The rational design of targeted therapies for leukemia requires the discovery of protein pathways in leukemia cells and the determination of their functional relevance in the systems biology of a specific leukemia type. Proteomics based on mass spectrometry has become a powerful method to detect the composition of complex protein mixtures. Acute promyelocytic leukemia (APL), characterized by translocation t(15;17) encoding the fusion protein PML-RARalpha, and the differentiation therapy of APL with retinoic acid (ATRA) serve as model system for effective targeted therapies of cancer. PML-RARalpha is activated via cleavage through neutrophil elastase and exerts its biological effects via targeting other proteins, such as inducing the reticulum-associated degradation of N-CoR.

We, therefore, hypothesize that the systematic identification of target proteins of PML-RARalpha in APL on a global proteome-wide level by proteomics-based techniques might be most relevant to understand the PML-RARalpha-induced pathogenesis on a post-genomic functional level.

The aim of this project is to identify the global protein expression changes 6 hours after induction of PML-RARalpha expression in a U937 cell line model with Zink-inducible expression of PML-RARalpha via two-dimensional gel electrophoresis using pH 3-10, pH 4-7, and pH 6-11 and MALDI-TOF mass spectrometry, and to determine the mechanism and function of one major discovered PML-RARalpha-induced pathway using siRNA, transfection, gelshift, ChIP, and promoter reporter assays with site-directed mutagenesis (specific aim 1).