HemaSphere

Leukemic stem cells (LSCs) sustain acute myeloid leukemia (AML) and are implicated in therapy resistance and relapse. Yet, it remains unknown how LSCs remodel the bone marrow niche. AML is known to alter stromal and vascular microenvironments, but these effects are difficult to separate from bulk leukemic burden and immune inflammation. Here, we use isogenic human AML xenografts with distinct LSC characteristics but comparable engraftment to define LSC-associated niche remodeling in vivo. Single-cell profiling revealed that LSC-high AML shifts the mesenchymal niche toward fibro-inflammatory states, expanding Fmod+ fibroblasts and Cd34+ perivascular fibroblast-like cells while suppressing osteolineage differentiation. The leukemic compartment remained heterogeneous, with a specific MEP-like LSC population expressing niche-remodeling ligands including TGFB1, IL1B, and ANGPT1. LSC-high AML activated a TGF{beta}-responsive, CREB3L1-controlled fibroblast trajectory, and perturbing TGF{beta} signaling or CREB3L1 activation reduced stromal support for AML cells. These findings identify a specific LSC subtype as a source of niche-remodeling cues that shape specialized leukemia-supportive niches.

Background: Relapse remains the principal cause of treatment failure in pediatric acute leukemia. Clinically validated minimal residual disease (MRD) assays - multiparameter flow cytometry, immunoglobulin/T-cell-receptor rearrangement sequencing, and targeted next-generation sequencing panels -- are highly sensitive but read out predefined features and do not reconstruct clonal architecture or link a cell's genotype to its functional state. We systematically reviewed what single-cell sequencing has revealed about clonal evolution, chemoresistance, the immune microenvironment, and post-therapeutic immune dynamics in this disease. Methods: This review followed PRISMA 2020. PubMed/MEDLINE, the Cochrane Library, and Google Scholar were searched to September 2026 for primary studies applying single-cell DNA, RNA, chromatin-accessibility, surface-protein, or immune-repertoire sequencing to pediatric acute leukemia samples or pediatric-derived models. Risk of bias in non-randomized studies was assessed with ROBINS-I. Because designs, platforms, and outcomes were heterogeneous, findings were synthesized narratively by biologic theme without meta-analysis, following the SWiM guideline. Results: Of 63 records identified, 10 studies were included (published 2016-2026; 6-159 patients or samples per study; up to ~700,000 cells profiled). Single-cell DNA sequencing traced minor NOTCH1-mutant diagnostic subclones into PTEN- or TP53-mutant relapse clones that bulk sequencing missed. Rare dormant, label-retaining cells and developmental-state-dependent drug sensitivity defined chemoresistant compartments. Remodeling of the bone marrow immune microenvironment identified patients with adverse outcome among those classified as MRD-low, and pre-infusion CAR T-cell type 2 functionality and post-transplant cytotoxic memory CD8+ T-cell subsets were associated with durable remission. Of the seven studies assessed with ROBINS-I, four were at moderate and three at serious overall risk of bias, driven chiefly by small, selected cohorts. Conclusions: Single-cell sequencing yields mechanistic insight that complements, rather than replaces, clinically validated MRD assays, and nominates candidate biomarkers for risk stratification, treatment selection, cell-therapy manufacturing, and post-transplant monitoring. The evidence remains preliminary; adequately powered, prospective, and harmonized studies are required before clinical adoption.

