Haematologica

Inflammation is a key driver of hematopoietic dysfunction in myeloid malignancies, but its role in the context of hypomethylating therapy remains incompletely understood. Although 5-Azacytidine is used posttransplant in high-risk myelodysplastic syndrome (MDS), only 50% of patients show a clinical response. We provide evidence that inherent inflammatory properties of healthy donor CD34+ stem cells exist that are likely to contribute to the "response" seen in MDS patients. These are linked to epigenetic priming of the myeloid niche, resulting in S100A8/A9-driven inflammatory program that promotes functionality of immature NK cells. Using in vitro differentiation systems, multi-omic profiling, and a S100A9-/- mouse model, we find that 5-AzaC modulates inflammatory transcriptional programs through epigenetic rewiring of upstream regulatory elements. Loss of S100A9 disrupts myeloid differentiation, impairs NK cell maturation, and alters key developmental regulators including CEBPB, JUN, and NFIL3. In vivo, 5-AzaC restores these defects and primes NK cells in a time- and context-dependent manner. Re-analysis of the published Australian MDS/CMML cohort shows that "responders" display increased S100A8/A9 expression together with enhanced IFN-{gamma}, IL6-JAK-STAT3, and TNF signaling. These findings suggest that inflammatory myeloid programs may serve as predictive biomarkers and therapeutic targets to enhance NK cell-mediated graft-versus-leukemia activity posttransplant. SummaryO_LIWe provide compelling evidence that inherent properties of healthy donor CD34+ hematopoietic stem cells (SCs) exist that are likely to contribute to the "response" seen upon pre-emptive posttransplant 5-AzaC therapy of patients with high-risk myelodysplastic syndrome (MDS). C_LIO_LIThese properties are linked to a distinct form of epigenetic plasticity at upstream-located transcription factor (TF) binding sites. This may indirectly contribute to acute S100A8/A9-driven inflammation, which is demonstrable in distinct monocyte subsets and, importantly, also in NK cells thereby determining the characteristics of inflammatory monocyte-NK cell crosstalk. C_LIO_LIMice with a targeted deletion of S100A9 fail to upregulate CEBPB / JUN and NFIL3 which results in impaired myeloid priming and dysfunctional NK cell maturation, respectively. C_LIO_LIRe-analysis of the Australian MDS/CMML cohort confirms that MDS patients that "respond" to 5-AzaC exhibit activated IFN-{gamma}, IL6-JAK-STAT3, and TNF-signaling pathways in the context of upregulated S100A8/A9 after six months of treatment. C_LIO_LIOur study indicates that screening of healthy donors SCs for specific inflammatory markers in early developing monocytes could be used as a marker to predict which donor will have the potential of generating a S100A8/A9-driven inflammatory response. This may help identify patients with MDS as well as AML who are likely to benefit from low-dose, short-term 5-AzaC therapy as early as day 7 after transplantation, potentially resulting in increased graft-versus-leukemia (GvL) activity. C_LI

Chimeric antigen receptor T-cell (CAR-T) therapy targeting CD19 has transformed outcomes for children with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL), yet the influence of molecular subtype on outcomes remains unclear. We evaluated the impact of cytogenetic and molecular signatures on complete response (CR), overall survival (OS), and leukemia-free survival (LFS) after CD19 CAR-T therapy in eighty-six pediatric patients with R/R B-ALL treated with tisagenlecleucel. CR was assessed 30 days after infusion. Cytogenetic data were available for 84 patients and molecular profiling for 62. Survival analyses included 72 patients who received CD19 CAR-T as the sole cellular therapy. Seventy-seven patients achieved CR (89.5%). Pre-infusion bone marrow blasts of [&ge;]20% were associated with lower CR rates (53.8% vs 95.9%, p<0.0001) and significantly reduced OS and LFS (both p<0.0001). Among molecular markers, RAS mutations correlated with inferior OS (p=0.0222) and LFS (0.0402). In multivariate analysis, bone marrow blasts >20% and RAS mutations independently predicted inferior OS. Post CAR-T, CD19 negative relapses showed almost twice higher prevalence of RAS mutations (66% vs 37.5%). These findings highlight RAS mutations as a key molecular predictor of outcome after CD19 CAR-T therapy and suggest emergence of unique risk stratification for patients receiving CD19-targeting therapy.

