Blood Advances

Hematopoietic stem and progenitor cell (HSPC) niches support lifelong production of blood and immune cells. Recently, we identified a gene signature unique to HSPC niche endothelial cells that is highly conserved across species and developmental time, and includes the scavenger receptors, stab1/2 and mrc1a. Whether these receptors support HSPC development in the niche remains unclear. To investigate this, we used chemical inhibition and CRISPR mutagenesis in zebrafish and found that loss of stab2, and to a lesser degree stab1, reduced the number of embryonic HSPCs labeled by runx1:mCherry. Subsequent analyses of runx1:mCherry; cd41:GFP double transgenic embryos revealed an imbalance in the HSPC pool in stab2 mutants, with reductions in sub-populations containing stem cells and erythroid progenitors, the latter of which was most decreased. Our findings suggest stabilin scavenger receptors support HSPC development in the fetal niche, which could inform clinical strategies for culturing and expanding HSPCs.

Embryonic hematopoiesis is essential for establishing lifelong blood and immune system function. During development, hematopoietic stem and progenitor cells (HSPCs) acquire intrinsic programs that persist into adulthood and can influence disease susceptibility, yet the molecular signals governing these early-life decisions remain poorly understood. Here, we investigated the role of developmental Transforming Growth Factor-{beta} (TGF-{beta}) signaling in regulating HSPC lineage bias and long-term hematopoietic outcomes. Using the zebrafish model, we found that transient embryonic TGF-{beta} signaling inhibition during the HSPC specification window altered their frequency and migration after emergence from the hemogenic endothelium and movement into the key secondary maturation and expansion niche. Single-cell transcriptomic analysis of embryonic HSPCs revealed repression of migration- and cytoskeleton-associated genes alongside dampened expression of myeloid/macrophage-related genes following ALK5 inhibition. Functionally, early ALK5 blockade reduced macrophage numbers and promoted an M2-like immunosuppressive transcriptional profile. These developmental perturbations produced sustained effects on hematopoietic and immune function into adulthood, including diminished inflammatory gene expression, reduced clonal complexity, and impaired regenerative capacity. Together, our findings identify embryonic TGF-{beta} signaling as a key developmental regulator of HSPC fate and immune programming, with potential implications for immune dysfunction and susceptibility to inflammatory-related disease later in life. PAPER HIGHLIGHTS- Transient developmental signaling perturbations reshape hematopoietic trajectories - Embryogenic TGF-{beta} signaling instructs HSPC lineage priming and macrophage specialization - Early HSPC programming establishes persistent inflammatory states, clonal diversity, and modifies regenerative capacity

Zebrafish combines the power of genetics and unparalleled in vivo imaging for investigating the dynamics of vertebrate hematopoietic development. Across species, the transcription factor Runx1 is essential for definitive hematopoiesis. We generated a zebrafish runx1-2A-creERT2 CRISPR knock-in for tamoxifen-regulated Cre recombinase Runx1 lineage tracing and characterized its activity using the ubi:Switch recombinase-dependent fluorescence reporter, microscopic live imaging and flow cytometry. Tamoxifen treatment beginning at gastrula stage labeled all expected Runx1 lineages in the early embryo, including neuroectodermal olfactory placode and Rohan-Beard neurons, primitive hematopoietic blood cells, and nascent hematopoietic stem and progenitor cells (HSPCs) in the dorsal aorta. Runx1 HSPCs colonized the larval caudal hematopoietic tissue and thymus from three to five days of development. Timed tamoxifen induction of Cre activity allowed separation of Runx1 primitive hematopoiesis from definitive HSPC emergence and larval stem cell niche colonization. Flow cytometry of kidney marrow and peripheral blood from adults treated with tamoxifen at gastrula stage revealed Runx1 embryonic hematopoietic cells contributed to adult hematopoietic precursors, myeloid, lymphoid, and peripheral blood lineages. Labeling of all blood lineages was also effective by tamoxifen treatment of 5-month-old adults. The zebrafish runx1-2A-creERT2 line provides a powerful tool for precise spatial and temporal analysis of Runx1 progenitor mechanisms in developmental and adult hematopoiesis. Key PointsO_LIzebrafish endogenous runx1-2A-creERT2 provides inducible Cre recombinase genetic analysis in all runx1 neuromesodermal and blood lineages C_LIO_LIzebrafish runx1-2A-creERT2 line enables in vivo spatial and temporal analysis of embryonic and adult hematopoiesis C_LI

