RESUMEN
OBJECTIVE: A team-based disease management approach that considers comorbid conditions, social drivers of health, and clinical guidelines improves diabetes care but can be costly and complex. Developing innovative models of care is crucial to improving diabetes outcomes. The objective of this analysis was to evaluate the efficacy of virtual interdisciplinary diabetes rounds in improving glycemic control. STUDY DESIGN: Retrospective cohort study using observational data from July 2018 to December 2021. METHODS: This study employed difference-in-differences analysis to compare change in hemoglobin A1c (HbA1c) in a group of patients whose providers received advice as part of virtual interdisciplinary rounds and a group of patients whose providers did not receive rounds advice. Patients with diabetes were identified for rounding (1) based on attribution to an accountable care organization along with an upcoming primary care appointment and an HbA1c between 8% and 9% or (2) via provider referral. RESULTS: The rounded group consisted of 481 patients and the comparison group included 1806 patients. There was a 0.3-point reduction in HbA1c (95% CI, 0.1-0.4) associated with rounds overall. In a subanalysis comparing provider adoption of recommendations among those rounded, provider adoption was associated with an HbA1c reduction of 0.5 points (95% CI, 0.1-0.9) at 6 months post rounds, although there was no significant difference in the full year post rounds. CONCLUSIONS: Interdisciplinary rounds can be an effective approach to proactively provide diabetes-focused recommendations. This modality allows for efficient, low-cost, and timely access to an endocrinologist and team to support primary care providers in diabetes management.
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Diabetes Mellitus Tipo 2 , Control Glucémico , Humanos , Hemoglobina Glucada , Estudios Retrospectivos , Diabetes Mellitus Tipo 2/terapia , EndocrinólogosRESUMEN
OBJECTIVE: To investigate healthcare professional staff and students' perception of wearing surgical masks before and after their experience with the COVID-19 pandemic, and to evaluate the impact on mask wearing behaviour in future influenza seasons. DESIGN: Cross-sectional study using anonymous survey. SETTING AND PARTICIPANTS: Healthcare students and staff from a healthcare academic institution in Southern California participated in the mask survey study. Survey results were collected from June to November 2021. A total of 305 respondents responded to the survey, with 173 being healthcare students and 132 being working healthcare staff. OUTCOMES: The study examined respondents' perceptions and hospital mask wearing behaviour before and after their COVID-19 pandemic experience, as well as during previous and future influenza seasons. RESULTS: Two hundred and sixty-four (86.6%) respondents agreed that wearing a surgical mask reduces infection and limits transmission of infectious disease, yet prior to the pandemic, only a small proportion wore a mask in the hospital or during patient care. After experiencing the COVID-19 pandemic, more respondents indicated that they would continue to wear a mask when they are in a hospital in general (n=145, 47.5%), during patient care (n=262, 85.9%), during influenza seasons throughout the hospital (n=205, 67.2%) and during influenza seasons during patient care (n=270, 88.5%). CONCLUSION: The pandemic experience has greatly influenced the health prevention behaviours of healthcare students and staff. After the pandemic, many respondents will continue to practice surgical mask wearing behaviour in the hospital, especially during face-to-face patient care. This demonstrates a significant change in health prevention perceptions among the current and the future generation of healthcare professionals.
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COVID-19 , Gripe Humana , COVID-19/epidemiología , COVID-19/prevención & control , California/epidemiología , Estudios Transversales , Atención a la Salud , Humanos , Gripe Humana/epidemiología , Gripe Humana/prevención & control , Máscaras , Pandemias/prevención & control , Estaciones del Año , EstudiantesRESUMEN
UNLABELLED: We report the safety, biodistribution, and internal radiation dosimetry of a new PET tracer, (18)F-AH111585, a peptide with a high affinity for the alpha(v)beta(3) integrin receptor involved in angiogenesis. METHODS: PET scans of 8 healthy volunteers were acquired at time points up to 4 h after a bolus injection of (18)F-AH111585. (18)F activity in whole blood and plasma and excreted urine were measured up to 4 h after injection. In vivo (18)F activities in up to 12 source regions were determined from quantitative analysis of the images. The cumulated activities subsequently calculated were then used to determine the internal radiation dosimetry, including the effective dose. RESULTS: Injection of (18)F-AH111585 was well tolerated in all subjects, with no serious or drug-related adverse events reported. The main route of (18)F excretion was renal (37%), and the 3 highest initial uptakes were by liver (15%); combined walls of the small, upper large, and lower large intestines (11%); and kidneys (9%). The 3 highest absorbed doses were received by the urinary bladder wall (124 microGy/MBq), kidneys (102 microGy/MBq), and cardiac wall (59 microGy/MBq). The effective dose was 26 microGy/MBq. CONCLUSION: (18)F-AH111585 is a safe PET tracer with a dosimetry profile comparable to other common (18)F PET tracers.
