RESUMEN
Aminophospholipids (aPL) such as phosphatidylserine are essential for supporting the activity of coagulation factors, circulating platelets, and blood cells. Phosphatidylthreonine (PT) is an aminophospholipid previously reported in eukaryotic parasites and animal cell cultures, but not yet in human tissues. Here, we evaluated whether PT is present in blood cells and characterized its ability to support coagulation. Several PT molecular species were detected in human blood, washed platelets, extracellular vesicles, and isolated leukocytes from healthy volunteers using liquid chromatography-tandem mass spectrometry. The ability of PT to support coagulation was demonstrated in vitro using biochemical and biophysical assays. In liposomes, PT supported prothrombinase activity in the presence and absence of phosphatidylserine. PT nanodiscs strongly bound FVa and lactadherin (nM affinity) but poorly bound prothrombin and FX, suggesting that PT supports prothrombinase through recruitment of FVa. PT liposomes bearing tissue factor poorly generated thrombin in platelet poor plasma, indicating that PT poorly supports extrinsic tenase activity. On platelet activation, PT is externalized and partially metabolized. Last, PT was significantly higher in platelets and extracellular vesicle from patients with coronary artery disease than in healthy controls. In summary, PT is present in human blood, binds FVa and lactadherin, supports coagulation in vitro through FVa binding, and is elevated in atherosclerotic vascular disease. Our studies reveal a new phospholipid subclass, that contributes to the procoagulant membrane, and may support thrombosis in patients at elevated risk.
Asunto(s)
Enfermedad de la Arteria Coronaria , Glicerofosfolípidos , Treonina/análogos & derivados , Tromboplastina , Animales , Humanos , Tromboplastina/metabolismo , Fosfatidilserinas/metabolismo , Liposomas/metabolismo , Plaquetas/metabolismo , Trombina/metabolismoRESUMEN
During transformation, myelodysplastic syndromes (MDS) are characterized by reducing apoptosis of bone marrow (BM) precursors. Mouse models of high risk (HR)-MDS and acute myelogenous leukemia (AML) post-MDS using mutant NRAS and overexpression of human BCL-2, known to be poor prognostic indicators of the human diseases, were created. We have reported the efficacy of the BCL-2 inhibitor, ABT-737, on the AML post-MDS model; here, we report that this BCL-2 inhibitor also significantly extended survival of the HR-MDS mouse model, with reductions of BM blasts and lineage negative/Sca1+/KIT+ (LSK) cells. Secondary transplants showed increased survival in treated compared to untreated mice. Unlike the AML model, BCL-2 expression and RAS activity decreased following treatment and the RAS:BCL-2 complex remained in the plasma membrane. Exon-specific gene expression profiling (GEP) of HR-MDS mice showed 1952 differentially regulated genes upon treatment, including genes important for the regulation of stem cells, differentiation, proliferation, oxidative phosphorylation, mitochondrial function, and apoptosis; relevant in human disease. Spliceosome genes, found to be abnormal in MDS patients and downregulated in our HR-MDS model, such as Rsrc1 and Wbp4, were upregulated by the treatment, as were genes involved in epigenetic regulation, such as DNMT3A and B, upregulated upon disease progression and downregulated upon treatment.
