RESUMO
The ETV6-RUNX1 onco-fusion arises in utero, initiating a clinically silent pre-leukemic state associated with the development of pediatric B-acute lymphoblastic leukemia (B-ALL). We characterize the ETV6-RUNX1 regulome by integrating chromatin immunoprecipitation- and RNA-sequencing and show that ETV6-RUNX1 functions primarily through competition for RUNX1 binding sites and transcriptional repression. In pre-leukemia, this results in ETV6-RUNX1 antagonization of cell cycle regulation by RUNX1 as evidenced by mass cytometry analysis of B-lineage cells derived from ETV6-RUNX1 knock-in human pluripotent stem cells. In frank leukemia, knockdown of RUNX1 or its co-factor CBFß results in cell death suggesting sustained requirement for RUNX1 activity which is recapitulated by chemical perturbation using an allosteric CBFß-inhibitor. Strikingly, we show that RUNX1 addiction extends to other genetic subtypes of pediatric B-ALL and also adult disease. Importantly, inhibition of RUNX1 activity spares normal hematopoiesis. Our results suggest that chemical intervention in the RUNX1 program may provide a therapeutic opportunity in ALL.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core , Leucemia-Linfoma Linfoblástico de Células Precursoras , Adulto , Criança , Humanos , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Fatores de Ligação ao Core , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Linfócitos B , Fusão GênicaRESUMO
Ligand-induced activation of Exchange Protein Activated by cAMP-1 (EPAC1) is implicated in numerous physiological and pathological processes, including cardiac fibrosis where changes in EPAC1 expression have been detected. However, little is known about how EPAC1 expression is regulated. Therefore, we investigated regulation of EPAC1 expression by cAMP in cardiac fibroblasts. Elevation of cAMP using forskolin, cAMP-analogues or adenosine A2B-receptor activation significantly reduced EPAC1 mRNA and protein levels and inhibited formation of F-actin stress fibres. Inhibition of actin polymerisation with cytochalasin-D, latrunculin-B or the ROCK inhibitor, Y-27632, mimicked effects of cAMP on EPAC1 mRNA and protein levels. Elevated cAMP also inhibited activity of an EPAC1 promoter-reporter gene, which contained a consensus binding element for TEAD, which is a target for inhibition by cAMP. Inhibition of TEAD activity using siRNA-silencing of its co-factors YAP and TAZ, expression of dominant-negative TEAD or treatment with YAP-TEAD inhibitors, significantly inhibited EPAC1 expression. However, whereas expression of constitutively-active YAP completely reversed forskolin inhibition of EPAC1-promoter activity it did not rescue EPAC1 mRNA levels. Chromatin-immunoprecipitation detected a significant reduction in histone3-lysine27-acetylation at the EPAC1 proximal promoter in response to forskolin stimulation. HDAC1/3 inhibition partially reversed forskolin inhibition of EPAC1 expression, which was completely rescued by simultaneously expressing constitutively active YAP. Taken together, these data demonstrate that cAMP downregulates EPAC1 gene expression via disrupting the actin cytoskeleton, which inhibits YAP/TAZ-TEAD activity in concert with HDAC-mediated histone deacetylation at the EPAC1 proximal promoter. This represents a novel negative feedback mechanism controlling EPAC1 levels in response to cAMP elevation.
Assuntos
AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Processamento de Proteína Pós-Traducional , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Amidas , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , Citocalasina D/metabolismo , Fibroblastos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Histonas/metabolismo , Humanos , Masculino , Piridinas , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Tiazolidinas/metabolismoRESUMO
ETV6-RUNX1 is associated with childhood acute B-lymphoblastic leukemia (cALL) functioning as a first-hit mutation that initiates a clinically silent pre-leukemia in utero. Because lineage commitment hierarchies differ between embryo and adult, and the impact of oncogenes is cell-context dependent, we hypothesized that the childhood affiliation of ETV6-RUNX1 cALL reflects its origins in a progenitor unique to embryonic life. We characterize the first emerging B cells in first-trimester human embryos, identifying a developmentally restricted CD19-IL-7R+ progenitor compartment, which transitions from a myeloid to lymphoid program during ontogeny. This developmental series is recapitulated in differentiating human pluripotent stem cells (hPSCs), thereby providing a model for the initiation of cALL. Genome-engineered hPSCs expressing ETV6-RUNX1 from the endogenous ETV6 locus show expansion of the CD19-IL-7R+ compartment, show a partial block in B lineage commitment, and produce proB cells with aberrant myeloid gene expression signatures and potential: features (collectively) consistent with a pre-leukemic state.