HDAC INHIBITORS IN COMBINATION WITH HYPOMETHYLATING AGENTS INDUCE SPECIFIC CHROMATIN MODIFICATION AND TRANSCRIPTIONAL ACTIVATION IN CBF/AML CELLS

Acute myeloid leukaemia (AML) characterized by the presence of AML1/ETO fusion protein, or by other rearrangements involving core binding factors, share a common leukemogenic mechanism, determining transcriptional repression of target genes involved in myeloid maturation via permanent recruitment complexes containing HDACs, DNMTs and co-repressor molecules. We demonstrated that butyrates are able as single agents to restore histone acetylation and to reinduce gene expression allowing granulocytic maturation in AML1/ETO pos Kasumi-1 cell line, as well as in primary CBF-AML blasts. Core histone proteins can be acetylated by histone acetyl transferases (HAT) or methylated by histone methyltransferases (HMT) at N-terminal lysines, at different residues which are specifically associated to the transcriptional state of the nearby chromatin. Transcriptionally active chromatin is characterized by histones with acetylated lysine tails, and methylated lysine 4 of histone H3, whereas dimethylated lysine 9 of H3 and deacetylated lysines in H4 and H3 seem associated with transcriptional silence . We investigated the acetylation and methylation pattern of lysine residues of histone H4 and H3 in our AML1/ETO pos cell model, prior and after exposure to butyrate as HDAC inhibitor alone or combined with azacitidine, as DNMTi. Cells were lysed after 6-12-72 and 96 hrs of culture and whole cell lysate was analysed by SDS-PAGE electrophoresis and Western blot, with antibodies specific to different lysine residues of the two histones. K5 and K16 residues of histone H4 were not acetylated in the absence of butyrate and their acetylation strongly increased after 6 h of butyrate administration. K8 and K12 residues showed a basal acetylation which was slightly enhanced by the cotreatment with HDACi and DNMTi . Lysine methylation of histone H3 was also evaluated. Butyrates determined a significant reduction of di-methylated K9 H3. Di-methylated K4 H3 was, on the other hand, unmodified after butyrate administration. Azacitidine was not influencing the lysine methylation. These histone epigenetic modifications were paralleled by an increase of p21/cip1 protein expression and a decrease of p27/kip1 protein expression. Appearance of a conspicuous amount of p57/kip2, an as yet uncharacterised oncosuppressor, after 72-96 h treatment with butyrates was also observed. Moreover, CEBP family expression and phosphorilation were induced. In parallel, AML1/ETO mRNA and protein expression was markedly reduced after D1 exposure.