Blood Advances

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

Abstract / SummarySurvival outcomes for pediatric Burkitt lymphoma (BL) substantially vary depending on geography (50-90%), which also serves as a proxy for the prevalence of Epstein-Barr virus (EBV) within the tumors. Although BL is considered an immunologically "cold" tumor with few tumor-infiltrating lymphocytes (TILs), their functional status has not been fully evaluated, especially for EBV-positive disease. Here, we characterize the exhaustion and activation profiles of T cells in the tumor microenvironment (TME) of EBV-positive BL using orthogonal methods, single-cell gene expression analysis, spectral flow cytometry, and immuno-histochemistry staining (IHC). We found that CD8+ TILs displayed a mosaic of immune inhibitory gene expression encoding, PD1, TIGIT, LAG3 and HAVCR2/TIM3. IHC validated the expression of PD1 and TIGIT on CD8+ TILs, as well as their respective ligands, PDL-1, PVR, and Nectin-2 on malignant B cells. Despite exhaustion-associated signatures, CD8+ TILs retain cytotoxic potential, expressing granules (i.e. Granzyme A, Perforin) and cytokines (i.e. IFN{gamma}) and demonstrate an increased uptake of metabolites such as glucose, arginine, and methionine. In peripheral blood, pediatric BL patients exhibited a significantly higher abundance of PD1+TIGIT+ CD8+ T cells compared to healthy children. Notably, these circulating T cells from BL patients express significantly lower levels of TOX, suggesting they are not irreversibly dysfunctional. Together, our results indicate that CD8+ T cells both in the TME and in circulation of children with BL are not terminally exhausted but remain poised for functional re-invigoration. These findings support the potential integration of immune checkpoint inhibitors into combination chemotherapeutic regimens to improve outcomes for these children. SignificanceEBV-positive BL tumors contain functional, metabolically active CD8+ T cells. Circulating PD1+TIGIT+CD8+ T cells found in BL patients blood are a biomarker for those in the tumor microenvironment.

Hematopoietic progenitors downstream of hematopoietic stem cells (HSC) are now recognized as the main drivers of day-to-day hematopoiesis. While embryonic and adult HSC fates have been studied in detail, less information exists on stages downstream from HSC, notably in the multipotent progenitor compartment. The early postnatal period represents an important growth phase of the animal and its immune system. Developing immune lineages must be generated in large numbers rapidly, and populate expanding organ niches. To shed light on this critical period, we focused our experiments on early postnatal Flt3+ hematopoietic progenitors, and combined genetic single progenitor barcoding using Polylox with Flt3-driven, inducible fate mapping. Key immune cell types, including T and B lymphocytes (lymphocytes), innate lymphocytes (ILC) 1-3, NK cells, and granulocytes and monocytes (myeloid) emerged from Flt3+ hematopoietic progenitors. Barcode analysis revealed that about 75% of Flt3+ hematopoietic progenitors had unipotent fates for lymphocytes, or ILC or myeloid cells, while the remaining fraction showed unprecedented fate combinations for these lineages. Focusing on ILC only, we uncovered clonal fate restriction towards ILC1, or ILC2, or ILC3 in tissues. These data indicate early tissue seeding by progenitors, and further differentiation towards discrete subsets in situ. In addition to these fate analyses, induction of fluorescent marker at this intermediate stage of hematopoiesis showed that Flt3+ progenitors generated a wave of progeny lasting for over one year. The washout of these cells over time provided kinetic data of cell turnover in major immune cell compartments (in the circulation and in tissues) in vivo. In conclusion, we tracked the fate of large numbers (in the order of hundreds) of Flt3+ progenitor clones in situ. These intermediate progenitors downstream of HSC displayed mostly lineage-restricted fates as well as strong fate complexity, thus serving as a source for early tissue seeding and durable immune lineage.

