Impact of PIGA Loss on Hematopoietic Stem Cell Function

The goal of this proposal is to identify the mechanisms underlying the expansion of phosphatidylinositol glycan type A (PIGA) gene-mutated hematopoietic cells in patients with paroxysmal nocturnal hemoglobinuria (PNH) disease. PIGA is a gene required for the biosynthesis of glycosylphosphatidylinositol (GPI) anchors. Patients with PNH disease develop somatic mutations in PIGA in their hematopoietic stem and progenitor cells (HSPCs). PIGA protein deficient (PIGA–) HSPCs lack all GPI-anchored proteins and clonally expand, causing life-threat- ening hemolytic anemia and thrombosis in PNH patients. Although progress has been made in treating hemolytic anemia associated with PNH, current treatments do not reverse PIGA– HSPC expansion and require life-long adherence to complement inhibitor therapy. The main challenge to developing more effective and curative ther- apies for PNH has been a gap in our understanding of the effects of PIGA loss on HSPC function and causes of clonal expansion of PIGA– HSPCs. Here, we propose to fill these gaps by defining the effects of PIGA loss on HSPC function. We recently developed a mouse model of hematopoietic-specific PIGA loss, in which we can compare the hematopoietic function of PIGA– and control HSPCs directly in a competitive environment within the same animal by taking advantage of X chromosome inactivation. Our preliminary data suggest that PIGA– hematopoietic stem cells (HSCs) produce greater numbers of mature blood cells than PIGA+ HSCs and have hyperactive Notch signaling that may contribute to the enhanced PIGA– HSPC expansion under conditions of immune bone marrow injury. We hypothesize that PIGA– HSPCs have a competitive advantage in immune- mediated bone marrow failure, mediated in part through increased Notch signaling. The objective of this proposal is to determine the effects of PIGA loss on HSPC function and identify mechanisms that underlie clonal expan- sion of PIGA– HSPCs. In Aim 1, we will determine how PIGA loss affects HSPC function, at steady state and in the context of immune-mediated bone marrow failure. Aim 2 will investigate the hypothesis that hyperactive Notch signaling stimulates PIGA– HSPC expansion in steady state conditions and during stress hematopoiesis associated with immune-mediated bone marrow injury. In Aim 3, we will use integrated single-cell protein and DNA sequencing and transcriptome profiling to determine factors that cause PIGA– clone expansion in patients. Our studies will address critical knowledge gaps in understanding the effects of PIGA loss on HSC function and reasons for competitive advantage and clonal expansion of PIGA– cells. Insights gained from these studies will guide future mechanism-based strategies to prevent and reverse PIGA– clone expansion and restore healthy hematopoiesis in immune-mediated bone marrow failure disorders to cure PNH. Project Number: 1R01HL174612-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Heart Lung and Blood Institute (NHLBI) | Principal Investigator: Daria Babushok | Institution: UNIVERSITY OF PENNSYLVANIA, PHILADELPHIA, PA | Award Amount: $786,456 | Activity Code: R01 | Study Section: Hemostasis, Thrombosis, Blood Cells and Transfusion Study Section[HTBT] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01HL17461201A1