Functional high-risk (FHR) multiple myeloma (MM) is defined as an unexpected, early relapse (ER) of disease in the absence of baseline molecular or clinical risk factors (RF), making FHR MM inherently dependent on which RFs were assessed at diagnosis, but also on what treatment patients received. To establish the true incidence of FHR, we analysed uniformly treated, transplant-eligible (TE) patients from the UK NCRI Myeloma-XI trial that had been profiled for IMS/IMWG defined high-risk cytogenetic aberrations (HRCA) and the SKY92 gene expression HR signature (GEP-HR). 135 TE MyXI patients meeting these criteria were studied, with a median follow-up of 88 months. 25 patients (18.5%) experienced ER, defined as relapse <18 months from maintenance randomization post-autologous stem-cell transplantation. Hereof, 15 (60%) were classified as IMS/IMWG-HR at diagnosis, of whom 8 were also GEP-HR. Another 6 patients were GEP-HR only and would have been missed by IMS/IMWG-HR. Among 4 patients with both IMS/IMWG- & GEP-standard risk (SR), 2 had isolated HR markers at diagnosis, leaving only 2 patients (8% of ER; 1.5% of all) truly meeting all FHR criteria. The combination of IMS/IMWG-HR and GEP-HR profiling identified 84% of ER, and differentiated long-term outcome across all 135 patients: co-occurring IMS/IMWG-HR and GEP-HR was associated with very short overall survival compared to the absence of both (HR=13.1, 95%-CI: 6.5-26.1, P<0.0001), followed by GEP-HR only (HR=5.1, 95%-CI: 2.4-11.1, P<0.0001) and IMS/IMWG-HR only (HR=3.2, 95%-CI: 1.6-6.2, P=0.0007). Our results support more comprehensive baseline diagnostic profiling to identify those at risk of ER upfront. ISRCTN49407852, NCT01554852.

Two gene therapy products have been FDA approved for sickle cell disease. Nearly all patients in the clinical trials that led to approval were either sickle hemoglobin gene homozygotes (sickle cell anemia) or had HbS-β0 thalassemia. HbSC disease, caused by compound heterozygosity for HbS and HbC genes, is the second most common genotype of sickle cell disease. Gene therapy has not been tested in HbSC disease patients who are severely symptomatic. We discuss the pathophysiology and clinical features of HbSC disease, and how gene therapy is likely to provide a curative option for some individuals. We also discuss the mechanism through which HbF and HbF-like HbA (HbAT87Q) might mitigate adverse clinical outcomes and end-organ damage in HbSC disease and other compound heterozygous sickle hemoglobinopathies.

Repeated bleeding into joints in hemophilia leads to chronic inflammation that plays a central role in the pathogenesis of hemophilic arthropathy (HA). Our recent studies revealed that factor VIIa (FVIIa) treatment releases extracellular vesicles from the endothelium (eEVs) and FVIIa-released eEVs exhibit anti-inflammatory and barrier protective functions. The present study was undertaken to investigate the effect of FVIIa-released eEVs on HA and the mechanism of their protective effect. Joint bleeding in hemophilia (F8-/- ) mice was induced by a needle puncture injury. Injured mice were treated with saline, control eEVs, or FVIIa-released eEVs, and the changes in the knee joints were analyzed by gross examination of knees as well as histological and immunohistochemical analysis. Joint tissues were examined for evidence of synovial hyperplasia, macrophage infiltration, neoangiogenesis, cartilage degeneration, and chondrocyte apoptosis. The data showed that treatment of mice with control eEVs had no significant effect on the development of HA whereas treatment with FVIIa-released eEVs markedly reduced all pathological features of joint bleed-induced HA. Incorporation of microRNA10a (miR10a) inhibitor into FVIIa-released eEVs abrogated the protective effect of FVIIa-released eEVs on HA. More importantly, loading miR10a mimic into control eEVs conferred a protective effect. Administration of miR10a containing FVIIa-released eEVs or control eEVs loaded with miR10a mimic were found to abrogate joint bleed-induced IL-6 production in the synovium. miR10a in eEVs had no effect on hemostasis. Cumulatively, our data indicates that EVs containing miR10a that effectively suppress synovial inflammation would have immense therapeutic value in treating HA.

