Megakaryocytic Differentiation Research Articles

Different inflammatory, fibrotic, and immunologic signatures between pre-fibrotic and overt primary myelofibrosis.

Primary myelofibrosis (PMF) is a myeloid proliferative neoplasm (MPN) characterized by bone marrow (BM) fibrosis. Pre-fibrotic PMF (pre-PMF) progresses to overt PMF. Megakaryocytes (MKs) play a primary role in PMF; however, the functions of MK subsets and those of other hematopoietic cells during PMF progression remain unclarified. Therefore, we analyzed BM aspirates in pre-PMFs, overt PMFs, and other MPNs using single-cell RNA sequencing (scRNA-seq). We identified 14 cell types with subsets, including hematopoietic stem and progenitor cells (HSPCs) and MKs. HSPCs in overt PMF were MK-biased and inflammation/fibrosis-enriched. Among MKs, the epithelial-mesenchymal transition (EMT)-enriched subset was abruptly increased in overt PMF. MKs in non-fibrotic/non-PMF MPN were MK differentiation-enriched, whereas those in fibrotic/non-PMF MPN were inflammation/fibrosis-enriched. Overall, the inflammation/fibrosis signatures of the HSPC, MK, and CD14+ monocyte subsets increased from pre-PMF to overt PMF. Cytotoxic and dysfunctional scores also increased in T and NK cells. Clinically, MK and HSPC subsets with high inflammation/fibrosis signatures were frequent in the patients with peripheral blood blasts ≥1%. scRNA-seq predicted higher cellular communications of MK differentiation, inflammation/fibrosis, immunologic effector/dysfunction, and tumor-associated signaling in overt PMF than pre-PMF. However, no decisive subset emerged during PMF progression. Our study demonstrated that HSPCs, monocytes, and lymphoid cells contribute to PMF progression, and subset specificity existed regarding inflammation/fibrosis and immunologic dysfunction. PMF progression may depend on multiple cell types' alterations, and EMTenriched MKs may be potential targets for the diagnosis and therapy of the progression.

Compound K Promotes Megakaryocytic Differentiation by NLRP3 Inflammasome Activation.

Platelets are essential blood components that maintain hemostasis, prevent excessive bleeding, and facilitate wound healing. Reduced platelet counts are implicated in various diseases, including leukemia, hepatitis, cancer, and Alzheimer's disease. Enhancing megakaryocytic differentiation is a promising strategy to increase platelet production. Compound K (CK), a major bioactive metabolite of ginsenosides from Panax ginseng, has demonstrated anti-cancer and neuroprotective properties. In this study, we investigated the effects of CK on megakaryocytic differentiation and apoptosis in chronic myeloid leukemia (CML) cell lines K562 and Meg-01. CK treatment significantly upregulated the mRNA expression of key megakaryocytic differentiation markers, including CD61, CD41, and CD42a, and promoted the formation of large, multinucleated cells in K562 cells. Additionally, flow cytometry analysis revealed that CK at 5 µM induced apoptosis, a critical process in thrombocytopoiesis, in both K562 and Meg-01 cells. RT2 Profiler PCR array analysis further identified a marked increase in the expression of genes associated with the activation of the NLRP3 inflammasome in CK-treated K562 and Meg-01 cells. This study is the first to demonstrate that CK promotes megakaryocytic differentiation and apoptosis through the activation of the ERK/EGR1 and NLRP3 inflammasome pathways. These findings suggest that CK may enhance platelet production, indicating its potential as a therapeutic candidate for platelet-related disorders and other associated diseases.

Oxazolidinone antibiotics impair ex vivo megakaryocyte differentiation from hematopoietic progenitor cells and their maturation into platelets.

