Erythroid PIM1 deficiency promotes hematopoietic aging via jun/jund activation and igkc-mediated proteostasis collapse

Abstract Anemia, the most prevalent hematological disorder in the elderly, commonly presents with accelerated aging phenotypes in severe cases—though the underlying mechanisms remain unclear. Using single-cell sequencing of whole bone marrow cells, we found that aged anemic mice exhibited downregulatedPIM1expression across all stages of erythroid development compared to aged non-anemic mice. Further analysis of public databases revealed that PIM1, which encodes a serine/threonine protein kinase, is highly expressed in normal erythroid cells relative to other tissues or hematopoietic lineages. These findings suggest that PIM1 may play a critical role in maintaining erythroid homeostasis in aging individuals. To probe the role of PIM1 in erythroid hematopoiesis during aging, we generated erythroid-restricted Pim1 knockout mice (Pim1fl/flGypacre) and analyzed young (3-month-old) and aged (≥18-month-old) cohorts. Phenotypic analysis showed age-specific effects: young knockouts had mild anemia with slightly reduced hemoglobin but normal marrow Ter119⁺ cells and spleen structure. In contrast, Aged PIM1 knockouts exhibited severe anemia characterized by reduced RBCs/hemoglobin, pale bone marrow, decreased Ter119+ cells, splenomegaly, and compensatory splenic erythropoiesis. In vitro cloning forming assays further showed severely impaired BFU-E and CFU-E colony formation in aged knockout marrow. These findings establish PIM1 as indispensable for stress erythropoiesis during aging. Comprehensive hematopoietic profiling further demonstrated that aged Pim1fl/flGypacre mice exhibited hallmark signatures of HSC aging, including skewed long-term to short-term HSC ratios, reduced common lymphoid progenitors, and elevated p16 expression in lineage-negative compartments. Behavioral assessments revealed significant neuromuscular decline, with weakened grip strength and impaired motor coordination. Transplantation studies using competitive repopulation assays definitively confirmed HSC functional impairment. Collectively, these findings demonstrate that erythroid-specific PIM1 deficiency non-cell-autonomously drives HSC aging demonstrate within the aged microenvironment. To elucidate mechanisms of anemia in aged Pim1fl/flGypacre mice, we conducted single-cell sequencing of marrow from young and aged Pim1fl/flGypacre and control mice. Unsupervised clustering identified 11 distinct cell populations. Aged knockouts exhibited reduced erythroid proportions compared to aged controls (consistent with flow cytometry), while young mice showed no differences. Erythroid-specific differential gene expression analysis revealed JUN and JUND among the top-10 upregulated transcripts. GO analysis revealed AP-1 pathway activation with JUN/JUND upregulation in PIM1-deficient aged erythroid progenitors. Functional studies confirmed JUN/JUND overexpression recapitulated the anemic phenotype (reduced Ter119+cells, splenic erythropoiesis), establishing their causal role in erythroid impairment, while separate mechanisms drive HSC aging. To assess systemic effects of PIM1 deficiency in aged mice, we analyzed single-cell sequencing data and found that aged (but not young) Pim1fl/flGypacre mice showed significantly reduced HSC proportions. Differential gene enrichment revealed markedly impaired proteolytic pathway activity specifically in aged HSCs-a characteristic aging signature absent in young knockouts. This age-restricted phenotype suggests erythroid PIM1 deficiency non-cell-autonomously promotes HSC aging through disrupted proteostasis. Plasma proteomics of aged Pim1fl/flGypacre mice identified significantly elevated IGKC levels. Functional validation showed that intraperitoneal IGKC administration in aged mice recapitulated key knockout phenotypes: anemia, splenomegaly, increased splenic nucleated erythrocytes, and downregulated proteolytic pathway genes in HSCs. Cell-cell interaction analysis revealed enhanced plasma cell-HSC communication with specific IGKC upregulation in knockout plasma cells, confirming IGKC's role in secondary HSC aging. Our findings establish a dual-pathway mechanism of hematopoietic aging caused by erythroid PIM1 deficiency: pathological JUN/JUND activation in erythroid progenitors directly impairs erythropoiesis, while triggering plasma cell-mediated IGKC secretion that disrupts HSC proteostasis. These findings identify the PIM1-JUN/JUND-IGKC axis as promising therapeutic targets for age-related hematological decline.