RESUMEN
Dendritic cells (DCs) are composed of multiple lineages of hematopoietic cells and orchestrate immune responses upon detecting the danger and inflammatory signals associated with pathogen and damaged tissues. Under steady-state, DCs are maintained at limited numbers and the functionally quiescent status. While it is known that a fine balance in the DC homeostasis and activation status is also important to prevent autoimmune diseases and hyperinflammation, mechanisms that control DC development and activation under stead-state remain not fully understood. Here we show that DC-specific ablation of CBL and CBL-B (CBL-/-CBL-B-/-) leads to spontaneous liver inflammation and fibrosis and early death of the mice. The mutant mice have a marked expansion of classic CD8α+/CD103+ DCs (cDC1s) in peripheral lymphoid organs and the liver. These DCs exhibit atypical activation phenotypes characterized by an increased production of inflammatory cytokines and chemokines but not the cell surface MHC-II and costimulatory ligands. While the mutant mice also have massive T cell activation, lymphocytes are not required for the disease development. The CBL-/-CBL-B-/- mutation enhances FLT3-mTOR signaling, due to defective FLT3 ubiquitination and degradation. Blockade of FLT3-mTOR signaling normalizes the homeostasis of cDC1s and attenuates liver inflammation. Our result thus reveals a critical role of CBLs in the maintenance of DC homeostasis and immune quiescence. This regulation could be relevant to liver inflammatory diseases and fibrosis in humans.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Células Dendríticas/inmunología , Proteínas Proto-Oncogénicas c-cbl/fisiología , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Presentación de Antígeno , División Celular , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Hepatitis Autoinmune/genética , Hepatitis Autoinmune/inmunología , Homeostasis , Subgrupos Linfocitarios/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mutación Puntual , Proteínas Proto-Oncogénicas c-akt/fisiología , Proteínas Proto-Oncogénicas c-cbl/deficiencia , Proteínas Proto-Oncogénicas c-cbl/genética , Sirolimus/farmacología , Tirosina Quinasa 3 Similar a fms/fisiologíaRESUMEN
PURPOSE: AXL has been shown to play a pivotal role in the selective response of FLT3-ITD acute myeloid leukemia (AML) cells to FLT3 tyrosine kinase inhibitors (TKI), particularly within the bone marrow microenvironment. EXPERIMENTAL DESIGN: Herein, we compared the effect of dual FLT3/AXL-TKI gilteritinib with quizartinib through in vitro models mimicking hematopoietic niche conditions, ex vivo in primary AML blasts, and in vivo with dosing regimens allowing plasma concentration close to those used in clinical trials. RESULTS: We observed that gilteritinib maintained a stronger proapoptotic effect in hypoxia and coculture with bone marrow stromal cells compared with quizartinib, linked to a dose-dependent inhibition of AXL phosphorylation. In vivo, use of the MV4-11 cell line with hematopoietic engraftment demonstrated that gilteritinib was more effective than quizartinib at targeting leukemic cells in bone marrow. Finally, FLT3-ITD AML patient-derived xenografts revealed that this effect was particularly reproducible in FLT3-ITD AML with high allelic ratio in primary and secondary xenograft. Moreover, gilteritinib and quizartinib displayed close toxicity profile on normal murine hematopoiesis, particularly at steady state. CONCLUSIONS: Overall, these findings suggest that gilteritinib as a single agent, compared with quizartinib, is more likely to reach leukemic cells in their protective microenvironment, particularly AML clones highly dependent on FLT3-ITD signaling.