Oxazolidinones (linezolid and tedizolid) adverse reactions include thrombocytopenia, the mechanism of which is still largely unknown. In cultured cells, oxazolidinones impair mitochondrial protein synthesis and oxidative metabolism. As mitochondrial activity is essential for megakaryocyte differentiation and maturation into platelets, we examined whether oxazolidinones impair these processes ex vivo and alter, in parallel, the activity of mitochondrial cytochrome c-oxidase (CYTOX; enzyme partly encoded by the mitochondrial genome) and cell morphology. Human CD34+ cells were isolated, incubated with cytokines (up to 14 days) and clinically relevant oxazolidinone concentrations or in control conditions, and used for (i) clonogenic assays [counting of megakaryocyte (CFU-Mk), granulocyte-monocyte (CFU-GM), burst-forming unit-erythroid (BFU-E) colonies]; (ii) the measure of the expression of megakaryocyte surface antigens (CD34 to CD41 and CD42); (iii) counting of proplatelets; (iv) the measurement of CYTOX activity; and (v) cell morphology (optic and electron microscopy). Oxazolidinones caused a significant decrease in BFU-E but not CFU-Mk or CFU-GM colonies. Yet, the megakaryocytic lineage was markedly affected, with a decreased differentiation of CD34+ into CD41+/CD42+ cells, an abolition of proplatelet formation and striking decrease in the numbers of large polylobulated nucleus megakaryocytes, with a complete loss of intracellular demarcation membrane system, disappearance of mitochondria, and suppression of CYTOX activity. These alterations were more marked in cells incubated with tedizolid than linezolid. These data suggest that oxazolidinones may induce thrombocytopenia by impairing megakaryocytic differentiation through mitochondrial dysfunction. Pharmacological interventions to prevent this toxicity might therefore be difficult as mitochondrial toxicity is most probably inherently linked to their antibacterial activity.

Linear podosomes display low Cdc42 activity for proplatelet elongation by megakaryocytes

Blood platelets result from differentiation of megakaryocytes (MKs) into the bone marrow. It culminates with the extension of proplatelets (PPT) through medullar sinusoids and release of platelets in the blood stream. Those processes are regulated by contact with the microenvironment mediated by podosomes. We previously demonstrated that contact of megakaryocytes to Collagen I fibers initiated the formation of linear podosomes required for proplatelets extension and release of mature platelets. MKs linear podosomes have the particularity of displaying mechanical pulling activity but, unlike other linear podosomes, they lack the ability of digesting the extracellular matrix (ECM), as we recently demonstrated. The Cdc42 small GTPase is required for actomyosin-dependent maturation of the demarcation membrane system (DMS), a membrane reservoir for PPT and platelets components. Cdc42 is a known protein of the podosomes core, and is instrumental to accurate platelets release into the sinusoids. Indeed, FRET analysis showed that Cdc42 activity was very high and central to DMS formation. Unexpectedly, even though we found the protein in linear podosomes, almost undetectable Cdc42 activity was detected in those structures. This observation suggests that Cdc42 could also act as scaffold to assemble proteins required for PPT formation/elongation along Collagen I fibers in MKs. Eventually, we demonstrated that linear podosomes appear as points of contact between Collagen I fibers and DMS membranes, to mechanically extend PPT along Collagen bundles, independently of Cdc42 activity.

Role of the mechanotransductor PIEZO1 in megakaryocyte differentiation.

From haematopoietic stem cells to megakaryocytes (Mks), cells undergo various mechanical forces that affect Mk differentiation, maturation and proplatelet formation. The mechanotransductor PIEZO1 appears to be a natural candidate for sensing these mechanical forces and regulating megakaryopoiesis and thrombopoiesis. Gain-of-function mutations of PIEZO1 cause hereditary xerocytosis, a haemolytic anaemia associated with thrombotic events. If some functions of PIEZO1 have been reported in platelets, few data exist on PIEZO1 role in megakaryopoiesis. To address this subject, we used an invitro model of Mk differentiation from CD34+ cells and studied step-by-step the effects of PIEZO1 activation by the chemical activator YODA1 during Mk differentiation and maturation. We report that PIEZO1 activation by 4 μM YODA1 at early stages of culture induced cytosolic calcium ion influx and reduced cell maturation. Indeed, CD41+CD42+ numbers were reduced by around 1.5-fold, with no effects on proliferation. At later stages of Mk differentiation, PIEZO1 activation promoted endomitosis and proplatelet formation that was reversed by PIEZO1 gene invalidation with a shRNA-PIEZO1. Same observations on endomitosis were reproduced in HEL cells induced into Mks by PMA and treated with YODA1. We provide for the first time results suggesting a dual role of PIEZO1 mechanotransductor during megakaryopoiesis.

