RESUMO
In an effort to identify novel drugs targeting fusion-oncogene-induced acute myeloid leukemia (AML), we performed high-resolution proteomic analysis. In AML1-ETO (AE)-driven AML, we uncovered a deregulation of phospholipase C (PLC) signaling. We identified PLCgamma 1 (PLCG1) as a specific target of the AE fusion protein that is induced after AE binding to intergenic regulatory DNA elements. Genetic inactivation of PLCG1 in murine and human AML inhibited AML1-ETO dependent self-renewal programs, leukemic proliferation, and leukemia maintenance in vivo. In contrast, PLCG1 was dispensable for normal hematopoietic stem and progenitor cell function. These findings are extended to and confirmed by pharmacologic perturbation of Ca++-signaling in AML1-ETO AML cells, indicating that the PLCG1 pathway poses an important therapeutic target for AML1-ETO+ leukemic stem cells.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Regulação Leucêmica da Expressão Gênica , Células-Tronco Hematopoéticas/patologia , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/patologia , Proteínas de Fusão Oncogênica/metabolismo , Fosfolipase C gama/metabolismo , Proteína 1 Parceira de Translocação de RUNX1/metabolismo , Animais , Autorrenovação Celular , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Células-Tronco Neoplásicas/metabolismo , Proteínas de Fusão Oncogênica/genética , Fosfolipase C gama/genética , Proteoma , Proteína 1 Parceira de Translocação de RUNX1/genética , Transcriptoma , Translocação GenéticaRESUMO
This protocol offers a detailed procedure for the in vitro differentiation of human pluripotent stem cells (hPSCs) to multipotent hematopoietic progenitors that arise from SOX17+ hemogenic endothelium, mimicking intra-embryonic, HOXA-positive, aorta-gonad mesonephros (AGM) hematopoiesis. The generated endothelium displays transcriptional similarities to cells sorted from human 5-week AGM, and CD45+CD34+RUNX1C+ progenitors share an accessible chromatin profile with adult hematopoietic stem cells and multipotent progenitors. Therefore, this protocol is suitable for the mechanistic study of human multipotent progenitor development and for modeling childhood leukemias. For complete details on the use and execution of this protocol, please refer to Nafria et al. (2020).
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Diferenciação Celular/fisiologia , Gônadas/citologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Mesonefro/citologia , Modelos Biológicos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/fisiologiaRESUMO
Acute myeloid leukemia (AML) is a hematopoietic malignancy caused by recurrent mutations in genes encoding transcriptional, chromatin, and/or signaling regulators. The t(8;21) translocation generates the aberrant transcription factor RUNX1-ETO (RUNX1-RUNX1T1), which by itself is insufficient to cause disease. t(8;21) AML patients show extensive chromatin reprogramming and have acquired additional mutations. Therefore, the genomic and developmental effects directly and solely attributable to RUNX1-ETO expression are unclear. To address this, we employ a human embryonic stem cell differentiation system capable of forming definitive myeloid progenitor cells to express RUNX1-ETO in an inducible fashion. Induction of RUNX1-ETO causes extensive chromatin reprogramming by interfering with RUNX1 binding, blocks differentiation, and arrests cellular growth, whereby growth arrest is reversible following RUNX1-ETO removal. Single-cell gene expression analyses show that RUNX1-ETO induction alters the differentiation of early myeloid progenitors, but not of other progenitor types, indicating that oncoprotein-mediated transcriptional reprogramming is highly target cell specific.