Abstract
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.
Highlights
The translocation t(8;21) is found in 10–15% of acute myeloid leukemia (AML), representing one of the most prevalent chromosomal aberrations associated with AML
RUNX1-ETO acts as dominant-negative inhibitor of RUNX1-dependent gene expression by recruiting the corepressor proteins NCoR and SMRT bound to the ETO moiety of the fusion protein [6,7,8]
NCoR and SMRT can interact with mSin3a and histone deacetylases (HDAC) [9,10], assembling a repressor complex which leads to transcriptional silencing of RUNX1 target genes like MDR1 [7], FOS [11], CDKN2A [12] and BCL2 [13]
Summary
The translocation t(8;21) is found in 10–15% of acute myeloid leukemia (AML), representing one of the most prevalent chromosomal aberrations associated with AML. It recruits the histone acetyl transferase (HAT) p300/CBP complex, facilitating histone acetylation and, more importantly, the acetylation of RUNX1-ETO itself This results in increased accessibility of regulatory elements and the recruitment of other activating transcription factors, and allows the transactivation of target genes, e.g. ID1, CDKN1A (p21) and EGR1 [14]. Kasumi-1 cells display a block in transition from the G1- to S-phase of the cell cycle, accompanied by reduced apoptosis and the induction of cellular senescence, in response to RUNX1-ETO RNAi [27] In line with these findings, Dunne et al [28] have demonstrated the impact of RUNX1-ETO silencing on the expression of genes associated with proliferation and differentiation, such as Insulin-like growth factor-binding protein 7 (IGFBP7) and Cathepsin G (CTSG), in Kasumi-1 and patient derived AML blasts with t(8;21) by oligonucleotide array and qRT-PCR
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