Chromosomal rearrangements without gene fusions have been implicated in leukemogenesis by causing deregulation of proto-oncogenes via relocation of cryptic regulatory DNA elements. AML with inv(3)/t(3;3) is associated with aberrant expression of the stem-cell regulator EVI1. Applying functional genomics and genome-engineering, we demonstrate that both 3q rearrangements reposition a distal GATA2 enhancer to ectopically activate EVI1 and simultaneously confer GATA2 functional haploinsufficiency, previously identified as the cause of sporadic familial AML/MDS and MonoMac/Emberger syndromes. Genomic excision of the ectopic enhancer restored EVI1 silencing and led to growth inhibition and differentiation of AML cells, which could be replicated by pharmacologic BET inhibition. Our data show that structural rearrangements involving the chromosomal repositioning of a single enhancer can cause deregulation of two unrelated distal genes, with cancer as the outcome.

The CEBPA gene is mutated in 9% of patients with acute myeloid leukemia (AML). Selective expression of a short 30 kDa C/EBPα translational isoform, termed p30, represents the most common type of CEBPA mutations 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 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 its down-regulation inhibited proliferation and restored differentiation in p30-dependent AML models. OICR-9429 is a novel 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.

We performed cytosine methylation sequencing on genetically diverse AML patients and found leukemic DNA methylation patterning is primarily driven by non-promoter regulatory elements and CpG shores. Enhancers displayed stronger differential methylation than promoters, consisting predominantly of hypomethylation. AMLs with dominant hypermethylation featured greater epigenetic disruption of promoters, while those with dominant hypomethylation displayed greater disruption of distal and intronic regions. Mutations in IDH and DNMT3A had opposing and mutually exclusive effects on the epigenome. Notably, co-occurrence of both mutations resulted in epigenetic antagonism, with most CpGs affected by either mutation alone no longer affected in double mutant AMLs. Importantly, this epigenetic antagonism precedes malignant transformation and can be observed in pre-leukemic LSK cells from Idh2R140Q or Dnmt3aR882H single and, Idh2R140Q/Dnmt3aR882H double mutant mice. Notably, IDH/DNMT3A double mutant AMLs manifested upregulation of RAS signaling signature and displayed unique sensitivity to MEK inhibition ex vivo as compared to AMLs with either single mutation.

One of the most studied transcription factors in hematopoiesis is the leucine zipper CCAAT-enhancer binding protein α (C/EBPα), which is mainly involved in cell fate decisions for myeloid differentiation. Its involvement in acute myeloid leukemia (AML) is diverse, with patients frequently exhibiting mutations, deregulation of gene expression, or alterations in the function of C/EBPα. In this review, we emphasize the importance of C/EBPα for neutrophil maturation, its role in myeloid priming of hematopoietic stem and progenitor cells, and its indispensable requirement for AML development. We discuss that mutations in the open reading frame of CEBPA lead to an altered C/EBPα function, affecting the expression of downstream genes and consequently deregulating myelopoiesis. The emerging transcriptional mechanisms of CEBPA are discussed based on recent studies. Novel insights on how these mechanisms may be deregulated by oncoproteins or mutations/variants in CEBPA enhancers are suggested in principal to reveal novel mechanisms of how CEBPA is deregulated at the transcriptional level.

Key Points The CEBPA locus harbors 14 enhancers of which distinct combinations are active in different CEBPA-expressing tissues. A +42-kb enhancer is required for myeloid-lineage priming to drive adequate CEBPA expression levels necessary for neutrophilic maturation.