Epstein-Barr virus (EBV) infects >90% of humans and is associated with both hematological and epithelial malignancies. Here, we analyzed 990 EBV genomes (319 newly sequenced and 671 from public databases) from patients with various diseases to comprehensively characterize genomic variations, including single nucleotide variations (SNVs) and structural variations (SVs). Although most SNVs were a result of conservative evolution and reflected the geographical origins of the viral genomes, we identified several convergent SNV hot spots within the central homology domain of EBNA3B, the transactivation domain of EBNA2, and the second transmembrane domain of LMP1. These convergent SNVs seem to fine-tune viral protein functionality and immunogenicity. SVs, particularly large deletions, were frequently observed in chronic active EBV disease (28%), EBV-positive diffuse large B-cell lymphoma (48%), extranodal natural killer/T-cell lymphoma (41%), and Burkitt lymphoma (25%), but were less common in infectious mononucleosis (11%), posttransplant lymphoproliferative disorder (7%), and epithelial malignancies (5%). In hematological malignancies, deletions often targeted viral microRNA clusters, potentially promoting viral reactivation and lymphomagenesis. Nondeletion SVs, such as inversions, were also prevalent, with several inversions disrupting the C promoter to suppress latent gene expression, thereby maintaining viral dormancy. Furthermore, recurrent EBNA3B deletions suggested that this viral transcription factor functions as a tumor suppressor. EBNA3B knockout experiments in vitro revealed downregulation of human tumor suppressors, including PTEN and RB1, which could explain the enhanced lymphomagenesis observed in EBNA3B-deficient lymphoblastoid cell line xenografts. Our findings highlight both disease-specific and general contributions of EBV genomic alterations to human cancers, particularly in hematological malignancies.

Loss-of-function (LoF) mutations frequently found in human cancers are generally intractable by classical small molecule inhibitor approaches. Among them are mutations affecting Polycomb-group (PcG) epigenetic regulators, enhancer of zeste homolog 2 (EZH2) and Additional sex combs like 1 (ASXL1), frequently found in hematological malignancies of myeloid or lymphoid lineage, and their concurrent mutations associates with particularly poor prognosis. Although there is a clear need to develop novel and effective treatments for these patients, the lack of appropriate disease models and mechanistic insights have significantly hindered the progress. Here, we show that genetic inactivation of Asxl1 and Ezh2 in murine hematopoietic stem/progenitor cells results in highly penetrant hematological malignancies as observed in corresponding human diseases. These PcG proteins regulate both coding and noncoding genomes, leading to marked reactivation of transposable elements (TEs) and DNA damage responses in PcG LoF-mutated cells, which create a novel vulnerability for poly(ADP-ribose) polymerase (PARP) inhibitor (PARPi)-induced synthetic lethality. Using both mouse models and primary patient samples, we demonstrate that Asxl1/Ezh2-mutated cells are highly sensitive to PARPis that induce excessive DNA damage and significantly extend disease latency. Intriguingly, the observed PARPi sensitivity can be specifically overridden by reverse transcriptase inhibitors that interrupt target site-primed reverse transcription and life cycle of TEs. This mechanism is contrastingly different from the current concept of BRCAness associated PARPi-induced synthetic lethality, which largely rely on deficient homologous recombination, and is independent on reverse transcriptase inhibitors. Together, this study reveals a novel application and mechanism of PARPi-induced synthetic lethal targeting of blood cancers with reactivated TEs such as those carrying PcG epigenetic mutations.

Acquired somatic mutations are incorporated in the classification and prognosis of myelodysplastic syndromes/neoplasms (MDSs). However, the predictive role of molecular features in MDS needs to be elucidated, especially in the lower-risk subtypes (LR-MDS), where treatment has become heterogeneous and predictive biomarkers are lacking. In this study, we investigated genetic markers associated with erythropoiesis-stimulating agents (ESAs) response in LR-MDS. A European cohort of 535 patients with LR-MDS was analyzed using targeted next-generation sequencing (t-NGS) to calculate molecular prognostic scores (International Prognostic Scoring System, molecular [IPSS-M]). The integration of IPSS-M score among the 2 known variables, serum erythropoietin (sEPO) and transfusion dependence (TD), refined the capability to predict response (area under the curve [AUC], 0.71 vs 0.63, P = .0004). Based on these 3 variables, a molecular predictive score, which we named ESA-PSS-M (-0.05 × [sEPO U/L] -4.5 × [IPSS-M score] -5 × [TD (yes = 1; no = 0)]; specificity 76%; sensitivity 57%), was generated and validated in an external cohort (n = 223 patients with LR-MDS). Despite the impact of IPSS-M score, no single mutated gene was linked to ESA response; however, when we stratified cases by sex at birth, the X-linked STAG2 gene mutations were significantly associated with ESA resistance in males with LR-MDS (odds ratio, 0.13; P = .003). To our knowledge, this is the first study based on a large multicenter cohort of patients suggesting that the integration of IPSS-M score and sex-specific mutations can characterize ESA resistance and guide first-line (1L) therapeutic choices for anemic LR-MDS (ie, ESAs vs luspatercept).

