Sufficient levels of fetal hemoglobin (HbF) can ameliorate the pathophysiologic basis of sickle cell disease and β-thalassemia postnatally. DNA methylation has long been posited to mediate silencing of HbF expression, but this has been controversial. Recent publications provide definitive evidence for the critical role of HBG gene promoter methylation in silencing and insight into the mechanisms involved. The data support a model in which methylation of CpG sites in the HBG promoters and repressive transcription factors recruit the MBD2-NuRD chromatin remodeling complex, which enforces silencing. These findings have implications for the treatment of β-globin disorders.

Graft-versus-host disease (GvHD) remains one of the major complications following allogeneic haematopoietic cell transplantation. Currently, immunosuppressants are used for GvHD prophylaxis and treatment in the majority of transplant recipients. Due to their systemic, non-specific mode of action, this treatment regimen is frequently associated with severe toxic side effects, opportunistic infections as well as cancer relapse when treating haematologic malignancies. By using short-term ex vivo modulation of haematopoietic cell transplants with the anti-human CD4 antibody MAX.16H5, we have developed a novel immune tolerance-inducing strategy enabling potent GvHD prevention. Functional in vitro assays and transcriptome profiling data suggest impaired TCR signalling and a shift towards an IL-10-dependent regulatory phenotype as the primary mechanism of action of anti-human CD4 antibody treatment, leading to a significantly reduced activation and proliferation of CD4+ and CD8+ T cells. A one-time incubation of haematopoietic transplants with MAX.16H5 prolongs survival of NSG mice and reduces signs of GvHD manifestation as effectively as repeated application with clinically applied immunosuppressants - making it a safe and effective immunotherapy for GvHD prevention.

Mutations in GATA1 that cause skipping of exon 2, which encodes the N-terminus, are associated with the myeloid leukemia of Down syndrome and Diamond-Blackfan anemia (DBA). To elucidate the molecular function of this N-terminal region, we employed single-cell RNA sequencing (scRNA-seq) on fetal liver cells from Gata1 mutant embryos that express only the short isoform of GATA1 (GATA1s) lacking the N-terminus of full-length GATA1 (GATA1FL). scRNA-seq revealed defects in erythropoiesis and aberrant upregulation of glycolytic genes, including PKM, which encodes pyruvate kinase to catalyze the final and irreversible step of glycolysis. Using precision nuclear run-on sequencing (PRO-seq) and cleavage under targets and release using nuclease (CUT&RUN) following acute GATA1 deletion in erythroid cells, we identified PKM as a direct target of GATA1. Substitution of GATA1FL with GATA1s induced histone lactylation at the PKM promoter, increased PKM expression and activity, and enhanced glycolytic flux in erythroid progenitors, without affecting mitochondrial respiration. Importantly, PKM expression is also significantly elevated in DBA patients with RPS19 mutations, which is associated with reduced levels of GATA1, further supporting a link between GATA1s-driven defective erythropoiesis and dysregulated glycolysis. Together, these findings reveal that GATA1 controls not only heme metabolism, but also glycolytic reprogramming.

By integrating short-read whole-genome sequencing and RNA sequencing (RNA-seq) data with long-read RNA-seq, we dissect the complex genomic architecture of PAX5 intragenic tandem multiplication, revealing that these complex rearrangements result in in-frame transcripts that likely encode proteins with altered domains.

Malaria, caused by Plasmodium parasites, accounts for ~250 million cases and over 600,000 deaths annually. One leading cause of mortality and morbidity is cerebral malaria (CM). Platelets mediate CM pathogenesis, although the exact mechanisms remain largely unknown. We examined if the mechanistic target of rapamycin (mTOR) pathway in platelets contributes to malaria pathogenesis. Our results demonstrate activation of the mTOR pathway in platelets ex vivo after co-incubation with Plasmodium falciparum-infected red blood cells and in vivo in Plasmodium berghei ANKA (PbA)-infected mice. When mTOR was specifically deleted in platelets (mTORplt-/-), mice with experimental cerebral malaria (ECM) had significantly increased survival. Survival differences were independent of parasitemia and thrombocytopenia. PbA-infected mTORplt-/- mice demonstrated significantly reduced platelet deposition in the brain resulting in improved cerebral blood flow and reduced brain vascular permeability. Plasma heme levels, generated during malaria, correlated significantly with intracerebral platelet accumulation in the PbA-infected mTORplt+/+ mice, but not in PbA-infected mTORplt-/- mice. In vitro experiments demonstrated that heme activates platelet mTOR downstream of Immunoreceptor Tyrosine-based Activation Motif (ITAM) signaling, predominantly through C-type lectin-like receptor 2. Blockage of heme-induced platelet activation with cobalt protoporphyrin significantly reduced platelet mTOR activation and decreased ECM-associated mortality. In conclusion, our findings demonstrate that platelet mTOR amplifies platelet activation responses induced by heme and deletion of platelet mTOR reduces platelet deposition in the brain, which we propose impedes symptomatic disease progression and malaria-associated mortality.