Mantle cell lymphoma (MCL) is a B-cell non-Hodgkin lymphoma characterized by heterogeneous clinical courses despite a common pathobiological initiating event. In this work we explore the genomic variants that characterize MCL and integrate transcriptomic data to comprehensively describe MCL biology. We performed whole exome sequencing (WES) on 28 tumor-normal pairs (lymph node and skin, respectively), as well as whole genome sequencing (WGS) and RNA sequencing on subsets of samples. We used established DNA and RNA analysis pipelines to detect single-nucleotide variants (SNV) and indels, structural variants, copy-number alterations, and RNA fusions. The canonical t(11;14)(q13;q32) CCND1::IGH translocation was detected in 8 of 10 WGS samples. Structural variant analysis additionally identified recurrent rearrangements involving KMT2A and PAFAH1B2. SNV and indel analyses revealed frequent mutations in ATM, TP53, CCND1, IGH, and NOTCH1. ATM exhibited diverse variant classes, including missense mutations, frameshift mutations, deletions, and duplications, while all detected NOTCH1 mutations were predicted loss-of-function frameshift variants. Copy-number analysis identified recurrent losses affecting DNA damage response genes, including TP53 and ATM, and recurrent gains involving transcriptional regulators and oncogenic signaling genes. Integrated pathway analysis demonstrated enrichment of transcriptional misregulation, DNA repair, PI3K/AKT signaling, and interleukin signaling pathways. We also identified recurrent alterations in candidate genes, including ASXL1, suggesting additional mechanisms of epigenetic dysregulation in MCL. Together, these findings provide a comprehensive description of somatic alterations in MCL and demonstrate that diverse genomic lesions converge on common pathways involved in genomic instability, transcriptional regulation, and tumor survival.

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

Certain aspects of lipid metabolism are known to regulate hematopoietic stem cell (HSC) function, including fatty acid oxidation and lipid uptake, but there is a limited understanding of the contribution of de novo fatty acid synthesis to HSC homeostasis. Here, we show that endogenous fatty acid synthesis is essential for HSC function. Conditional deletion of Acaca, the gene that encodes the rate-limiting enzyme for de novo fatty acid synthesis, acetyl-CoA carboxylase 1 (ACC1), in hematopoietic cells profoundly reduces HSC function, marked by a reduced ability to reconstitute irradiated mice after competitive transplantation. ACC1 deficiency reduced quiescence, increased uptake of extracellular lipids, and increased reactive oxygen species in HSCs. The loss of HSC function is partly caused by increased fatty acid oxidation as deletion of CPT1a, which is required for long-chain fatty acid oxidation, partially rescued HSC function. A balance between fatty acid synthesis and fatty acid oxidation is thus critical for the maintenance of HSC function.

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.

Haplo-identical hematopoietic cell transplantation (haploHCT) is an integral treatment paradigm for patients with leukemia. While overall survival (OS) post-haploHCT has steadily improved, relapse-free survival (RFS) remains relatively stagnant. Upon the discovery of killer immunoglobulin-like receptors (KIRs) on natural killer (NK) cells and their cognate human leukocyte antigen (HLA) ligands, algorithms have been developed to enhance graft versus leukemia effects. However, these algorithms fail to yield consistent predictions in patient outcomes. We utilized a combination of in silico protein folding and interactions to determine KIR:HLA reactivity in conjunction with in vitro acoustic force microscopy to measure cell avidity (CA) as a readout for KIR signal strength. CA was determined using monoallelic HLA expressing K562 cell lines, monoallelic KIR Jurkat cells, and peripheral blood NK cells. We extended the CA results and performed standard cytotoxicity assays as well. We discovered that HLA-B*35 interacts with KIR2DS4. We applied the newly discovered interaction to predict outcomes for HCT patients. Stratifying patients based on their HLA-B*35 positivity and donor KIR2DS4 status, we delineated a correlation to survival (P=0.061) when donors only had full-length KIR2DS4. Patients who received a haploHCT and NK cell addback from donors with only full-length KIR2DS4 had a significantly improved RFS (P=0.001) and OS (P=0.016) compared to truncated (KIR1D) and full-length KIR2DS4 donors. This was independently validated in a diverse 10/10 HLA matched European cohort with RFS (P=0.0255) and OS (P=0.0388). Thus, the identified novel KIR2DS4:HLA-B*35 interaction axis predicts patient survival, in both haplo-identical and fully matched, HCT and highlights that our current understanding of the KIR:HLA interactome is incomplete and requires remapping for enhanced therapeutic applications.

