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1.
Int J Hematol ; 115(2): 198-207, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34773575

RESUMO

INTRODUCTION: Primary myelofibrosis (PMF) is a clonal stem cell disorder characterized by myeloid dominant hematopoiesis and dysregulated proliferation of fibroblasts in the bone marrow. However, how these aberrant myeloid cells and fibroblasts are produced remains unclear. AIM AND METHODS: In this study, we examined in vivo engraftment kinetics of PMF patient-derived CD34+ cells in immunecompromised NOD/SCID/IL2rgKO (NSG) mice. Engrafted human cells were analyzed with flow cytometry, and proliferation of fibroblastic cells and bone marrow fibrosis were assessed with the histo-pathological examination. RESULTS: Transplantation of PMF patient-derived circulating CD34+ fractions into NSG newborns recapitulates clinical features of human PMF. Engraftment of human CD45+ leukocytes resulted in anemia and myeloid hyperplasia accompanied by bone marrow fibrosis by six months post-transplantation. Fibrotic bone marrow contained CD45-vimentin+ cells of both human and mouse origin, suggesting that circulating malignant CD34+ subsets contribute to myelofibrotic changes in PMF through direct and indirect mechanisms. CONCLUSION: A patient-derived xenotransplantation (PDX) model of PMF allows in vivo examination of disease onset and propagation originating from immature CD34+ cells and will support the investigation of pathogenesis and development of therapeutic modalities for the disorder.


Assuntos
Antígenos CD34/análise , Medula Óssea/patologia , Hematopoese , Células Mieloides/patologia , Mielofibrose Primária/patologia , Animais , Antígenos CD34/sangue , Células Cultivadas , Fibrose , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Mielofibrose Primária/sangue
2.
EBioMedicine ; 64: 103235, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33581643

RESUMO

BACKGROUND: Acute lymphoblastic leukaemia with mixed lineage leukaemia gene rearrangement (MLL-ALL) frequently affects infants and is associated with a poor prognosis. Primary refractory and relapsed disease due to resistance to glucocorticoids (GCs) remains a substantial hurdle to improving clinical outcomes. In this study, we aimed to overcome GC resistance of MLL-ALL. METHODS: Using leukaemia patient specimens, we performed bioinformatic analyses to identify target genes/pathways. To test inhibition of target pathways in vivo, we created pre-clinical therapeutic mouse patient-derived xenograft (PDX)-models by transplanting human MLL-ALL leukaemia initiating cells (LIC) into immune-deficient NSG mice. Finally, we conducted B-cell lymphoma-2 (BCL-2) homology domain 3 (BH3) profiling to identify BH3 peptides responsible for treatment resistance in MLL-leukaemia. FINDINGS: Src family kinases (SFKs) and Fms-like tyrosine kinase 3 (FLT3) signaling pathway were over-represented in MLL-ALL cells. PDX-models of infant MLL- ALL recapitulated GC-resistance in vivo but RK-20449, an inhibitor of SFKs and FLT3 eliminated human MLL-ALL cells in vivo, overcoming GC-resistance. Further, we identified BCL-2 dependence as a mechanism of treatment resistance in MLL-ALL through BH3 profiling. Furthermore, MLL-ALL cells resistant to RK-20449 treatment were dependent on the anti-apoptotic BCL-2 protein for their survival. Combined inhibition of SFKs/FLT3 by RK-20449 and of BCL-2 by ABT-199 led to substantial elimination of MLL-ALL cells in vitro and in vivo. Triple treatment combining GCs, RK-20449 and ABT-199 resulted in complete elimination of MLL-ALL cells in vivo. INTERPRETATION: SFKs/FLT3 signaling pathways are promising targets for treatment of treatment-resistant MLL-ALL. Combined inhibition of these kinase pathways and anti-apoptotic BCL-2 successfully eliminated highly resistant MLL-ALL and demonstrated a new treatment strategy for treatment-resistant poor-outcome MLL-ALL. FUNDING: This study was supported by RIKEN (RIKEN President's Discretionary Grant) for FI, Japan Agency for Medical Research and Development (the Basic Science and Platform Technology Program for Innovative Biological Medicine for FI and by NIH CA034196 for LDS. The funders had no role in the study design, data collection, data analysis, interpretation nor writing of the report.


