Acetylation of p53 Is Involved in Valproic Acid Induced Death of AML Cells

Abstract

Abstract 2461 Introduction:Impaired acetylation level of histone and non-histone proteins, due to increased histone deacetylase (HDAC) activity relates to pathological malignancies including leukemias. The tumor suppressor protein p53 is an important non-histone target of HDACs and regulates key cellular processes such as DNA repair, cell-cycle arrest, senescence and apoptosis. The p53 protein undergoes several post-translational modification and among them acetylation allows p53 to induce the expression of genes relevant to tumor suppression. In certain cases of leukemias, overexpression of HDACs has been associated with inactivation of p53 via deacetylation. Therefore, increasing the acetylation of p53 by inhibition of HDACs can be an effective approach to trigger the function of p53 in cancer cells. The anticonvulsant valproic acid (VPA) has been shown to be an efficient HDAC inhibitor (HDACI), able to induce apoptosis in acute myeloid leukemia (AML) cells, and has recently entered clinical trials as a potential therapeutic agent. Although VPA exerts strong anti-tumor activity against haematological malignancies, the molecular mechanism of events involved in VPA-mediated death of leukemia cells is largely unclear.Methods/results: To identify the potential downstream targets triggered by VPA in leukemia cells, the acetylation profile in total cell lysate was compared between VPA treated and untreated NB4 leukemia cell line. We observed increased acetylation of several proteins ranging from 20 KDa to 150 KDa after VPA treatment. Among them acetylation of p53 at lysine residue 382, critical for p53 function, was detected. This result motivated us to further elucidate the functional significance of p53 acetylation in leukemia cells. VPA mediated p53 acetylation resulted in more than two fold induction of several p53 target genes, such as p21, BAX, GADD45A. By knockdown of p53 using specific shRNA against mRNA of p53 we show that VPA mediated expression of p21 was independent of p53, in contrast VPA mediated expression of BAX required presence of p53. Activation of p53 by VPA involved increased expression of genes involved in cell-cycle arrest and apoptosis. Therefore we performed cell cycle analysis using BrdU and evaluated apoptosis by Annexin V staining after challenging the leukemia cells with VPA (0.5 mM, 1mM and 2mM). We observed a dose dependent decrease of cells entering S-phase and this was accompanied by increase of cells undergoing cell cycle arrest and apoptosis. VPA induced apoptosis and cell cycle arrest was significantly attenuated in p53 knock down cells, indicating p53 as an active player in VPA mediated killing of leukemic cells. To further address the clinical relevance of VPA mediated p53 signalling, we performed experiments with primary blasts isolated from AML patients (n = 10). Treatment with 1mM VPA imposed cytotoxic effect on all leukemia cells tested with varying intensities (6 high responsive and 4 low responsive). Acetylation of p53 was dramatically increased in the six patient samples which were highly sensitive to VPA in contrast to 4 patient samples which were less responsive. Furthermore increased acetylation of p53 in these blast samples was subsequently associated with increased mRNA expression of both p21 and BAX. Conclusion:In summary we demonstrate that p53 is an important player downstream of VPA signaling and suggest that induction of p53 acetylation by VPA plays a decisive role in imposing cytotoxic effect on AML cells. Disclosures:No relevant conflicts of interest to declare.