The oncogenic fusion protein RUNX1-ETO is a product of the t(8;21) translocation and consists of the hematopoietic transcriptional master regulator RUNX1 and the repressor ETO. RUNX1-ETO is found in 10-15% of acute myeloid leukemia and interferes with the expression of genes that are essential for myeloid differentiation. The neutrophil serine protease Cathepsin G is one of the genes suppressed by RUNX1-ETO, but little is known about its impact on the regulation of other lysosomal proteases. By lentiviral transduction of the t(8;21) positive cell line Kasumi-1 with an RUNX1-ETO specific shRNA, we analyzed long-term effects of stable RUNX1-ETO silencing on cellular phenotypes and target gene expression. Stable anti RUNX1-ETO RNAi reduces both proliferation and apoptosis in Kasumi-1 cells. In addition, long-term knockdown of RUNX1-ETO leads to an upregulation of proteolytic activity in Kasumi-1 cells, which may be released in vitro upon cell lysis leading to massive degradation of cellular proteins. We therefore propose that protein expression data of RUNX1-ETO-silenced Kasumi-1 cells must be analyzed with caution, as cell lysis conditions can heavily influence the results of studies on protein expression. Next, a mass spectrometry-based approach was used to identify protease cleavage patterns in RUNX1-ETO-depleted Kasumi-1 cells and Neutrophil Elastase has been identified as a RUNX1-ETO candidate target. Finally, proteolytic activity of Neutrophil Elastase and Cathepsin G was functionally confirmed by si/shRNA-mediated knockdown in Kasumi-1 cells.