Polypeptide blood group antigens, many of which are created by single exofacial amino acid (AA) substitutions, have varying immunogenicities. Why some AA substitutions are more immunogenic than others is little understood. Using AlphaFold2, an artificial intelligence system that predicts three-dimensional protein structure along with multiple other structure analysis programs, we investigated protein structure at sites of AA substitutions that create nine clinically-significant blood group antigens. Based on structure predictions, the AA substitutions that create the four most immunogenic of the nine antigens (K, Jka, Lua, E) were typically buried or partially buried in rigid, ordered protein regions, usually helices and β-strands. This was reflected by their lower mean relative solvent accessibility (RSA) than the five less immunogenic antigens (c, M, Fya, C, S; 0.13 versus 0.81; P=.003), and higher mean AlphaFold2 confidence score (92.5 versus 48.3; P=.001; scores <50 predict protein disorder). Substitutions creating the five least immunogenic antigens (c, Fya, M, C, S) were all predicted to be in flexible regions with high accessibility, either in surface-accessible loops (C, c) or disordered coils (Fya, M, S). Scatter plots revealed a positive linear correlation of immunogenicity with confidence score (R2=0.826; P=.0007) and percent helix/β-strand in 15-mers centered around the substitution sites (R2=0.763; P=.0021), and a negative linear correlation with RSA (R2=0.688; P=.0057). Therefore, based on an informatics analysis, the protein secondary and tertiary structure at AA substitution sites that create blood group antigens are significant correlates and potential determinants of immunogenicity.

Acute myeloid leukemia (AML) that evolves from myeloproliferative neoplasm (MPN) is known as post-MPN AML. Current treatments don't significantly extend survival beyond 12 months. BCL-xL has been found to be overexpressed in leucocytes from MPN patients, making it a potential therapeutic target. We investigated the role of BCL-xL in post-MPN AML and tested the efficacy of DT2216, a platelet-sparing BCL-xL proteolysis-targeting chimera (PROTAC), in preclinical models of post-MPN AML. We found that BCL2L1, the gene encoding BCL-xL, is expressed at higher levels in post-MPN AML patients compared to those with de novo AML. Single-cell multi-omics analysis revealed that leukemia cells harboring both MPN-driver and TP53 mutations exhibited higher BCL2L1 expression, elevated scores for leukemia stem cell, megakaryocyte development, and erythroid progenitor than wild-type cells. BH3 profiling confirmed a strong dependence on BCL-xL in post-MPN AML cells. DT2216 alone, or in combination with standard AML/MPN therapies, effectively degraded BCL-xL, reduced the apoptotic threshold, and induced apoptosis in post-MPN AML cells. DT2216 effectively eliminated viable cells in JAK2-mutant AML cell lines, induced pluripotent stem cell-derived hematopoietic progenitor cells (iPSC-HPCs), primary samples, and reduced tumor burden in cell line-derived xenograft model in vivo by degrading BCL-xL. DT2216, either as a single agent or in combination with azacytidine, effectively inhibited the clonogenic potential of CD34+ leukemia cells from post-MPN AML patients. In summary, our data indicate that the survival of post-MPN AML is BCL-xL dependent, and DT2216 may offer therapeutic advantage in this high-risk leukemia subset with limited treatment options.

Myelodysplastic syndromes (MDS) are myeloid malignancies often driven by mutations in genes encoding splicing factors (SFs). How these mutations drive the clonal expansion of MDS stem/progenitor cells to outcompete normal hematopoietic stem/progenitor cells (HSPCs) remains unexplained. Although a role for inflammatory processes in promoting clonal expansion of mutant HSPCs and MDS pathogenesis has been proposed, the specific mechanisms implicated remain incompletely understood. In this study, using human isogenic induced pluripotent stem cell (iPSC)-based models of SRSF2-mutant MDS and primary MDS patient cells, we show that the SRSF2 P95L mutation downregulates basal STAT1 expression. STAT1 downregulation dampens interferon (IFN) signaling in MDS stem/progenitor cells, which, unlike normal HSPCs, show resistance to suppression of clonogenic ability by IFNs. Treatment with the proteasome inhibitor bortezomib increased STAT1 protein levels and restored sensitivity of SRSF2-mutant cells to inflammatory stimuli. These results indicate that rewiring of STAT1 signaling by SRSF2 mutations blunts responsiveness to IFNs, conferring clonal fitness to SRSF2-mutant HSPCs against normal HSPCs in the presence of inflammatory stimuli. Our study provides a novel mechanistic link between SF mutations and inflammatory dysregulation and suggests proteasome inhibition as a potential strategy to treat MDS with SRSF2 mutations.