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

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 (CAR) T-cell therapy has transformed treatment for relapsed /refractory(r/r) lymphoid malignancies. Yet, these cellular immunotherapies are often associated with immune-related adverse events (irAEs), namely cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), that pose significant risks to patient safety and limit broader clinical implementation of CAR T-cell therapies. In the current study, we used proteomics technology to establish circulating protein signatures that would predict severe CRS and ICANS in r/r lymphoma patients that subsequently received CAR T-cell therapy. Initial discovery was performed using plasma samples collected preceding CAR T-cell infusion from 39 r/r lymphoma patients at MD Anderson Cancer Center. A 5-marker and 8-marker protein panel was developed for predicting Grade [≥] 2 CRS and ICANS respectively, yielding respective AUCs of 0.85 [95% CI: 0.72-0.98] and 0.91 [95% CI: 0.81-1.00]. Independent testing of the CRS and ICANS panel was performed in a cohort of 59 r/r lymphoma patients from the Moffitt Cancer Center, with resultant AUCs of 0.76 [95% CI: 0.63-0.89] and 0.67 [95% CI: 0.51-0.84] for the CRS and ICANS panel, respectively. Patients were further classified into low-, intermediate-, and high-risk groups based on panel score tertiles. In the combined dataset (MDACC + Moffitt), compared to patients in the low-risk group (reference), patients in the intermediate- and high-risk groups were 3.15 [95% CI: 0.92-12.71] and 13.84 [95% CI: 4.21-56.26] more likely to have Grade [≥] 2 CRS, and 1.21 [95% CI: 0.36-4.23] and 8.59 [95% CI: 2.87-29.09] more likely to have Grade [≥]2 ICANS. The protein biomarker panels provide a means to risk stratify patients who are at high risk for developing severe CRS and ICANS, to inform on the need for prophylactic interventions and improve patient outcomes.

PurposeClinical translation of CAR T cell therapies has accelerated, yet preclinical evidence still often originates from single-center studies lacking sufficient robustness. Preclinical confirmatory multicenter studies have been proposed to improve the translational success, but their feasibility in cellular therapies remains unexplored. MethodsWe performed a confirmatory multicenter study validating C-C-motive-receptor-8 (CCR8) overexpression in CAR T cells--a strategy previously shown to enhance solid tumor infiltration. In vitro experiments covering activation, cytotoxicity, and migration using three CAR constructs were conducted across two centers with harmonized materials, preregistered protocols, randomization, and blinding. ResultsThe data from the two centers confirmed key findings of the exploratory study: CCR8 overexpression in anti-EpCAM and anti-mesothelin CAR T cells leads to enhanced selective migration towards a CCL1-gradient, while not compromising antigen-specific T cell activatory capacity and cytotoxicity in vitro. The study furthermore broadened the applicability of CCR8 overexpression to anti-CEA CAR T cells. ConclusionsThis first-of-its-kind preclinical confirmatory CAR T study demonstrates the feasibility of a multicenter confirmation in cellular therapy, with technical and logistical challenges resolved through transparent communication between all parties involved. Both exploratory and confirmatory studies aim to downselect CAR candidates with the highest clinical success potential, as they compete for limited resources in preclinical research. It is therefore mandatory to clarify the extent of replications required to validate the experimental methodology and identify CAR candidates with most likelihood of success. TRANSLATIONAL RELEVANCEPreclinical evidence for novel CAR T cell therapeutic strategies relies mostly on exploratory single-center studies lacking robustness, with recent findings substantiating their limited predictive value for cellular therapies tested outside hematology. Here, the function of CCR8-armored CARs in vitro was confirmed in a preclinical confirmatory multicenter study, demonstrating the feasibility of such studies in adding value to the transition of preclinical concepts to clinical development. Our first-of-its-kind study may contribute to define new routes for preclinical testing and further raises the general question of what level of preclinical evidence is reasonably achievable in an academic context. It indicates the need for strong collaborative efforts to realize dedicated preclinical infrastructure for clinical translation of reprogrammed immune cellular therapeutics.

