RESUMEN
Affinity-optimized T cell receptors can enhance the potency of adoptive T cell therapy. Afamitresgene autoleucel (afami-cel) is a human leukocyte antigen-restricted autologous T cell therapy targeting melanoma-associated antigen A4 (MAGE-A4), a cancer/testis antigen expressed at varying levels in multiple solid tumors. We conducted a multicenter, dose-escalation, phase 1 trial in patients with relapsed/refractory metastatic solid tumors expressing MAGE-A4, including synovial sarcoma (SS), ovarian cancer and head and neck cancer ( NCT03132922 ). The primary endpoint was safety, and the secondary efficacy endpoints included overall response rate (ORR) and duration of response. All patients (N = 38, nine tumor types) experienced Grade ≥3 hematologic toxicities; 55% of patients (90% Grade ≤2) experienced cytokine release syndrome. ORR (all partial response) was 24% (9/38), 7/16 (44%) for SS and 2/22 (9%) for all other cancers. Median duration of response was 25.6 weeks (95% confidence interval (CI): 12.286, not reached) and 28.1 weeks (95% CI: 12.286, not reached) overall and for SS, respectively. Exploratory analyses showed that afami-cel infiltrates tumors, has an interferon-γ-driven mechanism of action and triggers adaptive immune responses. In addition, afami-cel has an acceptable benefit-risk profile, with early and durable responses, especially in patients with metastatic SS. Although the small trial size limits conclusions that can be drawn, the results warrant further testing in larger studies.
Asunto(s)
Antígenos de Neoplasias , Neoplasias de Cabeza y Cuello , Masculino , Humanos , Proteínas de Neoplasias , Antígenos HLA-A , Tratamiento Basado en Trasplante de Células y Tejidos , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodosRESUMEN
BACKGROUND: Gene-modified autologous T cells expressing NY-ESO-1c259, an affinity-enhanced T-cell receptor (TCR) reactive against the NY-ESO-1-specific HLA-A*02-restricted peptide SLLMWITQC (NY-ESO-1 SPEAR T-cells; GSK 794), have demonstrated clinical activity in patients with advanced synovial sarcoma (SS). The factors contributing to gene-modified T-cell expansion and the changes within the tumor microenvironment (TME) following T-cell infusion remain unclear. These studies address the immunological mechanisms of response and resistance in patients with SS treated with NY-ESO-1 SPEAR T-cells. METHODS: Four cohorts were included to evaluate antigen expression and preconditioning on efficacy. Clinical responses were assessed by RECIST v1.1. Engineered T-cell persistence was determined by qPCR. Serum cytokines were evaluated by immunoassay. Transcriptomic analyses and immunohistochemistry were performed on tumor biopsies from patients before and after T-cell infusion. Gene-modified T-cells were detected within the TME via an RNAish assay. RESULTS: Responses across cohorts were affected by preconditioning and intra-tumoral NY-ESO-1 expression. Of the 42 patients reported (data cut-off 4June2018), 1 patient had a complete response, 14 patients had partial responses, 24 patients had stable disease, and 3 patients had progressive disease. The magnitude of gene-modified T-cell expansion shortly after infusion was associated with response in patients with high intra-tumoral NY-ESO-1 expression. Patients receiving a fludarabine-containing conditioning regimen experienced increases in serum IL-7 and IL-15. Prior to infusion, the TME exhibited minimal leukocyte infiltration; CD163+ tumor-associated macrophages (TAMs) were the dominant population. Modest increases in intra-tumoral leukocytes (≤5%) were observed in a subset of subjects at approximately 8 weeks. Beyond 8 weeks post infusion, the TME was minimally infiltrated with a TAM-dominant leukocyte infiltrate. Tumor-associated antigens and antigen presentation did not significantly change within the tumor post-T-cell infusion. Finally, NY-ESO-1 SPEAR T cells trafficked to the TME and maintained cytotoxicity in a subset of patients. CONCLUSIONS: Our studies elucidate some factors that underpin response and resistance to NY-ESO-1 SPEAR T-cell therapy. From these data, we conclude that a lymphodepletion regimen containing high doses of fludarabine and cyclophosphamide is necessary for SPEAR T-cell persistence and efficacy. Furthermore, these data demonstrate that non-T-cell inflamed tumors, which are resistant to PD-1/PD-L1 inhibitors, can be treated with adoptive T-cell based immunotherapy. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01343043 , Registered 27 April 2011.
