Different roles of E proteins in t(8;21) leukemia: E2-2 compromises the function of AETFC and negatively regulates leukemogenesis

Significance AML1-ETO–containing transcription factor complex (AETFC) is a recently identified unique multiprotein complex through which AML1-ETO controls leukemogenesis. In this study, we found a functionally important AETFC heterogeneity defined by the three E proteins: while HEB and E2A facilitate leukemogenesis, E2-2 functions as a negative regulator. The underlying mechanism involves E2-2–mediated redistribution of AETFC to specific target genes, enabling a dendritic differentiation of the leukemic cells. Consistent with the fact that E2-2 is absent in AML1-ETO–expressing leukemic cell lines, E2-2 silencing/repression may occur as a cooperative event in some t(8;21) AML patients. An E2-2 target gene is identified as a predictor of relapse in the patients. This study thus provides insights into the mechanism of AML1-ETO–associated leukemogenesis and carries prognostic/therapeutic implications. The AML1-ETO fusion protein, generated by the t(8;21) chromosomal translocation, is causally involved in nearly 20% of acute myeloid leukemia (AML) cases. In leukemic cells, AML1-ETO resides in and functions through a stable protein complex, AML1-ETO–containing transcription factor complex (AETFC), that contains multiple transcription (co)factors. Among these AETFC components, HEB and E2A, two members of the ubiquitously expressed E proteins, directly interact with AML1-ETO, confer new DNA-binding capacity to AETFC, and are essential for leukemogenesis. However, the third E protein, E2-2, is specifically silenced in AML1-ETO–expressing leukemic cells, suggesting E2-2 as a negative factor of leukemogenesis. Indeed, ectopic expression of E2-2 selectively inhibits the growth of AML1-ETO–expressing leukemic cells, and this inhibition requires the bHLH DNA-binding domain. RNA-seq and ChIP-seq analyses reveal that, despite some overlap, the three E proteins differentially regulate many target genes. In particular, studies show that E2-2 both redistributes AETFC to, and activates, some genes associated with dendritic cell differentiation and represses MYC target genes. In AML patients, the expression of E2-2 is relatively lower in the t(8;21) subtype, and an E2-2 target gene, THPO, is identified as a potential predictor of relapse. In a mouse model of human t(8;21) leukemia, E2-2 suppression accelerates leukemogenesis. Taken together, these results reveal that, in contrast to HEB and E2A, which facilitate AML1-ETO–mediated leukemogenesis, E2-2 compromises the function of AETFC and negatively regulates leukemogenesis. The three E proteins thus define a heterogeneity of AETFC, which improves our understanding of the precise mechanism of leukemogenesis and assists development of diagnostic/therapeutic strategies.