Relapse remains a major barrier to survival in B-cell acute lymphoblastic leukemia (B-ALL). Both activation of B-cell signaling pathways and increased glucose consumption have been linked to chemo-resistance and relapse risk. Here, we connect these observations, showing that B-ALL cells with active signaling, marked by high phosphorylated ribosomal protein S6 (pS6+), are glucose dependent. Isotope tracing confirms that pS6+ cells are highly glycolytic and rely on glucose for de novo nucleotide synthesis. Uridine, but not other purines or pyrimidines, rescues pS6+ cells from glucose deprivation, highlighting uridine as essential for survival. Active mTOR signaling in pS6+ cells drives de novo pyrimidine synthesis by activating CAD (Carbamoyl phosphate synthetase 2, Aspartate transcarbamylase, and Dihydroorotase), which catalyzes the first steps of de novo pyrimidine synthesis. Inhibiting signaling abolishes glucose dependency and CAD phosphorylation. Primary pS6+ cells express high levels of pyrimidine synthesis proteins, including dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in pyrimidine synthesis. Increased DHODH expression correlates with relapse and poor event-free survival. Most B-ALL molecular subtypes exhibit DHODH activity. BAY-2402234, a DHODH inhibitor, effectively kills pS6+ cells in vitro, with IC50 values correlating with pS6 signaling strength across 14 B-ALL patient-derived xenografts (PDX). In vivo, DHODH inhibition prolongs survival and reduces leukemia burden in pS6+ B-ALL models. These findings link active signaling to pyrimidine dependency and relapse risk, highlighting DHODH inhibition as a promising therapeutic strategy for chemo-resistant B-ALL.

Long telomere length (TL) extends replicative capacity in vitro, and predisposes to clonal hematopoiesis. We characterized the cancer phenotype in 51 individuals from 24 families with mutant POT1, a negative regulator of telomerase elongation (median age 51, range 5-94). Hematologic malignancies were second in prevalence after melanoma (27%), and lymphoid subsets were more common. They clustered with history of sarcoma, thyroid cancer and chronic myeloproliferative neoplasms. UKB participants with pathogenic POT1 variants had long TL and higher lymphoid malignancy rates (45% by age 80, Hazard ratio 8.28, 95% CI, 5.29-13.0). Across cohorts, diagnoses encompassed acute lymphoblastic leukemia and Hodgkin lymphoma in children/young adults, and chronic lymphocytic leukemia/multiple myeloma in adults. They clustered in families manifesting as autosomal dominant pan-lymphoma with genetic anticipation at times. Lymphocyte TL was longer than granulocytes at baseline (age-adjusted mean +1 kb, P<0.0001), and was preserved longitudinally with aging. Ultra-long lymphocyte TL >99th percentile was more sensitive for identifying pathogenic variants (58% vs. 38% for granulocytes). Among asymptomatic POT1 variant carriers, 60% (12 of 20) had immunophenotype-detected B and/or T cell clonality with complete penetrance after age 65 (7 of 7). IGH CDR3 sequencing supported age-dependent pruning of the B cell repertoire, and cytogenetic and next-generation analyses uncovered preclinical clonal lymphoma-associated changes in nearly all POT1 variant carriers older than 60 (9 of 10). Our data identify extended cellular longevity due to long TL as an inherited risk factor for lymphoma explaining its syndromic association with solid tumors, and in some cases, myeloproliferative neoplasms.