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), which are 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 forkhead box protein O1 (Foxo1) and its upstream protein kinase, mechanistic target of rapamycin complex 2 (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 rapamycin-insensitive companion of mTOR (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 expression and identifies it as a promising target for iron-related disorders.

Cell-free RNA (cf-RNA) has emerged as a critical mediator of intercellular communication and a potential regulator 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 noncoding RNAs (sRNAs). Purified human and bovine Plg, isolated via lysine-affinity chromatography, were found to transport 25- to 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 (micromolar) affinity. Notably, Plg-sRNA complexes exhibited a distinct RNA profile compared to high-density lipoproteins, and their compositions were sensitive to hypercholesterolemic conditions. Functionally, removal of sRNA cargo from Plg via ribonuclease digestion significantly increased plasmin enzymatic activity and accelerated clot lysis, while also attenuating Plg-induced proinflammatory 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 cross talk 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 reduced NAD phosphate (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 in cells and in isolated membranes. OZ-induced p47phox phosphorylation was concentration dependent and preceded particle ingestion. Immunoglobulin G (IgG)- and complement protein fragment 3bi (C3bi)-opsonized zymosan similarly induced rapid phosphorylation and dephosphorylation of p47phox on Ser304 to Ser328, suggesting that IgG Fc-gamma receptors (FcγR) and complement receptor 3 (CR3) are involved in this process. Inhibitors of sarcome (Src) tyrosine kinase, spleen tyrosine kinase (Syk), phosphoinositide 3-kinase (PI3K), phospholipase C (PLC), phospholipase D (PLD), Ca2+, and protein kinase C beta 2 (PKCβ2) inhibited OZ-induced phosphorylation of p47phox. These results suggest that (1) OZ-induced p47phox phosphorylation on Ser304 to 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, Ca2+, and PKCβ2 control the phosphorylation of p47phox during phagocytosis.

NPM1 is a multifunctional phosphoprotein with key roles in ribosome biogenesis among its many functions. NPM1 gene mutations drive 30% of acute myeloid leukemia (AML) cases. The mutations disrupt a nucleolar localization signal and create a novel nuclear export signal, 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 evaluate the effects of isolated NPM1c mutations on the global proteome of preleukemic hematopoietic stem and progenitor cells (HSPCs) using conditional knockin 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 preleukemic Npm1cA/+ HSPCs display higher sensitivity to RNA polymerase I inhibitors, including actinomycin D (ActD), compared with Npm1+/+ cells. Combination treatment with ActD and venetoclax inhibited the growth and colony-forming ability of preleukemic and leukemic NPM1c+ cells, whereas low-dose ActD treatment was able to resensitize resistant NPM1c+ cells to venetoclax. Furthermore, using data from CRISPR dropout screens, we identified and validated TSR3, a 40S ribosomal maturation factor whose knockout 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 nonimmune target cells of inflammation, particularly organ-specific stem cells (SCs), also exhibit memory of previous inflammatory exposures. Previous inflammation experience imprints on the SCs and influences their regenerative potential and responses to subsequent inflammatory insults. This phenomenon has been observed in hematopoietic, intestinal, and skin epithelial SCs, with profound implications for tissue homeostasis, disease progression, and therapeutic strategies. Herein, we expand and develop the notion of inflammatory memory of SCs 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 spatiotemporal 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 hematologic diseases, hematopoietic SC transplantation, and cellular immunotherapies.

Immunodeficiency in telomere biology disorders (TBDs) has been described in pediatric patients with severe phenotypes, but is less characterized within the broader TBD spectrum. We collected complete blood counts, lymphocyte subsets, and infection history from 88 consecutive patients with TBD with a median age of 38 years (range, 6-76). Most patients were >18 years old (80/88; 90%) and harbored either a TERT (45%) or TERC germ line mutation (32%). Thirty-two patients (36%) experienced significant infections (opportunistic, recurrent, and/or requiring hospitalization); 47% had lymphopenia, and 3% severe neutropenia. Absolute lymphocyte counts (ALCs) of <0.96 and <1.1 × 103/μL, but not severe neutropenia, were associated with increased infection risk and lower overall survival, respectively. Decreased CD3+ T cells, both CD4+ and CD8+, were associated with bone marrow failure, increased infection risk, and reduced survival. Low CD3+ and CD4+ T cells were 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-cell lymphopenia, possibly a consequence of accelerated aging in the hematopoietic compartment. An ALC cutoff of <1.1 × 103/μL may be a useful biomarker to identify patients with an increased risk of infection, a major cause of death in patients with TBD.