Pediatric acute myeloid leukemia (AML) frequently involves chromosomal rearrangements, particularly KMT2A (MLL) gene fusions, with the KMT2A-MLLT4 (KM4-AML) fusion being highly aggressive and associated with poor prognosis. This study presents a comparative analysis of transcriptomic and epigenetic profiles between human leukemia models harboring KM4-AML and KMT2A-MLLT3 (KM3-AML) fusions. Transcriptomic signatures of KM4-AML models closely resembled those of patient samples, underscoring the model's clinical relevance. Epigenetic analysis revealed distinct chromatin accessibility and methylation patterns between KM4-AML and KM3-AML, particularly in enhancer regions linked to aggressive leukemia phenotypes. PROM1 was identified as a potential biomarker for KM4-AML, with its overexpression correlating with poor prognosis. Furthermore, high-throughput drug screening identified dopamine receptor antagonists (DRAs) as promising therapeutic candidates showing selective response for KM4-AML models. Docking simulations and NMR assays confirmed favorable binding interactions between DRAs and S100A8, S100A9 and ANXA6. These findings provide insights into the molecular mechanisms driving KM4-AML and offer potential therapeutic avenues for treatment.

Generating functional haematopoietic stem cells (HSCs) from human pluripotent stem cells remains a major goal for both regenerative medicine and the study of early human blood development. Achieving this requires a clear understanding of how HSC identity is established during embryogenesis and how these developmental cues can be recreated in vitro. Recent work from Prof. Hanna Mikkola's group has defined transcriptional signatures and niche interactions that mark the emergence and maturation of human HSCs in vivo. Complementary studies from Professor Elizabeth Ng's team have developed human pluripotent stem cell-based systems that recapitulate key stages of this ontogeny, generating haemogenic endothelium and HSC‑like cells capable of long‑term multilineage engraftment. In this perspective, drawing on the recent ISEH NIC webinar presented by Profs. Mikkola and Ng, we integrate insights from both developmental and engineered systems to outline shared principles of human HSC formation and highlight opportunities to refine in vitro strategies for producing fully functional HSCs. Teaser Abstract What can human development teach us about building haematopoietic stem cells in the laboratory? Inspired by presentations from Profs. Mikkola and Ng at the 2025 ISEH NIC webinar, this perspective highlights the developmental and engineering principles that underpin human HSC formation and considers how these insights are advancing efforts to generate durable, engraftable HSCs for translational and regenerative applications.

Chromatin remodeling by mammalian SWI/SNF complexes is essential for hematopoietic lineage specification and differentiation. Although the canonical BAF subunits ARID1A and ARID1B are recurrently altered in B-cell malignancies, their overlapping and stage-specific functions during B-cell development remain incompletely understood. Here, we used CD19-Cre-mediated deletion of Arid1a and Arid1b to define their roles in B-cell development and hematopoietic transformation in vivo. Loss of either Arid1a or Arid1b impaired B-cell differentiation, reduced immature and recirculating B-cell output, and compromised germinal center responses following antigen challenge, with more pronounced defects following combined deletion. Unexpectedly, combined Arid1a/Arid1b loss resulted in a lethal, transplantable myeloid leukemia rather than a B-cell malignancy. CD19-Cre lineage tracing and allele-recombination analysis identified a subset of multipotent progenitors carrying CD19-Cre activity, while combined deletion was associated with selective MPP3 expansion and impaired myelo-erythroid colony-forming capacity. Transcriptional profiling of Arid1a/Arid1b-deficient MPP2/3 cells revealed loss of HSPC- and lymphoid-associated programs together with aberrant activation of erythroid and megakaryocyte-erythroid transcriptional states, without acquisition of a conventional mature myeloid program. These findings reveal overlapping functions of ARID1A and ARID1B in B-cell development and support a broader role for canonical BAF complexes in maintaining hematopoietic lineage fidelity and constraining malignant transformation.

Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are driven by canonical JAK2, CALR, or MPL mutations that constitutively activate JAK-STAT signaling. Yet, the clinical reality reveals a sobering paradox: JAK inhibitors modify symptoms but fail to eradicate the malignant clone, reduce mutant allele burden, or reverse fibrosis. This therapeutic ceiling suggests that oncogenic signaling alone cannot explain disease persistence, progression, or transformation to secondary acute myeloid leukemia. Emerging evidence iMPLicates epitranscriptomics, the dynamic chemical modification of RNA, as an underexplored but fundamentally critical layer of regulatory control in MPN pathobiology. RNA modifications, particularly N6-methyladenosine (m[6]A), are orchestrated by specialized "writers" (METTL3/METTL14), "erasers" (FTO/ALKBH5), and "readers" (YTHDF/IGF2BP proteins) that collectively govern transcript stability, translation, splicing, and localization. In MPNs, this machinery is transcriptionally dysregulated rather than mutated, creating a feed-forward oncogenic loop wherein JAK-STAT signaling upregulates m[6]A writers, which in turn stabilize and translate key oncogenes (MYC, BCL2, JAK2 itself) while suppressing tumor suppressors. Moreover, metabolic shifts during disease progression, particularly IDH1/2 mutations that generate the oncometabolite 2-hydroxyglutarate, may alter α-ketoglutarate-dependent RNA demethylase activity and thereby provide a plausible link between metabolic remodeling and epitranscriptomic dysregulation during leukemic transformation. This review synthesizes the biological rationale, mechanistic evidence, and emerging therapeutic strategies targeting epitranscriptomic regulators in MPNs. We propose that epitranscriptomic remodeling represents both a driver of clonal fitness and a therapeutic vulnerability, positioning RNA modification pathways as promising frontiers for disease-modifying intervention in these recalcitrant malignancies.

The nuclear oncoprotein SET (patient "SE" translocation) has been implicated in the etiology of MLL (KMT2A) induced leukemia. Here we examine the details of this dependency in murine, primary hematopoietic cells. ChIP, RNAseq and immunoprecipitations demonstrated Set as downstream target of HoxA9 and confirmed a direct interaction with Mll. Set and Mll globally co-localized to promoter regions. Impairing Set expression phenocopied a knockdown of select Mll fusion targets with Set driving a pro-proliferative gene expression program that contained features indicative for Mll involvement. Molecularly, Set depletion caused dissociation of Mll from chromatin accompanied by a selective loss of RNA PolymeraseII Ser2-P. Concomitant with a function of Set as inhibitor of protein phosphatase 2A (PP2A), specific recruitment of PP2A to the Meis1 promoter, a known Mll target, inhibited transcription in reporter assays and in the natural chromatin environment. We identified Mitogen and stress induced kinase 1 (Msk1) as potential substrate protected by Set from dephosphorylation. Phosphorylated Msk1-P colocalized with Mll and disappeared from chromatin upon Set depletion. Biochemically, Msk-1 bound directly to Mll as well as to menin, a known Mll tethering factor. Loss of Set/Mll/Msk1 selectively affected H3K14 acetylation at promoters and this could be partially attributed to the reduced presence of the histone acetyltransferase Moz (Kat6a). Finally, we show that kinase inhibitors augment menin inhibitor activity in MLL leukemia cells, indicating a connection between cellular signaling and MLL-function.

