Coagulation is related to inflammation, but the key pathway, especially innate immune system and coagulation regulation, is not well understood and need to be further explored. Here, we demonstrated that neutrophil gelatinase-associated lipocalin (NGAL), an innate immune inflammatory mediator, is upregulated in patients with thrombosis. Furthermore, it contributes to the initiation and amplification of coagulation, hemostasis, and thrombosis. This occurs by enhancing tissue factor expression on the cell surface, potentiating various clotting factors such as thrombin, kallikrein, factor XIa (FXIa), and FVIIa, promoting thrombin-induced platelet aggregation, and inhibiting antithrombin. NGAL knockout led to strikingly prolonged clot reaction time and kinetic time in thromboelastography analysis, along with reduced thrombus generation angle and lower thrombus maximum amplitude, which were in line with remarkably prolonged activated partial thromboplastin time and prothrombin time. In several mouse hemostasis and thrombosis models, NGAL overexpression or IV administration promoted coagulation and hemostasis and aggravated thrombosis, whereas NGAL knockout or treatment with anti-NGAL monoclonal antibody significantly prolonged bleeding time and alleviated thrombus formation. Notably, NGAL knockout prolonged mouse tail bleeding time or artery occlusion time to over 40 or 60 minutes, respectively, resembling uncontrollable bleeding and clotting disorder seen in hemophilic mice. Furthermore, anti-NGAL monoclonal antibody treatment markedly reduced the formation of blood clots in inflammation-induced thrombosis models. Collectively, these findings unveil a previously unidentified role of NGAL in the processes of coagulation, hemostasis, and thrombosis, as well as the cross talk between innate immunity, inflammation, and coagulation. Thus, modulating NGAL levels could potentially help balance thrombotic and hemorrhagic risks.

Accumulation of free α-globin is a critical factor in the pathogenesis of β-thalassemia. Autophagy plays a crucial role in clearing toxic free α-globin, thereby reducing disease severity. However, the impact of natural mutations in autophagy-related genes (ATGs) on the phenotypic variability of β-thalassemia remains unclear. In this study, we systematically investigated the relationship between variants in ATGs and disease phenotypes in a cohort of 1022 patients with β-thalassemia, identifying 4 missense mutations in the autophagy and beclin 1 regulator 1 (AMBRA1) gene. Disruption of the Ambra1 gene in β-thalassemic mice was found to reduce autophagic clearance of α-globin in red blood cell precursors, exacerbating disease phenotypes. Functional characterization of the AMBRA1 gene and these mutations in patient-derived CD34+ cells, edited human umbilical cord blood-derived erythroid progenitor 2 (HUDEP-2) cells, and engineered HUDEP-2 β-thalassemic cells confirmed that AMBRA1 facilitates the autophagic clearance of free α-globin in human erythroid cells. Functional studies demonstrated that AMBRA1 missense mutants destabilize Unc-51-like kinase 1 protein, inhibit light chain 3 protein lipidation, and subsequently hinder autophagic flux, leading to increased α-globin deposition. Additionally, these mutations were associated with erythrotoxic effects in vitro, including increased intracellular reactive oxygen species levels, higher apoptosis rates, and impaired erythroid differentiation and maturation. This study sheds light on the molecular association between mutations in ATGs and the exacerbation of β-thalassemia, highlighting the potential role of the AMBRA1 gene as a promising diagnostic and therapeutic target for β-hemoglobinopathies.

The oral microbiota, second in abundance to the gut, is implicated in chronic systemic diseases, but its specific role in graft-versus-host disease (GVHD) pathogenesis has been unclear. Our study finds that mucositis-induced oral dysbiosis in patients after hematopoietic cell transplantation (HCT) associated with increased chronic GVHD (cGVHD), even in patients receiving posttransplant cyclophosphamide. In murine HCT models, oral dysbiosis caused by bilateral molar ligatures exacerbated cGVHD and increased bacterial load in the oral cavity and gut, with Enterococcaceae significantly increasing in both organs. In this model, the migration of Enterococcaceae to cervical lymph nodes both before and after transplantation activated antigen-presenting cells, thereby promoting the expansion of donor-derived inflammatory T cells. Based on these results, we hypothesize that pathogenic bacteria increase in the oral cavity might not only exacerbate local inflammation but also enhance systemic inflammation throughout the HCT course. Additionally, these bacteria translocated to the gut and formed ectopic colonies, further amplifying systemic inflammation. Furthermore, interventions targeting the oral microbiome mitigated murine cGVHD. Collectively, our findings highlight the importance of oral dysbiosis in cGVHD and suggest that modulation of the oral microbiome during transplantation may be an effective approach for preventing or treating cGVHD.