HFE-related hemochromatosis (HH) induces systemic iron overload. Although extensive studies indicate a pivotal role for iron homeostasis in αβ-T-cell immunity, its effect on γδ-T cells is unknown. Here we show a reversal of the Vδ2+/Vδ2- ratio in the γδ-T-cell compartment as a feature of hemochromatosis, which is associated with a Vδ2+ population that cannot be enriched by ZOL-stimulation, despite evidence of TCR-ligand formation and strong proliferative behavior. Already in vivo, ROS production and exhaustion marker expression are significantly increased on Vδ2+-T-cells in hemochromatosis compared with healthy individuals. Ex vivo, hemochromatosis-donor-derived Vδ2+-cells are hyporesponsive to TCR stimulation in terms of ROS production, but significantly increase their paramount expression of exhaustion markers further. Fas-Fas Ligand coexpression indicates their high susceptibility to activation-induced cell death. Consistent therewith FeSO4 alone induces Vδ2+ subset-specific proliferation in healthy PBMCs comparable to stimulation by ZOL, and blocking experiments identify FeSO4-induced proliferation as BTN3A1/TCR-mediated. Pyrophosphates are key for Vδ2+-TCR-ligand formation. Iron, by suppressing pyrophosphatase ALP promotes their stability. In light of this our data suggest that the transcriptional repression of pyrophosphatases - as under the conditions of iron overload in hemochromatosis in vivo - leads to the constitutive availability of stress-signaling Vδ2+-TCR ligand thus permanent TCR-triggering in Vδ2+-T-cells even under homeostatic conditions, ultimately leading to their subset-specific activation-induced cell death. A similar phenotype was observed in patients with iron overload due to inborn hemoglobinopathies, suggesting an inverted Vδ2+/Vδ2- ratio in the γδ-T-cell compartment as a hallmark of iron overload.

Potentially fatal non-ICANS neurotoxicities (NINTs) following BCMA-targeted CAR T-cell therapy have been linked to high CAR T-cell expansion and prolonged persistence. Cyclophosphamide in an effort to ablate CAR T-cells is an effective treatment strategy to mitigate steroid-refractory courses of NINTs.

Excessively restrictive inclusion and exclusion criteria in clinical trials are one of many barriers to clinical trial enrollment for patients with myelodysplastic syndromes/neoplasms (MDSs). Many organizations are developing efforts to increase clinical trial eligibility; yet, several recent publications focused on patients with MDS suggest that many patients with this disease may be excluded from clinical trials unnecessarily. Clinical trial eligibility should reflect the phase of the study and risks of the agent being studied. Phase 3 trials should be less restrictive than early-phase trials to represent the real-world population as closely as possible. We hypothesize that many clinical trials, particularly phase 3 trials, have unnecessarily restrictive eligibility criteria. This study aims to evaluate the most common eligibility criteria according to phase of trial and to determine whether criteria correspond with drug safety signals. We identified MDS clinical trials registered on ClinicalTrials.gov from 1 January 2000 to 1 September 2023 and analyzed the eligibility criteria of 191 therapeutic MDS trials. We found that categorical inclusion and exclusion criteria are remarkably similar in representation across trial phases. Additionally, only 13% of trials are concordant with drug safety signals, suggesting that the eligibility criteria are often arbitrary. On behalf of the icMDS (International Consortium for Myelodysplastic Syndromes), an association of international MDS experts, we provide a position statement on restrictive eligibility criteria for MDS clinical trials that should be avoided with the aim of removing barriers to clinical trial enrollment.

