Erythroleukemia Cells Research Articles

An Experimental and Theoretical Study of the 1,3-Dipolar Cyclo­addition of Alloxan-Derived Azomethine Ylides to Cyclopropenes

AbstractA diastereoselective synthesis of biologically interesting spirobarbiturates has been achieved via [3+2] cycloaddition of alloxan-derived azomethine ylides to 3-R-1,2-diphenylcyclopropenes. With this approach, a range of spirobarbiturate-3-azabicyclo[3.1.0]hexanes and spirobarbiturate-cyclopropa[a]pyrrolizines were obtained in moderate to good yields with excellent diastereoselectivities. DFT calculations (M11 density functional theory) were carried out to shed light on the molecular mechanism of 1,3-dipolar cycloaddition of alloxan-derived azomethine ylides to cyclopropenes. The cytotoxic activity of some obtained compounds against human erythroleukemia (K562) cell line was evaluated in vitro by MTS-assay.

Characterization of iron-doped crystalline silicon nanoparticles and their modification with citrate anions for in vivo applications

Objectives. This paper presents data on the development and study of the structural properties of iron-doped crystalline silicon (nc-Si/SiOx/Fe) nanoparticles obtained using the plasma-chemical method for application in magnetic resonance imaging diagnostics and treatment of oncological diseases. This work aimed to use a variety of analytical methods to study the structural properties of nc-Si/SiOx/Fe and their colloidal stabilization with citrate anions for in vivo applications.Methods. Silicon nanoparticles obtained via the plasma-chemical synthesis method were characterized by laser spark emission spectroscopy, atomic emission spectroscopy, Fouriertransform infrared spectroscopy, and X-ray photoelectron spectroscopy. The hydrodynamic diameter of the nanoparticles was estimated using dynamic light scattering. The toxicity of the nanoparticles was investigated using a colorimetric MTT test for the cell metabolic activity. Elemental iron with different Fe/Si atomic ratios was added to the feedstock during loading.Results. The particles were shown to have a large silicon core covered by a relatively thin layer of intermediate oxides (interface) and an amorphous oxide shell, which is silicon oxide with different oxidation states SiOx (0 ≤ x ≤ 2). The samples had an iron content of 0.8–1.8 at %. Colloidal solutions of the nanoparticles stabilized by citrate anions were obtained and characterized. According to the analysis of the cytotoxicity of the modified nanosilicon particles using monoclonal K562 human erythroleukemia cells, no toxicity was found for cells in culture at particle concentrations of up to 5 µg/mL.Conclusions. Since the obtained modified particles are nontoxic, they can be used in in vivo theranostic applications.

EloA promotes HEL polyploidization upon PMA stimulation through enhanced ERK1/2 activity

Megakaryocytes (MKs) are the unique non-pathological cells that undergo polyploidization in mammals. The polyploid formation is critical for understanding the MK biology, and transcriptional regulation is involved in the differentiation and maturation of MKs. However, little is known about the functions of transcriptional elongation factors in the MK polyploidization. In this study, we investigated the role of transcription elongation factor EloA in the polyploidy formation during the MK differentiation. We found that EloA was highly expressed in the erythroleukemia cell lines HEL and K562. Knockdown of EloA in HEL cell line was shown to impair the phorbol myristate acetate (PMA) induced polyploidization process, which was used extensively to model megakaryocytic differentiation. Selective over-expression of EloA mutants with Pol II elongation activity partially restored the polyploidization. RNA-sequencing revealed that knockdown of EloA decelerated the transcription of genes enriched in the ERK1/2 cascade pathway. The phosphorylation activity of ERK1/2 decreased upon the EloA inhibition, and the polyploidization process of HEL was hindered when ERK1/2 phosphorylation was inhibited by PD0325901 or SCH772984. This study evidenced a positive role of EloA in HEL polyploidization upon PMA stimulation through enhanced ERK1/2 activity.

Inner Nuclear Protein Matrin-3 Coordinates Hematopoietic Cell Transcription and Differentiation By Stabilizing Chromatin Architecture