Cancer patients face an increased risk of venous thromboembolism (VTE), and breakthrough thrombosis despite anticoagulation, with a six-month cumulative incidence of 5-8%. The management of these events is clinically challenging. Confirming suspected breakthrough thrombosis requires imaging review, ideally by comparison with post-index baseline studies, as residual thrombus is common and may mimic recurrence. When true breakthrough thrombosis is confirmed, several potential contributing factors should be assessed. Tumor extension can lead to mechanical vein compression and thrombus formation. Non-adherence is common among anticoagulated patients and should be evaluated through detailed medication history. Measurement of drug-specific plasma levels, when available, may assist in confirming non-adherence. In patients on LMWH, underlying prothrombotic conditions such as heparin-induced thrombocytopenia or acquired antithrombin-deficiency must also be considered. In patients receiving oral anticoagulants, drug-drug interactions and impaired gastrointestinal absorption should be excluded. Therapeutic strategies are guided by limited evidence, primarily from observational studies. Current practice generally favors switching to therapeutic low-molecular-weight heparin (LMWH) if the patient was on oral anticoagulation, escalating LMWH dosing by 25-33% if already on therapeutic LMWH, or increasing LMWH to weight-adjusted therapeutic dose if treatment was subtherapeutic. Despite treatment adjustments, recurrence and bleeding risks remain substantial. In this review, we outline common clinical scenarios of breakthrough thrombosis in cancer patients and critically appraise the available evidence to inform treatment decisions.

The occupation of the surface immunoglobulin antigen-binding site by oligomannose-type glycans (sIg-Mann) is a tumor-specific post-translational modification of classic follicular lymphoma (FL). SIg-Mann switches binding from antigen to dendritic cell-specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), known to be expressed on interfollicular macrophages and FL-associated follicular dendritic cells (FDCs). The interaction with DC-SIGN induces reorganization of sIg-Mann in wider and less dense clusters than anti-Ig, consistent with inefficient DC-SIGN-induced endocytosis and a low-level intracellular signaling. However, ligand-specific cell clusters form between sIg-Mann-expressing lymphoma and DC-SIGN-expressing cells, raising a need to understand the functional consequences of the interaction of DC-SIGN with sIg-Mann on primary FL cells. This engagement induces adhesion of FL cells to vascular cell adhesion molecule-1 (VCAM-1) via B-cell receptor proximal kinases and actin regulators in a fashion similar to anti-Ig, but without initiating apoptosis in vitro. Instead, antibody blockade of sIg-Mann contact with DC-SIGN expressed on FDC-derived YK6/SIGN cells inhibits adhesion and survival of primary FL cells in vitro. These data highlight that the specific interaction with DC-SIGN induces FL cell adhesion to VCAM-1, likely allowing FL cell retention in the lymph node, and survival of the FL cells. Adhesion and survival are inhibited by an anti-DC-SIGN blocking antibody, indicating a new early therapeutic approach against FL retention and survival in adaptive tumor tissue niches.

B cells expressing the IGHV4-34 gene sit at the centre of a web of B-cell functions, ranging from natural antibodies induced by infection through to autoimmunity and tumors. Tracking via the 9G4 monoclonal antibody (MoAb) specifically directed against the unique flag sequences in the first framework region (FR1) of IGHV4-34, allows insight into the evolving nature and multiple functions of B cells expressing a single IGHV gene. IGHV4-34+ B cells are found early in development, and expansion indicative of a superantigenic drive occurs with increasing age. IGHV4-34+ B cells secrete IgM in various hematologic conditions, including the mandatory association with cold agglutinins, and transient expansions of usage (not always overt agglutination) are seen in EBV, CMV, Mycoplasma pneumoniae and other infections. Perhaps connected to that, certain autoimmune conditions, such as systemic lupus erythematosus, show increases in IGHV4-34 expression which correlate with disease flares. Intriguingly there is preferential usage of IGHV4-34 in a wide range of lymphomas and in the IgG variant of chronic lymphocytic leukemia, suggestive of an autoantigenic drive. Structurally, both the interaction of the 9G4 MoAb, and the ability of the IGHV4-34-encoded protein to bind to N-acetyl lactosamine-containing sequences, rely on hydrophobic amino acids located in two positions of the looped FR1 sequence of the variable domain. This offers a focused site for blockade. IGHV4-34 is a paradigm of the plasticity of B cells, of interaction with potential autoantigens, and of their subversion in tumors.