Children with acute lymphoblastic leukemia (ALL) are treated with multiagent chemotherapy that causes profound changes to the immune system. There are limited data on how disease and therapy impact antigen-specific immune memory, leading to inconsistent guidelines on best practices for revaccination of this population. Here, to inform vaccine guidance, we investigated whether immunity derived from routine childhood measles and varicella zoster virus (VZV) vaccines is maintained during and after therapy for childhood ALL. We report that antibodies against measles and VZV were significantly reduced in children with ALL (n=45) compared to healthy controls (n=13), particularly in older children in whom a longer time had passed since their most recent vaccine dose. However, the avidity of the measles and VZV-specific antibodies was indistinguishable between groups. Despite changes to the composition of the T cell compartment, both overall and antigen-specific T cell function were preserved in children with ALL. These data provide compelling evidence for revaccination of children following ALL treatment. Intact T cell responses suggest that post-treatment revaccination would be effective.

TP53 mutations represent one of the strongest adverse prognostic factors in myelodysplastic neoplasms (MDS). While multi-hit TP53 (TP53multiHit) alterations uniformly lead to very poor outcomes, the prognostic relevance of monoallelic TP53 (TP53mono) mutations remains controversial. TP53 variants can cause loss-of-function, dominant-negative, or gain-of-function effects. We hypothesized that functional heterogeneity among TP53 variants contributes to the variable clinical behavior observed in monoallelic TP53-mutated MDS. Therefore, we analyzed pretreatment samples from 4,505 patients with MDS from two independent cohorts (IWG, n=3,173; J-MDS, n=1,332), including 271 patients with TP53mono and 499 with TP53multiHit. Functional annotation of TP53 variants was performed using a previously published phenotype score (PS) derived from saturation mutagenesis screens, capturing dominant-negative and loss-of-function effects. Median overall survival (OS) differed significantly by TP53 allelic state (TP53 wild-type (TP53wt) 42.4 months; TP53mono 22.9 months; TP53multiHit 9.2 months; p < 0.001). Within the TP53mono subgroup, functional annotation identified marked heterogeneity. Patients with high PS ([&ge;]7) showed significantly inferior OS compared with those with low PS (median OS: 13.8 vs. 39.2 months; HR 1.68, 95% CI 1.16-2.42; p = 0.006), particularly for IPSS-R and IPSS-M low-risk cases. Combining PS and variant allele frequency (VAF) further improved risk stratification. TP53mono patients with PS [&ge;]7 and VAF [&ge;]22% had outcomes comparable to TP53multiHit (median OS: 8.8, p = 0.2), whereas those with PS <7 and VAF <22% exhibited survival similar to TP53wt (median OS: 49.7, p = 0.9). Overall, functional annotation of TP53 variants refines prognostication in TP53mono-mutated MDS and may enhance individualized risk assessment.

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.

The RUNX1 transcription factor is a critical regulator of hematopoiesis and frequently mutated in myeloid malignancies. In the myeloproliferative neoplasm, chronic myeloid leukemia (CML), secondary somatic RUNX1 mutations and RUNX1::MECOM/EVI1, are associated with tyrosine kinase inhibitor (TKI) resistance and progression to the blast-phase (BP-CML). Research has predominantly focussed on transcriptional dysregulation mediated by RUNX1 mutations in myeloid malignancies, whilst post-transcriptional dysregulation remains comparatively unexplored. To address this, we used orthogonal organic phase separation (OOPS), to characterise the RNA-binding proteome of RUNX1 deficient BP-CML cells. RUNX1 depleted BP-CML cells exhibited significant alterations to RBP abundance involved in stress response pathways and translation/ribosome-biogenesis (RiBi). Furthermore, RUNX1 depletion or expression of RUNX1::EVI1 in BP-CML cells induced expression and RNA binding activity of SPATS2L, a component of stress granules (SG); membraneless cytoplasmic condensates protecting mRNAs from degradation, promoting survival under stress. Whilst RUNX1 depletion increased SG-assembly, SPATS2L depletion reduced SG-assembly in BP-CML cells and inhibited the growth and survival of multiple BP-CML cell lines. The translation inhibitor homoharringtonine (HHT), used historically in TKI-resistant CML, ablated SG-assembly in BP-CML cells with RUNX1 depletion, and, primary BP-CML cells with LOF/hypomorphic RUNX1 mutations (characterised by defective DNA-binding/CBF{beta}-interaction) were preferentially sensitised to HHT. Finally, suppressing SPATS2L expression induced by RUNX1 depletion, increased the HHT-sensitivity of RUNX1 depleted BP-CML cells, suggesting SPATS2L contributes to therapeutic resistance in CML with RUNX1 mutations. This study suggests that SPATS2L and SG induction could be critical to RUNX1-mutant leukemias, and, provides preliminary evidence for a mutationally-targeted approach in CML with RUNX1 aberrations.