Asunto(s)
Compuestos de Anilina/farmacología , Compuestos de Anilina/uso terapéutico , Benzotiazoles/farmacología , Benzotiazoles/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Compuestos de Fenilurea/farmacología , Compuestos de Fenilurea/uso terapéutico , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Pirazinas/farmacología , Pirazinas/uso terapéutico , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/fisiología , Línea Celular Tumoral , Hematopoyesis , Humanos , Tirosina Quinasa del Receptor AxlRESUMEN
The presence of FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) is one of the most frequent mutations in acute myeloid leukemia (AML) and is associated with an unfavorable prognosis. FLT3 inhibitors, such as midostaurin, are used clinically but fail to entirely eradicate FLT3-ITD + AML. This study introduces a new perspective and highlights the impact of RAC1-dependent actin cytoskeleton remodeling on resistance to midostaurin in AML. RAC1 hyperactivation leads resistance via hyperphosphorylation of the positive regulator of actin polymerization N-WASP and antiapoptotic BCL-2. RAC1/N-WASP, through ARP2/3 complex activation, increases the number of actin filaments, cell stiffness and adhesion forces to mesenchymal stromal cells (MSCs) being identified as a biomarker of resistance. Midostaurin resistance can be overcome by a combination of midostaruin, the BCL-2 inhibitor venetoclax and the RAC1 inhibitor Eht1864 in midostaurin-resistant AML cell lines and primary samples, providing the first evidence of a potential new treatment approach to eradicate FLT3-ITD + AML.
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Citoesqueleto de Actina/fisiología , Antineoplásicos/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Mutación , Estaurosporina/análogos & derivados , Tirosina Quinasa 3 Similar a fms/genética , Citoesqueleto de Actina/química , Apoptosis/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Pironas/farmacología , Quinolinas/farmacología , Estaurosporina/farmacología , Sulfonamidas/farmacología , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/fisiología , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Proteína de Unión al GTP rac1/fisiologíaRESUMEN
Osteosarcoma is the most common malignant bone tumor in adolescence and childhood. Metastatic osteosarcoma has a poor prognosis with an overall 5-year survival rate of approximately 20%. TAS-115 is a novel multiple receptor tyrosine kinase inhibitor that is currently undergoing clinical trials. Using the mouse highly lung-metastatic osteosarcoma cell line, LM8, we showed that TAS-115 suppressed the growth of subcutaneous grafted tumor and lung metastasis of osteosarcoma at least partially through the inhibition of platelet-derived growth factor receptor alpha, AXL, and Fms-like tyrosine kinase 3 phosphorylation. We also show that these signaling pathways are activated in various human osteosarcoma cell lines and are involved in proliferation. Our results suggest that TAS-115 may have potential for development into a novel treatment for metastatic osteosarcoma.
Asunto(s)
Osteosarcoma/metabolismo , Quinolinas/farmacología , Tiourea/análogos & derivados , Animales , Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Neoplasias Pulmonares , Ratones , Ratones Endogámicos C3H , Osteosarcoma/genética , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/metabolismo , Quinolinas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/fisiología , Transducción de Señal/efectos de los fármacos , Tiourea/metabolismo , Tiourea/farmacología , Tirosina Quinasa 3 Similar a fms/metabolismo , Tirosina Quinasa 3 Similar a fms/fisiología , Tirosina Quinasa del Receptor AxlRESUMEN
This work sought to confirm the human-like expression of exhaustion and senescence markers in a mouse model with a humanized immune system (HIS): the Balb/c Rag2KO IL2rgcKO SirpαNOD Flk2KO HLA-A2HHD (BRGSF-A2) mouse reconstituted with human CD34+ cord blood cells. With regard to senescence markers, the percentage of CD57+ T cells was higher in the bone marrow (BM) than in the spleen or blood. The same was true for KLRG1+ hCD8+ T cells. With regard to exhaustion markers, the percentage of programmed death 1 (PD-1+ ) T cells was higher in the BM than in the spleen or blood; the same was true for TIGIT+ hCD4+ cells. These tissue-specific differences were related to both higher proportions of memory T cells in BM and intrinsic differences in expression within the memory fraction. In blood samples from HIS mice and healthy human donors (HDs), we found that the percentage of KLRG1+ cells among hCD8+ T cells was lower in HIS compared to HDs. The opposite was true for CD4+ T cells. Unexpectedly, a high frequency of KLRG1+ cells was observed among naive T cells in HIS mice. CD57 expression on T cells was similar in blood samples from HIS mice and HDs. Likewise, PD-1 expression was similar in the two systems, although a relatively low proportion of HIS hCD4+ T cells expressed TIGIT. The BRGSF-A2 HIS mouse's exhaustion and senescence profile was tissue specific and relatively human like; hence, this mouse might be a valuable tool for determining the preclinical efficacy of immunotherapies.
