Maria Rosaria Imperato

Identification of a dynamic core transcriptional network in t(8;21) AML that regulates differentiation block and self-renewal.

Summary Oncogenic transcription factors such as RUNX1/ETO, which is generated by the chromosomal translocation t(8;21), subvert normal blood cell development by impairing differentiation and driving malignant self-renewal. Here, we use digital footprinting and chromatin immunoprecipitation sequencing (ChIP-seq) to identify the core RUNX1/ETO-responsive transcriptional network of t(8;21) cells. We show that the transcriptional program underlying leukemic propagation is regulated by a dynamic equilibrium between RUNX1/ETO and RUNX1 complexes, which bind to identical DNA sites in a mutually exclusive fashion. Perturbation of this equilibrium in t(8;21) cells by RUNX1/ETO depletion leads to a global redistribution of transcription factor complexes within preexisting open chromatin, resulting in the formation of a transcriptional network that drives myeloid differentiation. Our work demonstrates on a genome-wide level that the extent of impaired myeloid differentiation in t(8;21) is controlled by the dynamic balance between RUNX1/ETO and RUNX1 activities through the repression of transcription factors that drive differentiation.

The RUNX1-PU.1 axis in the control of hematopoiesis.

The differentiation from multipotent hematopoietic stem cells (HSC) to mature and functional blood cells requires the finely tuned regulation of gene expression at each stage of development. Specific transcription factors play a key role in this process as they modulate the expression of their target genes in an exquisitely lineage-specific manner. A large number of important transcriptional regulators have been identified which establish and maintain specific gene expression patterns during hematopoietic development. Hematopoiesis is therefore a paradigm for investigating how transcription factors function in mammalian cells, thanks also to the evolution of genome-wide and the next-generation sequencing technologies. In this review, we focus on the current knowledge of the biological and functional properties of the hematopoietic master regulator RUNX1 (also known as AML1, CBFA2, PEBP2aB) transcription factor and its main downstream target PU.1. We will outline their relationship in determining the fate of the myeloid lineage during normal stem cell development and under conditions when hematopoietic development is subverted by leukemic transformation.

Chronic FLT3-ITD Signaling in Acute Myeloid Leukemia Is Connected to a Specific Chromatin Signature

Summary Acute myeloid leukemia (AML) is characterized by recurrent mutations that affect the epigenetic regulatory machinery and signaling molecules, leading to a block in hematopoietic differentiation. Constitutive signaling from mutated growth factor receptors is a major driver of leukemic growth, but how aberrant signaling affects the epigenome in AML is less understood. Furthermore, AML cells undergo extensive clonal evolution, and the mutations in signaling genes are often secondary events. To elucidate how chronic growth factor signaling alters the transcriptional network in AML, we performed a system-wide multi-omics study of primary cells from patients suffering from AML with internal tandem duplications in the FLT3 transmembrane domain (FLT3-ITD). This strategy revealed cooperation between the MAP kinase (MAPK) inducible transcription factor AP-1 and RUNX1 as a major driver of a common, FLT3-ITD-specific gene expression and chromatin signature, demonstrating a major impact of MAPK signaling pathways in shaping the epigenome of FLT3-ITD AML.

A FOXO1-induced oncogenic network defines the AML1-ETO preleukemic program

Understanding and blocking the self-renewal pathway of preleukemia stem cells could prevent acute myeloid leukemia (AML) relapse. In this study, we show that increased FOXO1 represents a critical mechanism driving aberrant self-renewal in preleukemic cells expressing the t(8;21)-associated oncogene AML1-ETO (AE). Although generally considered as a tumor suppressor, FOXO1 is consistently upregulated in t(8;21) AML. Expression of FOXO1 in human CD34+ cells promotes a preleukemic state with enhanced self-renewal and dysregulated differentiation. The DNA binding domain of FOXO1 is essential for these functions. FOXO1 activates a stem cell molecular signature that is also present in AE preleukemia cells and preserved in t(8;21) patient samples. Genome-wide binding studies show that AE and FOXO1 share the majority of their binding sites, whereby FOXO1 binds to multiple crucial self-renewal genes and is required for their activation. In agreement with this observation, genetic and pharmacological ablation of FOXO1 inhibited the long-term proliferation and clonogenicity of AE cells and t(8;21) AML cell lines. Targeting of FOXO1 therefore provides a potential therapeutic strategy for elimination of stem cells at both preleukemic and leukemic stages.