The distinct features of neonatal megakaryocytes, high proliferation and inefficient platelet production, have clinical repercussions. A diminished capacity for stress thrombopoiesis, the response to acute drops in platelet counts, contributes to the high prevalence of thrombocytopenia in premature infants and to impaired platelet recovery after umbilical cord blood stem cell transplantation. High proliferation also promotes leukemogenesis in babies with Down Syndrome (DS). The transcriptional coactivator Mkl1/MrtfA participates in programming the ontogenic shift from fetal/neonatal to adult-type megakaryopoiesis; in this activity it is opposed by the DS-associated kinase Dyrk1a. In a screen for downstream ontogenic effectors in human progenitors, we identified the kinesin Kifc3 as a factor selectively decreased in adult megakaryocytes and whose knockdown in neonatal megakaryocytes induced adult-type morphogenesis with augmented platelet release. Kifc3 acts as a minus-end directed motor for centrosomal delivery of various cargos. Centrosomal release of Cep192 has recently been found induce cellular process extensions through actin remodeling, reminiscent of megakaryocyte platelet release. In our studies, Cep192 showed striking upregulation and dispersion in adult vs neonatal megakaryocytes, and Kifc3 knockdown recapitulated this effect in neonatal megakaryocytes. A role for Cep192 in promoting megakaryocyte morphogenesis, distinct from its role in centrosome biogenesis, was demonstrated in vitro and in vivo. In silico screening for Kifc3 inhibitors identified a small molecule that affected neonatal megakaryocytes similarly to Kifc3 knockdown, indicating feasibility for therapeutic argeting of the Kifc3-Cep192 pathway in clinical conditions associated with fetal-type megakaryopoiesis. Key PointsO_LIThe motor protein Kifc3 dictates megakaryocyte ontogeny in association with its control of the centrosomal actin-remodeling factor Cep192. C_LIO_LIKnockdown or small molecule targeting of Kifc3 enhances neonatal megakaryocyte morphogenesis and thrombopoiesis. C_LI

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.

Most patients with acute myeloid leukemia (AML) initially respond to standard chemotherapy. However, relapse and refractory disease remain common. The responses to targeted therapies are often transient and the efficacy of immunotherapy is limited. Although single-cell RNA sequencing (scRNA-seq) studies have provided insights into the cellular diversity and immune landscape of AML, many have primarily focused on limited, or newly diagnosed patient cohorts, leaving cellular dynamics across advanced disease incompletely defined. Here, we profiled 72 samples from AML patients across different disease stages using scRNA-seq and compared these against healthy donor samples. We observed selective enrichment of immature progenitor populations, along with widespread upregulation of oxidative phosphorylation in AML. The immune microenvironment of AML was characterized by CD8+ effector memory T cell expansion with reduced IL2-STAT5 and increased mTORC1 pathways and exhaustion markers, suggesting a functional imbalance. Several AML-specific genes were identified providing potential therapeutic opportunities. Cell communication analysis revealed reduced HLA interactions in relapsed/refractory samples compared to diagnosis samples, suggesting impaired antigen presentation and defective T cell priming. Together, these results improve the understanding of cellular and immune changes in AML during disease progression and provide a basis for new therapeutic strategies.