Transcription factor CEBPA is mutated in 10-15% of acute myeloid leukaemia (AML), a haematopoietic malignancy with high mortality. CEBPA mutations show a distinct pattern, and most patients are biallelic, carrying both an in frame C-terminal mutation and a frameshift N-terminal mutation on opposing alleles. Rare N-terminal germline cases have 100% penetrance to AML, all with an acquired a C-terminal mutation. This suggests a selective pressure from one CEBPA mutation to develop another. Our zebrafish models faithfully recapitulate the human disease. All biallelic mutant combinations die by 4-6 weeks of age, with pre-leukaemic haematopoietic stem cell (HSC) expansion. C-terminal and N-terminal mutants show phenotypic differences in myeloid primed HSC and differences in the myeloid differentiation block. RNA-Seq identified differentially expressed genes in opposing vectors. We identified phosphatase receptor PTPRJ as a candidate driver of clonal selection, with knock-out of ptprja in our fish accelerating pre leukaemic expansion of HSC in C-terminal mutants, decelerating it in N-terminal mutants. Cebpa mutant murine cells exhibit changes in differentiation and a clonal advantage with loss of Ptprj, which perturbs key signalling pathways. Our data suggest that PTPRJ contributes to the mechanism of leukaemogenesis in CEBPA mutant AML by driving the selective pressure from each mutation to develop the other.

Hematopoietic stem cells (HSCs) in the adult mouse can be prospectively isolated to near-purity through phenotypic markers, enabling detailed analysis of stem cell function. Homeobox B5 (Hoxb5) was previously identified as a definitive marker of long-term (LT) HSCs in adult bone marrow1. In contrast, fetal HSCs have not been purified to the same extent. Here, we show that Hoxb5 is expressed in fetal liver (FL) HSCs at embryonic day (E) 12.5-16.5 using a single-color tri-mCherry reporter driven by endogenous Hoxb5 regulation. Prospective purification by stringent multiparameter flow cytometry revealed Hoxb5 FL-HSCs to exhibit robust, multilineage reconstitution upon serial transplantation. Quantitative assays reveal that Hoxb5 enriches FL-HSCs to near-single-cell purity, analogous to its role in the adult bone marrow, underscoring its reliability in distinguishing LT-HSCs throughout hematopoietic ontogeny. Notably, Hoxb5 expression is not exclusive to FL-HSCs, as it is also detected across the fetal liver hematopoietic hierarchy and in fetal liver endothelial cells, suggesting developmental stage-specific regulation of its expression. In addition, single-cell RNA sequencing of FL-HSCs identified distinct transcriptional states defined by Hoxb5 expression. These findings establish Hoxb5 as a robust marker for enhancing the purification of fetal liver phenotypic HSCs (pHSC) and provide a framework for dissecting the molecular regulation of HSC ontogeny.

Haematopoietic stem cells (HSC), while usually quiescent, can rapidly divide following specific stimuli (mobilization). These HSC can seed additional niches, allowing for the swift generation of essential blood cells. However, studies in mice and humans have clearly demonstrated that cycling bone marrow (BM) HSC (cells in the G1/S/G2/M phases) engraft and reconstitute the haematopoietic system poorly compared with HSC in the G0 phase1. This raises the question why mobilized HSC, immediately following 3 or more cell divisions2, efficiently reconstitute the haematopoietic system. We studied this phenomenon in human HSC using scRNAseq analysis. We found that mobilized HSC rapidly start transcribing genes associated with quiescence, specific for the G0 phase of the cell cycle. We hypothesize that this rapid switch from actively dividing to quiescent cells combined with our extensive RNA expression data will allow us to better define pathways involved in this process.