Enhancing terminal erythroid differentiation in human embryonic stem cells through TRIB3 overexpression

Tribbles pseudokinase 3 (TRIB3) expression significantly increases during terminal erythropoiesis in vivo. However, we found that TRIB3 expression remained relatively low during human embryonic stem cell (hESC) erythropoiesis, particularly in the late stage, where it is typically active. TRIB3 was expressed in megakaryocyte-erythrocyte progenitor cells and its low expression was necessary for megakaryocyte differentiation. Thus, we proposed that the high expression during late stage of erythropoiesis could be the clue for promotion of maturation of hESC-derived erythroid cells. To our knowledge, the role of TRIB3 in the late stage of erythropoiesis remains ambiguous. To address this, we generated inducible TRIB3 overexpression hESCs, named TRIB3tet-on OE H9, based on a Tet-On system. Then, we analyzed hemoglobin expression, condensed chromosomes, organelle clearance, and enucleation with or without doxycycline treatment. TRIB3tet-on OE H9 cells generated erythrocytes with a high proportion of orthochromatic erythroblast in flow cytometry, enhanced hemoglobin and related protein expression in Western blot, decreased nuclear area size, promoted enucleation rate, decreased lysosome and mitochondria number, more colocalization of LC3 with LAMP1 (lysosome marker) and TOM20 (mitochondria marker) and up-regulated mitophagy-related protein expression after treatment with 2 μg/mL doxycycline. Our results showed that TRIB3 overexpression during terminal erythropoiesis may promote the maturation of erythroid cells. Therefore, our study delineates the role of TRIB3 in terminal erythropoiesis, and reveals TRIB3 as a key regulator of UPS and downstream mitophagy by ensuring appropriate mitochondrial clearance during the compaction of chromatin.

Harmine promotes megakaryocyte differentiation and thrombopoiesis by activating the Rac1/Cdc42/JNK pathway through a potential target of 5-HTR2A.

Harmine (HM), a β-carboline alkaloid extracted from plants, is a crucial component of traditional Chinese medicine (TCM) known for its diverse pharmacological activities. Thrombocytopenia, a common and challenging hematological disorder, often coexists with serious illnesses. Previous research has shown a correlation between HM and thrombocytopenia, but the mechanism needs further elucidation. The aim of this study was to clarify the mechanisms underlying the effects of HM on thrombocytopenia and to develop new therapeutic strategies. Flow cytometry, Giemsa staining, and Phalloidin staining were used to assess HM's impact on Meg-01 and HEL cell differentiation and maturation in vitro. A radiation-induced thrombocytopenic mouse model was employed to evaluate HM's effect on platelet production in vivo. Network pharmacology, molecular docking, and protein blotting were utilized to investigate HM's targets and mechanisms. The results demonstrated that HM dose-dependently promoted Meg-01 and HEL cell differentiation and maturation in vitro and restored platelet levels in irradiated mice in vivo. Subsequently, HM was found to be involved in the biological process of platelet production by upregulating the expressions of Rac1, Cdc42, JNK, and 5-HTR2A. Furthermore, the targeting of HM to 5-HTR2A and its correlation with downstream Rac1/Cdc42/JNK were also confirmed. In conclusion, HM regulates megakaryocyte differentiation and thrombopoiesis through the 5-HTR2A and Rac1/Cdc42/JNK pathways, providing a potential treatment strategy for thrombocytopenia.

Megakaryocyte maturation involves activation of the adaptive unfolded protein response.

Endoplasmic reticulum stress triggers the unfolded protein response (UPR) to promote cell survival or apoptosis. Transient endoplasmic reticulum stress activation has been reported to trigger megakaryocyte production, and UPR activation has been reported as a feature of megakaryocytic cancers. However, the role of UPR signaling in megakaryocyte biology is not fully understood. We studied the involvement of UPR in human megakaryocytic differentiation using PMA (phorbol 12-myristate 13-acetate)-induced maturation of megakaryoblastic cell lines and thrombopoietin-induced differentiation of human peripheral blood-derived progenitors. Our results demonstrate that an adaptive UPR is a feature of megakaryocytic differentiation and that this response is not associated with ER stress-induced apoptosis. Differentiation did not alter the response to the canonical endoplasmic reticulum stressors DTT or thapsigargin. However, thapsigargin, but not DTT, inhibited differentiation, consistent with the involvement of Ca2+ signaling in megakaryocyte differentiation.