There are few therapeutic options to treat patients with sickle cell disease (SCD), a blood disorder caused by mutations in the {beta}-globin gene that affects >7M individuals worldwide. Combining human genetics and high-throughput proteomics can help identify new drug targets. Here, we present results from a proteogenomic analysis of the plasma proteome in SCD patients. We measured the levels of 5,411 plasma proteins and tested their associations with common genetic variation in 343 SCD patients. After conditional analyses, we identified 560 protein quantitative trait loci (pQTL), including 58 (10%) that are novel. Many of these pQTL are not specific to SCD patients and associate with clinically relevant traits in non-SCD African Americans from the Million Veteran Program (e.g. hemoglobin concentration, triglycerides). The effect sizes of the pQTL is largely concordant between SCD and non-SCD individuals, although we found examples (e.g. APOL1, haptoglobin) with evidence of heterogeneity that suggests an interaction between the plasma proteome and the SCD genotype. Finally, we combine pQTL and genome-wide association study results for fetal hemoglobin (HbF) in a Mendelian randomization analysis to prioritize five proteins that may increase HbF production (ENPP5, LBP, NAAA, PT3X, ZP3).

BackgroundCompetitive transplantation is essential for defining intrinsic repopulating capacity of murine hematopoietic stem and progenitor cells (HSPCs), yet comparable assays for human cells have been limited by the lack of a robust in vivo platform. MethodsHere, we describe a novel competitive transplantation method in humanized NOD.Cg-KitW-41J Tyr + Prkdcscid Il2rgtm1Wjl/ThomJ (NBSGW) mice that enables simultaneous engraftment and longitudinal tracking of distinct human grafts within a shared microenvironment. ResultsUsing human leukocyte antigen-mismatched donor CD34+ cells, this method facilitates standard flow cytometry panels to track multiple donor cell chimerism, lineage output, and HSPC composition. The experimental framework may be adapted to different mouse models, conditioning strategies, donor sources, and treatments. ConclusionsOverall, this humanized competitive repopulation assay fills a critical translational gap and offers a flexible foundation for advancing mechanistic discovery in human hematopoietic biology and improving clinical strategies for stem cell transplantation.

Carbohydrates are classically catabolized by fermentation or oxidation, a choice that impacts many cellular functions including proliferation. Proliferating cells including somatic stem and progenitor cells are thought to favor fermentation over oxidation, and most proliferating cells in vitro depend on lactate production. However, it has not been tested if fermentation and oxidation are the universal obligatory terminal fates for carbohydrates in vivo because the key enzymes, lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH), have not been simultaneously deleted in any cell type. Here we show that both fermentation and oxidation are dispensable for the survival and function of hematopoietic stem cells (HSC). Combined LDHA and LDHB deletion to ablate LDH did not impair HSC function, suggesting that HSCs and rapidly proliferating hematopoietic progenitors surprisingly do not require fermentation. Combined LDHA, LDHB, and PDH deletion abolished both glucose oxidation and fermentation, but did not impair HSC function. Glycolysis was preserved, suggesting the operation of an alternative endpoint. LDH/PDH-deficient HSCs terminated glycolysis through pyruvate export. Pyruvate export by HSCs and progenitors was a physiological response to changing nutrient levels. Quadruple deletion of LDHA/B, PDH, and the pyruvate transporter MCT1 impaired HSC function. This suggested that an essential role of glycolysis termination is not to produce acetyl-CoA or lactate but to remove pyruvate. Therefore, in contrast to classical theories and to in vitro metabolism, carbohydrate metabolism in vivo does not require oxidation or fermentation but can terminate directly in pyruvate export, and this alternative pathway is sufficient to support stem cell function.

