Hematopoietic stem cells (HSCs) give rise to a diverse population of blood cells, including myeloid and lymphoid cells, platelets, and erythrocytes. These cell types play important roles in defending against disease, responding to tissue injury, and transporting vital cellular nutrients. Hematopoiesis, the generation of blood cells from HSCs, is a carefully orchestrated process that is highly conserved among vertebrates. In recent years, it has become apparent that HSCs are heterogenous and may be biased with respect to their production of different blood cells. Further, somatic mutations acquired over the course of an organism?s life may alter HSC output. If the mutations confer a survival or proliferative advantage, this may lead to the expansion of cells derived from a specific HSC (clonal expansion) and clonal hematopoiesis. In 2014, a clinical entity called clonal hematopoiesis of indeterminant potential (CHIP) was described in which specific genetic mutations appeared to be promoting HSC clonal expansion without any obvious hematologic abnormalities. Individuals with CHIP are at an increased risk for hematologic malignancy but also and more surprisingly at an increased risk for developing coronary heart disease (CHD) secondary to increased inflammation. At present, scientists do not fully understand how mutations in HSCs lead to increased inflammation nor know how to predict which individuals with CHIP will progress to malignancy. The overarching goal of the proposed work is to gain insight into HSC biology and immune cell development in unperturbed hematopoiesis and genetically- induced clonal hematopoiesis through single-cell analyses and lineage tracing.
In Aim 1, HSC output, immune cell development, and hematopoietic lineage relationships will be studied in unperturbed zebrafish. This will be accomplished with scGESTALT, a new method for in vivo cellular barcoding and fate mapping. Adult kidney marrows and thymi from scGESTALT zebrafish will be dissected and single-cell RNA sequencing will be performed to obtain transcriptional and ancestral information.
Aim 2 will assess the impact of mutations found in clonal hematopoiesis on HSC clonal output and immune cell development and differentiation. CRISPR-Cas9 will be used to mutate zebrafish orthologs of human ASXL1, NRAS, EZH2, and RUNX1 at genomic locations frequently mutated in patients. Transcriptional profiling and lineage tracing will be performed on kidney marrows and thymi from scGESTALT zebrafish with and without expanded clones. The ability of macrophages and lymphocytes to promote clonal expansion will be explored via targeted knockout/knockdown approaches. Identifying altered cells, developmental and transcriptional programs, and regulatory networks in clonal hematopoiesis will shed light on the contributing cells and mechanisms to both clonal expansion and increased inflammation. Such knowledge has the potential to better inform clinical management of CHIP and potentially point to strategies aimed at preventing the progression of CHIP to malignancy.
Clonal hematopoiesis of indeterminant potential (CHIP) is thought to occur in >10% of individuals over the age of 70 and has been associated with an increased risk of hematologic malignancy and cardiovascular disease. At present, it is unclear which individuals with CHIP will progress to malignancy and how inflammation contributes to this process. The proposed research will assess the impact of CHIP-associated mutations on hematopoietic stem cell clonal output and immune cell development and differentiation with the goal of identifying relevant cells and pathways that may be clinically targeted to prevent or delay leukemic transformation.
