3017 – A DISTINCT SUBSET OF LATENT LONG-TERM HUMAN HEMATOPOIETIC STEM CELLS RESISTS REGENERATIVE STRESS TO PRESERVES STEMNESS

The mechanisms governing how human hematopoietic stem cells (HSC) balance their capacity for maintaining long-term (LT) regeneration with meeting daily and/or stress demands are unknown. We previously linked INKA1-mediated PAK4 inhibition along with reduction of H4K16 acetylation (H4K16ac) to quiescence in leukemia stem cells. Here, we uncover mutually exclusive immunostaining for INKA1 and H4K16ac in cord blood HSC; supported by BioID and PLA data showing INKA1 interacts with the H4K16 deacetylase SIRT1. The proteomic data also pointed us to CD112, that enabled isolation of LT-HSC into transcriptionally distinct subsets that respond differently to regenerative stress: a CD112low subset (H4K16aclow, CDK6low, ROSlow, INKA1high) exhibited a transient restraint before contributing to hematopoietic reconstitution and a primed CD112high subset (H4K16achigh, CDK6high, ROShigh, INKA1low) that responded rapidly. Protein and scRNAseq pseudotime analysis of in vitro and in vivo (G-CSF-) activated HSC, respectively, confirmed upregulation of H4K16ac, PAK4, CDK6 and CD112 coinciding with cell-cycle priming. INKA1-overexpression (OE) or PAK4 knock-down (KD) induced transient restraint in reconstitution (@4w) with 20w engraftment reaching control levels; HSC frequency increased 4-8 fold as measures in secondary transplants. Thus, INKA1-OE or PAK4-KD enables HSC to resist early activation, thereby protracting their regenerative potential (a phenomenon we term latency). By elevating hematopoietic demand with 5-FU, the INKA1-OE imposed restraint was released engendering even further increased HSC frequency; in controls regenerative capacity was abolished. Collectively, our data uncover a new molecular axis for HSC self-renewal regulation and point to latency as an orchestrated physiological response that integrates quiescence control with HSC fate choices to maintain LT-HSC pool size.