The nucleus is spatially organized by chromosome and interchromatin functional components. Global reorganization of chromatin interactions and compartmentalization occurring during differentiation requires proper chromosome positioning, but the involvement of nuclear components in this process remains largely underexplored. In particular, blood cell development exemplifies a coordinated process accompanied by dramatic chromatin reorganization, thereby providing a model in which to interrogate chromatin dynamics during differentiation. Here, we show that an abundant inner nuclear protein Matrin-3 (Matr3) plays a critical role in the maintenance of chromatin structure and has a broad effect on erythroid cell differentiation by coordinating gene expression.First, we deleted the entire gene body by CRISPR/Cas9 in mouse erythroleukemia (MEL) cells. The Matr3 knockout (KO) cells proliferate normally and exhibit morphological changes on differentiation suggestive of accelerated maturation. Consistently, erythroid-specific genes were expressed at a higher level in MEL Matr3 KO cells than in parental cells. The consequences of Matr3 deletion were also determined in G1ER cells, in which differentiation is conditional on activation of GATA-1. To assess the global impact of Matr3 loss on erythroid cell maturation, we measured global RNA expression changes. Erythroid-specific genes were expressed at a much higher level upon differentiation of Matr3 KO cells. Differentiation is typically accompanied by specific changes in nuclear architecture. Using super-resolution microscopy, we observed that heterochromatin protein 1α (HP1α) was more dispersed and irregular in appearance in Matr3 KO cells, suggesting that Matr3 loss alters morphological boundaries of heterochromatin.Analysis of the interactions between different regions of chromatin identifies topologically associating domains and classifies the genome into two compartments (A and B). The A and B compartments correspond to the structures and characteristics of known euchromatin and heterochromatin, respectively. We next explored global chromatin structure using a high-throughput chromosome conformation capture (Hi-C) assay. In Matr3 KO cells, insulation at the domain boundaries was reduced, and the compartment strengths between the B compartments became stronger, while those between A-type domains were reduced. Remarkably, we found that these changes in cells lacking Matr3 were similar to changes in chromatin contact during differentiation. To access the genomic features at a higher resolution, we performed the assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq). Notably, the newly opened regions in Matr3 KO, as compared to parental, cells were enriched for GATA motifs, which are generally more accessible in differentiated erythroid cells.Architectural proteins function cooperatively to organize chromatin. Using affinity purification followed by mass spectrometry and immunoblotting, we found that Matr3 interacts with proteins involved in chromatin remodeling, such as CTCF and cohesin. To identify whether Matr3 loss alters chromatin occupancy of its interacting partners, we performed ChIP-seq for CTCF and the core cohesin component Rad21. In the absence of Matr3, occupancy of CTCF and Rad21 was perturbed in a subset of genomic regions. Moreover, destabilization of CTCF and cohesin binding correlated with altered transcription and accelerated erythroid differentiation. Most sites with disrupted CTCF and Rad21 binding during differentiation were also sensitive to the absence of the scaffold protein Matr3.Our data demonstrate that the nucleoplasmic protein Matr3 stabilizes the binding of the architectural proteins (CTCF and cohesin) to chromatin and serves to maintain chromatin structure. We speculate that Matr3 negatively regulates cell fate transitions by maintaining cellular state through fine-tuning the binding of CTCF/cohesin to chromatin and associated 3D interactions. Our work reveals a previously unrecognized role of Matr3 in chromatin organization and responses to developmental cues. DisclosuresNo relevant conflicts of interest to declare.

Small Molecule Kinase Inhibitor Sunitinib Specifically Targets Juvenile Myelomonocytic Leukemia Cells with SHP2(PTPN11) Mutation

Juvenile myelomonocytic leukemia (JMML) is a highly fatal malignant disease in early childhood. It is still unknown of the specific pathogenesis, and there is shortage of effective targeted therapeutic approaches. Gain of function SHP2 mutation encoded by PTPN11 gene is found in approximately 35% of JMML patients, which maybe contributed to its pathogenesis. JMML patients with SHP2 mutation have lower survival rate and higher recurrence rate. All of the above make development of new therapies imperative. Currently, there is no stable cell line that can accurately reflect the characteristics of JMML abnormal cells for research on JMML. In this study, we established two leukemia cell lines that depend on mutated SHP2 for survival, and discovered promising drugs that targeted mutated-SHP2-dependent oncogenic signaling pathway through drug screening method.HCD-57 cells are murine erythroleukemia cells that solely depend on exogenic erythropoietin (EPO) for survival. We constructed SHP2-D61Y and SHP2-E76K transformed HCD-57 cell lines through retroviral vectors, the survival of which dependent on mutated SHP2 mediated signaling pathway. Based on these cells, we established a drug screening platform and screened small molecule compound library containing 2862 FDA-approved drugs and 1707 kinase inhibitors. We performed cell viability, flow cytometry, Wright-Giemsa staining, and western blot to evaluate cells after drug treatment. To further assess therapeutic potential, we established in-vivo transplantation model that SHP2-D61Y transformed HCD-57 cells were implanted into immunodeficient NCG mice, and verified the effectiveness of the in-vitro screened drugs.We found that the survival and proliferation of HCD-57 cells transduced by SHP2-D61Y and SHP2-E76K no longer required EPO, but completely relied on the abnormal activation of signaling pathway mediated by mutated SHP2. Western blot results showed that the phosphorylation status of ERK1/2 and AKT of HCD-57 cells expressing SHP2 mutation were abnormally increased, consistent with SHP2-mutated JMML. Thus, we have obtained the leukemia cell lines that can represent the characteristics of activated signaling pathway in JMML with SHP2 mutation. Through drug screening, we observed that drug sunitinib (Sutent ®) selectively inhibits SHP2-mutated HCD-57 cell lines. CCK-8-based cell viability assay demonstrated a dose-dependent inhibition of SHP2-D61Y and SHP2-E76K transformed HCD-57 cell and no effects on the parental HCD-57 cells. Live cell counting with trypan blue revealed that the proliferation of SHP2-mutated HCD-57 cells was totally halted after one day upon treatment with 250 nM sunitinib, whereas the HCD-57 cells were unaffected. Wright-Giemsa staining demonstrated that SHP2-mutated HCD-57 cells showed no normal morphology change and no mitotic activity under sunitinib treatment, otherwise parental HCD-57 cells showed normal mitotic activity. Sunitinib induced apoptosis and cell cycle arrest at G1 phase in SHP2-mutated HCD-57 cells by flow cytometry, but had little effect on the parental HCD-57 cells. Sunitinib effectively downregulates the phosphorylation of ERK and AKT in SHP2-mutated cells, revealing the mechanism of sunitinib targeting SHP2-mutated cells. In addition, after transplantation of SHP2-D61Y transformed HCD-57 cells for 3 weeks, the spleen of NCG mice increased from an average of 45 mg to more than 300 mg; flow cytometry analysis showed that the implanted cells accounted for over 75% of the total nucleated cells in the bone marrow and spleen. Compared with the vehicle control, the number of monocytes in these mice was reduced to the normal range by treatment with sunitinib, and the spleen weights were reduced by about 50%. Histochemical staining showed disappearance of the myeloid infiltration in the spleen, liver and bone marrow. The above results all indicate that sunitinib has strong in-vivo anti-leukemia activity. Furthermore, western blot analysis showed that the administration of sunitinib significantly inhibited the phosphorylation expression level of AKT and ERK, indicating the effectivity of sunitinib in vivo.In conclusion, our data demonstrated that HCD-57 cell line is an effective tool for studying oncogenic signaling pathway and screening drugs that targeted JMML with SHP2 mutation. Sunitinib can be an effective drug for the targeted treatment of JMML in the future. DisclosuresNo relevant conflicts of interest to declare.

