PAX5-altered acute lymphoblastic leukemia (PAX5-alt ALL) is a recently recognized molecular subtype of B-ALL characterized by a distinct transcriptional signature and frequent PAX5 fusions (PAX5-r). While PAX5-alt ALL has been associated with intermediate outcomes, clinical data specifically investigating pediatric PAX5-r ALL remain limited. We collected and analyzed 159 pediatric cases of PAX5-r ALL treated between 2001-2024 within AIEOP-BFM ALL studies across Italy, Germany and Austria, revealing high-risk clinical features. Comparative analyses of patients consecutively enrolled in the AIEOP-BFM ALL 2017 study (PAX5-r, n=96 vs. non-PAX5-r, n=1948) confirmed higher rates of hyperleukocytosis at diagnosis (22.9% vs. 8%, p<0.001) and enrichment of IKZF1plus profile (14.7% vs. 7.8%, p=0.0015) in PAX5-r patients. No relevant differences in terms of minimal/measurable residual disease (MRD)-based treatment response and risk-group stratification were observed. PAX5-r patients had a 4-year EFS and OS of 72.6±5.7% and 95.4±2.3%, respectively. Four-year EFS were 100%, 63.4±9.7% and 63.3±10% for standard-risk, medium-risk (MR) and high-risk (HR) groups, respectively, indicating that the poor prognostic impact of PAX5-r applies only when end-of-induction MRD is positive (MR/HR). Whole transcriptome sequencing revealed high FLT3 median expression in PAX5-r ALL and high-throughput drug screening of patient-derived xenografts (PDXs) showed marked sensitivity to several FLT3 inhibitors. Gilteritinib showed potent ex vivo cytotoxicity and synergism with dexamethasone in PAX5-r PDX models. Collectively, this study investigated clinical and biological features of pediatric PAX5-r ALL highlighting its MRD-dependent unfavorable prognosis and identifying FLT3 overexpression as a novel, potential therapeutic target.

Heparin-induced thrombocytopenia (HIT) is a life-threatening prothrombotic disorder with substantial clinical mortality, characterized by pathologic antibody formation against platelet factor 4 (PF4)-heparin complexes. This study identified early pathologic mechanisms that initiate HIT immune complex formation and regulate thrombosis development. We found that repeated heparin administration over five days markedly enhanced platelet activation which implies an additional activating mechanism. Through plasma proteomic analysis, we observed that histidine-rich glycoprotein (HRG) levels were significantly reduced following successive heparin administration, and inversely related to platelet activation. Mechanistically, heparin and PF4 suppressed HRG expression via inhibiting the FGFR-ERK-Elk1 pathway whereby Elk1 directly binds to the HRG promoter region to regulate its liver expression. Physiological levels of HRG potently inhibited platelet activation, procoagulant activity, spreading, and aggregation, and significantly reduced thrombus formation under flow conditions. HRG bound to activated αIIbβ3 integrin in a zinc-dependent manner, thereby blocking its ligand-binding capacity. HRG also inhibited the formation of ultralarge PF4-heparin complexes (ULCs), disrupted the immune complexes (ULICs) comprising the HIT-like antibody KKO and ULCs, and blocked the binding of KKO to PF4/heparin on platelets. Furthermore, HRG attenuated ULCs-triggered autoantibody generation, inhibited the ULICs-induced neutrophil extracellular traps and microvascular thrombosis formation both in vitro and in a mouse model of HIT. In conclusion, our work establishes HRG as a pivotal regulator of pathologic immune responses and thrombosis in HIT, providing mechanistic insights into disease pathogenesis.

Historically the impact of hemophilia for female carriers was believed to be restricted to their male offspring. Recent studies have highlighted the increased bleeding experienced by many hemophilia carriers, challenging traditional concepts of X-linked inheritance. These data have resulted in a mindset shift in the haemostasis community, culminating in the 2021 ISTH update on classification of hemophilia carriers. Women and girls with reduced factor VIII (FVIII) /factor IX (FIX) levels <0.40 IU/mL are now recognised as women and girls with hemophilia (WGH). Furthermore, hemophilia carriers who experience excessive bleeding despite normal FVIII/FIX levels are termed symptomatic carriers. Despite these changes, the impact of historic misconceptions surrounding hemophilia carriership remains and many families and healthcare providers may still believe that hemophilia carriers are at no risk of increased bleeding. Education is key to address these ongoing challenges and engagement with both hemophilia communities and physician organisations is critical. Hemophilia treatment centers must endeavour to ensure access for all hemophilia carriers and availability of services appropriate to needs of hemophilia carriers and WGH throughout their lifespan. In contrast to past publications that focus on pregnancy, in this article we address bleeding in hemophilia carriers and WGH at different life stages, including the impact of delay on diagnosis and the management of reproductive tract and joint bleeding.

