Gprasp2 expression identifies a deeply quiescent hematopoietic stem cells subset with superior stemness and self-renewal

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Project Summary Hematopoietic stem cells (HSCs) are a rare pool of self-renewing, multipotent cells residing in the bone marrow that support lifelong blood production. HSCs can be transplanted into ablated recipients, where they can reconstitute all hematopoietic cell types, making them highly useful as a curative therapy for patients with hematopoietic diseases via HSC transplantation. To sustain lifelong blood output and meet emergency demands, HSCs tightly regulate a quiescent versus cycling state to protect from exhaustion. A deeply quiescent HSC subset has been identified that rarely contributes to steady-state hematopoiesis, preserving the long-term fidelity of the HSC pool. Upon transplantation, deeply quiescent HSCs display increased self-renewal and stemness, suggesting these HSCs may be highly relevant to transplant outcomes. However, little is known about the relationship between HSC quiescence and cell fate and few molecular markers of deeply quiescent HSCs exist. My goal is to identify novel molecular markers of deeply quiescent HSCs for mechanistic interrogation. We recently identified GPRASP2 (G-protein Coupled Receptor-associated (GPCR) Sorting Protein 2), an HSC- enriched protein involved in GPCR trafficking, as a regulator of HSC transplantation. Pilot single HSC immunofluorescence studies reveal heterogeneous HSC Gprasp2 expression and single-cell RNA sequencing (RNAseq) data show low Gprasp2-expressing HSCs are enriched in lineage-specific differentiation and cell cycle programs relative to Gprasp2high HSCs. We generated Gprasp2 reporter mice to interrogate HSC subsets based on Gprasp2 expression (Gprasp2low/Gprasp2high). Preliminary transplant data reveal Gprasp2high HSCs display slow blood repopulation kinetics and robust, lineage-balanced reconstitution compared to faster, less robust, and lymphoid-biased reconstitution by Gprasp2low HSCs. Bulk RNAseq and pathway analysis reveal Gprasp2high HSCs downregulate cell cycle and cellular responses to stimuli gene expression. Consistently, more Gprasp2high HSCs occupy G0 than Gprasp2low HSCs and display greater self-renewal during ex vivo culture. I hypothesize Gprasp2 is a novel marker and regulator of a deeply quiescent HSC subset that preferentially self- renews. In Aim 1, I will test if Gprasp2 marks a subset of deeply quiescent HSCs by assaying in vivo division history of Gprasp2low/high HSCs, quantifying exit from quiescence upon stimulation, and interrogating reconstitution kinetics post-transplant. In Aim 2, I will use serial transplantation and single-cell assays of self- renewal and cell fate to test if Gprasp2 predicts cell fate. In Aim 3, I will investigate if Gprasp2 is a driver of HSC self-renewal by modifying Gprasp2 expression and identify GPCRs and cell-surface receptors with known roles in hematopoiesis regulated by Gprasp2 via proximity labeling assays. Completion of these aims will produce data for peer-reviewed publication and give mechanistic targets as preliminary results for a K99/K00 application. The McKinney-Freeman laboratory at St. Jude is the optimal training environment for gold-standard HSC assays, proteomics approaches, and bioinformatics required for my career as an independent HSC investigator.