Establishment and Characterization of a Human Primary Myelofibrosis Cell Line

Aims: Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) characterized by clonal proliferation of stem cells and myeloid cells. JAK2, CARL, and MPL mutations could be detected in most patients, but 15% of PMF patients could be absent of these three gene mutations. PMF has shorter median survival time and poorer prognosis when compared with closely related MPNs, polycythemia vera (PV) and essential thrombocythemia (ET). Allogenic hematopoietic stem cells (allo-HSCT) is the only curative therapy for PMF patients, however transplantation-related complications and deaths were observed in more than 50% patients. Hydroxyurea and Ruxolitinib are commonly used drugs to improve clinical symptoms of PMF patients, but lack of anti-tumor activities. These drugs could not reverse bone marrow fibrosis or induce cytogenetic remission. Basic research work of PMF is limited as to the lack of human PMF cell line. Here, we aimed to establish and characterize a patient-derived human PMF cell line, which would provide a useful tool for PMF research and screenings for novel drugs.Methods: The bone marrow cells were obtained from a 61-year-old male patient, who was diagnosed as PMF for 2 years. Mono-nuclear cells were isolated and cultured in a continuous culture system. The cells were characterized by different methods, including STR profiling, morphology observation with Giemsa staining, flow cytometry, chromosome analysis, Epstein-Barr virus (EBV) detection, mycoplasma detection, whole-genome-exon sequencing, and cell proliferation assay.Results: The cells have continued to grow in liquid culture for more than 50 passages using the same culture conditions. Doubling time was about 48-72 hours. The STR profiling identified that the cultured cells and the patient's bone marrow cells were from the same origin. The cells are free from EBV and mycoplasma. Giemsa-Wrights staining showed primitive erythrocytes (Figure A), which was consistent with CD71 positive detected by flow cytometry. Chromosome analysis revealed a hyperdiploid karyotype, which was 47, XY,+12[1]/48,idem,+9[9]. Whole-genome-exon sequencing identified mutations of ASXL1, TP53, IKZF1, IDH1, FLT3, and TET1 (Figure B). Now this cell line, designated ZYXY-M2, is collected by China center for type culture collection (CCTCC).Discussion: Established cell lines are invaluable tools in cell biology. Right now, HEL and SET-2 are two cell lines that are most widely used in MPN-related researches. HEL is an erythroleukemia cell line which was derived from a patient with leukemia transformation of Hodgkin's disease, and SET-2 is a megakaryoblastic cell line which was derived from a patient with leukemic transformation of ET. Here, we established a cell line, ZYXY-M2, derived from a PMF patient and it would prove to be a valuable model for the study of PMF. ZYXY-M2 could help to explore the pathogenesis and development of PMF, novel drug screening, and may also help to set up xenograft PMF mouse models. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Synergistic Combination Activity of the Novel GSPT1 Degrader CC-90009 in Acute Myeloid Leukemia Models