Asunto(s)
Antígenos de Neoplasias/inmunología , Inmunoterapia Adoptiva , Proteínas de la Membrana/inmunología , Sarcoma Sinovial/inmunología , Sarcoma Sinovial/terapia , Linfocitos T/inmunología , Linfocitos T/metabolismo , Biomarcadores , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto , Citocinas/metabolismo , Citotoxicidad Inmunológica , Antígenos HLA-A/inmunología , Humanos , Inmunohistoquímica , Inmunoterapia Adoptiva/métodos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Sarcoma Sinovial/patología , Especificidad del Receptor de Antígeno de Linfocitos T , Resultado del Tratamiento , Microambiente Tumoral/inmunologíaRESUMEN
BACKGROUND: Human-induced pluripotent stem cells (hiPSCs) are a potentially invaluable resource for regenerative medicine, including the in vitro manufacture of blood products. HiPSC-derived red blood cells are an attractive therapeutic option in hematology, yet exhibit unexplained proliferation and enucleation defects that presently preclude such applications. We hypothesised that substantial differential regulation of gene expression during erythroid development accounts for these important differences between hiPSC-derived cells and those from adult or cord-blood progenitors. We thus cultured erythroblasts from each source for transcriptomic analysis to investigate differential gene expression underlying these functional defects. RESULTS: Our high resolution transcriptional view of definitive erythropoiesis captures the regulation of genes relevant to cell-cycle control and confers statistical power to deploy novel bioinformatics methods. Whilst the dynamics of erythroid program elaboration from adult and cord blood progenitors were very similar, the emerging erythroid transcriptome in hiPSCs revealed radically different program elaboration compared to adult and cord blood cells. We explored the function of differentially expressed genes in hiPSC-specific clusters defined by our novel tunable clustering algorithms (SMART and Bi-CoPaM). HiPSCs show reduced expression of c-KIT and key erythroid transcription factors SOX6, MYB and BCL11A, strong HBZ-induction, and aberrant expression of genes involved in protein degradation, lysosomal clearance and cell-cycle regulation. CONCLUSIONS: Together, these data suggest that hiPSC-derived cells may be specified to a primitive erythroid fate, and implies that definitive specification may more accurately reflect adult development. We have therefore identified, for the first time, distinct gene expression dynamics during erythroblast differentiation from hiPSCs which may cause reduced proliferation and enucleation of hiPSC-derived erythroid cells. The data suggest several mechanistic defects which may partially explain the observed aberrant erythroid differentiation from hiPSCs.
