RESUMEN
Osimertinib is a third-generation covalent EGFR inhibitor that is used in treating non-small cell lung cancer. First-generation EGFR inhibitors were found to elicit pro-differentiation effect on acute myeloid leukemia (AML) cells in preclinical studies, but clinical trials yielded mostly negative results. Here, we report that osimertinib selectively induced apoptosis of CD34+ leukemia stem/progenitor cells but not CD34- cells in EGFR-negative AML and chronic myeloid leukemia (CML). Covalent binding of osimertinib to CD34 at cysteines 199 and 177 and suppression of Src family kinases (SFK) and downstream STAT3 activation contributed to osimertinib-induced cell death. SFK and STAT3 inhibition induced synthetic lethality with osimertinib in primary CD34+ cells. CD34 expression was elevated in AML cells compared with their normal counterparts. Genomic, transcriptomic, and proteomic profiling identified mutation and gene expression signatures of patients with AML with high CD34 expression, and univariate and multivariate analyses indicated the adverse prognostic significance of high expression of CD34. Osimertinib treatment induced responses in AML patient-derived xenograft models that correlated with CD34 expression while sparing normal CD34+ cells. Clinical responses were observed in two patients with CD34high AML who were treated with osimertinib on a compassionate-use basis. These findings reveal the therapeutic potential of osimertinib for treating CD34high AML and CML and describe an EGFR-independent mechanism of osimertinib-induced cell death in myeloid leukemia. SIGNIFICANCE: Osimertinib binds CD34 and selectively kills CD34+ leukemia cells to induce remission in preclinical models and patients with AML with a high percentage of CD34+ blasts, providing therapeutic options for myeloid leukemia patients.
Asunto(s)
Acrilamidas , Compuestos de Anilina , Carcinoma de Pulmón de Células no Pequeñas , Indoles , Leucemia Mieloide Aguda , Neoplasias Pulmonares , Pirimidinas , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proteómica , Proliferación Celular , Neoplasias Pulmonares/metabolismo , Leucemia Mieloide Aguda/genética , Células Progenitoras Mieloides , Receptores ErbB/metabolismo , Antígenos CD34/metabolismo , Células Madre Neoplásicas/metabolismoRESUMEN
Long noncoding RNA-H19 (H19), an imprinted oncofetal gene, has a central role in carcinogenesis. Hitherto, the mechanism by which H19 regulates cancer stem cells, remains elusive. Here we show that breast cancer stem cells (BCSCs) express high levels of H19, and ectopic overexpression of H19 significantly promotes breast cancer cell clonogenicity, migration and mammosphere-forming ability. Conversely, silencing of H19 represses these BCSC properties. In concordance, knockdown of H19 markedly inhibits tumor growth and suppresses tumorigenesis in nude mice. Mechanistically, we found that H19 functions as a competing endogenous RNA to sponge miRNA let-7, leading to an increase in expression of a let-7 target, the core pluripotency factor LIN28, which is enriched in BCSC populations and breast patient samples. Intriguingly, this gain of LIN28 expression can also feedback to reverse the H19 loss-mediated suppression of BCSC properties. Our data also reveal that LIN28 blocks mature let-7 production and, thereby, de-represses H19 expression in breast cancer cells. Appropriately, H19 and LIN28 expression exhibits strong correlations in primary breast carcinomas. Collectively, these findings reveal that lncRNA H19, miRNA let-7 and transcriptional factor LIN28 form a double-negative feedback loop, which has a critical role in the maintenance of BCSCs. Consequently, disrupting this pathway provides a novel therapeutic strategy for breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , MicroARNs/genética , ARN Largo no Codificante/genética , Proteínas de Unión al ARN/genética , Neoplasias de la Mama/patología , Carcinogénesis/genética , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/biosíntesis , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , ARN Largo no Codificante/biosíntesis , Proteínas de Unión al ARN/biosíntesisRESUMEN
The truncated chromosome 22 that results from the reciprocal translocation t(9;22)(q34;q11) is known as the Philadelphia chromosome (Ph) and is a hallmark of chronic myeloid leukemia (CML). In leukemia cells, Ph not only impairs the physiological signaling pathways but also disrupts genomic stability. This aberrant fusion gene encodes the breakpoint cluster region-proto-oncogene tyrosine-protein kinase (BCR-ABL1) oncogenic protein with persistently enhanced tyrosine kinase activity. The kinase activity is responsible for maintaining proliferation, inhibiting differentiation, and conferring resistance to cell death. During the progression of CML from the chronic phase to the accelerated phase and then to the blast phase, the expression patterns of different BCR-ABL1 transcripts vary. Each BCR-ABL1 transcript is present in a distinct leukemia phenotype, which predicts both response to therapy and clinical outcome. Besides CML, the Ph is found in acute lymphoblastic leukemia, acute myeloid leukemia, and mixed-phenotype acute leukemia. Here, we provide an overview of the clinical presentation and cellular biology of different phenotypes of Ph-positive leukemia and highlight key findings regarding leukemogenesis.