The human PROMININ-1 (PROM-1, CD133), detected by its AC133 epitope, is expressed on the surface of leukemia-initiating cells of Philadelphia chromosome-positive (Ph+) leukemia cells. Ninety-five percent of chronic myeloid leukemia (CML) cases are Ph+. Ph+ acute lymphoblastic leukemia (ALL) accounts for twenty-five to thirty percent of adult ALL and is considered a high-risk ALL subgroup. Targeting the t(9;22)-fusion protein BCR::ABL1 with ABL1-directed kinase inhibitors (TKIs) is a proven therapeutic concept for CML and Ph+ ALL. Resistance against TKI comprises mutations in BCR::ABL1, but many patients exhibit a primary resistance with unknown mechanisms. Here, we investigated the impact of PROM-1 on the leukemogenesis and the therapy response of Ph+ leukemias. We applied flow cytometry, co-immunoprecipitation, immunoblotting, stem cell assays, and leukemia induction in vivo to cell lines, syngeneic mouse models, and human Ph+ long-term cultures. We found a direct relationship between BCR::ABL1 and PROM-1, a shift from CML towards ALL upon the loss of Prom-1, as well as a link between the response to TKIs and the PROM-1 expression. In contrast to solid tumors, resistance was accompanied by a progressive loss of PROM-1. Our data suggest that PROM-1 plays an essential role in both cell fate decision and induction of the BCR::ABL1-related leukemic phenotype, highlighting its impact on the drug response of Ph+ leukemias. Teaser abstract The Philadelphia chromosome (Ph) is the product of the t(9;22), which fuses the BCR gene on chromosome 22 to the ABL1 gene on chromosome 9. The result is the BCR::ABL1 oncogene, which is responsible for the induction of chronic myeloid leukemia (CML) and for about a third of acute lymphoblastic leukemia (Ph+ ALL). Only little is known about how t(9;22) induces either CML or ALL, and why there is a difference between the two diseases regarding their response to inhibitors that can inactivate BCR::ABL1. Here, we show that a gene called PROMININ-1 is involved in both processes: determining whether BCR::ABL1 induces CML or ALL, and explaining the weaker response of Ph+ ALL compared to CML to inhibitors of BCR::ABL1. We found that the reduction of PROMININ-1 increased the proportion of ALL induced by BCR::ABL1 in the mouse. We could not reproduce these findings in humans but distinguish CML from Ph+ ALL by the expression of PROMININ-1. Also resistance to BCR::ABL1 inhibitors was associated with low expression of PROMININ-1 in Ph+ ALL. These findings are in contrast to observations in solid tumors where a higher level of PROMININ-1 is accompanied by therapy resistance. Our findings contribute to a better understanding of the different types of Ph+ leukemia and their response to therapy, which will help patients better manage these diseases.

Aldehyde dehydrogenase 2 (ALDH2) is a key mitochondrial enzyme that detoxifies reactive aldehydes including formaldehyde, thereby mitigating their genotoxic effects. While ALDH2 deficiency exacerbates bone marrow (BM) failure in Fanconi anemia models, its role under exogenous genotoxic stress is less clear. In this study, we used Aldh2[-/-] mice to investigate how ALDH2 deficiency impacts hematopoiesis following treatment with busulfan (BSF), sublethal total body irradiation (TBI), or a combined (BSF+TBI) regimen. Under BSF or TBI monotherapy, Aldh2[-/-] mice exhibited moderate increases in DNA damage, measured by γH2AX expression in hematopoietic stem and progenitor cells (HSPCs), with a mild decline in BM cell engraftment, compared to Aldh2[+/+] controls. However, BSF+TBI treatment triggered more severe HSPC DNA damage confirmed by concordant γH2AX and 53BP1 elevation, and a sharper decline in mature hematopoietic cells. Notably, this dual stress significantly upregulated DNA repair and metabolic adaptation genes, including Ccno, Pold4, Taf1c, Npr2, and Aldh18a1. These results suggest that BSF+TBI treatment drives advanced DNA damage and compensatory gene expression shifts in the absence of ALDH2. We conclude that the impact of ALDH2 deficiency on hematopoiesis is dependent on the severity of exogenous stress. TEASER ABSTRACT: The ALDH2 rs671 loss-of-function polymorphism is carried by 30-40% of East Asian individuals, yet its hematopoietic consequences under clinical genotoxic stress remain poorly understood. Using Aldh2[-/-] mice, we demonstrate that ALDH2 deficiency increases DNA damage in hematopoietic stem and progenitor cells (HSPCs) following busulfan (BSF) or total body irradiation (TBI) monotherapy, without altering mature hematopoietic cell populations or long-term repopulating capacity. However, combined BSF+TBI treatment unmasks a clear, stress-severity-dependent phenotype: Aldh2[-/-] mice exhibit significantly greater HSPC apoptosis and DNA damage confirmed by concordant γH2AX and 53BP1 elevation, and more severe peripheral blood cytopenias. Transcriptomic analysis reveals compensatory upregulation of DNA repair pathways and Aldh18a1, suggesting a metabolic adaptation to mitigate genotoxic burden. These findings establish ALDH2 deficiency as a latent hematopoietic risk factor with direct implications for personalizing conditioning regimens in ALDH2 rs671 carriers undergoing hematopoietic stem cell transplantation.