Introduction: CC-90009 is a novel cereblon E3 ligase modulator (CELMoD ®) agent that is a first-in-class degrader of translation termination factor G1 to S phase transition 1 (GSPT1). CC-90009 has demonstrated antileukemic activity as a single agent and is currently under investigation in patients with acute myeloid leukemia (AML; NCT02848001). Treatment with CC-90009 led to rapid reductions in peripheral and bone marrow blasts, and demonstrated preliminary promising efficacy, including several complete remissions, in patients with relapsed or refractory AML. Here, we describe the identification and preclinical activity of select anti-AML agents as potential combination partners of CC-90009 to further improve its efficacy and therapeutic index. Based on these results, the combination activity of CC-90009 with venetoclax (VEN)/azacitidine (AZA) is being evaluated in a phase 1/2 trial in patients with AML (NCT04336982).Methods: A high-throughput cell viability screen was performed to identify synergistic partners of CC-90009. AML cell lines (HL-60, HNT-34, KG-1, ML-2, NOMO-1, MOLM-13, MV4-11, F-36P, OCI-AML2, OCI-AML3) were treated with CC-90009 in combination with > 70 compounds, including standard anti-AML agents, tyrosine kinase inhibitors (TKIs), unfolded protein response inducers, transcription inhibitors, and epigenetic agents. MOLM-13 and MV4-11 are fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) cell lines. Hits were validated in a colony formation (CF) assay using primary AML cells and bone marrow mononuclear cells (BMMC) from healthy donors. Synergy of the combination partners with CC-90009 was further assessed in AML patient-derived xenograft (PDX) models. Synergy between the isocitrate dehydrogenase 2 (IDH2) inhibitor enasidenib and CC-90009 was evaluated in a TF-1 erythroleukemia cell line overexpressing IDH2 R140Q mutant, and an IDH2 R140Q PDX model, AM7577.Results: Our high-throughput combination screen revealed multiple TKIs, epigenetic agents, and pro-apoptotic agents as potential combination partners of CC-90009 in AML cell lines. FLT3 inhibitors, including sunitinib, pexidartinib, midostaurin, lestaurtinib, crenolanib, and gilteritinib, synergized with CC-90009 to reduce viability in FLT3-ITD AML cell lines MV4-11 and MOLM-13. Similarly, the B-cell lymphoma 2 (BCL2) inhibitor VEN potentiated CC-90009-induced apoptosis and accelerated cell-autonomous killing. Reduction in levels of MCL-1, an anti-apoptotic factor, by CC-90009 most likely contributed to the synergy with VEN.We prioritized the evaluation of FLT3, BCL2, and IDH2 inhibitors as partners of CC-90009. In CF assays, midostaurin enhanced the inhibitory effect of CC-90009 in primary AML cells, without augmenting the effect of CC-90009 in CD34+ BMMC from healthy donors. Similarly, VEN enhanced the reduction in CF by CC-90009 in AML patient-derived BMMC without exacerbating the decrease in CF by CC-90009 in BMMC from healthy donors.We characterized FLT3 inhibitor/CC-90009 and BCL2 inhibitor/AZA/CC-90009 combinations in a FLT3-ITD PDX murine model, PDX1. The FLT3 inhibitor quizartinib significantly prolonged survival when combined with CC-90009 compared with either agent alone (P < 0.001). Similarly, VEN/AZA/CC-90009 combination markedly extended survival compared with single agents or VEN/AZA doublets (P < 0.001).The synergy between CC-90009 and epigenetic modulators was validated and further characterized in customized cell differentiation assays. Addition of CC-90009 to enasidenib, a mutant IDH2 inhibitor, enhanced differentiation and killing of CD34+ stem and progenitor cells, and increased differentiated CD235a+ erythroblasts, compared with enasidenib alone in a TF-1 cell line overexpressing IDH2 R140Q. Enasidenib/CC-90009 combination treatment reduced CD45+ malignant populations and increased differentiated CD14+ cells, leading to significantly prolonged animal survival in an IDH2 R140Q PDX model, AM7577, compared with either agent alone (P < 0.0001).Conclusion: Using a high-throughput combination screen, we identified rational combination partners that synergize with CC-90009 in in vitro and in vivo AML models. Collectively, these results support the clinical evaluation of CC-90009 in combination with FLT3, BCL2, and IDH2 inhibitors to further improve treatment outcomes for patients with AML. DisclosuresPierce: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Yao: Bristol Myers Squibb: Consultancy, Current equity holder in publicly-traded company, Ended employment in the past 24 months, Research Funding. Pace: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Wang: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Flandin-Blety: Bristol Myers Squibb: Current Employment. Benitez: Bristol Myers Squibb: Current Employment. Guarinos: Bristol Myers Squibb: Current Employment. Hoffmann: Bristol Myers Squibb: Current Employment. Carrancio: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Pourdehnad: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

A Severe Mouse Model of Alpha-Thalassemia to Study Abnormal Iron Metabolism and Erythropoiesis, Hematopoietic Stem Cell Behavior and Development of a Gene Therapy Approach for Its Treatment