MiR-1915-3p regulates megakaryocytic and erythroid differentiation by targeting SOCS4

BackgroundProper control of the lineage bias of megakaryocytic and erythroid progenitor cells (MEPs) is of significant importance, the disorder of which will lead to abnormalities in the number and function of platelets and erythrocytes. Unfortunately, the signaling pathways regulating MEP differentiation largely remain to be elucidated. This study aimed to analyze the role and the underlying molecular mechanism of miR-1915-3p in megakaryocytic and erythroid differentiation.MethodsWe utilized miRNA mimics and miRNA sponge to alter the expression of miR-1915-3p in megakaryocytic and/or erythroid potential cells; siRNA and overexpression plasmid to change the expression of SOCS4, a potential target of miR-1915-3p. The expression of relevant surface markers was detected by flow cytometry. We scanned for miR-1915-3p target genes by mRNA expression profiling and bioinformatic analysis, and confirmed the targeting by dual-luciferase reporter assay, western blot and gain- and lost-of-function studies. One-way ANOVA and t-test were used to analyze the statistical significance.ResultsIn this study, overexpression or knockdown of miR-1915-3p inhibited or promoted erythroid differentiation, respectively. Accordingly, we scanned for miR-1915-3p target genes and confirmed that SOCS4 is one of the direct targets of miR-1915-3p. An attentive examination of the endogenous expression of SOCS4 during megakaryocytic and erythroid differentiation suggested the involvement of SOCS4 in erythroid/megakaryocytic lineage determination. SOCS4 knockdown lessened erythroid surface markers expression, as well as improved megakaryocytic differentiation, similar to the effects of miR-1915-3p overexpression. While SOCS4 overexpression resulted in reversed effects. SOCS4 overexpression in miR-1915-3p upregulated cells rescued the effect of miR-1915-3p.ConclusionsmiR-1915-3p acts as a negative regulator of erythropoiesis, and positively in thrombopoiesis. SOCS4 is one of the key mediators of miR-1915-3p during the differentiation of MEPs.

Identification of Active Molecules against Thrombocytopenia through Machine Learning.

Thrombocytopenia, which is associated with thrombopoietin (TPO) deficiency, presents very limited treatment options and can lead to life-threatening complications. Discovering new therapeutic agents against thrombocytopenia has proven to be a challenging task using traditional screening approaches. Fortunately, machine learning (ML) techniques offer a rapid avenue for exploring chemical space, thereby increasing the likelihood of uncovering new drug candidates. In this study, we focused on computational modeling for drug-induced megakaryocyte differentiation and platelet production using ML methods, aiming to gain insights into the structural characteristics of hematopoietic activity. We developed 112 different classifiers by combining eight ML algorithms with 14 molecule features. The top-performing model achieved good results on both 5-fold cross-validation (with an accuracy of 81.6% and MCC value of 0.589) and external validation (with an accuracy of 83.1% and MCC value of 0.642). Additionally, by leveraging the Shapley additive explanations method, the best model provided quantitative assessments of molecular properties and structures that significantly contributed to the predictions. Furthermore, we employed an ensemble strategy to integrate predictions from multiple models and performed in silico predictions for new molecules with potential activity against thrombocytopenia, sourced from traditional Chinese medicine and the Drug Repurposing Hub. The findings of this study could offer valuable insights into the structural characteristics and computational prediction of thrombopoiesis inducers.

