Identification of epigenetic modifiers essential for growth and survival of AML1/ETO-positive leukemia
Ontology highlight
ABSTRACT: Aberrant gene expression patterns in acute myeloid leukemia (AML) with balanced chromosomal translocations are often associated with direct or indirect dysregulation of epigenetic modifiers. Previously, we have shown that the AML1/ETO (RUNX1/MTG8) fusion protein, encoded by the translocation (8;21)(q22;q22), leads to the epigenetic repression of its target genes. We aimed in this work to identify critical epigenetic modifiers, on which AML1/ETO-positive AML cells depend on for proliferation and survival using shRNA library screens and global transcriptomics approaches. Using shRNA library screens, we identified 41 commonly depleted genes in two AML1/ETO-positive cell lines Kasumi-1 and SKNO-1. Among them, several epigenetic regulators as DMAP1, SMYD1, SMYD2, BRD4, SETD8, SETD1A and HDAC8 were depleted. We validated genetically and pharmacologically DNMT1 and ATR using several AML1/ETO-positive and negative cell lines. We also showed in vivo differentiation of myeloblasts after treatment with the DNMT1-inhibitor decitabine in a patient with an AML1/ETO-positive AML. Bioinformatic analysis of global transcriptomics after AML1/ETO induction in 9/14/18-U937 cells identified 973 differentially expressed genes (DEGs) (648 up- and 325 downregulated). Pathway analysis revealed several interferon response pathways and metabolic pathways such as oxidative phosphorylation and hypoxia enriched after AML1/ETO induction. Three genes (PARP2, PRKCD and SMARCA4) were both downregulated after AML1/ETO induction, and also detected in the survival shRNA screens of both cell lines, decreasing survival and proliferation. In conclusion, using unbiased shRNA library screens and global transcriptomics, we have identified several driver epigenetic regulators for proliferation in AML1/ETO-positive AML. Some of these epigenetic regulators, such as DNMT1 and ATR, are susceptible to pharmacological inhibition by small molecules showing promising preclinical and clinical efficacy
Project description:Acute myeloid leukemia (AML) with the t(8;21)(q22;q22) chromosomal translocation is among the most common subtypes of AML and produces the AML1-ETO (AE) oncogenic fusion gene. AML1-ETO expression is critical to for t(8;21) AML leukemogenesis and maintenance. Post-transcriptional regulation of gene expression is often mediated through transcript 3'-untranslated regions (UTR). AML1-ETO uses the 3’UTR of the ETO gene, which is not normally expressed in hematopoietic cells. Therefore, the mechanisms regulating AML1-ETO via the 3’UTR are attractive therapeutic targets. In this study, we examine the regulation of AML1-ETO via the 3’UTR. We demonstrate that AML1-ETO primarily uses a 3.7kb isoform of the ETO 3’UTR in both t(8;21) patients and cell lines. Using a luciferase assay approach, we identify an AML1-ETO 3’UTR fragment between 2.8 and 3.4kb which is negatively regulated, increases expression upon inhibition of microRNA biogenesis, and contains a putative let-7 microRNA target site. We show that let-7b directly represses AML1-ETO through this site. An analysis of The Cancer Genome Atlas AML dataset shows that let-7b and let-7 family miRNA expression is significantly lower in t(8;21) AML than other AMLs. Finally, we demonstrate that let-7b-5p inhibits the proliferation of t(8;21) AML cell lines, rescues expression of AML1-ETO target genes, and promotes differentiation. Our findings establish AML1-ETO as a let-7b target gene and suggest that let-7 based therapeutics may be applied to t(8;21) AML.
Project description:AML1-ETO (Acute Myeloid Leukemia 1-Eight Twenty One) caused by the translocation t(8;21)(q22;q22) is a mutated transcription factor contributing to AML development. Although associated with a favorable prognosis, half of the patients fail to achieve long-term survival. We examined the role of the transcription factor Growth factor independence 1 (GFI1) in the initiation and progression of leukemia and exploited the use of a drug targeting GFI1 expression in the context of AML1-ETO associated AML. We could show that GFI1 is required for maintenance of AML1-ETO associated leukemia and that loss of GFI1 or targeting GFI1 expression impedes leukemia initiation and progression and could be a potential new therapeutic strategy for patients failing to respond to chemotherapy.
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:Somatic rearrangements of transcription factors are common abnormalities in the acute leukemias. With rare exception, however, the resultant protein products have remained largely intractable as pharmacologic targets. One example is AML1-ETO, the most common translocation reported in acute myeloid leukemia (AML). To identify AML1-ETO modulators, we screened a small molecule library using a chemical genomic approach. Gene expression signatures were used as surrogates for the expression versus loss of the translocation in AML1-ETO-expressing cells. The top classes of compounds that scored in this screen were corticosteroids and dihydrofolate reductase (DHFR) inhibitors. In addition to modulating the AML1-ETO signature, both classes induced evidence of differentiation, dramatically inhibited cell viability, and ultimately induced apoptosis via on-target activity. Furthermore, AML1-ETO-expressing cell lines were exquisitely sensitive to the effects of corticosteroids on cellular viability compared with nonexpressers. The corticosteroids diminished AML1-ETO protein in AML cells in a proteasome- and glucocorticoid receptor-dependent manner. Moreover, these molecule classes demonstrated synergy in combination with standard AML chemotherapy agents and activity in an orthotopic model of AML1-ETO-positive AML. This work suggests a role for DHFR inhibitors and corticosteroids in treating patients with AML1-ETO-positive disease.
