RESUMO
Increased food intake and lack of physical activity results in excess energy stored in adipocytes, and this imbalance contributes to obesity. New adipocytes are required for storage of energy in the white adipose tissue. This process of adipogenesis is widely studied in differentiating 3T3L1 preadipocytes in vitro. We have identified a key signaling kinase, protein kinase C delta (PKCδ), whose alternative splice variant expression is modulated during adipogenesis. We demonstrate that PKCδII splice variant promotes survival in differentiating 3T3L1 cells through the Bcl2 pathway. Here we demonstrate that resveratrol, a naturally occurring polyphenol, increases apoptosis and inhibits adipogenesis along with disruption of PKCδ alternative splicing during 3T3L1 differentiation. Importantly, we have identified a PKCδII splice variant inhibitor. This inhibitor may be a valuable tool with therapeutic implications in obesity.
Assuntos
Adipogenia , Processamento Alternativo , Apoptose , Proteína Quinase C-delta/antagonistas & inibidores , Estilbenos/química , Células 3T3-L1 , Animais , Diferenciação Celular , Regulação Enzimológica da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Polifenóis/química , Proteína Quinase C-delta/genética , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Resveratrol , TransfecçãoRESUMO
Philadelphia chromosome-negative myeloproliferative neoplasms, including polycythemia vera, essential thrombocytosis, and myelofibrosis, are disorders characterized by abnormal hematopoiesis. Among these myeloproliferative neoplasms, myelofibrosis has the most unfavorable prognosis. Furthermore, currently available therapies for myelofibrosis have little to no efficacy in the bone marrow and hence, are palliative. We recently developed a Janus kinase 2 (Jak2) small molecule inhibitor called G6 and found that it exhibits marked efficacy in a xenograft model of Jak2-V617F-mediated hyperplasia and a transgenic mouse model of Jak2-V617F-mediated polycythemia vera/essential thrombocytosis. However, its efficacy in Jak2-mediated myelofibrosis has not previously been examined. Here, we hypothesized that G6 would be efficacious in Jak2-V617F-mediated myelofibrosis. To test this, mice expressing the human Jak2-V617F cDNA under the control of the vav promoter were administered G6 or vehicle control solution, and efficacy was determined by measuring parameters within the peripheral blood, liver, spleen, and bone marrow. We found that G6 significantly reduced extramedullary hematopoiesis in the liver and splenomegaly. In the bone marrow, G6 significantly reduced pathogenic Jak/STAT signaling by 53%, megakaryocytic hyperplasia by 70%, and the Jak2 mutant burden by 68%. Furthermore, G6 significantly improved the myeloid to erythroid ratio and significantly reversed the myelofibrosis. Collectively, these results indicate that G6 is efficacious in Jak2-V617F-mediated myelofibrosis, and given its bone marrow efficacy, it may alter the natural history of this disease.
Assuntos
Janus Quinase 2/metabolismo , Mielofibrose Primária/tratamento farmacológico , Mielofibrose Primária/enzimologia , Inibidores de Proteínas Quinases/uso terapêutico , Bibliotecas de Moléculas Pequenas/uso terapêutico , Estilbenos/uso terapêutico , Substituição de Aminoácidos/genética , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Modelos Animais de Doenças , Hematopoese Extramedular/efeitos dos fármacos , Humanos , Hiperplasia , Janus Quinase 2/antagonistas & inibidores , Megacariócitos/efeitos dos fármacos , Megacariócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Células Mieloides/efeitos dos fármacos , Células Mieloides/patologia , Fosforilação/efeitos dos fármacos , Mielofibrose Primária/sangue , Mielofibrose Primária/fisiopatologia , Inibidores de Proteínas Quinases/farmacologia , Reticulina/efeitos dos fármacos , Reticulina/metabolismo , Fator de Transcrição STAT5/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Baço/efeitos dos fármacos , Baço/patologia , Baço/fisiopatologia , Esplenomegalia/complicações , Esplenomegalia/tratamento farmacológico , Esplenomegalia/patologia , Esplenomegalia/fisiopatologia , Estilbenos/farmacologiaRESUMO
In this study, we analyzed the structure-activity relationship properties of the small molecule Jak2 inhibitor G6. We synthesized a set of derivatives containing the native para-hydroxyl structure or an alternative meta-hydroxyl structure and examined their Jak2 inhibitory properties. We found that the para-hydroxyl derivative known as NB15 had excellent Jak2 inhibitory properties in silico, in vitro, and ex vivo when compared with meta-hydroxyl derivatives. These results indicate that NB15 is a potent derivative of the Jak2 inhibitor G6, and that maintaining the para-hydroxyl orientation of G6 is critical for its Jak2 inhibitory potential.
