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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. [ABSTRACT FROM AUTHOR]

Abstract Background Megakaryocytic 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. Methods The 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. Results KLF2 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. Conclusion This study implicated a regulatory role of KLF2 in megakaryocytic differentiation, which may suggest KLF2 as a target for illness with abnormal megakaryocytic differentiation.

Abstract 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.

Background: Megakaryocytic 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. Methods: The 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. Results: KLF2 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. Conclusion: This study implicated a regulatory role of KLF2 in megakaryocytic differentiation, which may suggest KLF2 as a target for illness with abnormal megakaryocytic differentiation. [ABSTRACT FROM AUTHOR]

Lethal toxin (LT) is the critical virulence factor of Bacillus anthracis, the causative agent of anthrax. One common symptom observed in patients with anthrax is thrombocytopenia, which has also been observed in mice injected with LT. Our previous study demonstrated that LT induces thrombocytopenia by suppressing megakaryopoiesis, but the precise molecular mechanisms behind this phenomenon remain unknown. In this study, we utilized 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced megakaryocytic differentiation in human erythroleukemia (HEL) cells to identify genes involved in LT-induced megakaryocytic suppression. Through cDNA microarray analysis, we identified Dachshund homolog 1 (DACH1) as a gene that was upregulated upon TPA treatment but downregulated in the presence of TPA and LT, purified from the culture supernatants of B. anthracis. To investigate the function of DACH1 in megakaryocytic differentiation, we employed short hairpin RNA technology to knock down DACH1 expression in HEL cells and assessed its effect on differentiation. Our data revealed that the knockdown of DACH1 expression suppressed megakaryocytic differentiation, particularly in polyploidization. We demonstrated that one mechanism by which B. anthracis LT induces suppression of polyploidization in HEL cells is through the cleavage of MEK1/2. This cleavage results in the downregulation of the ERK signaling pathway, thereby suppressing DACH1 gene expression and inhibiting polyploidization. Additionally, we found that known megakaryopoiesis-related genes, such as FOSB, ZFP36L1, RUNX1, FLI1, AHR, and GFI1B genes may be positively regulated by DACH1. Furthermore, we observed an upregulation of DACH1 during in vitro differentiation of CD34–megakaryocytes and downregulation of DACH1 in patients with thrombocytopenia. In summary, our findings shed light on one of the molecular mechanisms behind LT-induced thrombocytopenia and unveil a previously unknown role for DACH1 in megakaryopoiesis. [ABSTRACT FROM AUTHOR]

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.

Abstract Background Megakaryocytes (MKs) are platelet precursors, which arise from hematopoietic stem cells (HSCs). While MK lineage commitment and differentiation are accompanied by changes in gene expression, many factors that modulate megakaryopoiesis remain to be uncovered. Replication initiation determinant protein (RepID) which has multiple histone-code reader including bromodomain, cryptic Tudor domain and WD40 domains and Cullin 4-RING E3 ubiquitin ligase complex (CRL4) recruited to chromatin mediated by RepID have potential roles in gene expression changes via epigenetic regulations. We aimed to investigate whether RepID-CRL4 participates in transcriptional changes required for MK differentiation. Methods The PCR array was performed using cDNAs derived from RepID-proficient or RepID-deficient K562 erythroleukemia cell lines. Correlation between RepID and DAB2 expression was examined in the Cancer Cell Line Encyclopedia (CCLE) through the CellMinerCDB portal. The acceleration of MK differentiation in RepID-deficient K562 cells was determined by estimating cell sizes as well as counting multinucleated cells known as MK phenotypes, and by qRT-PCR analysis to validate transcripts of MK markers using phorbol 12-myristate 13-acetate (PMA)-mediated MK differentiation condition. Interaction between CRL4 and histone methylation modifying enzymes were investigated using BioGRID database, immunoprecipitation and proximity ligation assay. Alterations of expression and chromatin binding affinities of RepID, CRL4 and histone methylation modifying enzymes were investigated using subcellular fractionation followed by immunoblotting. RepID-CRL4-JARID1A-based epigenetic changes on DAB2 promoter were analyzed by chromatin-immunoprecipitation and qPCR analysis. Results RepID-deficient K562 cells highly expressing MK markers showed accelerated MKs differentiation exhibiting increases in cell size, lobulated nuclei together with reaching maximum levels of MK marker expression earlier than RepID-proficient K562 cells. Recovery of WD40 domain-containing RepID constructs in RepID-deficient background repressed DAB2 expression. CRL4A formed complex with histone H3K4 demethylase JARID1A in soluble nucleus and loaded to the DAB2 promoter in a RepID-dependent manner during proliferation condition. RepID, CRL4A, and JARID1A were dissociated from the chromatin during MK differentiation, leading to euchromatinization of the DAB2 promoter. Conclusion This study uncovered a role for the RepID-CRL4A-JARID1A pathway in the regulation of gene expression for MK differentiation, which can form the basis for the new therapeutic approaches to induce platelet production. Video Abstract

