Blood

Germline variants in DDX41 are the most frequent genetic predisposition to adult hematologic malignancies. The most common variants are truncating, implicating loss of function in the pathogenesis. However, non-truncating variants account for 30-40% of cases, and their impact on essential DDX41 functions remains unknown. We utilized a genetic complementation assay to assess the functionality of 10 recurrent germline non-truncating variants of DDX41. All variants restored viability to Ddx41-deficient hematopoietic progenitor cells at exogenous expression levels. In contrast, the hotspot mutant p.R525H, which is somatically acquired at disease onset in >50% of patients, failed to restore viability. CRISPR-based modeling in cell lines and mice revealed heterogeneity: some variants were non-functional at endogenous expression levels whereas others maintained complete functionality, supporting normal cell proliferation and even lifelong hematopoiesis in a homozygous setting. Notably, co-expression of p.R525H with some variants caused impaired hematopoietic progenitor cell viability, indicating a dominant-negative effect of p.R525H. In contrast, other variants, all classified as variants of unknown significance, were unaffected by the presence of p.R525H. A screen of 100 disease-associated variants confirmed that many non-truncating germline variants are susceptible to p.R525H-mediated dominant-negative effects, whereas wild-type DDX41 is not. These findings indicate that DDX41 variant curation is complicated by variable effects on functionality and variant-specific interactions with somatically-acquired DDX41 mutations. The dominant-negative effect of p.R525H provides a mechanistic basis for the conclusion of recent patient cohort analyses that co-occurrence with a somatic hotspot mutation is a reliable indicator of DDX41-driven disease in carriers of non-truncating variants.

Von Willebrand factor (VWF) is an essential contributor to hemostasis through its interaction with the platelet glycoprotein (GP) Ib receptor. VWF is cleaved by ADAMTS13 to limit its prothrombotic properties. Failure to do so can result in platelet-VWF complexes that occlude the microcirculation, as seen in thrombotic thrombocytopenic purpura (TTP). In this setting, plasmin becomes active to cleave VWF, forming a distinct plasmin-generated cleavage product of VWF (cVWF) that is detectable during acute attacks in patients with TTP and following therapeutic plasminogen activation in a mouse model of TTP. However, it remains unclear whether plasmin-mediated proteolysis of VWF alone accounts for the breakdown of platelet-VWF complexes. Using ristocetin-induced platelet agglutinations, we show that plasmin cleavage of VWF does not impair its platelet-binding capacity, whereas plasmin-mediated cleavage of GPIb reduces the ability of platelets released from agglutinates to bind VWF. Furthermore, platelets in suspension are relatively resistant to plasmin cleavage. We therefore propose that VWF binding may enhance GPIb cleavage by recruiting plasmin(ogen) to the platelet surface. In a TTP mouse model, plasminogen activation led to a VWF-dependent reduction in GPIb detectability, although to a lesser extent than observed in vitro. In patients with acute TTP, soluble GPIb levels were elevated, indicating increased GPIb shedding during attacks of thrombotic microangiopathy, although the extent to which this is plasmin-mediated remains unclear. Together, our findings demonstrate that plasmin cleavage of GPIb drives the disruption of ristocetin-induced agglutinates, while its contribution to the breakdown of platelet-VWF complexes in vivo appears limited.

