RESUMEN
Relapse of paediatric acute lymphoblastic leukaemia (ALL) may occur due to persistence of resistant cells with leukaemia-propagating ability (LPC). In leukaemia, the balance of B-cell lymphoma-2 (BCL-2) family proteins is disrupted, promoting survival of malignant cells and possibly LPC. A direct comparison of BCL-2 inhibitors, navitoclax and venetoclax, was undertaken on LPC subpopulations from B-cell precursor (BCP) and T-cell ALL (T-ALL) cases in vitro and in vivo. Responses were compared to BCL-2 levels detected by microarray analyses and Western blotting. In vitro, both drugs were effective against most BCP-ALL LPC, except CD34- /CD19- cells. In contrast, only navitoclax was effective in T-ALL and CD34- /CD7- LPC were resistant to both drugs. In vivo, navitoclax was more effective than venetoclax, significantly improving survival of mice engrafted with BCP- and T-ALL samples. Venetoclax was not particularly effective against T-ALL cases in vivo. The proportions of CD34+ /CD19- , CD34- /CD19- BCP-ALL cells and CD34- /CD7- T-ALL cells increased significantly following in vivo treatment. Expression of pro-apoptotic BCL-2 genes was lower in these subpopulations, which may explain the lack of sensitivity. These data demonstrate that some LPC were resistant to BCL-2 inhibitors and sustained remission will require their use in combination with other therapeutics.
Asunto(s)
Compuestos de Anilina/uso terapéutico , Antineoplásicos/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Sulfonamidas/uso terapéutico , Adolescente , Niño , Preescolar , Femenino , Humanos , Masculino , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Resultado del TratamientoRESUMEN
Hematopoietic stem/progenitor cells (HSPCs) reside in specialized bone marrow microenvironmental niches, with vascular elements (endothelial/mesenchymal stromal cells) and CXCR4-CXCL12 interactions playing particularly important roles for HSPC entry, retention, and maintenance. The functional effects of CXCL12 are dependent on its local concentration and rely on complex HSPC-niche interactions. Two Junctional Adhesion Molecule family proteins, Junctional Adhesion Molecule-B (JAM)-B and JAM-C, are reported to mediate HSPC-stromal cell interactions, which in turn regulate CXCL12 production by mesenchymal stromal cells (MSCs). Here, we demonstrate that another JAM family member, JAM-A, is most highly expressed on human hematopoietic stem cells with in vivo repopulating activity (p < .01 for JAM-A(high) compared to JAM-A(Int or Low) cord blood CD34(+) cells). JAM-A blockade, silencing, and overexpression show that JAM-A contributes significantly (p < .05) to the adhesion of human HSPCs to IL-1ß activated human bone marrow sinusoidal endothelium. Further studies highlight a novel association of JAM-A with CXCR4, with these molecules moving to the leading edge of the cell upon presentation with CXCL12 (p < .05 compared to no CXCL12). Therefore, we hypothesize that JAM family members differentially regulate CXCR4 function and CXCL12 secretion in the bone marrow niche. Stem Cells 2016;34:1664-1678.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Molécula A de Adhesión de Unión/metabolismo , Receptores CXCR4/metabolismo , Antígeno AC133/metabolismo , Antígenos CD34/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Adhesión Celular/efectos de los fármacos , Quimiocina CXCL12/farmacología , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Sangre Fetal/citología , Técnicas de Silenciamiento del Gen , Células HL-60 , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Células Jurkat , Unión Proteica/efectos de los fármacos , Nicho de Células Madre/efectos de los fármacosRESUMEN
Approximately 20% of children with acute lymphoblastic leukemia (ALL) relapse because of failure to eradicate the disease. Current drug efficacy studies focus on reducing leukemia cell burden. However, if drugs have limited effects on leukemia-initiating cells (LICs), then these cells may expand and eventually cause relapse. Parthenolide (PTL) has been shown to cause apoptosis of LIC in acute myeloid leukemia. In the present study, we assessed the effects of PTL on LIC populations in childhood ALL. Apoptosis assays demonstrated that PTL was effective against bulk B- and T-ALL cells, whereas the CD34(+)/CD19(-), CD34(+)/CD7(-), and CD34(-) subpopulations were more resistant. However, functional analyses revealed that PTL treatment prevented engraftment of multiple LIC populations in NOD/LtSz-scid IL-2Rγ(c)-null mice. PTL treatment of mice with established leukemias from low- and high-risk patients resulted in survival and restoration of normal murine hemopoiesis. In only 3 cases, disease progression was significantly slowed in mice engrafted with CD34(+)/CD19(-) or CD34(+)/CD7(-) and CD34(-) cells, but was not prevented, demonstrating that individual LIC populations within patients have different responses to therapy. These observations indicate that PTL may have therapeutic potential in childhood ALL and provide a basis for developing effective therapies that eradicate all LIC populations to prevent disease progression and reduce relapse.
