SPECIFIC DEGRADATION OF THE AML1-ETO/COREPRESSOR COMPLEX BY A NOVEL ORAL HYDROXAMIC ACID DERIVATIVE (2006)

Alterations in chromatin organization are a common shared mechanism\nin leukemogenesis. On this basis, many drugs affecting epigenetic\nmodulations have been proposed as therapy of hematopoietic neoplasms,\nmost of the times, unfortunately, without molecular selection\nof the types of disease more prone to respond to these agents. Nevertheless,\nhypomethylating agents have shown outstanding efficacy in\nmyelodysplastic syndromes and acute leukemias, whereas histone\ndeacetylase inhibitors (HDACi) seem the most powerful therapeutic\ntools in T cell cutaneous lymphomas. We think that HDACi could be\nessential in the treatment of some forms of acute myeloid leukemias, i.e\nCBF-AMLs. We analysed the effects of a new hydroxamic acid oral\nderivative, ITF2357 on AML1-ETO positive AML blast cells. We compared\nboth the efficacy of ITF2357 with that of SAHA, and its activity\non non CBF-AML blast cells. ITF2357and SAHA were employed at\nequimolar concentrations ( 0.1, 0.5, 1, 5 μM) in cell culture. Kasumi-1 and\nprimary AML1ETO pos cells, HL 60 cells and THP-1 cells were cultured in RPMI medium, 10% FCS, exposed at drugs for 3-6-24-48-72-96 hours.\nAt the end of culture, cell proliferation and apoptosis were evaluated in\nparallel with histone acetylation. ITF2357 0.1microM was able to block\nproliferation of CBF-AML cells and induce significative apoptosis (evaluated\nas Annexin V positivity) after 24 hours, whereas SAHA induced\nthe same rate of apoptosis and cell growth inhibition at 1 microM dose.\nNon CBF pos AML cells were significantly less sensitive to ITF2357 and\nSAHA inhibitory effects. When we evaluated histone H3 and H4 acetylation,\nmaximal acetylating activity of ITF2357 was demonstrated in\nAML1 ETO pos cells after 6 hours of exposure to the drug, was mantained\nup to 24 hrs, and then was decreasing. ITF2357 0.1microM was\nable to induce potent acetylation. SAHA induced significantly histone H3\nand H4 acetylation with a time schedule similar to the new hydroxamic\nderivative, but at doses 1 log higher. When we studied at confocal\nmicroscope the cellular distribution of the AML1ETO/DNMT/HDAC\ncomplex, we could demonstrate, after treatment of Kasumi-1 cells with\nITF2357 0.1 microM, a displacement of DNMT1and HDAC1 from\nnucleous to cytoplasm paralleled by degradation of AML1ETO protein.\nAt the same time, p300 was spotted inside the cell nucleous. The specificity\nof action of the HDACinhibitor on cells bearing the AML1ETO\noncogene was demonstrated Chromatin Immunoprecipitation (ChIP) of\nacetylated histone H4. Consistently with our evidences at immunofluorescent\nstaining, in Kasumi-1 cells, treatment with ITF2357 restored\nthe transcription of IL-3, whose gene is under direct control of\nAML1ETO. The pattern of methylation of the promoters of several\ngenes ( p15ink, e-cadherin, DAPkinase, IL-3) was analysed, but we could\nnot demonstrate by methylation specific PCR and sequencing any\ndecrease in CpG hypermethylation induced directly by treatment with\nITF2357. In conclusion, ITF 2357 is the most potent oral HDACi of the\nhydroxmic acid family tested so far in vitro on AML cells. Its activity\nreaches a maximum rapidly and effects are stable for 24 hours. ITF2357,\nbut also other HDACinhibitors show a significant specificity for\nAML1ETO cells in terms of biological effects and of molecular activity\non deflecting the stability of the DNA binding of DNMT/HDAC repressor\ncomplex. Our observations strongly sugget that therapy with HDACi\nshould be considered only for molecular subtypes of AML in which\nchromatin organizing protein are involved in the pathogenesis of the\ndisease and thus demonstrated HDACi specific activity may influence\nthe natural history of the leukmeia, most probably as we also demonstrated\nwith the support of hypomethylating agents. ITF2357 is in Phase\nII clinical trials for myeloma and inflammatory chronic diseases and\ndeserves to be considered as a targeted drug to implement therapy of\nCBF AMLs.