Asunto(s)
ADP-Ribosil Ciclasa 1/análisis , Antígenos CD34/análisis , Antineoplásicos/uso terapéutico , Aberraciones Cromosómicas , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Anciano , Separación Celular , Femenino , Citometría de Flujo , Humanos , Hibridación Fluorescente in Situ , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/inmunología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Masculino , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
Activating alleles of Janus kinase 2 (JAK2) such as JAK2(V617F) are central to the pathogenesis of myeloproliferative neoplasms (MPN), suggesting that small molecule inhibitors targeting JAK2 may be therapeutically useful. We have identified an aminopyrimidine derivative (CYT387), which inhibits JAK1, JAK2, and tyrosine kinase 2 (TYK2) at low nanomolar concentrations, with few additional targets. Between 0.5 and 1.5muM CYT387 caused growth suppression and apoptosis in JAK2-dependent hematopoietic cell lines, while nonhematopoietic cell lines were unaffected. In a murine MPN model, CYT387 normalized white cell counts, hematocrit, spleen size, and restored physiologic levels of inflammatory cytokines. Despite the hematologic responses and reduction of the JAK2(V617F) allele burden, JAK2(V617F) cells persisted and MPN recurred upon cessation of treatment, suggesting that JAK2 inhibitors may be unable to eliminate JAK2(V617F) cells, consistent with preliminary results from clinical trials of JAK2 inhibitors in myelofibrosis. While the clinical benefit of JAK2 inhibitors may be substantial, not the least due to reduction of inflammatory cytokines and symptomatic improvement, our data add to increasing evidence that kinase inhibitor monotherapy of malignant disease is not curative, suggesting a need for drug combinations to optimally target the malignant cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Benzamidas/farmacología , Neoplasias Hematológicas/tratamiento farmacológico , Hematopoyesis/efectos de los fármacos , Janus Quinasa 2/antagonistas & inhibidores , Trastornos Mieloproliferativos/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Animales , Apoptosis/genética , Apoptosis/inmunología , Línea Celular Tumoral , Citocinas/genética , Citocinas/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Ensayos de Selección de Medicamentos Antitumorales , Neoplasias Hematológicas/enzimología , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/inmunología , Hematopoyesis/genética , Hematopoyesis/inmunología , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 1/genética , Janus Quinasa 1/inmunología , Janus Quinasa 1/metabolismo , Janus Quinasa 2/genética , Janus Quinasa 2/inmunología , Janus Quinasa 2/metabolismo , Ratones , Ratones Endogámicos BALB C , Mutación Missense , Trastornos Mieloproliferativos/enzimología , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/inmunologíaRESUMEN
BCR-ABL is proposed to impair cell-cycle control by disabling p27, a tumor suppressor that inhibits cyclin-dependent kinases. We show that in cell lines p27 expression is inversely correlated with expression of SKP2, the F-box protein of SCF(SKP2) (SKP1/Cul1/F-box), the E3 ubiquitin ligase that promotes proteasomal degradation of p27. Inhibition of BCR-ABL kinase causes G(1) arrest, down-regulation of SKP2, and accumulation of p27. Ectopic expression of wild-type SKP2, but not a mutant unable to recognize p27, partially rescues cell-cycle progression. A similar regulation pattern is seen in cell lines transformed by FLT3-ITD, JAK2(V617F), and TEL-PDGFRbeta, suggesting that the SKP2/p27 conduit may be a universal target for leukemogenic tyrosine kinases. Mice that received transplants of BCR-ABL-infected SKP2(-/-) marrow developed a myeloproliferative syndrome but survival was significantly prolonged compared with recipients of BCR-ABL-expressing SKP2(+/+) marrow. SKP2(-/-) leukemic cells demonstrated higher levels of nuclear p27 than SKP2(+/+) counterparts, suggesting that the attenuation of leukemogenesis depends on increased p27 expression. Our data identify SKP2 as a crucial mediator of BCR-ABL-induced leukemogenesis and provide the first in vivo evidence that SKP2 promotes oncogenesis. Hence, stabilization of p27 by inhibiting its recognition by SCF(SKP2) may be therapeutically useful.
