Project description:Microarray gene profilling indentified snoRNAs are downstream target of Amino Enhancer of Split (AES) and are essential for AML1-ETO9a induced leukemia. Amino Enhancer of Split (Aes) is strongly induced by leukemia oncogenes AML1-ETO, PML-RARα and PLZF-RARα. With a conditional AES knockout mouse model we showed that AES is essential for AML1-ETO9a indeced leukemia. We performed gene expression microarray using mouse primary AML1-ETO9a transformed AES wildtype and knockout and showed that snoRNAs were downregulated in AES knockout cells. We found that SnoRNA induction is a common mechanism shared by distinct oncogenes including AML1-ETO, MYC and MLL-AF9. Suppression of C/D box snoRNA complexes or deletion of several single C/D box snoRNAs inhibit clonogenic growth of leukemia cells. These findings suggest that enhancement of snoRNA levels is a critical mechanism of leukemic transformation.
Project description:U937 AML cells that express an inducible AML1-ETO construct under the control of the tetracycline promoter. Microarrays used to discover an AML1-ETO signature for a GE-HTS screen to identify AML1-ETO modulators.
Project description: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 which 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 positive leukemic stem cells.
Project description:Kasumi-1 AML cells that were transfected in triplicate with AML1-ETO or luciferase siRNA constructs by either Amaxa nucleofection or Biorad siLentFect and incubated for 96 hours. Microarrays used to discover an AML1-ETO signature for a GE-HTS screen to identify AML1-ETO modulators.
Project description:Compare the gene expression profile among human CD34+ cord blood cells infected with MIGR1, MIGR1-AML1-ETO or MIGR1-AML1-ETO∆NHR1 AML1-ETO promotes the self-renewal of human hematopoietic stem/progenitor cells (HSPCs). We found deletion of NHR1 domain abrogates AML1-ETO induced expasion of HSPCs.
Project description:Approximately 20% of Acute Myelogenous Leukemia (AML) cases carry the t(8;21) translocation, which involves the AML1 and ETO genes, and express the resulting AML1/ETO fusion protein that functions as a transcriptional repressor by recruiting NCoR/SMRT/HDAC complexes to DNA. We used ChIP-chip to identify the determinants of AML1/ETO binding on a contiguous DNA region (chromosome 19). AML1/ETO binding regions are characterized by a specific sequence signature that includes the presence of the consensus binding sites for the AML1 and HEB transcription factors. We therefore assessed the binding patterns of AML1 and HEB on chromosome 19. A specific chromatin modification (tri-methylation of lysine 4 on histone 3 = 3MetK4) was also studied in U937 cells expressing AML1/ETO in order to correlate the identified binding profiles with active transcription sites. Keywords: ChIP-chip
Project description:Approximately 20% of Acute Myelogenous Leukemia (AML) cases carry the t(8;21) translocation, which involves the AML1 and ETO genes, and express the resulting AML1/ETO fusion protein that functions as a transcriptional repressor by recruiting NCoR/SMRT/HDAC complexes to DNA. We used microarrays to identify human promoters bound by AML1/ETO in U937 cells. Keywords: ChIP-chip
Project description:Approximately 20% of Acute Myelogenous Leukemia (AML) cases carry the t(8;21) translocation, which involves the AML1 and ETO genes, and express the resulting AML1/ETO fusion protein that functions as a transcriptional repressor by recruiting NCoR/SMRT/HDAC complexes to DNA. We used microarrays to identify genes differentially expressed in U937 cells expressing AML1/ETO compared to vector transfected U937 cells. Keywords: Transcriptional regulation
Project description:Approximately 20% of Acute Myelogenous Leukemia (AML) cases carry the t(8;21) translocation, which involves the AML1 and ETO genes, and express the resulting AML1/ETO fusion protein that functions as a transcriptional repressor by recruiting NCoR/SMRT/HDAC complexes to DNA. We used ChIP-chip to identify the determinants of AML1/ETO binding on a contiguous DNA region (chromosome 19). AML1/ETO binding regions are characterized by a specific sequence signature that includes the presence of the consensus binding sites for the AML1 and HEB transcription factors. We therefore assessed the binding patterns of AML1 and HEB on chromosome 19. A specific chromatin modification (tri-methylation of lysine 4 on histone 3 = 3MetK4) was also studied in U937 cells expressing AML1/ETO in order to correlate the identified binding profiles with active transcription sites. Keywords: ChIP-chip A U937 cell line that conditionally expresses HA-tagged AML1/ETO under the control of the mouse metallothionine promoter (U937-A1E) (Alcalay et al., J.Clin.Invest, 2003,112, 1751-1761) was used. Cell lines were treated for 8h with 100uM ZnSO4 to induce transgene expression in U937-A1E. We performed ChIP using anti-HA, anti-ETO, anti-AML1/RUNX1, anti-HEBor anti-3MetK4 antibodies. ChIP products were then PCR amplified, labeled with Cy3/Cy5 fluorescent dyes and hybridized to the NimbleGen custom made NGS_HG17_Chr.19Array. U937-Mt cells, which carry the empty vector, served as control (C) for non-specific antibody binding. Each sample identifier indicates Antibody_Cell line (example: HA_A1E = ChIP using anti HA antibody in U937-A1E cells; HA_C = ChIP using anti HA antibody in U937-Mt control cells)