Investigating hematopoietic stem cell dysfunction during sickle cell disease

Project Summary Chronic insults, such as inflammation and replicative stress, impair and exhaust blood-sustaining hematopoietic stem cells (HSCs), leading to dysfunction and selection for leukemia-associated mutations. We propose to study HSC insults in sickle cell disease (SCD), a chronic hemolytic anemia with a large inflammatory component and increased hematopoietic demand. Hematopoietic abnormalities in SCD include increased circulating hematopoietic stem and progenitors (HSPCs), increased numbers of BM HSPCs with perturbed phenotypes and damage to the bone marrow (BM) niche. Older SCD patients also mobilize HSPCs poorly and can become refractory to hydroxyurea. Mounting evidence indicates that SCD patients may have enhanced rates of clonal hematopoiesis, as well as MDS and AML at baseline and following allogeneic HSC transplantation or autologous HSC gene therapy. Considering that these are the only curative therapies for SCD, it is important to better understand and prevent SCD-induced insults to HSCs and their micro- environment. Thus, here we will examine the fitness of the HSC pool in individuals with SCD. Pilot data reveals a dramatic loss of both phenotypic and functional HSCs in middle-aged mouse models of SCD. HSCs in these mice also display evidence of increased cell cycling. Paradoxically, RNA-sequencing reveals senescence-related changes in SCD HSCs. Indeed, substantially more SCD HSPCs were positive for biomarkers of senescence in both mice and individuals with SCD, relative to age-matched controls. Based on our preliminary findings, we hypothesize that premature senescence driven by pathologic stress contributes to HSC dysfunction during SCD. In Aim 1 of this proposal, we will interrogate HSCs isolated from mice with SCD for molecular and functional hallmarks of senescence using high-resolution microscopy, single cell transcriptomics and quantitative assays for HSC function. We will also test if treating SCD mice with drugs that eliminate senescent cells can restore function to the HSC pool. In Aim 2, we will interrogate BM HSCs from pediatric SCD patients for molecular and functional hallmarks of senescence, relative to age- matched controls. We will define more precisely the age of onset of damage to the HSC pool in these young individuals with SCD using quantitative limiting dilution xenotransplantation and ex vivo single cell assays for lineage potential. HSPCs isolated from peripheral blood before and after plerixafor-mobilization will also be studied, as pilot studies suggest that plerixafor mobilizes senescent HSPCs in these patients. Aim 3 will focus on investigating mechanisms that contribute to premature senescence in SCD HSCs. Here, we will employ high-resolution microscopy, ex vivo culture, and single cell functional assays to specifically test the hypothesis that replicative stress and hemolysis contribute directly to premature senescence in SCD HSCs. We will also test if treatment with hemopexin, which eliminates free heme, can alleviate HSC senescence and restore function to the HSC pool in mice with SCD.