Asunto(s)
Genes abl , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Proteínas Quinasas Asociadas a Fase-S/genética , Animales , Secuencia de Bases , Trasplante de Médula Ósea , Ciclo Celular , Línea Celular Tumoral , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Cartilla de ADN/genética , Proteínas de Fusión bcr-abl , Expresión Génica , Leucemia Mielógena Crónica BCR-ABL Positiva/etiología , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Trastornos Mieloproliferativos/etiología , Trastornos Mieloproliferativos/patología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismoRESUMEN
A-kinase anchoring proteins (AKAPs) bind to protein kinase A (PKA) via an amphipathic helix domain that interacts with a dimerization/docking domain on the regulatory (R) subunit of PKA. Four other mammalian proteins (ROPN1, ASP, SP17, and CABYR) also contain a highly conserved RII dimerization/docking (R2D2) domain, suggesting all four proteins may interact with all AKAPs in a manner similar to RII. All four of these proteins were originally detected in the flagellum of mammalian sperm. In this report, we demonstrate that all four R2D2 proteins are expressed in a wide variety of tissues and three of the proteins SP17, CABYR, and ASP are located in motile cilia of human bronchus and fallopian tubes. In addition, we detect SP17 in primary cilia. We also provide evidence that ROPN1 and ASP bind to a variety of AKAPs and this interaction can be disrupted with anchoring inhibitor peptides. The interaction of SP17 and CABYR with AKAPs appears to be much more limited. None of the R2D2 proteins appears to bind cAMP, a fundamental characteristic of the regulatory subunits of PKA. These observations suggest that R2D2 proteins utilize docking interactions with AKAPs to accomplish their function of regulating cilia and flagella. Based on location, affinity for AKAPs and lack of affinity for cAMP, it appears that each R2D2 protein has a unique role in this process.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Subunidad RIIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Proteínas de Anclaje a la Quinasa A/genética , Secuencia de Aminoácidos , Animales , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión a Calmodulina , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cilios/metabolismo , Cilios/ultraestructura , AMP Cíclico/metabolismo , Subunidad RIIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Femenino , Humanos , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Filogenia , Estructura Terciaria de Proteína , Alineación de Secuencia , Cola del Espermatozoide/metabolismo , Cola del Espermatozoide/ultraestructura , Distribución Tisular , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
The JAK2(V617F) mutation is present in almost all patients with polycythemia vera (PV), large proportions of patients with essential thrombocythemia and idiopathic myelofibrosis, and less frequently in atypical myeloproliferative disorders (MPD). We show that transplantation of JAK2(V617F)-transduced bone marrow into BALB/c mice induces MPD reminiscent of human PV, characterized by erythrocytosis, granulocytosis, extramedullary hematopoiesis, and bone marrow fibrosis, but not thrombocytosis. Fluorescence-activated cell sorting of bone marrow and spleen showed proportional expansion of common myeloid progenitors, granulocyte-monocyte and megakaryocyte-erythrocyte progenitors. Megakaryocyte and late erythroid progenitors were dramatically increased, with only modest expansion of early erythroid progenitors. Erythropoietin (Epo) receptor expression was reduced on early, but normal on late erythroblasts. Serum levels of Epo and granulocyte colony-stimulating factor, but not granulocyte macrophage colony-stimulating factor, were reduced, whereas tumor necrosis factor-alpha was increased, possibly exerting a negative effect on JAK2(V617F)-negative hematopoiesis. These data suggest that erythrocytosis and granulocytosis in JAK2(V617F) mice are the net result of a complex interplay between cell intrinsic and extrinsic factors. There were no thromboembolic events and no animals succumbed to their disease, implicating additional factors in the manifestation of human disease. The disease was not transplantable and prolonged observation showed normalization of blood counts in most JAK2(V617F) mice, suggesting that the mutation may not confer self-renewal capacity.