Parvovirus B19 (B19V), a member of the genus Erythroparvovirus within the Parvoviridae family, infects human erythroid progenitor cells (EPCs) of bone marrow and fetal liver, and causes various hematological disorders. The minor capsid protein VP1 of B19V contains a unique N-terminal region (VP1u) that facilitates virus binding and internalization into EPCs via its receptor-binding domain (RBD). We previously identified tyrosine protein kinase receptor UFO (AXL) as a proteinaceous receptor for B19V infection of EPCs. In this study, we employed an ascorbate peroxidase 2 (APEX2)-based proximity labeling method to identify host proteins that are associated with B19V VP1u during entry. This analysis revealed human transferrin receptor 1 (hTfR) as a key host protein associated with VP1u. hTfR knockdown in UT7/Epo-S1 cells, a B19V-permissive human megakaryoblastoid leukemia cell line, showed significantly reduced B19V internalization and replication. Biolayer interferometry (BLI) assays confirmed a direct interaction between B19V VP1u and hTfR extracellular domain (ECD). Inhibition of VP1u interaction with hTfR ECD, either by a monoclonal antibody targeting the apical domain of the ECD or human ferritin, a natural ligand of hTfR that binds the apical domain, significantly reduced VP1u binding to hTfR, as well as B19V internalization and B19V replication in ex vivo-expanded EPCs. Furthermore, mutant RBD proteins that bear amino acid substitutions in the three helical domains nearly abolished RBD binding to hTfR and significantly reduced the ability to inhibit B19V infection of EPCs. Collectively, our findings establish hTfR as a B19V entry co-receptor that mediates B19V internalization into its natural host EPCs. SignificanceB19V causes severe hematological disorders, including transient aplastic crisis, chronic pure red cell aplasia, and hydrops fetalis, by selectively infecting erythroid progenitor cells (EPCs). Despite its clinical impact, no approved antivirals or vaccines exist, largely due to limited understanding of viral entry mechanisms. A unique feature of B19V is the externalization of the VP1 unique region (VP1u) from the viral capsid, which mediates receptor engagement. Our prior studies identified AXL as an attachment receptor for B19V. Here, we identify that human transferrin receptor 1 (hTfR) acts as a critical co-receptor that directly binds VP1u and promotes viral internalization. Inhibition of the VP1u-hTfR interaction by competitive binding of hTfR with either an anti-hTfR monoclonal antibody or human ferritin significantly reduces B19V internalization and replication in ex vivo-expanded EPCs, highlighting a link between VP1u binding to the apical domain of hTfR and viral internalization. RBD mutants that disrupt its interaction with hTfR barely inhibited B19V infection in EPCs. These findings support a receptor-switch model in which AXL mediates attachment and hTfR drives internalization. Defining these mechanisms provides a foundation for developing antiviral strategies targeting B19V entry into EPCs.

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

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

Waldenstrom macroglobulinemia (WM) is a rare indolent neoplasm characterized by presence of more than 10% lymphoid cells in BM that exhibit plasmacytoid or plasma cell differentiation that secretes an IgM monoclonal protein. This is a retrospective analysis of 89 patients of WM that describes the clinical and laboratory characteristics, treatment patterns and outcome of patients of WM. The median age of the entire cophort was 66 years with male predominance (67.4%). Most common presentations were symptoms pertaining to anemia (77.5%) and constitutional symptoms (33.7%). Median bone marrow lymphoplasmacytic cells were 41%. Positivity for MYD88 and CXCR4 mutations were seen in 81.8% and 2.4% cases. BR was the most common regimen used (52.8%). Overall response rates were seen at 87.8%. Median overall survival, progression free survival and time to next treatment is 8.49 years, 2.15 years and 3.88 years. BR regimen was associated with highest event free survival.

Background: Large language models (LLMs) perform well on standardized medical exam questions, but their reliability for complex hematology decision making is uncertain. We compared four general-purpose LLMs (GPT-4o, GPT-o3, Claude Sonnet 4, and DeepSeek-V3) with a Virtual MDS Panel (VMP), a coordinated multi-agent AI system in which domain-specialized, rule-bound software agents (WHO/ICC guidelines; IPSS-R/IPSS-M; NCCN) collaborate to generate tumor-board-level recommendations. Methods: Each model generated diagnostic, prognostic, and treatment recommendations for 30 myelodysplastic syndrome cases. Nine international MDS experts from five institutions, blinded to model identity, completed 3,000 structured ratings using 5-point Likert scales for diagnosis, prognosis, and therapy and classified errors by severity. Results: General-purpose LLMs achieved modest expert ratings (overall mean scores: 3.7 for GPT-o3, 3.2 for GPT-4o, 3.1 for DeepSeek, and 3.0 for Claude) and contained major factual errors in at least 24% of responses. The VMP increased the proportion of outputs rated 4 or higher to 87% (vs. 34-66% for general-purpose models), improved mean scores to 4.3 overall (4.3 for diagnosis, 4.4 for prognosis, and 4.1 for therapy), and reduced major errors to 8%. Conclusions: In this blinded evaluation of 30 complex MDS cases, general-purpose LLMs produced clinically important errors at rates that raise safety concerns for autonomous hematology decision making. The VMP, a rule-bound, multi-agent architecture, approached expert-level accuracy supporting its potential role as an effective decision-support tool for MDS in the future.