BackgroundVenous thromboembolism is a major cause of morbidity and mortality. Despite identification of risk factors, not all individuals with thrombophilia develop thrombosis. Understanding the multigenic factors modifying this incomplete penetrance would help guide patient care. MethodsThe zebrafish has a conserved hemostatic system and is amenable to large genetic studies. Loss of antithrombin III (At3) in zebrafish leads to an early consumptive coagulopathy and lethality in adulthood. Using this genetic background as a sensitized model we performed a dominant unbiased genome-wide N-ethyl-N-nitrosourea (ENU) mutagenesis screen followed by whole genome sequencing (WGS). We used survival studies, laser-mediated endothelial injury, and ex vivo protein assays to validate hits. ResultsENU-treated at3+/- males were crossed with at3+/- females to produce 4,030 total offspring (1.5x genome coverage). Four permanent lines transmitting a survival benefit beyond 7 months were identified and sequenced. A candidate screen of 63 known coagulation-related loci revealed a missense mutation, C504F, in a highly conserved residue of the prothrombin (F2) heavy chain, which was validated through genetic and biochemical studies. Evaluation of UK Biobank electronic health record (EHR) data was underpowered to detect interactions between F2 and AT3 due to minmal deleterious mutations. Mutations produced through genome editing revealed that heterozygosity for factor X and plasminogen also modified at3-/-, resulting in reduced lethality. The three remaining lines had no coagulation-related variants segregating with survival, suggesting the presence of novel modifier loci. ConclusionsUnbiased genome-wide screening identified a modifier of thrombosis. This demonstrated that re-balancing of hemostasis to mitigate thrombosis is conserved in zebrafish, including an unexpected role for fibrinolysis. This interaction was not detected even in a large human dataset, establishing the continued benefit of the zebrafish model. Finally, we found evidence for novel loci outside of the canonical coagulation cascade that may be new targets for diagnosis or treatment.

Polycomb repressive complexes PRC1 and PRC2 regulate diverse developmental processes, including the fetal-to-adult switch in hemoglobin production, a process whose reversal is a goal for the treatment of sickle cell disease and {beta}-thalassemia. PRC inhibitors show promise for various disorders, but use is limited because of pleiotropic PRC activities. We explored whether fetal hemoglobin (HbF) can be reactivated in adult erythroid cells by selective perturbations of PRC1 or PRC2 components without complete loss of PRC function. A high-density CRISPR-Cas9 mutagenesis screen identified a region in the EZH2 subunit where Cas9 induced exon 14 skipping (EZH2{Delta}14). EZH2{Delta}14, which lacks a portion of the CXC domain, relieves HbF repression while largely maintaining cellular fitness. EZH2{Delta}14 retains H3K27 methylation and repression of a PRC target gene subset. Experiments in cells derived from mice bearing human {beta}-globin genes confirm that pathways mediating EZH2 control of HbF expression can function in a mouse model of HBG switching. These findings demonstrate that partial disruption of PRC can yield selective phenotypes, highlighting the therapeutic potential of targeting non-enzymatic domains within chromatin-modifying complexes. Key PointsO_LICRISPR-Cas9 screen across PRC1 and a saturating mutagenesis screen of PRC2 found the EZH2 CXC domain a desirable target for HbF induction C_LIO_LIthe EZH2-CXC domain leads to exon 14 exclusion, resulting in de-repression of HbF but maintenance of cell fitness. C_LI

Therapy-driven genomic changes in multiple myeloma (MM) remain poorly defined. We analyzed whole-genome sequencing (WGS) data from relapsed/refractory MM (rrMM, N=386) and identified regional 1p31.1-p12 (hereafter 1pCEN, a region proximal to the centromere) loss-of-heterozygosity (LOH) as the only enriched aberration showing strong therapy-associated clonal selection (clonal timing rank fold-change = 3.7, P<2.2x10-16). This event showed enriched co-occurrence with 1qGain (OR = 2.3 (1.5-3.8), P=2x10-4) forming a recurrent "double-hit" in rrMM. To validate the clonal selection process, we examined three longitudinal cohorts (180 patients, 390 samples) and confirmed clonal expansion of 1pCEN and consistent prevalence of the 1pCEN+1q double-hit (20-24%). Survival analyses demonstrated significantly reduced progression-free survival in rrMM patients with this double-hit compared with those without. Comparison with a large newly diagnosed MM (ndMM) cohort confirmed previously-described 1p32 LOH is the prognostic locus at baseline, whereas 1pCEN is therapy-selected and largely independent of the 1p32 locus. Thus, 1pCEN+1q represents a recurrent double-hit event that clonally emerges in rrMM, conferring selective advantage under drug exposure and is distinct from the ndMM high-risk markers defined by current consensus guidelines. These findings nominate 1pCEN as a new genomic biomarker in rrMM and 1pCEN+1q may help patient stratification for therapeutic monitoring. Key PointsA therapy-driven common genomic double-hit (1p31.1-p12 LOH with 1q gain) clonally emerges in relapsed/refractory myeloma.

