Haematologica

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.

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

The Src homology 2 (SH2) domain-containing non-transmembrane protein-tyrosine phosphatases 1 and 2 (Shp1 and Shp2) have been implicated in regulating signaling from a variety of receptors and cell types, including the thrombopoietin (Tpo) receptor Mpl in megakaryocytes (MKs) and platelets. We previously showed that deletion of Shp1 and Shp2 in the MK/platelet lineage in mice using the Pf4-Cre transgene/loxP system impairs megakaryopoiesis and thrombopoiesis. However, we also observed unexpected phenotypes including a motheaten-like phenotype in Shp1-deficient mice and severe myelofibrosis in mice lacking both phosphatases. To determine whether these were lineage-specific effects, we utilized the Gp1ba-Cre transgenic mouse to delete loxP-flanked Shp1 and Shp2 in mice. Bone marrow-derived MKs from these mice expressed approximately 20-25% of Shp1 and Shp2, whereas platelets contain 5-10% of each phosphatase compared with controls. Minor MK/platelet defects were observed in mice lacking either Shp1 or Shp2 alone, however mice lacking both Shp1 and Shp2 exhibited macrothrombocytopenia, mild bleeding following tail injury, and impaired GPVI-mediated platelet aggregation and Syk phosphorylation, associated with reduction GPVI and integrin 2 subunit expression. Reduced Shp1 and Shp2 expression resulting in a significant reduction in ploidy, a block in MK maturation and proplatelet-producing MKs. Tpo-mediated Ras/MAPK signaling was reduced in Shp1/2-deficient MKs. Treatment of MKs with structurally distinct Shp2 allosteric inhibitors recapitulated key aspects of the Shp2-deficient phenotype, including aberrant megakaryopoiesis and reduced Mpl signaling. Our study highlights the synergistic functions of Shp1 and Shp2 in the MK/platelet lineage, and identifies Shp2 as a potential therapeutic target in myeloproliferative neoplasms. Key PointsO_LIDeletion of Shp1 and Shp2 in the MK/platelet lineage in mice results in macrothrombocytopenia and minor effects on platelet function. C_LIO_LIDefects can be partially explained by reduced Mpl signaling and aberrant megakaryopoiesis in the absence of Shp2 activity. C_LI

Paroxysmal Nocturnal Hemoglobinuria (PNH) is characterized by hemolysis due to the loss of GPI-anchored complement regulators. While terminal complement inhibitors improve survival, the precise intracellular mechanisms driving the destruction of PNH erythrocytes remain controversial. A recently proposed model suggests PNH cells undergo an inflammatory programmed cell death ("spectosis") driven by an NLRP3-Caspase-8 signaling cascade. Here, we use a whole packed cell lysis approach to map the cytoskeletal degradation of primary erythrocytes across a 22-patient PNH cohort. Our data show that membrane attack complex (MAC) pore formation drives targeted {beta}-spectrin fragmentation, which correlates with rapid intracellular potassium (K+) efflux. Notably, when probing these primary patient samples, we detected a complete absence of the NLRP3 protein and found no functional evidence of Caspase-8 activation during MAC pore formation. Furthermore, caspase inhibition did not alter cytoskeletal degradation or K+ efflux. Instead, our data demonstrate that MAC-induced membrane perforation permits a rapid influx of calcium, which activates calpain, the dominant calcium-dependent protease in erythrocytes. Rather than an inflammatory cascade, this calcium-dependent calpain activity executes the degradation of {beta}-spectrin. These findings challenge current models of PNH hemolysis. We show that the destruction of PNH erythrocytes is a consequence of the MAC-calcium-calpain axis, rather than an inflammatory programmed cell death event. Consequently, therapeutic strategies aimed at targeting the inflammasome or caspase signaling will likely offer no clinical benefit for PNH patients.

