Disruption of the NHR4 Domain Structure in AML1-ETO Abrogates Its Interaction with SON and Promotes AML1-ETO-Associated Leukemogenesis.

AML1-ETO is generated from t(8;21)(q22;q22), which is commonly associated with acute myeloid leukemia (AML), especially FAB M2 AML. While full length AML1-ETO fails to promote leukemia due to its detrimental effects on cell proliferation, an alternatively spliced isoform without its C-terminal NHR3 and NHR4 domains strongly induces leukemia. The NHR4 domain has a zinc-chelating topology and has been implicated in transcriptional repression through recruitment of the NCoR/SMRT complexes. Here, we report that disruption of the zinc-chelating structure in the NHR4 domain of AML1-ETO by replacing only one critical amino acid leads to rapid onset of leukemia, suggesting that the NHR4 domain is responsible for generating inhibitory effects on leukemia development. To identify the molecular mechanism of this leukemogenic effect, we searched for NHR4-interacting factors with yeast two hybrid screening, and identified a novel NHR4-interacting protein, SON, a potential DNA/RNA-binding protein. The N-terminal fragment of SON with 81 amino acids, which is encoded by exons 1 and 2, was sufficient to interact with the NHR4 domain of ETO. Interaction of endogenous SON and AML1-ETO was also detected in t(8;21) AML cell lines. A serine substitution of cysteine 663 which disrupts the zinc-chelating topology of the NHR4 domain completely abolished its interaction with SON, while retaining the interaction with another ETO-binding protein, N-CoR. Knockdown of SON by siRNA resulted in significant growth arrest. Disruption of the interaction between AML1-ETO and endogenous SON by overexpressing the SON N-terminal fragment rescued cells from AML1-ETO-induced growth arrest. These results suggest that SON is an indispensable factor for cell growth and the interaction between endogenous SON and AML1-ETO generates negative effects on cell growth. In t(8;21) AML patient-derived primary leukemic cells and cell lines, abnormal cytoplasmic localization of SON was detected, which may keep cells proliferating in the presence of full length AML1-ETO. Taken together, these results uncovered the crucial role of the NHR4 domain in determination of cellular fate during AML1-ETO-associated leukemogenesis and revealed the interaction of the NHR4 domain with SON, an essential protein for normal cell growth.