Assuntos
Apoptose/efeitos dos fármacos , Apoptose/genética , Resistencia a Medicamentos Antineoplásicos/genética , Rearranjo Gênico , Histona-Lisina N-Metiltransferase/genética , Proteína de Leucina Linfoide-Mieloide/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Sinergismo Farmacológico , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Pirimidinas/farmacologia , Pirróis/farmacologia , Esteroides/farmacologia , Esteroides/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Nat Cancer ; 2(3): 340-356, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-35121960

RESUMO

Aggressive therapy-resistant and refractory acute myeloid leukemia (AML) has an extremely poor outcome. By analyzing a large number of genetically complex and diverse, primary high-risk poor-outcome human AML samples, we identified specific pathways of therapeutic vulnerability. Through drug screens followed by extensive in vivo validation and genomic analyses, we found inhibition of cytosolic and mitochondrial anti-apoptotic proteins XIAP, BCL2 and MCL1, and a key regulator of mitosis, AURKB, as a vulnerability hub based on patient-specific genetic aberrations and transcriptional signatures. Combinatorial therapeutic inhibition of XIAP with an additional patient-specific vulnerability eliminated established AML in vivo in patient-derived xenografts (PDXs) bearing diverse genetic aberrations, with no signs of recurrence during off-treatment follow-up. By integrating genomic profiling and drug-sensitivity testing, this work provides a platform for a precision-medicine approach for treating aggressive AML with high unmet need.


Assuntos
Leucemia Mieloide Aguda , Proteínas Proto-Oncogênicas c-bcl-2 , Apoptose/genética , Proteínas Reguladoras de Apoptose/uso terapêutico , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética
4.
Trends Immunol ; 41(8): 706-720, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32631635

RESUMO

Rodent models for human diseases contribute significantly to understanding human physiology and pathophysiology. However, given the accelerating pace of drug development, there is a crucial need for in vivo preclinical models of human biology and pathology. The humanized mouse is one tool to bridge the gap between traditional animal models and the clinic. The development of immunodeficient mouse strains with high-level engraftment of normal and diseased human immune/hematopoietic cells has made in vivo functional characterization possible. As a patient-derived xenograft (PDX) model, humanized mice functionally correlate putative mechanisms with in vivo behavior and help to reveal pathogenic mechanisms. Combined with single-cell genomics, humanized mice can facilitate functional precision medicine such as risk stratification and individually optimized therapeutic approaches.


Assuntos
Hematopoese , Medicina de Precisão , Animais , Modelos Animais de Doenças , Hematopoese/imunologia , Humanos , Medicina de Precisão/tendências
5.
Life Sci Alliance ; 2(2)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30936185

RESUMO

The immune system encompasses acquired and innate immunity that matures through interaction with microenvironmental components. Cytokines serve as environmental factors that foster functional maturation of immune cells. Although NOD/SCID/IL2rgKO (NSG) humanized mice support investigation of human immunity in vivo, a species barrier between human immune cells and the mouse microenvironment limits human acquired as well as innate immune function. To study the roles of human cytokines in human acquired and innate immune cell development, we created NSG mice expressing hIL-7 and hIL-15. Although hIL-7 alone was not sufficient for supporting human NK cell development in vivo, increased frequencies of human NK cells were confirmed in multiple organs of hIL-7 and hIL-15 double knockin (hIL-7xhIL-15 KI) NSG mice engrafted with human hematopoietic stem cells. hIL-7xhIL-15 KI NSG humanized mice provide a valuable in vivo model to investigate development and function of human NK cells.


Assuntos
Diferenciação Celular , Técnicas de Introdução de Genes , Interleucina-15/sangue , Interleucina-15/genética , Interleucina-7/sangue , Interleucina-7/genética , Células Matadoras Naturais/fisiologia , Animais , Antígeno CD56/metabolismo , Feminino , Sangue Fetal/citologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Modelos Animais , Timo/citologia , Transcriptoma , Transplante Heterólogo
6.
EBioMedicine ; 41: 584-596, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30772305

