PROJECT SUMMARY/ABSTRACT This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT-CA- 24-029. Aging is associated with the accumulation of somatic mutations in hematopoietic stem cells (HSCs), leading to clonal hematopoiesis (CH) and increased risk for hematologic malignancies. This supplement project, led by Dr. Peter van Galen in collaboration with Drs. Jennifer Trowbridge and Ross Levine, focuses on the role of stem cell heterogeneity and inflammation in the clonal expansion of HSCs during aging. The long-term goal is to promote healthy aging of the blood system based on a molecular understanding of HSC biology. The objective of this application is to determine if inflammatory HSCs link aging to clonal expansion. The central hypothesis is that inflammatory HSCs increase with age and provide a reservoir for the clonal expansion of DNMT3A-mutated HSCs, and that the inflammatory HSC subset shares properties between humans and mice. The central hypothesis will be tested by pursuing three specific aims: 1) Quantify the emergence of clonal inflammatory HSC populations with age, 2) Inflammatory HSCs as a reservoir for DNMT3A-mutated clonal expansion, and 3) Define the conserved inflammatory HSC state by integrating mouse and human data. This work will involve comprehensive studies using cutting-edge single-cell technologies and a biobank of bone marrow samples established in the Van Galen lab. The analysis will encompass three age cohorts (young, middle-aged, and aged) and DNMT3A-mutated CH subjects. In addition, we will use advanced bioinformatics to dissect the concordance of HSC heterogeneity between mouse and human systems, focusing on the inflammatory HSC state. The research is innovative because it integrates HSC heterogeneity and clonal expansion, incorporating the aging-associated expansion of inflammatory HSCs to explain the fitness advantage conferred by DNMT3A mutations using a multidisciplinary approach that combines molecular biology, genetics, and bioinformatics. It extends beyond the current understanding by focusing on the interplay between DNMT3A mutations and inflammation, providing insights into the selective pressures that drive the clonal evolution of HSCs with age. The significance of this research lies in its potential to identify novel molecular mechanisms underlying age-associated hematologic diseases and to uncover new therapeutic targets. By deciphering the complex interactions between epigenetic dysregulation, inflammatory signaling, and aging, this project aims to pave the way for interventions that could mitigate the risk of hematologic malignancies in the aging population, ultimately improving health outcomes and quality of life.