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Azetidinas/farmacocinética , Péptidos/farmacocinética , Tomografía de Emisión de Positrones/métodos , Piridinas/farmacocinética , Radiometría/métodos , Radiofármacos/farmacocinética , Anciano , Arginina/química , Ácido Aspártico/química , Femenino , Glicina/química , Humanos , Masculino , Persona de Mediana Edad , Dosis de Radiación , Distribución TisularRESUMEN
UNLABELLED: The integrin alpha v beta3 receptor is upregulated on tumor cells and endothelium and plays important roles in angiogenesis and metastasis. Arg-Gly-Asp (RGD) peptide ligands have high affinity for these integrins and can be radiolabeled for PET imaging of angiogenesis or tumor development. We have assessed the safety, stability, and tumor distribution kinetics of a novel radiolabeled RGD-based integrin peptide-polymer conjugate, 18F-AH111585, and its feasibility to detect tumors in metastatic breast cancer patients using PET. METHODS: The biodistribution of 18F-AH111585 was assessed in 18 tumor lesions from 7 patients with metastatic breast cancer by PET, and the PET data were compared with CT results. The metabolic stability of 18F-AH111585 was assessed by chromatography of plasma samples. Regions of interest (ROIs) defined over tumor and normal tissues of the PET images were used to determine the kinetics of radioligand binding in tissues. RESULTS: The radiopharmaceutical and PET procedures were well tolerated in all patients. All 18 tumors detected by CT were visible on the 18F-AH111585 PET images, either as distinct increases in uptake compared with the surrounding normal tissue or, in the case of liver metastases, as regions of deficit uptake because of the high background activity in normal liver tissue. 18F-AH111585 was either homogeneously distributed in the tumors or appeared within the tumor rim, consistent with the pattern of viable peripheral tumor and central necrosis often seen in association with angiogenesis. Increased uptake compared with background (P = 0.002) was demonstrated in metastases in lung, pleura, bone, lymph node, and primary tumor. CONCLUSION: 18F-AH111585 designed to bind the alpha v beta3 integrin is safe, metabolically stable, and retained in tumor tissues and detects breast cancer lesions by PET in most anatomic sites.
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Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/metabolismo , Oligopéptidos/metabolismo , Péptidos/farmacocinética , Polietilenglicoles/farmacocinética , Tomografía de Emisión de Positrones/métodos , Adulto , Anciano , Femenino , Humanos , Tasa de Depuración Metabólica , Persona de Mediana Edad , Especificidad de Órganos , Péptidos/efectos adversos , Polietilenglicoles/efectos adversos , Tomografía de Emisión de Positrones/efectos adversos , Radiofármacos/efectos adversos , Radiofármacos/farmacocinética , Distribución TisularRESUMEN
We have developed a useful surrogate assay for monitoring the efficacy of FLT3 inhibition in patients treated with oral FLT3 inhibitors. The plasma inhibitory activity (PIA) for FLT3 correlates with clinical activity in patients treated with CEP-701 and PKC412. Using the PIA assay, along with in vitro phosphorylation and cytotoxicity assays in leukemia cells, we compared PKC412 and its metabolite, CGP52421, with CEP-701. While both drugs could effectively inhibit FLT3 in vitro, CEP-701 was more cytotoxic to primary samples at comparable levels of FLT3 inhibition. PKC412 appears to be more selective than CEP-701 and therefore less effective at inducing cytotoxicity in primary acute myeloid leukemia (AML) samples in vitro. However, the PKC412 metabolite CGP52421 is less selective than its parent compound, PKC412, and is more cytotoxic against primary blast samples at comparable levels of FLT3 inhibition. The plasma inhibitory activity assay represents a useful correlative tool in the development of small-molecule inhibitors. Our application of this assay has revealed that the metabolite CGP52421 may contribute a significant portion of the antileukemia activity observed in patients receiving oral PKC412. Additionally, our results suggest that nonselectivity may constitute an important component of the cytotoxic effect of FLT3 inhibitors in FLT3-mutant AML.