Aberrant activation of TAL1, a key oncogenic driver, defines a major subgroup comprising [~]30% of childhood T-lineage acute lymphoblastic leukemias (T-ALLs). We and others have shown that somatic non-coding mutations within upstream and intronic cis-regulatory regions of TAL1 contribute to transformation by creating binding sites for MYB and other transcription factors. Here we investigated cis-regulatory mechanisms mediated by somatic mutations occurring in an intergenic region located 29 kilobase pairs downstream of the canonical TAL1 transcription initiation site, implicated in 6% of TAL1-expressing T-ALLs. These somatic variants include i) complex indels resulting in de novo MYB transcription factor binding sites (TFBSs) and ii) internal tandem duplications (ITDs) encompassing canonical MYB TFBSs. Chromatin immunoprecipitation sequencing (ChIP-seq) revealed binding of the TAL1 core regulatory circuit (CRC) transcription factors MYB, GATA3, and RUNX1, resulting in enhancer activity mediated by sequences with the mutant allele. Strikingly, ChIP-seq peaks for the repressive H3K27me3 mark and the active H3K27ac mark co-existed across TAL1 regulatory sequences but enriched for different haplotypes. TAL1 transcription from the mutant haplotype initiated from a promoter located within exon 4 of the canonical TAL1 transcript, resulting in a short isoform normally expressed by hematopoietic stem cells (HSC). Interestingly, neither the isoform expression nor the enhancer activity could be predicted by the sequence-to-function deep learning artificial intelligence (AI) model AlphaGenome, emphasizing the importance of experimental validation. Our findings indicate that selection for cis-regulatory, non-coding variants leads to reactivation of enhancers normally active in HSC but silenced in differentiated lineages during normal hematopoietic cell development.

JAK2 is a key regulator of cytokine-mediated proliferative signaling in hematopoietic stem and progenitor cells. Activating mutations, most commonly JAK2 V617F, trigger aberrant cytokine signaling driving the pathogenesis of myeloproliferative neoplasms (MPNs). Phosphatidylinositol transfer proteins (PITPs) facilitate phosphoinositide synthesis by delivering phosphatidylinositol to lipid kinases, though their roles in oncogenic signaling have remained poorly defined. Here we show that PITP{beta} is critical for the development of JAK2V617F-driven MPN in mice. Deleting Pitp{beta} across the hematopoietic system, but not Pitp, prolonged 25-week survival of Jak2V617F mice from 10% to 85%. Loss of Pitp{beta} attenuated disease-associated splenomegaly and curtailed erythroid progenitors expansion both in vivo and in vitro. Mechanistically, PITP{beta} is necessary for AKT hyperactivation in hematopoietic progenitors, while STAT5 and ERK signaling remain unaffected. In alignment with this role, PITP{beta} promotes the production of PtdIns(3,4)P2, a phosphoinositide that sustains aberrant AKT signaling in Jak2V617F progenitors. Pharmacologic inhibition of AKT with the FDA-approved inhibitor capivasertib in Jak2V617F-transplanted mice similarly reduced splenomegaly and erythroid proliferation, mimicking the effects of Pitp{beta} loss. Collectively, these results identify a novel PITP{beta}-PtdIns(3,4)P2 signaling axis that selectively maintains pathological AKT activation in JAK2V617F-driven MPN, revealing a promising therapeutic vulnerability.