Asunto(s)
Eritropoyesis/genética , Sangre Fetal/citología , Regulación del Desarrollo de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Transcriptoma , Diferenciación Celular/genética , Análisis por Conglomerados , Eritroblastos/citología , Eritroblastos/metabolismo , Perfilación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Humanos , Células Madre Pluripotentes Inducidas/citologíaRESUMEN
Transforming growth factor ß (TGFß) is a potent inhibitor of hematopoietic stem and progenitor cell proliferation. However, the precise mechanism for this effect is unknown. Here, we have identified the transcription factor Gata2, previously described as an important regulator of hematopoietic stem cell function, as an early and direct target gene for TGFß-induced Smad signaling in hematopoietic progenitor cells. We also report that Gata2 is involved in mediating a significant part of the TGFß response in primitive hematopoietic cells. Interestingly, the cell cycle regulator and TGFß signaling effector molecule p57 was found to be upregulated as a secondary response to TGFß. We observed Gata2 binding upstream of the p57 genomic locus, and importantly, loss of Gata2 abolished TGFß-stimulated induction of p57 as well as the resulting growth arrest of hematopoietic progenitors. Our results connect key molecules involved in hematopoietic stem cell self-renewal and reveal a functionally relevant network, regulating proliferation of primitive hematopoietic cells.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/genética , Factor de Transcripción GATA2/genética , Células Madre Hematopoyéticas/efectos de los fármacos , Proteína Smad4/genética , Factor de Crecimiento Transformador beta/farmacología , Animales , Línea Celular , Proliferación Celular/genética , Células Cultivadas , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/metabolismo , Factor de Transcripción GATA2/metabolismo , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes/efectos de los fármacos , Redes Reguladoras de Genes/genética , Células HEK293 , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Modelos Genéticos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteína Smad4/metabolismoRESUMEN
The role of infection in erythropoietic dysfunction is poorly understood. In children with P. falciparum malaria, the by-product of hemoglobin digestion in infected red cells (hemozoin) is associated with the severity of anemia which is independent of circulating levels of the inflammatory cytokine tumor necrosis alpha (TNF-α). To gain insight into the common and specific effects of TNF-α and hemozoin on erythropoiesis, we studied the gene expression profile of purified primary erythroid cultures exposed to either TNF-α (10 ng/ml) or to hemozoin (12.5 µg/ml heme units) for 24 hours. Perturbed gene function was assessed using co-annotation of associated gene ontologies and expression of selected genes representative of the profile observed was confirmed by real time PCR (rtPCR). The changes in gene expression induced by each agent were largely distinct; many of the genes significantly modulated by TNF-α were not affected by hemozoin. The genes modulated by TNF-α were significantly enriched for those encoding proteins involved in the control of type 1 interferon signalling and the immune response to viral infection. In contrast, genes induced by hemozoin were significantly enriched for functional roles in regulation of transcription and apoptosis. Further analyses by rtPCR revealed that hemozoin increases expression of transcription factors that form part of the integrated stress response which is accompanied by reduced expression of genes involved in DNA repair. This study confirms that hemozoin induces cellular stress on erythroblasts that is additional to and distinct from responses to inflammatory cytokines and identifies new genes that may be involved in the pathogenesis of severe malarial anemia. More generally the respective transcription profiles highlight the varied mechanisms through which erythropoiesis may be disrupted during infectious disease.
Asunto(s)
Eritrocitos/citología , Eritropoyesis/fisiología , Enfermedades Hematológicas/etiología , Hemoproteínas/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Apoptosis/efectos de los fármacos , Células Cultivadas , Eritrocitos/metabolismo , Eritrocitos/parasitología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Hemoproteínas/metabolismo , Hemoproteínas/fisiología , Humanos , Inmunidad Celular , Interferones/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
We used the paradigmatic GATA-PU.1 axis to explore, at the systems level, dynamic relationships between transcription factor (TF) binding and global gene expression programs as multipotent cells differentiate. We combined global ChIP-seq of GATA1, GATA2, and PU.1 with expression profiling during differentiation to erythroid and neutrophil lineages. Our analysis reveals (1) differential complexity of sequence motifs bound by GATA1, GATA2, and PU.1; (2) the scope and interplay of GATA1 and GATA2 programs within, and during transitions between, different cell compartments, and the extent of their hard-wiring by DNA motifs; (3) the potential to predict gene expression trajectories based on global associations between TF-binding data and target gene expression; and (4) how dynamic modeling of DNA-binding and gene expression data can be used to infer regulatory logic of TF circuitry. This rubric exemplifies the utility of this cross-platform resource for deconvoluting the complexity of transcriptional programs controlling stem/progenitor cell fate in hematopoiesis.