Lethal toxin (LT) is the critical virulence factor of Bacillus anthracis, the causative agent of anthrax. One common symptom observed in patients with anthrax is thrombocytopenia, which has also been observed in mice injected with LT. Our previous study demonstrated that LT induces thrombocytopenia by suppressing megakaryopoiesis, but the precise molecular mechanisms behind this phenomenon remain unknown. In this study, we utilized 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced megakaryocytic differentiation in human erythroleukemia (HEL) cells to identify genes involved in LT-induced megakaryocytic suppression. Through cDNA microarray analysis, we identified Dachshund homolog 1 (DACH1) as a gene that was upregulated upon TPA treatment but downregulated in the presence of TPA and LT, purified from the culture supernatants of B. anthracis. To investigate the function of DACH1 in megakaryocytic differentiation, we employed short hairpin RNA technology to knock down DACH1 expression in HEL cells and assessed its effect on differentiation. Our data revealed that the knockdown of DACH1 expression suppressed megakaryocytic differentiation, particularly in polyploidization. We demonstrated that one mechanism by which B. anthracis LT induces suppression of polyploidization in HEL cells is through the cleavage of MEK1/2. This cleavage results in the downregulation of the ERK signaling pathway, thereby suppressing DACH1 gene expression and inhibiting polyploidization. Additionally, we found that known megakaryopoiesis-related genes, such as FOSB, ZFP36L1, RUNX1, FLI1, AHR, and GFI1B genes may be positively regulated by DACH1. Furthermore, we observed an upregulation of DACH1 during in vitro differentiation of CD34–megakaryocytes and downregulation of DACH1 in patients with thrombocytopenia. In summary, our findings shed light on one of the molecular mechanisms behind LT-induced thrombocytopenia and unveil a previously unknown role for DACH1 in megakaryopoiesis.

Background: Megakaryocytes (MKs) are platelet precursors, which arise from hematopoietic stem cells (HSCs). While MK lineage commitment and differentiation are accompanied by changes in gene expression, many factors that modulate megakaryopoiesis remain to be uncovered. Replication initiation determinant protein (RepID) which has multiple histone-code reader including bromodomain, cryptic Tudor domain and WD40 domains and Cullin 4-RING E3 ubiquitin ligase complex (CRL4) recruited to chromatin mediated by RepID have potential roles in gene expression changes via epigenetic regulations. We aimed to investigate whether RepID-CRL4 participates in transcriptional changes required for MK differentiation. Methods: The PCR array was performed using cDNAs derived from RepID-proficient or RepID-deficient K562 erythroleukemia cell lines. Correlation between RepID and DAB2 expression was examined in the Cancer Cell Line Encyclopedia (CCLE) through the CellMinerCDB portal. The acceleration of MK differentiation in RepID-deficient K562 cells was determined by estimating cell sizes as well as counting multinucleated cells known as MK phenotypes, and by qRT-PCR analysis to validate transcripts of MK markers using phorbol 12-myristate 13-acetate (PMA)-mediated MK differentiation condition. Interaction between CRL4 and histone methylation modifying enzymes were investigated using BioGRID database, immunoprecipitation and proximity ligation assay. Alterations of expression and chromatin binding affinities of RepID, CRL4 and histone methylation modifying enzymes were investigated using subcellular fractionation followed by immunoblotting. RepID-CRL4-JARID1A-based epigenetic changes on DAB2 promoter were analyzed by chromatin-immunoprecipitation and qPCR analysis. Results: RepID-deficient K562 cells highly expressing MK markers showed accelerated MKs differentiation exhibiting increases in cell size, lobulated nuclei together with reaching maximum levels of MK marker expression earlier than RepID-proficient K562 cells. Recovery of WD40 domain-containing RepID constructs in RepID-deficient background repressed DAB2 expression. CRL4A formed complex with histone H3K4 demethylase JARID1A in soluble nucleus and loaded to the DAB2 promoter in a RepID-dependent manner during proliferation condition. RepID, CRL4A, and JARID1A were dissociated from the chromatin during MK differentiation, leading to euchromatinization of the DAB2 promoter. Conclusion: This study uncovered a role for the RepID-CRL4A-JARID1A pathway in the regulation of gene expression for MK differentiation, which can form the basis for the new therapeutic approaches to induce platelet production. EhctJ7T1-GG5oU5fji2odB Video Abstract [ABSTRACT FROM AUTHOR]

Transplant of gene-modified autologous hematopoietic progenitors cells has emerged as a new therapeutic approach for Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency with microthrombocytopenia and abnormal lymphoid and myeloid functions. Despite the clinical benefits obtained in ongoing clinical trials, platelet restoration is suboptimal. The incomplete restoration of platelets in these patients can be explained either by a low number of corrected cells or by insufficient or inadequate WASP expression during megakaryocyte differentiation and/or in platelets. We therefore used in vitro models to study the endogenous WASP expression pattern during megakaryocytic differentiation and compared it with the expression profiles achieved by different therapeutic lentiviral vectors (LVs) driving WAS cDNA through different regions of the WAS promoter. Our data showed that all WAS promoter-driven LVs mimic very closely the endogenous WAS expression kinetic during megakaryocytic differentiation. However, LVs harboring the full-length (1.6-kb) WAS-proximal promoter (WW1.6) or a combination of the WAS alternative and proximal promoters (named AW) had the best behavior. Finally, all WAS-driven LVs restored the WAS knockout (WASKO) mice phenotype and functional defects of hematopoietic stem and progenitor cells (HSPCs) from a WAS patient with similar efficiency. In summary, our data back up the use of WW1.6 and AW LVs as physiological gene transfer tools for WAS therapy.