Alpha thalassemia (α-thal) is caused by insufficient production of the α-globin protein because of either deletional or non-deletional inactivation of endogenous α-globin genes. Clinical presentation of deletional α-thal varies from an asymptomatic condition (one inactivated α-globin gene) to a complete knockout (Hb Bart's Hydrops Fetalis). In patients with severe α-thal, a blood transfusion independent state is achievable through allogeneic bone marrow transplantation.The aims of this study are to develop a novel adult mouse model of α-thal and a gene therapy approach for this disease.We generated adult animals that do not produce α-globin chains (α-KO) through transplantation of homozygous B6.129S7-Hbatm1Paz/J fetal liver cells (FLC; isolated at E14.5) into WT recipient mice. These animals demonstrate a worsening phenotype, paradoxically showing elevated hematocrit, high reticulocyte count and a high number of red blood cells (RBC) which expressed only β-globin chains (HbH). RBC show aberrant morphology and aggregation of α- globin tetramers on RBC membranes. Due to severe inability of these RBC to deliver oxygen, the mice eventually succumb to anemia, showing splenomegaly and other organ pathologies, including vaso-occlusive events. These animals show iron deposition in the liver and kidney, in agreement with very low levels of hepcidin expression in the liver, and elevated erythropoietin (EPO) in the kidney.Interestingly, α-KO embryos show lower numbers of FLC compared to WT embryos, lower frequency of engraftable hematopoietic stem cells (HSC; Lin-Sca-1+c-kit+CD48-), decreased clonogenic potential (fewer class 4 CFUs) and elevated erythroferrone. Lethally irradiated mice transplanted with FLC-KO require 5-6x as many cells as those transplanted with FLC-WT for recovery, further suggesting some level of engraftment impairment. Our current hypothesis is that excessive hypoxia in the embryos impairs HSC function and stem cell fitness. Additional assays are in progress to assess the nature of this impairment.To generate a gene therapy tool to rescue these animals and eventually cure severe human α-thal patients, we screened multiple lentiviral vectors to identify the variant capable of producing the highest human α-globin protein per copy. The selection was conducted in murine erythroleukemia cells and human umbilical cord derived erythroid progenitor (HUDEP) cells, modified by knocking out all the human α-globin genes. We identified ALS20α, a vector where α-globin is under control of the β-globin promoter and its locus control region, as the most efficient vector. One copy of ALS20α produces exogenous α-globin at a level comparable to that produced by one endogenous α-globin gene. These results suggest that a relatively low VCN could result in dramatic therapeutic benefits. Transplantation of ALS20α transduced murine BM-KO results in correction of the disease phenotype in a dose-dependent manner. At VCN<1 we observe a delay in death proportional to the VCN value, while at VCN>1 we observe phenotypic normalization, including Hb, hepcidin and EPO levels.We tested ALS20α in CD34 cells isolated from four patients with both deletional and non- deletional HbH disease. We measured the change of β/α-globin mRNA ratio (β/αR) and protein level by HPLC in erythroblasts derived from these cultures. For the specimen with mutational HbH, the initial β/αR matches that of healthy controls, as the mutations do not eliminate the ability for the gene to produce aberrant mRNA transcripts, and decreased with increasing VCN. Erythroblasts with deletional HbH have a β/αR approximately 3x higher than normal cells, decreasing in a dose dependent manner with increasing VCN. HPLC detection of HbH (β4), a hallmark of HbH disease, is observed in hemolysis products from all non-transduced α−thal erythroblasts. A ~50% reduction of HbH is detected in the very same specimens upon integration of ALS20α (VCN between 1 and 2).In conclusion, we generated an adult mouse model of lethal α-thal and, in preliminary experiments, we rescue it with ALS20α. Furthermore, ALS20α successfully improves α-globin levels in patient cells. Further experiments are in progress to establish the consistency of our vector's expression in vivo, as well as to demonstrate its ability to transduce bona fide long-term HSCs. DisclosuresKattamis: Agios Pharmaceuticals: Consultancy; IONIS: Consultancy; VIFOR: Consultancy; CRISPR/Vertex: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria, Research Funding; Chiesi: Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy. Rivella: Celgene Corporation: Consultancy; Keros Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Disc Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees; MeiraGTx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Forma Theraputics: Consultancy; Incyte: Consultancy; Ionis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees.

The Y14-p53 regulatory circuit in megakaryocyte differentiation and thrombocytopenia

SummaryThrombocytopenia-absent radius (TAR) syndrome is caused by RBM8A insufficiency. We generated megakaryocyte-specific Rbm8a knockout (Rbm8aKOMK) mice that exhibited marked thrombocytopenia, internal hemorrhage, and splenomegaly, providing evidence that genetic deficiency of Rbm8a causes a disorder of platelet production. Rbm8aKOMK mice accumulated low-ploidy immature megakaryocytes in the bone marrow and exhibited defective platelet activation and aggregation. Accordingly, depletion of Y14 (RBM8A) in human erythroleukemia (HEL) cells compromised phorbol-ester-induced polyploidization. Notably, Y14/RBM8A deficiency induced both p53 and p21 in megakaryocytes and HEL cells. Treatment with a p53 inhibitor restored ex vivo differentiation of Rbm8aKOMK megakaryocytes and unexpectedly activated Y14 expression in HEL cells. Trp53 knockout partially restored megakaryocyte differentiation by reversing cell-cycle arrest and increased platelet counts of Rbm8aKOMK, indicating that excess p53 in part accounts for thrombocytopenia in TAR syndrome. This study provides evidence for the role of the Y14-p53 circuit in platelet production and a potential therapeutic strategy.

Sildenafil Citrate Downregulates PDE5A mRNA Expression in Women with Recurrent Pregnancy Loss without Altering Angiogenic Factors-A Preliminary Study.

In our previous study, we showed that sildenafil citrate (SC), a selective PDE5A blocker, modulated NK cell activity in patients with recurrent pregnancy loss, which correlated with positive pregnancy outcomes. It was found that NK cells had a pivotal role in decidualization, angiogenesis, spiral artery remodeling, and the regulation of trophoblast invasion. Thus, in the current study, we determined the effects of SC on angiogenic factor expression and production, as well as idNK cell activity in the presence of nitric synthase blocker L-NMMA. Methods: NK cells (CD56+) were isolated from the peripheral blood of 15 patients and 15 fertile women on MACS columns and cultured in transformation media containing IL-15, TGF-β, and AZA—a methylation agent—for 7 days in hypoxia (94% N2, 1% O2, 5% CO2). Cultures were set up in four variants: (1) with SC, (2) without SC, (3) with NO, a synthase blocker, and (4) with SC and NO synthase blocker. NK cell activity was determined after 7 days of culturing as CD107a expression after an additional 4h of stimulation with K562 erythroleukemia cells. The expression of the PDE5A, VEGF-A, PIGF, IL-8, and RENBP genes was determined with quantitative real-time PCR (qRT-PCR) using TaqMan probes and ELISA was used to measure the concentrations of VEGF-A, PLGF, IL-8, Ang-I, Ang-II, IFN–γ proteins in culture supernatants after SC supplementation. Results: SC downregulated PDE5A expression and had no effect on other studied angiogenic factors. VEGF-A expression was increased in RPL patients compared with fertile women. Similarly, VEGF production was enhanced in RPL patients’ supernatants and SC increased the concentration of PIGF in culture supernatants. SC did not affect the expression or concentration of other studied factors, nor idNK cell activity, regardless of NO synthase blockade.