Asunto(s)
Linaje de la Célula/genética , Regulación de la Expresión Génica , Genoma/genética , Hematopoyesis/genética , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Inmunoprecipitación de Cromatina , Células Eritroides/citología , Células Eritroides/metabolismo , Factor de Transcripción GATA1/metabolismo , Factor de Transcripción GATA2/metabolismo , Humanos , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Motivos de Nucleótidos/genética , Unión Proteica/genética , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismoRESUMEN
Gene expression in multiple individual cells from a tissue or culture sample varies according to cell-cycle, genetic, epigenetic and stochastic differences between the cells. However, single-cell differences have been largely neglected in the analysis of the functional consequences of genetic variation. Here we measure the expression of 92 genes affected by Wnt signaling in 1,440 single cells from 15 individuals to associate single-nucleotide polymorphisms (SNPs) with gene-expression phenotypes, while accounting for stochastic and cell-cycle differences between cells. We provide evidence that many heritable variations in gene function--such as burst size, burst frequency, cell cycle-specific expression and expression correlation/noise between cells--are masked when expression is averaged over many cells. Our results demonstrate how single-cell analyses provide insights into the mechanistic and network effects of genetic variability, with improved statistical power to model these effects on gene expression.
Asunto(s)
Expresión Génica , Estudios de Asociación Genética , Sitios de Carácter Cuantitativo/genética , Vía de Señalización Wnt/genética , Mapeo Cromosómico , Perfilación de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimorfismo de Nucleótido Simple , Análisis de la Célula IndividualRESUMEN
The stochastic nature of generating eukaryotic transcripts challenges conventional methods for obtaining and analyzing single-cell gene expression data. In order to address the inherent noise, detailed methods are described on how to collect data on multiple genes in a large number of single cells using microfluidic arrays. As part of a study exploring the effect of genotype on Wnt pathway activation, data were collected for 96 qPCR assays on 1440 lymphoblastoid cells. The description of methods includes preliminary data processing steps. The methods used in the collection and analysis of single-cell qPCR data are contrasted with those used in conventional qPCR.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Células Progenitoras Linfoides/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de la Célula Individual , Línea Celular , Interpretación Estadística de Datos , Humanos , Límite de Detección , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vía de Señalización WntRESUMEN
How the molecular programs of differentiated cells develop as cells transit from multipotency through lineage commitment remains unexplored. This reflects the inability to access cells undergoing commitment or located in the immediate vicinity of commitment boundaries. It remains unclear whether commitment constitutes a gradual process, or else represents a discrete transition. Analyses of in vitro self-renewing multipotent systems have revealed cellular heterogeneity with individual cells transiently exhibiting distinct biases for lineage commitment. Such systems can be used to molecularly interrogate early stages of lineage affiliation and infer rules of lineage commitment. In haematopoiesis, population-based studies have indicated that lineage choice is governed by global transcriptional noise, with self-renewing multipotent cells reversibly activating transcriptome-wide lineage-affiliated programs. We examine this hypothesis through functional and molecular analysis of individual blood cells captured from self-renewal cultures, during cytokine-driven differentiation and from primary stem and progenitor bone marrow compartments. We show dissociation between self-renewal potential and transcriptome-wide activation of lineage programs, and instead suggest that multipotent cells experience independent activation of individual regulators resulting in a low probability of transition to the committed state.
Asunto(s)
Linaje de la Célula/genética , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Células Madre Multipotentes/metabolismo , Animales , Antígenos CD34/genética , Antígenos CD34/metabolismo , Antígenos Ly/genética , Antígenos Ly/metabolismo , Células de la Médula Ósea/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular Transformada , Células Cultivadas , Análisis por Conglomerados , Citocinas/farmacología , Células Eritroides/metabolismo , Perfilación de la Expresión Génica/métodos , Immunoblotting , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , TranscriptomaRESUMEN
Unremitting blood cell production throughout the lifetime of an organism is reliant on hematopoietic stem cells (HSCs). A rare and relatively quiescent cell type, HSCs are, on entry into cell cycle fated to self-renew, undergo apoptosis or differentiate to progenitors (HPCs) that eventually yield specific classes of blood cells. Disruption of these HSC fate decisions is considered to be fundamental to the development of leukemia. Much effort has therefore been placed on understanding the molecular pathways that regulate HSC fate decisions and how these processes are undermined in leukemia. Transcription factors have emerged as critical regulators in this respect. Here we review the participation of zinc finger transcription factor GATA-2 in regulating normal hematopoietic stem and progenitor cell functionality, myelodysplasia and myeloid leukemia.