Asunto(s)
Proteínas de Fusión bcr-abl/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mieloide Aguda/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Carcinogénesis , Proliferación Celular , Inestabilidad Genómica , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Leucemia Mieloide Aguda/patología , Cromosoma Filadelfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Pronóstico , Proto-Oncogenes MasRESUMEN
The Notch signaling pathway has been implicated in the development of several leukemia and lymphoma. In order to investigate the relationship between Notch signaling and acute myeloid leukemia (AML), in this study, we expressed a recombinant Notch ligand protein, the DSL domain of the human Jagged1 fused with GST (GST-Jag1). GST-Jag1 could activate Notch signaling in the human promyelocytic leukemia cell line HL60, as shown by a reporter assay and the induced expression of Notch effector gene Hes1 and Hes5. However, GST-Jag1 had no effect on the proliferation and survival of HL60 cells. HL60 cells expressed both Notch ligands and receptors, and had a potential of reciprocal stimulation of Notch signaling between cells. We, therefore, blocked Notch signaling in cultured HL60 cells using a gamma-secretase inhibitor (GSI). We found that GSI inhibited the proliferation of HL60 cells significantly by blocking the cell-cycle progression in the G1 phase. Furthermore, GSI induced remarkably apoptosis of HL60 cells. These changes in GSI-treated HL60 cells correlated with the down-regulation of c-Myc and Bcl2, and the low phosphorylation of the Rb protein. These results suggested that reciprocal Notch signaling might be necessary for the proliferation and survival of AML cells, possibly through the maintenance of the expression of c-Myc and Bcl2, as well as the phosphorylation of the Rb protein.
Asunto(s)
Proliferación Celular , Leucemia Promielocítica Aguda/metabolismo , Receptores Notch/metabolismo , Proteína de Retinoblastoma/metabolismo , Transducción de Señal , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Apoptosis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Unión al Calcio/metabolismo , Ciclo Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Células HL-60 , Células HeLa , Proteínas de Homeodominio/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína Jagged-1 , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patología , Proteínas de la Membrana/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/metabolismo , Proteínas Serrate-Jagged , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Factor de Transcripción HES-1 , TransfecciónRESUMEN
Ex vivo expansion of hematopoietic progenitor cells (HPCs) is valuable for clinical application, however, traditional ex vivo culture negatively affects long-term hematopoietic reconstitution ability. In the hematopoietic system, the expression of Notch receptors and their ligands has been widely reported. Active Notch signal inhibits the differentiation of HSCs while promotes their expansion, suggesting that ex vivo expansion of hematopoietic progenitor cells could be enhanced by manipulating Notch signal pathways. In this article the Notch signal pathways, Notch signal and maintenance of hematopoietic progenitor cells, Notch signal and expansion of hematopoietic progenitor cells and molecular mechanism of Notch signal maintaining undifferentiation of hematopoietic progenitor cells were reviewed.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Receptores Notch/metabolismo , Animales , Células Madre Hematopoyéticas/citología , Humanos , Transducción de SeñalRESUMEN
In order to construct a pichia pastoris expression vector containing the extracellular domain of human Jagged1 and the Fc fragment of human IgG1 fusion gene, or containing only the Fc fragment of human IgG1 and to express them in pichia pastoris. The extracellular domain of human Jagged1 gene was cloned from normal human bone marrow cells. After DNA sequencing, the extracellular domain of Jagged1 gene was inserted into pIC-Fc vector constructed previously, which is Pichia pastoris expression vector containing only the Fc fragment of human IgG1. The constructed plasmid was transformed into yeast GS115 by means of electroporation. The recombinant transformants with a high copy number of the plasmid were selected on MD plate with G418. The expression of protein was induced by addition of methanol. Then, protein expression was analyzed by SDS-PAGE. The results indicated that the extracellular domain of human Jagged1 gene was effectively amplified. The DNA sequencing result showed that the constructed plasmid containing hJagged1(ext)-Fc fusion gene was the same as designed. The fusion protein was successfully expressed in Pichia pastoris. It is concluded that the hJagged1(ext) gene has been successfully cloned and expressed, which provides a new fusion protein for further experiments, the hJagged1(ext)-Fc fusion protein can be used as a new stimulator for proliferation of hematopoietic stem/progenitor cells in vitro.
Asunto(s)
Proteínas de Unión al Calcio/biosíntesis , Vectores Genéticos/genética , Fragmentos Fc de Inmunoglobulinas/biosíntesis , Inmunoglobulina G/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Proteínas de la Membrana/biosíntesis , Pichia/metabolismo , Proteínas de Unión al Calcio/genética , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Proteína Jagged-1 , Proteínas de la Membrana/genética , Pichia/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Serrate-Jagged , TransfecciónRESUMEN
AIM: To construct a Pichia pastoris expression vector PIC- hDll1(ext)-Fc, and to express the fusion protein containing the extracellular domain of human Delta-like1 and the Fc fragment of human IgG1 fusion gene in Pichia pastoris. METHODS: The extracellular domain of human Delta-like1 gene was amplified from pEF-BOSneo-hdll1(ext)-Fc by PCR. The expression vector was constructed by DNA recombination. The constructed plasmid was transformed into yeast GS115 by electroporation. The recombinant transformants with a high copy number of the plasmid were selected by using MD plate and G418. The expression of protein was induced by addition of methanol.Then analyzed protein expression by SDS-PAGE and Western blot. RESULTS: The extracellular domain of human Delta-like1 gene was effectively amplified. The DNA sequencing result showed that the constructed plasmid containing hDll(ext)-Fc fusion gene was the same as designed. The fusion protein was successfully expressed in Pichia pastoris. CONCLUSION: The hDll1(ext) gene has been successfully cloned and expressed in the form of Fc fusion protein, which provides a new fusion protein for further experiments.