Asunto(s)
Compuestos de Bifenilo/administración & dosificación , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de Unión al GTP Monoméricas/metabolismo , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/metabolismo , Nitrofenoles/administración & dosificación , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal/efectos de los fármacos , Células Madre/metabolismo , Sulfonamidas/administración & dosificación , Animales , Apoptosis/efectos de los fármacos , Médula Ósea/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica/métodos , Estimación de Kaplan-Meier , Ratones , Ratones Transgénicos , Proteínas de Unión al GTP Monoméricas/genética , Síndromes Mielodisplásicos/mortalidad , Piperazinas/administración & dosificación , Proteínas Proto-Oncogénicas c-bcl-2/genética , Células Madre/efectos de los fármacos , Transcriptoma/efectos de los fármacosRESUMEN
Minimal residual disease (MRD) in acute myeloid leukaemia (AML) poses a major challenge due to drug insensitivity and high risk of relapse. Intensification of chemotherapy and stem cell transplantation are often pivoted on MRD status. Relapse rates are high even with the integration of first-generation FMS-like tyrosine kinase 3 (FLT3) inhibitors in pre- and post-transplant regimes and as maintenance in FLT3-mutated AML. Pre-clinical progress is hampered by the lack of suitable modelling of residual disease and post-therapy relapse. In the present study, we investigated the nature of pro-survival signalling in primary residual tyrosine kinase inhibitor (TKI)-treated AML cells adherent to stroma and further determined their drug sensitivity in order to inform rational future therapy combinations. Using a primary human leukaemia-human stroma model to mimic the cell-cell interactions occurring in patients, the ability of several TKIs in clinical use, to abrogate stroma-driven leukaemic signalling was determined, and a synergistic combination with a mitogen-activated protein kinase (MEK) inhibitor identified for potential therapeutic application in the MRD setting. The findings reveal a common mechanism of stroma-mediated resistance that may be independent of mutational status but can be targeted through rational drug design, to eradicate MRD and reduce treatment-related toxicity.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Leucemia Mieloide Aguda , Modelos Biológicos , Tirosina Quinasa 3 Similar a fms , Adolescente , Adulto , Anciano , Hidrocarburos Aromáticos con Puentes/farmacología , Adhesión Celular/efectos de los fármacos , Niño , Preescolar , Quinasas MAP Reguladas por Señal Extracelular , Femenino , Humanos , Lactante , Recién Nacido , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Masculino , Persona de Mediana Edad , Neoplasia Residual , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
The PML/RARA fusion protein occurs as a result of the t(15;17) translocation in the acute promyelocytic leukemia subtype of human acute myeloid leukemia. Gain of chromosome 8 is the most common chromosomal gain in human acute myeloid leukemia, including acute promyelocytic leukemia. We previously demonstrated that gain of chromosome 8-containing MYC is of central importance in trisomy 8, but the role of the nearby TRIB1 gene has not been experimentally addressed in this context. We have now tested the hypothesis that both MYC and TRIB1 have functional roles underlying leukemogenesis of trisomy 8 by using retroviral vectors to express MYC and TRIB1 in wild-type bone marrow and in marrow that expressed a PML/RARA transgene. Interestingly, although MYC and TRIB1 readily co-operated in leukemogenesis for wild-type bone marrow, TRIB1 provided no selective advantage to cells expressing PML/RARA. We hypothesized that this lack of co-operation between PML/RARA and TRIB1 reflected a common pathway for their effect: both proteins targeting the myeloid transcription factor C/EBPα. In support of this idea, TRIB1 expression abrogated the all-trans retinoic acid response of acute promyelocytic leukemia cells in vitro and in vivo Our data delineate the common and redundant inhibitory effects of TRIB1 and PML/RARA on C/EBPα providing a potential explanation for the lack of selection of TRIB1 in human acute promyelocytic leukemia, and highlighting the key role of C/EBPs in acute promyelocytic leukemia pathogenesis and therapeutic response. In addition, the co-operativity we observed between MYC and TRIB1 in the absence of PML/RARA show that, outside of acute promyelocytic leukemia, gain of both genes may drive selection for trisomy 8.
Asunto(s)
Leucemia Mieloide Aguda/patología , Leucemia Promielocítica Aguda/patología , Animales , Proteínas Potenciadoras de Unión a CCAAT/fisiología , Cromosomas Humanos Par 8 , Humanos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Leucemia Mieloide Aguda/etiología , Leucemia Promielocítica Aguda/etiología , Ratones , Proteínas de Fusión Oncogénica , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas c-myc/fisiología , TrisomíaRESUMEN
Myelodysplastic syndrome (MDS) transforms into an acute myelogenous leukemia (AML) with associated increased bone marrow (BM) blast infiltration. Using a transgenic mouse model, MRP8[NRASD12/hBCL-2], in which the NRAS:BCL-2 complex at the mitochondria induces MDS progressing to AML with dysplastic features, we studied the therapeutic potential of a BCL-2 homology domain 3 mimetic inhibitor, ABT-737. Treatment significantly extended lifespan, increased survival of lethally irradiated secondary recipients transplanted with cells from treated mice compared with cells from untreated mice, with a reduction of BM blasts, Lin-/Sca-1(+)/c-Kit(+), and progenitor populations by increased apoptosis of infiltrating blasts of diseased mice assessed in vivo by technicium-labeled annexin V single photon emission computed tomography and ex vivo by annexin V/7-amino actinomycin D flow cytometry, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, caspase 3 cleavage, and re-localization of the NRAS:BCL-2 complex from mitochondria to plasma membrane. Phosphoprotein analysis showed restoration of wild-type (WT) AKT or protein kinase B, extracellular signal-regulated kinase 1/2 and mitogen-activated protein kinase patterns in spleen cells after treatment, which showed reduced mitochondrial membrane potential. Exon specific gene expression profiling corroborates the reduction of leukemic cells, with an increase in expression of genes coding for stem cell development and maintenance, myeloid differentiation, and apoptosis. Myelodysplastic features persist underscoring targeting of BCL-2-mediated effects on MDS-AML transformation and survival of leukemic cells.
