Human Erythroleukemia K562 Cells Research Articles

PI3K/HSCB axis facilitates FOG1 nuclear translocation to promote erythropoiesis and megakaryopoiesis.

Erythropoiesis and megakaryopoiesis are stringently regulated by signaling pathways. However, the precise molecular mechanisms through which signaling pathways regulate key transcription factors controlling erythropoiesis and megakaryopoiesis remain partially understood. Herein, we identified heat shock cognate B (HSCB), which is well known for its iron-sulfur cluster delivery function, as an indispensable protein for friend of GATA 1 (FOG1) nuclear translocation during erythropoiesis of K562 human erythroleukemia cells and cord-blood-derived human CD34+CD90+hematopoietic stem cells (HSCs), as well as during megakaryopoiesis of the CD34+CD90+HSCs. Mechanistically, HSCB could be phosphorylated by phosphoinositol-3-kinase (PI3K) to bind with and mediate the proteasomal degradation of transforming acidic coiled-coil containing protein 3 (TACC3), which otherwise detained FOG1 in the cytoplasm, thereby facilitating FOG1 nuclear translocation. Given that PI3K is activated during both erythropoiesis and megakaryopoiesis, and that FOG1 is a key transcription factor for these processes, our findings elucidate an important, previously unrecognized iron-sulfur cluster delivery independent function of HSCB in erythropoiesis and megakaryopoiesis.

PI3K/HSCB axis facilitates FOG1 nuclear translocation to promote erythropoiesis and megakaryopoiesis

Erythropoiesis and megakaryopoiesis are stringently regulated by signaling pathways. However, the precise molecular mechanisms through which signaling pathways regulate key transcription factors controlling erythropoiesis and megakaryopoiesis remain partially understood. Herein, we identified heat shock cognate B (HSCB), which is well known for its iron–sulfur cluster delivery function, as an indispensable protein for friend of GATA 1 (FOG1) nuclear translocation during erythropoiesis of K562 human erythroleukemia cells and cord-blood-derived human CD34+CD90+hematopoietic stem cells (HSCs), as well as during megakaryopoiesis of the CD34+CD90+HSCs. Mechanistically, HSCB could be phosphorylated by phosphoinositol-3-kinase (PI3K) to bind with and mediate the proteasomal degradation of transforming acidic coiled-coil containing protein 3 (TACC3), which otherwise detained FOG1 in the cytoplasm, thereby facilitating FOG1 nuclear translocation. Given that PI3K is activated during both erythropoiesis and megakaryopoiesis, and that FOG1 is a key transcription factor for these processes, our findings elucidate an important, previously unrecognized iron–sulfur cluster delivery independent function of HSCB in erythropoiesis and megakaryopoiesis.

Liver X receptor activation in chronic myelogenous leukemia cells yields distinct mass fingerprints by whole cell MALDI-TOF MS, which correspond to changes in cell viability, gene expression, and differentiation markers

Liver X receptor (LXR) agonists, including synthetic ligands or naturally-occurring oxysterols, exert promising lipid-modulating, anti-inflammatory, and anti-tumor effects. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) whole cell mass spectrometry (MS) shows promise in chronic disease and food science research, clinical diagnostics of cancer cells and bacterial pathogens, and drug/nutraceutical discovery due to its relatively low-cost, rapid, reliable, and high-throughput ability to identify unique cell phenotypes and differentiation states of immune cells that play essential roles in chronic metabolic and inflammatory disease pathophysiology. However, optimization of whole cell MALDI-TOF MS-based methods to assess cellular changes in response to pharmaceutical and/or nutraceutical treatment warrants further study. Thus, we investigated whether whole cell MALDI-TOF MS could detect cellular phenotype changes of human stem-like erythroleukemia K562 cells – a model for chronic myelogenous leukemia (CML) – induced by the LXR agonist TO-901317, a synthetic ligand that mimics effects of naturally-occurring oxysterols, which are oxidized cholesterol derivatives formed under conditions of high cellular cholesterol concentrations and in food products. TO-901317 dose- and time-dependently altered mRNA expression of cholesterol flux genes, including ATP-binding cassette transporter A1 (ABCA1), LDL-receptor (LDLR), HMG-CoA reductase (HMGCR), and reduced cellular cholesterol content by 22.9% at 24 hours. TO-901317 additionally dose- and time-dependently impacted K562 cell viability, number, size, and reduced mRNA expression of the anti-apoptotic gene BCL2L1 by 43.3%. These effects corresponded to induced gene expression of erythroid markers and hemoglobin content, suggesting that LXR activation may promote erythroid lineage differentiation. Whole cell MALDI-TOF MS mass fingerprinting revealed distinct proteomic phenotypes between TO-901317 vs. untreated cells, and identified histone 2A and histone H4 as uniquely expressed proteins. These findings not only elucidate a novel role for LXR agonists in a model of CML, but demonstrate that mass fingerprints obtained by whole cell MALDI-TOF mass spectrometry effectively distinguish between treatment-induced cell phenotypes.