RUNX1-ETO and RUNX1-EVI1 Differentially Reprogram the Chromatin Landscape in t(8;21) and t(3;21) AML

Summary Acute myeloid leukemia (AML) is a heterogeneous disease caused by mutations in transcriptional regulator genes, but how different mutant regulators shape the chromatin landscape is unclear. Here, we compared the transcriptional networks of two types of AML with chromosomal translocations of the RUNX1 locus that fuse the RUNX1 DNA-binding domain to different regulators, the t(8;21) expressing RUNX1-ETO and the t(3;21) expressing RUNX1-EVI1. Despite containing the same DNA-binding domain, the two fusion proteins display distinct binding patterns, show differences in gene expression and chromatin landscape, and are dependent on different transcription factors. RUNX1-EVI1 directs a stem cell-like transcriptional network reliant on GATA2, whereas that of RUNX1-ETO-expressing cells is more mature and depends on RUNX1. However, both types of AML are dependent on the continuous expression of the fusion proteins. Our data provide a molecular explanation for the differences in clinical prognosis for these types of AML.

C/EBPα overrides epigenetic reprogramming by oncogenic transcription factors in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogeneous disease caused by recurrent mutations in the transcription regulatory machinery, resulting in abnormal growth and a block in differentiation. One type of recurrent mutations affects RUNX1, which is subject to mutations and translocations, the latter giving rise to fusion proteins with aberrant transcriptional activities. We recently compared the mechanism by which the products of the t(8;21) and the t(3;21) translocation RUNX1-ETO and RUNX1-EVI1 reprogram the epigenome. We demonstrated that a main component of the block in differentiation in both types of AML is direct repression of the gene encoding the myeloid regulator C/EBPα by both fusion proteins. Here, we examined at the global level whether C/EBPα is able to reverse aberrant chromatin programming in t(8;21) and t(3;21) AML. C/EBPα overexpression does not change oncoprotein expression or globally displace these proteins from their binding sites. Instead, it upregulates a core set of common target genes important for myeloid differentiation and represses genes regulating leukemia maintenance. This study, therefore, identifies common CEBPA-regulated pathways as targets for therapeutic intervention.

Identification of common and distinct epigenetic reprogramming properties of RUNX1 fusion proteins in acute myeloid leukaemia

Abstract Background Regulation of gene expression by transcription factors such as RUNX1 is crucial for haemopoiesis. The most common RUNX1 translocation resulting in acute myeloid leukaemia is t(8;21), which forms RUNX1–ETO; a second translocation—t(3;21)—results in RUNX1–EVI-1. Although, these two fusion proteins have the same DNA binding domain, the prognoses of patients with these translocations differ greatly. Whether different RUNX1 fusion proteins deregulate the same genes is unknown. We sought to understand the differences in the epigenome that underlie these prognostic differences. Methods DNase-seq maps regions of open chromatin that represent active gene regulatory elements. By using this technique with RNA-seq, we were able to describe the epigenetic landscape in CD34+ purified, primary material from patients with RUNX1–EVI-1 and RUNX1–ETO leukaemias, and in healthy controls. We used ChIP-seq to map the binding sites of both normal RUNX1 and the fusion proteins. We integrated these analyses to determine transcription factor networks that characterise each type of leukaemia. Findings RUNX1–EVI-1, but not RUNX1–ETO, directly regulated a unique subset of genes required for stem-cell function. We found that these differences in binding sites of the two fusion proteins were associated with differences in the transcription factor complexes that collaborate with them, and were directly related to the differences in the epigenetic landscape of each leukaemia. RUNX1–EVI-1 knockdown restored differentiation of t(3;21) cells and this finding was associated with upregulation of genes crucial for myeloid differentiation, including C/EBPα. We showed that C/EBPα was necessary and sufficient for the response of t(3;21) cells to RUNX1–EVI-1 knockdown and that C/EBPα was commonly deregulated in both leukaemias. Interpretation The differences in the clinical outcomes of each RUNX1 mutant leukaemia was reflected by differences in their epigenetic landscape. This finding was driven by differences in the transcription factor networks in each type of leukaemia. Despite these differences, both leukaemias were dependent on downregulating C/EBP, thereby providing a common therapeutic route for both RUNX1–ETO and RUNX1–EVI-1 leukaemia. Funding Kay Kendall Leukaemia Fund, Bloodwise.