A recently published study from the laboratory has shown that acute myeloid leukemia patient samples have undergone different degrees of methylation throughout the CpG island of the gene. We observe three types, those that have a high degree of hypermethylation, those that have essentially no methylation and those that have an intermediate degree of methylation. In all cases the gene was not expressed. When there is no hypermethylation in patient samples at the p15INK4b locus CpG island we presume that there are other mechanisms that inhibit expression of the gene and for some acute myeloid cases we have determined an alternate pathway. In acute myeloid leukemia with inv(16) we have found that the INK4b silencing is caused by another mechanism. In these leukemias the inv(16)-encoded core binding factor beta-smooth muscle myosin heavy chain (CBFbeta-SMMHC) protein targets the promoter and displaces the transcription factor RUNX1 causing transcriptional repression. For the inv(16) acute myeloid leukemia patients, re-expression from the INK4b locus would not be predicted to occur using hypmethylating drugs. In our onging experiments we are determining repressive histone modifications that accompany the three different methylation states of p15INK4b DNA . Chromatin immunoprecipitation and DNA tiling microarrays (ChIP-on-chip) with 20bp resolution are being used to assess the distribution of histone modifications over a 1.2 megabase region of human chromosome 9 including p15INK4b and adjacent tumor suppressor genes p14ARF and p16INK4a. We found that acute myeloid leukemia cell lines with p15INK4b high and intermediate degrees of hypermethylation (Kasumi-1, KG-1, and KG-1a) have high levels of the repressive histone modification, trimethylation of lysine 27 of histone H3 (H3K27me3). Remarkably, this modification was found to span the entire INK4b-ARF-INK4a region while little binding was observed in adjacent regions of chromosome 9. Binding of EZH2, the polycomb associated H3K27 histone methyltransferase, co-localized with H3K27me3 distribution over the INK locus. H3K27me3 was not identified at this region in acute myeloid leukemia cell lines without p15INK4b DNA methylation (U937 and HL-60). Rather, histone modifications associated gene activation, trimethylation of lysine 4 of H3 (H3K4me3) and acetylation of lysine 9 (H3K9Ac), were found at the p15INK4b promoter in the latter cells. Enrichment of another repressive histone modification, trimethylation of histone H3 on lysine 9 (H3K9me3), did not correlate with the DNA methylation status of p15INK4b and appeared highest in exons 2 and 3 of p16INK4a in most cell lines. Since p15INK4b reactivation has been described as a component of a patients response to epigenetic therapies in acute myeloid leukemia treatment, we sought to determine the dynamics of histone modifications following treatment with the DNA methyltransferase (DNMT) inhibitor 5-aza-2-deoxycytidine and histone deacetylase (HDAC) inhibitor tricostatin A. So far only KG-1 cells with intermediate hypermethylation, have been examined. A reduction in p15INK4b DNA methylation and an increase in cell cycle arrest were observed following treatment with DNMT inhibitors. In contrast to other reports, treatment with HDAC inhibitors alone was capable of reducing p15INK4b DNA methylation, increasing mRNA expression and inducing cell cycle arrest suggesting that a repressive chromatin structure contributes to the DNA methylation in this cell line. Loss of DNA methylation was not sufficient for reactivation of p15INK4b expression as detectible expression was only observed following the combined treatment of DNMT and HDAC inhibitors. Reactivation was associated with an increase in the activation-associated histone modifications H3K4me3 and H3K9Ac at the promoter region and, unexpectedly, maintenance of the repressive modification H3K27me3. This bivalent histone modification pattern is characteristic of many developmentally poised genes in embryonic stem cells and correlates with the histone methylation status of p15INK4b that we found in CD34+ bone marrow progenitor/stem cells. This data supports the further use of HDAC inhibitors in addition to DNA demethylating agents for the treatment of AML.