Asunto(s)
Compuestos de Bifenilo/farmacología , Leucemia Mieloide Aguda/metabolismo , Nitrofenoles/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Sulfonamidas/farmacología , Proteínas ras/metabolismo , Animales , Antígenos Ly/metabolismo , Linaje de la Célula , Membrana Celular/metabolismo , Proliferación Celular , Transformación Celular Neoplásica , Trasplante de Células , Modelos Animales de Enfermedad , Citometría de Flujo , Regulación Leucémica de la Expresión Génica , Sistema de Señalización de MAP Quinasas , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Mitocondrias/metabolismo , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-kit/metabolismo , Células Madre/citologíaAsunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Proteínas con Dominio LIM/metabolismo , Linfoma de Células B Grandes Difuso , Tomografía Computarizada por Tomografía de Emisión de Positrones , Proteínas Proto-Oncogénicas/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Adulto , Anciano , Ciclofosfamida/administración & dosificación , Doxorrubicina/administración & dosificación , Femenino , Humanos , Linfoma de Células B Grandes Difuso/diagnóstico por imagen , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/mortalidad , Masculino , Persona de Mediana Edad , Prednisona/administración & dosificación , Rituximab/administración & dosificación , Vincristina/administración & dosificaciónRESUMEN
Acute promyelocytic leukemia is characterized by a chromosomal translocation involving the retinoic acid receptor alpha gene. To identify co-operating pathways to leukemogenesis, we crossed MRP8-PML/RARA transgenic mice with BXH-2 mice which harbor an endogenous murine leukemia virus that causes acute myeloid leukemia. Approximately half of the leukemias that arose in this cross showed features of acute promyelocytic leukemia. We identified 22 proviral insertion sites in acute promyelocytic-like leukemias and focused our analysis on insertion at Sox4, a HMG box transcription factor. Using a transplant model, co-operation between PML-RARα and Sox4 was confirmed with increased penetrance and reduced latency of disease. Interestingly, karyotypic analysis revealed cytogenetic changes suggesting that the factors combined to initiate but not complete leukemic transformation. The cooperation between these transcription factors is consistent with the paradigm of multiple routes to the disease and reinforces the concept that transcription factor networks are important therapeutic targets in myeloid leukemias.