Although B-cell acute lymphoblastic leukemia (B-ALL) is highly responsive to antigen-directed immunotherapies, treatment resistance remains a major barrier to achieving durable responses in patients. We recently developed a novel VpreB1 (CD179a)-directed antibody-drug conjugate with calicheamicin (VpreB1-ADC) that exploits the restricted expression of VpreB1 within the surrogate light chain in early B cells, including B-ALL. In the present work, we investigated mechanisms of resistance to the VpreB1-ADC. Mechanisms of resistance were evaluated using a TCF3::HLF B-ALL model, assessing target engagement parameters including VpreB1 surface expression and antibody internalization. The role of the multidrug resistance transporter ABCB1 (P-glycoprotein) was evaluated via pharmacologic inhibition, using tariquidar and zosuquidar, and enforced overexpression across multiple B-ALL cell lines. Sensitivity to alternative non-ABCB1 substrate payloads exatecan and PNU-159682 was also assessed. Resistant TCF3::HLF cells retained VpreB1 expression and efficient antibody internalization. Instead, resistance was driven by elevated ABCB1 expression and activity. ABCB1 inhibition with tariquidar or zosuquidar restored VpreB1-ADC sensitivity. Conversely, enforced ABCB1 overexpression conferred ADC resistance, which was reversed by ABCB1 inhibition. Cells with high ABCB1 activity remained fully sensitive to alternative payloads, including exatecan and PNU-159682, which are not ABCB1 substrates. ABCB1-mediated drug efflux drives intrinsic resistance to calicheamicin-conjugated ADCs in B-ALL. Combining ADCs with ABCB1 inhibitors or selecting payloads non-susceptible to ABCB1 efflux offer viable strategies to overcome resistance and optimize future ADC therapies.

Cell surface proteomics provides a direct topological assessment of the outer membrane of cells and enables the capture of low abundance proteins that may be missed by whole cell proteomics. Here we present an unbiased atlas of the whole cell and surface proteomes of 25 commonly used leukemic cell lines, encompassing both lymphoid and myeloid lineages, and a variety of driver mutations. Paired-wise analysis highlights recurrent surface proteins that are not detected by whole cell proteomics. Coupling this dataset to RNA-sequencing, we also discovered genes where protein and RNA abundances are discordant. In KMT2A-rearranged AML, CD70 expression was increased across cell lines and validated in primary patient samples, supporting CD70 as a candidate therapeutic target in this disease. Several proteins are enriched in the surface proteomes but lack surface annotation, adding to the growing list of potential non-canonical cell surface proteins. These findings reveal a substantial pool of proteins absent from conventional surface annotations, including RNA-binding proteins, an emerging class of candidate immunotherapeutic targets.

Background: Epigenetic deregulation is a defining feature of leukemia, and DNA methylation and chromatin accessibility profiles encode the cell of origin, lineage and clinical behaviour of the disease. Artificial intelligence (AI) and machine learning (ML) can extract predictive patterns from these high-dimensional data, but the published models have not been systematically appraised. We systematically reviewed AI models built on epigenomic data in leukemia for diagnosis, prognosis, relapse and treatment-response prediction, and measurable residual disease (MRD) detection. Methods: This review followed PRISMA 2020 and the SWiM guideline. PubMed was searched on 7 October 2026 with three structured queries, supplemented by a broader PubMed query, citation searching and web searching. Eligible studies were peer-reviewed primary studies that applied an ML or deep-learning algorithm to DNA methylation, chromatin-accessibility or histone-modification data from human leukemia samples and reported a quantitative diagnostic, prognostic, response or MRD outcome. Risk of bias and applicability were assessed with PROBAST, informed by PROBAST+AI. Owing to heterogeneity, results were synthesized narratively and in structured tables, without meta-analysis. Results: Of 68 unique database records and 32 reports from other sources, 30 studies (2010-2026; 22 since 2020) were included: 16 in acute myeloid leukemia, 4 in acute lymphoblastic leukemia, 2 spanning acute leukemia lineages, 3 in juvenile myelomonocytic leukemia and 5 in chronic lymphocytic leukemia; 10 were pediatric. DNA methylation was the input in 28 studies and chromatin accessibility in 2; no eligible study modelled histone marks. Tree ensembles (n = 9) and support vector machines (n = 5) were the most frequent algorithms. Diagnostic classifiers reported accuracy, concordance or F1 scores of 0.87-1.00, and two classifiers were applied to nanopore sequencing data, one returning acute leukemia subtype calls within about 2 hours. Prognostic epigenomic scores were associated with survival (reported hazard ratios 2.36-10.82), but discrimination fell on transport to independent cohorts (C-index 0.677 internally vs 0.529 in one external cohort). Fifteen studies validated models on an external epigenomic cohort, one prospectively. Overall risk of bias was high in 21 studies, unclear in 8 and low in 1, driven by the analysis domain (small samples, absent calibration, possible data leakage). Calibration was reported in 3 studies and code was available for 11. Conclusions: Epigenomic AI models classify leukemia subtypes with high reported accuracy and can deliver same-day diagnoses, and methylation-based scores add prognostic information. However, the evidence is dominated by retrospective, high-risk-of-bias development studies with limited external validation and almost no prospective or impact evaluation. Prospective, multi-center validation with calibrated, transparently reported models is required before clinical adoption.

