Human Erythroleukemia Cell Line Research Articles

Growth hormone is involved in GATA1 gene expression via STAT5B in human erythroleukemia and monocytic cell lines

GATAs are a family of transcription factors consisting of six members. Particularly, GATA1 and GATA2 have been reported to promote the development of erythrocytes, megakaryocytes, eosinophils, and mast cells. However, little information is available on the extracellular ligands that promote GATA1 expression. We evaluated whether growth hormone (GH) is an extracellular stimulator that participates in the signal transduction of GATAs, focusing on GATA1 expression in hematopoietic cell lineages. We used a reporter assay, RT-PCR, real-time quantitative PCR, and western blotting to evaluate GH-induced expression of GATA1 and GATA2 in the human erythroleukemic cell line K562 and the non-erythroid cell line U937. GATA1 expression in these hematopoietic cell lines increased at the transcriptional and protein levels in the presence of GH, and was inhibited by a STAT5 specific inhibitor. Cells transfected with activated STAT5B showed increased expression of GATA1. We identified functional STAT5B consensus sequences as binding site-158 bp from the transcription starting site in the GATA1 promoter region. These results suggest that GH directly induces GATA1 expression via GHR/JAK/STAT5 and is related to hematopoietic cell proliferation.

CDK9 phosphorylates RUNX1 to promote megakaryocytic fate in megakaryocytic-erythroid progenitors

AbstractThe specification of megakaryocytic (Mk) or erythroid (E) lineages from primary human megakaryocytic-erythroid progenitors (MEPs) is crucial for hematopoietic homeostasis, yet the underlying mechanisms regulating fate specification remain elusive. In this study, we identify RUNX1 as a key modulator of gene expression during MEP fate specification. Overexpression of RUNX1 in primary human MEPs promotes Mk specification, whereas pan-RUNX inhibition favors E specification. Although total RUNX1 levels do not differ between Mk progenitors (MkPs) and E progenitors (ErPs), there are higher levels of serine-phosphorylated RUNX1 in MkPs than ErPs, and mutant RUNX1 with phosphorylated-serine/threonine mimetic mutations (RUNX1-4D) significantly enhances the functional efficacy of RUNX1. To model the effects of RUNX1 variants, we use human erythroleukemia (HEL) cell lines expressing wild-type (WT), phosphomimetic (RUNX1-4D), and nonphosphorylatable (RUNX1-4A) mutants showing that the 3 forms of RUNX1 differentially regulate expression of 2625 genes. Both WT and RUNX1-4D variants increase expression in 40%, and decrease expression in another 40%, with lesser effects of RUNX1-4A. We find a significant overlap between the upregulated genes in WT and RUNX1-4D–expressing HEL cells and those upregulated in primary human MkPs vs MEPs. Although inhibition of known RUNX1 serine/threonine kinases does not affect phosphoserine RUNX1 levels in primary MEPs, specific inhibition of cyclin dependent kinase 9 (CDK9) in MEPs leads to both decreased RUNX1 phosphorylation and increased E commitment. Collectively, our findings show that serine/threonine phosphorylation of RUNX1 promotes Mk fate specification and introduce a novel kinase for RUNX1 linking the fundamental transcriptional machinery with activation of a cell type–specific transcription factor.

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

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

Diastereoselective 1,3-dipolar cycloaddition of cyclopropenes to acenaphthenequinone azomethine ylides

A diastereoselective 1,3-dipolar cycloaddition of azomethine ylides generated from acenaphthylene-1,2-dione (or aceantrylene-1,2-dione) and amino acids with 1,2-diphenylcyclopropenes has been developed. As a result, cyclopropa[a]pyrrolizidines and 3-azabicyclo[3.1.0]hexanes spiro-fused to acenaphthylen-1(2H)-one and aceanthrylen-1(2H)-one fragments were obtained for the first time. Based on DFT calculations at M11/cc-pVDZ level of theory, a concerted mechanism of cycloaddition was proposed to explain the observed diastereoselectivity. The cytotoxic activity of newly synthesized compounds against human erythroleukemia (K562) and human cervical carcinoma (HeLa) cell lines was evaluated in vitro by MTS-assay.