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Leucemia/tratamiento farmacológico , Leucemia/patología , Plasma/química , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Antineoplásicos/sangre , Antineoplásicos/farmacocinética , Carbazoles/sangre , Carbazoles/farmacocinética , Estudios de Casos y Controles , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/sangre , Inhibidores Enzimáticos/farmacocinética , Furanos , Humanos , Indoles/sangre , Indoles/farmacocinética , Concentración 50 Inhibidora , Leucemia/sangre , Farmacocinética , Estaurosporina/análogos & derivados , Estaurosporina/sangre , Estaurosporina/farmacocinéticaRESUMEN
Activating mutations in the FLT3 tyrosine kinase (TK) occur in approximately 35% of patients with acute myeloid leukemia (AML). Therefore, targeting mutated FLT3 is an attractive therapeutic strategy, and early clinical trials testing FLT3 TK inhibitors (TKI) showed measurable clinical responses. Most of these responses were transient; however, in a subset of patients blast recurrence was preceded by an interval of prolonged remission. The etiology of clinical resistance to FLT3-TKI in AML is unclear but is of major significance for the development of future therapeutic strategies. We searched for mechanisms of resistance in 6 patients with AML who had relapses upon PKC412 treatment. In an index AML patient, an algorithm of analyses was applied using clinical material. In vivo and in vitro investigation of primary blasts at relapse revealed persistent TK phosphorylation of FLT3 despite sufficient PKC412 serum levels. Through additional molecular analyses, we identified a single amino acid substitution at position 676 (N676K) within the FLT3 kinase domain as the sole cause of resistance to PKC412 in this patient. Reconstitution experiments expressing the N676K mutant in 32D cells demonstrated that FLT3-ITD-N676K was sufficient to confer an intermediate level of resistance to PKC412 in vitro. These studies point out that a genetically complex malignancy such as AML may retain dependence on a single oncogenic signal.
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Resistencia a Antineoplásicos/genética , Leucemia Mieloide/tratamiento farmacológico , Leucemia Mieloide/genética , Mutación Missense , Estaurosporina/análogos & derivados , Tirosina Quinasa 3 Similar a fms/genética , Enfermedad Aguda , Activación Enzimática/genética , Humanos , Leucemia Mieloide/enzimología , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/genética , Recurrencia , Estaurosporina/farmacología , Estaurosporina/uso terapéuticoRESUMEN
The majority of patients with systemic mast cell disease express the imatinib-resistant Asp816Val (D816V) mutation in the KIT receptor tyrosine kinase. Limited treatment options exist for aggressive systemic mastocytosis (ASM) and mast cell leukemia (MCL). We evaluated whether PKC412, a small-molecule inhibitor of KIT with a different chemical structure from imatinib, may have therapeutic use in advanced SM with the D816V KIT mutation. We treated a patient with MCL (with an associated myelodysplastic syndrome (MDS)/myeloproliferative disorder [MPD]) based on in vitro studies demonstrating that PKC412 could inhibit D816V KIT-transformed Ba/F3 cell growth with a 50% inhibitory concentration (IC50) of 30 nM to 40 nM. The patient exhibited a partial response with significant resolution of liver function abnormalities. In addition, PKC412 treatment resulted in a significant decline in the percentage of peripheral blood mast cells and serum histamine level and was associated with a decrease in KIT phosphorylation and D816V KIT mutation frequency. The patient died after 3 months of therapy due to progression of her MDS/MPD to acute myeloid leukemia (AML). This case indicates that KIT tyrosine kinase inhibition is a feasible approach in SM, but single-agent clinical efficacy may be limited by clonal evolution in the advanced leukemic phase of this disease.
