Liver sinusoidal endothelial cells (LSECs) are essential for maintaining liver function by actively sensing nutrients and producing angiocrine factors. LSECs also regulate systemic iron metabolism by secreting bone morphogenetic proteins (BMPs), key modulators of systemic iron homeostasis. However, the mechanism by which LSECs sense iron to regulate iron metabolism remains unclear. Here, we identify that the endothelial transcriptional factor Foxo1 and its upstream protein kinase, mTORC2, as critical iron-sensors. In response to iron, Foxo1 undergoes acute and dynamic nuclear translocation to activate the transcription of Bmp2 and Bmp6, thereby stimulating the synthesis of iron-regulatory hormone hepcidin in adjacent hepatocytes. Foxo1 directly binds evolutionally conserved Foxo binding sites within the Bmp2 and Bmp6 promoters to mediate this response. Mechanistically, iron triggers the lysosomal degradation of the mTORC2-specific component Rictor, enhancing Foxo1 activation. Endothelial-specific Foxo1 deletion reduces the expressions of hepatic Bmp2/6 and hepcidin, leading to systemic iron overload, whereas endothelial Rictor deletion increases the expressions of hepatic Bmp2/6 and hepcidin, producing an iron-deficient phenotype. Moreover, endothelial-targeted lipid nanoparticles expressing endothelial-specific and constitutively active Foxo1 alleviate iron overload in a murine model of hereditary hemochromatosis. Collectively, our study establishes the endothelial mTORC2-Foxo1 axis as an iron-responsive regulator of Bmp2 and Bmp6 expressions, and identifies it as a promising target for iron-related disorders.

Cell-free RNA (cf-RNA) have emerged as critical mediators of intercellular communication and potential regulators of hemostasis. In this study, plasminogen (Plg), the zymogen precursor of plasmin, was demonstrated to function as a secreted ribonucleoprotein that carries regulatory, extracellular small non-coding RNAs (sRNA). Purified human and bovine Plg, isolated via lysine-affinity chromatography, were found to transport 25-60 nucleotide-long sRNAs derived from both host and microbial sources. In vitro studies revealed that Plg accepted sRNA cargo from primary macrophages and bound candidate sRNAs with moderate (µM) affinity. Notably, Plg-sRNA complexes exhibited a distinct RNA profile compared to high-density lipoproteins (HDL), and their compositions were sensitive to hypercholesterolemic conditions. Functionally, removal of sRNA cargo from Plg via RNase digestion significantly increased plasmin enzymatic activity and accelerated clot lysis, while also attenuating Plg-induced pro-inflammatory cytokine expression in both mouse and human macrophages. These findings reveal a dual regulatory role for sRNAs in modulating both the fibrinolytic and immunogenic properties of Plg, offering novel insights into the crosstalk between cf-RNA biology and coagulation pathways. This work positions Plg-sRNA interactions as promising targets for therapeutic intervention in thrombotic and inflammatory diseases.

Neutrophils play a key role in innate immunity by killing microbes through phagocytosis and superoxide anion production by the phagocyte NADPH oxidase. The signaling pathways regulating NADPH oxidase activation in neutrophils have been extensively studied using soluble agonists, but are less understood during phagocytosis, a fundamental function of neutrophils. The aim of this study was to investigate the phosphorylation of the cytosolic NADPH oxidase protein p47phox in human neutrophils stimulated by serum-opsonized zymosan (OZ), which induces phagocytosis, using antibodies against phosphorylated sites. The results show that OZ induced rapid phosphorylation of p47phox on Ser304, Ser315, Ser320 and Ser328, followed by rapid dephosphorylation. Interestingly, despite the transient nature of p47phox phosphorylation, OZ-induced NADPH oxidase activity was sustained for a longer period of time in intact cells and in isolated membranes. OZ-induced p47phox phosphorylation at these sites was concentration dependent and preceded particle ingestion. IgG- and C3bi-opsonised zymosan similarly induced rapid phosphorylation and dephosphorylation of p47phox on Ser304, Ser315, Ser320 and Ser328, suggesting that FcγR and CR3 are involved in this process. Inhibitors of Src and Syk tyrosine kinases, PI3K, PLC, PLD, Ca++ and PKCβ2 inhibited OZ-induced phosphorylation of p47phox at these serine residues. These results suggest that: 1)-OZ-induced p47phox phosphorylation on Ser304, Ser315, Ser320 and Ser328 is required for the initiation of NADPH oxidase activation but not for its maintenance during phagocytosis; 2)-the membrane receptors FcγR and CR3 mediate this phosphorylation; and 3)-Src and Syk tyrosine kinases, PI3K, PLD, Ca++ and PKCβ2 control the phosphorylation of p47phox during phagocytosis.