Global mRNA translation is a defining functional property of hematopoietic stem cells (HSCs) and is increasingly recognized as a critical axis of dysregulation in myelodysplastic syndromes (MDS) and other clonal hematopoietic disorders. Yet the quantitative measurement of protein synthesis at single-cell resolution across phenotypically defined HSPC subpopulations, in parallel with apoptotic state, is technically challenging. Here we describe and validate a single-tube flow cytometry protocol that simultaneously quantifies global protein synthesis by O-propargyl-puromycin (OP-Puro) incorporation and intracellular cleaved Caspase-3 with cell immunophenotyping across the canonical CD34+ HSPC hierarchy in cryopreserved human cord blood (CB) CD34+ cells. The protocol enables quantitative assessment of key dynamic cell processes in defined subsets of primary hematopoietic cells on a standard flow cytometer. We apply this assay to a four-condition factor-omission analysis of the canonical SR1 + UM729 + dmPGE2 ex vivo expansion cocktail across three independent CB donors. The analysis assigns each compound a distinct functional profile: UM729 constrains protein synthesis and supports apoptotic priming across the hierarchy; SR1 maintains a pro-survival state without modulating translation; and dmPGE2 promotes HSC cycling and progressive exit from the primitive state, with minimal direct effect on the translation or apoptotic axes measured here. This analysis resolves three mechanistically distinct small-molecule signatures using a protocol directly transferable to clinical biobank specimens. We propose it as a functional-state analytic platform that may be useful for patient-derived CD34+ cells from MDS and other myeloid neoplasms in which translational dysregulation is a recognized pathological feature.

LZTR1 negatively regulates RAS family proteins via proteasomal degradation. Germline loss-of-function variants cause Noonan syndrome, with emerging evidence implicating LZTR1 in predisposition to childhood acute lymphoblastic leukemia (ALL), though its role in hematopoiesis remains poorly defined. Screening 1,587 children with ALL identified LZTR1 variants in 44 patients (2.8%). Germline variants were detected in 32 patients (2.0%), a frequency comparable to that observed in the general population (1.75%; 1,925/110,017; p=0.50). Somatic LZTR1 alterations were identified in 22 patients (1.4%) and were predominantly bi-allelic, arising through either a germline-plus-somatic or dual somatic configuration. They persisted at relapse. Despite enrichment in favorable-risk subtypes (ETV6::RUNX1, high-hyperdiploid, ERG/DUX4), bi-allelic LZTR1-mutated cases showed delayed minimal residual disease clearance and higher late relapse risk, identifying a subgroup unsuitable for treatment de-escalation. LZTR1 expression was increased in most wild-type leukemias, consistent with a compensatory response to aberrant RAS pathway activation. Bi-allelic LZTR1 inactivation abolished RAS regulation, leading to deregulated canonical RAS expression and ectopic expression of the non-canonical RIT1 protein, whose involvement in ALL has not previously been reported. These findings establish LZTR1 as a classical tumor suppressor in ALL via a two-hit model. Monoallelic alterations show insufficient signaling perturbation and low germline penetrance, whereas bi-allelic inactivation acts as a driver event linked to a high risk of late relapse despite favorable genomics.

Sickle cell disease (SCD) is caused by a point mutation in the {beta}-globin gene that promotes hemoglobin polymerization, leading to chronic hemolytic anemia, vaso-occlusive episodes, and progressive organ damage. The most efficacious therapies focus on reactivating fetal hemoglobin (HbF) expression to mitigate the pathological effects of sickle hemoglobin (HbS) polymerization. However, the predominantly used HbF inducer, hydroxyurea (HU), exhibits substantial interpatient variability in efficacy, and curative approaches such as gene therapy remain inaccessible to the vast majority of patients. Although all SCD patients share the same causative HBB glu7val mutation, differences in genetic background significantly influence disease severity and therapeutic response. We describe a SCD-specific induced pluripotent stem cell (iPSC) platform as a renewable and scalable preclinical model to interrogate treatment responses across the genetically diverse SCD patient population. By generating patient-specific iPSC-derived erythroblasts (iEry) representing distinct SCD genetic backgrounds, we demonstrate that this system faithfully recapitulates the heterogeneous HbF induction observed clinically in response to HU. Moreover, this platform enables the identification and evaluation of alternative therapeutic agents for HU non-responders and provides sufficient resolution to dissect drug-specific effects on erythroid differentiation and cellular phenotypes. Together, these findings support the use of iPSC-derived erythroid models as a versatile tool to advance precision therapeutic strategies for SCD. KEY POINTS- SCD iPSC-derived erythroid cells (iEry) reflect the diversity in HU-mediated HbF induction seen in SCD patients - SCD iEry recapitulate patient-specific treatment responses and can be used to identify therapeutic alternatives for HU non-responders - iEry provide a versatile platform to study the impact of novel HbF inducers on erythroid cell characteristics and differentiation parameters