Acute myeloid leukemia (AML) is a highly heterogeneous hematological malignancy that increasingly affects the elderly population, with its post-transcriptional landscape remaining largely elusive. Establishing a stable proteomics-based classification system and systematically screening age-related proteins and regulatory networks are crucial for understanding the pathogenesis and outcomes of AML. In this study, we leveraged a multi-omics cohort of 374 newly diagnosed AML patients, integrating proteome, phosphoproteome, genome, transcriptome, and drug screening data. Through similarity network fusion clustering, we established eight proteomic subtypes with distinct clinical and molecular properties, including S1 (CEBPA mutations), S3 (myelodysplasia-related AML), S4 (PML::RARA), S5 (NPM1 mutations), S6 (PML::RARA and RUNX1::RUNX1T1), S8 (CBFB::MYH11), S2 and S7 (mixed), aligning well with and adding actionable value to the latest World Health Organization nomenclature of AML. Hematopoietic lineage profiling of proteins indicated that megakaryocyte/platelet- and immune-related networks characterized distinct aging patterns in AML, which were consistent with our recent findings at the RNA level. Phosphosites also demonstrated distinct age-related features. The high protein abundance of megakaryocytic signatures was observed in S2, S3, and S7 subtypes, which were associated with advanced age and dismal prognosis of patients. A hematopoietic aging score with an independent prognostic value was established based on proteomic data, where higher scores correlated with myelodysplasia-related AML, NPM1 mutations, and clonal hematopoiesis-related gene mutations. Collectively, this study provides an overview of the molecular circuits and regulatory networks of AML during the aging process, advancing current classification systems and offering a comprehensive perspective on the disease.

Immunotherapy has transformed the treatment landscape of multiple myeloma (MM), a hematological cancer predominantly affecting older individuals. Yet, whether immune aging, shaped by intrinsic aging processes, genetics and external factors, impacts treatment efficacy remains unclear. To address this, we investigated the influence of age on the immune system in MM patients and explored whether immune aging associates with clinical outcomes in older patients. Using flow cytometry, we conducted high-dimensional profiling of T-cells and NK-cells in peripheral blood and bone marrow samples from 124 older (>65 years) and 145 younger (£65 years) newly diagnosed MM (NDMM) patients (ages 34-92 years) enrolled in the HOVON-143 and CASSIOPEIA/HOVON-131 trials. On average, older patients exhibited a more activated, differentiated, and senescent T-cell compartment than younger patients. Nonetheless, substantial inter-individual variation in T-cell subset frequencies within both age groups indicated that calendar age inadequately reflects an individual's immune status. We therefore developed an immune clock on high-dimensional phenotypic T-cell data to quantify each patient's 'immune age', revealing substantial variation in immune ages among patients of similar calendar age. Importantly, immune age appeared a stronger predictor of clinical outcomes than calendar age in older, non-fit NDMM patients receiving daratumumab-ixazomib-dexamethasone, even after adjusting for frailty and other established risk factors. Overall, these findings highlight immune age as a clinically relevant composite metric that better reflects a patient's immune status than their calendar age. Validating this methodology in other immunotherapy settings may improve our ability to predict immunotherapy efficacy in older patients with MM or other hematological cancers.

Platelets are specialized cells for hemostasis that circulate in close contact to the glycocalyx, an extracellular layer of interwoven glycoproteins, proteoglycans, and glycosaminoglycans that maintain vascular homeostasis. Platelets survey their circulating environment, balancing inhibitory signals that prevent inappropriate activation with activating signals that initiate thrombus formation. Disease can disrupt this delicate balance of endogenous inhibitory signaling, leading to an increased risk of thrombosis as in patients with inflammatory bowel disease (IBD). In this study, we demonstrate that physiological concentrations of hyaluronan (HA), an essential component of the glycocalyx, acts as an inhibitor of activation and aggregation in human platelets. Using a combination of affinity chromatography, and functional assays of platelets from humans and genetically modified mice, we identify layilin as the receptor for HA and an endogenous inhibitor of platelet activation. LAYN KO platelets display agonist-induced hyperactivation of αiiBβ3 and increased adhesion to fibrinogen under venous shear. Loss of layilin results in dysregulation of Rho GTPase family members-RAC1, Cdc42, RhoA, and Ras-like Rap1,- via layilin's binding partner, merlin, and downstream PAK1. Furthermore, IBD patient platelets contain reduced layilin protein levels correlating with heightened basal Rac1-GTP levels and increased reactivity. Finally, although IBD platelets show enhanced sensitivity to activation, pharmacological inhibition of RAC1 effectively reduces platelet hyperactivity in IBD patient platelets. These findings highlight a novel role for layilin and HA in the maintenance of platelet homeostasis that becomes disrupted in patients with IBD.

