ABSTRACT The goal of this 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. This stresses the cell and can drive adaptive changes that are required to rebalance proteostasis. 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. This helps maintain proteostasis by preventing the biogenesis of misfolded proteins, and it is required to maintain adult HSC self- renewal capacity. However, we have 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. In this regard, we have generated preliminary data demonstrating that Hsf1, a critical proteostasis sensor that dynamically remodels the proteostasis network in response to stress, is activated within aging HSCs where it is required to attenuate protein synthesis and preserve HSC self-renewal capacity. 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. We propose two aims to test this hypothesis. In the first aim, we will use Aarssti/sti mice, which have a defect in tRNA editing activity, to disrupt proteostasis in young and old adult mice. We will test whether proteostasis disruption accelerates clonal hematopoiesis and AML initiation during aging in the setting of Dnmt3aR878H and Tet2D/D mutations. In the second aim, we will test if normal age-related activation of Hsf1 creates a permissive context for AML initiation in aging HSCs. We will conditionally delete Hsf1 in young and old adult HSCs in the setting of a Dnmt3aR878H mutation with and without a cooperating NrasG12D mutation to determine if it contributes to the emergence of clonal hematopoiesis and increased incidence of AML in older adults. These studies will open new lines of investigation into a previously unappreciated link between age-related loss of proteostasis (a hallmark of aging) and leukemia initiation. Therapies that mitigate proteostasis dysfunction could preserve HSC clonal diversity later in life while reducing susceptibility to AML.