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 during treatment of B-cell acute lymphoblastic leukemia (B-ALL) is a harbinger of poor outcomes. Identifying biomarkers for subsequent relapse risk which are detectable at B-ALL diagnosis remains a priority. Off-target recombination-activating gene (RAG)-mediated structural variants (SVs) generate genomic instability that drives leukemogenesis and may underlie treatment resistance. Leveraging sequencing data in 1,496 pediatric B-ALL patients enriched for relapse status (relapse n=532; non-relapse n=964), we characterized RAG-mediated SVs across B-ALL molecular subtypes and examined their association with patient characteristics and their impact on clinical outcomes. Off-target RAG-mediated SVs were overall frequent, particularly in ETV6::RUNX1, ETV6::RUNX1-like, and Ph-like B-ALL subtypes, while increasing age-at-diagnosis was positively associated with burden of off-target RAG-mediated SVs (P<.001). Off-target RAG-mediated SVs with a recombination signal sequence (RSS) at one breakpoint, a hallmark of off-target RAG activity, were significantly more frequent at diagnosis in patients who subsequently relapsed (P=.001). This association remained significant in multivariable regression analysis (per SV odds ratio [OR]:1.08, 95%CI:1.04-1.12), in minimal residual disease (MRD)-negative patients (OR:1.09, 95%CI:1.04-1.14) and across subtypes. Excluding deletions, MRD-negative ETV6::RUNX1 patients with 3 off-target RAG-mediated SVs had a >3-fold risk of relapse (hazard ratio:3.47, 95%CI:1.86- 6.49). RAG-mediated SVs were also associated with relapse risk in T-cell ALL patients. Off-target RAG-mediated SV burden at diagnosis is a risk factor of relapse in pediatric ALL across molecular subtypes and independent of MRD status.

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.

Impaired cellular differentiation is a defining characteristic of myeloid malignancies and remains a major therapeutic challenge. The BRG1/Brahma-associated factor (BAF) chromatin remodeling complex, through the ATPases SMARCA4 and SMARCA2, maintains the stemness of leukemic blasts and thus represents a promising target for novel differentiation-based therapies. In a phase 1 study in advanced myeloid malignancies, the first-in-class dual SMARCA4/2 inhibitor FHD-286 combined with decitabine (DAC) was tolerated and produced an objective response rate of 12.8% (6/47) compared with no responses with FHD-286 monotherapy. To understand the basis of this activity, we integrated high-dimensional flow cytometry and single-cell genomic analyses of longitudinal bone marrow samples from responders and nonresponders. While FHD-286 monotherapy was predominantly associated with myeloid differentiation, responders to FHD-286+DAC combination therapy exhibited a range of myeloid and erythroid differentiation trajectories. FHD-286 potentiated the transcriptional impact of DAC, driving tumor clones to fully differentiate out of the immunophenotypically and transcriptionally defined blast compartment. Responders had a baseline transcriptional profile similar to that of CEBPA-mutant acute myeloid leukemia and showed further downregulation of CEBPA upon treatment. These findings reinforce tumor cell differentiation as a mechanism of response to pharmacologic SMARCA4/2 inhibition and support further evaluation of FHD-286+DAC in molecularly defined patient subsets.

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.

Sickle-hemoglobin-C (HbSC) sickle cell disease is characterized by RBC dehydration (xerocytosis), which promotes polymerization of HbS. HbSC causes substantial morbidity despite lower sickling potential than HbSS, suggesting a critical detrimental role of HbC in the disease pathophysiology. We derived HbCC mice by interbreeding our HbSC mice, which demonstrated a similar RBC phenotype of xerocytosis as humans with HbCC. We compared RBCs from HbCC, HbSC, and HbSS mice. Oxidized ferryl (Fe4+)-Hb, and its oxidative-denaturation, which results in hemichrome formation (Heinz-bodies), was most pronounced in HbCC>HbSC>HbSS, despite significantly higher reactive oxygen species in HbSS, illustrating a higher propensity of HbC to denaturation than HbS. RBC deformability followed a similar pattern, with Elongation Index lowest in HbCC<HbSC<HbSS. Next, we determined if RBC from HbSC patients on hydroxyurea showed improved membrane damage. Hydroxyurea treatment reduced Heinz-body formation and improved RBC deformability, despite negligible/modest fetal hemoglobin (HbF) induction, compared to non-hydroxyurea HbSC controls. The antioxidant quercetin showed a similar reduction in Heinz-body burden and improvement in RBC deformability as hydroxyurea, without affecting Hb or HbF concentration, reticulocyte count, or RBC xerocytosis. HbC-driven oxidative denaturation and membrane damage represent important contributors of RBC dysfunction in HbSC disease; hence, oxidative membrane injury could be targeted besides antisickling approaches.

