Expression of the Nuclear Corepressor Ski Predicts Response to Histone Deacetylase Inhibitor Valproic Acid in Cells of Acute Myeloid Leukemia.

Abstract 2410

Acute myeloid leukemia (AML) with deletion of chromosome 7 (−7) or 7q (del7q) has a poor prognosis. Using gene expression analysis, we previously identified the nuclear oncogene Ski as being up-regulated in AML, especially in AML with −7/del7q. We demonstrated that the transcriptional corepressor Ski acts as an inhibitor of vitamin A induced myeloid differentiation through interaction with N-CoR recruiting histone deacetylases (HDAC) (Ritter et al., Leukemia 2006). HDAC inhibitors such as valproic acid (VPA) promote histone acetylation, induce apoptosis and cell growth arrest in tumor cells (e.g. Kraemer et al., Trends Endocrinol Metab 2001). As Ski interacts with HDACs the aim of our investigation was to test the effect of HDAC inhibitors to cellular differentiation, apoptosis and Ski expression in primary AML cells. Treatment of the AML cell line HL60 expressing Ski with the HDAC inhibitor VPA enhances expression of the myeloid differentiation markers CD11b and CD11c as well as apoptosis. To address whether this effect is also observed in primary AML cells, we isolated mononuclear cells from blood or bone marrow of 12 AML patients (first diagnosis or relapse) and treated these cells with the HDAC inhibitor VPA. After harvesting, Ski protein expression was determined by Western blot. Flow cytometry was used to analyse expression of the differentiation markers CD11b and CD11c and apoptosis after propidium iodide staining. Of six AMLs with Ski protein expression, four responded either with differentiation or apoptosis, whereas none of six primary AMLs without Ski expression showed an effect to VPA compared to untreated control cells. To test whether other HDAC inhibitors would also reveal this effect we treated primary AML cells with further HDAC inhibitors (TSA, SAHA, LBH589) and confirmed our observation that HDAC inhibitors renders AML cells expressing Ski sensitive to differentiation.

In parallel, we observed that VPA down regulates Ski in AML cells as well as in melanoma cell lines with high Ski protein levels. Our goal was to elucidate the molecular background of Ski reduction by VPA. Treatment of melanoma cells expressing Ski with VPA and/or the proteasomal inhibitor MG132 revealed that decrease of Ski depends on proteasomal degradation. The ring finger protein Arkadia (RNF111) is an E3-ligase of Ski (Nagano et al., J Biol Chem 2007) and we tested whether Arkadia is involved in Ski reduction after VPA addition. First we demonstrated that Arkadia expression is inversely associated with Ski expression in several AML cell lines. Furthermore the expression of Arkadia is induced by VPA on protein and RNA level while Ski protein is down regulated in melanoma cells. We also found that knockdown of Arkadia using RNAi impairs reduction of Ski by VPA in melanoma cells.

Taken together our data suggest that high Ski expression in AML cells could be a molecular marker for VPA therapy. Thereby, VPA reduces the expression of the oncogene Ski as VPA induces expression of the E3-ligase Arkadia which abolish Ski by proteasomal degradation.