Diffuse B-cell lymphomas are the most prevalent canine hematologic malignancies, and their clinical presentation resembles that of human B-cell non-Hodgkin lymphomas (NHLs). However, a lack of a clear subtyping framework perpetuates imprecise treatment approaches that fail to address mechanisms of therapy resistance. Here, we employed deep phenotyping via serial sampling and longitudinal transcriptomics to evaluate intratumoral composition and therapy response in a 6-year-old neutered male goldendoodle with stage 5A marginal zone lymphoma (MZL). Following sequential treatment consisting of a single IV dose of oncolytic vesicular stomatitis virotherapy (VSV) and standard CHOP chemotherapy 30 days later, we performed scRNAseq on seven serial lymph node biopsies and spatial sequencing on a resection taken 10 days post-VSV treatment. B cells (>90%) comprised three transcriptionally distinct and temporally stable subpopulations, with specific copy number profiles and robust spatial organization despite disrupted lymph node architecture. Analysis across subpopulations revealed downregulation of the quiescence program including KLF2, suggesting similarity to KLF2-deficient human MZL. While VSV successfully reached target B cells, downstream transcriptional responses were predominantly localized to the T-cell compartment. T cells ([≤]6%) showed increased proliferation and upregulation of cytotoxicity markers post-VSV administration, enriching for leukocyte-mediated cytotoxicity pathways consistent with an anti-viral immune response. Ultimately, these results provide an in vivo proof-of-concept for the treatment paradigm in canine lymphoma, while emphasizing the need for improved subtyping frameworks and multidimensional treatment strategies targeting intratumoral heterogeneity.

Leukemic stem cells (LSCs) play a central role in disease progression, therapeutic resistance, and relapse in acute myeloid leukemia (AML). However, the identification and characterization of LSCs remain challenging because of their low abundance and their close phenotypic resemblance to normal hematopoietic stem and progenitor cells. Although patient-derived xenograft (PDX) models have provided important insights into AML biology and LSC heterogeneity, the relative engraftment potential of distinct CEBPA mutation subtypes and the immunophenotypic identity of LSCs in CEBPA N-terminal mutant AML (CEBPA-N-AML) remain poorly defined. To address these questions, we compared the engraftment characteristics of primary human CEBPA-mutated AML samples representing the major mutational subtypes using the highly permissive NSGS xenograft model. Primary CEBPA-N-AML samples exhibited markedly greater engraftment efficiency and leukemogenic potential than other CEBPA-mutated AML subtypes. Furthermore, we identified a CD366CD73CD123CD117CD371CD247 cell population that is highly enriched for functional LSCs in CEBPA-N-AML, demonstrating enhanced clonogenic activity, leukemia-initiating capacity, and long-term self-renewal. Collectively, our findings demonstrate that the leukemogenic potential of CEBPA-mutated AML is strongly influenced by mutation subtype, with CEBPA-N-AML exhibiting superior leukemia-propagating capacity in vivo. We further define a novel immunophenotypic LSC signature specific to CEBPA-N-AML, providing new insights into LSC heterogeneity in CEBPA-mutated AML and establishing a foundation for the development of LSC-directed therapeutic strategies.