Lysophosphatidic Acid Receptor 3 Activation Is Involved in the Regulation of Ferroptosis.

Ferroptosis, a unique form of programmed cell death trigged by lipid peroxidation and iron accumulation, has been implicated in embryonic erythropoiesis and aging. Our previous research demonstrated that lysophosphatidic acid receptor 3 (LPA3) activation mitigated oxidative stress in progeria cells and accelerated the recovery of acute anemia in mice. Given that both processes involve iron metabolism, we hypothesized that LPA3 activation might mediate cellular ferroptosis. In this study, we used an LPA3 agonist, 1-Oleoyl-2-O-methyl-rac-glycerophosphothionate (OMPT), to activate LPA3 and examine its effects on the ferroptosis process. OMPT treatment elevated anti-ferroptosis gene protein expression, including solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), heme oxygenase-1 (HO-1), and ferritin heavy chain (FTH1), in erastin-induced cells. Furthermore, OMPT reduced lipid peroxidation and intracellular ferrous iron accumulation, as evidenced by C11 BODIPY™ 581/591 Lipid Peroxidation Sensor and FerroOrange staining. These observations were validated by applying LPAR3 siRNA in the experiments mentioned above. In addition, the protein expression level of nuclear factor erythroid 2-related factor (NRF2), a key regulator of oxidative stress, was also enhanced in OMPT-treated cells. Lastly, we verified that LPA3 plays a critical role in erastin-induced ferroptotic human erythroleukemia K562 cells. OMPT rescued the erythropoiesis defect caused by erastin in K562 cells based on a Gly A promoter luciferase assay. Taken together, our findings suggest that LPA3 activation inhibits cell ferroptosis by suppressing lipid oxidation and iron accumulation, indicating that ferroptosis could potentially serve as a link among LPA3, erythropoiesis, and aging.

New Synthetic Isoxazole Derivatives Acting as Potent Inducers of Fetal Hemoglobin in Erythroid Precursor Cells Isolated from β-Thalassemic Patients.

Induction of fetal hemoglobin (HbF) is highly beneficial for patients carrying β-thalassemia, and novel HbF inducers are highly needed. Here, we describe a new class of promising HbF inducers characterized by an isoxazole chemical skeleton and obtained through modification of two natural molecules, geldanamycin and radicicol. After preliminary biological assays based on benzidine staining and RT-qPCR conducted on human erythroleukemic K562 cells, we employed erythroid precursors cells (ErPCs) isolated from β-thalassemic patients. ErPCs weretreated with appropriate concentrations of isoxazole derivatives. The accumulation of globin mRNAs was studied by RT-qPCR, and hemoglobin production by HPLC. We demonstrated the high efficacy of isozaxoles in inducing HbF. Most of these derivatives displayed an activity similar to that observed using known HbF inducers, such as hydroxyurea (HU) or rapamycin; some of the analyzed compounds were able to induce HbF with more efficiency than HU. All the compounds were active in reducing the excess of free α-globin in treated ErPCs. All the compounds displayed a lack of genotoxicity. These novel isoxazoles deserve further pre-clinical study aimed at verifying whether they are suitable for the development of therapeutic protocols for β-thalassemia.

Characterization of K562 cells: uncovering novel chromosomes, assessing transferrin receptor expression, and probing pharmacological therapies.

Human erythroleukemic K562 cells represent the prototypical cell culture model of chronic myeloid leukemia (CML). The cells are pseudo-triploid and positive for the Philadelphia chromosome. Therefore, K562 cells have been widely used for investigating the BCR/ABL1 oncogene and the tyrosine kinase inhibitor, imatinib-mesylate. Further, K562 cells overexpress transferrin receptors (TfR) and have been used as a model for targeting cytotoxic therapies, via receptor-mediated endocytosis. Here, we have characterized K562 cells focusing on the karyotype of cells in prolonged culture, regulation of expression of TfR in wildtype (WT) and doxorubicin-resistant cells, and responses to histone deacetylase inhibition (HDACi). Karyotype analysis indicates novel chromosomes and gene expression analysis suggests a shift of cultured K562 cells away from patient-derived leukemic cells. We confirm the high expression of TfR on K562 cells using immunofluorescence and cell-surface receptor binding radioassays. Importantly, high TfR expression is observed in patient-derived cells, and we highlight the persistent expression of TfR following doxorubicin acquired resistance. Epigenetic analysis indicates that permissive histone acetylation and methylation at the promoter region regulates the transcription of TfR in K562 cells. Finally, we show relatively high expression of HDAC enzymes in K562 cells and demonstrate the chemotoxic effects of HDACi, using the FDA-approved hydroxamic acid, vorinostat. Together with a description of morphology, infrared spectral analysis, and examination of metabolic properties, we provide a comprehensive characterization of K562 cells. Overall, K562 cell culture systems remain widely used for the investigation of novel therapeutics for CML, which is particularly important in cases of imatinib-mesylate resistance.

