Experimental Hematology

Background: The ability to repopulate all lineages of blood cells from a single hematopoietic stem cell (HSC) requires a constant balancing between maintenance of quiescence/self-renewal and the need for differentiation and adoption of specific lineage phenotypes. A classical interpretation suggests that during homeostasis, long- and then short-term HSCs differentiate into multipotent progenitors (MPPs), which then lack self-renewal capacity but can still differentiate into all downstream lineages. While much is known about the intrinsic and extrinsic regulators that promote HSC homeostasis, less is known about factors which influence HSC behavior following significant bone marrow (BM) injury, such as sublethal irradiation (SLI). Methods: Conditional knock-out of follistatin-like 1 (FSTL-1) was achieved by breeding mice with floxed Fstl1 sites to mice expressing CreERT2 under control of the ROSA locus, followed by systemic tamoxifen administration. ELISA and RT-PCR quantitated protein and RNA level loss of FSTL-1, respectively. HSC populations were assessed by flow cytometry under multiple conditions. Sublethal irradiation induced BM injury and blocking polyclonal FSTL-1 antibodies were used to recapitulate results seen with genetic ablation. Finally, single cell RNA sequencing defined both cell-specific expression of Fstl1 within the BM compartment, but also identified transcripts and pathways upregulated in the absence of FSTL-1. Results: These studies delineate a novel role for FSTL-1 in regulating HSC biology. In addition to selective expression of Fstl1 within HSCs, genetic deletion of Fstl1 increases Lineage negative;Sca-1+c-Kit+ (LSK) cell number and alters HSC homeostasis. These findings are further amplified in the BM in the context of recovery from sublethal irradiation. Importantly, the post-irradiation increase in BM cellularity seen with Fstl1 deletion is replicated by treatment with anti-FSTL-1 polyclonal antibodies. Finally, single cell RNA sequencing reveals distinct Fstl1 expression in specific LSK subpopulations, including HSCs and proliferating cells, and identifies a novel relationship between decreased FSTL-1 levels and enrichment of canonical metabolic pathways including oxidative phosphorylation, fatty acid metabolism, and glycolysis. Conclusions: Taken together, these results highlight a novel role for FSTL-1 in promoting HSC homeostasis. They also unlock an innovative way to promote HSPC recovery following BM injury, knowledge predicted to be of significant therapeutic value.

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.

Glutathione is a redox buffer, best known for preventing oxidative stress and ferroptosis, but rarely studied in stem cells. We studied glutathione function in hematopoietic stem cells (HSCs) by deleting the glutamate-cysteine ligase catalytic subunit (GCLC), which synthesizes glutathione, or glutathione peroxidase 4 (GPX4), which employs glutathione to prevent ferroptosis. GCLC deficiency eliminated glutathione from hematopoietic cells, severely impairing HSC function and hematopoiesis. In contrast, GPX4 deficiency did not affect HSC function and had little effect on hematopoiesis, even after vitamin E depletion, suggesting glutathione has ferroptosis-independent functions in HSCs. Consistent with this, GCLC deficiency increased reactive oxygen species levels in hematopoietic cells. But treatment with antioxidants or ferroptosis inhibitors gave little or no rescue of HSC function despite reducing ROS levels. Rather, GCLC-deficient, but not GPX4-deficient, HSCs exhibited unfolded protein accumulation, proteostatic stress, and cell death marked by elevated p53 levels and Annexin V staining. Deficiency for p53 prolonged the function of GCLC-deficient HSCs after transplantation. Glutathione is thus necessary in HSCs for proteostasis but may be dispensable for ferroptosis inhibition in vivo.

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.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy associated with a poor prognosis and an unmet need for more effective treatment options, particularly after relapse. One potential approach to address this need, which has been used successfully in solid tumors, is adoptive transfer of T cells engineered to express specific T cell receptors (TCRs). Particularly attractive are TCRs that can target epitopes derived from oncogenes on which cancer cells depend while sparing normal cells that do not harbor the mutation. One promising oncogenic target in AML is mutated nucleophosmin 1 (mtNPM1), present in up to 30% of AML cases. Most NPM1 mutations result in a common frameshifted sequence that generates peptides presented by several class I human leukocyte antigens (HLAs), such as HLA-A*11:01. In this study, we vaccinated HLA-A*11:01-transgenic mice with the mtNPM1 peptide AVEEVSLRK and isolated six functional TCRs from mtNPM1-reactive lymphocytes. When engineered into human T cells, two of these TCRs consistently mediated robust and specific lysis of an AML cell line and primary AML cells co-expressing HLA-A*11:01 and mtNPM1. Neither TCR recognized related human peptides, HLA-A*11:01-expressing normal peripheral blood mononuclear cells or stem/progenitor cells, or cells expressing diverse allogeneic HLAs, suggesting a low risk of cross-reactivity and alloreactivity. In an AML xenograft model, both TCRs controlled AML in hematopoietic organs, although extramedullary tumors emerged during treatment, potentially reflecting the aggressive behavior of the model that may not be representative of AML in patients. Together, these data suggest that the two TCRs represent strong candidates for further preclinical development in adoptive T cell therapy for NPM1-mutated, HLA-A*11:01-positive AML. These results also support the use of HLA-transgenic mice as a platform for generating TCRs targeting oncogene-derived epitopes in hematologic malignancies.