NPM1 is a multifunctional phosphoprotein with key roles in ribosome biogenesis amongst its many functions. NPM1 gene mutations drive 30% of acute myeloid leukemia (AML) cases. The mutations disrupt a nucleolar localization signal (NoLS) and create a novel nuclear export signal (NES), leading to cytoplasmic displacement of the protein (NPM1c). NPM1c mutations prime hematopoietic progenitors to leukemic transformation, but their precise molecular consequences remain elusive. Here, we first examine the effects of isolated NPM1c mutations on the global proteome of pre-leukemic hematopoietic stem and progenitor cells (HSPCs) using conditional knock-in Npm1cA/+ mice. We discover that many proteins involved in ribosome biogenesis are significantly depleted in these murine HSPCs, but also importantly in human NPM1-mutant AMLs. In line with this, we found that pre-leukemic Npm1cA/+ HSPCs display higher sensitivity to RNA polymerase I inhibitors, including Actinomycin D (ActD), compared to Npm1+/+ cells. Combination treatment with ActD and Venetoclax inhibited the growth and colony forming ability of pre-leukemic and leukemic NPM1c+ cells, whilst low-dose ActD treatment was able to re-sensitize resistant NPM1c+ cells to Venetoclax. Furthermore, using data from CRISPR dropout screens, we identified and validated TSR3, a 40S ribosomal maturation factor whose knock-out preferentially inhibited the proliferation of NPM1c+ AML cells by activating a p53-dependent apoptotic response. Similarly to low-dose ActD treatment, TSR3 depletion could partially restore sensitivity to Venetoclax in therapy-resistant NPM1c+ AML models. Our findings propose that targeted disruption of ribosome biogenesis should be explored as a therapeutic strategy against NPM1-mutant AML.

Immunological memory in adaptive and innate immune cells is well-characterized, enabling enhanced responses upon secondary challenges. However, it has only been recently appreciated that the non-immune target cells of inflammation, particularly organ-specific stem cells, also exhibit memory of prior inflammatory exposures. Previous inflammation experience imprints on the stem cells and influences their regenerative potential and responses to subsequent inflammatory insults. This phenomenon has been observed in hematopoietic, intestinal, and skin epithelial stem cells, with profound implications for tissue homeostasis, disease progression, and therapeutic strategies. Herein, we expand and develop the notion of inflammatory memory of stem cells and explore recent insights in the field. We discuss the emerging understanding of the molecular underpinnings and their potential clinical and biological implications. Inflammatory memory is driven by spatio-temporal changes in gene loci and transcription regulated by DNA and histones' epigenetic modifications, metabolic reprogramming, and chromatin accessibility changes. Understanding these mechanisms is critical for improving the outcomes of hematological diseases, hematopoietic stem cell transplantation (HSCT), and cellular immunotherapies.

Immunodeficiency in telomere biology disorders (TBDs) has been described in pediatric patients with severe phenotypes, but less characterized within the broader TBD spectrum. We collected complete blood counts, lymphocyte subsets, and infection history from 88 consecutive TBD patients with a median age of 37 years (range 6-76). Most patients were >18 years old (80/88; 90%) and harbored either a TERT (45%) or TERC (32%) germline mutation. Thirty-two patients (36%) experienced significant infections (opportunistic, recurrent, and/or requiring hospitalization); 47% had lymphopenia, and 3% severe neutropenia. Absolute lymphocyte counts (ALC) <0.96 and <1.1 x103/µL, but not severe neutropenia, associated with increased infection risk and lower overall survival, respectively. Decreased CD3+ T-cells, both CD4+ and CD8+, associated with BMF, increased infection risk, and reduced survival. Low CD3+ and CD4+ associated with solid cancers. Telomere length was shortened across the cohort without correlation with ALC or lymphocyte subsets. In a predominantly adult cohort of TBDs, immunodeficiency was marked by T lymphopenia, possibly a consequence of accelerated aging in the hematopoietic compartment. An ALC cut-off of <1.1 x103/µL may be a useful biomarker to identify patients with an increased risk of infection, a major cause of death of TBD patients.