Summary: Myelodysplastic syndrome (MDS), a largely incurable hematological malignancy, is driven by complex genetic and epigenetic alterations from an aberrant clone of hematopoietic stem/progenitor cells (HSPCs). Ubiquitin‐specific protease 7 (USP7) has been demonstrated to have an important oncogenic role in the development of several cancer types, but its role in MDS is unknown. Here, we demonstrate that USP7 expression is elevated in MDS cell lines and patient samples. The USP7‐selective small‐molecule inhibitors P5091 and P22077 inhibited cell proliferation and induced megakaryocytic differentiation in both cell lines and primary cells. Furthermore, pharmacological inhibition of USP7 markedly suppressed the growth of MDS cell lines in xenograft mouse models. To explore the mechanisms underlying the observed phenotypic changes, we employed RNA‐seq to compare the differences in genes after USP7 inhibitor treatment and found that gelsolin (GSN) expression was increased significantly after USP7 inhibitor treatment. Furthermore, knockdown of GSN attenuated the proliferation inhibition, apoptosis induction and megakaryocyte differentiation induced by USP7 inhibitors in MDS cells. Collectively, our findings identify previously unknown roles of USP7 and suggest that the USP7/GSN axis may be a potential therapeutic target in MDS. [ABSTRACT FROM AUTHOR]

Kaempferol-3-O-rhamnoside (KOR) has multiple potency involved in anti-cancer, anti-inflammatory and antibacterial actions. However, the potential roles of KOR and the analogues isolated from the leaves of Cyclocarya paliurus in anti-erythroleukemia remain unclear. In the present study, KOR and the two analogues (Kaempferol-3-O-(4″-O-acetyl-a-L-rhamnopyranoside) (KLR) and (kaempferol-3-O-α-L-(4″-E-p-coumaroyl) rhamnoside) (KCR) were isolated from leaves of Cyclocarya paliurus. Cell viability assay showed that KCR exerted an excellent anti-erythroleukemia activity. We observed that KCR not only significantly increased the percentage of G2 phase and apoptotic cells compared with control group, but also induced megakaryocytic differentiation in HEL and K562 cells by flow cytometry, indicating that KCR might inhibit cell proliferation through inducing differentiation-mediated apoptosis and cell cycle arrest. Mechanism investigation revealed that KCR treatment obviously increased phosphorylation levels of PKCδ and ERK1/2 as well as GATA1 expression. Taken together, these findings demonstrate that KCR induces megakaryocytic differentiation and suppresses leukemogenesis at least partly through activation of PKCδ/ERK1/2 signaling pathway in erythroleukemia cells. KCR may also serve as a promising natural compound for human erythroleukemia treatment.

Differentiation therapy is an alternative strategy used to induce the differentiation of blast cells toward mature cells and to inhibit tumor cell proliferation for cancer treatment. Nobiletin (NOB), a polymethoxyflavone phytochemical, is present abundantly in citrus peels and has been reported to possess anti-cancer activity. In this study, we investigated the anti-leukemic effects of NOB on cell differentiation and its underlying mechanisms in human chronic myeloid leukemia (CML) K562 cells. NOB (100 μM) treatment for 24 and 48 h significantly decreased viability of K562 cells to 54.4 ± 5.3% and 46.2 ± 9.9%, respectively. NOB (10–100 μM) significantly inhibited cell growth in K562 cells. Flow cytometry analysis and immunoblotting data showed that NOB (40 and 80 μM) could modulate the cell cycle regulators including p21, p27, and cyclin D2, and induce G1 phase arrest. NOB also increased the messenger RNA (mRNA) and protein expression of megakaryocytic differentiation markers, such as CD61, CD41, and CD42 as well as the formation of large cells with multi-lobulated nuclei in K562 cells. These results suggested that NOB facilitated K562 cells toward megakaryocytic differentiation. Furthermore, microarray analysis showed that expression of EGR1, a gene associated with promotion of megakaryocytic differentiation, was markedly elevated in NOB-treated K562 cells. The knockdown of EGR1 expression by small interference RNA (siRNA) could significantly attenuate NOB-mediated cell differentiation. We further elucidated that NOB induced EGR1 expression and CD61 expression through increases in MAPK/ERK phosphorylation in K562 cells. These results indicate that NOB promotes megakaryocytic differentiation through the MAPK/ERK pathway-dependent EGR1 expression in human CML cells. In addition, NOB when combined with imatinib could synergistically reduce the viability of K562 cells. Our findings suggest that NOB may serve as a beneficial anti-leukemic agent for differentiation therapy.