The involvement of krüppel-like transcription factor 2 in megakaryocytic differentiation induction by phorbol 12-myrestrat 13-acetate

BackgroundMegakaryocytic differentiation is a complicated process regulated by a series of transcription factors in a context- and stage-dependent manner. Recent studies have suggested that krüppel-like transcription factor 2 (KLF2) is involved in the control of embryonic erythroid precursor cell differentiation and maturation. However, the function and mechanism of KLF2 in regulating megakaryocytic differentiation remain unclear.MethodsThe expression patterns of krüppel-like transcription factors (KLFs) during megakaryocytic differentiation were identified from public databases. Phorbol 12-myristate 13-acetate (PMA) treatment of the myeloid-erythroid-leukemic cell lines K562 and HEL were used as cellular megakaryocytic differentiation models. A lentiviral transduction system was utilized to achieve the goal of amplifying or reducing KLF2. The expression of KLF2 was examined using real-time PCR and western blot. The impact of KLF2 on the megakaryocytic differentiation of K562 cells was examined by flow cytometry, Giemsa staining, Phalloidin staining and western blot. RNA-sequencing (RNA-seq) and chromatin immunoprecipitation-sequencing (ChIP-seq) technologies were used to identify the KLF2-regulated targets.ResultsKLF2 is increased in the maturation process of megakaryocytes. KLF2 overexpression accelerated the PMA-induced megakaryocytic differentiation, as reflected by an increased percentage of CD41/CD61 cells, an increased number of polyploid cells, and an elevated expression of P21 and P27. KLF2 knockdown exhibited the opposite results, indicating that KLF2 knockdown suppressed the megakaryocytic differentiation. Further, combination of the RNA-seq and ChIP-seq results suggested that chimerin 1 (CHN1) and potassium voltage-gated channel subfamily Q member 5 (KCNQ5) may be target genes regulated of KLF2. Both CHN1 and KCNQ5 knockdown could block the megakaryocytic differentiation to some content.ConclusionThis study implicated a regulatory role of KLF2 in megakaryocytic differentiation, which may suggest KLF2 as a target for illness with abnormal megakaryocytic differentiation.

Prospective genetic germline evaluation in a consecutive group of adult patients aged

Relevance of germline (GL) predisposition in myelodysplastic syndromes (MDSs) was stressed in both 2022 WHO and International Consensus classifications, but its incidence is probably underestimated, especially in young adult patients. We selected a cohort of 31 consecutive de novo MDS patients with unusual young age (<60 years). We performed exome sequencing (ES) on DNA extracted from noninvasive sources (peripheral blood and saliva), filtering for a panel of 344 genes specifically tailored for detecting GL variants related to clonal and nonclonal cytopenia. We observed at least one high- or low-confidence GL MDS variant in 7/31 (22.6%) and 9/31 (29.0%) of cases, respectively. Four of 31 patients (12.9%) confirmed having established MDS/AML predisposing disorders. We found heterozygous variants in genes involved in DNA repair/cancer predisposition (ATM, ATR, FANCM, PARN, BRCA1, BRCA2, CHEK2, MSH2) in 9/31 (29.0%) casesand variants affecting ribosome biogenesis (SBDS), hematopoietic stem cell (GATA2), and megakaryocyte (ANKRD26) differentiation in single cases. Two cases had variants in RBBP6, a gene previously described exclusively in familial myeloproliferative neoplasms. Lastly, four cases had variants in genes related to inherited anemias (CUBN and PIEZO1 genes). Our results showed that "young" MDS patients aged 40-60 years carried reported and unreported GL variants with an unexpectedly high proportion, and these events co-occurred with somatic mutations recurrent in myeloid neoplasms. We explored the "no man's land" of the young adult MDS cases adopting a practical and scalable diagnostic tool, capable to detect GL variants avoiding invasive methods.

FLI1 is associated with regulation of DNA methylation and megakaryocytic differentiation in FPDMM caused by a RUNX1 transactivation domain mutation

