Hematopoietic stem cell defects in Diamond-Blackfan Anemia - PROJECT SUMMARY Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure disorder caused mainly by heterozygous loss-of-function mutations in 20 out of 83 ribosomal protein (RP) genes, the most commonly affected one being RPS19. DBA usually presents with erythroid production failure during infancy, although clinical observations suggest impaired hematopoietic stem cell (HSC) function in some older DBA patients. Characterization of this suspected HSC defect is limited by difficulties in obtaining DBA patient CD34+ hematopoietic stem and progenitor cells (HSPCs) and the lack of ideal human cellular experimental models. I have used Cas9 to create RPS19 haploinsufficiency in healthy donor CD34+ HSPCs. My preliminary data show that RPS19+/- HSPCs exhibit impaired erythropoiesis and normal myelopoiesis in vitro and impaired bone marrow repopulation ability following xenotransplantation into immunodeficient mice, suggesting an HSC defect. Co-disruption of TP53 completely rescues the erythroid defect and partly alleviates the HSC defect. Furthermore, transcriptome analysis shows increased expression of PRC2 target genes in RPS19+/- HSPCs. This indicates reduced PRC2 activity, which epigenetically represses a gene program that is essential for HSC maintenance. Accordingly, my K99/R00 proposal investigates the hypothesis that RPS19 haploinsufficiency impairs HSPCs by inducing TP53 activity and reducing PRC2 activity. I will test this hypothesis by defining the effect of RPS19 haploinsufficiency on the lineage developmental potential of defined HSPC subpopulations (Aim 1) and describing the molecular mechanisms underlying activation of TP53 and reduction of PRC2 activities in RPS19+/- HSPCs (Aim 2). Lastly, I will utilize cutting-edge approaches, including single-cell studies, to describe alterations in transcriptome and translatome networks in RPS19+/- HSPCs (Aim 3). These investigations will elucidate a poorly understood aspect of DBA pathophysiology and provide new insights into the biology of HSCs. I will carry out the K99 phase of these aims in an exceptional research environment under the guidance of a senior mentorship team and a formal plan to receive training in advanced laboratory methods and various aspects of career development. My research, training plans and transition to independence achieved under the auspices of the K99/R00 mechanism will support my long-term goal of becoming an independent, NIH R01-funded physician-scientist with expertise in ribosomal biology, human hematopoiesis, bone marrow failure syndromes, bone marrow transplantation and gene therapy.