Asunto(s)
Transformación Celular Neoplásica/genética , Leucemia Mieloide Aguda/genética , Proteínas de Fusión Oncogénica/genética , Factores de Transcripción SOXC/genética , Animales , Médula Ósea/patología , Transformación Celular Neoplásica/metabolismo , Modelos Animales de Enfermedad , Expresión Génica , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Hígado/patología , Ganglios Linfáticos/patología , Ratones , Ratones Transgénicos , Proteínas de Fusión Oncogénica/metabolismo , Unión Proteica , Factores de Transcripción SOXC/metabolismo , Bazo/patologíaRESUMEN
INTRODUCTION: It is postulated that breast cancer stem cells (bCSCs) mediate disease recurrence and drive formation of distant metastases - the principal cause of mortality in breast cancer patients. Therapeutic targeting of bCSCs, however, is hampered by their heterogeneity and resistance to existing therapeutics. In order to identify strategies to selectively remove bCSCs from breast cancers, irrespective of their clinical subtype, we sought an apoptosis mechanism that would target bCSCs yet would not kill normal cells. Suppression of the apoptosis inhibitor cellular FLICE-Like Inhibitory Protein (c-FLIP) partially sensitizes breast cancer cells to the anti-cancer agent Tumour Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL). Here we demonstrate in breast cancer cell lines that bCSCs are exquisitely sensitive to the de-repression of this pro-apoptotic pathway, resulting in a dramatic reduction in experimental metastases and the loss of bCSC self-renewal. METHODS: Suppression c-FLIP was performed by siRNA (FLIPi) in four breast cancer cell lines and by conditional gene-knockout in murine mammary glands. Sensitivity of these cells to TRAIL was determined by complementary cell apoptosis assays, including a novel heterotypic cell assay, while tumour-initiating potential of cancer stem cell subpopulations was determined by mammosphere cultures, aldefluor assay and in vivo transplantation. RESULTS: Genetic suppression of c-FLIP resulted in the partial sensitization of TRAIL-resistant cancer lines to the pro-apoptotic effects of TRAIL, irrespective of their cellular phenotype, yet normal mammary epithelial cells remained refractory to killing. While 10% to 30% of the cancer cell populations remained viable after TRAIL/FLIPi treatment, subsequent mammosphere and aldefluor assays demonstrated that this pro-apoptotic stimulus selectively targeted the functional bCSC pool, eliminating stem cell renewal. This culminated in an 80% reduction in primary tumours and a 98% reduction in metastases following transplantation. The recurrence of residual tumour initiating capacity was consistent with the observation that post-treated adherent cultures re-acquired bCSC-like properties in vitro. Importantly however this recurrent bCSC activity was attenuated following repeated TRAIL/FLIPi treatment. CONCLUSIONS: We describe an apoptotic mechanism that selectively and repeatedly removes bCSC activity from breast cancer cell lines and suggest that a combined TRAIL/FLIPi therapy could prevent metastatic disease progression in a broad range of breast cancer subtypes.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Epitelio/metabolismo , Epitelio/patología , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/metabolismo , Ratones , Ratones Mutantes , ARN Interferente Pequeño , Receptor ErbB-2/metabolismoRESUMEN
Acute myeloid leukemia (AML) is a heterogeneous clonal disorder with a poor clinical outcome. Previously, we showed that overproduction of reactive oxygen species (ROS), arising from constitutive activation of NOX2 oxidase, occurs in >60% of patients with AML and that ROS production promotes proliferation of AML cells. We show here that the process most significantly affected by ROS overproduction is glycolysis. Whole metabolome analysis of 20 human primary AML showed that blasts generating high levels of ROS have increased glucose uptake and correspondingly increased glucose metabolism. In support of this, exogenous ROS increased glucose consumption while inhibition of NOX2 oxidase decreased glucose consumption. Mechanistically, ROS promoted uncoupling protein 2 (UCP2) protein expression and phosphorylation of AMPK, upregulating the expression of a key regulatory glycolytic enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3). Overexpression of PFKFB3 promoted glucose uptake and cell proliferation, whereas downregulation of PFKFB3 strongly suppressed leukemia growth both in vitro and in vivo in the NSG model. These experiments provide direct evidence that oxidase-derived ROS promotes the growth of leukemia cells via the glycolytic regulator PFKFB3. Targeting PFKFB3 may therefore present a new mode of therapy for this disease with a poor outcome. SIGNIFICANCE: These findings show that ROS generated by NOX2 in AML cells promotes glycolysis by activating PFKFB3 and suggest PFKFB3 as a novel therapeutic target in AML.