The JAK2V617F (JAK2VF) driver mutation is found in 95% of patients with polycythemia vera (PV), a progressive myeloproliferative neoplasm. Current treatments suppress excessive hematopoiesis but lack specificity for targeting JAK2VF cells, are unable to deplete mutant stem/progenitor cells and ultimately result in drug resistance. We discovered that the FDA-approved antibiotic, linezolid (LZD), ameliorates the PV phenotype across multiple model systems. LZD suppressed cell proliferation and STAT5 signaling, altered the cell cycle, and increased apoptosis of JAK2VF-harboring human erythroleukemia cells, but not in wild-type acute leukemia cells. Computational modelling indicated that LZD interacts specifically with mutant JAK2VF but not with wild-type JAK2 protein. We further showed that, in JAK2VF mice that faithfully recapitulate human PV, LZD mitigates disease burden by selectively targeting JAK2VF stem cells thereby normalizing spleen size and blood counts. LZD also inhibited hematopoietic colony formation by patient-derived peripheral blood mononuclear cells, with the more primitive progenitors being preferred targets. Importantly, LZD selectively decreased JAK2VF+ colony numbers, without impacting wild-type JAK2 colonies. In all, the data provide a firm foundation for evaluating LZD-like molecules as an effective therapy for PV and other myeloproliferative neoplasms. Key pointsO_LILinezolid acts as a JAK2V617FIZselective inhibitor in PV mouse models and PV patient samples while sparing wildIZtype hematopoiesis. C_LIO_LILinezolid acts directly on JAK2V617F hematopoietic stem cells. C_LI

Relapse and chemoresistance remain major challenges in paediatric acute myeloid leukaemia (PAML), particularly in KMT2A-rearranged (KMT2A-r) subtypes where conventional markers such as CD34 are often absent, complicating measurable residual disease (MRD) detection. Leukaemia stem/regenerating cells (LSC/LRC) drive disease initiation, progression, and relapse, sharing stemness and chemoresistance properties that make them critical therapeutic targets. Using high-dimensional spectral flow cytometry, we identified CD180, a Toll-like receptor-like surface protein, as highly expressed on blasts and stem-like populations in KMT2A-r AML, while near absent on normal haematopoietic stem cells (HSCs). PAML KMT2A-r exhibits an unconventional immunophenotype dominated by CD34-CD180 populations. Integrated single-cell transcriptomics and functional profiling revealed CD180high clusters enriched for quiescence, oxidative phosphorylation, and KMT2A/LSC stemness signatures. CD180 cells demonstrated robust leukaemia-initiating capacity in xenograft models and persisted through therapy, re-emerging at relapse with phenotypic plasticity. Epigenomic analysis showed CD180 is a direct transcriptional target of the KMT2A::MLLT3 fusion complex, regulated by intragenic enhancers and downregulated by menin and BET inhibitors. Longitudinal single-cell analysis confirmed persistence and clonal evolution of CD180 populations during treatment and relapse, underscoring their mechanistic role in chemoresistance and disease progression. In summary, CD180 marks dynamic, relapse-driving populations in KMT2A-r PAML, persists through therapy, and importantly is near absent on normal HSCs, offering a selective therapeutic window. These findings position CD180 as a clinically actionable biomarker for MRD detection and a compelling therapeutic target for eradicating chemoresistant, stem-like cells in paediatric AML. Main PointsO_LICD180 marks chemoresistant, relapse-driving stem-like blasts in KMT2A-r paediatric AML, overcoming CD34-based MRD limitations. C_LIO_LIAbsent on normal HSCs, CD180 is a KMT2A::MLLT3 target and actionable for MRD, relapse prediction, and CD180-directed therapies. C_LI NoveltyThis study introduces CD180 as a novel biomarker and therapeutic target in AML, particularly KMT2A-rearranged subtypes where conventional markers are often absent. Unlike MRD strategies focused on bulk blasts, CD180 marks chemoresistant, stem-like populations driving relapse, critical reservoirs poorly defined in paediatric AML. This work fills a major gap in prognostic assessment and therapy by enabling precise detection of relapse-driving cells and offering a selective therapeutic window.

