Epigenetic alterations marked/mediated by promoter methylation and chromatin modifications contribute to neoplastic phenotypes (disease pathogenesis, progression, resistance to therapy) by stably affecting the expression of critical genes and pathways. In CML, preliminary data suggest an important contribution of epigenetic processes marked by promoter DNA methylation in the progression of disease, as well as resistance to tyrosine kinase inhibitors (TKIs). Epigenetic processes may also affect immunity against CML neoplastic cells, for example by masking tumor antigens. Based on this, we propose the following hypotheses: (i) Epigenetic processes (DNA methylation, histone code alterations) contribute to disease progression (CP to AP and BP) in CML, as well as to mutation-independent resistance to TKIs, by affecting the function of critical pathways through stable modulation of gene expression, (ii) Targeting epigenetic silencing by DNA methylation inhibitors and histone modifying drugs will complement TKI therapy in advanced CML. To address these hypotheses, we propose the following specific aims: (1) Global analysis of the CML epigenome. Using Methylated CpG Island (MCA) and Chromatin Immunoprecipitation coupled with deep sequencing (on the Solexa platform), we will determine common epigenetic abnormalities in CML cells (compared to normal CD34+ cells) at various phases (CP, AP, BP) and after resistance to TKIs develop. We will validate selected loci using standard technology and gene expression analysis, and use a pathway approach to studying functional epigenetic anomalies in CML. In addition to unsupervised approaches to pathway identification, we will also examine specifically the PISK pathway (an interaction with project 4) and the JAhC/STAT pathway (an interaction with project 5) and potential immunomodulators such as tumor antigens (in an interaction with project 2). (2) Predictive and prognostic testing of selected epigenetic marks in prospective cohorts. In preliminary data, we have Identified a potential DNA methylation signature of progression in CML, as well as TKI resistance. In this aim, we will test the prognostic/predictive value of this gene panel in prospective cohorts of patients with early chronic phase CML enrolled on clinical trials of TKI (an interaction with project 1), as well as trials of stem cell transplantation for TKI resistant CML at all phases (an interaction with project 2). In subsequent years, genes/pathways identified in aim 1 will be tested in the same cohorts to potentially improve on the predictive power of these markers. (3) Pharmacodynamic analysis of epigenetic parameters in patients treated with epigenetic acting drugs. In this specific aim, we will analyze samples from patients enrolled on a clinical trial ofthe hypomethylating agent decitabine in combination with the TKI dasatinib (collaboration with project 1), and a clinical trial of azacitidine to stimulate anti-CML immunity (project 2). We will correlate epigenetic patterns at baseline with likelihood of response, as well as epigenetic modulation (i.e. pharmacodynamic measurement) with likelihood of response to the drug/stem cell transplant intervention. This project will provide definitive and global data on epigenetic alterations in CML, pathways affected, prognostic significance as well as integration of epigenetic acting drugs in the treatment of this disease.
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