Yin Yang 1 (YY1) is a multifunctional transcription factor involved in chromatin organization and gene regulation. While the mechanisms by which YY1 controls B cell lineage development have been explored at the transcriptional level, little is known at the post-transcriptional level. To address this, we performed integrated analyses combining bulk transcriptomics, miRNA profiling, and single-cell transcriptomics to identify YY1-dependent regulatory networks at the pro-B cell stage. YY1 knockout (KO) in pro-B cells leads to reduced B-lineage commitment and activation of genes associated with alternative hematopoietic fates. Our integrative analyses suggest that these changes are organized in part through coordinated miRNA-mRNA regulatory networks. In wild-type (WT) pro-B cells, miRNAs such as miR-10a-5p, miR-674-5p, and miR-21a-5p maintain B cell identity by suppressing RNAs linked to other lineages. In contrast, in YY1 KO pro-B cells increased expression of miRNAs including miR-15b-5p, miR-342-5p, and miR-34a-5p target pathways required for B cell development, proliferation, and survival thus reducing B lineage identity. Together, these findings suggest that YY1 preserves B cell identity through coordinated regulation of transcriptional and post-transcriptional programs, with YY1-dependent miRNA networks contributing to the maintenance of B cell-associated gene expression and restriction of alternative lineage programs. Among these, miR-15b-5p emerged as a central regulator predicted to regulate Bcl6b and E2f family transcription factors that control proliferative and transcriptional programs critical for early B cell development. At single-cell resolution, YY1 KO pro-B cells grown on OP9-DL4 feeders that provide Notch signaling displayed marked transcriptional heterogeneity and loss of pro-B lineage commitment. Pseudotime analysis revealed a branched developmental landscape, with cells diverging toward monocyte, macrophage, and dendritic cell fates rather than a consistent B cell differentiation trajectory. Collectively, these findings establish YY1 as a central regulator of lineage commitment integrating transcriptional and miRNA-mediated post-transcriptional mechanisms to regulate B cell identity.

CD8 T cells are key mediators of anti-tumor immunity, yet their differentiation states and clonal architecture within the acute myeloid leukemia (AML) bone marrow microenvironment remain incompletely defined. We performed high dimensional immune profiling of bone marrow samples from 46 newly diagnosed AML patients using mass cytometry to probe the microenvironment and T cell states. We used single-cell RNA and T cell receptor (TCR) sequencing to define CD8 T cell phenotypes, transcriptional states, and clonal architecture. AML-associated CD8 T cells were skewed toward terminally differentiated effector memory cells re-expressing CD45RA (TEMRAs), while canonically exhausted T cell populations were rare. Functional studies revealed that TEMRA CD8 T cells robustly produced IFN{gamma} and TNF upon in vitro stimulation. Clonal expansion was concentrated within the TEMRA compartment. Comparison of TCR sequences with known viral epitopes identified a small subset of expanded viral-specific clonotypes, while the majority of highly expanded clonotypes had unknown specificity and may include AML-specific T cells. Differential gene expression analysis revealed shared cytotoxic effector programs during clonal expansion but diverging activation signatures: viral-specific clones upregulated canonical T cell receptor signaling genes, whereas expanded clones of unknown specificity exhibited enrichment of natural killer (NK) receptor-associated genes. These genes comprised the leukemia-associated NK-immune (LaNKi) score which was elevated in an independent AML dataset compared to pan cancer tumor infiltrating lymphocytes. These findings define the CD8 T cell differentiation landscape and clonal architecture in AML marrow at diagnosis and suggest that T cell dysfunction in AML diverges from classical activation and exhaustion paradigms.

Proliferating cells must acquire nucleotides to support DNA replication, yet how cells meet these nucleotide demands for proliferation under physiological conditions remains understudied. Here, we investigated how physiological nutrient availability shapes nucleotide acquisition strategies in a mouse model of B-cell acute lymphoblastic leukemia (B-ALL). To assess how environmental nutrients impact nucleotide metabolism, we formulated a mouse plasma-like medium (MPM) that reproduces the circulating metabolite composition of plasma from mice with B-ALL and assessed how this influenced nucleotide metabolism relative to standard culture conditions, where nucleotide acquisition has historically been studied. We find that leukemia cells cultured in MPM acquire nucleotides through salvage pathways, and that select nucleotide salvage pathways are required for proliferation under physiological conditions. Of note, this dependency on nucleotide salvage in plasma-like conditions was not caused by precursor metabolite limitation for de novo synthesis. Instead, we found that physiological folate levels are insufficient to support deoxynucleotide triphosphate (dNTP) synthesis for genome replication, leading to DNA replication stress and impaired proliferation when nucleotide salvage is disrupted. Consistently, dietary folate restriction exacerbates the impaired leukemia progression phenotype of nucleotide salvage-deficient B-ALL cells. Together, these findings demonstrate that access to folates is an endogenous limitation for nucleotide synthesis in plasma-like nutrient conditions, increasing the relevance of nucleotide salvage pathways for leukemia progression. More broadly, this work highlights how micronutrient abundance can influence metabolic dependencies and reveals that folate levels shape nucleotide metabolism under physiological conditions.