Children with Down syndrome (DS) have a high risk of GATA1-associated myeloid leukemia (ML-DS) before age 4. Somatic N-terminal GATA1 mutations (GATA1s) are necessary, but not sufficient, for ML-DS, but their significance at birth for individual babies and whether mutations occur after birth is unclear. To address these questions, we performed a prospective study of DS newborns using next-generation sequencing-based GATA1 mutation analysis with detailed hematologic and clinical evaluation and follow-up for the window of ML-DS risk. Of 450 DS neonates, 113 (25%) had GATA1s mutations of which 20/113 (17.7%) were multiple and 59 (52%) were clinically silent. Variant allele frequency (VAF) varied from 0.3-89%. VAF positively correlated (p<0.0001) with % blasts, leukocytes, dyserythro- and dysmegakaryopoiesis scores and clinical disease and negatively with hemoglobin, although only 4/113 were anemic. GATA1s mutations were detected from 28 weeks(w) gestation; the highest frequency (45%) was at 34-35w while mutation frequency in early fetal samples (<20w) was only <4% (2/57). GATA1s clones (VAF, % blasts) fell rapidly post-natally becoming undetectable by 6 months (6m) except in neonates who developed ML-DS. 7/110 surviving neonates (6.4%) developed ML-DS at a median age of 17.5m. GATA1s clone size at birth was the only predictor of subsequent ML-DS. No neonates lacking GATA1s mutations acquired mutations after birth or developed ML-DS. Taken together, the fetal environment is essential for GATA1s mutation selection and expansion of GATA1s clones. Rates of leukemic transformation of GATA1s clones detected at birth are low but clones that persist beyond 6 months transformed.

Mutations in TP53 are mutually exclusive with other known drivers of myeloid transformation and define a distinct molecular subtype within de novo Acute Myeloid Leukemia (AML) that is associated with a complex karyotype, resistance to chemotherapy, and poor prognosis. Although TP53 defects are rare in de novo AML, biallelic mutations are a defining molecular feature of erythroleukemia. The genetic alterations that cooperate with defective TP53 to transform erythroid progenitors remain unknown. We found that loss of BAP1 (BRCA1 Associated Protein-1) co-occurs in one-third of patients with TP53-mutated AML, is associated with an erythroid-primed gene expression signature, and confers an additional adverse effect on overall survival. BAP1 is a tumor suppressor involved in the DNA damage response as well as epigenetic regulation through histone H2AK119 de-ubiquitination. While Bap1KO mice develop myelodysplasia with prominent dyserythropoiesis, combined deletion of Bap1 and Trp53 caused transplantable erythroleukemia, and occasionally mixed AML, mirroring the heterogeneity of human disease. Bulk and single-cell RNA-seq coupled to ChIP-seq in hematopoietic progenitors revealed that Bap1 loss triggers a proinflammatory response and cooperates with Trp53 deficiency to transform erythroid-primed multipotent progenitors. Mechanistically, genomic instability led to the development of erythroleukemia, while epigenetic deregulation caused myelomonocytic skewing suggesting a dichotomous and context dependent role for BAP1. We also demonstrate that BAP1 deficient erythroleukemia is dependent on BCL2L1 expression and is sensitive to BCL-xL inhibitors in vivo.