Co-infusion of ex-vivo generated megakaryocytic progenitors with hematopoietic stem/progenitor cells (HSC/HPC) may contribute to a faster platelet recovery upon umbilical cord blood (UCB) transplantation. A two stage protocol containing cell expansion and megakaryocyte (Mk) differentiation was established using human UCB CD34+-enriched cells. The expansion stage used a pre-established protocol supported by a human bone marrow mesenchymal stem cells (MSC) feeder layer and the differentiation stage used TPO (100 ng/mL) and IL-3 (10 ng/mL). 18% of culture-derived Mks had higher DNA content (>4 N) and were able to produce platelet-like particles. The proliferation extent of CD34+ cells obtained in the expansion stage (FI-CD34+), rather than expansion duration, determined as a key parameter for efficient megakaryocytic differentiation. A maximum efficiency yield (EY) of 48 ± 7.7 Mks/input CD34+ cells was obtained for a FI-CD34+ of 17 ± 2.5, where a higher FI-CD34+ of 42 ± 13 resulted in a less efficient megakaryocytic differentiation (EY of 22 ± 6.7 and 19 ± 4.6 %CD41).

Plant homeodomain finger 2 (PHF2) is a JmjC family histone demethylase that demethylates H3K9me2, a repressive gene marker. PHF2 was found to play a role in the differentiation of several tissue types such as osteoblast and adipocyte differentiation. We report here that PHF2 plays a role in the epigenetic regulation of megakaryocytic (MK) and erythroid differentiation. We investigated PHF2 expression during MK and erythroid differentiation in K562 and human CD34+ progenitor (hCD34+) cells. Our data demonstrate that PHF2 expression is down regulated during megakaryopoiesis and erythropoiesis. PHF2 has a negative role inMK and erythroid differentiation of K562 cells; knockdown of PHF2 promotes MK and erythroid differentiation of hCD34+ cells. Similarly, we found that p53 expression is also down-regulated during MK and erythroid differentiation, which parallels PHF2 expression. PHF2 binds to the p53 promoter and regulates the expression of p53 by demethylatingH3K9me2in the promoter region of p53. Taken together, our data show that PHF2 is a negative epigenetic regulator of MK and erythroid differentiation, and that one of the pathways through which PHF2 affects MK and erythroid differentiation is via regulation of p53 expression. [ABSTRACT FROM AUTHOR]

Prolonged isolated thrombocytopenia (PT) is a severe complication in patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Whether the megakaryoctic potential of hematopoietic stem cells (HSCs) in bone marrow is intact and what factors drive the pathological process of PT remain elusive. A retrospective study in patients (n = 285) receiving HSCT revealed that the occurrence of PT was approximately 8% and the number of platelets and megakaryocytes in PT patients is much lower compared with control subjects. To test whether the deficiency of thrombopoiesis was caused by the activities of HSCs, the megakaryocytic differentiation potential of HSCs before or after transplantation was assessed. Interestingly, a substantial decrease of megakaryocytic differentiation was observed 2 weeks after transplantation of HSCs in all of the allo-HSCT recipients. However, 4 weeks after transplantation, the ability of HSCs to generate CD41 + CD42b + megakaryocytes in successful platelet engraftment patients recovered to the same level as those of HSCs before implantation. In contrast, HSCs derived from PT patients throughout the postimplantation period exhibited poor survival and failed to differentiate properly. A protein array analysis demonstrated that multiple inflammation-associated cytokines were elevated in allo-HSCT recipients with PT. Among them, insulin-like growth factor–binding protein 1 and regulated on activation, normal T cell expressed and secreted were found to significantly suppress the proliferation and megakaryocytic differentiation of HSCs in vitro. Our results suggested that the occurrence of PT may be attributed, at least partially, to the damage to HSC function caused by inflammation-associated cytokines after HSCT. These findings shed light on the mechanism underlying HSC megakaryocytic differentiation in PT patients and may provide potential new strategies for treating PT patients after HSCT. [ABSTRACT FROM AUTHOR]