Hyperleukocytosis (white blood cell [WBC] count >100 000/uL) at diagnosis is an important prognostic risk factor in pediatric acute lymphoblastic leukemia (ALL), though its significance with contemporary therapy is unclear. We analyzed 1 826 pediatric ALL patients from a multi-institution cohort to determine whether hyperleukocytosis independently predicts outcomes using multivariable Cox proportional hazard modeling. Hyperleukocytosis occurred in 211 patients (12%), with 121 having B-ALL, and showed no prognostic significance in T-ALL patients. In B-ALL, 5-year event-free survival (EFS) was 65% versus 89% for non-hyperleukocytosis patients, and overall survival (OS) was 78% versus 93%. After adjustment for age, cytogenetic risk, central nervous system disease status, and treatment site, hyperleukocytosis remained an independent predictor of end-of-induction minimal residual disease (MRD) positivity (odds ratio 2.53 [95% confidence interval [CI]: 1.71-3.94; p<0.001]), inferior EFS (hazard ratio [HR] 2.44; 95% CI: 1.77-3.38; p<0.001) and inferior OS (HR 2.00; 95% CI: 1.29-3.12; p=0.002). A continuous dose-response relationship was observed between WBC count and these outcomes. Survival associations persisted across all cytogenetic risk categories and MRD strata. Despite risk-adapted therapy with treatment intensification for high-risk features, hyperleukocytosis identifies an aggressive B-ALL phenotype with persistently inferior outcomes, suggesting these patients may benefit from novel therapeutic approaches.

Amino acid deprivation with L-asparaginase is a cornerstone of treatment in acute lymphoblastic leukaemia (ALL), but clinical challenges limit its use in adults. Deficiency in the enzyme argininosuccinate synthase (ASS1-low) confers arginine auxotrophy, defining a dependence on extracellular arginine, and represents an analogue metabolic vulnerability that is targetable through arginine deprivation. We analysed transcriptomic data across >550 adult B-ALL cases to establish clinico-biologic characteristics of low ASS1 expression identifying the Philadelphia chromosome-positive (Ph+) ALL subgroup as a stereotypical arginine auxotroph within molecularly diverse B-ALL. Functionally, arginine deprivation with pegargiminase induced robust apoptosis in both Ph+ ALL cell lines and primary samples in an ASS1-dependant manner and was highly effective as a monotherapy treatment in independent Ph+ ALL patient derived xenografts. Mechanistically, arginine deprivation induces endoplasmic reticulum stress mediated apoptosis which was orthogonal to tyrosine kinase inhibition (TKI) mediated outcomes. Using in vitro and in vivo models of non-genetically mediated TKI-resistance, we demonstrate pegargiminase and TKI combinations robustly eradicates TKI-resistant leukaemia. Thus, we establish ASS1 deficiency (ASS1-low) as a therapeutically actionable vulnerability in Ph+ ALL and a strategy to bypass TKI-resistance, supporting clinical evaluation of arginine deprivation as a novel adjunct to chemotherapy-free treatment.

Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like episodes (MELAS) syndrome is primarily caused by the heteroplasmic m.3243A>G/MT-TL1 pathogenic variant. Patients exhibit elevated circulating cell-free mtDNA (cf-mtDNA) in plasma, which acts as a damage-associated molecular pattern. Using patient-derived fibroblasts and neuronal progenitors, as well as transmitochondrial cytoplasmic hybrids (cybrids), we show that mutant cells release higher levels of cf-mtDNA than wild-type controls, demonstrating that the m.3243A>G pathogenic variant drives mtDNA release. Mechanistically, increased mitochondrial oxidative stress promotes mtDNA oxidation and fragmentation, leading to Ca2+ overload and subsequent mtDNA extrusion. This, in turn, triggers inflammasome activation and pyroptosis, resulting in the secretion of pro-inflammatory cytokines and the activation of innate immune pathways. Pharmacological inhibition of the Mitochondrial Calcium Uniporter (MCU) or Voltage-Dependent Anion Channel (VDAC) reduced mtDNA release, confirming their involvement. Overall, our findings reveal a previously unrecognized mechanism in MELAS linking mitochondrial dysfunction to innate immune activation, with potential implications for therapeutic intervention. TeaserMELAS mutation drives mtDNA release, triggering inflammation via oxidative stress, calcium imbalance, and inflammasome activation

