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Bone marrow fibrosis is the most extensive matrix remodeling of the microenvironment and can include de novo formation of bone (osteosclerosis). Spatiotemporal information on the contribution of distinct bone marrow niche populations to this process is incomplete. We demonstrate that fibrosis‐inducing hematopoietic cells cause profibrotic reprogramming of perivascular CXCL12‐abundant reticular (CAR) progenitor cells, resulting in loss of their hematopoiesis‐support and upregulation of osteogenic and pro‐apoptotic programs. In turn, peritrabecular osteolineage cells (OLCs) are activated in an injury‐specific, Wnt‐dependent manner, comparable to skeletal repair. OLCs fuel bone marrow fibrosis through their expansion and skewed differentiation, resulting in osteosclerosis and expansion of Ly6a+ fibroblasts. NCAM1 expression marks peritrabecular OLCs and their expansion into the central marrow is specific for fibrosis in mice and patients. Peritrabecular stromal β‐catenin expression is linked to fibrosis in patients, and inhibition of Wnt signaling reduces bone marrow fibrosis and osteosclerosis, possibly being a clinically relevant therapeutic target.

Osteoporosis (OP) could lead to the alteration of bone marrow microenvironment and non-homeostasis of hematopoiesis, which could increase the incidence of hematologic malignancies. However, whether myeloid-biased hematopoiesis occurred and contributed to the leukemogenesis under the condition of OP remains unclear. Results: This study successfully induced a mouse model for OP by hindlimb unloading, which shows increased myeloid cells and decreased B cells in the peripheral blood (PB). Furthermore, our study demonstrates that the myeloid-biased subset of HSPCs (hematopoietic stem and progenitor cells) with reduced differentiation and apoptosis, including multipotent progenitor 3 (MPP3) and granulocyte-monocyte progenitors (GMPs), were expanded in the OP mice. The expansion of myeloid-biased HSPCs contributes to the accumulation of HSPCs in the bone marrow and increased myeloid cells in the PB of OP mice. In the expanded pool of HSPCs, OP mice specifically enriched subsets were identified and profiled by single cell RNA-seq, including subHSCs from primitive HSCs, MPP3-1 from MPP3, GMP5 from GMPs, MkP2 from megakaryocyte progenitors and EryP1 from erythrocyte progenitors. Meanwhile, those OP-HU mice enriched subsets shared significantly up- and down-regulated genes enriched in chromatin modification and cell differentiation and apoptosis such as Bromodomain-containing protein 4 (Brd4), encoding an important chromatin remodeling protein, and Proteinase 3 (Prtn3). Moreover, the specific transcription factors corresponding to the expansion of subHSCs, MPP3-1, GMP5 and EryP1 in OP-HU mice were identified as Zfp951, Nfic, Maz and Ezh2. Finally, inhibition of BRD4 in vivo could partially restore the phenotype of OP-HU mice and the expression of genes regulating HSPC expansion, differentiation and apoptosis. Conclusions: First of all, our study shows that OP could induce the unbalanced hematopoiesis and enhances the myeloid-biased hematopoiesis. Secondly, OP mice enriched subsets of HSPCs were identified and characterized with enhanced chromatin remodeling, reduced differentiation and resistance to apoptosis. Finally, this study demonstrate that Brd4 regulated gene programs endow the myeloid-biased subsets of HSPCs with tumor cell-like characters in OP mice, which may increase the incidence of the leukemic evolution. This study sheds light on the importance for the prevention of myeloid leukemogenesis in human with OP.

The maintenance of a basal immunoinflammatory signature in hematopoietic stem/progenitor cells (HSPCs) constitutes a fundamental regulatory axis governing hematopoietic competence and immune effector generation. While epigenetic repressors constrain this inflammatory phenotype, the molecular amplifiers that preserve this critical state remain undefined. Through integrated single-cell transcriptomic/epigenomic profiling and functional interrogation, we identify Setd2-mediated H3K36me3 as an indispensable epigenetic amplifier sustaining baseline inflammation in murine HSPCs. Setd2 ablation specifically eliminated interferon (IFN)-enriched HSPC subpopulations and attenuated inflammatory signaling cascades. Functionally, Setd2-deficient HSPCs exhibited impaired IFNγ responsiveness, compromised B-lymphopoiesis, and diminished reconstitution capacity due to Lin−c-Kit+Sca1high cell depletion. Paradoxically, Setd2 loss conferred resistance to IFNγ-induced HSPCs exhaustion, which may contribute to the maintenance of Setd2-deficient HSPCs in our myelodysplastic syndrome (MDS) model under the inflammatory milieu. Mechanistically, Setd2 sustained chromatin accessibility and enhancer (H3K27ac) activity at inflammatory gene loci. This work delineates a critical link between Setd2-mediated chromatin regulation, baseline inflammation, HSPC function, and immune competence, providing insights into inflammatory dysregulation in hematopoietic malignancies like MDS.