PROJECT SUMMARY/ABSTRACT Clonal hematopoiesis of indeterminate potential (CHIP) broadly describes the clonal expansion of blood cells derived from hematopoietic stem cells (HSCs) with somatic pre-leukemic mutation(s). CHIP is strongly linked to aging and confers to an increased risk for blood cancers, non-hematological diseases, and all-cause mortality. ASXL1 is one of the commonly mutated genes in CHIP. ASXL1 mutations are predominantly nonsense or frameshift mutations. In particular, the hotspot frameshift mutation at codon 646 (corresponding to codon G643 in mice-Asxl1tm) represents a pathological CHIP mutation with high risk to drive myeloid diseases. We recently discovered that Asxl1tm/+ bone marrow (BM) cells and HSCs underwent significant expansion in old/aged recipients (20-24 months) but not in young recipients (2-4 months old). This expansion was associated with alterations in epigenetic landscape. Further characterization of young vs old BM microenvironment (BMM) and serum revealed local and systemic inflammation and expansion of mesenchymal stromal cells (MSCs) and endothelial cells (ECs), two important regulatory components of BM niche. MSC expansion was likely due to enhanced MSC survival, while both accumulation of senescent cells and increased survival attributed to EC expansion. Consistent with our observation, treatment of ABT-263, a potent inhibitor of anti-apoptotic proteins Bcl-2 and Bcl-xl, reduced the senescent MSCs in vitro. Moreover, ABT-263 effectively removed accumulated MSCs, moderately reduced number of ECs, and partially mitigated the expansion of phenotypic HSCs in the old mice. CITE-Seq analysis of RNA and ~120 immune cell surface proteins at single cell level identified aging- associated cellular and molecular changes in old BM cells, including expansion of inflammatory neutrophil subsets and overexpression of IL-1β. These alterations can be mitigated by dietary supplementation of nicotinamide riboside (NR), a NAD+ precursor. In addition, human myeloid leukemia cells with ASXL1 mutations were sensitive to GSK525762 (GSK), a pan-BET inhibitor. Based on our preliminary results, we hypothesize that aged BMM promotes the expansion of Asxl1tm/+ HSCs. Targeting aged BMM and Asxl1tm/+ hematopoietic cells through NR, ABT263, and/or GSK may prevent and/or inhibit Asxl1tm/+ HSC expansion. In this grant application, we propose the following aims to test our hypothesis: 1) To investigate how aged BMM interacts with Asxl1tm/+ HSCs to promote their expansion; and 2) To determine whether targeting aged BMM and Asxl1tm/+ hematopoietic cells alter Asxl1tm/+ HSC expansion in old recipients. Our proposal is in response to the SHINE Program from NIA (PAS-22-096), aiming to provide new insights into the pathogenesis, prevention, and potential treatment of nonmalignant hematologic diseases.