Abstract Mitochondrial DNA (mtDNA) heteroplasmy, the coexistence of multiple mtDNA variants within cells, accumulates with age and is associated with hematological malignancies and mortality. However, whether predicted deleterious heteroplasmies causally contribute to cancer or merely represent passenger mutations remains unresolved. Here, leveraging ~36,000 first-degree relative pairs from the UK Biobank and All of Us Research Program cohorts, we deconvolute overall heteroplasmy metrics into those that are shared across family members (representing inherited variants) and those that are not (representing de novo variants) to establish a Mendelian randomization framework for assessing causality. We show that shared heteroplasmies exhibit strong purifying selection, with reduced predicted deleteriousness compared to not shared variants, and that 90% of an individual's deleterious heteroplasmy burden is somatically acquired. Critically, shared deleterious heteroplasmy burden, fixed at conception and thus temporally upstream of potential confounders, is significantly associated with hematological malignancies (RR=2.81, 95% CI 1.29-6.13), with effect sizes concordant with the not shared heteroplasmy burden. Furthermore, shared deleterious heteroplasmy specifically associates with high-risk clonal hematopoiesis of indeterminate potential (CHIP), particularly spliceosome mutations, suggesting mitochondrial dysfunction promotes clonal expansion of specific CHIP subtypes. Finally, we identify ultra-rare individual mtDNA variants associated with hematological malignancies, a hallmark of driver mutations. These findings establish mtDNA heteroplasmies, including inherited variants, as causal contributors to hematological malignancy risk and demonstrate that most disease-relevant burden is acquired during life, identifying potential opportunities for prevention and therapeutic intervention in individuals at elevated risk for hematological cancer, particularly of myeloid origin.

The regulation of human hematopoietic stem cell (HSC) function through its native environment is virtually unknown. Cross-species chimeras, particularly humanized mice, are essential tools for investigating human HSC function in vivo. However, conventional models often require toxic conditioning that impairs niche and donor cell function, or simplify niche complexity. We utilize NSGW41 mice, which harbor a KIT receptor mutation, to achieve robust human leukocyte engraftment without prior treatment. We demonstrate that KIT-proficient human HSCs possess a clear advantage, effectively outcompeting endogenous murine stem cells and progenitors to establish stable, multilineage human hematopoiesis. Crucially, the murine niche undergoes significant plastic adaptation in response to humanization. We identify that mesenchymal stromal cells (MSCs) expand and undergo a transcriptional shift, transitioning from a mixed adipo- or osteo-primed state toward predominantly Lepr+ adipo-primed HSC-supporting cells. This adaptation is vital; the depletion of Lepr+ MSCs or the targeted deletion of Stem Cell Factor (SCF) from these cells leads to the mobilization or loss of human HSC engraftment, respectively. These findings provide compelling evidence for functional cross-species niche-HSC communication, identifying Lepr+MSCs as primary regulators of human HSC maintenance in xenotransplantation models. By mapping this molecular dialogue, our work establishes a physiological in vivo platform to study human HSC biology and evaluate niche-targeted therapeutic interventions to improve transplantation outcomes.