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Ácido Aspártico/genética , Leucemia de Mastocitos/tratamiento farmacológico , Leucemia de Mastocitos/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-kit/genética , Estaurosporina/análogos & derivados , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Humanos , Inmunofenotipificación , Leucemia de Mastocitos/metabolismo , Leucemia de Mastocitos/patología , Persona de Mediana Edad , Mutación/genética , Fosforilación/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Estaurosporina/farmacocinética , Estaurosporina/uso terapéuticoRESUMEN
Fms-like tyrosine kinase 3 (FLT3) receptor mutations as internal tandem duplication (ITD) or within the kinase domain are detected in up to 35% of patients with acute myeloid leukemia (AML). N-benzoyl staurosporine (PKC412), a highly effective inhibitor of mutated FLT3 receptors, has significant antileukemic efficacy in patients with FLT3-mutated AML. Mutation screening of FLT3 exon 20 in AML patients (n = 110) revealed 2 patients with a novel mutation (Y842C) within the highly conserved activation loop of FLT3. FLT3-Y842C-transfected 32D cells showed constitutive FLT3 tyrosine phosphorylation and interleukin 3 (IL-3)-independent growth. Treatment with PKC412 led to inhibition of proliferation and apoptotic cell death. Primary AML blasts bearing FLT3-Y842C mutations showed constitutive FLT3 and signal transducer and activator of transcription 5 (STAT-5) tyrosine phosphorylation. Ex vivo PKC412 treatment of primary blasts resulted in suppression of constitutive FLT3 and STAT-5 activation and apoptotic cell death. Inspection of the FLT3 structure revealed that Y842 is the key residue in regulating the switch from the closed to the open (= active) conformation of the FLT3 activation loop. Overall, our data suggest that mutations at Y842 represent a significant new activating mutation in AML blasts. Since FLT3 tyrosine kinase inhibitors (TKIs) such as PKC412 are currently being investigated in clinical trials in AML, extended sequence analysis of FLT3 may be helpful in defining the spectrum of TKI-sensitive FLT3 mutations in AML.
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Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , Mutación/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Ciclo Celular , Línea Celular , Proteínas de Unión al ADN/metabolismo , Activación Enzimática/genética , Regulación de la Expresión Génica , Humanos , Ratones , Proteínas de la Leche/metabolismo , Modelos Moleculares , Fosfotirosina/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/química , Proteínas Tirosina Quinasas Receptoras/química , Factor de Transcripción STAT5 , Transducción de Señal , Transactivadores/metabolismo , Tirosina/genética , Tirosina/metabolismo , Tirosina Quinasa 3 Similar a fmsRESUMEN
Leukemic cells from 30% of patients with acute myeloid leukemia (AML) have an activating mutation in the FLT3 (fms-like tyrosine kinase) gene, which represents a target for drug therapy. We treated 20 patients, each with mutant FLT3 relapsed/refractory AML or high-grade myelodysplastic syndrome and not believed to be candidates for chemotherapy, with an FLT3 tyrosine kinase inhibitor, PKC412 (N-benzoylstaurosporine), at a dose of 75 mg 3 times daily by mouth. The drug was generally well tolerated, although 2 patients developed fatal pulmonary events of unclear etiology. The peripheral blast count decreased by 50% in 14 patients (70%). Seven patients (35%) experienced a greater than 2-log reduction in peripheral blast count for at least 4 weeks (median response duration, 13 weeks; range, 9-47 weeks); PKC412 reduced bone marrow blast counts by 50% in 6 patients (2 of these to < 5%). FLT3 autophosphorylation was inhibited in most of the Corresponding patients, indicating in vivo target inhibition at the dose schedule used in this study. PKC412 is an oral tyrosine kinase inhibitor with clinical activity in patients with AML whose blasts have an activating mutation of FLT3, suggesting potential use in combination with active agents, such as chemotherapy.