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.

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.

Ex vivo expansion of mobilized peripheral blood (mPB) hematopoietic stem cells (HSCs) represents a promising approach to advance cell and gene therapy strategies yet is hampered by loss of stem cell function when applying commonly used culture protocols. We performed in-depth characterization of mPB expansion cultures by single cell RNA sequencing, which highlighted differentiation trajectories with preservation of lineage fidelity in committed progenitors. Defining a putative HSC cluster allowed an estimation of transduction efficiency in ex vivo cultures, which correlated with long-term gene marking in xenografts and patients enrolled in a gene therapy study. We then developed a clinically translatable, GMP-compliant process to expand lentivirus (LV)-transduced HSCs from mPB of pediatric patients and adult donors, by biologically informed protocol improvements of cytokine supplementation, media choice, timing of LV transduction and combinations of small molecules preventing the activation of differentiation programs. Our optimized process outperforms validated state-of-the-art cord blood expansion protocols when applied to mPB. LV integration site analysis and genomic barcode-based clonal tracking provided definitive proof for symmetric HSC self-renewal divisions occurring during ex vivo culture. These results warrant clinical testing of this HSC transduction/expansion process in an upcoming clinical gene therapy trial for autosomal recessive osteopetrosis (EU CT 2024-518972-30). One Sentence SummaryA mobilized peripheral blood HSC expansion protocol optimized for gene therapy allows robust polyclonal long-term engraftment of LV-transduced cells.

Background In clinical trials for alopecia areata (AA) the treatment effect (percentage of hair loss) is estimated using the Severity of Alopecia Tool (SALT) score. Trials in patients with severe AA (>=50% hair loss) employed a local rating of the SALT score performed at trial sites by different investigators. However, in mild-to-moderate AA (<= 50% hair loss) where SALT scores are lower, potential inter rater variability and margin of error may compromise the results. Objectives To compare Centralised and Local measurement of hair loss in mild moderate AA. Methods In a Phase 2 clinical trial a centralised measurement of hair loss was performed from photographic images taken using a standardised protocol and professional camera equipment. Local scoring was also undertaken at screening/baseline for eligibility. We assessed: the repeatability of the central system (screening vs baseline values), the reproducibility of the central versus the local rating system and the potential impact of each method on the endpoints using a Monte-Carlo simulation method. Results There was good agreement and consistency of scoring with Central rating. This provided much smaller margins of error, 50% lower than Local rating. The simulations demonstrated that substituting Local rating for Central rating would result in a reduction of the likelihood of a statistically significant outcome by at least 50% depending on the SALT score defined clinical response endpoint. Conclusions Central rating is most appropriate in the Phase 2 learning stage of clinical development and provides an accurate representation of the quantity of hair loss, minimising error and ensuring consistency in measurements.