RESUMO

BACKGROUND: Graft-versus host disease (GVHD) is a complication of stem cell transplantation associated with significant morbidity and mortality. Non-specific immune-suppression, the mainstay of treatment, may result in immune-surveillance dysfunction and disease recurrence. METHODS: We created humanised mice model for chronic GVHD (cGVHD) by injecting cord blood (CB)-derived human CD34+CD38-CD45RA- haematopoietic stem/progenitor cells (HSPCs) into hIL-6 transgenic NOD/SCID/Il2rgKO (NSG) newborns, and compared GVHD progression with NSG newborns receiving CB CD34- cells mimicking acute GVHD. We characterised human immune cell subsets, target organ infiltration, T-cell repertoire (TCR) and transcriptome in the humanised mice. FINDINGS: In cGVHD humanised mice, we found activation of T cells in the spleen, lung, liver, and skin, activation of macrophages in lung and liver, and loss of appendages in skin, obstruction of bronchioles in lung and portal fibrosis in liver recapitulating cGVHD. Acute GVHD humanised mice showed activation of T cells with skewed TCR repertoire without significant macrophage activation. INTERPRETATION: Using humanised mouse models, we demonstrated distinct immune mechanisms contributing acute and chronic GVHD. In cGVHD model, co-activation of human HSPC-derived macrophages and T cells educated in the recipient thymus contributed to delayed onset, multi-organ disease. In acute GVHD model, mature human T cells contained in the graft resulted in rapid disease progression. These humanised mouse models may facilitate future development of new molecular medicine targeting GVHD.


Assuntos
Doença Enxerto-Hospedeiro/imunologia , Interleucina-6/genética , Macrófagos/imunologia , Linfócitos T/imunologia , Doença Aguda , Animais , Animais Recém-Nascidos , Doença Crônica , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/mortalidade , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Queratinócitos/citologia , Queratinócitos/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Taxa de Sobrevida , Linfócitos T/metabolismo , Transcriptoma
7.
J Immunol ; 200(9): 3291-3303, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29581358

RESUMO

Disturbed balance between immune surveillance and tolerance may lead to poor clinical outcomes in some malignancies. In paired analyses of adenocarcinoma and normal mucosa from 142 patients, we found a significant increase of the CD4/CD8 ratio and accumulation of regulatory T cells (Tregs) within the adenocarcinoma. The increased frequency of Tregs correlated with the local infiltration and extension of the tumor. There was concurrent maturation arrest, upregulation of programmed death-1 expression, and functional impairment in CD8+ T cells (CTLs) isolated from the adenocarcinoma. Adenocarcinoma-associated Tregs directly inhibit the function of normal human CTLs in vitro. With histopathological analysis, Foxp3+ Tregs were preferentially located in stroma. Concurrent transcriptome analysis of epithelial cells, stromal cells, and T cell subsets obtained from carcinomatous and normal intestinal samples from patients revealed a distinct gene expression signature in colorectal adenocarcinoma-associated Tregs, with overexpression of CCR1, CCR8, and TNFRSF9, whereas their ligands CCL4 and TNFSF9 were found upregulated in cancerous epithelium. Overexpression of WNT2 and CADM1, associated with carcinogenesis and metastasis, in cancer-associated stromal cells suggests that both cancer cells and stromal cells play important roles in the development and progression of colorectal cancer through the formation of a tumor microenvironment. The identification of CTL anergy by Tregs and the unique gene expression signature of human Tregs and stromal cells in colorectal cancer patients may facilitate the development of new therapeutics against malignancies.


Assuntos
Adenocarcinoma/imunologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias Colorretais/imunologia , Linfócitos T Reguladores/imunologia , Evasão Tumoral/imunologia , Idoso , Feminino , Humanos , Imunidade nas Mucosas/imunologia , Vigilância Imunológica/imunologia , Mucosa Intestinal/imunologia , Masculino , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1
8.
Sci Transl Med ; 9(413)2017 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-29070697

RESUMO

Numerous variant alleles are associated with human acute myeloid leukemia (AML). However, the same variants are also found in individuals with no hematological disease, making their functional relevance obscure. Through NOD.Cg-PrkdcscidIl2rgtmlWjl/Sz (NSG) xenotransplantation, we functionally identified preleukemic and leukemic stem cell populations present in FMS-like tyrosine kinase 3 internal tandem duplication-positive (FLT3-ITD)+ AML patient samples. By single-cell DNA sequencing, we identified clonal structures and linked mutations with in vivo fates, distinguishing mutations permissive of nonmalignant multilineage hematopoiesis from leukemogenic mutations. Although multiple somatic mutations coexisted at the single-cell level, inhibition of the mutation strongly associated with preleukemic to leukemic stem cell transition eliminated AML in vivo. Moreover, concurrent inhibition of BCL-2 (B cell lymphoma 2) uncovered a critical dependence of resistant AML cells on antiapoptotic pathways. Co-inhibition of pathways critical for oncogenesis and survival may be an effective strategy that overcomes genetic diversity in human malignancies. This approach incorporating single-cell genomics with the NSG patient-derived xenograft model may serve as a broadly applicable resource for precision target identification and drug discovery.