Clonal hematopoiesis of indeterminate potential (CHIP) driven by somatic mutations in TET2 and DNMT3A is present in >10% of adults over 60 and confers substantial risk for hematologic malignancy and cardiovascular disease, yet the majority of patients with CHIP do not progress to disease. Analyzing 1,020,538 individuals across three biobanks (UK Biobank, All of Us, BioVU), we show that a discrete subset of enzymatically disruptive mutations, TET2 loss-of-function variants and the DNMT3A R882 hotspot, account for the majority of clinical risk in these genes and exhibit the strongest clonal fitness advantage. Because DNMT3A and TET2 encode enzymes that modulate DNA methylation, we reasoned that peripheral blood methylation patterns should reflect the functional impact of individual mutations, enabling a direct readout of enzymatic dysfunction in CHIP patients. We developed and validated methylation-based activity scores for TET2 and DNMT3A as patient specific biomarkers that quantify enzymatic activity. These scores capture functional heterogeneity across mutation subtypes, predict disease risk comparably to clinical risk scores such as the Clonal Hematopoiesis Risk Score and the AHA PREVENT cardiovascular risk model. Integrating the activity score with the clinical models substantially improves prediction of incident cytopenia, myeloid neoplasm, and major adverse cardiovascular events. These findings establish that TET2 and DNMT3A CHIP pathogenicity is proportional to the degree of enzymatic disruption conferred by specific variants, and nominate methylation-based activity scores as a functional biomarker for individualized CHIP risk stratification and monitoring therapeutic response.

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 intestinal microbiome influences immune recovery and long-term outcomes after allogeneic hematopoietic stem cell transplantation (allo-SCT). While reduced bacterial diversity and depletion of immunomodulatory microbial metabolites during peri-engraftment have been linked to acute graft-versus-host disease (aGvHD) and mortality, it remains unclear whether microbiome recovery after engraftment and immune reconstitution is better reflected by bacterial diversity or by microbial metabolic output. We aimed to define microbiome recovery in the late post-transplant period and test whether a metabolite-based biomarker improves the prediction of clinical outcomes, including overall survival (OS) and chronic (c) GvHD. In this two-center longitudinal observational study, serial stool samples were collected from pre-transplant baseline to day +100 after allo-SCT in a discovery cohort (n = 20, Technical University Munich University Hospital (TUM)) and an independent validation cohort (n = 100, University Hospital Regensburg (UKR)). Gut microbiome composition was assessed by 16S rRNA gene amplicon sequencing, with metagenomic profiling in selected patients, and stool metabolites were quantified using targeted mass spectrometry. Patients were classified as RECOVERY or NO RECOVERY based on changes in bacterial richness between baseline and the post-transplant period. To capture microbial metabolic output, the previously established Immune-Modulatory Metabolite Risk Index (IMM-RI), comprising butyric, propionic, and isovaleric acids, desaminotyrosine and indole-3-carboxaldehyde, was adapted to the late post-transplant period (IMM-RI post-TX). Bacterial alpha diversity frequently improved by day +100; however, this did not consistently indicate restoration of baseline community structure and was not paralleled by recovery of stool metabolite profiles. Accordingly, RECOVERY status showed a limited association with survival or transplant-related mortality (TRM). In contrast, IMM-RI post-TX low-risk identified patients with preserved butyrate-associated biosynthetic capacity and was significantly associated with improved OS in both cohorts (UKR: HR 0.2052, 95% CI 0.07703 - 0.5466, p < 0.0001). In the validation cohort, IMM-RI post-TX low-risk was significantly associated with reduced relapse-related mortality. Interestingly, stool butyric-, propionic and valeric acid concentrations were increased in cGvHD of the skin, indicating context-dependent metabolite effects. These findings suggest that metabolite profiling outperforms bacterial diversity for predicting outcomes after allo-SCT and support microbial metabolites as promising biomarkers for risk stratification and actionable candidates for precision microbiome interventions after allo-SCT.

