DNA METHYLATION PROFILE OF PRE-LEUKEMIC AND LEUKEMIC HEMATOPOIETIC STEM CELLS FROM ACUTE MYELOID LEUKEMIA DETECTED BY A SINGLE CELL DNA METHYLATION ASSAY

Abstract

ThiThe acute myeloid leukemias (AML) are clonal hematopoietic disorders characterized by myeloaccumulation of immature myeloid cells. The AML categorization define AML with Favorable, Intermediate I, Intermediate II and Adverse risk according the karyotype anomaly, genetic alterations, overall patient survival, post- treatment relapse risk and patients' response to treatment. Although all the AML pathogenesis knowledge the mechanisms involved in leukemogenesis process still unknown. Thus, this project aims to identify the methylation pattern of genes involved in the process of self-renewal and pluripotency of normal hematopoietic stem cells (HSC), pre-leukemic, leukemic stem cells (LSC) and myeloblasts from acute myeloid leukemia (AML) patients positive for DNMT3, TET2 and IDH1/2 mutations. Due to the low frequency of these cells subpopulations in peripheral blood of AML patients we intend to develop an effective methodology to detect the overall methylation profile with low DNA concentration. For this purpose the method "Reduced Representation Bisulfite Sequencing" (RRBS) was chosen to obtain the DNA methylation profile of a cell mapping (Mapping single-cell DNA methylation). The Single Cell DNA Methylation Assay test will provide the genes differential methylation profile among HSC, LSC and myeloblast and will enable to discriminate the genes involved in self-renewal and pluripotency processes in AML patients with intermediate risk.The differential methylation profile data of HSC, LSC and myeloblasts generated will be further analyzed in association with chromatin remodeling data previous obtained. The analysis of the methylation data and chromatin remodeling of the same samples will provide a better understanding of the epigenetic signature of HSC, LSC and myeloblast from AML patients positive for DNMT3a, IDH1/2 and TET2 mutations. These results will elucidate the network of genes involved in the regulation of normal and leukemic cell self-renewal and pluripotency. The results of this study will contribute to describe the molecular mechanisms involved in AML leukemogenesis and to design new and more efficient therapies for patients.