Dual inhibition of tubulin and DNMT by the novel letermovir derivative H62 blocks leukemia.

Retinoic acid (RA), a bioactive metabolite of vitamin A, plays roles in early embryogenesis and hematopoietic development. However, its precise function in directing the hematopoietic lineage outcomes of human pluripotent stem cells (hPSCs) remains unclear. Here, we uncovered a distinct, stage-specific role for RA as a lineage-specifying modulator during late-stage hematopoietic differentiation, rather than as a promoter of hematopoietic progenitor generation. Using a stepwise hPSC differentiation system, we demonstrated that RA exerted minimal or inhibitory effects when applied during early mesoderm or hemogenic endothelial stages. In contrast, RA treatment during days 13-15 significantly enhanced progenitor maturation, proliferation, and functional output. Notably, RA acted cooperatively with external cytokines to modulate lineage fate. In the presence of erythropoietin (EPO), RA strongly promoted erythroid differentiation by activating EPOR signaling and upregulating erythroid transcriptional programs, including GATA1, KLF1, and globin gene expression. Conversely, under GM-CSF/M-CSF stimulation, RA biased progenitor differentiation toward macrophages, consistent with its role as an amplifier of the prevailing cytokine-directed lineage fate rather than an independent suppressor of erythropoiesis. These effects were highly dose- and context-dependent, with low-dose RA optimally enhancing lineage bias without cytotoxicity. Importantly, RA modulated the transcriptional and proliferative dynamics of committed progenitors. Taken together, our findings reveal a previously unrecognized role of RA as a versatile and tunable modulator of hematopoietic lineage fate that offers a novel strategy for in vitro blood cell engineering. This study advances approaches for lineage-specific blood production relevant to disease modeling, drug screening, and regenerative medicine.

Aberrant splicing of MBD1 reshapes the epigenome to drive convergent myeloerythroid defects in MDS.

Erythroid enucleation is the final stage of erythroid terminal differentiation and involves the separation of an orthochromatic erythroblast into two daughter cells - a pyrenocyte containing the extruded nucleus, and a reticulocyte, which will become a red blood cell. Our previous work has identified CDK9 as a regulator of erythroid enucleation that appears to act independently of its known role in regulating RNA polymerase II transcription, suggesting the potential for a new CDK9 role. Using a co-immunoprecipitation and mass spectrometry approach, we here identified the interactome of CDK9 in differentiating erythroblasts. We show that CDK9 interacts with a RanGTP-importin-β complex during erythroid terminal differentiation, and inhibition of importin-β in erythroblasts blocks erythroid enucleation. Using imaging analysis and functional assays of enucleating erythroblasts, we show that CDK9 and importin-β colocate at a crucial site of activity opposite to the nucleus before nuclear extrusion and we describe a novel finding that physically links CDK9 and importin-β activity prior to calmodulin and Ca2+ signalling, and subsequent F-actin activity, to achieve enucleation.

Glucocorticoid receptor (GR) is a critical regulator of erythroid progenitor proliferation, while Nemo-like kinase (NLK) is reported to be hyperactivated in Diamond-Blackfan anemia (DBA), suggesting a possible cross talk. Here, we demonstrate that NLK directly interacts with multiple domains of GR and promotes its ubiquitin-mediated proteasomal degradation. Co-immunoprecipitation assays confirmed endogenous NLK-GR interaction in K562 cells, enhanced by proteasomal inhibition. NLK overexpression downregulated levels of GR in a kinase-dependent manner across HEK293T, K562, and MEL cells, an effect reversed by MG132 or a ubiquitination-defective mutant. NLK directly phosphorylated GR at Ser226, as shown by in vitro kinase assays and site-specific immunoblotting. Conversely, NLK depletion reduced basal GR phosphorylation while increasing total GR. We identified OTS167 as a direct NLK inhibitor through cellular thermal shift and kinase assays. OTS167 suppressed NLK autophosphorylation and decreased Ser226 phosphorylation of GR, stabilizing GR protein. Functionally, among all the inhibitors tested, OTS167 maximally inhibited proliferation of K562 and MEL cells by 40-90%. OTS167 also induced erythroid differentiation in K562 and MEL by increasing CD71/TER119 expression and benzidine-positive cells by 60-80%, while NLK overexpression inhibited hemin-induced benzidine staining by 25%. In primary human CD34+ cells, NLK and GR exhibited inverse temporal expression during erythropoiesis. OTS167 or dexamethasone expanded CD71+ and CFU-E populations and enhanced proliferation (Ki67+) across BFU-E, CFU-E, and proerythroblast stages. Conversely, dexamethasone upregulated NLK, suppressing GR and suggesting a feedback loop. Thus, NLK-mediated GR downregulation constrains erythropoiesis, and its inhibition by OTS167 promotes erythroid expansion, revealing a targetable pathway in erythroid disorders.