Protein arginine methyltransferase 1 (PRMT1) serves pivotal roles in various cellular processes. However, its role in megakaryocytic differentiation has not been clearly reported. The aim of the present study was to explore the role of the PRMT‑RNA binding motif protein 15 (RBM15) axis in human MK differentiation and the feasibility of targeting PRMT1 for leukemia treatment. In the present study, PRMT1 was overexpressed and the RBM15 protein was knocked down in human umbilical cord blood cluster of differentiation (CD)34+ cells and the cells were then cultured in megakaryocytic differentiation medium. Flow cytometry was used to analyze CD41 and CD42 double‑positive cells, as well as the protein expression levels of PRMT1 and RBM15. The results demonstrated that human cord blood CD34+ cells differentiate into mature MKs in high thrombopoitin medium, as demonstrated by CD41 and CD42 expression. Overexpression of PRMT1 in human umbilical cord blood CD34+ cells blocked the maturation of megakaryocytic cells. Knockdown of RBM15 by short hairpin RNA produced less mature MKs. PRMT1 inhibitor rescued PRMT1‑blocked megakaryocytic differentiation. These results provide evidence for a novel role of PRMT1 in the negative regulation of megakaryocytic differentiation. PRMT1 may be a therapeutic target for leukemia treatment. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) infection can cause severe, life-threatening events, and no specific treatments of DENV infection are currently approved. Although thrombocytopenia is frequently observed in dengue patients, its pathogenesis is still not fully understood. Previous studies have suggested that DENV-induced thrombocytopenia occurs through viral-replication-mediated megakaryopoiesis inhibition in the bone marrow; however, the exact mechanism for megakaryopoiesis suppression remains elusive. In this study, a reductionist approach was applied, in which C57B/6J mice were inoculated with recombinant DENV-envelope protein domain III (DENV-EIII) instead of the full viral particle. Our results demonstrated that DENV-EIII-suppressed megakaryopoiesis is similar to those observed with DENV infection. Furthermore, in agreement with ourin vivoanalyses, DENV-EIII sufficiently suppressed the megakaryopoiesis of progenitor cells from murine bone marrow and human cord bloodin vitro. Additional analyses suggested that autophagy impairment and apoptosis are involved in DENV-EIII-mediated suppression of megakaryopoiesis. These data suggest that, even without viral replication, the binding of DENV-EIII to the cell surface is sufficient to suppress megakaryopoiesis. [ABSTRACT FROM AUTHOR]

Anagrelide is a treatment option for patients with essential thrombocythemia. Although the clinical efficacy of anagrelide has been established, there is limited knowledge of the molecular mechanism underlying its effect. Here, we evaluated the effect of anagrelide on primary megakaryocytic progenitors from cord blood-derived CD34-positive cells. Anagrelide treatment reduced the expression of megakaryocytic markers (CD41 and CD61). Microarray analysis was performed to characterize gene profiles altered by exposure to anagrelide. The analysis demonstrated upregulation and downregulation (>2-fold) of eight and 34 genes, respectively, in anagrelide-treated megakaryocyte progenitors. This included genes encoding prototypical megakaryocytic proteins, such as PPBP, PF4, and GP6. Gene ontology analysis of genes suppressed by anagrelide treatment revealed significant enrichment of genes involved in platelet activation and degranulation. Expression levels of transcription factors involved in megakaryocyte commitment/differentiation were further evaluated by quantitative RT-PCR, demonstrating significant downregulation of FLI1 and TAL1 in anagrelide-treated megakaryocyte progenitors. Knockdown of TAL1 in primary megakaryocyte progenitors confirmed significant downregulation of FLI1 and megakaryocytic genes. Anagrelide had no significant effect on the surface expression of erythroid markers or on the expression of transcription factors involved in erythroid commitment/differentiation. In conclusion, anagrelide suppresses megakaryocytic differentiation, partly through decreasing the expression of megakaryocytic transcription factors. [ABSTRACT FROM AUTHOR]

In our previous investigations we have shown that platelets and their precursors express nicotinic α7 acetylcholine receptors (nAChRα7) that are involved in platelet function andin vitrodifferentiation of the megakaryoblastic cell line MEG-01. In this study, we were interested in the expression analysis of additional nAChR and the effects of nicotine in anex vivomodel using megakaryocytic cells differentiated from cord blood derived CD34+cells (CBMK) and anin vivomodel using blood samples from smokers. CBMK were differentiated with thrombopoietin (TPO) for up to 17 days. Quantitative real-time PCR (QRT-PCR), Western blot analysis and flow cytometry were used to investigate nAChR expression (nAChRα7, nAChRα4, nAChRβ2) and nicotine effects. In blood samples of 15 nonsmokers and 16 smokers platelet parameters (count, mean platelet volume – MPV and platelet distribution width – PDW) were determined as indicators for changes ofin vivomegakaryopoiesis. Platelet function was determined by the use of whole blood aggregometry and flow cytometry. The functional role of nAChR was evaluated using specific antagonists in aggregometry.CHRNA7, CHRNA4andCHRNB2gene transcripts and the corresponding proteins could be identified in CBMK during all stages of differentiation. Platelets contain nAChRα7 and nAChRβ2 but not nAChRα4. Nicotine had no effect on TPO-induced differentiation of CBMK. There was no significant difference in all platelet parameters of the smokers compared to the nonsmokers. In line with this, cholinergic gene transcripts as well as the encoded proteins were equally expressed in both the study groups. Despite our observation of nAChR expression in megakaryopoiesis and platelets, we were not able to detect effects of nicotine in ourex vivoandin vivomodels. Thus, the functional role of the nAChR in these cells remains open. [ABSTRACT FROM AUTHOR]