Project description:The formation of the AML1-ETO fusion protein, resulting from the t(8;21) translocation, is considered to be among the t(8;21) acute myeloid leukemia (AML) initiating events. However, the mechanisms of the oncogenic activity of AML1-ETO remains unclear. In this study, we found that AML1-ETO triggers the heterochromatic silencing of UBXN8 by recognizing the AML1 binding sites and recruiting chromatin remodeling enzymes to the UBXN8 promoter region. Decitabine, a specific inhibitor of DNA methylation, upregulated the expression of UBXN8 in AML1-ETO+ AML cell lines. Overexpression of UBXN8 inhibited the proliferation and colony-forming ability and promoted cell cycle arrest in t(8;21) AML cell lines. Enhancement of UBXN8 level can significantly inhibit the tumor proliferation of AML1-ETO+ cells in vivo. Thus, our results indicated that epigenetic silencing of UBXN8 via its promoter region methylation mediated by the AML1-ETO fusion protein contributes to the leukemogenesis of t(8;21)AML. These results demonstrated the feasibility and effectiveness of the pharmacological disruption of AML1-ETO/HDACs/DNMTs complex and that the UBXN8 targeting maybe an potential therapeutic strategy for t(8;21)AML.
Project description:Purpose: The goal was to compare transcriptome profile of FACS-purified bone marrow AML1/ETO positive LSPCs from AML patients treated with rhIL-33 or controls (untreated). Methods: The transcriptomic analysis of AML1/ETO positive LSPCs were assessed in biological replicates upon treatment treated rhIL-33 or controls using Illumina NextSeq 500. Results: We mapped around 30 million sequence reads per sample to the human genome (GRCh38 - hg38) and identified differentially expressed transcripts in FACS-purified bone marrow AML1/ETO positive LSPCs from AML patients treated with rhIL-33 or controls (untreated). Differentially expressed genes between LSPCs treated with rhIL-33 or controls, were identified with a fold change ≥1.5 and FDR p-value <0.05. Conclusions: Our study represents the first detailed RNA-seq analysis of FACS-purified bone marrow AML1/ETO positive LSPCs from AML patients treated with rhIL-33 or controls. Our results show that IL-33 treatment induces the regulation of metabolic process, cell cycle, regulation of NF-κΒ, MAPK and canonical Wnt signaling pathways, as well as the regulation of stem cell division and maintenance.
Project description:The AML1-ETO fusion protein, a transcription factor generated by the t(8;21) translocation in acute myeloid leukaemia (AML), dictates a leukemic program by increasing self-renewal and inhibiting differentiation. Here we demonstrate that the histone demethylase JMJD1C functions as a co-activator for AML1-ETO and is required for its transcriptional program. JMJD1C is directly recruited by AML1-ETO to its target genes and regulates their expression by maintaining low H3K9me2 levels. Analyses in JMJD1C knockout mice also establish a JMJD1C requirement for AML1-ETOâs ability to increase proliferation. We also show a critical role for JMJD1C in the survival of multiple human AML cell lines, suggesting that it is required for leukemic programs in different AML cell types through its association with key transcription factors. Examination of RNA expression when Kasumi-1 cells are treated with control shRNA or two different JMJD1C shRNAs; in duplicate. Please note that the 'shAML1_ETO_vs_shControl.all_gene_exp.tb.txtl' was generated comparing control and shRNA treated RNA abundance-using previously published data [GSE43834; GSM1071857 and GSM1071852].
Project description:AML1-ETO is regarded as an initial event and plays a pivotal role in t(8;21) acute myeloid leukemia (AML) which is a highly heterogeneous disease. DNA methylation patterns are frequently deregulated in t(8;21) AML, though little is known of the mechanisms through which specific gene sets become aberrantly methylated. Here, We found that the promoter DNA methylation signature of t(8;21) AML blasts differs from those of normal CD34+ bone marrow cells and other AMLs. This signature contains 408 differentially methylated genes, many of which are genes involved in cancer pathway and myeloid leukemia. Database systematic survey and differential methylated regions (DMR) analysis were performed. These study demonstrated that a novel hypermethylated zinc finger containing protein-THAP10 is a target gene and can be epigenetic silenced by AML1-ETO at transcriptional level in t(8;21) AML and silencing of THAP10 by AML1-ETO predicts a poor clinical outcome. Our findings also identified that THAP10 is a bona fide target of miR-383 which can be epigenetic activated by AML1-ETO. In this study, we provided evidence that epigenetic silencing of THAP10 is the mechanistic link between AML1-ETO fusion proteins and tyrosine kinase cascades. In addition, we showed that THAP10 is a nuclear protein which inhibits myeloid proliferation inhibition and promotes differentiation both in vitro and in vivo. Altogether, our results revealed an unexpected and important link between fusion protein AML1-ETO, mir-383, THAP10 and tyrosine cascades in t(8;21) AML.
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:Acute myeloid leukaemia (AML) is caused by mutations in transcriptional and epigenetic regulator genes impairing myeloid differentiation. The t(8;21)(q22;q22) translocation generates the leukemogenic RUNX1-ETO fusion protein which interferes with the hematopoietic master regulator RUNX1. We previously showed that maintenance of t(8;21) AML is dependent on RUNX1-ETO as its depletion causes extensive changes in transcription factor binding and gene expression as well as myeloid differentiation. How changes in gene expression and binding events are connected within a transcriptional network is unclear. To this end, we assigned cis-regulatory elements to each other using promoter-capture chromosomal conformation assays in the presence and absence of RUNX1-ETO. From these data we constructed a RUNX1-ETO dependent dynamic transcriptional network maintaining AML. Integration of these data with gene expression and transcription factor binding data shows that RUNX1-ETO participates in interactions and that differential cis-element interactions are driven by alterations in the binding of RUNX1-ETO regulated transcription factors.