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.

Background CD19-directed CAR-T cell therapy can induce durable remissions in chronic lymphocytic leukemia (CLL), but response rates are lower than in other B-cell malignancies, in part because CLL is characterized by T-cell dysfunction, defective immune synapse formation, and impaired target-cell co-stimulation. Lenalidomide is an immunomodulatory drug with the potential to act on both sides of the CAR-T/CLL interface by improving T-cell fitness and modifying malignant B-cell susceptibility to immune engagement. Methods We are conducting an open-label, non-randomized phase I/II clinical trial VTB-CLL002 (ClinicalTrials.gov identifier: NCT06762431) evaluating the safety and efficacy of CD19 CAR-T cell therapy combined with concomitant lenalidomide in patients with relapsed or refractory CLL and small lymphocytic lymphoma followed by lenalidomide maintenance. The primary endpoint was safety. The secondary endpoint included overall response rate (ORR), complete response (CR), progression-free survival (PFS) and overall survival (OS). Results Twelve patients were treated. The median age was 60 years and the median number of prior lines of therapy was 2. All patients were BTK inhibitor-naive, and all had measurable disease at the time of infusion. CAR-T manufacturing was successful in all patients. All treated patients achieved complete remission, with a median time to response of 1 month. CAR T-cells expansion was observed in all patients, with a median peak expansion of 137 cells/L and a median time to peak expansion of 14 days. CAR T-cells remained detectable at the last follow-up in all patients, with persistence documented up to 24 months. At dose levels 2-3, eight of nine patients had ongoing MRD-negative responses at the time of analysis. Toxicity was clinically meaningful. Cytokine release syndrome (CRS) occurred in all patients, with severe CRS observed in 2 of 12 patients. ICANS occurred in 5 of 12 patients, including severe ICANS in 4 of 12 patients. One patient developed late grade 4 ICANS temporally associated with lenalidomide reintroduction and secondary CAR-T expansion. Early and late immune effector cell-associated hematotoxicity were common. In mechanistic studies, lenalidomide enhanced CAR-T proliferation and cytotoxicity, shifted CAR-T cells toward effector-associated phenotypes, reduced selected exhaustion markers during repeated antigen challenge, and increased IL-2 and IFN-{gamma} secretion. Lenalidomide also increased CAR-T/CLL conjugate formation and upregulated CD54/ICAM-1 on CLL target cells without broad induction of CD80, CD86, or CD40, consistent with improved adhesive target-cell engagement rather than classical co-stimulation. Transcriptomic profiling supported enhanced Th1/cytotoxic and T-cell activation-associated programs with lower T reg -associated genes in lenalidomide-treated CAR-T cells. Conclusions Lenalidomide-augmented CD19 CAR-T therapy demonstrated strong early clinical activity in relapsed/refractory CLL, characterized by deep responses, durable CAR-T persistence, and substantial incidence of immune effector-associated toxicities. These findings support further evaluation of lenalidomide as a rational CAR-T partner in CLL and suggest that its activity may involve both improved CAR-T fitness and enhanced target-cell engagement. Future studies should optimize lenalidomide timing and dosing to preserve response depth while reducing delayed immune-effector toxicity.