Asunto(s)
Biomarcadores/análisis , Senescencia Celular , Proteínas de Unión al ADN/fisiología , Antígeno HLA-A2/fisiología , Subunidad gamma Común de Receptores de Interleucina/fisiología , Receptores Inmunológicos/fisiología , Linfocitos T/inmunología , Tirosina Quinasa 3 Similar a fms/fisiología , Adulto , Anciano , Animales , Femenino , Voluntarios Sanos , Humanos , Leucocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones Noqueados , Persona de Mediana Edad , Receptores Inmunológicos/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismoRESUMEN
FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that is expressed almost exclusively in the hematopoietic compartment. Its ligand, FLT3 ligand (FL), induces dimerization and activation of its intrinsic tyrosine kinase activity. Activation of FLT3 leads to its autophosphorylation and initiation of several signal transduction cascades. Signaling is initiated by the recruitment of signal transduction molecules to activated FLT3 through binding to specific phosphorylated tyrosine residues in the intracellular region of FLT3. Activation of FLT3 mediates cell survival, cell proliferation, and differentiation of hematopoietic progenitor cells. It acts in synergy with several other cytokines to promote its biological effects. Deregulated FLT3 activity has been implicated in several diseases, most prominently in acute myeloid leukemia where around one-third of patients carry an activating mutant of FLT3 which drives the disease and is correlated with poor prognosis. Overactivity of FLT3 has also been implicated in autoimmune diseases, such as rheumatoid arthritis. The observation that gain-of-function mutations of FLT3 can promote leukemogenesis has stimulated the development of inhibitors that target this receptor. Many of these are in clinical trials, and some have been approved for clinical use. However, problems with acquired resistance to these inhibitors are common and, furthermore, only a fraction of patients respond to these selective treatments. This review provides a summary of our current knowledge regarding structural and functional aspects of FLT3 signaling, both under normal and pathological conditions, and discusses challenges for the future regarding the use of targeted inhibition of these pathways for the treatment of patients.
Asunto(s)
Tirosina Quinasa 3 Similar a fms/fisiología , Animales , Antineoplásicos/farmacología , Células Madre Hematopoyéticas/fisiología , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Transducción de Señal , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/genéticaAsunto(s)
Biomarcadores de Tumor/genética , Epigénesis Genética , Factor de Transcripción GATA1/genética , Leucemia Mieloide Aguda/patología , Mutación , Proteínas Nucleares/genética , Tirosina Quinasa 3 Similar a fms/genética , Animales , Femenino , Factor de Transcripción GATA1/fisiología , Regulación Neoplásica de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/etiología , Leucemia Mieloide Aguda/genética , Ratones , Ratones Noqueados , Nucleofosmina , Pronóstico , Secuencias Repetidas en Tándem , Tirosina Quinasa 3 Similar a fms/fisiologíaRESUMEN
Neuron damage contributes to ischemic brain injury. Although FMS-like tyrosine kinase-3 (FLT3) plays a critical role in neuron survival, its function and molecular mechanism in cerebral ischemia/reperfusion injury is unclear. In the present study, we exposed SH-SY5Y cells to oxygen and glucose deprivation/reoxygenation (OGD/R) to mimic ischemia/reperfusion injury. We found that FLT3 and MAPK14/p38α expression increased in OGD/R-treated cells. FLT3 silence significantly increased OGD/R-induced SH-SY5Y cell survival, inhibited reactive oxygen species production. Also, we observed that FLT3 silence suppressed OGD/R-induced SH-SY5Y cell apoptosis, apoptosis related protein Bax level and caspase-3 activity was decreased and Bcl-2 expression was increased in FLT3 silence SH-SY5Y cell treated with OGD/R. Furthermore, FLT3 depletion induced MAPK14/p38α inhibition in SH-SY5Y cultures after OGD/R exposure. These findings suggest that MAPK14/p38α overexpression reverses the action of FLT3 silence in OGD/R-induced SH-SY5Y cells. They also provide the first evidence that FLT3 silence has a neuroprotective role in OGD/R-induced SH-SY5Y cell damage. These data provide insight about potential neuroprotective molecular for ischemia/reperfusion injury.