Familial platelet disorder with associated myeloid malignancies (FPDMM) is an autosomal dominant disease caused by heterozygous germline mutations in RUNX1. It is characterized by thrombocytopenia, platelet dysfunction, and a predisposition to hematological malignancies. Although FPDMM is a precursor for diseases involving abnormal DNA methylation, the DNA methylation status in FPDMM remains unknown, largely due to a lack of animal models and challenges in obtaining patient-derived samples. Here, using genome editing techniques, we established two lines of human induced pluripotent stem cells (iPSCs) with different FPDMM-mimicking heterozygous RUNX1 mutations. These iPSCs showed defective differentiation of hematopoietic progenitor cells (HPCs) and megakaryocytes (Mks), consistent with FPDMM. The FPDMM-mimicking HPCs showed DNA methylation patterns distinct from those of wild-type HPCs, with hypermethylated regions showing the enrichment of ETS transcription factor (TF) motifs. We found that the expression of FLI1, an ETS family member, was significantly downregulated in FPDMM-mimicking HPCs with a RUNX1 transactivation domain (TAD) mutation. We demonstrated that FLI1 promoted binding-site-directed DNA demethylation, and that overexpression of FLI1 restored their megakaryocytic differentiation efficiency and hypermethylation status. These findings suggest that FLI1 plays a crucial role in regulating DNA methylation and correcting defective megakaryocytic differentiation in FPDMM-mimicking HPCs with a RUNX1 TAD mutation.

Epigenetic regulation of megakaryopoiesis and platelet formation.

Platelets, produced by megakaryocytes, play unique roles in physiological processes, such as hemostasis, coagulation, and immune regulation, while also contributing to various clinical diseases. During megakaryocyte differentiation, the morphology and function of cells undergo significant changes due to the programmed expression of a series of genes. Epigenetic changes modify gene expression without altering the DNA base sequence, effectively affecting the inner workings of the cell at different stages of growth, proliferation, differentiation, and apoptosis. These modifications also play important roles in megakaryocyte development and platelet biogenesis. However, the specific mechanisms underlying epigenetic processes and the vast epigenetic regulatory network formed by their interactions remain unclear. In this review, we systematically summarize the key roles played by epigenetics in megakaryocyte development and platelet formation, including DNA methylation, histone modification, and non-coding RNA regulation. We expect our review to provide a deeper understanding of the biological processes underlying megakaryocyte development and platelet formation and to inform the development of new clinical interventions aimed at addressing platelet-related diseases and improving patients' prognoses.

Neutrophil-specific expression of JAK2-V617F or CALRmut induces distinct inflammatory profiles in myeloproliferative neoplasia

BackgroundNeutrophils play a crucial role in inflammation and in the increased thrombotic risk in myeloproliferative neoplasms (MPNs). We have investigated how neutrophil-specific expression of JAK2-V617F or CALRdel re-programs the functions of neutrophils.MethodsLy6G-Cre JAK2-V617F and Ly6G-Cre CALRdel mice were generated. MPN parameters as blood counts, splenomegaly and bone marrow histology were compared to wild-type mice. Megakaryocyte differentiation was investigated using lineage-negative bone marrow cells upon in vitro incubation with TPO/IL-1β. Cytokine concentrations in serum of mice were determined by Mouse Cytokine Array. IL-1α expression in various hematopoietic cell populations was determined by intracellular FACS analysis. RNA-seq to analyse gene expression of inflammatory cytokines was performed in isolated neutrophils from JAK2-V617F and CALR-mutated mice and patients. Bioenergetics of neutrophils were recorded on a Seahorse extracellular flux analyzer. Cell motility of neutrophils was monitored in vitro (time lapse microscopy), and in vivo (two-photon microscopy) upon creating an inflammatory environment. Cell adhesion to integrins, E-selectin and P-selection was investigated in-vitro. Statistical analysis was carried out using GraphPad Prism. Data are shown as mean ± SEM. Unpaired, two-tailed t-tests were applied.ResultsStrikingly, neutrophil-specific expression of JAK2-V617F, but not CALRdel, was sufficient to induce pro-inflammatory cytokines including IL-1 in serum of mice. RNA-seq analysis in neutrophils from JAK2-V617F mice and patients revealed a distinct inflammatory chemokine signature which was not expressed in CALR-mutant neutrophils. In addition, IL-1 response genes were significantly enriched in neutrophils of JAK2-V617F patients as compared to CALR-mutant patients. Thus, JAK2-V617F positive neutrophils, but not CALR-mutant neutrophils, are pathogenic drivers of inflammation in MPN. In line with this, expression of JAK2-V617F or CALRdel elicited a significant difference in the metabolic phenotype of neutrophils, suggesting a stronger inflammatory activity of JAK2-V617F cells. Furthermore, JAK2-V617F, but not CALRdel, induced a VLA4 integrin-mediated adhesive phenotype in neutrophils. This resulted in reduced neutrophil migration in vitro and in an inflamed vessel. This mechanism may contribute to the increased thrombotic risk of JAK2-V617F patients compared to CALR-mutant individuals.ConclusionsTaken together, our findings highlight genotype-specific differences in MPN-neutrophils that have implications for the differential pathophysiology of JAK2-V617F versus CALR-mutant disease.