Asunto(s)
Apoptosis/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Sesquiterpenos/farmacología , Adolescente , Animales , Antiinflamatorios no Esteroideos/farmacología , Células de la Médula Ósea/patología , Supervivencia Celular/efectos de los fármacos , Niño , Preescolar , Modelos Animales de Enfermedad , Femenino , Humanos , Lactante , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , FN-kappa B/metabolismo , Trasplante de Neoplasias/mortalidad , Trasplante de Neoplasias/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Factores de Riesgo , Prevención Secundaria , Trasplante HeterólogoAsunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Benzoquinonas/farmacología , Supervivencia Celular/efectos de los fármacos , Niño , Relación Dosis-Respuesta a Droga , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Humanos , Lactamas Macrocíclicas/farmacología , Terapia Molecular Dirigida , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Transducción de Señal/efectos de los fármacosRESUMEN
Optimization of therapy for childhood acute lymphoblastic leukemia (ALL) requires a greater understanding of the cells that proliferate to maintain this malignancy because a significant number of cases relapse, resulting from failure to eradicate the disease. Putative ALL stem cells may be resistant to therapy and subsequent relapses may arise from these cells. We investigated expression of CD133, CD19, and CD38 in pediatric B-ALL. Cytogenetic and molecular analyses demonstrated that karyotypically aberrant cells were present in both CD133(+)/CD19(+) and CD133(+)/CD19(-) subfractions, as were most of the antigen receptor gene rearrangements. However, ALL cells capable of long-term proliferation in vitro and in vivo were derived from the CD133(+)/CD19(-) subfraction. Moreover, these CD133(+)/CD19(-) cells could self-renew to engraft serial nonobese diabetic-severe combined immunodeficient recipients and differentiate in vivo to produce leukemias with similar immunophenotypes and karyotypes to the diagnostic samples. Furthermore, these CD133(+)/CD19(-) ALL cells were more resistant to treatment with dexamethasone and vincristine, key components in childhood ALL therapy, than the bulk leukemia population. Similar results were obtained using cells sorted for CD133 and CD38, with only the CD133(+)/CD38(-) subfraction demonstrating xenograft repopulating capacity. These data suggest that leukemia-initiating cells in childhood B-ALL have a primitive CD133(+)/CD19(-) and CD38(-) phenotype.