Ikaros family zinc finger 1 (IKZF1) deletions occur commonly in patients with B-cell acute lymphoblastic leukemia (B-ALL) and predict poor outcomes. Ikaros isoform 6 (IK6) is a functionally defective splicing isoform of IKZF1. We aimed to explore the effect of IK6 on the biological functions of acute lymphoblastic leukemia (ALL) cells and its mechanism. We constructed SUP-B15 (human acute lymphoblastic leukemia cell line) cells (Ph-positive) with IK6 knocked down. Western blot was used to detect the expression of IK6 protein. Cell Counting Kit-8 (CCK-8) was used to detect cell proliferation and drug sensitivity. The propidium iodide (PI) assay method was used to detect the cell cycle. The Annexin V-PI assay method was used to detect cell apoptosis. The target cells were cocultured with human stromal cells, and cell migration ability was detected using the Transwell method. RNA sequencing results of SUP-B15 cells interfering with IK6 were analyzed using gene set enrichment analysis (GSEA) software. SUP-B15 cells with IK6 knockdown lost their proliferation advantage and exhibited a decreased proportion in the G0/G1 phase and an increased proportion in the S phase, with downregulation of CDK4, P27, and BCL-2. The sensitivity to dexamethasone increased, but there was no significant change in susceptibility to imatinib and dasatinib. The migration ability of SUP-B15 cells with IK6 knockdown significantly decreased when cocultured with human stromal cells. gene set enrichment analysis (GSEA) in IK6 knockdown SUP-B15 cells showed several signaling pathways altered, which are involved in cell adhesion and migration-related pathways, such as the leukocyte transendothelial migration signaling pathway, the vascular endothelial growth factor (VEGF) signaling pathway, the cell function-related mitogen-activated protein kinase (MAPK) signaling pathway, and so on. IK6 expression affects the biological characteristics of ALL cells and may change the sensitivity of cells to dexamethasone. These alterations may relate to some signaling pathways (such as the MAPK pathway).

Hematopoietic stem cells (HSCs) are important for human health and clinical therapy. A heterogeneous pool of HSCs sustain blood production through life by balancing self-renewal and multilineage differentiation. Aging is associated with a decline in the function of the HSC pool and hematopoietic perturbations. Additionally, aging correlates with clonal hematopoiesis, driven by the accumulation of genetic mutations in this long-lived cell population that can lead to altered cell function and ultimately to leukemic transformation. Alongside their natural role in sustaining hematopoiesis, healthy HSCs are also used clinically for their regenerative capacity in stem cell transplantation, in which they reconstitute the entire adult blood system and can cure a range of blood disorders. Within these contexts, the term fitness is regularly used but often poorly defined. In this perspective, we summarize two distinct types of HSC fitness, clonal fitness and stem cell fitness. We then introduce mechanisms known to shape each and discuss the therapeutic implications for modulating HSC fitness mechanisms.

Hematopoietic stem cells (HSCs) self-renew and generate all blood cell lineages. During cytokinesis, the midbody is asymmetrically inherited by one daughter cell or symmetrically released, but whether these patterns affect HSC fate remains unclear. We developed a fusion protein of humanized monomeric Kusabira-Orange 2 (hmKO2) and MgcRacGAP to trace midbody inheritance and daughter-cell fate by time-lapse imaging. Using this system, we found that midbody release was associated with delayed progression to the next division but not with HSC lineage potential, suggesting that the midbody remnant may regulate cell-cycle progression.

Mast cell-derived histamine restrains hematopoietic stem cell activity by shaping the bone marrow niche. Blocking histamine signaling with the United States Food and Drug Administration (FDA)-approved antihistamine cetirizine expands the stem and progenitor pool and accelerates hematopoietic recovery after transplant, pointing to a repurposable strategy for boosting blood regeneration in patients.