Clonal hematopoiesis (CH) can predict treatment outcomes in solid cancers, but the prevalence and clinical impact of CH in previously untreated, aggressive large B-cell lymphomas (LBCL) remains poorly characterized. Moreover, CH may be detected in cell-free DNA (cfDNA)-based biomarker assays, but the longitudinal dynamics of CH mutations during and after immunochemotherapy in LBCL remain unknown. Hence, we characterized the spectrum of CH mutations in 247 patients with high-risk primary LBCL from three phase II clinical trials. We identified CH mutations in 23% of patients. Compared with healthy controls, TET2 mutations were enriched in peripheral blood (PB) samples of LBCL patients (P=0.01). CH clone size showed high concordance between PB and cfDNA (R2=0.59, P<0.0001) but was not associated with circulating tumor DNA (ctDNA) concentration (P-values 0.25-0.86). In contrast with prior reports in the context of recurrent lymphoma, CH mutation status was not associated with overall survival (OS) (P=0.78) or progression-free survival (PFS) (P=0.89) in our primary LBCL cohort. CH clones showed variable longitudinal dynamics during immunochemotherapy, with 18% of evaluable patients developing mutant CH clones, mostly with TP53/PPM1D mutations. Together, CH mutations can be readily detected in cfDNA of lymphoma patients, but they do not predict survival in primary high-risk LBCL.

Uncontrolled proliferation of myeloid progenitor cells in acute myeloid leukemia (AML) is counteracted in most patients by toxic and often ineffective systemic treatments. Poly (ADP-ribose) polymerase inhibitors (PARPi) show subtype-restricted activity - potent in RUNX1-RUNX1T1 and PML-RAR[a] fusions, limited in KMT2A-rearranged (KMT2A-r) disease - but the lack of molecular understanding has hampered their clinical implementation. We combined single-cell and single-molecule assays on DNA replication intermediates and DNA damage signalling with therapy response readouts to investigate the role of fork plasticity factors in response to PARPi and AML standard-of-care (cytarabine, araC). In PARPi-sensitive AML models, PARP inhibition deregulates RECQ1-mediated fork restart, initially triggering fork acceleration and later fork breakage within the same S phase. Conversely, PARPi resistant KMT2A-r AML lines are protected by PrimPol-dependent DNA synthesis and its inactivation promptly induces fork breakage and PARPi sensitivity. Strikingly, PrimPol overexpression in PARPi-sensitive AML models prevents fork collapse and PARPi/araC therapy response, both in vitro and in vivo, identifying PrimPol as novel predictive biomarker and therapeutic target in AML. Our data uncover novel tissue-specific mechanisms of action for PARPi and pinpoint replication fork plasticity as key molecular determinant of AML therapy response. HighlightsO_LIFork plasticity is a key molecular determinant of treatment response in leukemia. C_LIO_LIPARP inhibition triggers fork breakage via deregulated restart of reversed forks. C_LIO_LIBypassing fork reversal, PrimPol limits therapy-induced DNA damage and cytotoxicity in AML. C_LIO_LIPrimPol drives resistance to cytarabine and PARP inhibition in vitro and in vivo. C_LI