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Isquemia Encefálica/metabolismo , Daño por Reperfusión/metabolismo , Tirosina Quinasa 3 Similar a fms/metabolismo , Apoptosis , Caspasa 3 , Hipoxia de la Célula/genética , Línea Celular , Línea Celular Tumoral , Supervivencia Celular , Glucosa/metabolismo , Humanos , Proteína Quinasa 14 Activada por Mitógenos , Neuronas , Fármacos Neuroprotectores , Oxígeno/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Especies Reactivas de Oxígeno , Proteína X Asociada a bcl-2 , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/fisiologíaAsunto(s)
Autorrenovación de las Células , Leucemia Experimental/etiología , Células Madre Neoplásicas/patología , Proteína p53 Supresora de Tumor/fisiología , Tirosina Quinasa 3 Similar a fms/fisiología , Animales , Proliferación Celular , Leucemia Experimental/patología , Ratones , Ratones Noqueados , Células Madre Neoplásicas/metabolismo , Secuencias Repetidas en TándemRESUMEN
Fms-like tyrosine kinase 3 (FLT3) is a type III kinase that is highly expressed in seminal plasma of infertile men. FLT3 activation can be blocked by inhibition of its phosphorylation using the nontoxic and selective inhibitor, quizartinib. We investigated the function of FLT3 and the corresponding effects of quizartinib in mouse spermatozoa. Spermatozoa were treated with different concentrations (0.1, 1, 10, 20, and 30⯵M) of quizartinib for 90â¯minâ¯at 37⯰C in 5% CO2 in air. FLT3 was detected in capacitated and non-capacitated spermatozoa. While the level of FLT3 was unaffected, the levels of phospho-FLT3 were significantly altered in spermatozoa by quizartinib. Exposure of spermatozoa to higher concentrations of quizartinib significantly altered sperm viability, motility, motion kinematics, levels of intracellular ATP, and capacitation status. Fertilization and early embryonic development were suppressed by quizartinib. This may have occurred as a consequence of decreased protein kinase A (PKA) activity and tyrosine phosphorylation. The inhibition of FLT3 by quizartinib may affect the fertilization and embryonic development by reducing tyrosine phosphorylation through a PKA-dependent pathway. Our data implicate FLT3 as a biomarker for diagnosis and prognosis of male fertility. In addition, quizartinib has potential for development as a new contraceptive agent.
Asunto(s)
Infertilidad Masculina/genética , Tirosina Quinasa 3 Similar a fms/fisiología , Adenosina Trifosfato/metabolismo , Animales , Benzotiazoles/farmacología , Biomarcadores/metabolismo , Infertilidad Masculina/metabolismo , Masculino , Ratones , Compuestos de Fenilurea/farmacología , Fosforilación/efectos de los fármacos , Análisis de Semen , Capacitación Espermática/efectos de los fármacos , Capacitación Espermática/genética , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Espermatozoides/fisiología , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
Genetic rearrangements involving FLT3 are rare and only recently have been detected in myeloid/lymphoid neoplasms associated with eosinophilia (MLN-eos) and chronic myeloproliferative disorders. Here we report two cases with FLT3 fusions in patients demonstrating mixed features of myelodysplastic/myeloproliferative neoplasms. In the first case, FLT3 was fused with a new fusion partner MYO18A in a patient with marrow features most consistent with atypical chronic myeloid leukemia; the second case involving ETV6-FLT3 fusion was observed in a case with bone marrow features most consistent with chronic myelomonocytic leukemia. Notably, we observed that samples from both patients demonstrated FLT3 inhibitor (quizartinib and sorafenib) sensitivity in ex vivo drug screening assay.