Synthesis, Characterization, and Biological Activity of Water-Soluble, Dual Anionic and Cationic Ruthenium–Arene Complexes Bearing Imidazol(in)ium-2-dithiocarboxylate Ligands

An efficient synthetic protocol was devised for the preparation of five cationic ruthenium-arene complexes bearing imidazol(in)ium-2-dithiocarboxylate ligands from the [RuCl2(p-cymene)]2 dimer and 2 equiv of an NHC·CS2 zwitterion. The reactions proceeded cleanly and swiftly in dichloromethane at room temperature to afford the expected [RuCl(p-cymene)(S2C·NHC)]Cl products in quantitative yields. When the [RuCl2(p-cymene)]2 dimer was reacted with only 1 equiv of a dithiolate betaine under the same experimental conditions, a set of five bimetallic compounds with the generic formula [RuCl(p-cymene)(S2C·NHC)][RuCl3(p-cymene)] was obtained in quantitative yields. These novel, dual anionic and cationic ruthenium-arene complexes were fully characterized by various analytical techniques. NMR titrations showed that the chelation of the dithiocarboxylate ligands to afford [RuCl(p-cymene)(S2C·NHC)]+ cations was quantitative and irreversible. Conversely, the formation of the [RuCl3(p-cymene)]- anion was limited by an equilibrium, and this species readily dissociated into Cl- anions and the [RuCl2(p-cymene)]2 dimer. The position of the equilibrium was strongly influenced by the nature of the solvent and was rather insensitive to the temperature. Two monometallic and two bimetallic complexes cocrystallized with water, and their molecular structures were solved by X-ray diffraction analysis. Crystallography revealed the existence of strong interactions between the azolium ring protons of the cationic complexes and neighboring donor groups from the anions or the solvent. The various compounds under investigation were highly soluble in water. They were all strongly cytotoxic against K562 cancer cells. Furthermore, with a selectivity index of 32.1, the [RuCl(p-cymene)(S2C·SIDip)]Cl complex remarkably targeted the erythroleukemic cells vs mouse splenocytes.

Transcriptomic profile of human erythroleukemia cells in response to Sargassum fusiforme polysaccharide and its structure analysis.

Sargassum fusiforme (S. fusiforme) has been used as an ingredient in Chinese herbal medicine for thousands of years. However, there are a limited number of studies concerning its therapeutic mechanism. High performance gel permeation chromatography (HPGPC) analysis showed that the average molecular weight of the S. fusiforme polysaccharide, SFPS 191212, is 43 kDa. SFPS 191212 is composed of mannose, rhamnose, galactose, xylose, glucose, and fucose (at a molar ratio: 2.1 : 2.9 : 1.8 : 15.5 : 4.6 : 62.5) with α- and β-configurations. The present research evaluated the anti-tumor potential of the S. fusiforme polysaccharide in human erythroleukemia (HEL) cells in vitro. To explore the SFPS 191212's apoptosis mechanism in HEL cells, transcriptome analysis was performed on HEL cells that were incubated with SFPS 191212. The inhibitory effect of SFPS 191212 on HEL cell growth was also analyzed. It was found that SFPS 191212 inhibited HEL cell proliferation, reduced cell viability in a concentration-dependent manner, and induced an insignificant toxic effect on normal human embryonic lung (MRC-5) cells. Compared with the control group, transcriptome analysis identified a total of 598 differentially expressed genes (DEGs), including 243 up-regulated genes and 355 down-regulated genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on all DEGs, and 900 GO terms and 52 pathways were found to be significantly enriched. Finally, 23 DEGs were randomly selected and confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). Moreover, SFPS 191212 down-regulated the PI3K/Akt signal transduction pathway. Our results provide a framework for understanding the effect of SFPS 191212 on cancer cells and can serve as a resource for delineating the anti-tumor mechanisms of S. fusiforme.

Deficiencies in the DNA Binding Protein ARID3a Alter Chromatin Structures Important for Early Human Erythropoiesis.

ARID3a is a DNA-binding protein important for normal hematopoiesis in mice and for in vitro lymphocyte development in human cultures. ARID3a knockout mice die in utero with defects in both early hematopoietic stem cell populations and erythropoiesis. Recent transcriptome analyses in human erythropoietic systems revealed increases in ARID3a transcripts implicating potential roles for ARID3a in human erythrocyte development. However, ARID3a transcript levels do not faithfully reflect protein levels in many cells, and the functions and requirements for ARID3a protein in those systems have not been explored. We used the erythroleukemic cell line K562 as a model to elucidate functions of ARID3a protein in early human erythropoiesis. ARID3a knockdown of hemin-stimulated K562 cells resulted in lack of fetal globin production and modifications in gene expression. Temporal RNA sequencing data link ARID3a expression with the important erythroid regulators Gata1, Gata2, and Klf1 Ablation of ARID3a using CRISPR-Cas9 further demonstrated it is required to maintain chromatin structures associated with erythropoietic differentiation potential. These data demonstrate that the ARID3a protein is required for early erythropoietic events and provide evidence for the requirement of ARID3a functions for proper maintenance of appropriate chromatin structures.

Anticancer Activity and In Silico ADMET Properties of 2,4,5-Trisubstitutedthiazole Derivatives.