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Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/enzimología , Mutación/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Estaurosporina/análogos & derivados , Estaurosporina/uso terapéutico , Adulto , Anciano , Recuento de Células Sanguíneas , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Femenino , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Masculino , Persona de Mediana Edad , Fosfotirosina/metabolismo , Estaurosporina/efectos adversos , Estaurosporina/sangre , Estaurosporina/farmacocinética , Tirosina Quinasa 3 Similar a fmsRESUMEN
Human stem cell leukemia-lymphoma syndrome usually presents itself as a myeloproliferative disorder (MPD) that evolves to acute myeloid leukemia and/or lymphoma. The syndrome associated with t(8;13)(p11;q12) results in expression of the ZNF198-fibroblast growth factor receptor (FGFR) 1 fusion tyrosine kinase. Current empirically derived cytotoxic chemotherapy is inadequate for treatment of this disease. We hypothesized that small-molecule inhibitors of the ZNF198-FGFR1 fusion would have therapeutic efficacy. We characterized the transforming activity of ZNF198-FGFR1 in hematopoietic cells in vitro and in vivo. Expression of ZNF198-FGFR1 in primary murine hematopoietic cells caused a myeloproliferative syndrome in mice that recapitulated the human MPD phenotype. Transformation in these assays, and activation of the downstream effector molecules PLC-gamma, STAT5, and phosphatidylinositol 3-kinase/AKT, required the proline-rich domains, but not the ZNF domains, of ZNF198. A small-molecule tyrosine kinase inhibitor, PKC412 (N-benzoyl-staurosporine) effectively inhibited ZNF198-FGFR1 tyrosine kinase activity and activation of downstream effector pathways, and inhibited proliferation of ZNF198-FGFR1 transformed Ba/F3 cells. Furthermore, treatment with PKC412 resulted in statistically significant prolongation of survival in the murine model of ZNF198-FGFR1-induced MPD. Based in part on these data, PKC412 was administered to a patient with t(8;13)(p11;q12) and was efficacious in treatment of progressive myeloproliferative disorder with organomegaly. Therefore, PKC412 may be a useful therapy for treatment of human stem cell leukemia-lymphoma syndrome.
Asunto(s)
Trastornos Mieloproliferativos/tratamiento farmacológico , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Estaurosporina/análogos & derivados , Estaurosporina/farmacología , Animales , Línea Celular , Cromosomas Humanos Par 13 , Cromosomas Humanos Par 8 , Modelos Animales de Enfermedad , Femenino , Variación Genética , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/química , Receptores de Factores de Crecimiento de Fibroblastos/genética , Transformación Genética , Translocación Genética , Dedos de ZincRESUMEN
PURPOSE: Mutant FLT-3 receptor tyrosine kinase is a client protein of the molecular chaperone heat shock protein 90 and is commonly present and contributes to the leukemia phenotype in acute myelogenous leukemia (AML). LAQ824, a cinnamyl hydroxamate histone deacetylase inhibitor, is known to induce acetylation and inhibition of heat shock protein 90. Here, we determined the effects of LAQ824 and/or PKC412 (a FLT-3 kinase inhibitor) on the levels of mutant FLT-3 and its downstream signaling, as well as growth arrest and cell-death of cultured and primary human AML cells. EXPERIMENTAL DESIGN: The effect of LAQ824 and/or PKC412 treatment was determined on the levels of FLT-3 and phosphorylated (p)-FLT-3, on downstream pro-growth and pro-survival effectors, e.g., p-STAT5, p-AKT, and p-extracellular signal-regulated kinase (ERK) 1/2, and on the cell cycle status and apoptosis in the cultured MV4-11 and primary AML cells with mutant FLT-3. RESULTS: Treatment with LAQ824 promoted proteasomal degradation and attenuation of the levels of FLT-3 and p-FLT-3, associated with cell cycle G(1)-phase accumulation and apoptosis of MV4-11 cells. This was accompanied by attenuation of p-STAT5, p-AKT, and p-ERK1/2 levels. STAT-5 DNA-binding activity and the levels of c-Myc and oncostatin M were also down-regulated. Cotreatment with LAQ824 and PKC412 synergistically induced apoptosis of MV4-11 cells and induced more apoptosis of the primary AML cells expressing mutant FLT-3. This was also associated with more attenuation of p-FLT-3, p-AKT, p-ERK1/2, and p-STAT5. CONCLUSIONS: The combination of LAQ824 and PKC412 is highly active against human AML cells with mutant FLT-3, which merits in vivo studies of the combination against human AML.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Inhibidores Enzimáticos/administración & dosificación , Inhibidores de Histona Desacetilasas , Ácidos Hidroxámicos/administración & dosificación , Leucemia Mieloide Aguda/tratamiento farmacológico , Mutación , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Estaurosporina/análogos & derivados , Estaurosporina/antagonistas & inhibidores , Apoptosis , Western Blotting , Ciclo Celular , Línea Celular Tumoral , ADN/química , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Sinergismo Farmacológico , Exones , Citometría de Flujo , Fase G1 , Humanos , Proteínas de la Leche/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , ARN Mensajero/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT5 , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Transactivadores/metabolismo , Tirosina Quinasa 3 Similar a fmsRESUMEN