CTDSPL2: A Comprehensive Review from Molecular Structure to Clinical Applications.

Erythroid differentiation requires precise regulation of transcription factor binding to chromatin targets as hematopoietic progenitors relinquish multipotency and activate lineage programs. GATA2 maintains progenitor identity and is thought to be progressively silenced as GATA1 levels rise. However, the precise changes in GATA2 chromatin binding kinetics during this transition remain undefined. Here, we combined live-cell single-molecule imaging in cell lines and primary mouse progenitors with CUT&Tag chromatin profiling to define GATA2 activity during erythropoiesis. Single-molecule tracking resolved two interaction modes: short-lived (<1s) searching interactions and long-lived (>5 s) binding. Surprisingly, early erythroid differentiation was characterized by a transitory strengthening of long-lived GATA2 chromatin engagement. This manifested as increased residence time of GATA2 bound to chromatin in G1E-ER4 cells and an expansion of the long-lived bound population in HPC7 cells and primary mouse progenitors. This transitory phase of enhanced engagement declined upon further differentiation. Genome-wide mapping identified regulatory elements selectively occupied by GATA2 during this early transition state, revealing promoter-proximal sites enriched for GATA/RUNX motifs and distal elements containing composite GATA/E-box signatures. Together, our imaging and chromatin profiling indicate that GATA2 chromatin engagement is kinetically remodeled at the onset of differentiation, with early recruitment targets partitioning into distinct promoter- and enhancer-associated subclasses. These results support a model in which transcription factor kinetics constitute a dynamic chromatin engagement layer that characterizes the GATA2-to-GATA1 transition.

Sickle cell disease (SCD) is caused by a point mutation in the β-globin gene that promotes hemoglobin polymerization, leading to chronic hemolytic anemia, vaso-occlusive episodes, and progressive organ damage. The most efficacious therapies focus on reactivating fetal hemoglobin (HbF) expression to mitigate the pathological effects of sickle hemoglobin (HbS) polymerization. However, the predominantly used HbF inducer, hydroxyurea (HU), exhibits substantial interpatient variability in efficacy, and curative approaches such as gene therapy remain inaccessible to the vast majority of patients. Although all SCD patients share the same causative HBB glu7val mutation, differences in genetic background significantly influence disease severity and therapeutic response. We describe a SCD-specific induced pluripotent stem cell (iPSC) platform as a renewable and scalable preclinical model to interrogate treatment responses across the genetically diverse SCD patient population. By generating patient-specific iPSC-derived erythroblasts (iEry) representing distinct SCD genetic backgrounds, we demonstrate that this system faithfully recapitulates the heterogeneous HbF induction observed clinically in response to HU. Moreover, this platform enables the identification and evaluation of alternative therapeutic agents for HU non-responders and provides sufficient resolution to dissect drug-specific effects on erythroid differentiation and cellular phenotypes. Together, these findings support the use of iPSC-derived erythroid models as a versatile tool to advance precision therapeutic strategies for SCD.