Recently, a series of 15 compounds with 2,4,5-trisubstitutedthiazole scaffold having 2- amino/amido/ureido functional groups attached with 5-aryl and 4-carboxylic acid/ester groups (1-15) were reported from our research group as novel potential inhibitors of carbonic anhydrase III (CA III) enzyme. Several research studies revealed the potential role of CA inhibitors as anticancer agents, giving us the impetus to further explore these compounds for their potential as anticancer agents. The objective of this study is to investigate the potential of 2,4,5-trisubstitutedthiazole derivatives (1-15) for their possible cytotoxic activity (in vitro), and to calculate (in silico) the absorption, distribution, metabolism, excretion and toxicity (ADMET) properties to evaluate the drug-likeness of these compounds. Cytotoxic activity (in vitro) was carried out on two breast cancer cell lines (MCF7 and MDA231), and the lymphoblastoid human erythroleukemia cell line (K562) using 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. Doxorubicin was used as a positive control. ADMET properties were calculated (in silico) using the QikProp module of Schrodinger. Compounds 6 and 9 with a phenylureido group at 2-position, and a methyl-carboxylate moiety at 4-position having para-tolyl and benzyl moiety, respectively at the 5-position of the thiazole ring showed significant cytotoxicity against all the three cell lines. In particular, compound 6 with para-tolyl group at 5-position exhibited the most potent inhibitory effect on the viability of MCF7, MDA231 and K562 cells, with IC50 values of 22, 26 and 11 μM, respectively. Notably, all the highly active compounds possess a phenyluriedo group at 2-- position with a methyl ester group at 4-position, indicating the probable role of these substituents in the target interaction and inducing cytotoxicity. Interestingly, compounds 1-4 and 10-13 with a free amino group at 2-position did not show any cytotoxic effect on the K562 cell line, while exhibiting mild to moderate cytotoxicity against the MCF7 and MDA231 cell lines. However, none of the tested compounds showed any activity against normal human dermal fibroblast cells indicating the safety/tolerability of the examined concentrations. Furthermore, these compounds also exhibited satisfactory ADMET properties (in silico), without violating Lipinski's rule of five. The most active compounds 6 and 9 predicted to have good oral absorption and low human serum protein binding, exhibiting no reactive functional group and probable CNS activity compared with 95% of the known oral drugs as predicted (in silico) by QikProp. Thus, compounds 6 and 9 can be considered as lead molecules for further modification and discovery of novel anticancer agents with nanomolar potency.

Involvement of autophagy in diosgenin‑induced megakaryocyte differentiation in human erythroleukemia cells.

Natural agents have been used to restart the process of differentiation that is inhibited during leukemic transformation of hematopoietic stem or progenitor cells. Autophagy is a housekeeping pathway that maintains cell homeostasis against stress by recycling macromolecules and organelles and plays an important role in cell differentiation. In the present study, an experimental model was established to investigate the involvement of autophagy in the megakaryocyte differentiation of human erythroleukemia (HEL) cells induced by diosgenin [also known as (25R)-Spirosten-5-en-3b-ol]. It was demonstrated that Atg7 expression was upregulated from day 1 of diosgenin-induced differentiation and was accompanied by a significant elevation in the conversion of light chain 3 A/B (LC3-A/B)-I to LC3-A/B-II. Autophagy was modulated before or after the induction of megakaryocyte differentiation using 3-methyladenine (3-MA, autophagy inhibitor) and metformin (Met, autophagy initiation activator). 3-MA induced a significant accumulation of the LC3 A/B-II form at day 8 of differentiation. It was revealed that 3-MA had a significant repressive effect on the nuclear (polyploidization) and membrane glycoprotein V [(GpV) expression] maturation. On the other hand, autophagy activation increased GpV genomic expression, but did not change the nuclear maturation profile after HEL cells treatment with Met. It was concluded that autophagy inhibition had a more prominent effect on the diosgenin-differentiated cells than autophagy activation.

Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope

Base editors are RNA-guided deaminases that enable site-specific nucleotide transitions. The targeting scope of these Cas-deaminase fusion proteins critically depends on the availability of a protospacer adjacent motif (PAM) at the target locus and is limited to a window within the CRISPR-Cas R-loop, where single-stranded DNA (ssDNA) is accessible to the deaminase. Here, we reason that the Cas9-HNH nuclease domain sterically constrains ssDNA accessibility and demonstrate that omission of this domain expands the editing window. By exchanging the HNH nuclease domain with a monomeric or heterodimeric adenosine deaminase, we furthermore engineer adenine base editor variants (HNHx-ABEs) with PAM-proximally shifted editing windows. This work expands the targeting scope of base editors and provides base editor variants that are substantially smaller. It moreover informs of potential future directions in Cas9 protein engineering, where the HNH domain could be replaced by other enzymes that act on ssDNA.