With the incorporation of effective therapies for myelofibrosis (MF), accurately predicting outcomes after allogeneic hematopoietic cell transplantation (allo-HCT) is crucial for determining the optimal timing for this procedure. Using data from 5,183 MF patients who underwent first allo-HCT between 2005 and 2020 at EBMT centers, we examined different machine learning (ML) models to predict overall survival (OS) after transplant. The cohort was divided into a training set (75%) and a test set (25%) for model validation. A Random Survival Forests (RSF) model was developed based on 10 variables: patient age, comorbidity index, performance status, blood blasts, hemoglobin, leukocytes, platelets, donor type, conditioning intensity, and graft-versus-host disease prophylaxis. Its performance was compared with a four-level Cox regression-based score and other ML-based models derived from the same dataset, and with the CIBMTR score. The RSF outperformed all comparators, achieving better concordance indices across both primary and post-essential thrombocythemia/polycythemia vera MF subgroups. The robustness and generalizability of the RSF model was confirmed by Akaike's Information Criterion and time-dependent Receiver Operating Characteristic (ROC) Area Under the Curve (AUC) metrics in both sets. While all models were prognostic for non-relapse mortality, the RSF provided better curve separation, effectively identifying a high-risk group comprising 25% of patients. In conclusion, ML enhances risk stratification in MF patients undergoing allo-HCT, paving the way for personalized medicine. A web application (https://gemfin.click/ebmt) based on the RSF model offers a practical tool to identify patients at high risk for poor transplantation outcomes, supporting informed treatment decisions and advancing individualized care.

Chronic myeloid leukemia (CML) is rare in children and adolescents. Although outcomes have dramatically improved owing to tyrosine kinase inhibitors (TKIs) in the last two decades, there are still many challenges related to the management of pediatric CML, including the impact of TKIs on growth deceleration and unknown long-term adverse effects as well as defining the role of treatment-free remission. Unlike adult CML, which is driven by evidence-based guidelines, management of pediatric CML is often extrapolated from adult guidelines. However, pediatric CML differs from adult CML in many ways, presenting with different biological, molecular, and most importantly, host factors that make it necessary for a different treatment approach. Following the initial approval of first-generation imatinib for pediatric CML in 2003, three TKIs, all second-generation TKIs (2G-TKIs), have been approved, including dasatinib, nilotinib and bosutinib, which has greatly expanded therapeutic options but also added complexity to treatment determination. The expanded treatment options also call into question the treatment choice for pediatric CML, long-term efficacy and safety profiles of these TKIs. We present three cases commonly encountered in pediatric CML, their challenges and relevant issues as well as recommended managements.

Venous thromboembolism, which includes deep vein thrombosis (DVT) and pulmonary embolism, is a common cardiovascular disorder associated with significant morbidity and mortality. Current treatment options primarily involve anticoagulants, which reduce the risk of fatal events and DVT recurrence but increase the risk of bleeding, particularly in patients requiring prolonged thromboprophylaxis. Growing evidence characterizes DVT as a complex inflammation-driven process rather than a merely coagulation-dependent thrombosis, with endothelial cells, neutrophils, and platelets playing major roles in its initiation. Recent studies demonstrate that these cell types undergo profound metabolic reprogramming in response to stasis, hypoxia, and inflammatory stimuli, including shifts in glycolysis, the pentose phosphate pathway, and redox balance. This review summarizes current insights into these metabolic adaptations, examines evidence from preclinical DVT models showing that targeting metabolic pathways can reduce venous thrombus formation without impairing hemostasis, and highlights potential metabolic targets for intervention. By modulating metabolic pathways that underlie the prothrombotic and proinflammatory phenotypes, it may be possible to prevent DVT initiation or limit its progression while reducing the reliance on anticoagulants and the risk of associated bleeding complications. This metabolism-centered perspective opens new avenues for the development of safer, more effective treatments for DVT.

We comprehensively profiled the landscape of immunoglobulin (IG) and T-cell receptor (TR) rearrangements at diagnosis in 1212 acute lymphoblastic leukemia (ALL) patients (573 children, 639 adults) diagnosed in Germany between 2017 and 2022.Our findings indicate that immunogenetic maturity in ALL patients is age-dependent, with younger patients exhibiting more mature profiles. In fact, 68.7% of pediatric B-ALL and 85.7% T-ALL patients carried IGK, or complete TRB and/or TRD rearrangements, respectively; compared to 39.0% and 67.3% in adults (B-ALL: p<2.2e-16, T-ALL: p=6.7e-03). Additionally, children carried more IG/TR markers compared to adults (mean 6/patient versus 4/patient, respectively; p=2.5e-38). Only 0.5% of pediatric patients lacked markers, contrasted with 6.7% of adults.IGH clonal evolution was most pronounced among pro-B ALL cases (60.9%), with the V-to-DJ mechanism driving pro-B evolution (78.6%), while V-replacement dominated other immunophenotypes. Additionally, we observed that the presence of expanded accompanying T-cell clones of unknown significance in B-ALL patients increased with age.This next-generation sequencing (NGS)-based study offers an unprecedented characterization of IG/TR rearrangement patterns across ALL subtypes and age groups. It highlights the higher immunogenetic maturity in children, which may be explained by the infection-driven abnormal activity of the IG/TR recombination machinery in pediatric ALL.

