The t(8;21) translocation fuses the DNA-binding domain of the hematopoietic master regulator RUNX1 to the ETO protein. The resultant RUNX1/ETO fusion protein is a leukemia-initiating transcription factor that interferes with RUNX1 function. The result of this interference is a block in differentiation and, finally, the development of acute myeloid leukemia (AML). To obtain insights into RUNX1/ETO-dependant alterations of the epigenetic landscape, we measured genome-wide RUNX1- and RUNX1/ETO-bound regions in t(8;21) cells and assessed to what extent the effects of RUNX1/ETO on the epigenome depend on its continued expression in established leukemic cells. To this end, we determined dynamic alterations of histone acetylation, RNA Polymerase II binding and RUNX1 occupancy in the presence or absence of RUNX1/ETO using a knockdown approach. Combined global assessments of chromatin accessibility and kinetic gene expression data show that RUNX1/ETO controls the expression of important regulators of hematopoietic differentiation and self-renewal. We show that selective removal of RUNX1/ETO leads to a widespread reversal of epigenetic reprogramming and a genome-wide redistribution of RUNX1 binding, resulting in the inhibition of leukemic proliferation and self-renewal, and the induction of differentiation. This demonstrates that RUNX1/ETO represents a pivotal therapeutic target in AML.

Acute myeloid leukemia (AML) is a heterogeneous disease caused by a variety of mutations in transcription factors, epigenetic regulators and signaling molecules. To determine how different mutant regulators establish AML subtype-specific transcriptional networks we performed a comprehensive global analysis of cis-regulatory element activity and interaction, transcription factor occupancy and gene expression patterns in purified leukemic blast cells. Here, we focussed on specific sub-groups of patients carrying mutations in genes encoding transcription factors ( RUNX1, CEBPA) and signaling molecules ( FTL3-ITD, RAS, NPM1). Integrated analyses of these data demonstrates that each mutant regulator establishes a specific transcriptional and signaling network unrelated to that seen in normal cells, sustaining the expression of unique sets of genes required for AML growth and maintenance.

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 t(4;11)(q21;q23) fuses mixed-lineage leukemia (MLL) to AF4, the most common MLL-fusion partner. Here we show that MLL fused to murine Af4, highly conserved with human AF4, produces high-titer retrovirus permitting efficient transduction of human CD34 cells, thereby generating a model of t(4;11) pro-B acute lymphoblastic leukemia (ALL) that fully recapitulates the immunophenotypic and molecular aspects of the disease. MLL-Af4 induces a B ALL distinct from MLL-AF9 through differential genomic target binding of the fusion proteins leading to specific gene expression patterns. MLL-Af4 cells can assume a myeloid state under environmental pressure but retain lymphoid-lineage potential. Such incongruity was also observed in t(4;11) patients in whom leukemia evaded CD19-directed therapy by undergoing myeloid-lineage switch. Our model provides a valuable tool to unravel the pathogenesis of MLL-AF4 leukemogenesis.

SummaryAcute 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.

SummaryOncogenic transcription factors such as the leukemic fusion protein RUNX1/ETO, which drives t(8;21) acute myeloid leukemia (AML), constitute cancer-specific but highly challenging therapeutic targets. We used epigenomic profiling data for an RNAi screen to interrogate the transcriptional network maintaining t(8;21) AML. This strategy identified Cyclin D2 (CCND2) as a crucial transmitter of RUNX1/ETO-driven leukemic propagation. RUNX1/ETO cooperates with AP-1 to drive CCND2 expression. Knockdown or pharmacological inhibition of CCND2 by an approved drug significantly impairs leukemic expansion of patient-derived AML cells and engraftment in immunodeficient murine hosts. Our data demonstrate that RUNX1/ETO maintains leukemia by promoting cell cycle progression and identifies G1 CCND-CDK complexes as promising therapeutic targets for treatment of RUNX1/ETO-driven AML.

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Background: Nasal polyposis is a multifactorial disease characterized by a chronic eosinophilic inflammation of the sinus mucosa, often associated with asthma and aspirin sensitivity. We have recently shown that the presence of IgE antibodies to Staphylococcus aureus enterotoxins (SAEs) was related to the severity of eosinophilic inflammation in nasal polyp tissue. In this study, we therefore aimed to determine, whether aspirin sensitivity was related to an immune response to SAEs, and how both criteria would be related to eosinophilic inflammation. Methods: 40 subjects with nasal polyposis (NP) were classified as aspirin-sensitive (n = 13, ASNP) or aspirin-tolerant (n = 27, ATNP) based on a bronchial aspirin challenge test. Homogenates prepared from nasal polyp tissue and inferior nasal turbinates from healthy subjects (n = 12) were analyzed for concentrations of IL-5 by enzyme immunoassay and for ECP, total and IgE to a mix of SAEs (A, C, TSST-1) using the ImmunoCAP system. Results: Concentrations of IL-5, ECP, total IgE, and IgE to an SAE mix were significantly increased in ASNP compared with ATNP patients and controls. In addition, a subgroup analysis showed an increase in eosinophilic markers in ATNP-SAE(+) compared to ATNP-SAE(–). This relationship, however, was not found in ATNP-SAE(+) and ATNP-SAE(–) subjects, indicating that SAE immune response is overlapped or not relevant in this condition. Conclusions: Aspirin sensitivity was associated with increased concentrations of eosinophil-related mediators, as well as IgE antibodies to SAEs in nasal polyp tissue. However, a direct impact of S. aureus could not be established. It seems that aspirin sensitivity and immune reactions to SAEs are independently related to eosinophilic inflammation.