Selective Requirement of MYB for Oncogenic Hyperactivation of a Translocated Enhancer in Leukemia

Abstract

In acute myeloid leukemia (AML) with inv(3)(q21;q26) or t(3;3)(q21;q26), a translocated GATA2 enhancer drives oncogenic expression of EVI1. We generated an EVI1-GFP AML model and applied an unbiased CRISPR/Cas9 enhancer scan to uncover sequence motifs essential for EVI1 transcription. Using this approach, we pinpointed a single regulatory element in the translocated GATA2 enhancer that is critically required for aberrant EVI1 expression. This element contained a DNA-binding motif for the transcription factor MYB, which specifically occupied this site at the translocated allele and was dispensable for GATA2 expression. MYB knockout as well as peptidomimetic blockade of CBP/p300-dependent MYB functions resulted in downregulation of EVI1 but not of GATA2. Targeting MYB or mutating its DNA-binding motif within the GATA2 enhancer resulted in myeloid differentiation and cell death, suggesting that interference with MYB-driven EVI1 transcription provides a potential entry point for therapy of inv(3)/t(3;3) AMLs. Significance: We show a novel paradigm in which chromosomal aberrations reveal critical regulatory elements that are nonfunctional at their endogenous locus. This knowledge provides a rationale to develop new compounds to selectively interfere with oncogenic enhancer activity. This article is highlighted in the In This Issue feature, p. 2659

The leukemic oncogene EVI1 hijacks a MYC super-enhancer by CTCF-facilitated loops

“…These commonalities suggest a shared mechanism for EVI1 activation in all 3q26-rearranged leukemias, whereby an active hematopoietic enhancer is hijacked by a CTCF-mediated loop with the EVI1 promoter. To validate this hypothesis, we targeted the EVI1 CTCF binding site in MUTZ3-EVI1-eGFP, an inv(3) cell line engineered with eGFP as a reporter for EVI1 31 . In this model, mutations of the CTCF motif in a fraction of cells resulted in the loss of eGFP and EVI1 expression (Supplementary Fig.…”

“…Leukemias with 3q26 rearrangements depend on EVI1: interfering with EVI1 causes growth inhibition, differentiation, and ultimately death of leukemic cells 15,31 . Our data demonstrate mechanistic similarities between the distinct enhancer-driven EVI1+ leukemias, suggesting that therapy for one subtype may be effective for all these AMLs.…”

“…Generation of EVI1 expression cell model. The plasmids to clone T2A-eGFP in frame with EVI1 were designed and described by my colleagues as follows 31 . The repair template was generated using Gibson Assembly (NEB).…”

“…Cells were incubated at 37 °C and 5% CO 2 and passaged every 3-4 days to 100,000 cells/ml. A previously generated MUTZ3 EVI1-eGFP cell line was cultured in αMEM (HyClone) with 20% fetal calf serum (FCS, Gibco) and 20% conditioned 5637 medium 31 . Unique biological materials are available upon request by contacting the corresponding author.…”

MECOM-related disorder: Radioulnar synostosis without hematological aberration due to unique variants

“…In humans, disruption of MECOM can cause (or is associated with) diseases. Typically, MECOM germline variants can cause RUSAT and/or bone marrow failure 4,5,9,10 ; its inversion or translocation causes acute myeloid leukemia, 21 and its overexpression is related to a poor outcome in patients with acute myeloid leukemia. 22 So far, the correlation between MECOM and human diseases was mainly investigated by focusing on the phenotypes of hematopoiesis.…”