EPIGENETIC MODIFICATIONS IN A T(8;21) ACUTE MYELOID LEUKAEMIA CELL LINE CAUSED BY ADMINISTRATION OF TWO HDAC INHIBITORS, VPA AND SAHA

Introduction. The acute myeloid leukaemia (AML) cell line Kasumi-1 is characterized by the translocation t(8;21), manifested with the expression of AML1/ETO fusion protein, responsible for HDAC recruitment, determining transcriptional repression of target genes involved in myeloid maturation. We demonstrated that butyrates are able to restore histone acetylation and to induce granulocytic maturation, and we recently exhibited the selective anti-leukemic activity of low dose ITF2357 on AML1/ETO-positive cells. Transcriptionally active chromatin is characterized by histones with acetylated lysine tails. We investigated the acetylation of specific lysine residues on histone H4 and H3 after exposure to HDAC inhibitors (HDACi) members of different chemical family. We also analyzed the effects of these HDACi on the expression of Interleukine-3 (IL3) gene, target of AML1/ETO, and the amount of histones acetylation on IL3 promoter. Methods. Kasumi-1 cells were treated with VPA 2 mM or SAHA 1 M. Cells were lysed after different times of culture to extract cell proteins for the analysis by Western blotting and mRNA for the relative quantification in Real Time PCR or to immunoprecipates chromatin bound to acetylated H4 or H3. Results. VPA or SAHA treatment increased H4 and H3 total acetylation, with a more rapid kinetics for SAHA respect to VPA. Moreover, both HDACi were able to induce, even with a different trend, a marked increase of acetylation on 5, 8 and 16 lysine residues of H4 and lysine 9 of H3, but also in case of SAHA, on lysine 12 of H4. In particular VPA, even inducing transient modifications, presents a late effect but more lasting than SAHA, that acts at earlier times. These epigenetic modifications were paralleled by a resumption of transcriptional activity. In fact, we showed that VPA and SAHA induced the riexpression of IL3 gene, with a fast effect of SAHA and a delayed but more effective effect of VPA. This differential expression depended on a different amount of acetylated H4 and H3 on IL3 promoter. SAHA determined a rapid and simultaneous increase of acetylated H4 on 5, 8 and 12 lysine residues and acetylated H3 on 27 lysine residue, by which could be depend its rapid but transient effect on histones acetylation and IL3 expression. Whereas VPA induced a contemporary acetylation of H4 on 8, 12 and 16 lysine residues and H3 on 27 lysine residue, that could be justify its sustained effect for more times. Conclusion. These results suggest that VPA seems to have a more relevant effect respect to SAHA, inducing expression of gene silenced by AML1/ETO, such as IL3, and increasing H4 and H3 acetylation on IL3 promoter.