Older patients with diffuse large B cell lymphoma (DLBCL) present unfavorable genetic and microenvironmental alterations. In this phase 2 trial, we assessed the efficacy and safety of zanubrutinib in combination with rituximab and lenalidomide (ZR2) in patients with de novo DLBCL aged ≥75 years (NCT04460248). Forty patients were enrolled, and the primary endpoint was complete response rate, which was 65.0% (95% confidence interval [CI]: 48.3-78.9) at the end of induction treatment. The 2-year progression-free and overall survival rates were 67.1% (95% CI: 50.1-79.4) and 82.4% (95% CI: 66.5-91.2). The most common grades 3 and 4 hematologic adverse event (AE) was neutropenia (n = 14; 35.0%). The most common grades 3 and 4 non-hematologic AEs were increased alanine transaminase (n = 5; 12.5%) and aspartate transaminase level (n = 5; 12.5%), and pulmonary infection (n = 5; 12.5%). No events of atrial fibrillation were observed. Importantly, the efficacy of ZR2 was more dependent on tumor microenvironmental than genetic alterations, in association with up-regulation of Class I and II human leukocyte antigen, increased number and function of conventional type 1 dendritic cells. Pre-existing expansion of intra-tumoral CD8+T cells and treatment-induced clonal T-cell receptor (TCR) repertoire contributed to better clinical outcome. TCR sequencing of the peripheral blood mononuclear cell samples from patients with durable remission detected the expanded T cell clones 3 years post-treatment. These findings thus provided better understanding of T-cell immunological memory effect on immunotherapy as ZR2, and a paradigm shift in the era of mechanism-based targeted therapy of aggressive lymphoma.

The EZH2 histone methyltransferase inhibitors tazemetostat and valemetostat have recently received approval for clinical use in follicular lymphoma and adult T-cell leukemia/lymphoma, respectively. In follicular lymphoma, the expression of AID is responsible for increased mutational signatures and genomic instability. Because EZH2 inhibitors induce epigenetic and transcriptional changes in normal and lymphoma B cells, we studied whether these inhibitors could alter the pattern of AID-mediated chromosomal translocations. Here we show that treatment with EZH2 inhibitors did not significantly change AID expression or AID-dependent chromosomal translocation frequency when used as monotherapyin either CH12F3 mouse B cells or MEC-1 human B cells. In contrast, when combined with PI3Kδ inhibition, which enhances AID expression, EZH2 inhibition significantly increased the frequency of chromosomal translocations compared to either EZH2 or PI3Kδ inhibition alone both in mouse CH12F3 cells and human MEC-1 cells. EZH2 inhibition also further enhanced translocation formation in mouse B cells that were Ligase4 deficient. Mechanistically, EZH2 inhibition in B cells depletes the repressive histone modification H3K27me3 while concurrently enhancing the active histone modification H3K27ac, thereby selectively increasing transcriptional activity and facilitating chromosomal translocation formation in the presence of high AID activity or Ligase4 deficiency. These findings highlight the impact of drugs that induce epigenetic changes to influence chromosomal translocations and demonstrate the genetic safety of EZH2 inhibitors as monotherapy while highlighting the increased risk of genomic instability when used in cells prone to translocations, such as B cells with high AID levels or DNA-repair deficiency.

Detection of KIT p.D816V is a cornerstone in the diagnosis and classification of mast cell activation syndromes (MCAS) and mastocytosis. However, KIT p.D816V may be undetected due to a low mutated cell burden in blood and bone marrow (BM) of many patients, particularly among those without skin lesions. These findings underscore the need for ultra-sensitive molecular techniques for the detection of KIT p.D816V in these clinical settings. Here, we evaluated for the first time the sensitivity and specificity of a novel Flow-SuperRCA® KIT p.D816V assay, compared to conventional ASOqPCR, through the analysis of 548 blood and BM samples from 337 adults MCAS and mastocytosis patients. Our results demonstrated greater sensitivity of the new technique vs ASOqPCR (limit-of-detection: 0.001% vs 0.01% VAF), with higher rates of positivity for KIT p.D816V in whole-blood and/or BM of 64% of monoclonal-MCAS (MMAS) and 55% of BM mastocytosis patients (p<.0001). Notably, the sensitivity of the new assay went even beyond that of ASOqPCR in purified BM mast cells (p<.0001). Additionally, clonality was newly identified in 18% of patients previously diagnosed with non-clonal-MCAS, who presented with a unique cutaneous MCA-related symptomatic profile. These results confirm the high-specificity and ultra-sensitivity of the Flow-SuperRCA® assay for the detection of KIT p.D816V, emerging as a well-suited whole-blood and whole-BM test for the diagnostic screening and classification of MCAS and mastocytosis patients. These findings further highlight the clonal nature of an unprecedently high fraction of patients who presented with anaphylaxis and MCAS, with important pathogenic, diagnostic and clinical implications.