Terminal erythroid differentiation involves dramatic cellular remodeling that culminates in the expulsion of the nucleus, a process known as enucleation. While enucleation is conserved across mammals and is crucial for the generation of fully functional erythrocytes, the mechanisms governing this process have remained largely unknown, in part because the absence of genetic material in mature, enucleated red blood cells hinders genetic experimentation. Here, we performed a pooled, forward-genetic CRISPR-Cas9 screen in enucleated red blood cells derived from primary human hematopoietic stem cells to identify genes required for enucleation. We found that Chloride Intracellular Channel 3 (CLIC3) and Vesicle-associated membrane protein 8 (VAMP8) are both necessary for terminal erythroid differentiation, yet likely act through different mechanisms. Knockdown of CLIC3 led to a delay in erythroblast differentiation, culminating in impaired enucleation. We found that the knockdown cells had increased p53 and p21 and exhibited cell cycle alterations, suggesting CLIC3 plays a crucial role in coordinating cell cycle progression during erythropoiesis. In comparison, VAMP8-depleted cells initially appear to undergo accelerated differentiation but then display a specific defect in enucleation. Transcriptional analysis of the VAMP8-knockdown cells suggested dysregulation of pathways for vesicle trafficking and actin binding, and imaging of late-stage erythroblasts revealed impaired nuclear polarization and disorganized actin. This work provides a new approach for functional genomics in enucleated cells and reveals novel factors important for terminal erythroid differentiation and enucleation. Key pointsO_LIA CROPseq-based CRISPR-Cas9 screen enables functional genomics in enucleated primary human red blood cells. C_LIO_LIChloride Intracellular Channel 3 (CLIC3) and Vesicle Associated Membrane Protein 8 (VAMP8) were identified as critical for terminal erythroid differentiation and enucleation, likely acting through two distinct mechanisms. C_LI

Myeloproliferative neoplasms (MPNs) are characterized by progressive myelofibrosis that drives morbidity and mortality. Liquid biopsy approaches to noninvasively monitor fibrotic progression remain limited. We performed comparative transcriptomic profiling of CD45-depleted platelet-enriched and CD45+ leukocyte-enriched fractions from matched peripheral blood samples of 76 individuals (27 primary myelofibrosis, 17 polycythemia vera, 14 essential thrombocythemia, 18 healthy controls). Platelet RNA sequencing was performed in 2018-2020 on Illumina HiSeq 4000, while WBC RNA sequencing was conducted in 2023 on Illumina NovaSeq 6000 from cryopreserved CD45+ enriched fractions of specimens obtained at the identical time and from the same blood sample as the platelet RNA. Despite comparable library preparation protocols and higher sequencing depth in WBC samples, platelet transcriptomes exhibited 5.1-fold more differential expression in myelofibrosis (3,453 versus 681 genes, adjusted p<0.05, |log2FC|>1). Platelet signatures were enriched for proteostasis pathways including endoplasmic reticulum stress and unfolded protein response, reflecting megakaryocyte dysfunction in the fibrotic bone marrow niche. WBC signatures predominantly featured immune activation and proliferative pathways, indicating systemic inflammatory responses. Multinomial LASSO classification demonstrated superior performance of platelet-based models for myelofibrosis diagnosis (AUROC 0.85) compared to WBC-based (AUROC 0.77) or clinical models (AUROC 0.59). Combined platelet+WBC models did not improve performance (AUROC 0.80), indicating complementary but non-additive information. These findings establish platelet transcriptomic profiling as a superior noninvasive biomarker platform for monitoring myelofibrosis in MPNs, capturing megakaryocyte-driven fibrogenesis with greater sensitivity than peripheral leukocyte-based approaches. HighlightsUsing matched WBC and platelet RNA-seq from MPN patients, we identify myelofibrosis-associated transcriptomic signatures specifically enriched in platelets. Multinomial LASSO modeling highlights platelet-derived gene expression as a dominant and predictive biomarker of myelofibrosis, outperforming clinical parameters and WBC signatures. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/714941v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1d695aborg.highwire.dtl.DTLVardef@fc250forg.highwire.dtl.DTLVardef@1e52e8eorg.highwire.dtl.DTLVardef@15378e3_HPS_FORMAT_FIGEXP M_FIG C_FIG