Asunto(s)
Proliferación Celular , Glucólisis , Leucemia Mieloide Aguda/patología , Fosfofructoquinasa-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Médula Ósea/patología , Línea Celular Tumoral , Femenino , Regulación Leucémica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Leucemia Mieloide Aguda/metabolismo , Masculino , Metabolómica , Ratones , NADPH Oxidasa 2/metabolismo , Fosfofructoquinasa-2/genética , Cultivo Primario de Células , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Hyperactivation of Ras is one of the most common abnormalities in acute myeloid leukemia. In experimental models, Ras inhibits myeloid differentiation, which is characteristic of leukemia; however, the mechanism through which it disrupts hematopoiesis is poorly understood. In multipotent FDCP-mix cells, Ras inhibits terminal neutrophil differentiation, thereby indefinitely extending their proliferative potential. Ras also strongly promotes the sensitivity of these cells to granulocyte-macrophage colony-stimulating factor (GM-CSF). Using this model, we have dissected the signaling elements downstream of Ras to determine their relative contribution to the dysregulation of hematopoiesis. Cells expressing Ras mutants selectively activating Raf (Ras*T35S) or phosphatidylinositol 3-kinase (Ras*Y40C) did not significantly affect differentiation or proliferative capacity, whereas Ras*E37G (which selectively activates RalGEFs) perpetuated proliferation and blocked neutrophil development in a manner similar to that of Ras. Correspondingly, expression of constitutively active versions of these effectors confirmed the overriding importance of Ral guanine nucleotide exchange factors. Cells expressing Ras demonstrated hyperactivation of Ral, which itself was able to exactly mimic the phenotype of Ras, including hypersensitivity to GM-CSF. Conversely, dominant negative Ral promoted spontaneous neutrophil development. Ral, in turn, appears to influence differentiation through multiple effectors. These data show, for the first time, the importance of Ral in regulating differentiation and self-renewal in hematopoietic cells.
Asunto(s)
Diferenciación Celular , Genes ras , Leucemia Mieloide/fisiopatología , Factor de Intercambio de Guanina Nucleótido ral/fisiología , Proteínas ras/fisiología , Animales , Western Blotting , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Proliferación Celular , Células Clonales , Relación Dosis-Respuesta a Droga , Activación Enzimática , Regulación Leucémica de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Leucemia Mieloide/genética , Ratones , Mutación , Neutrófilos/citología , Neutrófilos/metabolismo , Retroviridae/genética , Factor de Intercambio de Guanina Nucleótido ral/genéticaRESUMEN
Recent studies in breast cancer cell lines have shown that oncostatin M (OSM) not only inhibits proliferation but also promotes cell detachment and enhances cell motility. In this study, we have looked at the role of OSM signaling in nontransformed mouse mammary epithelial cells in vitro using the KIM-2 mammary epithelial cell line and in vivo using OSM receptor (OSMR)-deficient mice. OSM and its receptor were up-regulated approximately 2 d after the onset of postlactational mammary regression, in response to leukemia inhibitory factor (LIF)-induced signal transducer and activator of transcription-3 (STAT3). This resulted in sustained STAT3 activity, increased epithelial apoptosis, and enhanced clearance of epithelial structures during the remodeling phase of mammary involution. Concurrently, OSM signaling precipitated the dephosphorylation of STAT5 and repressed expression of the milk protein genes beta-casein and whey acidic protein (WAP). Similarly, during pregnancy, OSM signaling suppressed beta-casein and WAP gene expression. In vitro, OSM but not LIF persistently down-regulated phosphorylated (p)-STAT5, even in the continued presence of prolactin. OSM also promoted the expression of metalloproteinases MMP3, MMP12, and MMP14, which, in vitro, were responsible for OSM-specific apoptosis. Thus, the sequential activation of IL-6-related cytokines during mammary involution culminates in an OSM-dependent repression of epithelial-specific gene expression and the potentiation of epithelial cell extinction mediated, at least in part, by the reciprocal regulation of p-STAT5 and p-STAT3.