Assuntos
Leucemia Mieloide Aguda/genética , Mutação/genética , Transdução de Sinais/genética , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Carcinogênese/genética , Carcinogênese/patologia , Células Clonais , Feminino , Genômica , Humanos , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Análise de Sequência de DNA , Análise de Célula Única , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Blood ; 127(6): 722-34, 2016 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-26702062

RESUMO

Induction of specific immune response against therapy-resistant tumor cells can potentially improve clinical outcomes in malignancies. To optimize immunotherapy in the clinic, we aimed to create an in vivo model enabling us to analyze human cytotoxic T-lymphocyte (CTL) responses against human malignancies. To this end, we developed NOD/SCID/IL2rgKO (NSG) mice expressing the HLA class I molecules HLA-A*0201 and A*2402. In the bone marrow (BM) and spleen of HLA class I transgenic (Tg) NSG mice transplanted with cord blood hematopoietic stem cells (HSCs), we found human memory CD8(+) T cells and antigen-presenting cells. To evaluate antigen-specific human CTL responses, we immunized HLA class I Tg NSG mice using polyinosinic:polycytidylic acid mixed Wilms tumor 1 (WT1) peptides, with or without WT1 peptide-loaded autologous dendritic cells. After immunization, the frequencies of HLA-restricted WT1-specific CTLs increased significantly in the spleen. Next, we transplanted the WT1-specific T-cell receptor (WT1-TCR) gene-transduced human HSCs into HLA class I Tg NSG newborn mice. WT1 tetramer-positive CD8(+) T cells differentiated from WT1-TCR-transduced HSCs in the recipients' BM, spleen, and thymus. Upon stimulation with WT1 peptide in vitro, these CTLs produced interferon-γ and showed lytic activity against leukemia cells in an antigen-specific, HLA-restricted manner. HLA class I Tg NSG xenografts may serve as a preclinical model to develop effective immunotherapy against human malignancies.


Assuntos
Linfócitos T CD8-Positivos/fisiologia , Diferenciação Celular , Antígeno HLA-A2/genética , Antígeno HLA-A24/genética , Células-Tronco Hematopoéticas/fisiologia , Animais , Animais Recém-Nascidos , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Feminino , Antígeno HLA-A2/metabolismo , Antígeno HLA-A24/metabolismo , Humanos , Subunidade gama Comum de Receptores de Interleucina/genética , Células K562 , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Proteínas WT1/metabolismo
10.
Blood ; 125(6): 967-80, 2015 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-25538041

RESUMO

Translocation of the mixed-lineage leukemia (MLL) gene with AF4, AF9, or ENL results in acute leukemia with both lymphoid and myeloid involvement. We characterized leukemia-initiating cells (LICs) in primary infant MLL-rearranged leukemia using a xenotransplantation model. In MLL-AF4 patients, CD34(+)CD38(+)CD19(+) and CD34(-)CD19(+) cells initiated leukemia, and in MLL-AF9 patients, CD34(-)CD19(+) cells were LICs. In MLL-ENL patients, either CD34(+) or CD34(-) cells were LICs, depending on the pattern of CD34 expression. In contrast, in patients with these MLL translocations, CD34(+)CD38(-)CD19(-)CD33(-) cells were enriched for normal hematopoietic stem cells (HSCs) with in vivo long-term multilineage hematopoietic repopulation capacity. Although LICs developed leukemic cells with clonal immunoglobulin heavy-chain (IGH) rearrangement in vivo, CD34(+)CD38(-)CD19(-)CD33(-) cells repopulated recipient bone marrow and spleen with B cells, showing broad polyclonal IGH rearrangement and recipient thymus with CD4(+) single positive (SP), CD8(+) SP, and CD4(+)CD8(+) double-positive (DP) T cells. Global gene expression profiling revealed that CD9, CD32, and CD24 were over-represented in MLL-AF4, MLL-AF9, and MLL-ENL LICs compared with normal HSCs. In patient samples, these molecules were expressed in CD34(+)CD38(+) and CD34(-) LICs but not in CD34(+)CD38(-)CD19(-)CD33(-) HSCs. Identification of LICs and LIC-specific molecules in primary human MLL-rearranged acute lymphoblastic leukemia may lead to improved therapeutic strategies for MLL-rearranged leukemia.


