RUNX1-ETO Depletion in t(8;21) AML Leads to C/EBP alpha- and AP-1-Mediated Alterations in Enhancer-Promoter Interaction

Acute myeloid leukemia (AML) is associated with mutations in transcriptional and epigenetic regulator genes impairing myeloid differentiation. The t(8;21) (q22;q22) translocation generates the RUNX1-ETO fusion protein, which interferes with the hematopoietic master regulator RUNX1. We previously showed that the maintenance of t(8;21) AML is dependent on RUNX1-ETO expression. Its depletion causes extensive changes in transcription factor binding, as well as gene expression, and initiates myeloid differentiation. However, how these processes are connected within a gene regulatory network is unclear. To address this question, we performed Promoter-Capture Hi-C assays, with or without RUNX1-ETO depletion and assigned interacting cis-regulatory elements to their respective genes. To construct a RUNX1- ETO-dependent gene regulatory network maintaining AML, we integrated cis-regulatory element interactions with gene expression and transcription factor binding data. This analysis shows that RUNX1-ETO participates in cis-regulatory element interactions. However, differential interactions following RUNX1- ETO depletion are driven by alterations in the binding of RUNX1-ETO-regulated transcription factors

CEPBA in normal blood cell development and in myeloid malignancies

Expression levels of the myeloid transcription factor _CEBPA_ in the bone marrow are critical to maintain the balance between normal hematopoiesis and the onset of malignancy. What maintains constant levels of _CEBPA_ in normal hematopoiesis and how these levels are perturbed to trigger leukemogensis are less understood. In this thesis, a critical myeloid-specific regulatory element controls Cebpa expression levels in both mice and humans, was discovered. In the murine hematopoietic system, this regulatory element engages with Cebpa to prime neutrophilic differentiation. Genetic knockout of this regulatory element using CRISPR technology in mouse zygotes resulted in loss of mature neutrophils in the peripheral blood and also perturbed the myeloid progenitor system in the bone marrow. In a subtype of acute myeloid leukemia (AML), _CEBPA_ expression levels are low when relatively compared to other AMLs. What is the fundamental cause of low _CEBPA_ expression levels in AML? This question was approached in two ways: either by potential DNA mutations occuring in the regulatory regions of _CEBPA_ or epigenetic deregulation caused by AML-related oncoproteins. The CEBPA locus was screened for mutations in 300 AML patients. Only one patient harboured a biallelic deletion of the whole _CEBPA_ locus (220kb). However, it is more likely that _CEBPA_ expression levels are deregulated by oncoproteins that bind the identified regulatory element, reverses its active chromatin state and disturbs the chromatin loop between the enhancer and _CEBPA_. The thesis reports the identification of the regulatory element that primes the entry of myelopoiesis in the bone marrow and identifies a mechanism by which oncoproteins block myeloid differentiation in AML by disturbing the transcriptional regulatory mechanism of _CEBPA_ in the bone marrow

An autonomous CEBPA enhancer specific for myeloid-lineage priming and neutrophilic differentiation

Neutrophilic differentiation is dependent on CCAAT enhancer-binding protein α (C/EBPα), a transcription factor expressed in multiple organs including the bone marrow. Using functional genomic technologies in combination with clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 genome editing and in vivo mouse modeling, we show that CEBPA is located in a 170-kb topological-associated domain that contains 14 potential enhancers. Of these, 1 enhancer located +42 kb from CEBPA is active and engages with the CEBPA promoter in myeloid cells only. Germ line deletion of the homologous enhancer in mice in vivo reduces Cebpa levels exclusively in hematopoietic stem cells (HSCs) and myeloid-primed progenitor cells leading to severe defects in the granulocytic lineage, without affecting any other Cebpa-expressing organ studied. The enhancer-deleted progenitor cells lose their myeloid transcription program and are blocked in differentiation. Deletion of the enhancer also causes loss of HSC maintenance. We conclude that a single +42-kb enhancer is essential for CEBPA expression in myeloid cells only

Pharmacological targeting of the Wdr5-MLL interaction in C/EBPα N-terminal leukemia

The CEBPA gene is mutated in 9% of patients with acute myeloid leukemia (AML). Selective expression of a short (30-kDa) CCAAT-enhancer binding protein-α (C/EBPα) translational isoform, termed p30, represents the most common type of CEBPA mutation in AML. The molecular mechanisms underlying p30-mediated transformation remain incompletely understood. We show that C/EBPα p30, but not the normal p42 isoform, preferentially interacts with Wdr5, a key component of SET/MLL (SET-domain/mixed-lineage leukemia) histone-methyltransferase complexes. Accordingly, p30-bound genomic regions were enriched for MLL-dependent H3K4me3 marks. The p30-dependent increase in self-renewal and inhibition of myeloid differentiation required Wdr5, as downregulation of the latter inhibited proliferation and restored differentiation in p30-dependent AML models. OICR-9429 is a new small-molecule antagonist of the Wdr5-MLL interaction. This compound selectively inhibited proliferation and induced differentiation in p30-expressing human AML cells. Our data reveal the mechanism of p30-dependent transformation and establish the essential p30 cofactor Wdr5 as a therapeutic target in CEBPA-mutant AML