The role of PALLD-STAT3 interaction in megakaryocyte differentiation and thrombocytopenia treatment.

Impaired differentiation of megakaryocytes constitutes the principal etiology of thrombocytopenia. The signal transducer and activator of transcription 3 (STAT3) is a crucial transcription factor in regulating megakaryocyte differentiation, yet the precise mechanism of its activation remains unclear. PALLD, an actin-associated protein, has been increasingly recognized for its essential functions in multiple biological processes. This study revealed that megakaryocyte/plateletspecific knockout of PALLD in mice exhibited thrombocytopenia due to diminished platelet biogenesis. In megakaryocytes, PALLD deficiency led to impaired proplatelet formation and polyploidization, ultimately weakening their differentiation for platelet production. Mechanistic studies demonstrated that PALLD bound to STAT3 and interacted with its DNA-binding domain (DBD) and Src homology 2 (SH2) domain via Immunoglobulin domain 3 (Ig3). Moreover, the absence of PALLD attenuated STAT3 Y705 phosphorylation and impeded STAT3 nuclear translocation. Based on the PALLD-STAT3 binding sequence, we designed a peptide C-P3, which can facilitate megakaryocyte differentiation and accelerate platelet production in vivo. In conclusion, this study highlights the pivotal role of PALLD in megakaryocyte differentiation and proposes a novel approach for treating thrombocytopenia by targeting the PALLD-STAT3 interaction.

Daucosterol regulates JAK2-STAT3 signaling pathway to promote megakaryocyte differentiation

Immune thrombocytopenia (ITP) is an autoimmune disease caused by the loss of immune tolerance to platelet autoantigens, resulting in reduced platelet production and increased platelet destruction. Impaired megakaryocyte differentiation and maturation is a key factor in the pathogenesis and treatment of ITP. Sarcandra glabra, a plant of the Chloranthaceae family, is commonly used in clinical practice to treat ITP, and daucosterol (Dau) is one of its active ingredients. However, whether Dau can treat ITP and the key mechanism of its effect are still unclear. In this study, we found that Dau could effectively promote the differentiation and maturation of megakaryocytes and the formation of polyploidy in the megakaryocyte differentiation disorder model constructed by co-culturing Dami and HS-5 cells. In vivo experiments showed that Dau could not only increase the number of polyploidized megakaryocytes in the ITP rat model, but also promote the recovery of platelet count. In addition, through network pharmacology analysis, we speculated that the JAK2-STAT3 signaling pathway might be involved in the process of Dau promoting megakaryocyte differentiation. Western blot results showed that Dau inhibited the expression of P-JAK2 and P-STAT3. In summary, these results provide a basis for further studying the pharmacological mechanism of Dau in treating ITP.

A Carbon 21 Steroidal Glycoside with Pregnane Skeleton from Cynanchum atratum Bunge Promotes Megakaryocytic and Erythroid Differentiation in Erythroleukemia HEL Cells through Regulating Platelet-Derived Growth Factor Receptor Beta and JAK2/STAT3 Pathway.