Hematopoietic stem cells (HSCs) generated from induced pluripotent stem cells (iPSCs) offer a promising patient-specific alternative to allogeneic transplantation, yet current differentiation protocols fail to fully recapitulate in vivo HSC maturation. During mouse development, yolk sac (YS)-derived macrophages populate the aorta-gonad-mesonephros (AGM) region at the time of HSC emergence, but the mechanisms by which they support ex vivo hematopoietic stem and progenitor cell (HSPC) generation remain poorly defined. Bulk RNA sequencing revealed that mature AGM CD206 macrophages upregulate pro-inflammatory cytokines and the adhesion molecule F4/80. Using F4/80 knockout embryos, we identify a previously unreported, niche-specific role for F4/80 in restraining the frequency and colony-forming activity of HSPC subsets in the AGM, while supporting endothelial cell maintenance; this effect was absent in the YS. Lineage-tracing with a Cdh5-CreERT2;Rosa26LSL-tdTomato pulse-chase system confirmed that both CD206 and CD206- AGM cells originate from early YS-derived endothelial precursors, with no evidence of local macrophage generation within the AGM. Functional co-culture assays further demonstrated that the ability of CD206 macrophages to enhance the progenitor potential of hemogenic endothelium is AGM-specific and not an intrinsic, ontogeny-determined property, as YS macrophages failed to confer the same benefit even when paired with AGM endothelial cells, and AGM macrophages were ineffective with YS endothelium. Differential expression and NicheNet ligand-receptor interaction analyses identified a small set of AGM-restricted macrophage genes - including Mmp2, Nrep, Ccl2, and Cxcl16 - which are predicted to interact with both endothelial and cluster cells during endothelial-to-hematopoietic transition. Together, these findings establish that AGM macrophages acquire niche-specific transcriptional and functional properties upon entry into the aortic microenvironment, independent of their YS origin, and identify candidate macrophage-derived factors and a novel regulatory role for F4/80 in shaping HSPC output. These insights may guide the refinement of iPSC-based HSC differentiation protocols through the targeted, temporally controlled addition of macrophage-associated signals.

Chronic myelomonocytic leukemia (CMML) is an aggressive hematologic malignancy characterized by excess inflammatory signaling and clonal myeloproliferation. The relative contribution of neutrophils (PMNs) to the inflammatory milieu in CMML is poorly understood. In this study we sought to understand whether neutrophil extracellular trap (NET) formation, a key mediator of neutrophilic inflammation, is dysregulated in CMML and can be therapeutically targeted with a novel peptide inhibitor of NETosis called neonatal NET-inhibitory factor (nNIF). Here, we demonstrate that baseline NET formation is aberrantly increased in primary CMML PMNs transcriptionally primed for NETosis, and that soluble factors produced during CMML NET formation promote clonogenicity in CMML CD34+ hematopoietic cells matched to the same patient. Further, we show that nNIF and clinical agents under investigation in CMML effectively inhibit NETosis, warranting further study of NET inhibitory agents in this rare disease with limited treatment options.

Background Monoclonal gammopathy of thrombotic significance (MGTS) is a recently described chronic prothrombotic condition characterized by monoclonal anti-PF4 antibodies that are detected above the polyclonal antibody background in patient sera (i.e. present as monoclonal gammopathy of undetermined significance, MGUS). Due to conflicting data in the published literature on antibody clonality in heparin-induced thrombocytopenia (HIT), we evaluated clonality and abundance of anti-PF4 antibodies in HIT, including investigating whether an MGUS, if present in HIT, represents the causative anti-PF4 antibody. Methods Blood samples from 15 patients with HIT were subject to Platelet Factor 4-dependent antigen-based and functional tests. The unmanipulated serum antibody repertoire and isolated anti-PF4 antibodies were subjected to mass spectrometric evaluation. Results Two of the 15 HIT patients had an IgG MGUS. Notably, anti-PF4 antibodies were not synonymous with the MGUS antibody in either of the two patients. Eight of the 15 patients demonstrated monoclonal anti-PF4 antibodies, however, none of the anti-PF4 antibodies were detectable as an MGUS upon evaluation of the entire serum antibody repertoire, reflecting their low abundance. In the seven patients with multiple anti-PF4 antibodies, non-monoclonality was confirmed by analysis of deglycosylated antibody heavy chains. Conclusions Anti-PF4 HIT antibodies are monoclonal in approximately 50% of HIT patients, however, antibody abundance is low such that they are not detectable over the polyclonal IgG background (i.e. are MGUS-negative), differentiating HIT from MGTS. This observation helps explain the transient nature of HIT relative to the persistent prothrombotic state seen in MGTS.