Diffuse large B-cell lymphoma (DLBCL), the most common aggressive lymphoma, encompasses histologically similar but genetically distinct cancers. Recent genetic studies have defined at least six molecular subtypes, yet none account for Epstein-Barr virus (EBV), despite 5-15% of DLBCLs being EBV-associated. By reanalyzing published whole-exome and RNA-sequencing data from 481 tumors, we identified 19 EBV-positive cases. These were significantly enriched in the BN2 subtype (6/19), while most (11/19) remained unclassified. In BN2 tumors, several subtype-defining mutations were reduced in frequency among EBV-positive cases, supporting the hypothesis that EBV oncogenes substitute for specific cellular alterations and may confound DLBCL classification algorithms. Extending our analysis to cell lines, we found that the widely used Val cell line harbors the B95-8 laboratory EBV strain; other EBV-positive lines appeared authentic but modeled only non-BN2 subtypes and expressed an atypical viral latency III program, whereas some DLBCL tumors expressed the atypical latency III program and others latency I or II. Together, these findings demonstrate that EBV-positive DLBCL, like DLBCL itself, is not a single disease, and that current in vitro models only partially capture its biological heterogeneity. Key pointsO_LIEBV-positive DLBCL is not a single disease and EBV status can impact genetic-based classifications. C_LIO_LICurrent EBV-positive DLBCL cell lines do not adequately capture tumor complexity; we determined that Val is a problematic cell line. C_LI

Red blood cell (RBC) alloimmunization is a clinically significant complication in transfused patients whose immunological determinants remain incompletely understood. Type I interferon (IFN-I) signaling drives RBC alloimmunization in murine models, and systemic lupus erythematosus (SLE) is characterized by constitutive IFN-I hyperactivation alongside elevated alloimmunization rates. We analyzed three publicly available SLE RNA-seq cohorts (GSE72509, GSE112087, GSE122459; whole blood and PBMC; total n = 150 SLE) in a pre-specified discovery-replication-validation design. A 14-gene IFN-I signature score was computed per sample; differential expression, gene set enrichment analysis, and Spearman correlation were performed independently per cohort. IFN-I scores were significantly elevated in SLE versus healthy controls in all three cohorts (p < 0.01 each). IFN-high SLE patients showed 665 differentially expressed genes, with enrichment of alloimmunization-associated and plasmablast differentiation gene sets confirmed by GSEA. The alloimmunization signature score correlated significantly with IFN-I score across all three independent cohorts ({rho} = +0.77, +0.51, +0.60; all FDR q < 0.05); Tfh differentiation showed no association in any cohort. To our knowledge, this represents the first human transcriptomic evidence that IFN-I pathway activity in SLE is coupled to alloimmunization-associated immune programs in vivo. These findings identify IFN-I score as a candidate biomarker of alloimmunization susceptibility in SLE and provide translational rationale for prospective studies incorporating transfusion outcome data.

Clonal hematopoiesis of indeterminate potential (CHIP) is an age-associated condition linked to chronic inflammation and an increased risk of cardiovascular diseases and hematological malignancies. People with HIV (PWH) exhibit a higher prevalence of CHIP than the general population, but the mechanisms underlying this association remain unclear. In particular, it is unknown whether the excess burden of CHIP reflects earlier emergence of mutant clones, altered clonal expansion dynamics, or differences in selective pressures acting on hematopoietic stem cells. We reconstructed longitudinal trajectories of CHIP variant allele frequency (VAF) in 52 PWH using serial peripheral blood samples spanning up to 25 years from the Swiss HIV Cohort Study. We used spline-based modelling to estimate clone size and growth dynamics, and dynamic time warping to identify common trajectory patterns. Associations between clonal dynamics and longitudinal immune parameters were assessed using linear mixed-effects models. Trajectories in PWH were compared with publicly available longitudinal CHIP data from the SardiNIA population cohort. We identified heterogeneous clonal dynamics consistent with known gene-specific fitness patterns. Larger clone size was associated with lower CD4 T-cell count and lower CD4/CD8 ratio. Compared with the general population cohort, PWH showed higher VAF across the observed age range and steeper early trajectory increases, while long-term expansion rates were broadly similar. Greater variability in clonal dynamics among PWH suggests a stronger contribution of host environmental factors to clonal fitness. These findings support a model in which HIV-associated immune dysregulation alters the hematopoietic fitness landscape, contributing to earlier detectable clonal expansion and increased burden of CHIP in PWH.