Asunto(s)
Antígenos CD/metabolismo , Glicoproteínas/metabolismo , Células Madre Neoplásicas/metabolismo , Péptidos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Antígeno AC133 , ADP-Ribosil Ciclasa 1/metabolismo , Adolescente , Animales , Antígenos CD19/metabolismo , Proliferación Celular , Niño , Preescolar , Reordenamiento Génico de Linfocito T/fisiología , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Madre Neoplásicas/patología , Fenotipo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Treating refractory pediatric acute lymphoblastic leukemia (ALL) remains a challenge despite impressive remission rates (>90%) achieved in the last decade. The use of innovative immunotherapeutic approaches such as anti-CD19 chimeric antigen receptor T cells does not ensure durable remissions, because leukemia-propagating cells (LPCs) that lack expression of CD19 can cause relapse, which signifies the need to identify new markers of ALL. Here we investigated expression of CD58, CD97, and CD200, which were previously shown to be overexpressed in B-cell precursor ALL (BCP-ALL) in CD34+/CD19+, CD34+/CD19-, CD34-/CD19+, and CD34-/CD19- LPCs, to assess their potential as therapeutic targets. Whole-genome microarray and flow cytometric analyses showed significant overexpression of these molecules compared with normal controls. CD58 and CD97 were mainly co-expressed with CD19 and were not a prerequisite for leukemia engraftment in immune deficient mice. In contrast, expression of CD200 was essential for engraftment and serial transplantation of cells in measurable residual disease (MRD) low-risk patients. Moreover, these CD200+ LPCs could be targeted by using the monoclonal antibody TTI-CD200 in vitro and in vivo. Treating mice with established disease significantly reduced disease burden and extended survival. These findings demonstrate that CD200 could be an attractive target for treating low-risk ALL, with minimal off-tumor effects that beset current immunotherapeutic approaches.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Animales , Antígenos CD19 , Niño , Citometría de Flujo , Humanos , Ratones , Neoplasia Residual , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológicoRESUMEN
A significant number of children with T-lineage acute lymphoblastic leukemia (T-ALL) fail to respond to therapy and experience early relapse. CD99 has been shown to be overexpressed on T-ALL cells and is considered to be a reliable detector of the disease. However, the relevance of CD99 overexpression in ALL has not been investigated in a functional context. The aim of this study was to investigate the functional capacity of CD99+ cells in childhood ALL and determine the suitability of CD99 as a therapeutic target. Flow cytometric analyses confirmed higher expression of CD99 in ALL blasts (81.5±22.7%) compared to normal hemopoietic stem cells (27.5±21.9%) and T cells (3.1±5.2%, P≤0.004). When ALL cells were sorted and assessed in functional assays, all 4 subpopulations (CD34+/CD99+, CD34+/CD99-, CD34-/CD99+ and CD34-/CD99-) could proliferate in vitro and establish leukemia in NSG mice. Leukemia propagating cell frequencies ranged from 1 in 300 to 1 in 7.4x104 but were highest in the CD34+/CD99- subpopulation. In addition, all four subpopulations had self-renewal ability in secondary NSG mice. Cells in each subpopulation contained patient specific TCR rearrangements and karyotypic changes that were preserved with passage through serial NSG transplants. Despite high levels of CD99 antigen on the majority of blast cells, leukemia initiating capacity in vivo was not restricted to cells that express this protein. Consequently, targeting CD99 alone would not eliminate all T-ALL cells with the ability to maintain the disease. The challenge remains to develop therapeutic strategies that can eliminate all leukemia cells with self-renewal capacity in vivo.
Asunto(s)
Antígeno 12E7/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Adolescente , Animales , Antígenos CD34/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Proliferación Celular , Células Cultivadas , Niño , Preescolar , Femenino , Citometría de Flujo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Hibridación Fluorescente in Situ , Cariotipo , Masculino , Ratones , Ratones Endogámicos NOD , Leucemia-Linfoma Linfoblástico de Células T Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismoRESUMEN
A significant proportion of children with T-cell acute lymphoblastic leukemia (T-ALL) continue to fail therapy. Consequently, characterization of the cells that proliferate to maintain the disease should provide valuable information on the most relevant therapeutic targets. We have used in vitro suspension culture (SC) and nonobese diabetic-severe combined immune deficient (NOD/SCID) mouse assays to phenotypically characterize and purify T-ALL progenitor cells. Cells from 13 pediatric cases were maintained in vitro for at least 4 weeks and expanded in 8 cases. To characterize the progenitors, cells were sorted for expression of CD34 and CD4 or CD7 and the subfractions were evaluated in vitro and in vivo. The majority of cells capable of long-term proliferation in vitro were derived from the CD34+/CD4- and CD34+/CD7- subfractions. Moreover, the CD34+/CD4- or CD7- cells were the only subfractions capable of NOD/SCID engraftment. These T-ALL cells successfully repopulated secondary and tertiary recipients with equivalent levels of engraftment, demonstrating self-renewal ability. The immunophenotype and genotype of the original leukemia cells were preserved with serial passage in the NOD/SCID mice. These data demonstrate the long-term repopulating ability of the CD34+/CD4- and CD34+/CD7- subfractions in T-ALL and suggest that a cell with a more primitive phenotype was the target for leukemic transformation in these cases.