Presence of the activating length mutation (LM) in the juxtamembrane domain or point mutation in the kinase domain of FMS-like tyrosine kinase-3 (FLT-3) mediates ligand-independent progrowth and prosurvival signaling in approximately one-third of acute myelogenous leukemia (AML). PKC412, an inhibitor of FLT-3 kinase activity, is being clinically evaluated in AML. Present studies demonstrate that treatment of human acute leukemia MV4-11 cells (containing a FLT-3 LM) with the heat shock protein 90 inhibitor 17-allylamino-demethoxy geldanamycin (17-AAG) attenuated the levels of FLT-3 by inhibiting its chaperone association with heat shock protein 90, which induced the poly-ubiquitylation and proteasomal degradation of FLT-3. Treatment with 17-AAG induced cell cycle G(1) phase accumulation and apoptosis of MV4-11 cells. 17-AAG-mediated attenuation of FLT-3 and p-FLT-3 in MV4-11 cells was associated with decrease in the levels of p-AKT, p-ERK1/2, and p-STAT5, as well as attenuation of the DNA binding activity of STAT-5. Treatment with 17-AAG, downstream of STAT5, reduced the levels of c-Myc and oncostatin M, which are transactivated by STAT5. Cotreatment with 17-AAG and PKC412 markedly down-regulated the levels of FLT-3, p-FLT-3, p-AKT, p-ERK1/2, and p-STAT5, as well as induced more apoptosis of MV4-11 cells than either agent alone. Furthermore, the combination of 17-AAG and PKC412 exerted synergistic cytotoxic effects against MV4-11 cells. Importantly, 17-AAG and PKC412 induced more loss of cell viability of primary AML blasts containing FLT-3 LM, as compared with those that contained wild-type FLT-3. Collectively, these in vitro findings indicate that the combination of 17-AAG and PKC412 has high level of activity against AML cells with FLT-3 mutations.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Proteínas de la Leche , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Rifabutina/análogos & derivados , Rifabutina/farmacología , Estaurosporina/análogos & derivados , Estaurosporina/farmacología , Apoptosis/efectos de los fármacos , Benzoquinonas , Cisteína Endopeptidasas/metabolismo , ADN de Neoplasias/metabolismo , Proteínas de Unión al ADN/metabolismo , Sinergismo Farmacológico , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacología , Fase G1/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Lactamas Macrocíclicas , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Complejos Multienzimáticos/metabolismo , Complejo de la Endopetidasa Proteasomal , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Tirosina Quinasas Receptoras/metabolismo , Rifabutina/administración & dosificación , Factor de Transcripción STAT5 , Transducción de Señal/efectos de los fármacos , Estaurosporina/administración & dosificación , Transactivadores/metabolismo , Ubiquitina/metabolismo , Tirosina Quinasa 3 Similar a fmsRESUMEN
Protein cargoes that contain a classic nuclear localization signal (NLS) are transported into the nucleus through binding to a heterodimeric receptor comprised of importin/karyopherin alpha and beta. An evolutionarily conserved auto-inhibitory sequence within the N-terminal importin beta binding (IBB) domain of importin alpha regulates NLS-cargo binding to the NLS binding pocket on importin alpha. In this study, we have used site-directed mutagenesis coupled with in vitro binding assays and in vivo analyses to investigate the intramolecular interaction of the N-terminal IBB domain and the NLS binding pocket of Saccharomyces cerevisiae importin alpha, Srp1p. We find that mutations within the IBB domain that decrease the binding affinity of the auto-inhibitory sequence for the NLS binding pocket impact importin alpha function in vivo. In addition, the severity of the in vivo phenotype is directly correlated to the reduction of auto-inhibition measured in vitro, suggesting that the in vivo phenotypes are directly related to the loss of auto-inhibitory function. We exploit a conditional auto-inhibitory mutant, srp1-55, to study the in vivo functional overlap between the N-terminal IBB domain of importin alpha and other factors implicated in NLS-cargo release, Cse1p and Nup2p. We propose that the N-terminal IBB domain of importin alpha and Cse1p function together in NLS-cargo release, whereas Nup2p contributes to cargo release/importin alpha recycling through a distinct mechanism.