The immunosuppressive bone marrow microenvironment is an important contributor to the limited success of immunotherapy in acute myeloid leukemia (AML), but the cellular interactions underlying AML immune evasion are incompletely understood. We therefore generated a single-cell spatial transcriptomic and proteomic atlas using 148 bone biopsies from 113 individuals comprising pediatric and adult AML at diagnosis and non-leukemic controls. We observed an expansion of regulatory T cells (Tregs) in AML, with stronger colocalization between Tregs and macrophages compared to non-leukemic bone marrow. Distinct cellular neighborhoods were enriched for myeloid progenitor-like cells together with macrophages and T cells, which correlated with higher macrophage and T cell immune checkpoint expression. Moreover, these neighborhoods were associated with specific AML subtypes, especially KMT2A-rearranged and RUNX1::RUNX1T1 AML. These spatial patterns were validated by identification of malignant cells via in situ fusion detection in RUNX1::RUNX1T1 cases. Functional experiments revealed that macrophages and AML cells not only actively recruit Tregs, but also promote naive T cell differentiation into Tregs. Spatially informed ligand-receptor analysis predicted the involvement of the Galectin-9 - CD44/TIM-3 axis in this immunosuppressive crosstalk, which was supported by in vitro inhibition of CD44 and/or TIM-3 preventing macrophage- and AML-induced Treg differentiation. Collectively, this comprehensive spatial map of the AML bone marrow identified tripartite crosstalk between AML, macrophages, and T cells mediated by the Galectin-9 - CD44/TIM-3 axis as a key component of the immunosuppressive microenvironment. Targeting Galectin-9 - CD44/TIM-3 interactions may be a promising strategy to overcome immune evasion and enhance immunotherapeutic success in AML. HighlightsO_LISpatial transcriptomic and proteomic atlas of pediatric and adult acute myeloid leukemia (AML) bone marrow C_LIO_LIIncreased colocalization of macrophages and regulatory T cells (Tregs) in AML C_LIO_LIMacrophages and AML cells induce differentiation of naive T cells to Tregs in vitro, which can be prevented by inhibition of CD44 or TIM-3 C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/743431v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@5887f4org.highwire.dtl.DTLVardef@45d17corg.highwire.dtl.DTLVardef@1bc3264org.highwire.dtl.DTLVardef@9059aa_HPS_FORMAT_FIGEXP M_FIG C_FIG

Glucocorticoids (GCs) are foundational to B-cell acute lymphoblastic leukemia (B-ALL) therapy, yet how glucocorticoid receptor (GR) activation interacts with lineage regulatory circuitry to shape therapeutic response remains incompletely understood. Using LEF1 to interrogate canonical Wnt regulatory circuitry, we show that GR activation reorganizes GR+LEF1 elements into recruitment, retention, and dissociation states. LEF1 dissociates from Wnt-responsive regulatory elements while being recruited to GRE-rich GR-associated elements that progressively acquire chromatin accessibility, H3K27ac, BRD4 and BRG1. RUNX1 and AP-1 show class-dependent redistribution across LEF1-defined states, extending organization beyond LEF1. GR+LEF1 reorganization is observed in patient-derived B-ALL. GR+LEF1 elements lose accessibility in GC-resistant B-ALL and during diagnosis-to-relapse evolution, with recruitment elements enriched for remodeling. Perturbation of resistance-associated GR+LEF1 elements at BIM and BMF alters GC-induced apoptotic signaling. Together, these findings identify Wnt/LEF1 circuitry as an interface through which therapeutic GR activation reorganizes the B-ALL regulatory landscape into states associated with glucocorticoid response and resistance.

Acute myeloid leukemia (AML) is a clinically heterogeneous disease. Although the genetic abnormalities associated with poor prognosis are well defined, how they drive unfavorable outcomes remains unclear. Using published gene-expression and dependency datasets, we searched for cell-surface protein-coding genes associated with poor survival and required for AML growth, prioritizing this class of proteins for its accessibility to biologics. This search identified CD59, a GPI-anchored protein with a canonical role in complement regulation, whose high mRNA expression correlates with adverse-risk genetics and stemness signatures. CD59 silencing impaired proliferation across genetically diverse AML cell lines, reduced leukemic burden, and extended survival in cell xenograft models. Moreover, CD59 expression was enriched on leukemic stem cells (LSCs), and its depletion impaired LSC self-renewal and primary AML engraftment in vivo while sparing normal hematopoiesis. Mechanistically, these effects reflected a non-canonical role for CD59 in sustaining Ras-MAPK signaling, whereby its loss depleted inner-leaflet phosphatidylserine and impaired Ras and c-Raf membrane recruitment and activation. rILYd4, a recombinant fragment of the bacterial toxin intermedilysin that binds and degrades CD59, recapitulated these effects and sensitized cells to venetoclax in vivo. These findings reveal CD59 as a critical regulator of Ras-MAPK signaling required for AML growth and nominate its rILYd4-mediated degradation as a therapeutic strategy.