Asunto(s)
Leucemia-Linfoma de Células T del Adulto/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Células Madre/inmunología , Células Madre/patología , Adolescente , Animales , Técnicas de Cultivo de Célula , Proliferación Celular , Separación Celular , Células Cultivadas , Niño , Preescolar , Femenino , Reordenamiento Génico de la Cadena gamma de los Receptores de Antígenos de los Linfocitos T/genética , Genotipo , Humanos , Inmunofenotipificación , Lactante , Leucemia-Linfoma de Células T del Adulto/diagnóstico , Leucemia-Linfoma de Células T del Adulto/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Only some acute lymphoblastic leukemia (ALL) cells are thought to be capable of proliferating to maintain the leukemic clone, and these cells may be the most relevant to target with treatment regimens. We have developed a serum-free suspension culture (SC) system that supported growth of B-ALL cells from 33 patients for up to 6 weeks. ALL cells from 28 cases (85%) were expanded in this system, and growth was superior in SC than in long-term bone marrow culture. To characterize ALL progenitors, cells were sorted for expression of CD34 and CD10 or CD19 and the subfractions assayed in SC and in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Cells capable of long-term proliferation in vitro and NOD/SCID repopulation were derived only from the CD34(+)/CD10(-) and CD34(+)/CD19(-) subfractions, and these cells could engraft secondary recipients. The engrafted cells had the same immunophenotype and karyotype as was seen at diagnosis, suggesting they had differentiated in vivo. These results demonstrate that ALL cells capable of long-term proliferation in vitro and in vivo are CD34(+)/CD10(-)/CD19(-). This suggests that cells with a more immature phenotype, rather than committed B-lymphoid cells, may be the targets for transformation in B-ALL.
Asunto(s)
Células Madre Neoplásicas/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Adolescente , Animales , Antígenos CD19/análisis , Antígenos CD34/análisis , Técnicas de Cultivo de Célula , Proliferación Celular , Niño , Preescolar , Supervivencia de Injerto , Humanos , Inmunofenotipificación , Lactante , Ratones , Ratones SCID , Trasplante de Neoplasias , Neprilisina/análisis , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Antithymocyte globulin (ATG) is widely used in the treatment of aplastic anaemia (AA) and a response occurs in 60-80% of patients. However, its exact mechanism of action in the treatment of AA has yet to be determined. Previously, we have shown that ATG increases colony growth from purified bone marrow CD34+ cells of AA patients in vitro, and decreases stem cell apoptosis and the expression of soluble Fas receptor after ATG therapy in vivo. The aim of this study was to further examine the association of ATG with AA haemopoietic progenitor cells. We describe here that ATG bound directly to CD34+ cells. Forty-six patients and 20 normal control subjects were studied. ATG bound to CD34+ cells in normal control subjects (mean 90.38%) as determined by flow cytometry. The mean percentage of CD34+ cells binding to ATG was 59.90% in untreated aplastic patients, 83.24% in partial responders, 58.3% in non-responders and 62.73% in relapsed patients. In completely recovered patients, ATG binding was indistinguishable from control subjects. The functionality of AA patients' haemopoietic progenitor cells was assessed using colony assays. These results demonstrate the direct binding of ATG to CD34+ cells and suggest that differences in its binding to AA CD34+ cells could reflect functional differences in the haemopoietic stem cell compartment throughout the disease process.