Established from the calvaria of newborn macrophage colony-stimulating factor (M-CSF)-deficient mice, OP9 is a stromal cell line that used as a feeder layer to support the in vitro differentiation of pluripotent stem cells into various hematopoietic lineage cells, including granulocytes, erythrocytes, lymphocytes, and megakaryocytes. However, as a primary culture cell line, OP9 can be used as stromal cells for only 1 month. Therefore, to obtain functional OP9 cells, numerous M-CSF-deficient newborn mice must be sacrificed. These limitations in some ways restrict the application of OP9 cells in longterm and largescale experiments. In this study, we used human papillomavirus 16 E6 and E7 genes to generate immortalized OP9 stromal cells, designated I-OP9 cells, and then tested their ability to support the megakaryocytic differentiation of pluripotent stem cells in vitro . I-OP9 cells have similar morphology and properties as do parental OP9 cells, and, as expected, have an extended lifespan and can support megakaryocytic differentiation. Our data suggest that the method used in this study, including establishing I-OP9 cells, enables the possibility to enlarge and lengthen the scale of the experiment and, more critically, provides a humanistic approach for preparing stromal cells that support the hematopoietic differentiation of pluripotent stem cells in vitro . [ABSTRACT FROM AUTHOR]

Introduction of a polyploidy inducer is a promising strategy to achieve a high level of polyploidization during megakaryocytic (MK) differentiation. Here, we report that a multi-kinase inhibitor, BMS-777607, is a potent polyploidy inducer for elevating high ploidy cell formation in the MK-differentiated CHRF-288-11 (CHRF) cells. Our result showed that BMS-777607 strongly inhibited cell division without affecting cell viability when detected at day 1 after treatment. As a consequence, the high ploidy (≥8N) cells were accumulated in culture for 8 days, with an increase from 16.2 to 75.2 % of the total cell population. The elevated polyploidization was accompanied by the increased expression level of MK marker, CD41 (platelet glycoprotein IIb/IIIa, GPIIb/IIIa), suggesting that BMS-777607 promoted both polyploidization and commitment of MK-differentiated CHRF cells. Platelet-like fragments (PFs) were released by mature CHRF cells. Based on a flow cytometry assay, it was found that the PFs produced from BMS-777607-treated cells tended to have larger size and higher expression of GPIIb/IIIa, a receptor for platelet adhesion. Taken together, these results suggested that BMS-777607 promoted MK differentiation of CHRF cells and increased the functional property of platelet-like fragments. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) have been proven to be important activators for various cellular activities, including cell differentiation. Several reports showed the necessity of ROS during cell differentiation of the megakaryocytic (MK) lineage. In this study, we employed near ultraviolet (near-UV) irradiation to generate endogenous oxidative stress in an MK differentiation process of K562 cells with phorbol 12-myristate 13-acetate (PMA) induction. A significant increase in the intracellular ROS level was detected on day 1 after near-UV irradiation. In the initial stage of differentiation, a shifted fraction of G and G phase cells was obtained using near-UV irradiation, giving an increased percentage of G phase cells (up from 31.1 to 68.7%). The near-UV irradiation-induced upregulation of the p21 gene, which is a cell cycle inhibitor, suggested that the G phase cells were prevented from undergoing cell division. It was found that the percentage of high ploidy (8N and 16N) cells was enhanced significantly at the later stage of the K562 cell culture with near-UV irradiation. Moreover, time-lapse analysis showed that near-UV irradiation encouraged the expression of CD41, a specific surface marker of megakaryocytes. This is the first report that the elevated oxidative stress through the near-UV irradiation promoted the MK differentiation of PMA-induced K562 cells. [ABSTRACT FROM AUTHOR]

The human chronic myelogenous cell line K562 has been used extensively as a model for the study of leukemia differentiation. We show here that treatment of K562 cells with caffeic acid phenethyl ester (CAPE) induced a majority of cells to differentiate towards the megakaryocytic lineage. Microscopy analysis showed that K562 cells treated with CAPE exhibited characteristic features of physiological megakaryocytic differentiation, including the presence of vacuoles and demarcation membranes. Differentiation of K562 cells treated with CAPE was also accompanied by a net increase in megakaryocytic markers. The transcriptional activity of lactosylceramide α-2,3-sialyltransferase (GM3 synthase) and synthesis of ganglioside GM3 were increased by CAPE treatment. The promoter analysis of GM3 synthase demonstrated that CAPE induced the expression of GM3 synthase mRNA via activation of the cAMP response element-binding protein (CREB), transcription factor in nucleus. Interestingly, the inhibition of ganglioside GM3 synthesis by D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propranol (D-PDMP) and GM3 synthase-siRNA blocked the CAPE-induced expression of the megakaryocytic markers and differentiation of K562 cells. Taken together, these results suggest that CAPE induces ganglioside GM3-mediated megakaryocytic differentiation of human chronic myelogenous cells. [ABSTRACT FROM AUTHOR]