Leukocyte Adhesion Deficiency syndrome type III (LAD-III) is characterized by recurrent infections and is caused by FERMT3 gene mutations. FERMT3 encodes two isoforms, a standard kindlin-3 and a longer splicing variant, which differs by the addition of four residues Ile-Pro-Arg-Arg (IPRR) in the pleckstrin homology (PH) domain (kindlin-3-IPRR). Previous studies suggested that kindlin-3-IPRR was dysfunctional in inducing neutrophil adhesion, attributed to altered phospholipid binding of the IPRR-containing PH domain. Here we show that kindlin-3-IPRR is fully functional in activating β2 integrins and promotes neutrophil adhesion. The IPRR insert enhances phospholipid binding in vitro and promotes association of kindlin-3 with the plasma membrane. By analyzing RNA sequencing datasets of neutropoiesis, we show that the kindlin-3 short isoform lacking IPRR is highly expressed in neutrophil precursors but downregulated in mature neutrophils. In contrast, a higher proportion of the long isoform was detected in hematopoietic stem cells (HSCs) and mature neutrophils. Kindlin-3-IPRR supports robust adhesion of HSCs. We propose that kindlin-3-IPRR may be crucial in conditions requiring rapid neutrophil mobilization. Indeed, kindlin-3 splicing is altered in neutrophils from patients undergoing stress myelopoiesis after exposure to granulocyte colony-stimulating factor (G-CSF) or HSC transplantation (HSC-T) compared with healthy donors. During stress myelopoiesis induced by G-CSF treatment, the kindlin-3 long isoform is selectively upregulated in mature neutrophils, coinciding with enhanced β2 integrin activation. These findings support a model in which alternative splicing of FERMT3 dynamically fine-tunes integrin activation and leukocyte adhesion during hematopoietic stress, with potential implications for monitoring hematopoietic recovery after HSC transplantation.

Venous thromboembolism (VTE) provoked by transient risk factors is generally considered low risk, supporting short-term anticoagulation. However, this paradigm is based on heterogeneous definitions of provoking factors. In a prospective cohort study, we evaluated recurrence risk using the International Society on Thrombosis and Haemostasis (ISTH) classification of major and minor transient risk factors. Patients with symptomatic deep vein thrombosis and/or pulmonary embolism who completed ≥3 months of anticoagulation were followed after treatment discontinuation. Patients with cancer, major thrombophilia, pregnancy-associated VTE, or indication for extended anticoagulation were excluded. The primary outcome was symptomatic recurrent VTE. Cumulative incidence was estimated by Kaplan-Meier analysis. The study was terminated early due to unexpectedly high recurrence risk among patients with major provoking factors. Between 2019 and 2025, 242 patients were followed for a median of 23.9 months; 72 had major and 170 minor transient risk factors. Recurrent VTE occurred in 13 patients with major and 7 with minor risk factors. At 24 months, recurrence risk was 9.5% (95% CI, 6.0-14.0) overall, 19.7% (11.0-30.2) after major, and 5.0% (2.2-9.5) after minor risk factors. No recurrence occurred in 81 women with hormone-associated VTE; after their exclusion, the recurrence risk among patients with other minor risk factors increased to 9.1% (95% CI 4.0-17.0). Recurrence after provoked VTE is not uniformly low and varies substantially by the type of transient risk factor. These findings challenge current treatment paradigms and support individualized decisions regarding anticoagulation duration.

Acute myeloid leukemia (AML) is an aggressive blood cancer with a 5-year overall survival rate of ~30%. Although immunotherapies engaging T cells demonstrate remarkable success in treating many solid tumors and blood cancers, they show little to no efficacy in treating AML. Therefore, immunotherapies are traditionally underappreciated and underdeveloped in AML. Through a drug re-purpose screen, we identified and validated that combined MEK and HDAC inhibitions via trametinib and quisinostat (TQ) potently inhibited the growth of mouse and human NRAS;ASXL1-AML (NA-AML), MLLr, and NPM1 mutated AML cells in vitro. In NA-AML mice, TQ drastically slowed down AML progression and prolonged their survival. The survival benefits of TQ largely relied on T cell functions. We show that TQ synergized to downregulate immune checkpoint ligands and upregulate STAT1- and CIITA-mediated expression of MHC-I and MHC-II in NA-AML cells. In addition, TQ treatment significantly reprogrammed transcriptome and epigenetic landscape of T cells, activated STAT1 signaling, and upregulated genes and pathways promoting activation, survival, and cytotoxicity of CD4 and CD8 T cells. A cytotoxic cluster was thus expanded in central memory and effector memory T cells in TQ-treated NA-AML mice. More importantly, TQ directly acted on AML-associated mouse and human T cells, reverting them from a dysfunctional state to an active state. In leukemia:T cell co-cultures, TQ-treated T cells demonstrated greatly improved MHC-dependent leukemia killing. Our findings suggest that the dual actions of TQ on NA-AML and T cells enhance leukemia recognition and anti-leukemia killing of endogenous T cells, leading to effective AML clearance.