In inflammation, circulating neutrophils indirectly damage the skeleton by inducing formation of bone-resorbing osteoclasts. However, neutrophil progenitors in marrow have no known physiological function. A bone-protective role for the neutrophil lineage was recently suggested when a profound defect in bone structure was observed in mice with neutropenia due to Granulocyte Colony Stimulating Factor (G-CSF) deletion coupled with STAT3 hyperactivation in bone cells. Here, we tested the existence of this protective effect by manipulating neutrophil progenitors in bone marrow by Ly6G antibody (aLy6G) treatment. Two protocols revealed an inverse relationship between marrow neutrophil progenitors and osteoclasts. Two weeks of aLy6G treatment increased marrow immature neutrophils by 25% and halved osteoclast mRNA markers in cortical bone. In contrast, coupling six weeks of aLy6G with anti-rat IgG2a to maintain antigenicity reduced marrow pre-neutrophils by 50%. This doubled trabecular osteoclast surface, halved trabecular bone mass, and significantly reduced high density bone mass both in control mice, and in mice with bone-specific STAT3 hyperactivation. In culture, isolated pre-neutrophils dose-dependently inhibited osteoclastogenesis independent of direct contact. We conclude that neutrophil progenitors directly inhibit osteoclast formation by releasing soluble factors. This identifies a novel action of hematopoietic cells in marrow to protect bone structure.

Platelet shape and volume changes are early mechanical events contributing to platelet activation and thrombosis. Here, we identify single-nucleotide polymorphisms in leucine-rich repeat-containing 8 (LRRC8) protein subunits that form the volume-regulated anion channel (VRAC), which are independently associated with altered mean platelet volume. LRRC8A is required for functional VRAC in megakaryocytes (MKs) and regulates platelet volume; adhesion; and agonist-stimulated activation, aggregation, adenosine triphosphate (ATP) secretion, and calcium mobilization. MK-specific LRRC8A conditional knockout mice have reduced laser injury-induced cremaster arteriolar thrombus formation and prolonged FeCl3 induced carotid arterial thrombosis without prolonged bleeding times. Mechanistically, platelet LRRC8A mediates swell-induced cytosolic ATP release to amplify agonist-stimulated calcium-phosphoinositide 3-kinase-protein kinase B signaling. Small-molecule LRRC8 channel inhibitors recapitulate defects observed in LRRC8A-null platelets in vitro and in vivo. These studies identify the mechanoresponsive LRRC8 channel complex as an ATP release channel in platelets, which positively regulates platelet function and thrombosis, providing a proof of concept for a novel antithrombotic drug target.

Disease-defining signatures in lymphomas, driven by intricate molecular mechanisms, have advanced molecular taxonomies, refined classification, and may guide clinical management; however, the role of these signatures in driving disease hallmarks including subtype-specific organotropism remains largely unexplored. Primary mediastinal large B-cell lymphoma (PMBCL) is an exemplary lymphoma characterized by disease manifestations in the thymic niche, unique genetic alterations and immune escape. Here, we identified IRF4-C99R mutations uniquely occurring in PMBCL through mutational meta-analysis of large-scale datasets. Our functional studies, integrating multi-omics approaches with genome editing in PMBCL cells, revealed that IRF4-C99R contributes to a differentiation block phenotype. Specifically, we showed that IRF4-C99R reduces its binding to the ISRE motif within PRDM1, which encodes a key transcriptional regulator of B-cell differentiation, resulting in decreased PRDM1 expression. Additionally, IRF4-C99R suppresses TNIK, a key IFNγ pathway regulator, by impairing ISRE motif binding, thereby reducing IFNγ signaling and increasing thymus and activation-regulated chemokine (TARC) expression, which drives TARC-mediated chemotaxis of T regulatory cells. We also revealed that IRF4-C99R upregulates Ephrin Type-B Receptor 1 (EPHB1) through non-canonical AICE motif binding, and showed that overexpression of EPHB1 in an immunocompetent syngeneic lymphoma model influenced organotropism to favor thymic localization, without affecting tumor burden in other organs. IRF4-C99R mutation-induced phenotypes were validated in primary PMBCL tissues using single-nuclei RNA sequencing, confirming that the molecular mechanisms observed in vitro align with the pathophysiology of PMBCL in patients. Together, these findings demonstrate how a single genetic mutation orchestrates the coordinated regulation of hallmark traits including thymus-specific tropism in PMBCL.