We previously characterized the zinc finger protein gene HZF1 [also known as ZNF16 (zinc finger protein 16)] and demonstrated its important roles in erythroid and megakaryocytic differentiation of K562 cells. In the present study, we investigated its effect on erythroid and megakaryocytic differentiation of HSPCs (haemopoietic stem/progenitor cells).We observed up-regulation of ZNF16 during erythroid and megakaryocytic differentiation of the CD34+ HSPCs, and demonstrated that ZNF16 promotes erythroid and megakaryocytic differentiation by gain-of-function and loss-of-function experiments. Using a luciferase reporter and ChIP assays ZNF16 was demonstrated to bind to the c- KIT gene promoter and inhibit its expression in K562 cells. Enforced expression and knockdown of ZNF16 down-regulated and up-regulated the expression of the c-KIT gene in K562 cells and HSPCs respectively. Significantly decreased levels of the c-Kit protein were observed following erythroid and megakaryocytic differentiation of K562 and CD34+ cells. The knockdown of c-KIT partially rescued the differentiation inhibition caused by ZNF16 knockdown. The knockdown of c-KIT also blocked the activity of the c-Raf/MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK/c-Jun signal pathway and reduced further the level of HEY1 (hes-related family bHLH transcription factor with YRPW motif 1), a repressor of GATA1 (GATA-binding protein 1) transcription, which finally up-regulated the expression of GATA1, a central regulator of erythroid and megakaryocytic differentiation. In conclusion the results of the present study demonstrate that ZNF16 plays an important role in erythropoiesis and megakaryocytopoiesis via its regulation of the c-Kit/c- Raf/MEK/ERK/c-Jun/HEY1/GATA1 cascade. [ABSTRACT FROM AUTHOR]

Abstract: The human myelogenous cell line, K562 has been extensively used as a model for the study of megakaryocytic (MK) differentiation, which could be achieved by exposure to phorbol 12-myristate 13-acetate (PMA). In this study, real-time PCR analysis revealed that the expression of catalase (cat) was significantly repressed during MK differentiation of K562 cells induced by PMA. In addition, PMA increased the intracellular reactive oxygen species (ROS) concentration, suggesting that ROS was a key factor for PMA-induced differentiation. PMA-differentiated K562 cells were exposed to hydrogen peroxide (H2O2) to clarify the function of ROS during MK differentiation. Interestingly, the percentage of high-ploidy (DNA content >4N) cells with H2O2 was 34.8±2.3% at day 9, and was 70% larger than that without H2O2 (21.5±0.8%). Further, H2O2 addition during the first 3 days of PMA-induced MK differentiation had the greatest effect on polyploidization. In an effort to elucidate the mechanisms of enhanced polyploidization by H2O2, the BrdU assay clearly indicated that H2O2 suppressed the division of 4N cells into 2N cells, followed by the increased polyploidization of K562 cells. These findings suggest that the enhancement in polyploidization mediated by H2O2 is due to synergistic inhibition of cytokinesis with PMA. Although H2O2 did not increase ploidy during the MK differentiation of primary cells, we clearly observed that cat expression was repressed in both immature and mature primary MK cells, and that treatment with the antioxidant N-acetylcysteine effectively blocked and/or delayed the polyploidization of immature MK cells. Together, these findings suggest that MK cells are more sensitive to ROS levels during earlier stages of maturation. [Copyright &y& Elsevier]

Myeloid sarcoma (MS) is defined as a tumour mass consisting of myeloid blasts that occurs at an anatomical site other than bone marrow. MS with megakaryocytic differentiation (MSmgk) is extremely rare and its clinicopathological features have not been well described. We reviewed 11 cases in 11 patients of extramedullary mass-forming malignant tumours composed of immature non-lymphoid haematopoietic cells expressing CD41 with or without concurrent bone marrow lesions. The patients consisted of seven men and four women (1.75:1 male-to-female ratio). The mean and median ages at diagnosis were 50 and 62 years, respectively, ranging from 2 to 78 years. Extramedullary mass lesions were solitary in three cases (27%) and multiple in eight cases (73%). Tumour locations were lymph nodes (6 cases), subcutaneous tissue (3 cases), intramuscular (1 case), and bone (1 case). Seven of the 11 patients (64%) had a history of myelodysplastic syndrome (MDS) or myeloproliferative neoplasm (MPN). Three patients (27%) developed MS during remissions of acute myelogenous leukaemia, and one patient had a recurrence of MS at other sites. Follow-up data were available for four cases. Tumour cells were positive for CD41, CD33, CD34, MPO, and CD68 in 11 (100%), three (27%), seven (64%), four (36%), and seven (64%) cases, respectively. Cytogenetic analysis was successfully performed in two cases. Complex but inconsistent abnormalities were evident. When compared with cases of MS without megakaryocytic differentiation, the survival of MSmgk was significantly shorter (p=0.0033). Compared to MS without megakaryocytic differentiation, MSmgk is more likely to follow MDS/MPN, to involve multiple sites, and to be associated with poorer outcomes. More detailed studies, including genomic or gene expression analyses, could confirm the characteristics of MSmgk., (Copyright © 2021 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Abstract: Endocytosis is pivotal for uptake of fibrinogen from plasma into megakaryocytes and platelet α-granules. Due to the complex adaptor and cargo contents in endocytic vehicles, the underlying mechanism of fibrinogen uptake is not yet completely elucidated. In this study, we investigated whether the endocytic adaptor protein Disabled-2 (DAB2) mediates fibrinogen uptake in an adaptor-specific manner. By employing primary megakaryocytes and megakaryocytic differentiating human leukemic K562 cells as the study models, we found that fibrinogen uptake is associated with the expression of integrin αIIbβ3 and DAB2 and is mediated through clathrin-dependent manner. Accordingly, constitutive and inducible knockdown of DAB2 by small interfering RNA reduced fibrinogen uptake for 53.2±9.8% and 59.0±10.7%, respectively. Culturing the cells in hypertonic solution or in the presence of clathrin inhibitor chlorpromazine abrogated clathrin-dependent endocytosis and diminished the uptake of fibrinogen. Consistent with these findings, 72.2±0.2% of cellular DAB2 was colocalized with clathrin, whereas 56.4±4.1% and 54.6±2.0% of the internalized fibrinogen were colocalized with clathrin and DAB2, respectively. To delineate whether DAB2 mediates fibrinogen uptake in an adaptor-specific manner, K562 stable cell lines with knockdown of the adaptor protein-2 (AP-2) or double knockdown of AP-2/DAB2 were established. The AP-2 knockdown cells elicited normal fibrinogen uptake activity but the uptake of collagen was diminished. In addition, collagen uptake was further reduced in DAB2/AP-2 knockdown cells. These findings thereby define an adaptor-specific mechanism in the control of fibrinogen uptake and implicate that DAB2 is the key adaptor in the clathrin-associated endocytic complexes to mediate fibrinogen internalization. [Copyright &y& Elsevier]