Diorganotin(IV) N-methyl-N-phenethyldithiocarbamate Compounds Induce Cytotoxicity via Apoptosis in K562 Human Erythroleukaemia Cells

Imatinib mesylate (IM), a leading treatment for chronic myeloid leukaemia (CML), has sparked worries about the possibility of CML patients developing a resistance to it. As a result, researchers are becoming more interested in organotin(IV) compounds due to their strong potential to be developed as anticancer agents and employed as an option to address the issues regarding IM-resistance therapy. Generally, this study is to determine the cytotoxicity induced by diorganotin(IV) dithiocarbamate compounds in K562 human erythroleukaemia cells. The two novel diorganotin(IV) compounds namely diphenyltin(IV) N-methyl-N-phenethyldithiocarbamate (C1) and dibutyltin(IV) N-methyl-N-phenethyldithiocarbamate (C2) were assessed their cytotoxicity via MTT [3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and mode of cell death via Annexin V-FITC/PI assay with the duration treatment of 24 h. Both compounds displayed strong cytotoxicity in K562 cells. At concentration of 4.2 µM for C1 and 1.6 µM for C2, both compounds were able to induce 49.70% and 46.83% apoptotic events, respectively. The changes in cells' morphological can also be seen 24 h after being exposed to the compounds at their respective IC50 doses. The findings demonstrated that the morphology of the cells was similar to apoptotic features, including cell shrinkage and the production of apoptotic bodies, meanwhile, the low levels of necrotic cells (<1%) also can be seen via cell lysis. In conclusion, both compounds possess the potential as antileukaemia drugs nevertheless, further studies on their action mechanism are required to ratify their qualities and suitability in the research of anticancer drugs development.

Abstract 4040: Asciminib activates erythroid differentiation in K562 cell line

Abstract Background: Asciminib an allosteric tyrosine kinase inhibitor (TKI) was recently approved by the FDA for managing Philadelphia positive chronic myeloid leukemia (CML) patients in chronic phase who had failed to respond to previous TKIs. In this study, we used human erythroleukemic K562 cell line to investigate the role of asciminib as an inducer of erythroid differentiation. Additionally, imatinib, which has previously been reported as a potential inducer of erythroid differentiation, was also tested on these cells, both independently as well as in combination, in order to investigate the degree of erythroid differentiation in a K562 cell line. Methodology: In this study, we investigated the effect of asciminib (5-20nM) with and without imatinib (50-200nM) in differentiating the erythroleukemic cell line K562 towards erythroid lineage. K562 cells were cultured over a 12-day period in basic (IMDM) media and an EPO-based differentiation media to select optimal medium conditions. In addition, an in-vitro drug cytotoxicity experiment (XTT) was used to determine the preliminary toxicity of asciminib with IMDM media and EPO-based differentiation media CD235a and CD71 surface marker expressions were analyzed by flow cytometry at all time-points to determine the degree of differentiation achieved by each of the conditions Moreover, to analyze and identify hemoglobin producing cells ensuing differentiation, benzidine staining was performed at indicated time points. Results: Our results suggest that asciminib, imatinib or a combination of both drugs in EPO-based differentiation media results in efficient erythroid differentiation with high levels of GPA expression (approx. 90%) and significant upregulation of globin genes (p value = 0.0001). These results were further confirmed by benzidine staining, where drug addition and dose escalation significantly increased benzidine-positive cells. Our results also demonstrated that asciminib was able to achieve maximum differentiation at a much earlier time point as compared to imatinib. Moreover, asciminib, when combined with another TKI such as imatinib, can also aid in lowering the drug concentrations being used, hence resulting in lessening the toxicity of the drug when used alone required at high doses. Conclusion: The results from this study highlighted the novel role of asciminib in erythroid differentiation using an in-vitro K562 model that can give high-levels of surface markers and globin expression. Potential clinical application of asciminib in hematological diseases that are associated specifically with a failure in the expression of globin genes should be evaluated. Keywords:K562, erythroid differentiation, asciminib, imatinib, glycophorin A, transferrin receptor, hemoglobin Citation Format: Hammad Hassan, Sarah Yousuf, Syed Muhammad Ahmed, Karim Ruknuddin, Hadiqa Raees, Sadia Habib, Fizza Iftikhar, Afsar Ali Mian, Elnasir Lalani. Asciminib activates erythroid differentiation in K562 cell line. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4040.