Asunto(s)
Apoptosis/genética , Diferenciación Celular/genética , Células Epiteliales/fisiología , Glándulas Mamarias Animales/fisiología , Oncostatina M/fisiología , Animales , Muerte Celular/genética , Células Cultivadas , Células Epiteliales/metabolismo , Femenino , Regulación de la Expresión Génica , Lactancia/genética , Lactancia/metabolismo , Glándulas Mamarias Animales/metabolismo , Ratones , Ratones Noqueados , Leche/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Oncostatina M/metabolismo , Subunidad beta del Receptor de Oncostatina M/genética , Subunidad beta del Receptor de Oncostatina M/metabolismo , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Myelodysplastic syndromes (MDS) comprise a heterogeneous group of disorders characterized by ineffective hematopoiesis, with an increased propensity to develop acute myelogenous leukemia (AML). The molecular basis for MDS progression is unknown, but a key element in MDS disease progression is loss of chromosomal material (genomic instability). Using our two-step mouse model for myeloid leukemic disease progression involving overexpression of human mutant NRAS and BCL2 genes, we show that there is a stepwise increase in the frequency of DNA damage leading to an increased frequency of error-prone repair of double-strand breaks (DSB) by nonhomologous end-joining. There is a concomitant increase in reactive oxygen species (ROS) in these transgenic mice with disease progression. Importantly, RAC1, an essential component of the ROS-producing NADPH oxidase, is downstream of RAS, and we show that ROS production in NRAS/BCL2 mice is in part dependent on RAC1 activity. DNA damage and error-prone repair can be decreased or reversed in vivo by N-acetyl cysteine antioxidant treatment. Our data link gene abnormalities to constitutive DNA damage and increased DSB repair errors in vivo and provide a mechanism for an increase in the error rate of DNA repair with MDS disease progression. These data suggest treatment strategies that target RAS/RAC pathways and ROS production in human MDS/AML.
Asunto(s)
Daño del ADN , Reparación del ADN , Inestabilidad Genómica , Leucemia Mieloide/genética , Especies Reactivas de Oxígeno/metabolismo , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Genes bcl-2 , Genes ras , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Ratones , Ratones TransgénicosRESUMEN
Histone deacetylase inhibitors (HDI) increase gene expression through induction of histone acetylation. However, it remains unclear whether increases in specific gene expression events determine the apoptotic response following HDI administration. Herein, we show that a variety of HDI trigger in hematopoietic cells not only widespread histone acetylation and DNA damage responses but also actual DNA damage, which is significantly increased in leukemic cells compared with normal cells. Thus, increase in H2AX and ataxia telangiectasia mutated (ATM) phosphorylation, early markers of DNA damage, occurs rapidly following HDI administration. Activation of the DNA damage and repair response following HDI treatment is further emphasized by localizing DNA repair proteins to regions of DNA damage. These events are followed by subsequent apoptosis of neoplastic cells but not normal cells. Our data indicate that induction of apoptosis by HDI may result predominantly through accumulation of excessive DNA damage in leukemia cells, leading to activation of apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Inhibidores de Histona Desacetilasas , Animales , Apoptosis/efectos de la radiación , Proteínas de la Ataxia Telangiectasia Mutada , Butiratos/farmacología , Proteínas de Ciclo Celular/metabolismo , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Daño del ADN/efectos de la radiación , Proteínas de Unión al ADN/metabolismo , Rayos gamma , Células HL-60 , Histonas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Células K562 , Ratones , Ratones Transgénicos , Especificidad de Órganos/efectos de los fármacos , Péptidos Cíclicos/farmacología , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , Estaurosporina/farmacología , Transfección , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Addressing the global burden of cancer, understanding its diverse biology, and promoting appropriate prevention and treatment strategies around the world has become a priority for the United Nations and International Atomic Energy Agency (IAEA), the WHO, and International Agency for Research on Cancer (IARC). The IAEA sponsored an international prospective cohort study to better understand biology, treatment response, and outcomes of diffuse large B-cell lymphoma (DLBCL) in low and middle-income countries across five UN-defined geographical regions. We report an analysis of biological variation in DLBCL across seven ethnic and environmentally diverse populations. In this cohort of 136 patients treated to a common protocol, we demonstrate significant biological differences between countries, characterized by a validated prognostic gene expression score (p < .0001), but International Prognostic Index (IPI)-adjusted survivals in all participating countries were similar. We conclude that DLBCL treatment outcomes in these populations can be benchmarked to international standards, despite biological heterogeneity.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/epidemiología , Adulto , Anciano , Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Biomarcadores de Tumor , Ciclofosfamida/uso terapéutico , Doxorrubicina/uso terapéutico , Femenino , Salud Global , Humanos , Estimación de Kaplan-Meier , Linfoma de Células B Grandes Difuso/diagnóstico , Linfoma de Células B Grandes Difuso/genética , Masculino , Persona de Mediana Edad , Vigilancia de la Población , Prednisona/uso terapéutico , Pronóstico , Rituximab , Resultado del Tratamiento , Vincristina/uso terapéuticoRESUMEN