Myeloid sarcoma (MyS) is defined as an extramedullary tumor-forming neoplasm consisting of immature myeloid cells with/without maturation. We experienced a case involving a 68-year-old Japanese male patient who had been followed-up for four years with a diagnosis of chronic idiopathic myelofibrosis/primary myelofibrosis (PMF) and noticed a painful mass in his left axilla. A wedge biopsy characterized the lesion as MyS that displayed megakaryoblastic/megakaryocytic differentiation. As his complete blood count included a few myeloid blasts (1% of WBC) and a bone marrow biopsy detected fibrosis without evidence of acute myelogenous leukemia (AML), a diagnosis of extramedullary blastic transformation of PMF was made, which was confirmed later by V617F mutation in Janus kinase-2 in both initial bone marrow biopsy and axillary tumor biopsy specimens. The patient died of pneumonia eight months after developing the axillary tumor. At autopsy, multiple MyS masses were detected in his soft tissue, but his bone marrow only contained fibrosis. Although MyS rarely develops before the leukemic transformation of PMF, no evidence of AML could be found in the patient's bone marrow at any point during the course of his disease. Thus, it is possible that the blasts in his peripheral blood were derived from the remaining MyS. Furthermore, the present case indicates that extramedullary blastic transformation, which is occasionally seen in CML, can also occur in PMF. Therefore, it is important to recognize that there is a wide variation in the pathogeneses of MyS and PMF. [ABSTRACT FROM AUTHOR]

Induction of the differentiation of human leukemia cells is a useful strategy in treatment of human leukemia. However, the molecular mechanisms involved in leukemia cell differentiation have not been fully elucidated. Interleukin 6 (IL-6) is a pleiotropic cytokine acting on a variety of cell types, and plays important roles in hematopoiesis. GATA binding protein 1 (GATA-1) is an important transcription factor involved in either megakaryocytic or erythrocytic differentiation. Herein we report that Rab7b, a late endosome/lysosome-localized myeloid small GTPase, promotes phorbol-12-myristate-13-acetate (PMA)-induced megakaryocytic differentiation by increasing nuclear factor κB (NF-κB)-dependent IL-6 production and subsequently enhancing the association of activated signal transducer and activator of transcription 3 (STAT3) with GATA-1. By using PMA-induced megakaryocytic differentiation of leukemia cells as a model, we investigated the roles of Rab7b in megakaryocytic differentiation. We find that Rab7b can potentiate PMA-induced upregulation of megakaryocytic markers, production of IL-6, and activation of NF-κB. Inhibitor of NF-κB and neutralizing antibodies for IL-6 or the IL-6 signaling receptor gp130 can block the effects of Rab7b in megakaryocytic differentiation. In Rab7b-silenced cells, PMA-induced activation of NF-κB, IL-6 production, and megakaryocytic differentiation are impaired. Furthermore, we demonstrate that IL-6-induced activation of STAT3 and the subsequent association of STAT3 with GATA-1 may contribute to PMA-induced and Rab7b-mediated transcriptional upregulation of megakaryocytic differentiation markers. Therefore, our data suggest that Rab7b may play important roles in megakaryopoiesis by activating NF-κB and promoting IL-6 production. Our study also indicates that the IL-6-induced association of STAT3 with GATA-1 may regulate megakaryocytic differentiation. [ABSTRACT FROM AUTHOR]