Assuntos
Antígenos CD34/genética , Regulação Leucêmica da Expressão Gênica , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Animais , Antígeno CD24/genética , Criança , Pré-Escolar , Feminino , Rearranjo Gênico , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Imunofenotipagem , Lactente , Masculino , Camundongos , Camundongos Endogâmicos NOD , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Receptores de IgG/genética , Tetraspanina 29/genética
11.
PLoS One ; 8(5): e62506, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23667482

RESUMO

BACKGROUND: Definite identification of the cell types and the mechanism relevant to cardiomyogenesis is essential for effective cardiac regenerative medicine. We aimed to identify the cell populations that can generate cardiomyocytes and to clarify whether generation of donor-marker(+) cardiomyocytes requires cell fusion between BM-derived cells and recipient cardiomyocytes. METHODOLOGY/PRINCIPAL FINDINGS: Purified BM stem/progenitor cells from green fluorescence protein (GFP) mice were transplanted into C57BL/6 mice or cyan fluorescence protein (CFP)-transgenic mice. Purified human hematopoietic stem cells (HSCs) from cord blood were transplanted into immune-compromised NOD/SCID/IL2rγ(null) mice. GFP(+) cells in the cardiac tissue were analyzed for the antigenecity of a cardiomyocyte by confocal microscopy following immunofluorescence staining. GFP(+) donor-derived cells, GFP(+)CFP(+) fused cells, and CFP(+) recipient-derived cells were distinguished by linear unmixing analysis. Hearts of xenogeneic recipients were evaluated for the expression of human cardiomyocyte genes by real-time quantitative polymerase chain reaction. In C57BL/6 recipients, Lin(-/low)CD45(+) hematopoietic cells generated greater number of GFP(+) cardiomyocytes than Lin(-/low)CD45(-) mesenchymal cells (37.0+/-23.9 vs 0.00+/-0.00 GFP(+) cardiomyocytes per a recipient, P = 0.0095). The number of transplanted purified HSCs (Lin(-/low)Sca-1(+) or Lin(-)Sca-1(+)c-Kit(+) or CD34(-)Lin(-)Sca-1(+)c-Kit(+)) showed correlation to the number of GFP(+) cardiomyocytes (P<0.05 in each cell fraction), and the incidence of GFP(+) cardiomyocytes per injected cell dose was greatest in CD34(-)Lin(-)Sca-1(+)c-Kit(+) recipients. Of the hematopoietic progenitors, total myeloid progenitors generated greater number of GFP(+) cardiomyocytes than common lymphoid progenitors (12.8+/-10.7 vs 0.67+/-1.00 GFP(+) cardiomyocytes per a recipient, P = 0.0021). In CFP recipients, all GFP(+) cardiomyocytes examined coexpressed CFP. Human troponin C and myosin heavy chain 6 transcripts were detected in the cardiac tissue of some of the xenogeneic recipients. CONCLUSIONS/SIGNIFICANCE: Our results indicate that HSCs resulted in the generation of cardiomyocytes via myeloid intermediates by fusion-dependent mechanism. The use of myeloid derivatives as donor cells could potentially allow more effective cell-based therapy for cardiac repair.


Assuntos
Células da Medula Óssea/citologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Miócitos Cardíacos/citologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Fusão Celular , Linhagem da Célula , Sangue Fetal/citologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/citologia , Miócitos Cardíacos/metabolismo
12.
Sci Transl Med ; 5(181): 181ra52, 2013 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-23596204