Asunto(s)
Factor de Transcripción GATA2/metabolismo , Células Madre Hematopoyéticas/metabolismo , Leucemia Mieloide/patología , Síndromes Mielodisplásicos/patología , Animales , Diferenciación Celular/genética , Supervivencia Celular/genética , Transformación Celular Neoplásica/genética , Factor de Transcripción GATA2/genética , Células Madre Hematopoyéticas/patología , Humanos , Mutación/genéticaRESUMEN
Evidence suggests the transcription factor GATA-2 is a critical regulator of murine hematopoietic stem cells. Here, we explore the relation between GATA-2 and cell proliferation and show that inducing GATA-2 increases quiescence (G(0) residency) of murine and human hematopoietic cells. In human cord blood, quiescent fractions (CD34(+)CD38(-)Hoechst(lo)Pyronin Y(lo)) express more GATA-2 than cycling counterparts. Enforcing GATA-2 expression increased quiescence of cord blood cells, reducing proliferation and performance in long-term culture-initiating cell and colony-forming cell (CFC) assays. Gene expression analysis places GATA-2 upstream of the quiescence regulator MEF, but enforcing MEF expression does not prevent GATA-2-conferred quiescence, suggesting additional regulators are involved. Although known quiescence regulators p21(CIP1) and p27(KIP1) do not appear to be responsible, enforcing GATA-2 reduced expression of regulators of cell cycle such as CCND3, CDK4, and CDK6. Enforcing GATA-2 inhibited human hematopoiesis in vivo: cells with highest exogenous expression (GATA-2(hi)) failed to contribute to hematopoiesis in nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice, whereas GATA-2(lo) cells contributed with delayed kinetics and low efficiency, with reduced expression of Ki-67. Thus, GATA-2 activity inhibits cell cycle in vitro and in vivo, highlighting GATA-2 as a molecular entry point into the transcriptional program regulating quiescence in human hematopoietic stem and progenitor cells.
Asunto(s)
Ciclo Celular , Factor de Transcripción GATA2/fisiología , Células Madre Hematopoyéticas/citología , Animales , Apoptosis , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Trasplante de Células Madre de Sangre del Cordón Umbilical , Estradiol/farmacología , Sangre Fetal/citología , Factor de Transcripción GATA2/biosíntesis , Factor de Transcripción GATA2/genética , Regulación de la Expresión Génica/genética , Genes Sintéticos , Genes cdc , Humanos , Interleucina-3/farmacología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Receptores de Estrógenos/efectos de los fármacos , Receptores de Estrógenos/genética , Proteínas Recombinantes de Fusión/fisiología , Fase de Descanso del Ciclo Celular , Tamoxifeno/farmacología , Transcripción GenéticaRESUMEN
The zinc finger transcription factor GATA-2 has been implicated in the regulation of hematopoietic stem cells. Herein, we explored the role of GATA-2 as a candidate regulator of the hematopoietic progenitor cell compartment. We showed that bone marrow from GATA-2 heterozygote (GATA-2(+/-)) mice displayed attenuated granulocyte-macrophage progenitor function in colony-forming cell (CFC) and serial replating CFC assays. This defect was mapped to the Lin(-)CD117(+)Sca-1(-)CD34(+)CD16/32(high) granulocyte-macrophage progenitor (GMP) compartment of GATA-2(+/-) marrow, which was reduced in size and functionally impaired in CFC assays and competitive transplantation. Similar functional impairments were obtained using a RNA interference approach to stably knockdown GATA-2 in wild-type GMP. Although apoptosis and cell-cycle distribution remained unperturbed in GATA-2(+/-) GMP, quiescent cells from GATA-2(+/-) GMP exhibited altered functionality. Gene expression analysis showed attenuated expression of HES-1 mRNA in GATA-2-deficient GMP. Binding of GATA-2 to the HES-1 locus was detected in the myeloid progenitor cell line 32Dcl3, and enforced expression of HES-1 expression in GATA-2(+/-) GMP rectified the functional defect, suggesting that GATA-2 regulates myeloid progenitor function through HES-1. These data collectively point to GATA-2 as a novel, pivotal determinant of GMP cell fate.