Loss of Cyb5R3 Function Silences Fetal Hemoglobin and Hematocrit Responses to Hydroxyurea in Patients with Sickle Cell Anemia

BACKGROUND: Hydroxyurea (HU) is the first FDA-approved and most widely prescribed drug for sickle cell (SS) patients due to its beneficial effects (decreased Hb polymerization, pain crises and hospitalization) and relative safety profile. While the clinical benefits of HU have been found to associate with increased nitric oxide (NO) and fetal hemoglobin (HbF) in most SS patients, there has been considerable interpatient variability in HbF response to the drug. From this standpoint, HU is imperfectly understood mechanistically. Cytochrome b5 reductase 3 (Cyb5R3), also known as methemoglobin reductase, is an enzyme known to modulate redox signaling in erythrocytes and vascular cells by catalyzing one electron transfer reactions using reduced nicotinamide adenine dinucleotide (NADH) as an electron donor. Functionally, membrane-associated CYB5R3 exerts antioxidant properties by modulating heme iron reduction, plasma membrane coenzyme Q reduction, elongation and unsaturation of fatty acids, cholesterol biosynthesis and drug metabolism, while the soluble form of CYB5R3 reduces red blood cell methemoglobin. Unremitting oxidation of the soluble guanylate cyclase (sGC) heme iron in the absence of sufficient Cyb5R3 activity in vascular smooth muscle has been shown to explain some NO resistance that characterizes pulmonary arterial hypertension and other vasculopathy associated with SCD. More than 40 variants of Cyb5R3 have been identified. Of particular interest is a Cyb5R3 variant (T117S) that contains a threonine to serine substitution in the flavoprotein, which lessens the reductase's activity by roughly 50% compared to wild-type. This variant occurs with high frequency (0.23 minor allele) in persons of African ancestry but is rare (<1%) in all other races. Nothing is known about the role of Cyb5R3 in the anemia of sickle cell disease. We hypothesized that Cyb5R3 may act as a modifier of HbF induction by HU in sickle cell anemia. METHODS: Male and female SS patients (n=633, 12-70 years of age) from the Treatment of Pulmonary Hypertension and Sickle Cell Disease with Sildenafil Therapy (walk-PHaSST) trial were recruited for genetic analysis (University of Pittsburgh IRB Protocol Study 19060255). Genotyping of rs1800457 (T117S SNP) was performed using Taqman genotyping assay. One-way ANOVA and Holms Sidak were used for multiple comparisons of HbF levels by Cyb5R3 genotype and HU treatment status. Intrapatient correlation analyses of HbF and hematocrit (HCT) were conducted using Pearsons r. RESULTS: Comparisons of HbF levels by Cyb5R3 genotype (CC, wild-type; CG, heterozygous wild-type and T117S; GG, homozygous T117S) and HU treatment status (treated vs. untreated) at time of study enrollment (baseline) show that HU is a less potent HbF inducer in the GG group (4.32 mean diff, P=0.344) versus the CC and CG patient groups (CC: 6.027 mean diff, P<0.0001 and CG: 6.914 mean diff, P<0.0001). Correlation analyses showed that as HbF levels increased among HU-untreated patients, the trend was for HCT to decrease. This was true for all SS patients, irrespective of Cyb5R3 genotype (CC: r = -0.336, P<0.0001, n=320; CG: r = -0.437 P<0.0001, n=120; GG: r = -0.253, P=0.136, n=36). However, with HU therapy, the inverse relationship between HbF and HCT was less negative in SS patients carrying at least one copy of the wild-type Cyb5R3 gene (CC or CG). In these individuals, increasing levels of HbF (up to 10%) correlated with positive gains in HCT at HbF greater than 5-6% (CC: r = -0.161, P=0.097, n=107) and 6-7% (CG: r = -0.186, P=0.25, n=40). In patients homozygous for T117S, HCT continued trending downward as HbF increased (GG: r = -0.419, P=0.228, n=10). These data indicate that the positive relationship between HbF and HCT is subject to uncoupling by loss of Cyb5R3 function. In vitro studies with human erythroleukemic K562 cells reveal loss of gamma globin induction by HU treatment when wild-type Cyb5R3 is knocked down by siRNA, indicating a requirement for Cyb5R3 in HU-stimulated HbF induction. SUMMARY andCONCLUSION: Loss of Cyb5R3 activity negatively impacts HbF and HCT in HU treated SS patients. We conclude that the T117S variant may be a useful marker for predicting which SS patients will incur beneficial HbF and HCT responses to HU, thereby improving clinical decision-making about best drug choices for preventing HbS polymerization and treating anemia in SS patients.

Triphenyltin(IV) dithiocarbamate compound induces genotoxicity and cytotoxicity in K562 human erythroleukemia cells primarily via mitochondria-mediated apoptosis.