Recent advances have transformed the treatment landscape for relapsed and refractory follicular lymphoma. While chemotherapy has long served as the backbone of treatment, the availability of novel targeted, immunomodulatory and immunotherapeutic approaches is challenging its relevance. These approaches have focused on targeting epigenetic regulators, components of the B-cell receptor or its downstream intracellular pathways, and the follicular lymphoma tumor microenvironment. The recent development of bispecific antibodies and chimeric antigen receptor T-cell therapies, which target both tumor-associated and host-specific antigens, has enabled a redirection of the immune system, enhancing the innate anti-tumor immune response. Rational combinations of these strategies are actively being evaluated in the relapsed and refractory setting and will inevitably move forward into earlier lines of treatment. The success of these approaches has led to numerous and parallel options for patients and clinicians. The emerging challenge now lies in how best to approach each individual patient with relapsed or refractory follicular lymphoma, addressing complex decision-making that considers a patient's prior treatment history, goals of care, clinical and biological characteristics of recurrence, as well as personal preferences. Understanding the implications of refractory and transformed disease, as well as the timing and biology of relapse will be critical to support a more personalized treatment approach in the modern era.

Blood clotting is triggered in hemostasis and thrombosis when the membrane-bound tissue factor (TF)/factor VIIa (FVIIa) complex activates factor X (FX). There are no structures of TF/FVIIa on membranes, with or without FX. Using cryo-EM to address this gap, we assembled TF/FVIIa complexes on nanoscale membrane bilayers (nanodiscs), bound to XK1 and an antibody fragment. XK1 is a FX mimetic whose protease domain is replaced by the first Kunitz-type (K1) domain of tissue factor pathway inhibitor, while 10H10 is a non-inhibitory, anti-TF antibody. We determined a cryo-EM structure of a TF/FVIIa/XK1/10H10/nanodisc complex with a resolution of 3.7 Å, allowing us to model all the protein backbones. TF/FVIIa extends perpendicularly from the membrane, interacting with a "handle shaped" XK1 at two locations: the K1 domain docks into FVIIa's active site, while the γ-carboxyglutamate-rich (GLA) domain binds to TF's substrate-binding exosite. The FX and FVIIa GLA domains also contact each other and the membrane surface. Except for a minor contact between the first epidermal growth factor (EGF)-like domain of XK1 and TF, the rest of the FX light chain does not interact with TF/FVIIa. The structure reveals a previously unrecognized, membrane-dependent allosteric activation mechanism between FVIIa and TF where a serine-rich loop in TF that partially obscures the TF exosite must undergo a shift to allow access of the FX GLA domain to its final binding location on the membrane-bound TF/FVIIa complex. This mechanism also provides a novel explanation for the otherwise puzzling phenomenon of TF encryption/decryption on cell surfaces.

Venous thromboembolism (VTE) is a frequent (annual incidence of 1-2 per 1,000) and potentially life-threatening (case-fatality rate up to 10%) disease. VTE is associated with serious short-term and long-term complications, including a recurrence rate approaching 20% within five years. Anticoagulant therapy, the mainstay of VTE treatment, drastically reduces the risk of early VTE recurrence, but it exposes patients to a substantial risk of bleeding. We analysed the genomic architecture of VTE recurrence using data from 6,355 patients across eight cohorts (including 1,775 recurrences), enriched by subgroup analyses, according to sex and clinical manifestation of first VTE, which led to the identification of 28 molecular markers. Through genome-wide association studies, we identified one locus associated with VTE recurrence: GPR149/MME. Among all variants known to be associated with first VTE, KNG1 and FGG were associated with recurrence. Additionally, Mendelian Randomization analyses identified seven proteins as risk factors for recurrence: elevated plasma levels of coagulation factor XI, coagulation factor VIII, von Willebrand factor, BGAT and GOLM2; decreased levels of PCSK9 and pro-IL16. Subgroup analyses revealed 18 molecular determinants associated with VTE recurrence, with notable differences between subgroups. For example, the exonic variant SLC4A1 p.Glu40Lys was significantly associated in pulmonary embolism patients (Hazard Ratio (HR)=3.13, P=5.9×10-11) but showed no effect in deep vein thrombosis patients (HR=0.91, P=0.72). These findings emphasize the role of specific genetic loci and protein pathways in influencing VTE recurrence and provide valuable insights into potential therapeutic targets. Further research is needed to clarify the biological mechanisms driving these associations.