Assuntos
Benzilaminas/química , Benzilaminas/farmacologia , Janus Quinase 2/antagonistas & inibidores , Modelos Moleculares , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Hidroxilação , Camundongos , Camundongos Transgênicos , Estrutura Molecular , Estilbenos/química , Estilbenos/farmacologia , Relação Estrutura-AtividadeRESUMO
We recently developed a Janus kinase 2 (Jak2) small-molecule inhibitor called G6 and found that it inhibits Jak2-V617F-mediated pathologic cell growth in vitro, ex vivo, and in vivo. However, its ability to inhibit Jak2-V617F-mediated myeloproliferative neoplasia, with particular emphasis in the bone marrow, has not previously been examined. Here, we investigated the efficacy of G6 in a transgenic mouse model of Jak2-V617F-mediated myeloproliferative neoplasia. We found that G6 provided therapeutic benefit to the peripheral blood as determined by elimination of leukocytosis, thrombocytosis, and erythrocytosis. G6 normalized the pathologically high plasma concentrations of interleukin 6 (IL-6). In the liver, G6 eliminated Jak2-V617F-driven extramedullary hematopoiesis. With respect to the spleen, G6 significantly reduced both the splenomegaly and megakaryocytic hyperplasia. In the critically important bone marrow, G6 normalized the pathologically high levels of phospho-Jak2 and phospho-signal transducer and activator of transcription 5 (STAT5). It significantly reduced the megakaryocytic hyperplasia in the marrow and completely normalized the M/E ratio. Most importantly, G6 selectively reduced the mutant Jak2 burden by 67%on average, with virtual elimination of mutant Jak2 cells in one third of all treated mice. Lastly, clonogenic assays using marrow stem cells from the myeloproliferative neoplasm mice revealed a time-dependent elimination of the clonogenic growth potential of these cells by G6. Collectively, these data indicate that G6 exhibits exceptional efficacy in the peripheral blood, liver, spleen, and, most importantly, in the bone marrow, thereby raising the possibility that this compound may alter the natural history of Jak2-V617F-mediated myeloproliferative neoplasia.
Assuntos
Neoplasias da Medula Óssea/tratamento farmacológico , Neoplasias da Medula Óssea/genética , Transformação Celular Neoplásica/genética , Janus Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Estilbenos/uso terapêutico , Substituição de Aminoácidos/fisiologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Neoplasias da Medula Óssea/patologia , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Janus Quinase 2/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Fenilalanina/genética , Inibidores de Proteínas Quinases/farmacologia , Estilbenos/farmacologia , Valina/genéticaRESUMO
Using structure-based virtual screening, we previously identified a novel stilbenoid inhibitor of Jak2 tyrosine kinase named G6. Here, we hypothesized that G6 suppresses Jak2-V617F-mediated human pathological cell growth in vitro and in vivo. We found that G6 inhibited proliferation of the Jak2-V617F expressing human erythroleukemia (HEL) cell line by promoting marked cell cycle arrest and inducing apoptosis. The G6-dependent increase in apoptosis levels was concomitant with increased caspase 3/7 activity and cleavage of PARP. G6 also selectively inhibited phosphorylation of STAT5, a downstream signaling target of Jak2. Using a mouse model of Jak2-V617F-mediated hyperplasia, we found that G6 significantly decreased the percentage of blast cells in the peripheral blood, reduced splenomegaly, and corrected a pathologically low myeloid to erythroid ratio in the bone marrow by eliminating HEL cell engraftment in this tissue. In addition, drug efficacy correlated with the presence of G6 in the plasma, marrow, and spleen. Collectively, these data demonstrate that the stilbenoid compound, G6, suppresses Jak2-V617F-mediated aberrant cell growth. As such, G6 may be a potential therapeutic lead candidate against Jak2-mediated, human disease.
Assuntos
Ciclo Celular/efeitos dos fármacos , Janus Quinase 2/metabolismo , Leucemia Eritroblástica Aguda/enzimologia , Mutação de Sentido Incorreto , Inibidores de Proteínas Quinases/farmacologia , Estilbenos/farmacologia , Substituição de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Janus Quinase 2/genética , Leucemia Eritroblástica Aguda/tratamento farmacológico , Leucemia Eritroblástica Aguda/genética , Camundongos , Camundongos Mutantes , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Janus kinase 2 (JAK2) plays a crucial role in the pathomechanism of myeloproliferative disorders and hematologic malignancies. A somatic mutation of JAK2 (Val617Phe) was previously shown to occur in 98% of patients with polycythemia vera and 50% of patients with essential thrombocythemia and primary myelofibrosis. Thus, effective JAK2 kinase inhibitors may be of significant therapeutic importance. Here, we applied a structure-based virtual screen to identify novel JAK2 inhibitors. One JAK2 inhibitor in particular, G6, demonstrated remarkable potency as well as specificity, which makes it as a potential lead candidate against diseases related to elevated JAK2 tyrosine kinase activity.