The novel di-and triphenyltin(IV) dithiocarbamate compounds represented as RnSnL2 (where R=C4H9, C6H5; n=2,3; L=N,N-dithiocarbamate), Ph2Sn(N,N-diisopropyldithiocarbamate) (OC1), Ph3Sn(N,N-diisopropyldithiocarbamate) (OC2), Ph2Sn(N,N-diallyldithiocarbamate) (OC3), Ph3Sn(N,N-diallyldithiocarbamate) (OC4), and Ph2Sn(N,N-diethyldithiocarbamate) (OC5) were assessed for their cytotoxicity in K562 human erythroleukemia cells. All compounds inhibited the growth of cells at low micromolar concentrations (<10μM), and the mechanism underlying their antiproliferative effects on K562cells was apoptosis, as corroborated by the exposure of plasma membrane phosphatidylserine. OC2, which showed the most promising antiproliferative activity, was selected for further analyses. The results demonstrated that OC2 induced apoptosis in K562cells via an intrinsic mitochondrial pathway triggered upon DNA damage, an early apoptotic signal. Subsequently, OC2 produced excessive intracellular reactive oxygen species. The role of oxidative stress was corroborated by the significant reduction in GSH levels and percentage of apoptosis in NAC-pretreated cells. OC2 could arrest the cell cycle progression in the S phase. These new findings elucidate the antiproliferative potential of OC2 in the K562 human erythroleukemia cells and warrant further investigation, specifically to determine the exact signaling pathway underlying its antileukemic efficacy.

LRF Promotes Indirectly Advantageous Chromatin Conformation via BGLT3-lncRNA Expression and Switch from Fetal to Adult Hemoglobin.

The hemoglobin switch from fetal (HbF) to adult (HbA) has been studied intensively as an essential model for gene expression regulation, but also as a beneficial therapeutic approach for β-hemoglobinopathies, towards the objective of reactivating HbF. The transcription factor LRF (Leukemia/lymphoma-related), encoded from the ZBTB7A gene has been implicated in fetal hemoglobin silencing, though has a wide range of functions that have not been fully clarified. We thus established the LRF/ZBTB7A-overexpressing and ZBTB7A-knockdown K562 (human erythroleukemia cell line) clones to assess fetal vs. adult hemoglobin production pre- and post-induction. Transgenic K562 clones were further developed and studied under the influence of epigenetic chromatin regulators, such as DNA methyl transferase 3 (DNMT3) and Histone Deacetylase 1 (HDAC1), to evaluate LRF’s potential disturbance upon the aberrant epigenetic background and provide valuable information of the preferable epigenetic frame, in which LRF unfolds its action on the β-type globin’s expression. The ChIP-seq analysis demonstrated that LRF binds to γ-globin genes (HBG2/1) and apparently associates BCL11A for their silencing, but also during erythropoiesis induction, LRF binds the BGLT3 gene, promoting BGLT3-lncRNA production through the γ-δ intergenic region of β-type globin’s locus, triggering the transcriptional events from γ- to β-globin switch. Our findings are supported by an up-to-date looping model, which highlights chromatin alterations during erythropoiesis at late stages of gestation, to establish an “open” chromatin conformation across the γ-δ intergenic region and accomplish β-globin expression and hemoglobin switch.

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.

Cytotoxicity of the gold complex based on mercaptotriazole – A comparison with the conventional cyanide electrolyte

The study presents the 3-(4,5-dımethylthıazol-2-yl)-2,5-dıphenyltetrazolıum bromıde (MTT) assay results on human erythroleukemia K562 ​cell line to compare the toxicity of electrolytes based on the mercaptotriazole gold complex (Au-MT) and gold potassium cyanide. Solutions immediately after mixing and after three months at different concentrations were compared, with a prolonged aging period, to a year, for the electrolytes at pH ​= ​9. Lower toxicity of new organic gold complex at pH ​= ​4, 7, and 12 than of alkaline cyanide electrolyte, but higher at pH ​= ​2, was determined. The solution of the Au-MT at pH ​= ​9 had comparable, but lower cell toxicity in the whole investigated period.

Cilostazol-mediated reversion of γ-globin silencing is associated with a high level of HbF production: A potential therapeutic candidate for β-globin disorders

Reversal of fetal hemoglobin (HbF) silencing is an attractive therapeutic intervention for β-thalassemia and sickle cell anemia. The current study proposes the therapeutic of repurposing of cilostazol, an FDA-approved antithrombotic agent, as a promising HbF inducer. Preliminary, we report that cilostazol induced erythroid differentiation and hemoglobinization of human erythroleukemia K562 cells. The erythroid differentiation was accompanied by increased expression of γ-globin mRNA transcripts and HbF production. Cilostazol induced erythroid differentiation and HbF production, without significantly affecting proliferation and viability of hemoglobin producing cells at maximum erythroid inducing concentration. Moreover, we investigated the effect of cilostazol on human β- and γ-globin transgenes in in vivo β-YAC transgenic mice, harboring human β-locus along with β-LCR. A good in vitro correlation was found with substantial up-regulation in fetal globin mRNA; whereas, the β-globin gene expression was not significantly changed. F-cells, analysis in the peripheral blood of cilostazol-treated mice, revealed a significant increase in the F-cells population as compared with sham control groups. Together, these findings support the potential of cilostazol as an HbF inducer, which can be evaluated further to develop a new HbF inducer.