The human T cell repertoire is generated through recombination of a multitude of T cell receptor gene segments, yielding a complex array of T cell clones. T cell receptor beta (TRB) V gene segment defined, T cell clonal frequency when organized in correspondence with the respective V gene segment positions on the TRB genomic locus yields a periodic, undulating curve in the spatial domain. Using the genomic distance from the TRB-D1 segment to the TRB-V1-29 segments, spectral analysis was performed utilizing Lomb-Scargle periodogram (modified Fourier analysis) to obtain spectral power curves quantifying the TRB V clonal frequencies from 12 allogeneic stem cell transplant donors (baseline) and recipients (<100 days or b/w 100-365) using a variety of analytic software. Patients either underwent HLA matched related bone marrow or HLA matched unrelated blood stem cell transplantation, using PT-CY for the former and ATG for the latter. Spectral Power curves revealed dominant spectral peaks at wavelengths ranging from 113-335 millicycles/kb in the 12 donors, with consistent frequency domain spectral patterns, supporting similar use of V segments across healthy individuals. PT-CY recipients had power spectra closely corresponding to the respective donors, however the ATG recipients had relatively dispersed spectra, with spectral centroid shifted towards higher frequencies compared to donors and a reciprocal decline in the low frequency indices. Spectral power was concentrated in the <3 kb and 3-12 kb wavelengths in both groups, consistent with the periodicity observed in the relative V gene segment usage across the repertoire. The analyses reported here demonstrate that the healthy SCT donors have a spectral signature occupying short to intermediate wavelegnths in the frequency domain, PT CY recipients recover with donor-like spectra on the average, however ATG recipients tend to shift towards higher frequencies. These findings are consistent with a normal organized distribution of TRB V segment usage in healthy individuals, with PT-CY preserving this organization more consistently than ATG. Spectral (Fourier) analysis of TRB sequencing data provides a repertoire wide summary of T cell clonal distribution.

Stem cell factor (SCF) from Leptin Receptor-expressing (LepR+) mesenchymal stromal cells and endothelial cells is required for hematopoietic stem and progenitor cell maintenance in bone marrow. Consistent with this, Scf is highly expressed by LepR+ cells and at lower levels by endothelial cells in bone marrow. However, endothelial cells outside the bone marrow produce circulating SCF, raising the question of whether the SCF that promotes stem and progenitor cell maintenance is produced inside or outside bone marrow. We found that Scf deletion in spleen and liver endothelial cells reduces SCF levels in the blood without affecting stem or progenitor numbers in the bone marrow. Moreover, deletion of Scf from mesenchymal cells in the long bones, but not vertebrae, of Prx1Cre; Scf fl/fl mice depletes stem and progenitor cells in long bones but not vertebrae. SCF thus acts locally within the bones in which it is made to promote stem and progenitor cell maintenance.

Somatic mutations in the de novo DNA methyltransferase DNMT3A are frequent in clonal hematopoiesis, but how they alter the function of differentiated myeloid cells remains unclear. Here, we used isogenic human embryonic stem cell-derived macrophage models to define the consequences of DNMT3A dysfunction during myeloid differentiation. DNMT3A-mutant macrophages selectively upregulated bivalent Polycomb target genes, accompanied by DNA hypomethylation, reduced H3K27me3 and increased promoter accessibility. Despite broad epigenetic changes across bivalent promoters, transcriptional derepression occurred at a subset of loci characterized by lower baseline DNA methylation and higher H3K27me3, identifying pre-existing chromatin state as a determinant of transcriptional response. This Polycomb-associated program was conserved in murine macrophages and enriched for genes involved in cell migration and wound repair. DNMT3A-mutant macrophages exhibited enhanced migration and preferential early recruitment to injured tissue. These findings link clonal hematopoiesis-associated epigenetic alterations to selective transcriptional reprogramming and altered function of differentiated myeloid cells.

