INVESTIGATING THE ROLE OF COHESIN MUTATIONS AND EPIGENETIC HETEROGENEITY IN PRE-LEUKEMIC HEMATOPOIETIC STEM CELL CLONAL EVOLUTION

Abstract

Acute myeloid leukemia is a highly heterogeneous disease characterized by the accumulation of poorly differentiated myeloid progenitors. Mutations in epigenetic regulators are highly recurrent in AML, underscoring the importance of the epigenome in affecting this disease. Past research in our lab has identified an evolutionary process through which, and has tracked regulatory chromatin signatures that contribute to leukemia pathogenesis; however, the regulatory programs and epigenetic factors contributing to AML relapse remains largely unexplored. To model pre-leukemic mutations, we utilize CRISPR/Cas9 to genetically modify a cohesin complex subunit (STAG2) in cord blood-derived CD34+ cells and include a homology donor encoding GFP to track the edited cells. Chromatin accessibility in STAG2-edited cells is measured using ATAC-seq, and epigenetic state is assessed both in bulk and at the single cell level. Following with past studies of cohesin mutations in CD34+ cells, genome editing of the endogenous STAG2 locus results in defects in erythroid differentiation, enhanced self-renewal, and retention of stem cell surface markers. STAG2 knockout cells display subtle yet distinct alterations in chromatin accessibility when compared to control cells. Single-cell ATAC-seq reveals distinct regulatory signatures and epigenetic heterogeneity in both primary patient-derived pHSCs and STAG2-engineered cells and identifies RUNX and GATA family members as critical factors in STAG2-edited CD34+ cells. Ongoing work involves characterizing cis and trans regulatory elements responsible for driving epigenetic heterogeneity in pHSCs. The successful completion of these studies will clarify the role of pre-leukemic epigenetic mutations in leukemia evolution and further elucidate the functional consequences of cohesin deficiency in hematopoietic stem cells.