Fully Retargeted, Myeloma-Specific Oncolytic Measles Viruses.

We previously reported that attenuated measles viruses have potent anti-tumor activity in primary myeloma cells and myeloma xenograft models. The viruses are selectively oncolytic because they cause extensive cell to cell fusion via CD46, a membrane complement regulatory protein that is more highly expressed on myeloma cells than on normal cells. A phase I clinical trial is planned at Mayo Clinic using an oncolytic measles virus engineered to express the thyroidal sodium iodide symporter gene (MV-NIS). Expected toxicities of MV-NIS include a mild measles-like illness due to residual infection of normal host cells via either of the two natural virus receptors, CD46 and SLAM. To facilitate the development of fully retargeted oncolytic measles viruses we recently developed the pseudoreceptor system “STAR”, which allows rescue and propagation of targeted viruses with modified H proteins that no longer bind to the native measles receptors (Nakamura T. et. al., Nature Biotechnology, 2005). Using the STAR system, we have now generated fully retargeted oncolytic measles viruses against two targets, CD38 or HM1.24 that are highly expressed on the surface of multiple myeloma cells. CD38 is expressed abundantly on myeloma plasma cells but is also expressed in a variety of normal human cells, such as myeloid progenitors and activated T cells. The HM1.24 antigen is highly expressed on terminally differentiated B-cells (including myeloma cells) and is not found on liver, spleen, kidney, or heart. To facilitate evaluation of the receptor specificity of the recombinant viruses, they were each constructed with an additional transgene insert coding for green fluorescent protein (GFP). Efficient viral incorporation of each of the recombinant H proteins was demonstrated by immunoblotting of pelleted virions. CHO cells were transfected with each of the targeted receptors and then infected with each of the targeted viruses. As expected, each virus demonstrated the predicted host range properties and was blind to CD46 and SLAM receptors, but efficiently entered cells through the respective targeted receptor. In addition, these retargeted viruses demonstrated myeloma-specific infection, syncytium formation and cytotoxicity for multiple myeloma cell lines RPIM8226 and KAS-6/1, but not on receptor-negative control cells such as K562 human erythroleukemic cells. These fully retargeted oncolytic measles viruses are promising candidates that may provide the basis for safer and more effective virotherapy in multiple myeloma.

Use of trans‐3′5'4‐trihydroxystilbene (Resveratrol) as an adjunct in the treatment of Sickle Cell Disease

Sickle Cell Disease (SCD) and □-thalassemia are among the most common forms of hemoglobinopathy and life-threatening genetic diseases worldwide. So far, the only approved drug for these disorders is the ribonucleotide reductase inhibitor hydroxyurea (HU) falls short of being curative. The efficacy of HU in the management of thalassemia and SCD is generally attributed to its limited ability to boost the levels of fetal hemoglobin (Hb F,α2γ2), which decreases sickling by interfering with the polymerization of hemoglobin in patients with SCD and can partially correct the globin chain imbalance in □-thalassemia patients, while at the same time facilitating O2 transport to myoglobin in peripheral tissues. The naturally occurring dietary polyphenol resveratrol (trans-3′5'4-trihydroxystilbene, RV), has also been postulated to induce Hb F production via a biochemical mechanism like HU. Preclinical studies using the human erythroleukemic K562 cell line suggested RV can induce Hb F production in a more robust manner than HU. However, clinical studies on the efficacy and effectiveness of RV are limited and conflicting. This study sought to evaluate the clinical efficacy and safety of RV in managing SCD compared to HU. A systematic literature review was conducted in peer-reviewed, published full-text articles investigating RV in the management of SCD. Eligible studies were required to report explicitly, including participants diagnosed with β-thalassemia or SCD anemia or cultured cells with the SCD or thalassemia disease and where HU was included as a control. The analysis included thalassemia, RV type, RV dosage, duration of treatment, and outcome, Hb F induction and antioxidant activity. Additionally, a pilot study of RV in the management of adults with SCD who received treatment at National Hospital Abuja, Nigeria, was attempted but discontinued due to the COVID 19 pandemic. A case study of a single 23-year-old female SCD patient who had been taking RV [1000mg daily p.o.] for more than 6 years was included. Outcome variables evaluated include number of hospitalization episodes of pain crisis, need for blood transfusion, possible side effects and RBC indices. The early results of this metanalysis show that RV was as effective as HU in inducing production of Hb F in adult SCD patients and reducing hospital treatment days post RV treatment. Four primary treatment and curative therapies that have shown great potential and are currently used to prevent and treat acute and chronic complications in SCD, HU and blood transfusions, are the most well-established disease-modifying therapies used to prevent and treat complications associated with SCD. Other lesser used therapies include stem cell transplantation or the allogeneic hematopoietic stem cell transplant (HSCT), and the investigational gene therapy is the only known curative therapy that exists regarding SCD. The results of this study indicate that RV is an effective inducer of Hb F production in adults and shows promise as a cost effective and useful adjunct for the treatment of hemoglobinopathies including SCD and thalassemia.