Extramedullary acute myeloid leukemia (eAML) represents a clinically challenging manifestation of acute myeloid leukemia (AML), but its molecular drivers remain poorly defined. We performed targeted sequencing in 85 eAML biopsies, representing one of the largest molecular analyses of eAML to date. We detected mutations in RAS or RAS-modifying genes (RASMUT; NRAS, KRAS, PTPN11, CBL, and NF1) in 41% of cases, representing a significant enrichment compared to bone marrow (BM) samples of more than 1300 AML patients not selected for eAML. Analysis of paired eAML and BM specimens revealed expansion and/or de-novo appearance of RASMUT clones at the extramedullary site. Functional studies using primary murine leukemia cells and CRISPR/Cas9-engineered isogenic human leukemia cell lines demonstrated that RASMUT increase the migration and invasion of leukemic cells compared to RAS-wildtype controls. Consistently, RASMUT cells showed increased infiltration into the chorioallantoic membrane of chicken embryos and demonstrated enhanced extramedullary growth after injection into immunocompromised mice. RNA sequencing revealed increased expression of junctional adhesion molecule-like (JAML) and activation of PI3K/AKT signaling in RASMUT cells. JAML silencing and pharmacologic AKT inhibition reversed the RASMUT-driven effects on leukemic cell migration, demonstrating a causal role of the JAML-PI3K/AKT axis in RASMUT-driven eAML formation. In conclusion, these findings delineate the molecular landscape of extramedullary AML and show that RASMUT are enriched within this AML subform. They further demonstrate that RASMUT actively contribute to leukemic tissue infiltration through activation of a RASMUT-JAML-PI3K/AKT axis, highlighting AKT signaling as a potential therapeutic vulnerability in RASMUT-associated eAML.

Fetal hemoglobin (HbF) prevents the polymerization of sickle hemoglobin (HbS). HbF, measured usually as a percent of total hemoglobin (%HbF), is inversely associated with the severity of sickle cell disease (SCD) but fails to capture the distribution of HbF concentrations within red blood cells (RBCs). The relative proportion of HbF and HbS within a RBC is reflected by the HbF:HbS ratio whereas HbF/F-cell quantifies the absolute amount of HbF/RBC. While correlated, HbF:HbS ratio and HbF/F-cell are not interchangeable. In the context of mean corpuscular hemoglobin (MCH), HbF/F-cell approximates whether sufficient HbF is present to inhibit HbS polymerization. We examined the association of mean HbF/F-cell with sub-phenotypes of sickle cell disease in three independent cohorts. Both %HbF and HbF/F-cell were significantly associated with multiple clinical and laboratory features of SCD; however, HbF/F-cell demonstrated stronger associations with clinical severity measures across cohorts. Higher HbF/F-cell was associated with fewer clinical events, reduced hemolysis, and mortality. Changes in HbF/F-cell after hydroxyurea treatment were associated with ~11-13% reduction in acute events in patients with <1 pg increase and >60% reduction with a >5 pg increase in HbF/F-cell. For each pg increase in HbF/F-cell there was ~6% reduction in the rate of acute events. As a surrogate for the distribution of HbF concentrations among F-cells, HbF/F-cell adds physiologically relevant insights that could guide prognosis and treatment

Sickle cell disease (SCD) is caused by the production of an abnormal adult hemoglobin that generates sickle-shaped red blood cells (RBCs). Transplantation of autologous genetically corrected hematopoietic stem/progenitor cells (HSPCs) represents a promising therapy. Persistent fetal hemoglobin expression improves SCD. Here, we engineered the fetal HBG1/2 promoters by replacing the BCL11A repressor binding site (BS) with a TAL1:GATA1 motif recognized by transcriptional activators. We exploited the prime editing nuclease (PEn) that efficiently installed the TAL1:GATA1 motif in K562 cells, outperforming the original PE. Non-homologous end joining (NHEJ) and/or alternative-end joining (alt-EJ) pathway inhibition enhanced precise editing. However, this strategy was poorly efficient in patients HSPCs. Alternatively, we used CRISPR/Cas9 nuclease to either disrupt the BCL11A BS via NHEJ and/or alt-EJ or to replace it with the TAL1:GATA1 motif via homology-directed repair (HDR) using a donor ssODN template. NHEJ and alt-EJ inhibition improved product purity, reducing InDels and achieving superior precise editing efficiency compared to PEn in K562 and HSPCs. HDR-edited HSPCs preserved clonogenic capacity and differentiated into RBCs showing elevated HBG expression and correction of the sickling phenotype. These results demonstrate that replacing the BCL11A BS with a TAL1:GATA1 motif is a potent strategy for reactivating HBG1/2 to treat SCD.