Inherited genetic variants that modulate platelet function contribute significantly to thrombotic disorders, yet their mechanisms and clinical implications remain underexplored. Two genome-wide association studies identified an AàG variant (rs10886430) in the first intron of G protein-coupled-receptor kinase 5 (GRK5), found in homozygosity in ~5 million Americans. The homozygous GRK5 GG genotype has an increased risk of stroke and venous thromboembolism, but the mechanistic link between this variant and thrombotic risk has remained unclear. To investigate this, we identified three GG individuals. GRK5 protein levels in GG platelets were 90% lower than in AA controls. The significant reduction in GRK5 levels in GG platelets led to elevated platelet responsiveness to thrombin and a PAR1 agonist but not to a PAR4 agonist. These findings were corroborated in GRK5-/- iPSC-derived megakaryocytes, transgenic Grk5-deficient murine platelets, and AA platelets exposed to a GRK5 inhibitor. We demonstrated that PAR1 internalization was reduced in GG platelets, leading to enhanced PAR1 signaling. Under venous shear in an endothelialized microfluidic system, GG platelets exhibited increased accumulation, which was reversed by PAR1 inhibition with vorapaxar. In an arterial murine thrombosis model following human platelet infusion, GG platelets also showed enhanced thrombus formation in vivo. This study provides the first experimental evidence directly linking a highly prevalent human GRK5 variant to defective PAR1 regulation and increased thrombotic risk. Together, these findings establish that the GRK5 GG genotype confers increased thrombotic potential through impaired PAR1 desensitization, providing mechanistic insight that connects human genetics, thrombin receptor signaling, and thrombotic disease.

The coagulation-anticoagulation balance is tightly regulated by endothelial-derived factors, which have not been clearly defined. Here, we report that clusterin, a component of Weibel-Palade bodies (WPBs), plays a crucial role in maintaining hemostatic equilibrium by sta bilizing von Willebrand factor (VWF) multimers in plasma. Clusterin was identified by proteomic analysis as a component of endothelial secretome under both chemical and physical conditions and demonstrated as a WPB component via immunostaining and co-transfection assays. Notably, a significant reduction of clusterin protein level was observed in type 2A von Willebrand disease (VWD) patient plasma. Furthermore, loss of clusterin in mice led to hemorrhagic diathesis and impaired thrombosis, accompanied by reduced high molecular weight (HMW) VWF levels. These defects were rescued by exogenous clusterin administration, underscoring its therapeutic potential. Mechanistically, clusterin binds to the D4N domain of VWF, which competitively inhibits ADAMTS13-mediated proteolysis under shear stress, and thereby preserves HMW VWF multimers essential for hemostasis. This study redefines WPBs as hubs for regulatory proteins and establishes clusterin as a key modulator of VWF multimer quality, offering a paradigm shift in targeting coagulation dysfunction through multimer stabilization rather than protein replacement. Our findings bridge a critical gap in understanding endothelial-driven coagulation homeostasis and suggest a potential therapeutic strategy targeting VWF multimer quality for bleeding disorder diseases.

In 2014, the International Myeloma Working Group (IMWG) expanded multiple myeloma diagnostic criteria to include a serum free light chain (sFLC) ratio of ≥100 as a standalone myeloma-defining biomarker, based on studies suggesting ∼80% risk of progression to overt myeloma at 2 years. However, subsequent studies demonstrate a substantially lower risk of progression, with population-based registry data showing a 2-year risk as low as 30.4% in this group. Importantly, subsequent data showed that >70% of patients with an sFLC ratio ≥100 have 24-hour monoclonal proteinuria <200 mg, a subgroup with particularly low progression risk (13.5% at 2 years) and minimal risk of irreversible renal failure. Furthermore, with the IMWG diagnostic amendment, an sFLC ratio ≥100 is currently included within the composite end point of progression-free survival in early-intervention clinical trials of high-risk smoldering multiple myeloma (SMM), which poses a risk of misclassifying biochemical changes as clinically meaningful events. We propose immediate revision of the diagnostic criteria to remove sFLC ratio ≥100 as a standalone myeloma-defining event and exclusion of patients with an sFLC ratio ≥100 from trials of newly diagnosed myeloma. These patients should be included in prospective studies on therapeutic interventions in high-risk SMM as well as active surveillance with modern imaging to define their natural history in the contemporary era.