Mechanisms and consequences of sickle cell disease-induced cycling in hematopoietic stem cells

项目总结

Project Summary Sickle cell disease (SCD) is a painful debilitating life-long condition resulting from mutations in the gene encoding hemoglobin β subunit, causing abnormal hemoglobin polymerization leading to hemolysis, repeated vasooclusion, and chronic systemic inflammation resulting in substantial global morbidity and early mortality. Curative therapy for SCD relies on hematopoietic stem cell (HSC) transplantation, however the damaging effects of SCD pathophysiology on HSCs remain uncharacterized and we seek to fill this gap in current knowledge. Our preliminary studies show that HSCs display increased cycling in a transgenic SCD mouse model upon aging, and myeloid lineage biased in vitro differentiation of SCD patient derived HSCs. As a postdoctoral fellow in the McKinney-Freeman laboratory, I will investigate the functional consequences and molecular mechanisms underlying SCD mediated HSC cycling in a murine model and translate these findings to human HSC during SCD. In Aim 1, I will use colony formation assays, limiting dilution primary and secondary HSC transplantation, and serial exposure to chemotherapy to assess the detrimental impact of SCD-induced cycling on HSC frequency and function. In Aim 2, I will probe the molecular and epigenetic dysregulation underlying increased HSC cycling during SCD. HSCs isolated from SCD and control mice will be subjected to bulk RNA-seq and ATAC-seq to define transcriptional dysregulation correlated with changes in gene promoter accessibility meditating increased cycling. Finally, I will translate our findings to humans by interrogating cell cycle dysregulation in HSCs isolated from SCD patients (Aim 3). SCD patient-derived bone marrow aspirates will be profiled for frequency of phenotypic HSCs and alterations in cell cycle status by flow cytometry and ex vivo EdU incorporation. Lineage potential and hematopoietic output of SCD HSCs will be analyzed by colony formation and in vitro single HSC differentiation assays. Lastly, SCD HSC repopulating potential and self-renewal will be examined by limiting dilution primary and secondary transplantation into human cytokine expressing immune deficient mice. Together the proposed studies serve to deepen our understanding of a previously unexplored aspect of HSC biology by examining the impact of SCD. Greater understanding of SCD mediated mechanisms of HSC impairment become exceptionally important as autologous HSC transplantation following gene editing or gene therapy for SCD improve and increase in frequency. These aims draw on my prior training in cancer biology of hematologic malignancies but also provide abundant novel training opportunities in the field of HSC biology and for professional career development. The McKinney-Freeman lab and St. Jude Children’s Research Hospital are ideal environments in which to receive training in the study HSC biology, combining state-of-the-art institutional resources, career development resources, and excellent mentorship with the singular goal of advancing to an independent academic primary investigator position.