Prolonged cytopenias are a frequent complication of chimeric antigen receptor (CAR) T-cell therapies and are associated with increased infection risk and non-relapse mortality. Although inflammatory cytokines released during CAR-T cell activation have been implicated in immune effector cell-associated hematotoxicity (ICAHT), their direct effects on human hematopoietic stem and progenitor cells' (HSPCs) function remains incompletely understood. Here, we established a reductionist model of CAR-T-associated hematotoxicity using conditioned media (CM) derived from activated CD19 CAR-T cells. Sustained exposure of human HSPCs to CAR-T-derived inflammatory secretome impaired HSPC expansion and reduced long-term repopulating capacity in xenotransplantation assays. In contrast, short-term exposure did not abrogate HSPC function, indicating that brief inflammatory signals can initiate durable reprogramming events, with functional consequences emerging during subsequent proliferative expansion. Mechanistically, CAR-T CM induced IFN gamma- (IFNg) and TNF alpha- (TNFa) responsive transcriptional programs in HSPCs and promoted inflammatory myeloid skewing without evidence of apoptosis-dependent stem cell loss. Combined inhibition of IFNg and TNFa restored HSPC expansion, normalized lineage output, reversed inflammatory transcriptional signatures, and rescued in vivo repopulating capacity without impairing CAR-T cytotoxic activity. These findings demonstrate that CAR-T-derived inflammatory signaling can directly impair human HSC function and identify dual IFNg/TNFa blockade as a potential strategy to mitigate CAR-T-associated hematotoxicity while preserving antitumor efficacy.

Despite extensive sequencing, the genetic etiology of sporadic angiosarcoma remains poorly defined (1-3). Maffucci syndrome, characterized by vascular tumors and elevated cancer risk, is driven by mosaic gain-of-function mutations in IDH1/2 (4,5), though these have not been reported in sporadic angiosarcoma. We identify recurrent, low-variant allele frequency hotspot mutations in IDH1/2 in over half of sporadic angiosarcomas. Mutations were validated by Sanger sequencing and immunohistochemistry. Mutant IDH1 endothelial cells promote tumorigenesis through non-cell-autonomous mechanisms, secreting 2-hydroxyglutarate (2-HG) to increase growth factor and endothelial-to-mesenchymal transition gene expression, activate pAkt/pERK signaling, induce DNA methylation changes, and promote anchorage-independent growth, which are reversed by the mutant IDH1 inhibitor ivosidenib. Patients with mosaic IDH1 mutations show reduced serum 2-HG and marked tumor regression following ivosidenib treatment. The clinical efficacy of ivosidenib in vascular tumors with subclonal IDH1 mutations suggests that low VAF IDH1/2 mutations may be a targetable vulnerability in sporadic angiosarcoma. (6,7) Statement of SignificanceWe identify recurrent, low-VAF IDH1/2 mutations in angiosarcoma and provide evidence that these subclonal mutations promote tumorigenesis through non-cell-autonomous mechanisms. Vascular tumors driven by subclonal IDH1 mutations responded dramatically to ivosidenib, thus revealing a novel treatment for a subset of vascular tumors.

Primary light-chain amyloidosis (pAL) is caused by plasma cell (PC) clones that secrete misfolded free light chains that deposit. Anti-CD38 antibody daratumumab is the first-line therapy, while ~10-30% of patients exhibit suboptimal responses (very good partial response, VGPR), and baseline predictors and resistance mechanisms remain under investigation. We generated a single-cell bone marrow atlas with B cell receptor and transcriptome sequencing from a cohort of 30 patients with pAL treated with daratumumab-bortezomib-dexamethasone, including 11 paired pre-/post-treatment samples. Among 27 outcome-evaluable patients, 10 demonstrated suboptimal responses before cycle 6 or the start of subsequent therapy. Among patients with t(11;14), compared with good responders, suboptimal responders' amyloidogenic PCs exhibited lower baseline protein-translation and cell-cell-adhesion gene expression programs, but higher endoplasmic reticulum stress programs. With treatment, mitotic programs were upregulated and gave rise to additional pathogenic PC states. Suboptimal responders also demonstrated two PC-centered immune processes that were enhanced relative to baseline: (i) an inflammatory PTGES2/3-PTGER2/4 axis driven by PTGS2-expressing myeloid-derived suppressor cell-like CD38-negative CD14-positive monocytes that expanded with treatment; and (ii) an immunosuppressive non-classical MHC I axis, in which PCs exerted inhibitory interactions (HLA-E-KLRK1, HLA-G-LILRB1, HLA-F-LILRB1). Consistent with these cell-cell interactions, myeloid cells and NK cells showed functional impairment, while T cells were more exhausted; all three cell types exhibited increased interferon-gamma responses in suboptimal versus good responders. This atlas reveals amyloidogenic PCs' resistance to daratumumab and an inflammatory-immunosuppressive niche driven by prostaglandin and non-classical MHC I, underpinning suboptimal responses.