Hemorrhage causes millions of deaths and hundreds of billions of dollars in medical costs every year, and a large percentage of trauma bleeding associated deaths occur in the pre-hospital setting. Bleeding is typically treated with transfused blood products, but this is difficult in the prehospital setting due to limitations in transportation and storage, especially in rural and remote military settings. Advancements in cold-stored platelets and lyophilized blood products have the potential to address some of these limitations. However, devising novel products that continue to improve shelf life, portability, scalability, cost, and safety for patients experiencing bleeding in pre-hospital settings could greatly improve treatment options and patient outcomes. This review primarily focuses on rationale design of material-based approaches to develop novel hemostatic agents that strive to meet limitations of current blood products, especially for use in the pre-hospital setting. Key topics of consideration include how material design can lead to identification of effective therapies that stop bleeding as well as strategies to iterate on existing designs to enhance healing after cessation of bleeding. Improving performance and functionality of existing and emerging materials could be achieved through the incorporation of transglutaminases, growth factors, cellular components, or inorganic molecules. Finally, consideration of patient specific factors that influence bleeding, such as patient sex and age, through evaluation of therapies in specific populations and/or design of materials targeted for specific patient populations is a key area for development of next generation hemostatic materials.

Heparin-induced thrombocytopenia (HIT) is initiated by antibodies that recognize large antigenic complexes composed of multiple molecules of cationic platelet factor 4 (PF4) and polyanions such as unfractionated heparin (UFH) that bind to each other primarily through electrostatic interactions. We asked whether the formation and stability of these HIT antigenic or ultralarge immune complexes (ULICs) would be inhibited by biocompatible synthetic polycationic molecules shown previously to dissociate unfractionated heparin from antithrombin III and to inhibit polyphosphates. Members of this family of molecules, designated Universal Heparin Reversal Agents (UHRAs), inhibited formation and dissociated preformed ultralarge PF4-UFH complexes (ULCs), dissociated ULICs composed of the HIT-like monoclonal antibody KKO and ULCs, blocked binding of human HIT IgG antibodies to PF4/heparin, binding of KKO to platelets, KKO-induced adhesion of platelets to activated human endothelium under flow, and microvascular thrombosis induced by KKO in a mouse model of HIT. These data suggest that UHRAs might provide a rationale intervention that acts at an early step in the pathogenesis of HIT to enhance the benefits and lessen the risks of non-heparin anticoagulants. Destabilization of immune complexes using polycationic inhibitors might also find a role in management of other polyanion-PF4-antibody-mediated conditions, including vaccine-induced thrombocytopenia/thrombosis, post-viral, and autoimmune HIT.

Hematopoietic stem cells (HSCs) exhibit significant age-related phenotypic and functional alterations. Although single-cell technologies have elucidated age-related compositional changes, prospective identification of aging-associated HSC subsets has remained challenging. In this study, utilizing Clusterin (Clu)-GFP reporter mice, we demonstrated that Clu expression faithfully marks age-associated myeloid/platelet-biased HSCs throughout life. Clu-GFP expression clearly segregates a novel age-associated HSC subset that overlaps with but is distinct from those previously identified using antibodies against aging maker proteins or reporter systems of aged HSC signature genes. Clu-positive (Clu+) HSCs emerge as a minor population in the fetus and progressively expand with age. Clu+ HSCs display not only an increased propensity for myeloid/platelet-biased differentiation but also a unique behaviour in the BM, favouring self-renewal over differentiation into downstream progenitors. In contrast, Clu-negative (Clu-) HSCs exhibit lineage-balanced differentiation, which predominates in the HSC pool during development but becomes underrepresented as aging progresses. Both subsets maintain long-term self-renewal capabilities even in aged mice but contribute differently to hematopoiesis. The predominant expansion of Clu+ HSCs largely drives the age-related changes observed in the HSC pool. Conversely, Clu- HSCs preserve youthful functionality and molecular characteristics into old age. Consequently, progressive changes in the balance between Clu+ and Clu- HSC subsets account for HSC aging. Our findings establish Clu as a novel marker for identifying aging-associated changes in HSCs and provide a new approach that enables lifelong tracking of the HSC aging process.