Proteomics of AML1/ETO Target Proteins: AML1–ETO Targets a C/EBP–NM23 Pathway

Abstract

Introduction The rational design of targeted therapies for acute myeloid leukemia (AML) requires the discovery of novel protein pathways in the systems biology of a specific AML subtype. We have shown that in the AML subtype with translocation t(8;21), the leukemic fusion protein AML1-ETO inhibits the function of transcription factors PU.1 and C/EBPα via direct protein-protein interaction. In addition, recently using proteomics, we have also shown that the AML subtypes differ in their proteome, interactome, and post-translational modifications. Methods We, therefore, hypothesized that the systematic identification of target proteins of AML1-ETO on a global proteome-wide level will lead to novel insights into the systems biology of t(8;21) AML on a post-genomic functional level. Thus, 6 h after inducible expression of AML1-ETO, protein expression changes were identified by two-dimensional gel electrophoresis and subsequent mass spectrometry analysis. Results Twenty-eight target proteins of AML1-ETO including prohibitin, NM23, HSP27, and Annexin1 were identified by MALDI-TOF mass spectrometry. AML1-ETO upregulated the differentiation inhibitory factor NM23 protein expression after 6 h, and the NM23 mRNA expression was also elevated in t(8;21) AML patient samples in comparison with normal bone marrow. AML1-ETO inhibited the ability of C/EBP transcription factors to downregulate the NM23 promoter. These data suggest a model in which AML1-ETO inhibits the C/EBP-induced downregulation of the NM23 promoter and thereby increases the protein level of differentiation inhibitory factor NM23. Conclusions Proteomic pathway discovery can identify novel functional pathways in AML, such as the AML1-ETO-C/EBP-NM23 pathway, as the main step towards a systems biology and therapy of AML.