Haematologica

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

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

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

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

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. Key PointsO_LIRAS mutations independently predict unfavorable survival after CAR-T CD19 in pediatric B-ALL. C_LIO_LIRAS mutations increase risk of CD19 negative relapse after CAR-T CD19 therapy in pediatric B-ALL. C_LI

FLT3 inhibitors have improved outcomes in acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD), but responses are not durable. Notably, FLT3 inhibitors clear blasts from the blood, but not the bone marrow, a hypoxic niche. We investigated effects of hypoxia and the key nutrient glutamine on FLT3 inhibitor response. FLT3-ITD AML cell lines and patient blasts were cultured with FLT3 inhibitors under normoxia (21%) or hypoxia (<1% O2) with or without glutamine or the glutaminase inhibitor telaglenastat (CB-839). Cytotoxicity was measured in WST-1 assays and drug combination effects by Chou-Talalay analysis. Protein expression was measured by immunoblotting, turnover and proteasomal degradation by cycloheximide chase with and without MG-132, and mRNA expression by RT-qPCR. Effect of the ubiquitin ligase c-CBL was tested by siRNA knockdown. FLT3 inhibitor ICs were 3-5-fold higher in hypoxia than normoxia, associated with FLT3-ITD and p-STAT5 downregulation and accelerated FLT3-ITD proteasomal degradation (half-life, 1.0 vs. 2.5 hours). c-CBL expression increased in hypoxia, and c-CBL knockdown restored FLT3-ITD expression and FLT3 inhibitor sensitivity. Glutamine deprivation or telaglenastat treatment abrogated c-CBL upregulation in hypoxia and preserved FLT3-ITD and p-STAT5 expression and FLT3 inhibitor sensitivity. Telaglenastat synergized with FLT3 inhibitors in hypoxia, supporting clinical testing.

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.

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

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

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

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.

Human cytomegalovirus (CMV) infection represents a significant risk factor for transplant recipients, including patients undergoing hematopoietic stem cell transplantation (HSCT). Interestingly, several studies have reported an association between early CMV reactivation and a reduced risk of leukemia relapse, particularly in acute myeloid leukemia (AML). Given that CMV profoundly shapes the natural killer (NK) cell compartment, a contribution of CMV-primed NK cells to this effect has been proposed. To explore this mechanism, we analyzed the relationship between NK cell functionality and CMV reactivation in the context of AML. Consistent with observations in peripheral blood, CMV-seropositive HSCT recipients displayed expanded NKG2Cpos NK cell populations within the bone marrow, characterized by high Granzyme B expression. CMV replication was associated with elevated plasma IFN{gamma} levels, which in vitro rendered AML cells more susceptible to apoptosis when co-cultured with peripheral blood mononuclear cells. Importantly, IFN{gamma} treatment modulated NK cell responses by inducing a variety of NK cell ligands including HLA-E on primary bone marrow-derived blasts and AML cell lines. In line with this, the activation of CMV-associated NKG2Cpos NK cells was enhanced upon stimulation with IFN{gamma}-pretreated AML cells. In summary, our findings demonstrate that CMV replication induces a transient increase in IFN{gamma} levels that influences both AML and NK cells, ultimately enhancing AML cell susceptibility to NK cell-mediated cytotoxicity initiated through the NKG2C-HLA-E axis. ImportancePrevious studies suggested that CMV reactivation after HSCT may reduce leukemia relapse in AML patients, but the underlying mechanism remained unclear. Here, we show that CMV replication induces IFN{gamma} release, which sensitizes AML cells to NK cell-mediated killing. This effect involves upregulation of HLA-E on AML cells and activation of expanded NKG2Cpos NK cells within the bone marrow. Our findings uncover a novel IFN{gamma}-dependent link between CMV replication and enhanced NK cell cytotoxicity in AML, suggesting that combining IFN{gamma} treatment with NK cell-based immunotherapy or NKG2A blockade could reduce post-HSCT relapse, even in CMV-negative patients.

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

Haematopoiesis and differentiation of immune cells from haematopoietic stem and progenitor cells (HSPCs) are essential to core aspects of health and disease. A key player in haematopoiesis and HSPC differentiation is the cytoskeleton, which governs cell division and lineage bias. Despite insights using mouse models, regulation of haematopoiesis by the septin cytoskeleton is mostly unknown. Septins are unconventional filament forming proteins best known for roles in cell division and host defence. To investigate septin-mediated host defence in vivo, we generated septin-deficient zebrafish models for infection with Mycobacterium marinum. Unexpectedly, septin-deficient larvae were protected from mycobacterial infection due to significantly increased macrophage numbers, reduced cell death, and enhanced inflammatory responses. Underlying this, we found that septin-deficient larvae produce significantly more HSPCs and show myeloid lineage bias, establishing a requirement for septins in haematopoiesis. In agreement with classical HSPC hierarchy, increased myeloid production in septin-deficient larvae is at the expense of erythroid lineage production. Our findings that septins play a role in haematopoiesis is consistent with hallmarks of haematological disorders in which septin dysfunction has been implicated, including acute myeloid leukaemia, myelodysplastic syndrome, and platelet disorder Bernard-Soulier syndrome. These results highlight zebrafish as a new model to investigate septin-mediated haematopoiesis and application of septin-based medicines to treat blood disorders.

The BCL-2 inhibitor venetoclax (VEN) in combination with hypomethylating agents (HMAs) has improved treatment responses in acute myeloid leukaemia (AML), but the mechanisms underlying their synergy remain unclear. We investigated the role of DNA demethylation in the enhanced cytotoxicity of VEN-HMA combinations. Using AML cell lines, we compared the effects of azacitidine (AZA), decitabine (DAC), cytarabine (ARA-C) and the DNMT1-selective inhibitor GSK-3685032 (GSK5032) with VEN. As expected, VEN showed strong synergy with AZA, DAC, and the DNA-damaging agent ARA-C, but not with GSK5032, despite the latter inducing extensive DNA demethylation. Genome-wide methylation profiling confirmed that loss of DNA methylation did not correlate with increased cytotoxicity or synergy with VEN. Moreover, combining GSK5032 with ARA-C did not enhance cytotoxicity, indicating that DNA demethylation and DNA damage do not act additively. Instead, synergy was consistently associated with the DNA damage-inducing properties of AZA, DAC, and ARA-C. Extensive DNA demethylation tended to antagonize VEN activity, suggesting that the epigenetic effects of HMAs may limit their synergistic potential. Overall, our findings demonstrate that DNA damage-related cytotoxicity, rather than DNA demethylation, is the dominant mechanism driving VEN-HMA synergy and provide evidence that VEN-mediated cytotoxicity arises primarily from genotoxic stress, supporting refinement of treatment strategies.

Multiple myeloma remains a fatal, incurable disease. Most therapies are targeted to the cancer cell or T cell engagement. Little is known about the supporting myeloma microenvironment and its contribution to tumor fitness. Here, we expand upon the observation of human mast cells in the NSG-hIL6 myeloma patient derived xenograft mouse model to show mast cells decrease time to engraftment, promote increased myeloma engraftment and cause myeloma bone disease. We identify 10 mast cell secreted factors that together improve the survival of patient myeloma cells in vitro. Our results highlight the versatility of the NSG-hIL6 model to study microenvironmental interactions between human bone marrow cells and myeloma and confirm prior suggestions that clinical signs of disease, such as osteolytic lesions, may at least partially be related to non-malignant bone marrow microenvironmental cells, such as mast cells.

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

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