Asunto(s)
Leucemia Mieloide/genética , Enfermedades Mielodisplásicas-Mieloproliferativas/genética , Tirosina Quinasa 3 Similar a fms/genética , Benzotiazoles/farmacología , Médula Ósea/patología , Eosinofilia/genética , Humanos , Leucemia Mieloide/fisiopatología , Leucemia Mielomonocítica Crónica/genética , Linfoma/genética , Masculino , Persona de Mediana Edad , Miosinas/genética , Compuestos de Fenilurea/farmacología , Proteínas Proto-Oncogénicas c-ets/genética , Recombinación Genética/genética , Proteínas Represoras/genética , Sorafenib/farmacología , Tirosina Quinasa 3 Similar a fms/fisiología , Proteína ETS de Variante de Translocación 6RESUMEN
OBJECTIVE: To explore the clinical value of PKC412 (midostaurin) in treatment of AML patients with FLT3-. METHODS: The bone marrow or peripheral blood were collected and heparinized from 21 newly diagnosed FLT3- AML patients, then the mononuclear cells from bone marrow or peripheral blood were isolated by density-gradient method. The sensitivity of leukemia cells to PKC412 of 8 concentration in vitro was detected by ATP-bioluminescence-tumor chemosensitivity assay (ATP-TCA), and the relationship among sensitivity results in vitro, risk stratification and therapeutic efficacy was analyzed. RESULTS: The leukemia cells of 21 patients with AML displayed different sensitivities to PKC412 in vitro. The rate of sensitivity in vitro was 42.9%, and sensitive concentration in vitro were between 1 µmol/L and 5 µmol/L. There was no significant relationship between risk stratification and sensitivity results of PKC412 in vitro. There was also no significant relationship between clinical efficacy and sensitivity results of PKC412 in vitro. The survival of patients in low-risk and intermediate-risk groups was better than that of patients in high-risk groups (P=0.015). CONCLUSION: PKC412 can be one of the effective therapeutic method for AML patients without FLT3 mutation. The sensitivity of leukemia cells to PKC412 may become a prognostic marker for evaluating clinical efficacy of PKC412, which is independent of other factors.
Asunto(s)
Leucemia Mieloide Aguda/tratamiento farmacológico , Estaurosporina/análogos & derivados , Tirosina Quinasa 3 Similar a fms/fisiología , Línea Celular Tumoral , Humanos , Mutación , Estaurosporina/farmacologíaRESUMEN
Acute myeloid leukemia (AML) is induced by the cooperative action of deregulated genes that perturb self-renewal, proliferation, and differentiation. Internal tandem duplications (ITDs) in the FLT3 receptor tyrosine kinase are common mutations in AML, confer poor prognosis, and stimulate myeloproliferation. AML patient samples with FLT3-ITD express high levels of RUNX1, a transcription factor with known tumor-suppressor function. In this study, to understand this paradox, we investigated the impact of RUNX1 and FLT3-ITD coexpression. FLT3-ITD directly impacts on RUNX1 activity, whereby up-regulated and phosphorylated RUNX1 cooperates with FLT3-ITD to induce AML. Inactivating RUNX1 in tumors releases the differentiation block and down-regulates genes controlling ribosome biogenesis. We identified Hhex as a direct target of RUNX1 and FLT3-ITD stimulation and confirmed high HHEX expression in FLT3-ITD AMLs. HHEX could replace RUNX1 in cooperating with FLT3-ITD to induce AML. These results establish and elucidate the unanticipated oncogenic function of RUNX1 in AML. We predict that blocking RUNX1 activity will greatly enhance current therapeutic approaches using FLT3 inhibitors.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/fisiología , Leucemia Mieloide Aguda/etiología , Tirosina Quinasa 3 Similar a fms/fisiología , Animales , Células Cultivadas , Subunidad alfa 2 del Factor de Unión al Sitio Principal/antagonistas & inhibidores , Proteínas de Homeodominio/genética , Humanos , Ratones , Procesamiento Proteico-Postraduccional , Factores de Transcripción/genéticaRESUMEN