Background There are no licensed treatments for patients with mild to moderate patchy alopecia areata (AA). Objectives To evaluate the efficacy, safety and dose response of STS01, a novel nanoparticle controlled release, topical formulation of dithranol/Prosilic. Methods In a phase 2, double blind study, adult patients with mild to moderate AA (guideline 10% to 50% of scalp hair loss) were randomly assigned to STS01 at doses of 0.25%, 0.5%, 1%, 2% or placebo, daily for 6 months. The primary endpoints included the proportion of patients achieving a >=30% improvement in Severity of Alopecia Tool (SALT) score, and percentage change from baseline in SALT score. This minimum level of improvement is generally accepted as an indicator of the population likely to progress to complete regrowth Results A total of 155 patients were randomized and treated (placebo, n=32; STS01 groups, n=30 to 31). STS01 1% met the primary efficacy endpoint of >=30% SALT score improvement compared to placebo: 75.9% (95% CI, 60.3 to 91.4%) vs 36.7% (95% CI, 19.4 to 53.9%) at 6 months; p=0.0037. The least squares (LS) mean percentage change in SALT score from baseline to end of treatment showed a clear dose response relationship; STS01 0.5% was the minimally effective dose and 2% the maximum tolerated dose, and there was a statistically significant improvement in the STS01 1% group (minus 55.0% vs +0.6% with placebo; p<0.01). Significant improvements (p<0.05) in LS mean percentage changes from baseline in SALT scores were demonstrated in the STS01 1% group at 2 months (-28.6% vs 12.8%), 4 months (-57.2% vs 1.5%), and 6 months (minus 67.0% vs 0.6%). Clinical Global Impression improvement was reported in 72.0% of patients with STS01 1% vs 41.7% with placebo (p<0.05). The most commonly reported treatment emergent adverse events were skin irritation reactions, but were mostly mild (STS01: 56.7% to 71.0%; placebo: 21.9%) or moderate (STS01:13.3% to 35.5%; placebo: 0%) and manageable by reduced frequency of application. There were 15 skin-related discontinuations with STS01 (12.2%) and 2 (6.3%) with placebo. Conclusions STS01 demonstrated a clear dose response, with STS01 1% dose optimally more effective than placebo for hair regrowth with minimal tolerance concerns in mild to moderate patchy AA. Skin irritation reactions were generally manageable and there were no new safety signals. Further characterisation of the STS01 1% dose is planned in a phase 3 study. Chief Investigator AGM reports fees from Soterios Ltd. Chief Statistician DMF is an employee of Soterios Ltd. All other authors were Principal Investigators in the trial and their clinics were reimbursed for the work involved. Most also had sponsorship in the form of consultancies, investigational roles or lecturing roles on behalf of other Dermatological pharmaceutical companies

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.

Prognostic stratification in multiple myeloma (MM) relies on staging systems that assign patients to fixed categories at diagnosis and discard the temporal information that accumulates during treatment. We developed a dynamic multimodal framework that predicts residual overall survival using observation windows ranging from 1 to 18 months post-diagnosis. The model integrates DeepInsight-transformed gene expression representation, longitudinal laboratory measurement trajectories across 10 analytes, and treatment history for three drug classes through an adaptive fusion mechanism that accounts for missing clinical observations. On the MMRF CoMMpass cohort (n = 752), five-fold cross-validation yielded a concordance index (C-index) of 0.773 {+/-} 0.024 and a time-dependent AUC at a 1-year prediction horizon (tdAUC1yr) of 0.789 {+/-} 0.021, outperforming all evaluated baseline methods including DeepSurv (0.633 {+/-} 0.095) and random survival forests (0.636 {+/-} 0.024) on matched cross-validation splits. Modality ablation identified longitudinal laboratory measurements as the strongest individual contributor (C-index 0.693); the DeepInsight spatial encoding of gene expression yielded higher discrimination than a multilayer perceptron (MLP) baseline operating on the same features (0.624 vs. 0.596). Kaplan-Meier analysis showed significant prognostic group separation at all primary landmarks (log-rank p < 0.001; hazard ratios 3.46-3.93). A distilled student model retaining only the DeepInsight representation and five baseline clinical features achieved C-index 0.672 and tdAUC1yr 0.740 on an independent microarray cohort (GSE24080, n = 507) without retraining. Interpretability analysis identified prognostic associations consistent with established myeloma biology, including ubiquitin-proteasome pathway genes, endoplasmic reticulum stress markers, and Interferon Alpha Response pathway enrichment.

T cell lymphomas (TCL) are a heterogeneous collection of malignancies whose origins and pathogenesis are poorly understood and for which few efficacious therapeutic options exist. Here, we conduct single-cell transcriptomic profiling spanning eight TCL entities and describe entity-associated programmes. We predict the cell of origin for these tumours through an integrative analysis of transcriptome and T cell receptor (TCR) maturation states. By identifying tumours with TCR states ranging from the pre-TCR through non-productive and productive TCR alpha and beta chain rearrangements we shed new light on their developmental origins. Furthermore, we apply our drug2cell computational drug target predictions with drug screens using patient-derived cell models, systematically benchmarking the performance of drug2cell and validating compounds and targets. This process identifies SYK inhibitors as a therapeutic opportunity and prioritises TIM3 for immunotherapy based on combined spatial transcriptomics analysis. Overall, our data provide a resource for diagnostics and therapies for tumours of critical unmet need.