Infectious complications remain a leading cause of early mortality in patients with acute myeloid leukemia (AML), yet the contribution of qualitative neutrophil abnormalities to infectious risk remains poorly understood. We prospectively evaluated circulating neutrophil phenotypes and oxidative burst reserve in 27 consecutive patients with newly diagnosed AML treated at two tertiary referral centers. Neutrophil subsets were characterized by multiparameter flow cytometry according to CD16/CD62L expression and compared with those of healthy controls (n = 10) and AML patients in complete remission after induction therapy (n = 10). At diagnosis, patients exhibited expansion of activated-like N1 and immature N2B subsets, contraction of the predominant homeostatic N2A population, and impaired oxidative burst reserve (all p < 0.05). Both phenotypic abnormalities and oxidative burst partially recovered after induction therapy. Infection-related mortality occurred in 22.2% of patients. After adjustment for age, the log-transformed N1/N2A ratio remained independently associated with infection-related mortality (adjusted odds ratio, [OR] 4.90; 95% CI, 1.17-20.49; p = 0.029). The ratio also demonstrated moderate discriminatory performance (area under the curve [AUC], 0.774; 95% CI, 0.585-0.963; p = 0.049), and the optimal cutoff (-2.022) identified patients with significantly inferior infection-related survival (hazard ratio [HR], 12.83; 95% CI, 2.62-62.90; log-rank p = 0.0017). In addition, higher log(N1/N2A) values were associated with lower oxidative burst reserve (Spearman r = -0.45; p = 0.018). These findings suggest that neutrophil phenotypic remodeling reflects innate immune dysfunction in AML and that the log-transformed N1/N2A ratio may serve as a practical biomarker for early identification of patients at increased risk of infection-related mortality.

Hematopoietic stem cells (HSCs) are essential for the reconstitution of the hematopoietic and immune systems and are widely used in transplantation and emerging cell-based therapies. Cryopreservation is a critical technology enabling long-term storage, banking, and distribution of HSC grafts and immuno-oncology cell products. However, the cryopreservation process exposes stem cells and progenitors to multiple sources of cryoinjury, including intracellular ice formation, osmotic shock, solute effects, and ice recrystallization, which can compromise post-thaw viability, recovery, and functional potency. For decades, dimethyl sulfoxide (DMSO) has remained the gold-standard cryoprotective agent (CPA) due to its ability to reduce osmotic stress, limit intracellular ice formation, and stabilize cellular structures. Despite its widespread use, it is associated with dose- and time-dependent cytotoxicity and adverse infusion-related reactions, motivating efforts to reduce or replace its use. This review summarizes key cryobiological principles underlying HSC preservation, including the importance of optimized CPA exposure, controlled cooling, rapid thawing, and storage below glass transition temperature. We also discuss established and emerging permeating and nonpermeating CPAs, including sugars, polymers, and carbohydrate-based ice recrystallization inhibitors (IRI), several of which have shown to improve post-thaw outcomes and engraftment in preclinical models. Finally, we review strategies to mitigate DMSO toxicity, including reduced-DMSO formulations and newly developed "all-in-one" DMSO-free cryosolutions. Collectively, these advances are driving the evolution of safer and more effective cryopreservation strategies for HSC transplantation and next-generation cellular therapeutics.

Acute myeloid leukemia (AML) exhibits heterogeneous outcomes and lacks reliable prognostic markers. As a critical regulator of cell fate, the prognostic value of calcium signaling in AML requires investigation. This study aimed to construct a calcium-related gene (CRG)-based prognostic model for AML. Differential analysis on RNA-seq data was conducted for AML from The Cancer Genome Atlas and Gene Expression Omnibus (GEO). Intersecting differentially expressed genes and CRGs yielded AML-associated differentially expressed CRGs (DECRGs). A prognostic model was developed using univariate/multivariate Cox regression and least absolute shrinkage and selection operator (LASSO) and validated in a GEO dataset. Bioinformatics analyses explored the links between risk groups and immune characteristics, genomic mutations, and drug sensitivity. Key genes' effects on cell proliferation, apoptosis, and differentiation were verified in vitro using CCK-8 assay, colony formation assay, and flow cytometry. The 13-DECRG-based model distinguished high- and low-risk patients in both training and validation cohorts, with high-risk patients showing a worse prognosis. The risk score was an independent prognostic factor. Immune analysis revealed a unique immune microenvironment for the high-risk group. CAMK2A overexpression inhibited cell proliferation and colony-forming ability, promoted cell apoptosis, and induced an increased proportion of CD11b- and CD14-positive cells. In vitro experiments indicated CAMK2A-induced suppression of AML cells' malignant phenotype by activating the P53 signaling pathway. An AML CRG-based model with favorable risk stratification performance was constructed. In vitro experiments revealed CAMK2A-induced inhibition of the malignant phenotype via suppressing proliferation, promoting apoptosis, and facilitating myeloid differentiation in AML cells. This study provides novel evidence for understanding CRGs in AML as well as the potential functions of CAMK2A.