Internal tandem duplication (ITD) mutation in Fms-like tyrosine kinase 3 gene (FLT3/ITD) represents an unfavorable genetic change in acute myeloid leukemia (AML) and is associated with poor prognosis. Metabolic alterations have been involved in tumor progression and attracted interest as a target for therapeutic intervention. However, few studies analyzed the adaptations of cellular metabolism in the context of FLT3/ITD mutation. Here, we report that FLT3/ITD causes a significant increase in aerobic glycolysis through AKT-mediated upregulation of mitochondrial hexokinase (HK2), and renders the leukemia cells highly dependent on glycolysis and sensitive to pharmacological inhibition of glycolytic activity. Inhibition of glycolysis preferentially causes severe ATP depletion and massive cell death in FLT3/ITD leukemia cells. Glycolytic inhibitors significantly enhances the cytotoxicity induced by FLT3 tyrosine kinase inhibitor sorafenib. Importantly, such combination provides substantial therapeutic benefit in a murine model bearing FLT3/ITD leukemia. Our study suggests that FLT3/ITD mutation promotes Warburg effect, and such metabolic alteration can be exploited to develop effective therapeutic strategy for treatment of AML with FLT3/ITD mutation via metabolic intervention.
Asunto(s)
Glucólisis/genética , Repeticiones de Microsatélite , Terapia Molecular Dirigida , Proteínas de Neoplasias/genética , Tirosina Quinasa 3 Similar a fms/genética , Adenosina Trifosfato/metabolismo , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular , Transformación Celular Neoplásica , Desoxiglucosa/farmacología , Glucólisis/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Hexoquinasa/biosíntesis , Hexoquinasa/genética , Humanos , Hidrocarburos Bromados/farmacología , Leucemia Experimental/tratamiento farmacológico , Ratones , Ratones Endogámicos BALB C , Mitocondrias/enzimología , Proteínas de Neoplasias/fisiología , Niacinamida/análogos & derivados , Niacinamida/farmacología , Niacinamida/uso terapéutico , Compuestos de Fenilurea/farmacología , Compuestos de Fenilurea/uso terapéutico , Propionatos/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sorafenib , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/fisiologíaRESUMEN
Current therapy for acute myeloid leukemia (AML) primarily includes high-dose cytotoxic chemotherapy with or without allogeneic stem cell transplantation. Targeting unique cellular metabolism of cancer cells is a potentially less toxic approach. Monotherapy with mitochondrial inhibitors like metformin have met with limited success since escape mechanisms such as increased glycolytic ATP production, especially in hyperglycemia, can overcome the metabolic blockade. As an alternative strategy for metformin therapy, we hypothesized that the combination of 6-benzylthioinosine (6-BT), a broad-spectrum metabolic inhibitor, and metformin could block this drug resistance mechanism. Metformin treatment alone resulted in significant suppression of ROS and mitochondrial respiration with increased glycolysis accompanied by modest cytotoxicity (10-25%). In contrast, 6-BT monotherapy resulted in inhibition of glucose uptake, decreased glycolysis, and decreased ATP with minimal changes in ROS and mitochondrial respiration. The combination of 6-BT with metformin resulted in significant cytotoxicity (60-70%) in monocytic AML cell lines and was associated with inhibition of FLT3-ITD activated STAT5 and reduced c-Myc and GLUT-1 expression. Therefore, although the anti-tumor and metabolic effects of metformin have been limited by the metabolic reprogramming within cells, the novel combination of 6-BT and metformin targets this bypass mechanism resulting in reduced glycolysis, STAT5 inhibition, and increased cell death.
