Project Summary/Abstract The goal of this proposal is to understand how the tetraspanin family member CD53 protects hematopoietic stem cells (HSCs) from inflammatory stress. While important for normal immune system function, inflammatory signaling can impair HSC function and promote the development of hematopoietic malignancies. In preliminary data, we identified CD53 as a critical regulator of HSC function in the context of inflammatory stress. CD53 is a member of the tetraspanin family of transmembrane proteins that organize multi-protein networks to regulate a wide variety of cellular processes such as proliferation, migration, and survival. While normally expressed at very low levels in HSCs, CD53 is markedly upregulated in response to multiple stressors including inflammatory cytokines, toll like receptor agonists and mobilizing agents. Using our newly-generated Cd53-/- mouse, we found that loss of CD53 causes a significant reduction in HSC repopulating ability and increased cycling in the face of inflammatory stress. RNA sequencing and proximity labeling studies suggest that CD53 promotes HSC quiescence in response to inflammation via activation of “DREAM,” a transcriptional repressor complex involving the Rb-like family members p107/Rbl1 and p130/Rbl2 that inhibits the expression of cell cycle genes in response to p53 and p21 activation. Based on this data, we hypothesize that CD53 promotes DREAM complex-mediated repression of cell cycle-related genes in HSCs in response to inflammatory stress, thereby promoting HSC quiescence and protecting HSC function. Notably, CD53 expression is markedly increased in HSCs deficient for Tet2 or Dnmt3a. Mutations in these epigenome regulators are commonly associated with age-related clonal hematopoiesis (CH), which involves inflammation-driven expansion of mutant HSCs and increased risk of leukemic transformation. We predict that CD53 may thus promote the clonal advantage of mutant HSCs in CH. Using a combination of proteomic, transcriptomic and in vivo HSC functional tools, we will: 1) Determine the role of CD53 in promoting HSC function in response to inflammatory stimuli; 2) Determine how CD53 regulates HSC cycling and DREAM complex activity; and 3) Determine whether elevated CD53 promotes the clonal advantage of mutant HSCs. We will perform overexpression studies to elucidate the effects of sustained CD53 expression on HSC function, and DREAM knockout mice will be used to determine the role of this complex in mediating the effects of CD53 on HSCs. We will determine the mechanisms by which CD53 regulates HSC cycling and DREAM activation using proximity ligation assays to characterize CD53- interacting partners. Finally, we will perform functional studies and chimeric modeling experiments using Cd53 and Dnmt3a knockout mice to determine whether CD53 promotes the clonal expansion of mutant HSCs. Together, our proposed studies will describe a novel mechanism that enables HSCs to resist inflammatory stres...