Measurable (or minimal) residual disease (MRD) predicts relapse in patients with acute myeloid leukemia (AML). However, the biological and spatial characteristics of the AML bone marrow (BM) microenvironment (BMME) in which MRD cells survive remain largely unexplored; in particular, little is known of the BMME in TP53 mutant (TP53mut) AML. Here, we applied sequential immunofluorescence to whole BM biopsy specimens obtained from patients with TP53 wild-type (TP53WT) AML and TP53mut AML at diagnosis and in morphological complete remission (CR) to generate a comprehensive spatial map of the hematopoietic and BMME components. We identified TP53mut leukemia cells based on high p53 expression and delineated their spatial organization relative to stromal and immune niches. Biopsy-based cell composition analysis revealed marked B-cell depletion and an increased abundance of regulatory T-cells (Tregs) in TP53mut BM at CR. Unlike TP53WT BM, TP53mut BM at CR exhibited persistent TP53mut erythroid and immature leukemia cell clusters, spatially segregated from T-cell clusters, in perisinusoidal niches, suggesting niche-level immune evasion. Spatial profiling further revealed that Tregs characterized by FOXP3 upregulation were enriched near TP53mut MRD cells, indicating a locally enhanced immunosuppressive activity. Single-cell RNA sequencing-based cell-cell communication analysis identified erythroid-T-cell interactions mediated by the GDF15-CD48 axis as a potential mechanism of T-cell suppression, suggesting that the erythroid differentiation of TP53mut AML cells enhances local immunosuppression. Collectively, our results show a spatially organized immunosuppressive BMME in TP53mut AML and highlight the potential of spatial proteomics to identify actionable MRD niches in leukemias. Key pointsO_LITP53 mutant erythroid and immature leukemia cells form spatial clusters segregated from T-cells in complete remission. C_LIO_LIAn erythroblast-centered immunosuppressive niche characterizes TP53 mutant leukemia cells. C_LI

Severe hemolysis is a life-threatening condition with limited therapeutic options. Although haptoglobin and hemopexin sequester hemoglobin and heme, these protective systems are rapidly saturated during acute hemolysis, leading to the accumulation of cytotoxic free heme. In this study, we identify scavenger receptor BI (SR-BI) as a critical mediator of free heme clearance. SR-BI binds heme and facilitates its hepatic uptake under pathological conditions. Mice lacking hepatic SR-BI exhibit impaired heme clearance and increased susceptibility to heme- and hemolysis-induced lethality. Pharmacological upregulation of hepatic SR-BI via imatinib or adenoviral delivery confers protection against heme toxicity. Using a humanized model of sickle cell disease (SCD), we further demonstrate that sickle hepatopathy significantly reduces hepatic SR-BI expression compared to non-SCD littermates, potentially increasing vulnerability to heme-induced injury. Notably, adenoviral-mediated SR-BI upregulation rescues SCD mice from heme toxicity. These findings reveal a previously unrecognized mechanism of heme detoxification via hepatic SR-BI and identify a promising therapeutic target for hemolytic disorders. One-Sentence SummaryIdentification of scavenger receptor BI as a targetable scavenger of heme in hemolysis