Asunto(s)
Factor de Transcripción GATA2/fisiología , Células Progenitoras de Granulocitos y Macrófagos/citología , Animales , Línea Celular , Factor de Transcripción GATA2/genética , Factor de Transcripción GATA2/metabolismo , Perfilación de la Expresión Génica , Genotipo , Células Progenitoras de Granulocitos y Macrófagos/fisiología , Ratones , Ratones Mutantes , Unión Proteica , Interferencia de ARNRESUMEN
Lineage specification and cellular maturation require coordinated regulation of gene expression programs. In large part, this is dependent on the activator and repressor functions of protein complexes associated with tissue-specific transcriptional regulators. In this study, we have used a proteomic approach to characterize multiprotein complexes containing the key hematopoietic regulator SCL in erythroid and megakaryocytic cell lines. One of the novel SCL-interacting proteins identified in both cell types is the transcriptional corepressor ETO-2. Interaction between endogenous proteins was confirmed in primary cells. We then showed that SCL complexes are shared but also significantly differ in the two cell types. Importantly, SCL/ETO-2 interacts with another corepressor, Gfi-1b, in red cells but not megakaryocytes. The SCL/ETO-2/Gfi-1b association is lost during erythroid differentiation of primary fetal liver cells. Genetic studies of erythroid cells show that ETO-2 exerts a repressor effect on SCL target genes. We suggest that, through its association with SCL, ETO-2 represses gene expression in the early stages of erythroid differentiation and that alleviation/modulation of the repressive state is then required for expression of genes necessary for terminal erythroid maturation to proceed.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células Eritroides/metabolismo , Eritropoyesis , Megacariocitos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular , Células Cultivadas , Células Eritroides/citología , Regulación de la Expresión Génica , Ratones , Mutación/genética , Proteínas Nucleares/genética , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Represoras/genética , Proteína 1 de la Leucemia Linfocítica T Aguda , Factores de Transcripción/genética , Transcripción Genética/genéticaRESUMEN
The chimaeric protein Bcr/Abl, the hallmark of chronic myeloid leukaemia, has been connected with several signalling pathways, such as those involving protein kinase B/Akt, JNK (c-Jun N-terminal kinase) or ERKs (extracellular-signal-regulated kinases) 1 and 2. However, no data about the p38 MAPK (mitogen-activated protein kinase) have been reported. Here, we present evidence showing that Bcr/Abl is able to modulate this signalling pathway. Transient transfection experiments indicated that overexpression of Bcr/Abl in 293T cells is able to activate p38 MAPK or induce p73 stabilization, suggesting that c-Abl and Bcr/Abl share some biological substrates. Interestingly, the control exerted by Bcr/Abl on the p38 MAPK pathway was not only mediated by the tyrosine kinase activity of Bcr/Abl, as the use of STI571 demonstrated. In fact, Bcr alone was able to induce p38 MAPK activation specifically through MKK3 (MAP kinase kinase 3). Supporting these observations, chronic myeloid leukaemia-derived K562 cells or BaF 3 cells stably transfected with Bcr/Abl showed higher levels of phosphorylated p38 MAPK compared with Bcr/Abl-negative cells. While Bcr/Abl-negative cells activated p38 MAPK in response to Ara-C (1-beta-D-arabinofuranosylcytosine), Bcr/Abl-positive cells were unable to activate p38 MAPK, suggesting that the p38 MAPK pathway is not sensitive to Abl-dependent stimuli in Bcr/Abl-positive cells. Our results demonstrate that the involvement of Bcr/Abl in the p38 MAPK pathway is a key mechanism for explaining resistance to Ara-C, and could provide a clue for new therapeutic approaches based on the use of specific Abl inhibitors.