BACKGROUND: In spite of the recent discovery of genetic mutations in most myelodysplasic (MDS) patients, the pathophysiology of these disorders still remains poorly understood, and only few in vivo models are available to help unravel the disease. METHODS: We performed global specific gene expression profiling and functional pathway analysis in purified Sca1+ cells of two MDS transgenic mouse models that mimic human high-risk MDS (HR-MDS) and acute myeloid leukemia (AML) post MDS, with NRASD12 and BCL2 transgenes under the control of different promoters MRP8NRASD12/tethBCL-2 or MRP8[NRASD12/hBCL-2], respectively. RESULTS: Analysis of dysregulated genes that were unique to the diseased HR-MDS and AML post MDS mice and not their founder mice pointed first to pathways that had previously been reported in MDS patients, including DNA replication/damage/repair, cell cycle, apoptosis, immune responses, and canonical Wnt pathways, further validating these models at the gene expression level. Interestingly, pathways not previously reported in MDS were discovered. These included dysregulated genes of noncanonical Wnt pathways and energy and lipid metabolisms. These dysregulated genes were not only confirmed in a different independent set of BM and spleen Sca1+ cells from the MDS mice but also in MDS CD34+ BM patient samples. CONCLUSIONS: These two MDS models may thus provide useful preclinical models to target pathways previously identified in MDS patients and to unravel novel pathways highlighted by this study.
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Perfilación de la Expresión Génica/métodos , Leucemia Mieloide/genética , Síndromes Mielodisplásicos/genética , Transducción de Señal/genética , Enfermedad Aguda , Animales , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Leucemia Mieloide/patología , Ratones , Ratones Transgénicos , Síndromes Mielodisplásicos/patología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de RiesgoRESUMEN
As a part of an international study on the molecular analysis of Diffuse Large B-cell Lymphoma (DLBCL), a robust protocol for gene expression analysis from RNA extraction to qRT-PCR using Formalin Fixed Paraffin Embedded tissues was developed. Here a study was conducted to define a strategy to validate the previously reported 6-gene (LMO2, BCL6, FN1, CCND2, SCYA3 and BCL2) model as predictor of prognosis in DLBCL. To avoid variation, all samples were tested in a single centre and single platform. This study comprised 8 countries (Brazil, Chile, Hungary, India, Philippines, S. Korea, Thailand and Turkey). Using the Kaplan-Meier and log rank test on patients (n=162) and two mortality risk groups (with those above and below the mean representing high and low risk groups) confirmed that the 6-gene predictor score correlates significantly with overall survival (OS, p<0.01) but not with event free survival (EFS, p=0.18). Adding the International Prognostic Index (IPI) shows that the 6-gene predictor score correlates significantly with high IPI scores for OS (p<0.05), whereas those with low IPI scores show a trend not reaching significance (p=0.08). This study defined an effective and economical qRT-PCR strategy and validated the 6-gene score as a predictor of OS in an international setting.
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Biomarcadores de Tumor/genética , Fijadores/química , Formaldehído/química , Perfilación de la Expresión Génica/métodos , Linfoma de Células B Grandes Difuso/genética , Adhesión en Parafina , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fijación del Tejido/métodos , Transcriptoma , Anciano , Asia , Biopsia , Supervivencia sin Enfermedad , Europa (Continente) , Femenino , Perfilación de la Expresión Génica/normas , Predisposición Genética a la Enfermedad , Humanos , Estimación de Kaplan-Meier , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/mortalidad , Linfoma de Células B Grandes Difuso/patología , Masculino , Persona de Mediana Edad , Fenotipo , Valor Predictivo de las Pruebas , Modelos de Riesgos Proporcionales , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , América del Sur , Factores de TiempoRESUMEN
We have previously shown that a specific promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA) DNA vaccine combined with all-trans retinoic acid (ATRA) increases the number of long term survivors with enhanced immune responses in a mouse model of acute promyelocytic leukemia (APL). This study reports the efficacy of a non-specific DNA vaccine, pVAX14Flipper (pVAX14), in both APL and high risk myelodysplastic syndrome (HR-MDS) models. PVAX14 is comprised of novel immunogenic DNA sequences inserted into the pVAX1 therapeutic plasmid. APL mice treated with pVAX14 combined with ATRA had increased survival comparable to that obtained with a specific PML-RARA vaccine. Moreover, the survival advantage correlated with decreased PML-RARA transcript levels and increase in anti-RARA antibody production. In HR-MDS mice, pVAX14 significantly improved survival and reduced biomarkers of leukemic transformation such as phosphorylated mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1. In both preclinical models, pVAX14 vaccine significantly increased interferon gamma (IFNγ) production, memory T-cells (memT), reduced the number of colony forming units (CFU) and increased expression of the adapter molecule signalling to NF-κB, MyD88. These results demonstrate the adjuvant properties of pVAX14 providing thus new approaches to improve clinical outcome in two different models of myeloid malignancies, which may have potential for a broader applicability in other cancers.