Modelling Congenital Dyserythropoietic Anemia Type II through Gene Editing in Hematopoietic Stem and Progenitor Cells

Congenital dyserythropoietic anemias (CDAs) are a group of inherited anemias that affect the development of the erythoid lineage. They are characterized by ineffective erythropoiesis with distinct morphologic abnormalities of erythroblasts, a degree of hemolysis, and secondary hemochromatosis. Patients usually present with congenital anemia, jaundice, splenomegaly, and an absolute reticulocyte count inadequate for the degree of anemia. CDA type II (CDAII) is the most frequent type. CDAII patients show anemia of variable degrees, and 20% are transfusion dependent. The most specific finding of CDAII marrow is the presence of more than 10% mature binucleated erythroblasts with two nuclei at the same erythroid maturation stage. Treatment of CDAII patients may involve blood transfusions, iron chelation therapy and splenectomy. The only described definitive therapy is allogeneic bone marrow transplantation, which implies additional side effects for these patients. Therefore, new therapeutic approaches are needed. CDA II is caused by mutations in the SEC23B gene. SEC23B is part of coat protein complex II (COPII). COPII is involved in protein processing and Golgi-reticulum trafficking. However, how mutations in SEC23B cause CDA II is not known yet. Therefore, studying the role of SEC23B in the erythropoiesis will help to elucidate the underlying mechanism of CDA II and to develop new therapeutic approaches for the disease. We have developed a CDA II model in human cells through the introduction of genomic mutations in the gene using the CRISPR/Cas9 gene editing system. Different single guides RNAs (sgRNA) targeting the start of the coding sequence of human SEC23B gene were designed and tested in human erythroleukemia K562 cell line and in healthy human cord blood hematopoietic stem and progenitors (hCB-CD34+). The gene editing outcome at SEC23B gene was assessed at: i) genomic level through Sanger sequencing, Inference of CRISPR Edits (ICE) and Next-Generation Sequencing (NGS). ii) Protein level through Western-blot analysis. iii) Functional level through morphological analysis and erythroid differentiation either in vitro or in vivo in human hematopoietic chimeras in NOD.Cg-KitW-41JTyr+PrkdcscidIl2rgtm1Wjl/ThomJ (NBSGW) mice. K562 cells were nucleofected with three different sgRNAs, as ribonucleoprotein (RNP), independently or in combination. Afterwards, seventy five K562 clones were established from the cells nucleofected with the most efficient sgRNA or with the combination of the three sgRNAs. Forty per cent of them showed a high efficiency of knock-out (higher than 50% of alleles). Eight SEC23BKO clones were selected for further analysis. All of those eight clones showed a reduction in SEC23B protein and six of them had a lower proliferation than control cells and morphological abnormalities, such as presence of bi/multinucleated cells. Moreover, when CB-CD34+ cells were nucleofected with the most efficient sgRNA or with the combination of the three sgRNAs, up to 80% of knock-out efficiency and close to 90% reduction of SEC23B protein were obtained. Interestingly, when those gene edited hematopoietic progenitors were differentiated in vitro to erythroid cells, their terminal differentiation was hampered, with a reduce percentage of enucleated cells and the presence of high number of bi/multinucleated cells. Similarly, the in vivo erythroid differentiation of these gene edited progenitors two months after HSPC transplant into NBSGW mice showed again an impairment of terminal erythroid differentiation with an increment in the percentage of erythroid bi/multinucleated cells without altering other human hematopoietic lineages. In summary, CRISPR/Cas9 system has been used to model CDA II in a human cell line and in human hematopoietic progenitors through the knock-out of SEC23B gene. Our system reproduced the most relevant feature characteristic of CDA II pathology. This gene editing based CDA II model will allow the study of how mutations in SEC23B cause CDA II and the development of new therapeutic strategies to cure this disease. Disclosures Tornador: Bloodgenetics: Current Employment. Sanchez:Bloodgenetics: Current Employment. Segovia:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from the Company., Patents &amp; Royalties, Research Funding.

Pro-apoptotic activity of novel synthetic isoxazole derivatives exhibiting inhibitory activity against tumor cell growth in vitro.