Understanding how specific VWF variants disrupt endothelial processing and function is central to elucidating von Willebrand disease (VWD) pathophysiology. However, current in vitro systems lack either the endothelial specificity or the genetic flexibility required for systematic variant characterization. Here, we present a genetically defined VWF-knockout cord-blood-derived endothelial colony-forming cell (VWF-KO cbECFC) model that enables controlled reintroduction of VWF variants in a physiologically relevant endothelial context. Using a patient with type 3 VWD carrying the homozygous pathogenic variant p.M771V and a second homozygous variant of uncertain significance p.R2663P as a reference, we demonstrate that expression of p.M771V in VWF-KO cbECFCs reproduces the patients intracellular processing defect and loss of high-molecular-weight multimers, whereas p.R2663P behaves as a benign allele. These findings establish the models ability to accurately distinguish pathogenic from non-pathogenic variants. Comparative analyses with HEK293 cells show that VWF-KO cbECFCs provide superior subcellular resolution, reliably forming authentic Weibel-Palade bodies (WPBs) and faithfully revealing ER retention phenotypes that remain ambiguous in non-endothelial systems. The proliferative capacity of cbECFCs further enables scalable and reproducible experimentation, overcoming major limitations associated with patient-derived ECFCs. Looking ahead, the VWF-KO cbECFC platform offers broad potential for VWF and VWD research. Its endothelial identity and genetic flexibility make it suitable for investigating VWF biosynthesis and trafficking, secretion dynamics, WPB biology, angiogenic processes, and shear-dependent VWF function. This system therefore provides a versatile foundation for mechanistic studies, systematic variant assessment, and future translational applications.

Long-term follow-up of the CASSIOPEIA trial (NCT02541383) demonstrated superior progression-free survival (PFS) with daratumumab, both in combination with bortezomib, thalidomide, and dexamethasone during induction and consolidation, and during maintenance therapy, in transplant- eligible patients newly diagnosed with multiple myeloma (MM). However, outcomes among CASSIOPEIA patients remain heterogeneous across treatment groups. Measurable residual disease (MRD) is a strong indicator of the depth and duration of therapeutic response and is independently associated with both PFS and overall survival (OS), but it does not fully capture the biological diversity of MM. We performed a risk prediction analysis based on transcriptomic subgroups in CASSIOPEIA patients. A subset of 628 patients was characterized using RNA sequencing and consensus clustering identified five transcriptomic subtypes of MM. Long-term follow-up allowed the definition of three transcriptomic risk categories, with estimated 72-month PFS rates of 70%, 51%, and 27% for low, intermediate, and high-risk groups, respectively, among patients who received daratumumab in at least one treatment phase. In these patients, MRD negativity rates after consolidation and six months later were significantly higher in the low and high-risk groups compared with the intermediate-risk group. In the high-risk group, MRD status was not associated with PFS or OS. This suggests that, although daratumumab administered during both the induction/consolidation and maintenance phases improves the clinical outcomes of patients with activation of NSD2 or overexpressing members of the MAF family, highly aggressive minor clones may rapidly expand. These findings emphasize the need for novel therapeutic strategies in this high-risk population.

Pediatric platelet disorders are commonly classified according to specific structural or functional abnormalities, yet it remains unclear how well these diagnoses capture overall hemostatic phenotype. Here, we combined quantitative single-cell platelet measurements with spatially resolved plasma clotting analysis to characterize pediatric patients with dense granule deficiency, platelet function defects, immune thrombocytopenia, and other inherited platelet disorders. Quantitative fluorescence microscopy revealed reduced dense granule abundance not only in dense granule deficiency but also in several patients from other diagnostic groups. Measurements of platelet adhesion, spreading, and calcium signaling identified substantial functional diversity, with individual patients exhibiting distinct combinations of abnormalities that were not predicted by diagnostic category. Unexpectedly, plasma clotting analysis frequently revealed hypercoagulable behavior, including accelerated fibrin clot growth and spontaneous fibrin formation, despite clinical diagnoses associated with platelet-related bleeding disorders. Hypercoagulable phenotypes occurred across multiple diagnostic groups and did not show a simple relationship with platelet functional abnormalities. Together, these findings reveal previously unrecognized complexity in pediatric platelet disorders and suggest that platelet and plasma pathways contribute independently to hemostatic variability. These findings argue that pediatric platelet disorders are best viewed as multidimensional functional phenotypes rather than isolated platelet defects and motivate broader integration of platelet and coagulation measurements in future studies.