To analyze erythropoietin (EPO) secretion and bone marrow proliferation under conditions of renal anemia with varying degrees of kidney injury. First, A cross-sectional analysis compared serum erythropoietin (EPO) and erythropoietin receptor (EPOR) levels between patients with early-stage chronic kidney disease (CKD stages 1-2) and those with advanced CKD (stage 5) who had not undergone hemodialysis or received EPO therapy (CKD5+nonHD). Bone marrow hyperplasia of two patients from each group was analyzed using Wright-Giemsa staining. Second, two C57BL/6 mouse models of progressive renal anemia were established using adenine gavage with or without unilateral pedicle ligation (8M‑N: control; 8M‑M: adenine+sham; 8M‑NM: adenine+ligation). Renal and hepatic EPO expression, bone marrow EPOR and CD71 levels, CpG island methylation in the EPO promoter, and erythroid differentiation by flow cytometry were analyzed. etc. Compared to CKD stage 1-2 patients, the CKD5+nonHD group showed significantly lower hemoglobin, eGFR, and higher creatinine and BUN levels (p<0.001). Serum EPO concentrations showed no significant differences between patient groups or experimental mouse groups with varying degrees of kidney injury (p>0.05). However, the proportion of nucleated cells to mature erythrocytes and erythroid precursors to bone marrow nucleated cells were markedly reduced in CKD5+nonHD patients compared to CKD1-2 patients (p<0.05). In animal models with progressive kidney injury (normal controls, adenine+sham, and adenine+unilateral pedicle ligation), serum creatinine, renal fibrosis as assessed by MASSON/PASM staining, CpG island methylation in the EPO promoter and hepatic EPO expression were gradually elevated (p<0.05). In contrast, hemoglobin, hematocrit, and marrow EPOR/CD71 levels and the proportions of Ter119+ nucleated erythroid cells declined progressively with worsening renal injury (8M-N>8M-M>8M-NM, p<0.001), and the proportions of CD44low/CD711ow late erythroid precursors within Ter119+ nucleated erythroid cells was significantly reduced in 8M-NM (p<0.001). In renal anemia, compensatory EPO production from extrarenal tissues may partially offsets the reduction in systemic EPO levels. However, bone marrow proliferation, particularly erythroid hematopoiesis, is significantly impaired, highlighting the critical impact of reduced erythropoiesis on anemia progression.

Stag2 dependent chromatin remodeling enforces the erythroid-specific Gata1 cistrome.

Long non-coding RNAs (lncRNAs) have emerged as important regulators of developmental processes. Recent studies have established roles for lncRNAs in human and murine erythroid regulation, yet additional regulators remain to be discovered. To identify lncRNA candidates involved in human erythroid regulation, we established a pooled genome-editing screen strategy using human embryonic stem cells (hESCs). Long Intergenic Non-Protein Coding RNA 1089 (LINC01089) was selected for functional investigation. We found that reduced LINC01089 expression impaired erythroid differentiation. Transcriptomic profiling revealed consistent downregulation of genes related to hemoglobin assembly, heme biosynthesis, and membrane maturation, suggesting that LINC01089 supports coordinated erythroid transcriptional programs. In particular, progressive reduction of HBB expression emerged as a key transcriptional anchor. Enrichment analyses of upregulated genes identified recurrent focal adhesion signatures, suggesting a potential link between LINC01089 and focal adhesion kinase (FAK)-related signaling. Given prior evidence linking LINC01089 to FAK phosphorylation, we performed a pilot FAK-inhibition experiment, producing a partial shift toward wild-type HBB expression and supporting FAK/phosphorylated FAK (pFAK) signaling as a potential contributing axis in the impaired transcriptional programs. Together, our findings identify LINC01089 as a novel lncRNA linked to coordinated heme–globin transcriptional programs in human erythroid differentiation, with possible involvement of the FAK/pFAK axis.

Erythropoiesis is a tightly regulated process involving rapid cell proliferation with orderly differentiation to ensure production of millions of RBCs. TGF-β1 is a key regulator of erythropoiesis, however, the mechanisms via which it regulates erythropoiesis are not well elucidated. Using myelodysplastic syndromes patient samples, we show that elevated TGF-β1 and SMAD2 signaling correlates with the degree of anemia. Functional studies in primary human HSPCs demonstrate that TGF-β1 exerts a bifurcated effect - suppressing proliferation and inducing premature erythroid differentiation - both of which are rescued by clinical-stage TGFBR1 inhibitor. Through integrative RNA-seq, ChIP-seq, and Micro-C analyses, we find TGF-β1 activates β-globin LCR, driving early differentiation, while concurrently disrupting the MYC enhancer-promoter interaction to block proliferation. We validate erythropoiesis defects in vivo by performing single-cell RNA-seq in a TGF-β1 overexpressing mouse model. Overall, we show that TGF-β1/SMAD2 signaling re-wires chromatin to regulate erythropoiesis by affecting the β-LCR and MYC super enhancer regions.