Germline gain-of-function (GOF) mutations in the signal transducer and activator of transcription 1 (STAT1) gene cause a dominantly inherited inborn error of immunity (IEI) characterized by chronic mucocutaneous candidiasis, autoimmunity, severe opportunistic infections and an increased risk of malignancy. Allogeneic hematopoietic stem cell (HSC) transplantation (HSCT) is curative but is associated with increased risk of morbidity and mortality in STAT1 GOF patients compared to other IEI. To develop a curative, autologous alternative to HSCT, we evaluated gene editing strategies in STAT1 GOF model cell lines, primary T cells, and patient-derived HSCs. Universal and mutation-specific strategies using CRISPR/Cas-mediated homology-directed repair (HDR) were limited by low efficacy (<25%), poor viability, and a lack of allele-specificity. In contrast, adenine base editing corrected the recurrent and highly pathogenic p.T385M mutation with upwards of 90% efficiency in patient T cells and HSCs without significant unintended on- or off-target genomic aberrations. Gene editing functionally restored total STAT1 expression (p<0.0217), STAT1 phosphorylation (p<0.0056), interferon-stimulated gene expression (OAS1; p=0.0005) and improved IL-17 production (p<0.0001). Edited HSCs retained multilineage differentiation capacity and sustained engraftment with persistence of the corrected allele at 16 weeks in humanized immunodeficient mice. These data demonstrate efficient and precise correction of STAT1 GOF mutations by base editing, with maintenance of the correction through long-term engraftment in vivo. This represents the first application of gene editing to correct a dominant gain-of-function mutation causing immunodeficiency, with potential applicability to other genetic disorders associated with heterozygous and gain-of-function mutations.

Mitochondrial dynamics is a key regulator of cellular homeostasis, orchestrating metabolic reprogramming that fuels tumor progression and treatment resistance. In multiple myeloma (MM), however, the functional relevance of mitochondrial remodeling has not been fully defined. Using ultrastructural analyses, we reveal that MM cells display a highly fragmented mitochondrial network, a phenotype further exacerbated in both cell lines and primary MM cells resistant to proteasome inhibitors. Transcriptomic profiling across multiple patient-derived datasets consistently demonstrated upregulation of DNM1L gene, which encodes the mitochondrial fission GTPase DRP1, particularly in relapsed and refractory MM, and revealed a significant association with inferior overall survival. Disrupting mitochondrial fission, either through genetic targeting of DNM1L or pharmacologic inhibition of DRP1 with the selective small molecule inhibitor Drpitor1a, resulted in pronounced mitochondrial dysfunction, impaired oxidative phosphorylation, and potent anti-myeloma activity in vitro, culminating in a hybrid cell death program with a predominant apoptotic component accompanied by ferroptotic features. These effects were recapitulated in vivo in a bortezomib-resistant xenograft model, where either DNM1L depletion or DRP1 inhibition produced similar outcomes. Mechanistically, the transcription factor c-MYC upregulated DNM1L expression, and DRP1-dependent mitochondrial fragmentation sustained MYC-driven oxidative metabolism and lipid synthesis. Altogether, these findings establish aberrant mitochondrial fission as a pathogenic hallmark of MM and highlight DRP1 inhibition as a promising therapeutic approach, especially for relapsed or refractory disease.

Autoimmune neutropenia (AiN) in adults is rare when compared to children. It occurs predominantly in female patients and in individuals with other autoimmune conditions. Anti‑neutrophil antibodies are detected in ~50% of patients with AiN, and it is not clear whether this reflects testing limitations or cell-mediated (rather than antibody-mediated) mechanisms of neutrophil destruction. Unlike the commonly self‑limited pediatric disease, adult AiN is generally chronic. The degree of neutropenia and the clinical severity is highly variable, and most patients remain asymptomatic and free of infection despite absolute neutrophil counts (ANC) persistently lower than 500 /uL. As most patients do well despite their neutropenia, observation and supportive care are generally preferred over active treatment. Where necessary, empiric treatment recommendations have stemmed from small numbers of cases. Therapy focuses on boosting endogenous neutrophil production, reducing immune-mediated destruction, or eliminating pathogenic autoantibodies. Clinical response is highly variable, and second-line therapies, including T‑cell-directed approaches, can be effective, supporting a role for cell-mediated mechanisms of neutrophil destruction in a subset of patients. Emerging strategies, such as plasma cell-targeted therapies, modulation of cytokine signaling pathways, and complement blockade also show promise. Collaboration and discussion around this rare condition is critical to enhance treatment algorithms.