Clonal hematopoiesis (CH) is an age-associated condiion defined by over-representation of hematopoietic stem cells (HSCs) and their progeny carrying somatic mutations or variants that confer a selective advantage. CH is associated with increased risk of hematologic malignancies (1), cardiovascular disease and inflammatory bone loss (2, 3). Chronic inflammation is increasingly recognized as a central mediator of CH-mutant hematopoietic stem and progenitor cell (HSPC) expansion underlying CH (4). DNA methyltransferase 3a ( Dnmt3a )-mutant cells produce higher levels of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) (5), and blocking these pathways reduces the competitive advantage of Dnmt3a -mutant HSPCs (4, 6). The upstream mediators initiating inflammatory signaling in CH are unknown. Strong candidates are S100A8 and S100A9, members of the S100 calcium-binding protein family that regulate inflammatory signaling in the hematopoietic system. These proteins form a heterodimer complex and activate innate immune signaling through receptors including Toll-like receptor 4 (TLR4) and the receptor for advanced glycation end products (RAGE) (7). S100A8/A9 signaling promotes production of pro-inflammatory cytokines and inflammasome activation leading to poor prognosis in myelodysplastic syndrome and myeloproliferative neoplasms (8, 9). Across multiple myeloid malignancies, neutrophils are the primary bone marrow (BM) source of this alarmin (8, 10, 11) and pharmacologic inhibition of S100A9 with tasquinimod reduces disease severity without disrupting normal hematopoiesis (10, 12). Given the role of S100A8/A9 in establishing an inflammatory milieu, here we investigated the role of S100A8/A9 in Dnmt3a -mutant hematopoiesis. We identify neutrophils as a major source of elevated S100A8/A9 in the BM of Dnmt3a -mutant mice and this increase correlates with production of the inflammatory cytokines TNFα and IL-6. We show that tasquinimod reduces TNFα and IL-6 levels and selectively reduces the Dnmt3a -mutant HSPC compartment.

Key PointsO_ST_ABSQuestionC_ST_ABSDoes the circadian timing of stem cell infusion influence the risk of aGVHD after allo-PBSCT? FindingsIn this randomized prospective clinical trial that included 198 patients, infusion stem cell at 12:00 pm at noon was associated with a significantly lower incidence and less severity of aGVHD compared with infusion at 6:00 pm, without influencing engraftment or relapse. MeaningScheduling stem cell infusion at an earlier time-of-day may reduce aGVHD risk after allo-PBSCT. IMPORTANCEAcute graft-versus-host disease (aGVHD) remains a major complication following allogeneic peripheral blood stem cell transplantation (allo-PBSCT), compromising patient survival and quality of life. OBJECTIVETo evaluate the effect of stem cell infusion time-of-day on aGVHD after allo-PBSCT. DESIGNA multicenter, randomized, open-label, phase 3 clinical trial was conducted from March 18, 2024, through June 11, 2025, with follow-up through December 31, 2025 (median, 462 days among survivors). SETTINGSix transplantation centers in China. PARTICIPANTSPatients aged 12 to 60 years with malignant hematologic diseases undergoing first allo-PBSCT were screened; 198 eligible patients were randomized. INTERVENTIONSPatients were randomly assigned in a 1:1 ratio to receive stem cell infusion at either 12:00 pm at noon ({+/-} 0.5 hour) or 6:00 pm ({+/-} 0.5 hour). MAIN OUTCOMES AND MEASURESThe primary end point was the cumulative incidence of grade II-IV aGVHD within 100 days after transplantation. Secondary end points included grade III-IV aGVHD, hematopoietic recovery, transplant-related mortality (TRM), relapse, and survival outcomes. RESULTSAmong 198 randomized patients (median age, 38 years; 119 [60.1%] male), grade II-IV aGVHD within 100 days occurred in 11 of 99 patients (11.1%) in the 12:00 pm group and 22 of 99 patients (23.2%) in the 6:00 pm group. The cumulative incidences of grade II-IV and III-IV aGVHD were significantly lower in the 12:00 pm group (II-IV: 11.1% [95% CI, 5.9%-18.2%] vs 23.2% [95% CI, 15.4%-32.0%], P = 0.029, hazard ratio, 2.18 [95% CI, 1.06-4.48]; III-IV: 2.0% [95% CI, 0.4%-6.5%] vs 12.2% [95% CI, 6.7%-19.5%], P = 0.006, hazard ratio, 6.25 [95% CI, 1.39-28.15]). There were no significant differences in hematopoietic recovery, TRM, or relapse between groups. The estimated probability of GVHD-free, relapse-free survival (GRFS) at 360 days favored the 12:00 pm group (66.7% [95% CI, 56.2%-75.2%] vs 56.5% [95% CI, 46.1%-65.5%]). CONCLUSIONS AND RELEVANCEStem cell infusion at 12:00 pm was associated with a lower incidence and severity of aGVHD compared with infusion at 6:00 pm, without influencing engraftment or relapse. Optimization of infusion timing may represent a simple strategy to reduce aGVHD risk. TRIAL REGISTRATIONClinicalTrials.gov Identifier: NCT06294678.