RESUMO

Leukemia stem cells (LSCs) that survive conventional chemotherapy are thought to contribute to disease relapse, leading to poor long-term outcomes for patients with acute myeloid leukemia (AML). We previously identified a Src-family kinase (SFK) member, hematopoietic cell kinase (HCK), as a molecular target that is highly differentially expressed in human primary LSCs compared with human normal hematopoietic stem cells (HSCs). We performed a large-scale chemical library screen that integrated a high-throughput enzyme inhibition assay, in silico binding prediction, and crystal structure determination and found a candidate HCK inhibitor, RK-20449, a pyrrolo-pyrimidine derivative with an enzymatic IC50 (half maximal inhibitory concentration) in the subnanomolar range. A crystal structure revealed that RK-20449 bound the activation pocket of HCK. In vivo administration of RK-20449 to nonobese diabetic (NOD)/severe combined immunodeficient (SCID)/IL2rg(null) mice engrafted with highly aggressive therapy-resistant AML significantly reduced human LSC and non-stem AML burden. By eliminating chemotherapy-resistant LSCs, RK-20449 may help to prevent relapse and lead to improved patient outcomes in AML.


Assuntos
Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Adulto , Idoso , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Transplante de Medula Óssea , Cristalografia por Raios X , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Hematopoese/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Masculino , Camundongos , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/patologia , Proteínas Proto-Oncogênicas c-hck/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-hck/química , Proteínas Proto-Oncogênicas c-hck/metabolismo , Pirimidinas/uso terapêutico , Pirróis/uso terapêutico , RNA Interferente Pequeno/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Células Tumorais Cultivadas , Adulto Jovem
13.
Cell Stem Cell ; 10(6): 753-758, 2012 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-22704516

RESUMO

A porcine model of severe combined immunodeficiency (SCID) promises to facilitate human cancer studies, the humanization of tissue for xenotransplantation, and the evaluation of stem cells for clinical therapy, but SCID pigs have not been described. We report here the generation and preliminary evaluation of a porcine SCID model. Fibroblasts containing a targeted disruption of the X-linked interleukin-2 receptor gamma chain gene, Il2rg, were used as donors to generate cloned pigs by serial nuclear transfer. Germline transmission of the Il2rg deletion produced healthy Il2rg(+/-) females, while Il2rg(-/Y) males were athymic and exhibited markedly impaired immunoglobulin and T and NK cell production, robustly recapitulating human SCID. Following allogeneic bone marrow transplantation, donor cells stably integrated in Il2rg(-/Y) heterozygotes and reconstituted the Il2rg(-/Y) lymphoid lineage. The SCID pigs described here represent a step toward the comprehensive evaluation of preclinical cellular regenerative strategies.


Assuntos
Marcação de Genes , Terapia Genética , Subunidade gama Comum de Receptores de Interleucina/genética , Imunodeficiência Combinada Severa/terapia , Animais , Modelos Animais de Doenças , Feminino , Humanos , Subunidade gama Comum de Receptores de Interleucina/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Masculino , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/imunologia , Suínos , Linfócitos T/imunologia , Linfócitos T/metabolismo
14.
J Immunol ; 188(12): 6145-55, 2012 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-22611244

RESUMO

Although physiological development of human lymphoid subsets has become well documented in humanized mice, in vivo development of human myeloid subsets in a xenotransplantation setting has remained unevaluated. Therefore, we investigated in vivo differentiation and function of human myeloid subsets in NOD/SCID/IL2rγ(null) (NSG) mouse recipients transplanted with purified lineage(-)CD34(+)CD38(-) cord blood hematopoietic stem cells. At 4-6 mo posttransplantation, we identified the development of human neutrophils, basophils, mast cells, monocytes, and conventional and plasmacytoid dendritic cells in the recipient hematopoietic organs. The tissue distribution and morphology of these human myeloid cells were similar to those identified in humans. After cytokine stimulation in vitro, phosphorylation of STAT molecules was observed in neutrophils and monocytes. In vivo administration of human G-CSF resulted in the recruitment of human myeloid cells into the recipient circulation. Flow cytometry and confocal imaging demonstrated that human bone marrow monocytes and alveolar macrophages in the recipients displayed intact phagocytic function. Human bone marrow-derived monocytes/macrophages were further confirmed to exhibit phagocytosis and killing of Salmonella typhimurium upon IFN-γ stimulation. These findings demonstrate the development of mature and functionally intact human myeloid subsets in vivo in the NSG recipients. In vivo human myelopoiesis established in the NSG humanized mouse system may facilitate the investigation of human myeloid cell biology including in vivo analyses of infectious diseases and therapeutic interventions.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Células Mieloides/citologia , Células Mieloides/imunologia , Células Mieloides/metabolismo , Transplante Heterólogo/imunologia , Animais , Citometria de Fluxo , Humanos , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Microscopia Confocal , Fagocitose/imunologia , Transplante Heterólogo/métodos
15.
Blood ; 119(12): 2768-77, 2012 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-22279057

