High Mobility Group A1 (HMGA1) Chromatin Regulators Maintain Quiescent, Regenerative HSC By Repressing IGF1 Networks That Promote Stem Cell Aging

Introduction: Chromatin architecture and cell state are key determinants of stem cell function, although mechanisms governing these properties in hematopoietic stem cells (HSC) are only beginning to emerge. The High Mobility Group A1 (HMGA1) chromatin regulator gene is induced by growth factors and highly expressed during embryogenesis and in HSCs, but silenced in differentiated tissues (Resar et al, Cancer Res 2018). In acute leukemia, myeloproliferative neoplasms, and solid tumors, HMGA1 becomes re-expressed where it modulates chromatin structure to activate enhancers and stem cell gene networks, leading to aberrant proliferation and differentiation (Li et al, Blood 2022; Chia et al, JCI 2023). HMGA1 also drives self-renewal by amplifying Wnt networks in gut stem cells (Xian et al, Nature Comm 2017). However, the role of HMGA1 in hematopoiesis is unknown. We therefore sought to: 1) Define HMGA1 function in HSC, 2) Determine whether activating HMGA1 networks will enhance regenerative potential. Methods: We compared HMGA1 expression across hematopoietic stem and progenitor cells (HSPCs) and differentiated progeny in humans and mice. Next, we generated mice with global Hmga1 knock-out (Hmga1-KO) or hematopoietic-specific Hmga1 deletion (vav-Hmga1-KO) to compare HSC function with clonogenic assays, aging, competitive bone marrow transplantation (cBMT), 5-fluorouracil (5-FU), and irradiation (IR). To identify underlying mechanisms, we performed single cell RNA sequencing (scRNAseq) of bone marrow (BM)-derived Lin-, Sca+, c-kit+ (LSK) cells from adult mice. HMGA1 pathways were identified via gene set enrichment analysis (GSEA). To study HMGA1 in human HSPC, we used CRISPR to inactivate HMGA1 in CD34+ cells. Rescue experiments were performed to assess the role of HMGA1 gene targets. Results: HMGA1 is enriched in HSC, multipotent progenitors (MPP), and megakaryocyte-erythroid progenitors (MEP), with decreasing levels after differentiation, suggesting that HMGA1 functions in stem and early progenitors. However, steady state hematopoiesis is unperturbed in HSC with Hmga1-KO until 90 weeks of age, after which thrombocytopenia ensues. HSPC lacking HMGA1 exhibit decreased clonogenicity with serial replating. Similarly, in cBMT experiments, Hmga1-KO HSC are outcompeted by HSC with intact Hmga1, with most pronounced effects after serial transplantation. Survival after 5-FU is decreased in mice with Hmga1-KO HSC. Following IR, platelet count recovery is impaired in vav-Hmga1-KO mice, while irradiated HSPC exhibit reduced clonogenicity with decreases in total CFU, granulocyte-monocyte (GM)-CFU and megakaryocyte (Mk)-CFU. In human CD34+ cells, CRISPR-mediated HMGA1 inactivation decreases total CFU, GM-CFU, and Mk-CFU, and these defects become more pronounced with serial replating. To elucidate mechanisms underlying HMGA1, we compared scRNAseq in LSK cells from mice with intact or deficient Hmga1, which demonstrated that HMGA1 is required to maintain the most quiescent and regenerative HSC (marked by high Procr expression), whereas Hmga1 loss pushes HSC into cell cycle, resulting in expansion of more proliferative, less regenerative HSC and early progenitors. GSEA from single cell transcriptomes revealed that HMGA1 activates pro-survival gene networks, such as TNFα via NFκB, while down-regulating stem cell aging pathways repressed by the insulin growth factor 1 (IGF1). In hematopoietic cell lines, HMGA1 occupies the IGF1 enhancer and increases chromatin accessibility. Importantly, IGF1 mediates youthful stem cell function in diverse tissues, including HSC and BM-derived mesenchymal stem cells (MSC). Moreover, IGF1 rescues clonogenicity in HSC with HMGA1 deficiency. Experiments to assess in vivo rescue of human HSPC by IGF1 are underway. Conclusions: HMGA1 deficiency impairs HSC regenerative capacity under conditions of stress hematopoiesis by depleting quiescent HSC. Mechanistically, HMGA1 activates transcriptional networks involved in survival signals, while repressing IGF1 transcriptional networks associated with stem cell aging. Further, IGF1 rescues regenerative capacity in HSPC with HMGA1 deficiency. Our findings highlight HMGA1 as a critical factor in stress hematopoiesis and suggest that activating HMGA1 networks with IGF1 could enhance youthful regenerative capacity and repress aging pathways following BMT, IR, 5-FU, or normal aging.