In order to develop potential anticancer agents stimulating apoptosis, novel 3,4-isoxazolediamide and 4,5,6,7-tetrahydro-isoxazolo-[4,5-c]-pyridine derivatives have been synthetized. The original structures of geldanamycin and radicicol, which are known natural heat shock protein (HSP) inhibitors, were deeply modified because both of them exhibit several drawbacks, such as poor solubility, hepatotoxicity, intrinsic chemical instability or deprivation of the in vivo activity. This novel class of synthetic compounds containing the isoxazole nucleus exhibited potent and selective inhibition of HSP90 in previous studies. Biological assays (focusing on in vitro antiproliferative effects and pro-apoptotic activity) in human erythroleukemic K562 cells (as a model system referring to tumor cells grown in suspension), glioblastoma U251-MG and glioblastoma temozolomide (TMZ)-resistant T98G cell lines (two model systems referring to tumor cells grown attached to the flask), were performed. Almost all isoxazole derivatives demonstrated significant antiproliferative and pro-apoptotic activities, showing induction of both early and late apoptosis of K562 cells. Different effects were observed on the glioma U251-MG and T98G cells, depending on the structure of the analogues. Antiproliferative and pro-apoptotic activities in K562 cells were associated with the activation of the erythroid differentiation program. The present study demonstrated that 3,4-isoxazolediamide and 4,5,6,7-tetrahydro-isoxazolo-[4,5-c]-pyridine derivatives should be considered for in vivo studies focusing on the development of anticancer drugs acting, at least partially, via activation of apoptosis.

Effect of the triphenylphosphonium cation on the biological properties of new rhenium and technetium-99m fac-[M(CO)3(NSN)]±-type complexes: Synthesis, structural characterization, in vitro and in vivo studies

Triphenylphosphonium (TPP) cations have been used for the development of tumor or myocardial diagnostic radiopharmaceuticals. In this work, the development of new [99mTc][Tc(CO)3(N,S,N)]+ complexes of the (N,S,N) tridentate chelator benzimidazol-2-yl-methylthioethylamine (L), and its triphenylphosphonium (TPP) cation derivative L-TPP is described. The TPP-moiety was conjugated at the Nτ-benzimidazol position of L. The reaction of the chelators L and L-TPP with a suitable precursor [Re(sol)3(CO)3]+ yielded single products of ReL+ and ReL-TPP2+. The complexes were characterized by spectroscopic methods and furthermore the structure of ReL was elucidated by X-ray crystallography. The respective 99mTc-radiotracers were synthesized in high yield, their lipophilicity was measured and both exhibited high stability in cysteine, histidine solutions as well as in rat plasma. The in vitro cell studies in human erythroleukemia K-562 and glioblastoma U-87MG tumor cells showed that the tracer 99mTcL+ exhibited significantly higher cellular uptake, while 99mTcL-TPP2+ exhibited significantly higher mitochondrial accumulation. The tracers 99mTcL+ and 99mTcL-TPP2+ exhibited fast blood elimination and excretion via the hepatobiliary and the renal routes after intravenous administration in healthy mice. Tracer 99mTcL+ exhibited higher myocardial uptake and renal excretion, while 99mTcL-TPP2+ exhibited primarily hepatobiliary excretion. These data confirm the high mitochondrial accumulation of 99mTcL-TPP2+ in vitro and show its potential as a candidate for tumor imaging.

Synthesis and Erythroid Induction Activity of New Thiourea Derivatives.

The use of medicinal agents to augment the fetal hemoglobin (HbF) accretion is an important approach for the treatment of sickle-cell anemia and β-thalassemia. HbF inducers have the potential to reduce the clinical symptoms and blood transfusion dependence in the patients of β- hemoglobinopathies. The current study was aimed to examine the erythroid induction potential of newly synthesized thiourea derivatives. Thiourea derivatives 1-27 were synthesized by using environmentally friendly methods. Compounds 3, 10 and 22 were found to be new. The structures of synthesized derivatives were deduced by using various spectroscopic techniques. These derivatives were then evaluated for their erythroid induction using the human erythroleukemic K562 cell line, as a model. The benzidine-H2O2 assay was used to evaluate erythroid induction, while HbF expression was studied through immunocytochemistry using the Anti-HbF antibody. Cytotoxicity of compounds 1-27 was also evaluated on mouse fibroblast 3T3 cell line and cancer Hela cell line using MTT assay. All the compounds (1-27) have not been reported for their erythroid induction activity previously. Compounds 1, 2, and 3 were found to be the potent erythroid inducing agents with % induction of 45± 6.9, 44± 5.9, and 41± 6.1, at 1.56, 0.78, and 0.78 μM concentrations, respectively, as compared to untreated control (12 ± 1 % induction). Furthermore, compound 1, 2, and 3 significantly induced fetal hemoglobin the expression up to 4.2-fold, 4.06-fold, and 3.52-fold, respectively, as compared to untreated control. Moreover, the compounds 1-4, 6-9, 11, 12, 15, 17, 19, 22, 23, and 25 were found to be non-cytotoxic against the 3T3 cell line. This study signifies that the compounds reported here may serve as the starting point for the designing and development of new fetal hemoglobin inducers for the treatment of β- hemoglobinopathies.