RESUMO

In recent years, advances in the humanized mouse system have led to significantly increased levels of human hematopoietic stem cell (HSC) engraftment. The remaining limitations in human HSC engraftment and function include lymphoid-skewed differentiation and inefficient myeloid development in the recipients. Limited human HSC function may partially be attributed to the inability of the host mouse microenvironment to provide sufficient support to human hematopoiesis. To address this problem, we created membrane-bound human stem cell factor (SCF)/KIT ligand (KL)-expressing NOD/SCID/IL2rgKO (hSCF Tg NSG) mice. hSCF Tg NSG recipients of human HSCs showed higher levels of both human CD45(+) cell engraftment and human CD45(+)CD33(+) myeloid development compared with NSG recipients. Expression of hSCF/hKL accelerated the differentiation of the human granulocyte lineage cells in the recipient bone marrow. Human mast cells were identified in bone marrow, spleen, and gastrointestinal tissues of the hSCF Tg NSG recipients. This novel in vivo humanized mouse model demonstrates the essential role of membrane-bound hSCF in human myeloid development. Moreover, the hSCF Tg NSG humanized recipients may facilitate investigation of in vivo differentiation, migration, function, and pathology of human mast cells.


Assuntos
Células-Tronco Hematopoéticas/citologia , Mastócitos/citologia , Camundongos Transgênicos , Células Mieloides/citologia , Regiões Promotoras Genéticas , Fator de Células-Tronco/metabolismo , Animais , Diferenciação Celular/fisiologia , Separação Celular , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Análise de Sequência com Séries de Oligonucleotídeos , Quimeras de Transplante/fisiologia , Tolerância ao Transplante/fisiologia
16.
Proc Natl Acad Sci U S A ; 107(29): 13022-7, 2010 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-20615947

RESUMO

Whereas humanized mouse models have contributed significantly to human immunology research, human T cells developing in mouse thymic environment fail to demonstrate HLA-restricted function. To achieve HLA-restricted human immune response, we created an immune-compromised non-obese diabetic/SCID/IL2rg(null) strain (NSG) with homozygous expression of HLA class I heavy chain and light chain (NSG-HLA-A2/HHD). Transplantation of purified Lin-CD34+CD38- human hematopoietic stem cells into NSG-HLA-A2/HHD newborns resulted in the development of human CD4+ and CD8+ TCR alphabeta+ T cells and CD4-CD8- and CD8+ TCR gammadelta+ cells in recipient bone marrow and spleen. Human cytotoxic T lymphocytes (CTLs) become functionally mature, as evidenced by the production of granzyme corresponding to phenotypic transition from naïve to effector memory CTLs. In these recipients, human Th17 cells developed along with Th1 and Th2 cells. Epstein-Barr virus (EBV) infection in the humanized NSG-HLA-A2/HHD recipients resulted in the formation of lymphoproliferative lesions consisting mainly of human B cells with scattered human T cells. Human CTLs developing in the recipients recognized EBV-derived peptides in an HLA-restricted manner and exerted HLA-restricted cytotoxicity against EBV-infected human B cells. The HLA-expressing humanized mouse with functional HLA-restricted T cells and consistent representation of rare T-cell subsets overcomes a major constraint in human immunology, and serves as a useful model for investigation of human immune responses against pathogens and for the development of therapeutic strategies against human diseases.


Assuntos
Antígeno HLA-A2/imunologia , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Animais Recém-Nascidos , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/virologia , Diferenciação Celular/imunologia , Proliferação de Células , Separação Celular , Citotoxicidade Imunológica , Infecções por Vírus Epstein-Barr/imunologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Herpesvirus Humano 4/imunologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Especificidade da Espécie , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/virologia , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologia
17.
Sci Transl Med ; 2(17): 17ra9, 2010 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-20371479