Chimeric antigen receptor T (CAR-T) cell therapy has revolutionized treatment for B-cell malignancies, yet over 60% of patients relapse within one year, often due to insufficient CAR-T persistence. While mouse and primary cell models have been instrumental in advancing CAR-T therapy, they frequently fail to predict clinical outcomes, underscoring the need for more translationally relevant models. To address this limitation, we conducted the first systematic evaluation of CAR structure-function relationships in an immunocompetent nonhuman primate (NHP) model. We engineered an array of 20 CD20-targeted CARs with distinct combinations of hinge, transmembrane, and costimulatory domains. Following ex vivo characterization, we administered pooled autologous CAR-T arrays to three NHPs and tracked CAR abundance longitudinally using a novel digital droplet PCR assay. Ex vivo, CAR-T cells incorporating the MyD88-CD40 costimulatory domain exhibited markedly distinct functional profiles, including increased activation, unique cytokine secretion, tonic signaling, and resistance to exhaustion. In vivo, MyD88-CD40 CARs expanded dramatically, comprising up to 100% of peripheral CAR-T cells and significantly outperforming canonical CD28- and 4-1BB-based CARs. This expansion was associated with robust B-cell depletion across all animals. MyD88-CD40 CARs, particularly those with a CD28 hinge and transmembrane domain, demonstrated superior trafficking to secondary lymphoid tissues and persistence through study endpoint, unlike other CARs which waned by day 28. Our findings highlight the value of NHP models for screening CAR designs and identify MyD88-CD40 CARs as candidates with unmatched potency. The unique functional attributes conferred by this domain may provide key insights into features that drive enhanced CAR-T cell activity.

Aberrant activation of BCL11B ("BCL11B-a") defines a subtype of lineage ambiguous leukemias with T-lymphoid and myeloid features, co-occurring activating FLT3 mutations, and a stem/progenitor immunophenotype and gene expression profile. As with other lineage ambiguous leukemias, optimal treatment is unclear and there are limited targeted therapeutic options. Here, we investigated the efficacy of BCL-2 and FLT3 inhibition with venetoclax and gilteritinib, respectively, in preclinical models of BCL11B-a leukemia. Despite variation in response to single agent therapies, the combination of venetoclax plus gilteritinib (VenGilt) was highly effective in all models evaluated. BH3 profiling suggested that resistance to venetoclax monotherapy was due to the tumor-intrinsic dependence on additional BCL-2 family proteins prior to drug treatment. Longitudinal single cell RNA-seq analysis identified mitochondrial pathways and a pro-lymphoid gene expression signature as potential drivers of rare cell survival on VenGilt therapy. These data support clinical evaluation of venetoclax in combination with gilteritinib in BCL11B-a lineage ambiguous leukemias.

Integration of torque teno mini virus (TTMV) generating the TTMV::RARA fusion represents a newly recognized subtype of acute promyelocytic leukemia (APL) that merits detailed investigation. We present the first comprehensive characterization of its epidemiologic profile, clinical presentation, virologic characteristics, and underlying molecular mechanisms. Our findings indicate that TTMV::RARA is more prevalent in pediatric patients and represents the second most common retinoic acid receptor fusion after PML::RARA. Affected patients exhibit a high incidence of extramedullary involvement, particularly myeloid sarcoma. Cytogenetic abnormalities involving i(17)(q10) or 7q22 were identified in 52.0% of cases, largely in a mutually exclusive manner. Co-occurring mutations in epigenetic regulators were present in 76.9% of patients. Although most patients achieved initial remission, relapse was common and associated with rapid acquisition of all-trans retinoic acid (ATRA) resistant mutation and secondary chemoresistance. Venetoclax-containing regimens demonstrated encouraging clinical efficacy. Phylogenetic analysis indicated that patient-derived TTMV strains clustered into a distinct clade. TTMV integration consistently occurred within RARA intron 2, involving a consensus fragment of 510-610 bp encompassing the viral promoter and ORF2 N-terminus, likely mediated by microhomology-driven recombination. Tandem RUNX1-binding motifs within the integrated viral promoter may underlie the myelotropism of these TTMV strains and facilitate transcriptional activation of TTMV::RARA. The chimeric protein retains at least the first 56 N-terminal residues of ORF2 and remains transcriptionally responsive to pharmacological concentrations of ATRA. These findings establish TTMV::RARA-APL as a distinct leukemia entity, laying the foundation for future studies on virus-mediated leukemogenesis and therapeutic strategies.