The NHR2 Oligomerization Domain of AML1/ETO as a Novel Therapeutic Target Structure in t(8;21) Positive Leukemias.

Abstract

About 12% of all de novo acute myeloid leukemias are characterized by the translocation t(8;21) which generates the oncogenic fusion protein AML1/ETO. AML1/ETO is found in high molecular weight complexes (HMWC) which are crucial for the block in myeloid differentiation of AML1/ETO transformed cells. Essential for HMWC formation is the alpha-helical nervy homology region 2 (NHR2) within ETO which serves as an interacting surface for oligomerization as well as association with members of the ETO protein family. Based on the observation that the integrity of the NHR2 domain is essential for transcriptional repressor activity of ETO, we designed a peptide (NC128) aimed to selectively interfere with AML1/ETO oligomerization. Confocal laser scanning microscopy revealed colocalization of NC128 with AML1/ETO in the nucleus. In protein-protein interaction studies we could demonstrate that the NHR2 domain in NC128 was necessary and sufficient for proper binding to AML1/ETO. Expression of NC128 in AML1/ETO positive cells led to inhibition of AML1/ETO HMWC formation as demonstrated by size-exclusion chromatography. NC128 expression in the AML1/ETO growth dependent Kasumi-1 cells restores transcription of AML1/ETO target genes. Among others, the expression of PU.1, a master regulator of myeloid differentiation, was significantly upregulated in the presence of NC128. In Kasumi-1 cells as well as in U937-AML1/ETO cells expression of NC128 almost completely overcomes the block of cytokine mediated differentiation induced by AML1/ETO. In the presence of NC128, Kasumi-1 cells lose their progenitor cell characteristics and upregulate marker of early monocytic differentiation. Likewise, NC128 expressing Kasumi-1 and SKNO-1 cells are arrested in cell cycle progression with a peak in the G1 phase of the cell cycle. The binding affinity of NHR2 peptide mutants to ETO directly correlates with the intensity of growth inhibition. A derivative of NC128 which retained all NHR2 amino acids (N89) maintained full binding capacity to ETO as well as antiproliferative effects, whereas a mutant of N89 lacking 7 C-terminal amino acids (N82) significantly lost binding capacity and its antiproliferative effect. Next a codon optimized expression construct was developed in order to increase the cellular expression levels of N89. Compared to N89, expression of this construct enhances growth arrest suggesting that the NHR2 peptides act in a dose dependent manner. To investigate the influence of NC128 on primary cells, human CD34 progenitor cells were immortalized with AML1/ETO. Expression of NC128 in these cells resulted in growth arrest, loss of CD34 expression and an increased rate of apoptosis. In order to directly deliver the peptide to AML1/ETO expressing cells, the NHR2 domain was fused to the HIV-1 Tat protein transduction domain. The bacterial expressed peptides were purified by affinity chromatography and were proven to be stable in serum-containing medium for several hours. Upon protein transduction into mammalian cells, recombinant NHR2 peptides could be detected in cellular lysates. Furthermore, we already can show by co-immunoprecipitation experiments that the transducible peptides are able to specifically interact with ETO protein. Our results propose that selective interference with NHR2-mediated oligomerization could provide a promising strategy for the inhibition of the oncogenic properties of AML1/ETO. Based on our results, we are aiming to develop screening strategies for both peptides and small molecule substances to interfere with NHR2 mediated AML1/ETO complex formation.