Asunto(s)
Proteínas de Fusión bcr-abl/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Benzamidas , Línea Celular , Línea Celular Tumoral , Citarabina/antagonistas & inhibidores , Citarabina/metabolismo , Citarabina/farmacología , Proteínas de Unión al ADN/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Proteínas de Fusión bcr-abl/fisiología , Genes Supresores de Tumor , Humanos , Mesilato de Imatinib , Riñón/química , Riñón/citología , Riñón/embriología , Riñón/metabolismo , Proteínas Nucleares/metabolismo , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-abl/metabolismo , Pirimidinas/farmacología , Proteína Tumoral p73 , Proteínas Supresoras de Tumor , Células U937/enzimología , Células U937/metabolismo , Células U937/patologíaAsunto(s)
Proteínas de Fusión bcr-abl/genética , Regulación Leucémica de la Expresión Génica , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Animales , Células de la Médula Ósea/metabolismo , Proteínas de Fusión bcr-abl/biosíntesis , Perfilación de la Expresión Génica , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Ratones , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Transcripción GenéticaAsunto(s)
Análisis Mutacional de ADN/métodos , Disqueratosis Congénita/diagnóstico , Anemia de Fanconi/diagnóstico , Disqueratosis Congénita/genética , Electroforesis en Gel de Poliacrilamida/métodos , Anemia de Fanconi/genética , Humanos , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo Conformacional Retorcido-SimpleRESUMEN
OBJECTIVE: Resistance to imatinib mesylate monotherapy is clearly a barrier to successful treatment of chronic myeloid leukemia (CML) patients. In some patients, resistance arises due to powerful selective pressure on rare cells that carry amplified copies of the BCR-ABL fusion oncogene or point mutations in the Bcr-Abl tyrosine kinase domain that affect binding of the drug to the oncoprotein. However, in a proportion of patients neither mechanism operates, and resistance appears to be a priori, existing prior to exposure to the drug. These mechanisms of imatinib resistance are poorly understood and may be heterogeneous. MATERIALS AND METHODS: We have previously described such innate resistance to imatinib in subclones of a myeloid leukemia cell line, KCL22, in which imatinib exposure inhibits the activity of Bcr-Abl and yet fails to induce apoptosis. We describe here whole-genome expression analysis of imatinib-sensitive and -resistant cells derived from the original KCL22 line, using Affymetrix microarray analysis. RESULTS: We detected differential expression of 39 genes that correlate with the imatinib-resistant phenotype. The resistant cells overexpress several genes associated with the suppression of apoptosis or with conferral of a transformed phenotype. CONCLUSION: Amongst the differentially-expressed genes correlating with imatinib resistance, several suggest the activation of alternative pathway(s) that maintain viability and growth independently of Bcr-Abl kinase activity. Given the high rate of primary imatinib resistance in blast crisis, the potential of activating such alternative pathways appears to correlate with disease progression.
Asunto(s)
Antineoplásicos/uso terapéutico , Perfilación de la Expresión Génica , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Piperazinas/uso terapéutico , Pirimidinas/uso terapéutico , Benzamidas , Línea Celular Tumoral , Resistencia a Antineoplásicos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/genéticaRESUMEN
Imatinib mesylate (Gleevec) (formerly STI571) has secured a definitive role in the treatment of chronic myeloid leukemia (CML) due to its specificity and efficacy. Although some patients become resistant to the drug, it may still be possible to control the leukemia with imatinib-containing regimens. Front-line treatment with such combinations may indeed minimize the risk that resistance, and hence relapse, occurs. In this review, we discuss the published data on in vitro studies that address this question in a variety of models, and attempt to predict efficacious combinations for future clinical trials. These represent regimens where imatinib is combined with conventional chemotherapeutic drugs or with inhibitors of other key signal transduction molecules that may be preferentially activated in CML cells.