Transfusion-dependent hemolytic anemia caused by compound heterozygosity due to mutations in the erythroid Krüppel-like factor 1 (KLF1) gene is a rare and severe blood disorder. The clinical manifestations of the patient are mainly related to erythroid cells. Moreover, the roles of the identified KLF1 mutations in the pathophysiology of this disease remain unclear due to the lack of an appropriate study model. The advent of genome editing technology combined with the generation of patient-specific induced pluripotent stem cells (iPSCs) may provide a better understanding of the molecular mechanisms underlying this disease in an in vitro system and offer a novel therapeutic approach in the future. KLF1-mutant iPSCs were generated from patients with compound heterozygosity of KLF1 mutations, and the mutation was corrected through the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system together with a single-stranded oligodeoxynucleotide donor template (ssODN). The obtained iPSC lines were differentiated towards erythroid cells, and the disease-related phenotypes were examined. Erythroid cells derived from KLF1-mutated iPSCs had lower proliferative capacity, showed delayed maturation, and expressed lower level of the KLF1-related gene, CD44. These results were consistent with some of the phenotypes observed in the patients. After CRISPR/Cas9 gene editing, the corrected iPSCs retained pluripotency, exhibited a normal karyotype, and had undetectable off-target mutations. Importantly, some of the defects were partially restored after genetic correction of the KLF1 gene. KLF1-iPSCs presented disease-related phenotypes of compound heterozygous KLF1 mutations, which could be mediated by gene editing through CRISPR/Cas9 and ssODN. This study offers a useful strategy for studying the underlying disease mechanisms of rare diseases, which could be applied to the development of novel treatments for inherited blood disorders in the future.

Erythropoiesis is a meticulously regulated process influenced by a multitude of factors. Prior research has demonstrated that hypoxic conditions (5% O2) facilitate erythroid differentiation. Nevertheless, although our preliminary data indicate the involvement of VEGF/VEGFR2 in this process, the precise regulatory mechanism in erythropoiesis under hypoxia remains inadequately understood. In this study, an in vitro model of inhibited erythroid differentiation was developed using CD34+ hematopoietic stem cells treated with a VEGFR2-neutralizing antibody. Additionally, the signaling axis was modulated under hypoxia conditions employing the mTOR inhibitor rapamycin and TTI1-siRNA. The findings revealed that hypoxia significantly upregulated VEGF/VEGFR2 expression and promoted erythroid differentiation. Conversely, VEGFR2 inhibition resulted in a substantial decrease in erythroid surface markers and hemoglobin synthesis. Proteomics analysis suggested that TTI1 and mTOR are pivotal components of this signaling axis, with their suppression effectively impeding erythropoiesis. Under hypoxic conditions, VEGFR2 may regulate erythroid differentiation of CD34+ cells through a mechanism involving the TTI1-mTORC1 signaling pathway.

PGC-1\u03b1 agonism via oral administration of ZLN005 induces fetal hemoglobin and is antisickling in sickle mice

Heme, an iron-containing cofactor, is synthesized in mitochondria by an eight-enzyme pathway. Although cells were thought to manage heme autonomously, more than 1000 proteins contribute to its production, transport, and regulation. During terminal erythroid differentiation, mitochondria are discarded, yet hemoglobin production continues, which implies a cell-nonautonomous heme supply. We show that, under stress, erythroblasts import heme through the permease heme-responsive gene 1 (HRG1), which localizes to the plasma membrane and accumulates during stress erythropoiesis, the emergency program that expands red cell output. HRG1 loss impaired heme uptake, inhibited terminal erythroid differentiation, and caused anemia. In β-thalassemic mice, partial HRG1 loss reduces ineffective erythropoiesis, underscoring the importance of balanced heme import. These findings reveal intercellular heme sharing and identify HRG1 as a potential therapeutic target in hemoglobinopathies.