Hematopoietic stem cells (HSCs) arise from the hemogenic endothelium in the dorsal aorta through the endothelial-to-hematopoietic transition, a tightly regulated developmental process requiring coordinated signaling from the developing hematopoietic niche. Wnt and Notch signaling are essential for HSC specification, with Wnt functioning upstream of Notch; however, the molecular mechanisms by which these pathways are coordinated during HSC development remain poorly understood. Here, we identify Disheveled 2 (Dvl2), a key intracellular Wnt scaffold protein, as an essential regulator of HSC identity specification in zebrafish. Loss of dvl2 impaired HSC specification and reduced Notch signaling in the dorsal aorta, despite normal canonical Wnt activity in the developing endothelium, suggesting that Dvl2 regulates HSC specification independently of endothelial beta-catenin signaling. Hemogenic endothelial-specific expression of the Notch intracellular domain rescued HSC defects in dvl2 morphants, demonstrating that Dvl2 functions upstream of Notch activation in the hemogenic endothelium during HSC specification. In addition, loss of dvl2 reduced primitive macrophages and neutrophil populations. Restoration of primitive myeloid cells through gata1a knockdown rescued both endothelial Notch signaling and HSC defects, demonstrating that Dvl2 is also required for the supportive immune cells that induce HSC identity. Together, these findings reveal that Dvl2 coordinates hemogenic endothelial and immune niche cues to control HSC identity specification.

Homozygosity for the HBB codon 8/9 (+G) frameshift (c.27dup (p.Ser10ValfsTer14)) causes transfusion-dependent {beta}-thalassaemia and is common in South Asia. Prime editing can reverse this insertion without double-strand breaks or donor DNA, but its efficiency depends on pegRNA design. We derived Sendai-reprogrammed iPSCs from a homozygous patient, optimised PEmax editing (spacer, pegRNA extension, secondary nick, MLH1dn) and compared patient, PEmax-treated and control iPSC-derived erythroid cells by flow cytometry, colony assays, RT-qPCR, western blotting and cation-exchange HPLC. Patient iPSCs had a normal 46,XY karyotype, expressed pluripotency markers, formed all three germ layers and were Sendai-free by passage 15. A PAM-disrupting spacer with a +68 nicking sgRNA gave the highest intended-edit frequency 8.4%; MLH1dn added little. PEmax-treated cultures matured and formed colonies more like control than patient cultures, restored HBB transcript (about 4-fold control iPSC-derived cells) and detectable {beta}-globin protein, and contained an HPLC fraction consistent with HbA ( undetectable in patient cells). Fetal haemoglobin (about 80%) and embryonic globin remained predominant. Prime editing corrects HBB c.27dupG in patient iPSCs and restores {beta}-globin expression, with partial restoration of adult haemoglobin in a fetal/embryonic-type erythroid background. Validation in additional donors, haematopoietic stem cells and in vivo is required.

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.

Many studies unraveled the reciprocal regulation between hematopoietic stem cells (HSC) and the bone marrow (BM) niche by using transgenic knock-out or reporter mice, often overlooking the pathophysiological relevance and the existence of compensatory mechanisms. Here, we exploit the th3 murine model of a genetic anemia, beta-thalassemia (BThal), to investigate the dual impact of chronically reduced thrombopoietin (TPO) production on HSC and megakaryocytic BM niche. Decreased systemic TPO downregulates stemness programs in HSC and impairs their commitment towards megakaryocytes (Mk). We uncover dysmegakaryopoiesis in the BM from BThal patients and mice with defective Mk maturation and reduced production of niche factors, altering the HSC-niche crosstalk. Notably, in vivo stimulation of the TPO signaling restores HSC quiescence, Mk maturation and niche support. These findings demonstrate that TPO regulates HSC through both direct and niche-mediated routes, underscoring the value of using a genetic disease model to disentangle general molecular mechanisms.

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.

TNF is the primary age-related cytokine responsible for hyperreactive platelet formation. In mouse models, increased TNF induced abnormal megakaryocyte development and platelet activity. Here, we extend these findings to demonstrate that TNF drives this thrombotic phenotype through expansion of the hematopoietic stem and progenitor cell (HSPCs) compartment. Using HSPC-specific TNF receptor labeling and single cell RNA sequencing, we found that TNF receptors are absent from megakaryocytes and their progenitors (MkPs) indicating that TNF does not directly act on these cells. Chronic TNF exposure expanded HSPCs in the bone marrow and extramedullary tissues - and these expanded HSPCs retained functional repopulation capacity. Using species-specificity TNF receptor activation, we further demonstrate that TNFR1 signaling is sufficient to induce platelet hyperreactivity independent of HSPC expansion. Unlike emergency hematopoiesis, chronic TNF promoted megakaryopoiesis through the canonical hematopoietic hierarchy as demonstrated by lineage-tracing studies. Mechanistically, chronic TNF induced a distinct transcriptional program in single cell RNA sequencing of HSPCs and megakaryocytes. Together, these findings establish that chronic TNF promotes hyperreactive platelet formation not through direct effects on megakaryocytes or MkPs but by expanding and transcriptionally reprogramming HSPCs, thereby imprinting a TNF dependent program that persists through megakaryopoiesis and ultimately produces hyperreactive platelets.