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

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphoma (PTCL) are aggressive hematological malignancies requiring novel therapeutic strategies. The majority of T-ALL and PTCL tumors display metabolic activation and addiction to endogenous serine/glycine synthesis (SSP), providing opportunities for targeted therapy with the clinically used antidepressant sertraline, inhibiting SSP enzymes SHMT1/2. However, sertraline monotherapy only induces cell cycle arrest and has limited efficacy in suppressing disease progression in vivo. MethodsDrug synergy of sertraline combined with clinically used proteasome inhibitors carfilzomib and bortezomib was evaluated. Drug effects on cell cycle, proliferation and apoptosis were assessed in T-ALL, PTCL and healthy blood cells using flow cytometry assays. Proteomic, lipidomic and metabolic analyses on drug treated T-ALL cells were performed to elucidate the molecular mechanisms underlying drug synergy, followed by validation of changes of interest, metabolic rescues and shRNA-knockdown of SSP enzymes in T-ALL cells. In vivo therapeutic efficacy and immune remodelling were evaluated in an immunocompetent MYCN-overexpressing PTCL mouse model. ResultsSertraline acted synergistically with clinically used proteasome inhibitor carfilzomib to induce cell cycle arrest and apoptosis in T-ALL and PTCL cells with SSP activity, with minimal effects on SSP-inactive T-ALL cells or healthy blood cells. Adding carfilzomib also enhanced the therapeutic efficacy of sertraline in an aggressive MYCN PTCL model. Sertraline rewired cell metabolism towards increased cholesterol uptake and biosynthesis in SSP-active T-ALL cells, and this effect was not obtained by other means of SSP inhibition. In contrast to sertraline, carfilzomib promoted cholesterol efflux. Moreover, carfilzomib reduced total lipid levels, further restricting nutrients in sertraline - carfilzomib treated cells. Additionally, the drug combination impaired mitochondrial respiration and elevated reactive oxygen species (ROS) levels and DNA damage in SSP-active tumor cells, which was rescued by citrate supplementation. Interestingly, these metabolic changes were associated with microenvironmental changes in our mouse model, where the drug combination elevated natural killer T-cells, neutrophils and eosinophils. ConclusionsOur study identifies synergy of sertraline - carfilzomib combination treatment mediated through metabolic impairment and is associated with remodelling of the immune microenvironment. This invites for further clinical investigation of this drug combination as a therapeutic strategy for SSP-active T-cell malignancies.

Background: The clinical course of acute leukemias and related hematologic neoplasms is defined by aggressive clonal proliferation, severe hematopoietic disruption, and recurrent molecular alterations. Although serial bone marrow biopsies, multiparameter flow cytometry, and next-generation sequencing remain standard surveillance modalities, procedural invasiveness, high costs, and operational turnaround times limit their frequency. Minimally invasive, scalable biomarkers integrating routine hematologic indices, targeted gene expression, circulating oncoprotein levels, and patient compliance metrics may provide an accessible framework for dynamic monitoring. Methods: In this longitudinal cohort study of 66 patients across 272 serial encounters (discrete follow-up intervals: days 0, 14, 41, 71, and 78), we assessed the prognostic value of peripheral complete blood counts (white blood cell count [WBC], hemoglobin [Hb]), molecular indicators (FLT3, NPM1, and DNMT3A transcript levels; targeted soluble FLT3 quantification), and medication adherence. The primary clinical endpoint was longitudinal progression to critical deterioration or disease-related mortality. Generalized linear mixed-effects models (GLMM) with patient-specific random intercepts were constructed to account for intra-subject repeated measures. Predictors were standardized per 1-SD increment. Model performance was assessed via the Akaike Information Criterion (AIC) and the Concordance index (C-index). Results: Leukocyte expansion correlated inversely with hemoglobin concentration (r=-0.877, p<0.001), reflecting bone marrow infiltration and secondary erythroid suppression. Multivariable mixed-effects modeling confirmed that elevated WBC count (adjusted odds ratio [aOR] = 1.52 per 1-SD increase; 95% CI: 1.24-1.86; p<0.001) and reduced medication adherence (aOR = 0.58; 95% CI: 0.44-0.76; p<0.001) independently predicted adverse clinical transition. Integrating targeted molecular markers (FLT3 expression and soluble protein levels) with routine hematologic indices and adherence demonstrated superior calibration and discrimination over single-domain models ({Delta}AIC[&ge;]116; Harrells C-index = 0.81). Conclusions: Readily accessible peripheral hematologic indices, targeted molecular quantification, and medication adherence provide complementary prognostic information regarding disease activity. Integrating these low-burden parameters into clinical workflows offers an efficient, scalable paradigm for early risk stratification in hematologic malignancies.