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Muerte Celular/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Metformina/uso terapéutico , Tioinosina/análogos & derivados , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Sinergismo Farmacológico , Sangre Fetal/citología , Glucólisis/efectos de los fármacos , Humanos , Secuencias Invertidas Repetidas , Leucemia Mieloide Aguda/genética , Factor de Transcripción STAT5/antagonistas & inhibidores , Tioinosina/uso terapéutico , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/fisiologíaAsunto(s)
Inhibidores Enzimáticos/farmacología , Leucemia Mieloide Aguda/patología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Factor de Transcripción STAT5/metabolismo , Quinasa Syk/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/fisiología , Secuencia de Aminoácidos , Proliferación Celular , Humanos , Leucemia Mieloide Aguda/metabolismo , Homología de Secuencia de Aminoácido , Tirosina Quinasa 3 Similar a fms/químicaRESUMEN
The novel FMS-like tyrosine kinase 3 (FLT3)-N676K point mutation within the FLT3 kinase domain-1 was recently identified in 6 % of de novo acute myeloid leukemia (AML) patients with inv(16). Because FLT3-N676K was encountered almost exclusively in inv(16) AML, we investigated the transforming potential of FLT3-N676K, the cooperation between FLT3-N676K and core binding factor ß-smooth muscle myosin heavy chain (CBFß-SMMHC) (encoded by the inv(16) chimeric gene CBFB-MYH11) in inducing acute leukemia, and tested the sensitivity of FLT3-N676K-positive leukemic cells to FLT3 inhibitors. Retroviral expression of FLT3-N676K in myeloid 32D cells induced AML in syngeneic C3H/HeJ mice (n = 11/13, median latency 58 days), with a transforming activity similar to FLT3-internal tandem duplication (ITD) (n = 8/8), FLT3-TKD D835Y (n = 8/9), and FLT3-ITD-N676K (n = 9/9) mutations. Three out of 14 (21.4 %) C57BL/6J mice transplanted with FLT3-N676K-transduced primary hematopoietic progenitor cells developed acute leukemia (latency of 68, 77, and 273 days), while no hematological malignancy was observed in the control groups including FLT3-ITD. Moreover, co-expression of FLT3-N676K/CBFß-SMMHC did not promote acute leukemia in three independent experiments (n = 16). In comparison with FLT3-ITD, FLT3-N676K induced much higher activation of FLT3 and tended to trigger stronger phosphorylation of MAPK and AKT. Importantly, leukemic cells carrying the FLT3-N676K mutant in the absence of an ITD mutation were highly sensitive to FLT3 inhibitors AC220 and crenolanib, and crenolanib even retained activity against the AC220-resistant FLT3-ITD-N676K mutant. Taken together, the FLT3-N676K mutant is potent to transform murine hematopoietic stem/progenitor cells in vivo. This is the first report of acute leukemia induced by an activating FLT3 mutation in C57BL/6J mice. Moreover, further experiments investigating molecular mechanisms for leukemogenesis induced by FLT3-N676K mutation and clinical evaluation of FLT3 inhibitors in FLT3-N676K-positive AML seem warranted.