Hematopoiesis sustains lifelong blood production and immunity and is strongly influenced by biological sex. Males and females differ in their hematopoietic composition and susceptibility to infection and hematologic disease. Because current models cannot readily disentangle the independent contributions of sex chromosomes versus hormones, the mechanisms underlying sexual dimorphism remain poorly understood. Zebrafish have proven to be a powerful, translatable system for studying hematopoiesis, and offer a unique opportunity to isolate hormonal contributions due to their absence of sex chromosomes. However, whether sex is regulated in the zebrafish hematopoietic system remained unknown. Here, we discovered pronounced sexual dimorphism in the adult zebrafish marrow, with compositional differences peaking at reproductive age. Transcriptome analysis of marrows revealed conservation of sex-specific programs between zebrafish and human datasets. Upon immune challenge with lipopolysaccharide, all major blood lineages exhibited sex-biased changes in their composition and transcriptomes with the largest responses in erythroid and myeloid cell types. Among these sex differences was an unexpected immunoregulation of the erythroid lineage across maturation states. These findings establish biological sex as a major regulator of zebrafish hematopoiesis and immunity, paralleling humans, and lay the groundwork necessary for future mechanistic studies on sexual dimorphism in hematologic health and disease.

Germline mutations in the RNA helicase DDX41 are the most common genetic predisposition to myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), representing 5-7% of AML. Over 70% of patients acquire a somatic mutation at specific hotspots (R525H/G530D) in the helicase domain of DDX41 in trans, which is linked to disease progression. DDX41 has diverse cellular functions, including RNA splicing, ribosome biogenesis, R-loop resolution and inflammation. However, the mechanisms by which somatic DDX41 mutations drive MDS/AML pathogenesis are yet to be determined. Here, we systematically evaluated the function of pathogenic, missense germline and somatic DDX41 mutations. We report that DDX41 somatic mutations are gain of function alleles and fail to rescue AML cell growth, distinct from germline DDX41 mutations. DDX41 somatic mutations R525H and G530D drive widespread splicing changes in isogenic AML cell lines, consistent in genetically engineered murine bone marrow HSPCs and CD34+ cells from DDX41-mutant MDS/AML patients. Mechanistically, DDX41-R525H exhibits increased RNA binding affinity compared to wildtype, with aberrantly spliced RNA targets overlapping with enriched binding. Strikingly, aberrant splicing of SEPTIN7 is enriched exclusively in CD34+ HSPCs of patients harboring both germline and somatic DDX41 mutations but absent in samples with germline mutation alone or lacking DDX41 mutations altogether. SEPTIN7 mis-splicing introduces a premature stop codon resulting in its downregulation at the protein level. SEPTIN7 is a known regulator of cytokinesis, HSC polarity and repopulation capacity. Our results identify a novel mis-spliced candidate, SEPTIN7, whose deregulation implicates a mechanism by which the DDX41 somatic mutations promote dysplastic hematopoiesis.

Hematopoietic stem and progenitor cells (HSPC) depend on interactions with nearby stromal cells to support their homeostatic functions and mount responses to perturbations. The murine bone marrow (BM) niche can be profiled using single-cell RNA sequencing (scRNA-seq), but there is considerable inconsistency in cell type identification, and uncertainty over how populations can be isolated prospectively by flow cytometry. Here, we produced new scRNA-seq reference datasets using purified populations of murine BM stromal cells, which we used to create a bespoke classifier for stromal cells. Our NicheScribe approach is consistent, accurate, and generalizable to query datasets irrespective of methods used for cell isolation. Moreover, consistent annotation permits comparative analyses of the same stromal cells across different bone types and perturbations like inflammation, neoplasia, and aging. Our work provides new tools for consistent cell type annotation in the murine BM niche and new insights for combined molecular and functional analyses.

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.

The mammalian BRG-/BRM-associated factor (BAF) chromatin remodeling complexes are crucial regulators of hematopoietic stem and progenitor cell (HSPC) fate. Expression of the ncBAF subcomponent, BRD9, is altered in both normal aging and myeloid malignancies. We find that in adult human HSPC, BRD9 regulates the differentiation of myeloid progenitors. Upon degradation of BRD9 in these cells, ncBAF is displaced from the chromatin, leading in turn to enhanced recruitment of cBAF to bona fide CEBPA target sites. With the shift from ncBAF to cBAF, HSPC differentiation results in increased generation of myeloid-derived suppressor cells (MDSC) that secrete elevated levels of IL-8. Soluble factors from these myeloid cells, which have lost BRD9, enhance the stemness of breast cancer cells in vitro. In contrast, bromodomain inhibition of BRD9 results in minimal alterations of ncBAF binding and myeloid differentiation, indicating that BRD9 acts as a barrier to human MDSC differentiation in a bromodomain-independent manner.

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.