ERCC6L2 disease (ED) is an inherited bone marrow failure (BMF) syndrome that progresses almost exclusively to erythroid, TP53-mutated myeloid malignancy, but the somatic evolution underlying the progression in patients is unknown. We characterized the genetic landscape of 29 ED patients with longitudinal sampling, integrating whole-genome and single-cell RNA sequencing, and compared the findings with Shwachman-Diamond syndrome (SDS), another TP53 mutation-prone disease, to define somatic evolution across disease stages. ED was defined by early, intense, and recurrent selection of TP53-mutant clones: TP53 mutations were more frequent, more often multiple, and showed steeper age-related expansion than in SDS; all TP53-negative ED patients were children. The burden of multiple TP53 mutations exceeded that of sporadic TP53-mutated myeloid neoplasia and was evident before malignancy. Missense mutations dominated the BMF phase, whereas non-missense mutations were seen in patients with malignancy, consistent with biallelic second hits, marking a stage-dependent shift in p53 inactivation. Beyond TP53, the landscape was strikingly restricted, with PPM1D as the only recurrent driver gene. Notably, despite the DNA-repair function of ERCC6L2, no ED-specific mutational signature emerged; but instead reflected age-associated processes. Malignant progression was accompanied by a complex karyotype, recurrent chromosome 5, 7, and 12p losses enriched in erythroid progenitors, and kataegis, consistent with branched, erythroid-predominant clonal evolution. Our findings establish ED as a paradigm of preleukemic dependency on p53-pathway attenuation. As progression is driven predominantly by p53 and captured longitudinally from the earliest clones, ED offers a model of how TP53-mutated leukemia arises and evolves, with relevance beyond a rare syndrome.

CBFA2T3-GLIS2 (C/G) defines a high-risk pediatric AML subtype in which de novo enhancer rewiring sustains an aggressive leukemic transcriptional state. The early epigenomic mechanisms reinforcing this program and linking it to leukemic survival remain incompletely defined. Here, we identify enhancer-proximal hypermethylation as a noncanonical, transcription-permissive feature of C/G AML. Using primary C/G-positive AML samples and a developmentally relevant C/G-transformed cord blood hematopoietic stem/progenitor cell model, we integrated DNA methylation, chromatin state, fusion occupancy, and gene expression across early and leukemia-stage contexts. Early C/G-associated hypermethylated regions localized near H3K27ac/H3K4me1-marked active or primed cis-regulatory elements, were enriched for ERG/ETS-like motifs, and showed related architecture in leukemia-stage contexts. DNMT3B was prominently increased in C/G contexts, and its loss selectively altered methylation-associated gene expression without measurably changing global 5mC. Genetic DNMT3B loss increased venetoclax sensitivity in vitro, while pharmacologic DNMT3B inhibition with Nanaomycin-A similarly enhanced venetoclax response. During venetoclax treatment, DNMT3B-deficient xenografts showed reduced disease burden and prolonged survival. To assess whether this interaction extended to clinically used hypomethylating agents, azacitidine or decitabine plus venetoclax induced apoptosis-related transcriptional programs and mitochondrial depolarization. These findings identify hypermethylated active cis-regulatory elements as C/G-associated states linked to transcriptional dysregulation and venetoclax response.