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

Alterations in chromatin organization are a common shared mechanism in leukemogenesis. On this basis, many drugs affecting epigenetic modulations have been proposed as therapy of hematopoietic neoplasms, most of the times, unfortunately, without molecular selection of the types of disease more prone to respond to these agents. Nevertheless, hypomethylating agents have shown outstanding efficacy in myelodysplastic syndromes and acute leukemias, whereas histone deacetylase inhibitors (HDACi) seem the most powerful therapeutic tools in T cell cutaneous lymphomas. We think that HDACi could be essential in the treatment of some forms of acute myeloid leukemias, i.e CBF-AMLs. We analysed the effects of a new hydroxamic acid oral derivative, ITF2357 on AML1-ETO positive AML blast cells. We compared both the efficacy of ITF2357 with that of SAHA, and its activity on non CBF-AML blast cells. ITF2357and SAHA were employed at equimolar concentrations ( 0.1, 0.5, 1, 5 μM) in cell culture. Kasumi-1 and primary 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. At the end of culture, cell proliferation and apoptosis were evaluated in parallel with histone acetylation. ITF2357 0.1microM was able to block proliferation of CBF-AML cells and induce significative apoptosis (evaluated as Annexin V positivity) after 24 hours, whereas SAHA induced the same rate of apoptosis and cell growth inhibition at 1 microM dose. Non CBF pos AML cells were significantly less sensitive to ITF2357 and SAHA inhibitory effects. When we evaluated histone H3 and H4 acetylation, maximal acetylating activity of ITF2357 was demonstrated in AML1 ETO pos cells after 6 hours of exposure to the drug, was mantained up to 24 hrs, and then was decreasing. ITF2357 0.1microM was able to induce potent acetylation. SAHA induced significantly histone H3 and H4 acetylation with a time schedule similar to the new hydroxamic derivative, but at doses 1 log higher. When we studied at confocal microscope the cellular distribution of the AML1ETO/DNMT/HDAC complex, we could demonstrate, after treatment of Kasumi-1 cells with ITF2357 0.1 microM, a displacement of DNMT1and HDAC1 from nucleous to cytoplasm paralleled by degradation of AML1ETO protein. At the same time, p300 was spotted inside the cell nucleous. The specificity of action of the HDACinhibitor on cells bearing the AML1ETO oncogene was demonstrated Chromatin Immunoprecipitation (ChIP) of acetylated histone H4. Consistently with our evidences at immunofluorescent staining, in Kasumi-1 cells, treatment with ITF2357 restored the transcription of IL-3, whose gene is under direct control of AML1ETO. The pattern of methylation of the promoters of several genes ( p15ink, e-cadherin, DAPkinase, IL-3) was analysed, but we could not demonstrate by methylation specific PCR and sequencing any decrease in CpG hypermethylation induced directly by treatment with ITF2357. In conclusion, ITF 2357 is the most potent oral HDACi of the hydroxmic acid family tested so far in vitro on AML cells. Its activity reaches a maximum rapidly and effects are stable for 24 hours. ITF2357, but also other HDACinhibitors show a significant specificity for AML1ETO cells in terms of biological effects and of molecular activity on deflecting the stability of the DNA binding of DNMT/HDAC repressor complex. Our observations strongly sugget that therapy with HDACi should be considered only for molecular subtypes of AML in which chromatin organizing protein are involved in the pathogenesis of the disease and thus demonstrated HDACi specific activity may influence the natural history of the leukmeia, most probably as we also demonstrated with the support of hypomethylating agents. ITF2357 is in Phase II clinical trials for myeloma and inflammatory chronic diseases and deserves to be considered as a targeted drug to implement therapy of CBF AMLs.