Erythroleukemia is a rare form of acute myeloid leukemia (AML). Its molecular pathogenesis remains vague, and this disease has no specific therapeutic treatments. Previously, our group isolated a series of Carbon 21 (C-21) steroidal glycosides with pregnane skeleton from the root of Cynanchum atratum Bunge. Among them, we found that a compound, named BW18, can induce S-phase cell cycle arrest and apoptosis via the mitogen-activated protein kinase (MAPK) pathway in human chronic myeloid leukemia K562 cells. However, its anti-tumor activity against erythroleukemia remains largely unknown. In this study, we aimed to investigate the anti-erythroleukemia activity of BW18 and the underlying molecular mechanisms. Our results demonstrated that BW18 exhibited a good anti-erythroleukemia activity in the human erythroleukemia cell line HEL and an in vivo xenograft mouse model. In addition, BW18 induced cell cycle arrest at the G2/M phase and promoted megakaryocytic and erythroid differentiation in HEL cells. Furthermore, RNA sequencing (RNA-seq) and rescue assay demonstrated that overexpression of platelet-derived growth factor receptor beta (PDGFRB) reversed BW18-induced megakaryocytic differentiation in HEL cells, but not erythroid differentiation. In addition, the network pharmacology analysis, the molecular docking and cellular thermal shift assay (CETSA) revealed that BW18 could inactivate Janus tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway, which might mediate BW18-induced erythroid differentiation. Taken together, our findings elucidated a novel role of PDGFRB in regulating erythroleukemia differentiation and highlighted BW18 as an attractive lead compound for erythroleukemia treatment.

An intricate regulatory circuit between FLI1 and GATA1/GATA2/LDB1/ERG dictates erythroid vs. megakaryocytic differentiation.

During hematopoiesis, megakaryocytic erythroid progenitors (MEPs) differentiate into megakaryocytic or erythroid lineages in response to specific transcriptional factors, yet the regulatory mechanism remains to be elucidated. Using the MEP‑like cell line HEL western blotting, RT‑qPCR, lentivirus‑mediated downregulation, flow cytometry as well as chromatin immunoprecipitation (ChIp) assay demonstrated that the E26 transformation‑specific (ETS) transcription factor friend leukemia integration factor 1 (Fli‑1) inhibits erythroid differentiation. The present study using these methods showed that while FLI1‑mediated downregulation of GATA binding protein 1 (GATA1) suppresses erythropoiesis, its direct transcriptional induction of GATA2 promotes megakaryocytic differentiation. GATA1 is also involved in megakaryocytic differentiation through regulation of GATA2. By contrast to FLI1, the ETS member erythroblast transformation‑specific‑related gene (ERG) negatively controls GATA2 and its overexpression through exogenous transfection blocks megakaryocytic differentiation. In addition, FLI1 regulates expression of LIM Domain Binding 1 (LDB1) during erythroid and megakaryocytic commitment, whereas shRNA‑mediated depletion of LDB1 downregulates FLI1 and GATA2 but increases GATA1 expression. In agreement, LDB1 ablation using shRNA lentivirus expression blocks megakaryocytic differentiation and modestly suppresses erythroid maturation. These results suggested that a certain threshold level of LDB1 expression enables FLI1 to block erythroid differentiation. Overall, FLI1 controlled the commitment of MEP to either erythroid or megakaryocytic lineage through an intricate regulation of GATA1/GATA2, LDB1 and ERG, exposing multiple targets for cell fate commitment and therapeutic intervention.

Long non-coding RNA NORAD regulates megakaryocyte differentiation and proplatelet formation via the DUSP6/ERK signaling pathway

Megakaryopoiesis and platelet production is a complex process that is underpotential regulation at multiple stages. Many long non-coding RNAs (lncRNAs) are distributed in hematopoietic stem cells and platelets. lncRNAs may play important roles as key epigenetic regulators in megakaryocyte differentiation and proplatelet formation. lncRNA NORAD can affect cell ploidy by sequestering PUMILIO proteins, although its direct effect on megakaryocyte differentiation and thrombopoiesis is still unknown. In this study, we demonstrate NORAD RNA is highly expressed in the cytoplasm during megakaryocyte differentiation. Interestingly, we identified for the first time that NORAD has a strong inhibitory effect on megakaryocyte differentiation and proplatelet formation from cultured megakaryocytes. DUSP6/ERK1/2 pathway is activated in response to NORAD knockdown during megakaryocytopoiesis, which is achieved by sequestering PUM2 proteins. Finally, compared with the wild-type control mice, NORAD knockout mice show a faster platelet recovery after severe thrombocytopenia induced by 6 Gy total body irradiation. These findings demonstrate lncRNA NORAD has a key role in regulating megakaryocyte differentiation and thrombopoiesis, which provides a promising molecular target for the treatment of platelet-related diseases such as severe thrombocytopenia.