Multifunctional protein 4.1R regulates the asymmetric segregation of Numb during terminal erythroid maturation

The asymmetric cell division of stem or progenitor cells generates daughter cells with distinct fates that balance proliferation and differentiation. Asymmetric segregation of Notch signaling regulatory protein Numb plays a crucial role in cell diversification. However, the molecular mechanism remains unclear. Here, we examined the unequal distribution of Numb in the daughter cells of murine erythroleukemia cells (MELCs) that undergo DMSO-induced erythroid differentiation. In contrast to the cytoplasmic localization of Numb during uninduced cell division, Numb is concentrated at the cell boundary in interphase, near the one-spindle pole in metaphase, and is unequally distributed to one daughter cell in anaphase in induced cells. The inheritance of Numb guides this daughter cell toward erythroid differentiation while the other cell remains a progenitor cell. Mitotic spindle orientation, critical for distribution of cell fate determinants, requires complex communication between the spindle microtubules and the cell cortex mediated by the NuMA-LGN-dynein/dynactin complex. Depletion of each individual member of the complex randomizes the position of Numb relative to the mitotic spindle. Gene replacement confirms that multifunctional erythrocyte protein 4.1R (4.1R) functions as a member of the NuMA-LGN-dynein/dynactin complex and is necessary for regulating spindle orientation, in which interaction between 4.1R and NuMA plays an important role. These results suggest that mispositioning of Numb is the result of spindle misorientation. Finally, disruption of the 4.1R-NuMA-LGN complex increases Notch signaling and decreases the erythroblast population. Together, our results identify a critical role for 4.1R in regulating the asymmetric segregation of Numb to mediate erythropoiesis.

Identification of Spiro-Fused [3-azabicyclo[3.1.0]hexane]oxindoles as Potential Antitumor Agents: Initial In Vitro Evaluation of Anti-Proliferative Effect and Actin Cytoskeleton Transformation in 3T3 and 3T3-SV40 Fibroblast.

Novel heterocyclic compounds containing 3-spiro[3-azabicyclo[3.1.0]hexane]oxindole framework (4a, 4b and 4c) have been studied as potential antitumor agents. The in silico ADMET (adsorption, distribution, metabolism, excretion and toxicity) analysis was performed on 4a–c compounds with promising antiproliferative activity, previously synthetized and screened against human erythroleukemic cell line K562 tumor cell line. Cytotoxicity of 4a–c against murine fibroblast 3T3 and SV-40 transformed murine fibroblast 3T3-SV40 cell lines were evaluated. The 4a and 4c compounds were cytotoxic against 3T3-SV40 cells in comparison with those of 3T3. In agreement with the DNA cytometry studies, the tested compounds have achieved significant cell-cycle perturbation with higher accumulation of cells in G0/G1 phase. Using confocal microscopy, we found that with 4a and 4c treatment of 3T3 cells, actin filaments disappeared, and granular actin was distributed diffusely in the cytoplasm in 82–97% of cells. The number of 3T3-SV40 cells with stress fibers increased to 7–30% against 2% in control. We discovered that transformed 3T3-SV40 cells after treatment with compounds 4a and 4c significantly reduced the number of cells with filopodium-like membrane protrusions (from 86 % in control cells to 6–18% after treatment), which indirectly suggests a decrease in cell motility. We can conclude that the studied compounds 4a and 4c have a cytostatic effect, which can lead to a decrease in the number of filopodium-like membrane protrusions.

Nitric Oxide Synthase Dependency in Hydroxyurea Inhibition of Erythroid Progenitor Growth.

Hydroxyurea (HU) causes nitric oxide (NO) bioactivation, acting as both a NO donor and a stimulator of NO synthase (NOS). To examine whether HU effects are NO mediated by chemical degradation or enzymatic induction, we studied human and mouse erythroid cells during proliferation, apoptosis, and differentiation. The HU and NO donor demonstrated persisted versus temporary inhibition of erythroid cell growth during differentiation, as observed by γ- and β-globin gene expression. HU decreased the percentage of erythroleukemic K562 cells in the G2/M phase that was reversed by N-nitro l-arginine methyl ester hydrochloride (L-NAME). Besides activation of endothelial NOS, HU significantly increased apoptosis of K562 cells, again demonstrating NOS dependence. Administration of HU to mice significantly inhibited colony-forming unit-erythroid (CFU-E), mediated by NOS. Moreover, burst-forming-units-erythroid (BFU-E) and CFU-E ex vivo growth was inhibited by the administration of nitrate or nitrite to mice. Chronic in vivo NOS inhibition with L-NAME protected the bone marrow cellularity despite HU treatment of mice. NO metabolites and HU reduced the frequency of NOS-positive cells from CFU-E and BFU-E colonies that was reverted by NOS inhibition. HU regulation of the G2/M phase, apoptosis, differentiation, cellularity, and NOS immunoreactive cells was NOS dependent. Inhalation of NO therapy as well as strategies to increase endogenous NO production could replace or enhance HU activity.

A Positive Regulatory Feedback Loop between EKLF/KLF1 and TAL1/SCL Sustaining the Erythropoiesis.

The erythroid Krüppel-like factor EKLF/KLF1 is a hematopoietic transcription factor binding to the CACCC DNA motif and participating in the regulation of erythroid differentiation. With combined use of microarray-based gene expression profiling and the promoter-based ChIP-chip assay of E14.5 fetal liver cells from wild type (WT) and EKLF-knockout (Eklf−/−) mouse embryos, we identified the pathways and direct target genes activated or repressed by EKLF. This genome-wide study together with the molecular/cellular analysis of the mouse erythroleukemic cells (MEL) indicate that among the downstream direct target genes of EKLF is Tal1/Scl. Tal1/Scl encodes another DNA-binding hematopoietic transcription factor TAL1/SCL, known to be an Eklf activator and essential for definitive erythroid differentiation. Further identification of the authentic Tal gene promoter in combination with the in vivo genomic footprinting approach and DNA reporter assay demonstrate that EKLF activates the Tal gene through binding to a specific CACCC motif located in its promoter. These data establish the existence of a previously unknow positive regulatory feedback loop between two DNA-binding hematopoietic transcription factors, which sustains mammalian erythropoiesis.