RESUMO

Human acute myeloid leukemia (AML) originates from rare leukemia stem cells (LSCs). Because these chemotherapy-resistant LSCs are thought to underlie disease relapse, effective therapeutic strategies specifically targeting these cells may be beneficial. Here, we report identification of a primary human LSC gene signature and functional characterization of human LSC-specific molecules in vivo in a mouse xenotransplantation model. In 32 of 61 (53%) patients with AML, either CD32 or CD25 or both were highly expressed in LSCs. CD32- or CD25-positive LSCs could initiate AML and were cell cycle-quiescent and chemotherapy-resistant in vivo. Normal human hematopoietic stem cells depleted of CD32- and CD25-positive cells maintained long-term multilineage hematopoietic reconstitution capacity in vivo, indicating the potential safety of treatments targeting these molecules. In addition to CD32 and CD25, quiescent LSCs within the bone marrow niche also expressed the transcription factor WT1 and the kinase HCK. These molecules are also promising targets for LSC-specific therapy.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Leucemia/terapia , Células-Tronco Neoplásicas/metabolismo , Animais , Transplante de Medula Óssea/métodos , Ciclo Celular , Linhagem da Célula , Separação Celular , Desenho de Fármacos , Citometria de Fluxo , Regulação Leucêmica da Expressão Gênica , Humanos , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Camundongos , Camundongos SCID , Receptores de IgG/biossíntese , Transplante Heterólogo
18.
Nat Biotechnol ; 28(3): 275-80, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20160717

RESUMO

Cancer stem cells have been proposed to be important for initiation, maintenance and recurrence of various malignancies, including acute myeloid leukemia (AML). We have previously reported that CD34+CD38- human primary AML stem cells residing in the endosteal region of the bone marrow are relatively chemotherapy resistant. Using a NOD/SCID/IL2rgamma(null) mouse model of human AML, we now show that the AML stem cells in the endosteal region are cell cycle quiescent and that these stem cells can be induced to enter the cell cycle by treatment with granulocyte colony-stimulating factor (G-CSF). In combination with cell cycle-dependent chemotherapy, G-CSF treatment significantly enhances induction of apoptosis and elimination of human primary AML stem cells in vivo. The combination therapy leads to significantly increased survival of secondary recipients after transplantation of leukemia cells compared with chemotherapy alone.


Assuntos
Ciclo Celular/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo
19.
Cell Stem Cell ; 3(1): 8-10, 2008 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-18593553

RESUMO

Ex vivo hematopoiesis from embryonic sources offers exciting promises in basic research and medicine. In this issue of Cell Stem Cell, Ledran et al. (2008) describe human embryonic stem cell (hESC)-derived hematopoiesis, while Taoudi et al. (2008) define the origin of definitive hematopoietic stem cells (HSCs) from the mouse aorta-gonad-mesonephros (AGM) region.


Assuntos
Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Células Estromais/citologia , Células Estromais/fisiologia , Animais , Antígenos CD/fisiologia , Sangue , Caderinas/fisiologia , Diferenciação Celular , Humanos , Antígenos Comuns de Leucócito/fisiologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID
20.
Nat Biotechnol ; 25(11): 1315-21, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17952057

RESUMO

Acute myelogenous leukemia (AML) is the most common adult leukemia, characterized by the clonal expansion of immature myeloblasts initiating from rare leukemic stem (LS) cells. To understand the functional properties of human LS cells, we developed a primary human AML xenotransplantation model using newborn nonobese diabetic/severe combined immunodeficient/interleukin (NOD/SCID/IL)2r gamma(null) mice carrying a complete null mutation of the cytokine gamma c upon the SCID background. Using this model, we demonstrated that LS cells exclusively recapitulate AML and retain self-renewal capacity in vivo. They home to and engraft within the osteoblast-rich area of the bone marrow, where AML cells are protected from chemotherapy-induced apoptosis. Quiescence of human LS cells may be a mechanism underlying resistance to cell cycle-dependent cytotoxic therapy. Global transcriptional profiling identified LS cell-specific transcripts that are stable through serial transplantation. These results indicate the potential utility of this AML xenograft model in the development of novel therapeutic strategies targeted at LS cells.


Assuntos
Divisão Celular , Resistencia a Medicamentos Antineoplásicos , Leucemia Mieloide Aguda/metabolismo , Modelos Biológicos , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Animais , Apoptose , Medula Óssea , Perfilação da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Mutantes , Transplante de Neoplasias , Células-Tronco Neoplásicas/efeitos dos fármacos , Osteoblastos
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