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Adyuvantes Inmunológicos/farmacología , Vacunas contra el Cáncer/farmacología , Leucemia Promielocítica Aguda/tratamiento farmacológico , Síndromes Mielodisplásicos/tratamiento farmacológico , Neoplasias Experimentales/tratamiento farmacológico , Tretinoina/farmacología , Vacunas de ADN/farmacología , Animales , Anticuerpos/sangre , Secuencia de Bases , Vacunas contra el Cáncer/inmunología , Regulación Neoplásica de la Expresión Génica , Genes ras , Memoria Inmunológica/efectos de los fármacos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/inmunología , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patología , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Ratones Transgénicos , Datos de Secuencia Molecular , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/inmunología , Síndromes Mielodisplásicos/metabolismo , Síndromes Mielodisplásicos/patología , Neoplasias Experimentales/genética , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Transducción de Señal/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Factores de Tiempo , Carga Tumoral/efectos de los fármacos , Vacunación , Vacunas de ADN/inmunologíaRESUMEN
We have previously demonstrated that two prognostic features of myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML), mutant NRAS and over-expressing BCL-2, cooperate physically and functionally in vivo. Screening of MDS patient bone marrow (BM) identified NRAS:BCL-2 co-localization in 64% cases, correlating with percentage BM blasts, apoptotic features and disease status (p<0.0001). Localization of the complex at the plasma membrane or the mitochondria correlated with disease and apoptosis features in MDS patients, whilst caspase-9 mediated mechanism was elucidated in vivo and in vitro. The intensity and localization of the RAS:BCL-2 complex merits further evaluation as a novel biomarker of MDS.
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Apoptosis , Síndromes Mielodisplásicos/metabolismo , Síndromes Mielodisplásicos/patología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas ras/metabolismo , Animales , Apoptosis/genética , Membrana Celular/metabolismo , Progresión de la Enfermedad , Genes ras , Humanos , Ratones , Ratones Transgénicos , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Síndromes Mielodisplásicos/genética , Unión Proteica , Proteínas Proto-Oncogénicas c-bcl-2/genética , Distribución Tisular/fisiologíaRESUMEN
It is well established that lysosomes play an active role during the execution of cell death. A range of stimuli can lead to lysosomal membrane permeabilization (LMP), thus inducing programmed cell death without involvement of the classical apoptotic programme. However, these lysosomal pathways of cell death have mostly been described in vitro or under pathological conditions. Here we show that the physiological process of post-lactational regression of the mammary gland is accomplished through a non-classical, lysosomal-mediated pathway of cell death. We found that, during involution, lysosomes in the mammary epithelium undergo widespread LMP. Furthermore, although cell death through LMP is independent of executioner caspases 3, 6 and 7, it requires Stat3, which upregulates the expression of lysosomal proteases cathepsin B and L, while downregulating their endogenous inhibitor Spi2A (ref. 8). Our findings report a previously unknown, Stat3-regulated lysosomal-mediated pathway of cell death under physiological circumstances. We anticipate that these findings will be of major importance in the design of treatments for cancers such as breast, colon and liver, where cathepsins and Stat3 are commonly overexpressed and/or hyperactivated respectively.