We previously demonstrated that reduced intrinsic electron transport chain (ETC) activity predicts and promotes sensitivity to the B-cell lymphoma 2 (BCL-2) antagonist, venetoclax (Ven), in multiple myeloma (MM). Heme, an iron-containing prosthetic group and metabolite, is fundamental to maintaining ETC activity. Interrogation of the cyclin D1 group 2 subgroup of MM from the Relating Clinical Outcomes in MM to Personal Assessment of Genetic Profile (CoMMpass) trial (NCT01454297), which can be used as a proxy for Ven-sensitive MM (VS MM), shows reduced expression of the conserved heme biosynthesis pathway gene signature. Consistent with this, we identified that VS MM exhibits reduced heme biosynthesis and curiously elevated hemin (oxidized heme) uptake. Supplementation with hemin or protoporphyrin IX (heme lacking iron) promotes Ven resistance, whereas targeting ferrochetalase, the penultimate enzyme involved in heme biosynthesis, increases Ven sensitivity in cell lines and primary MM cells. Mechanistically, heme-mediated activation of prosurvival rapidly accelerated fibrosarcoma-rat sarcoma virus-mitogen-activated protein kinase (MEK) signaling and metabolic rewiring, increasing de novo purine synthesis, were found to contribute to heme-induced Ven resistance. Cotargeting BCL-2 and myeloid cell leukemia-1 suppresses heme-induced Ven resistance. Interrogation of the Multiple Myeloma Research Foundation CoMMpass study of patients shows increased purine and pyrimidine biosynthesis to corelate with poor progression-free survival and overall survival. Elevated heme and purine biosynthesis gene signatures were also observed in matched relapse refractory MM, underscoring the relevance of heme metabolism in therapy-refractory MM. Overall, our findings reveal, for the first time, a role for extrinsic heme, a physiologically relevant metabolite, in modulating proximity to the apoptotic threshold with translational implications for BCL-2 antagonism in MM therapy.

This article has been retracted; please see Elsevier's Article Correction, Retraction and Removal Policy (Article withdrawal | Elsevier policy).This article has been retracted at the request of the Editors.Within the paper, image duplications were identified in Figures 2 and 6 and supplemental Figure 4. Image duplications were also identified between Figure 1 and supplemental Figure 4 from this paper and a 2019 publication in another journal. In each case, the duplicated image was modified between versions, such as via rotation and/or shifting the field of view.The authors state that the duplications were image handling errors and that the adjustments were made to improve visual comparison and do not affect their conclusions.No authors approve the retraction.

Diffuse large B-cell lymphoma (DLBCL) is a clinically and molecularly heterogeneous disease. The increasing recognition and targeting of genetically defined DLBCLs highlight the need for robust classification algorithms. We previously characterized recurrent genetic alterations in DLBCL and identified 5 discrete subtypes, clusters 1 to 5 (C1-C5), with unique mechanisms of transformation, immune evasion, candidate treatment targets, and different outcomes after standard first-line therapy. Herein, we validate the C1 to C5 DLBCL taxonomy in an independent data set and use the expanded series of 699 primary DLBCLs to develop a probabilistic molecular classifier and confirm its performance in an independent test set. Using our previously assigned cluster labels as a reference, we systematically compared multiple machine learning models and strategies for input feature dimensionality reduction with a newly developed performance metric that captured the relationship between accuracy and confidence of class assignments. The winning neural network model, DLBclass, assigned all cases in the training/validation and independent test sets with 91% and 89% accuracies, respectively. In the 75% of cases with confidence >0.7, DLBclass assignments were accurate in 97% of the training/validation set and 98% of the test set. DLBclass enables robust prospective classification of single cases for inclusion in genetically guided clinical trials or practice and represents a framework for the development of genomics-based classification methods in other cancers.

Pediatric acute myeloid leukemia frequently harbors fusion oncogenes associated with poor prognosis, including KMT2A, NUP98, and GLIS2 rearrangements. Although murine models have demonstrated their leukemogenic activities, the steps from a normal human cell to leukemic blasts remain unclear. Here, we precisely reproduced the inversion of chromosome 16 resulting in the ETO2::GLIS2 fusion in human induced pluripotent stem cells (iPSCs). iPSC-derived ETO2::GLIS2-expressing hematopoietic cells showed differentiation alterations in vitro and efficiently induced in vivo development of leukemia that closely phenocopied human acute megakaryoblastic leukemia (AMKL), reflected by flow cytometry and single-cell transcriptomes. Comparison of iPS-derived cells with patient-derived cells revealed altered chromatin accessibility at early and later bona fide leukemia stages, with aberrantly higher accessibility and expression of the osteogenic homeobox factor DLX3 that preceded increased accessibility to ETS factors. DLX3 overexpression in normal CD34+ cells increased accessibility to ETS motifs and reduced accessibility to GATA motifs. A DLX3 transcriptional module was globally enriched in both ETO2::GLIS2 AMKL and some aggressive pediatric osteosarcoma. Importantly, DLX3 knockout abrogated leukemia initiation in this ETO2::GLIS2 iPSC model. Collectively, the characterization of a novel human iPSC-derived AMKL model revealed that hijacking of the osteogenic homeobox transcription factor DLX3 is an essential early step in chromatin changes and leukemogenesis driven by the ETO2::GLIS2 fusion oncogene.