Large genomically annotated acute myeloid leukaemia (AML) datasets exist, but population-based contemporary cohorts remain scarce. Here we report clinicopathological, genomic, and outcome data from Danish AML patients. 2,512 AML patients were identified between 2015-2022, of whom 33.8% had available NGS data (NGS+). In patients [≤]70 years, baseline characteristics and outcomes were comparable between NGS+ and NGS- groups. In patients >70 years, more NGS+ patients received intensive treatment, but survival was similar among intensively treated patients. The distribution of mutations varied significantly by age and sex, with older age and male sex exhibiting higher frequencies of adverse-risk gene mutations. In intensively treated NGS+ patients, ELN2017 stratified 5-year OS: 58.4% (favorable), 43.4% (intermediate), and 28.2% (adverse), with hazard ratios (HRs) of 0.63 (favorable) and 1.45 (adverse) relative to intermediate. ELN2022 yielded corresponding OS rates of 56.9%, 51.8%, and 29.7%, with HRs of 0.78 and 1.86. The two models had comparable predictive performance for OS in a time-dependent model. In conclusion, outcomes of intensively treated AML patients were comparable irrespective of NGS status, underscoring the representativeness of the REFORM-AML database for the Danish AML population. Age and male sex correlated with adverse-risk mutations, and both ELN2017 and ELN2022 robustly predicted survival.

Purpose: Adults with high- or very high-risk acute leukemia (AL) or myelodysplastic syndrome (MDS) face substantial relapse risk after allogeneic hematopoietic stem-cell transplantation. We evaluated single-unit cord blood (CB) transplantation after ex vivo expansion with UM171 in this population. Patients and Methods: Two prospective, single-arm phase II trials at four centers enrolled 64 adults with high- or very high-risk AL or MDS; 60 received a UM171-expanded CB transplant and comprised the analysis population. CB units were preferentially selected at a 5/8 HLA match to maximize the graft versus leukemia effect. Patients received intermediate- or high-intensity conditioning with tacrolimus/mycophenolate mofetil graft-versus-host-disease (GVHD) prophylaxis. Endpoints included safety, feasibility, non-relapse mortality (NRM), relapse-free survival (RFS), overall survival (OS), GVHD, GVHD-free relapse-free survival (GRFS), chronic GVHD-free relapse free survival (CRFS). Results: Thirty-two percent of patients had undergone previous transplantation, 17% of patients with AL were not in remission and 24% of those with AML/MDS had TP53 mutations. Of 62 patients who remained eligible for transplantation, 60 had a graft successfully manufactured and infused. Median times to neutrophil and platelet engraftment were 17 and 38 days, respectively. NRM was 5.1% at day 100 and 15.2% at 1 year. Two-year cumulative incidence of relapse was 22.3%. Two-year OS and RFS were 63.9% and 60.4%, respectively. Grade III-IV acute GVHD incidence was 20.3% at 1 year and moderate-to-severe chronic GVHD incidence was 6.8% at 2 years. Conclusion: UM171-expanded CB transplantation was feasible and provided prompt engraftment, durable disease control, and infrequent clinically significant chronic GVHD in adults with high- and very high-risk AL/MDS. Comparative studies are warranted to define its role relative to contemporary donor platforms.

The circulating blood proteome provides a systemic readout of disease biology and holds promise for advancing diagnostics and disease monitoring in pediatric leukemia. Here, we profiled 3072 proteins in diagnostic serum from 54 children with acute lymphoblastic leukemia (ALL), 21 with acute myeloid leukemia (AML), and 12 healthy controls using the Olink Proximity Extension Assay. We observed profound alterations in circulating protein levels in leukemia patients compared with controls and identified immunophenotype-specific proteins, including SIGLEC15 in B-cell precursor ALL (BCP-ALL), NOTCH1 in T-ALL, and CEBPA in AML, all which remained high even in patients with low (<20%) or no peripheral blood blasts. Within BCP-ALL, molecular subtypes were reflected in the circulating proteome; for example, DSC2 and PTPRK were elevated in ETV6::RUNX1-positive cases, while IL-6R and ADAM8 were higher in High Hyperdiploid cases. Angiogenic growth factors decreased across all leukemia patients compared with controls, suggesting a fragile peripheral vasculature at diagnosis. Integration with external datasets revealed the likely cellular source of abundant proteins and examination of an external cohort validated our subtype-specific findings. Together, these results define shared and distinct proteomic signatures across pediatric acute leukemias and highlight candidate biomarkers for diagnostics and disease monitoring.

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.