BMSC-derived exosomal ANTXR1 inhibits erythroid differentiation by suppressing the TLR4/MyD88/NF-κB signaling pathway.

First-generation genome editing therapies have largely focused on correcting or compensating for pathogenic variants. However, as these approaches enter the clinic, emerging biological constraints limit maximal therapeutic impact. Because globin genes are activated late during erythroid differentiation, genome-corrected hematopoietic stem and progenitor cells (HSPCs) gain little selective advantage in the bone marrow. Here, we establish a strategy that links therapeutic genome edits to an erythroid fitness-enhancing allele to amplify the output of clinically relevant cells. We develop a multiplex base editing strategy that couples fetal hemoglobin (HbF) reactivation with erythroid lineage expansion. Introduction of a naturally occurring erythropoietin receptor truncation (tEPOR) associated with benign erythrocytosis increased erythroid cell production without impairing viability or differentiation. Combinatorial editing of tEPOR together with the BCL11A erythroid enhancer and HBG1/2 promoters in healthy donor, sickle cell disease, and β-thalassemia HSPCs synergistically increased erythroid proliferation and HbF expression beyond single base-edited or Casgevy-treated controls. Multiplex base-edited HSPCs retained long-term lineage repopulation and engraftment capacity in vivo, establishing a modular strategy that pairs disease correction with lineage amplification to improve therapeutic potency.

Erythropoiesis is tightly regulated by lineage-specific transcription factors that govern erythroid commitment, proliferation, and differentiation. A core erythroid transcriptional network, together with non-DNA-binding cofactors, occupies regulatory regions of genes essential for erythroid development. This process is further shaped by epigenetic mechanisms, including histone post-translational modifications and long-range chromatin interactions. CCCTC-binding factor (CTCF) is a multifunctional regulator with a central role in three-dimensional chromatin organization. Although CTCF has been implicated in hematopoietic differentiation and leukemogenesis, its specific function in erythropoiesis remains poorly defined. Here, we investigated the role of CTCF during erythroid differentiation using two complementary models: pluripotent K562 leukemia cells and primary human CD34+ hematopoietic stem/progenitor cells, each induced toward the erythroid lineage by distinct stimuli. In both systems, CTCF silencing impaired erythroid differentiation by repression of key erythroid transcription factor genes, including LMO2, KLF1, MYB, and ETS1. This repression was associated with enrichment of repressive histone marks at CTCF-binding sites within their regulatory regions. Moreover, CTCF cooperated with cohesin to establish and stabilize long-range chromatin interactions at these loci. These results provide new insight into how CTCF-dependent chromatin regulation contributes to normal erythroid development and suggest that perturbation of this regulatory axis may have implications for hematopoietic disorders and malignancies.

Terminal erythroid differentiation involves dramatic cellular remodeling that culminates in the expulsion of the nucleus, a process known as enucleation. While enucleation is conserved across mammals and is crucial for the generation of fully functional erythrocytes, the mechanisms governing this process have remained largely unknown, in part because the absence of genetic material in mature, enucleated red blood cells hinders genetic experimentation. Here, we performed a pooled, forward-genetic CRISPR-Cas9 screen in enucleated red blood cells derived from primary human hematopoietic stem cells to identify genes required for enucleation. We found that Chloride Intracellular Channel 3 (CLIC3) and Vesicle-associated membrane protein 8 (VAMP8) are both necessary for terminal erythroid differentiation, yet likely act through different mechanisms. Knockdown of CLIC3 led to a delay in erythroblast differentiation, culminating in impaired enucleation. We found that the knockdown cells had increased p53 and p21 and exhibited cell cycle alterations, suggesting CLIC3 plays a crucial role in coordinating cell cycle progression during erythropoiesis. In comparison, VAMP8-depleted cells initially appear to undergo accelerated differentiation but then display a specific defect in enucleation. Transcriptional analysis of the VAMP8-knockdown cells suggested dysregulation of pathways for vesicle trafficking and actin binding, and imaging of late-stage erythroblasts revealed impaired nuclear polarization and disorganized actin. This work provides a new approach for functional genomics in enucleated cells and reveals novel factors important for terminal erythroid differentiation and enucleation.