Germline GATA2 deficiency is a pleiotropic condition 1-8 characterized by numerous phenotypes, including monocytopenia, immunodeficiency, microbial susceptibilities, and high rates of myeloid malignancies 1,8. Accurate curation of germline GATA2 variants is critical for patient care and requires well-defined phenotypes associated with GATA2 deficiency. The many phenotypes attributed to the condition render a simple description of GATA2 deficiency difficult, complicating the development of GATA2 variant curation rules. Therefore, the Myeloid Malignancy Variant Curation Expert Panel (MM-VCEP) sought to define GATA2 deficiency based on a statistical comparison of phenotype data. To do so, the MM-VCEP systematically analyzed phenotype data and applied statistical comparisons to define the phenotypic features of GATA2 deficiency to inform germline variant curation. The MM-VCEP assembled an international cohort of 339 people with clinically diagnosed GATA2 deficiency from 16 centers in seven countries. The 73 phenotypes of these individuals were compared statistically to those of control participants from the UK Biobank (UKBB). We compared single phenotypes as well as combinations of two, three, and four phenotypes in people with clinically diagnosed GATA2 deficiency to UKBB controls. We defined GATA2 deficiency as any of the 2,903 combinations of two or three phenotypes with log10Odds Ratio ≥3 (OR ≥ 1000). This definition of GATA2 deficiency will inform gene-specific phenotypic criteria used in GATA2 variant curation guidelines that will facilitate standardized variant curation by clinical laboratories worldwide.

Talquetamab is the first and only approved bispecific antibody targeting G protein-coupled receptor class C group 5 member D (GPRC5D) for treatment of relapsed/refractory multiple myeloma based on results from the phase 1/2 MonumenTAL-1 study. Here, we report efficacy and ongoing safety from MonumenTAL-1 with 3 years of follow-up. Patients naïve to T-cell redirection therapy (TCR) received talquetamab 0.4 mg/kg weekly (n=143) or 0.8 mg/kg every other week (n=154); a separate cohort received prior TCR (n=78, either talquetamab dose). Median follow-up was 38, 31, and 30 months in the 3 cohorts, respectively, as of September 2024. Overall response rate was 67-74% and complete response or better rate was 33-42%. Median progression-free survival was 7.5, 11.2, and 7.7 months, and median overall survival (OS) was 34.0 months, not reached, and 28.3 months (36-month OS rates 49.3%, 60.8%, and 44.6%), in the 3 cohorts, respectively. The most common adverse events (AEs) were cytokine release syndrome (73-79%; grade 3/4, 0.6-2.1%), taste changes (72-76%), and infections (61-78%; grade 3/4, 21-26%). Ataxia/balance disorders occurred in 5.3% of patients (no grade 4/5 events). Dose reduction and discontinuation rates due to AEs remained low; no patients died due to talquetamab-related AEs. With 3 years of follow-up, talquetamab continued to demonstrate high rates of deep and durable responses. The long-term safety profile was comparable to previous results and continued to show lower risk of high-grade infections relative to approved BCMA-targeting bispecific antibodies. NCT03399799 (phase 1) and NCT04634552 (phase 2).

Ex vivo expansion of human hematopoietic stem cells (HSCs) holds promise for overcoming their limited availability, a major barrier to broader clinical application. Although recent advances in culture systems can increase HSC numbers, these conditions frequently impair self-renewal and induce myeloid bias, and the underlying molecular mechanisms remain poorly understood. Here, we performed single-cell multiome sequencing (scMultiome-seq) on human umbilical cord blood-derived CD34⁺ hematopoietic stem and progenitor cells to co-profile transcriptional and epigenetic adaptations within the same cells during ex vivo culture. Our analyses revealed reduced transcriptional and epigenetic HSC signatures, accompanied by markedly increased activity of myeloid-associated transcription factor motifs, providing molecular insight into the functional decline and myeloid bias of cultured HSCs. We further observed substantial functional heterogeneity among phenotypically defined HSCs following culture. To address these limitations, we established a niche-mimetic culture system that integrates intrinsic and extrinsic bone marrow regulatory cues, including pharmacologic inhibition of the m6A reader YTHDF2 using the small molecule Y13-27, a three-dimensional microenvironment, and N-cadherin-mediated adhesion. This condition (3D-NcadP-Y) robustly preserved long-term repopulating capacity. When combined with the self-renewal agonist UM729, the resulting platform (3D-NcadP-Y-UM) uniquely enabled the expansion of serially transplantable long-term HSCs with balanced multilineage potential. scMultiome-seq and cellular analyses demonstrated that this condition preserves transcriptional and epigenetic long-term HSC signatures, maintains multilineage-associated transcription factor motifs, and limits excessive cell-cycle activation. Together, these findings elucidate molecular mechanisms underlying culture-induced HSC dysfunction and establish a niche-mimetic strategy for expanding functional human long-term HSCs while preserving key features of stemness.