ABSTRACT This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT-CA- 23-045. The goal of the parent proposal is to understand how loss of protein homeostats (proteostasis) in aging blood-forming hematopoietic stem cells (HSCs) applies a selective pressure that promotes leukemia initiation. Loss of proteostasis is one of the least understood hallmarks of aging, particularly as it relates to malignant transformation. As an organism ages, misfolded proteins can accumulate in post-mitotic cells, or in cells that are largely quiescent. HSCs are particularly susceptible to a loss of proteostasis. Adult HSCs have low rates of protein synthesis relative to more frequently dividing lineage-committed blood progenitors. A low protein synthesis rate helps maintain proteostasis by preventing the biogenesis of misfolded proteins, and it is required to maintain adult HSC self-renewal capacity. However, we discovered that aged HSCs experience significant protein stress in vivo, and proteostasis must be actively maintained through changes in gene expression and stress-response pathways to sustain HSC self-renewal activity and longevity. These data indicate that aged HSCs must actively maintain proteostasis to remain functional, and loss of proteostasis may create a selective pressure that promotes clonal hematopoiesis and leukemia initiation. Based on these data, we hypothesize that a loss of proteostasis and pressure to maintain proteostasis in aging HSCs promotes clonal hematopoiesis and acute myeloid leukemia (AML) initiation in older adults. In the parent award (U01CA267031), we set out to determine if proteostasis disruption promotes clonal hematopoiesis and AML initiation during aging. Now, in collaboration with Dr. Eric Bennett, we generated a new genetic mouse model (Rps23K60R) with a mutation in the decoding region of the ribosome that enhances translation fidelity and mitigates proteostasis disruption in aging HSCs. These animals are viable, grossly normal, fertile and can live beyond 3 years of age and have functional HSCs as determined in transplantation studies. Using this new mouse model, our goal is to extend our parental award to directly test if preserving proteostasis in HSCs restricts the emergence of clonal hematopoiesis and impedes AML initiation. We will test whether delaying proteostasis disruption prevents clonal hematopoiesis and AML initiation during aging in the setting of a Dnmt3aR878H mutation. We will also examine if enhanced translation fidelity in Rps23K60R animals impedes AML initiation in aging HSCs using Dnmt3aR878H mutant animals with a cooperating NrasG12D mutation. Research outcomes from this supplemental research will determine if enhancing translation fidelity restrains the emergence of clonal hematopoiesis and reduces the incidence of AML in older animals. These studies will open new lines of investigation into a previously unappreciated link between age- related proteosta...