU.1 transcription factor is a key regulator of hematopoietic development, but its role at each hematopoietic stage remains unclear. In particular, the expression of PU.1 in hematopoietic stem cells (HSCs) could simply represent "priming" of genes related to downstream myelolymphoid lineages. By using a conditional PU.1 knock-out model, we here show that HSCs express PU.1, and its constitutive expression is necessary for maintenance of the HSC pool in the bone marrow. Bone marrow HSCs disrupted with PU.1 in situ could not maintain hematopoiesis and were outcompeted by normal HSCs. PU.1-deficient HSCs also failed to generate the earliest myeloid and lymphoid progenitors. PU.1 disruption in granulocyte/monocyte-committed progenitors blocked their maturation but not proliferation, resulting in myeloblast colony formation. PU.1 disruption in common lymphoid progenitors, however, did not prevent their B-cell maturation. In vivo disruption of PU.1 in mature B cells by the CD19-Cre locus did not affect B-cell maturation, and PU.1-deficient mature B cells displayed normal proliferation in response to mitogenic signals including the cross-linking of surface immunoglobulin M (IgM). Thus, PU.1 plays indispensable and distinct roles in hematopoietic development through supporting HSC self-renewal as well as commitment and maturation of myeloid and lymphoid lineages.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Hematopoéticas/citologia , Proteínas Proto-Oncogênicas/fisiologia , Transativadores/fisiologia , Animais , Medula Óssea/metabolismo , Células Cultivadas , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Fígado/embriologia , Fígado/metabolismo , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Transativadores/deficiência , Transativadores/genéticaRESUMO
The BCR/ABL fusion protein is found in more than 90% of patients with chronic myeloid leukemia (CML) as well as in a subset of patients with acute B-cell leukemia. We have previously described a transgenic model for an inducible and reversible acute B-cell leukemia caused by p210 BCR/ABL. Here, we describe a new model of an inducible BCR/ABL disease by directing the expression of the oncogene to megakaryocytic progenitor cells within the murine bone marrow using the tetracycline-responsive expression system under the control of human CD34 regulatory elements. The predominant feature was the development of a chronic thrombocytosis. The condition progressed with the development of splenomegaly accompanied by lymphadenopathy in some mice. Affected animals demonstrated a dramatic increase in the number of megakaryocytes in the bone marrow and the spleen. Immunohistochemistry demonstrated that the reporter gene was expressed in hematopoietic stem cells (HSCs), common myeloid progenitor (CMP) cells, as well as in megakaryocytic/erythroid progenitor cells (MEPs). Although these mice did not display the increase in granulopoiesis commonly found in chronic myeloid leukemia (CML), the phenotype closely resembles a myeloproliferative disorder affecting the megakaryocytic lineage observed in some patients with the BCR/ABL P210 translocation.
Assuntos
Antígenos CD34/genética , Proteínas de Fusão bcr-abl/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reguladores/fisiologia , Megacariócitos/patologia , Transtornos Mieloproliferativos/etiologia , Animais , Modelos Animais de Doenças , Proteínas de Fusão bcr-abl/genética , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Doenças Linfáticas , Camundongos , Camundongos Transgênicos , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Esplenomegalia , Tetraciclina/farmacologia , TrombocitoseRESUMO
Human CD34 (hCD34)-positive cells are used currently as a source for hematopoietic transplantation in humans. However, in steady-state murine hematopoiesis, hematopoietic stem cells (HSCs) with long-term reconstitution activity are found almost exclusively in the murine CD34 (mCD34)-negative to low fraction. To evaluate the possible differences in hCD34 and mCD34 gene expression in hematopoiesis, we made transgenic mouse strains with human genomic P1 artificial chromosome clones spanning the entire hCD34 genomic locus. In all transgenic mouse strains, a vast majority of phenotypic and functional HSC populations including mCD34(-/lo) express the hCD34 transgene. These data strongly support the notion that hCD34(+) human bone marrow cells contain long-term HSCs that can maintain hematopoiesis throughout life.
Assuntos
Antígenos CD34/genética , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Animais , Antígenos CD34/metabolismo , Diferenciação Celular , Regulação para Baixo , Citometria de Fluxo , Genótipo , Humanos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Modelos Genéticos , Fenótipo , Especificidade da Espécie , Fatores de Tempo , TransgenesRESUMO
The elements regulating gene expression in hematopoietic stem cells are still poorly understood. We previously reported that a 141-kilobase (kb) human CD34 transgene confers properly regulated human CD34 expression in transgenic mice. A construct with only the human CD34 promoter and 3' enhancer region is not sufficient, suggesting that critical distal elements are necessary for expression of the human CD34 gene. To further localize such elements, we analyzed deletion constructs of the human CD34 gene and evaluated their function in transgenic mice. Constructs harboring as little as 18 kb of 5' and 26 kb of 3' human CD34 flanking sequence conferred human expression in tissues of transgenic mice with a pattern similar to that of the 141-kb human transgene. In contrast, a construct harboring 10 kb of 5' and 17 kb of 3' human CD34 flanking sequence gave no expression. These data demonstrate that regions between 10 to 18 kb upstream and/or 17 to 26 kb downstream of the human CD34 gene contain critical elements for human CD34 expression in vivo. Further functional analysis of these regions in transgenic mice will be crucial for understanding CD34 gene expression in hematopoietic stem and progenitor cells.
Assuntos
Região 5'-Flanqueadora/genética , Antígenos CD34/genética , Regulação da Expressão Gênica/genética , Células-Tronco Hematopoéticas/metabolismo , Sequências Reguladoras de Ácido Nucleico , Animais , Antígenos CD34/biossíntese , Células da Medula Óssea/metabolismo , Diferenciação Celular , Ensaio de Unidades Formadoras de Colônias , Sistemas Computacionais , Biblioteca Gênica , Genótipo , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , TransgenesRESUMO
The human CD34 gene is expressed on early progenitor and stem cells in the bone marrow. Here we report the isolation of the human CD34 locus from a human P1 artificial chromosome (PAC) library and the characterization and evaluation of this genomic fragment for expression of reporter genes in stable cell lines and transgenic mice. We show that a 160-kb fragment spanning 110 kb of the 5' flanking region and 26 kb of the 3' flanking region of the CD34 gene directs expression of the human CD34 gene in the bone marrow of transgenic mice. The expression of human CD34 transgenic RNA in tissues was found to be similar to that of the endogenous murine CD34 gene. Colony-forming cell assays showed that bone marrow cells staining positive for human CD34 consist of early progenitor cells in which expression of CD34 decreased with cell maturation. In order to test the construct for its ability to express heterologous genes in vivo, we used homologous recombination in bacteria to insert the tetracycline-responsive transactivator protein tTA. Analysis of transgenic human CD34-tTA mice by cross breeding with a strain carrying Cre recombinase under control of a tetracycline-responsive element demonstrated induction of Cre expression in mice in a pattern consistent with the expression of the human CD34 transgene.