Asunto(s)
Leucemia Experimental/genética , Mutación Missense , Mutación Puntual , Tirosina Quinasa 3 Similar a fms/genética , Sustitución de Aminoácidos , Animales , Antineoplásicos/uso terapéutico , Bencimidazoles/uso terapéutico , Benzotiazoles/uso terapéutico , Trasplante de Médula Ósea , Transformación Celular Neoplásica/genética , Regulación Leucémica de la Expresión Génica , Predisposición Genética a la Enfermedad , Vectores Genéticos , Humanos , Leucemia Experimental/tratamiento farmacológico , Leucemia Experimental/enzimología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Células Madre Neoplásicas/trasplante , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/fisiología , Compuestos de Fenilurea/uso terapéutico , Piperidinas/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Procesamiento Proteico-Postraduccional/genética , Quimera por Radiación , Retroviridae , Secuencias Repetidas en Tándem , Transgenes , Ensayo de Tumor de Célula Madre , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/fisiologíaRESUMEN
Activating mutations of FMS-like tyrosine kinase 3 (FLT3), notably internal tandem duplications (ITDs), are associated with a grave prognosis in acute myeloid leukemia (AML). Transforming FLT3ITD signal transduction causes formation of reactive oxygen species (ROS) and inactivation of the protein-tyrosine phosphatase (PTP) DEP-1/PTPRJ, a negative regulator of FLT3 signaling. Here we addressed the underlying mechanisms and biological consequences. NADPH oxidase 4 (NOX4) messenger RNA and protein expression was found to be elevated in FLT3ITD-positive cells and to depend on FLT3ITD signaling and STAT5-mediated activation of the NOX4 promoter. NOX4 knockdown reduced ROS levels, restored DEP-1 PTP activity and attenuated FLT3ITD-driven transformation. Moreover, Nox4 knockout (Nox4(-/-)) murine hematopoietic progenitor cells were refractory to FLT3ITD-mediated transformation in vitro. Development of a myeloproliferative-like disease (MPD) caused by FLT3ITD-transformed 32D cells in C3H/HeJ mice, and of a leukemia-like disease in mice transplanted with MLL-AF9/ FLT3ITD-transformed murine hematopoietic stem cells were strongly attenuated by NOX4 downregulation. NOX4-targeting compounds were found to counteract proliferation of FLT3ITD-positive AML blasts and MPD development in mice. These findings reveal a previously unrecognized mechanism of oncoprotein-driven PTP oxidation, and suggest that interference with FLT3ITD-STAT5-NOX4-mediated overproduction of ROS and PTP inactivation may have therapeutic potential in a subset of AML.
Asunto(s)
Transformación Celular Neoplásica , Leucemia Mieloide Aguda/patología , NADPH Oxidasas/fisiología , Proteínas Tirosina Fosfatasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Tirosina Quinasa 3 Similar a fms/fisiología , Animales , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , NADPH Oxidasa 4 , NADPH Oxidasas/genética , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/análisis , Secuencias Repetidas en Tándem , Tirosina Quinasa 3 Similar a fms/análisisRESUMEN
OBJECTIVES: Acute myeloid leukemia (AML) cells are highly resistant to therapy. The presumed molecular basis of this resistance is the effect of tumor necrosis factor alpha (TNF-α) and other cytokines on endothelial adhesion molecule expression. The aim of this study was to test the hypothesis that cytokines and soluble adhesion molecules correlate in AML. METHODS: Baseline serum levels of 17 cytokines and 5 soluble adhesion molecules were measured in 53 AML patients using biochip array technology. Age, leukocyte count, secondary AML, CRP, FLT3-ITD and remission were variables. Statistical analysis was performed in R version 3.1.2. RESULTS: VCAM-1 correlated with ICAM-1 (P < 0.0001), E-selectin (P < 0.0001), leukocyte count (P = 0.0005) and TNF-α (P = 0.0035). E-selectin correlated with leukocyte count (P < 0.0001), P-selectin (P = 0.0032) and MCP-1 (P = 0.0119). CRP correlated with IL-6 (P < 0.0001), leukocyte count negatively correlated with IL-7 (P = 0.0318). FLT3-ITD was associated with higher E-selectin (P = 0.0010) and lower IL-7 (P = 0.0252). Secondary AML patients were older. Failure of induction therapy was associated with significantly higher CRP and lower P-selectin. Leukocyte count (P < 0.0001), FLT3-ITD (P = 0.0017) and secondary AML (P = 0.0439) influenced the principal component. CONCLUSIONS: Leukemic cells can modulate the microenvironment. Cytokine, adhesion molecule levels and leukocyte count correlate in AML. Understanding these mechanisms may form the basis of novel therapeutic approaches.