PROJECT SUMMARY Acute myeloid leukemia (AML) occurs mostly in adults 65 years and older and is associated with age-related clonal hematopoiesis (CH). This condition that results from the clonal expansion of mutationally-marked hematopoietic stem and progenitor cells (HSPC). These HSPC expansions often feature somatic mutations in leukemia-associated genes such as the epigenetic regulator TET2. TET2-mutant CH is a risk factor for AML, and CH progression to AML is promoted by acquiring cooperating mutations such as constitutively active FLT3ITD. Yet, it is unclear how aging and TET2 mutation interact to drive the evolution of CH to leukemia. We seek to reveal the epigenetic mechanisms for how CH evolves to leukemia in an aging microenvironment, laying the foundation for therapeutic strategies to block this evolution and prevent leukemia in the aging US population. To this end, we will leverage our team's complementary expertise in intra-tumor heterogeneity and computational epigenomics; aging, cancer evolution, and leukemogenesis; inflammation and hematopoietic stem cell (HSC) biology; and mouse HSC proliferation and functional heterogeneity. Our preliminary results in mice show that HSPC epigenetic heterogeneity increases in old age. This process occurs in an aged bone marrow (BM) microenvironment characterized by inflammation and altered HSPC support. The rise of epigenetic and transcriptomic heterogeneity in HSC with Tet2 mutation (Tet2MT) and Flt3ITD precedes leukemic transformation. We hypothesize that aging and TET2 mutation cooperatively enhance epigenetic heterogeneity and evolvability of HSPC, thus contributing to clonal expansion and leukemogenesis. In Aim 1, we will determine the combined impact of aging and Tet2MT on epigenetic heterogeneity and gene regulation in HSPC by examining the somatic epigenomic landscape of Tet2MT HSPC from young (2-3 months) and old (22 months) mice, including single-cell (sc) transcriptomes by scRNA-seq, open-chromatin profiles by snATAC-seq, and DNA methylomes by RRBS. We expect to define epigenetic configurations in old Tet2MT HSPC associated with genes in self-renewal, quiescence, and responses to inflammation and stress. In Aim 2, we will define epigenetic configurations of Tet2MT HSPC that are adaptive in the aged context. We will transplant genetically barcoded HSPC into young or old mice and assess subclone expansion/contraction to determine if an age-dependent selection is for all Tet2MT HSPC or only a subset. We will use scRNA-seq and snATAC-seq to define molecular signatures and epigenetic heterogeneity of barcoded HSPC that are positively selected in the aged context. In Aim 3, we will define the epigenetic configurations of Tet2MT HSPC that are permissive to Flt3ITD-induced transformation in the aged context. We will transduce the Flt3ITD gene into young or aged Tet2MT or wild-type HSC and then transplant these genetically barcoded cells into young or old host mice. We will assess the epigenetic ...