Distinctive Roles of Nitric Oxide Synthase Isoforms in Hydroxyurea-Mediated Cytostasis of Erythroid Progenitors

Chemotherapeutic hydroxyurea (HU) is used in the therapy of sickle cell anemia, thalassemia and myeloproliferative neoplasms according to its stimulation of fetal hemoglobin and myelosuppression. We reported that most nitric oxide (NO) in mammals is produced by the reduction of nitrate and nitrite. The NO properties of HU have been widely explored, while its enzymatic NO synthase (NOS) dependence is not completely defined. We have already demonstrated concomitant inhibition of erythroid cell proliferation by HU and NO donors. In this study, we investigated the NOS-mediation in HU regulation of proliferation, differentiation and cell cycle in HEL92.1.7 human erythroleukemic cell line and / or erythroid progenitors of Nos2- or Nos3-null mice. HU induced a dose-dependent increase in NOS2 and NOS3 protein levels and their enzymatic activity by increasing production of NO and citrulline in erythroleukemic cells. Inhibition of the NOS enzymes by pan-specific L-NAME, NOS2 inhibitor 1400W, or NOS2/NOS3 inhibitor DPI was sufficient to prevent the HU-mediated decrease in HEL92.1.7 proliferation assessed by Ki67 immunofluorescence. HU increased the frequency of cells in S-phase (66.58±2.44 vs. 55.18±1,06%) of the cycle at the expense of G0/G1 (17.07±1.67 vs. 28.55±1.01%) due to blocked DNA synthesis. The combined treatment of HU with 10μM DPI decreased the number of cells in G0/G1 (3.34±0.98 vs. 17.07±1.67%) and increased in S (72.3±0.96 vs. 66.58±%) and G2/M (24.36±0.19 vs. 16.35±2.28%) compared with HU alone. Moreover, NOS3 knock-down by shRNA decreased the percentage of cells in S phase from 69,19±2.68 to 34.23±1.07% (p<0.001) and increased in G2/M from 15.62±2.17 to 34.71±3.76 (p<0.001). HU-induced apoptosis was prevented by NOS2 (1400W) inhibition, determined by annexin V, decreasing the number of cells in early and late apoptosis in erythroleukemic cells. In accordance, NOS3 knock-down was not able to rescue HU-induced apoptosis. Furthermore, by performing in vitro enzymatic assay we showed that regulation of NOS2 activity seems to be achieved by direct interaction with HU leading to a dose- and time-dependent increase in NO and citrulline products. HU increased the expression of Nos1/2 isoforms by 20%, and Nos3 by 10% in mouse erythroid progenitors (mERP) of wild-type mice. Bone marrow cells isolated from Nos2- or Nos3-null mice treated orally with HU showed decreased protein nitrosylation in bone marrow compared to HU-treated wild-type mice. In ex vivo colony formation assay, both Nos2-/- and Nos3-/- bone marrow cells formed significantly more late erythroid colonies (CFU-E, 34- and 24-fold, respectively, p<0.001), and Nos2-/- also yielded more of the early erythroid (BFU-E, 9- and 7-fold, p<0.01) and granulocyte/macrophage (CFU-GM, 14- and 15-fold, p<0.01) colonies compared to wild-type mice untreated or HU-treated. HU treatment of mERP, of Nos2-/- and Nos3-/- origin, demonstrated decreased frequency of S-phase cells to levels comparable to untreated wild-type mice. Nos2 and Nos3 deficiency was able to rescue Caspase-3 expression in the presence of HU in mERP. Nevertheless, HU increased the quantity of late apoptotic cells in both Nos2 and Nos3 deficient mice. Our results demonstrated that HU induces the enzymatic activity of NOS proteins that mediate the cytostatic effect. NOS2/3 enzymes are involved in HU-inhibition of proliferation of human erythroleukemic cells and ex vivo differentiation of mERP. The observed NOS enzymes were fully and partially involved in HU-induced apoptosis of HEL92.1.7 cells and mERP, respectively. Uncovering all aspects of HU mechanism of action may help to increase the responsiveness or reduce the negative effects of cytotoxic chemotherapy.

Four New Diterpenoids from the South China Sea Soft Coral Sinularia nanolobata and DFT-Based Structure Elucidation.

Three new cembranoids (1-3) and a new casbanoid (4), along with three known analogues (5-7), have been isolated from the soft coral Sinularia nanolobata collected off Ximao Island. The structures, including the absolute configurations of new compounds, were established using extensive spectroscopic data analysis, time-dependent density functional theory/electronic circular dichroism (TDDFT-ECD) calculations, and the comparison with spectroscopic data of known compounds. In the in vitro bioassay, compounds 1 and 5 exhibited moderate cytotoxic activities against human erythroleukemia (HEL) cell lines, with IC50 values of 37.1 and 42.4 μM, respectively.

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.

11H-Benzo[4,5]imidazo[1,2-a]indol-11-one as a New Precursor of Azomethine Ylides: 1,3-Dipolar Cycloaddition Reactions with Cyclopropenes and Maleimides.

The possibility of generating azomethine ylides from 11H-benzo[4,5]imidazo[1,2-a]indol-11-one and amino acids is shown for the first time. Based on the cycloaddition reactions of these azomethine ylides with cyclopropenes and maleimides, cyclopropa[a]pyrrolizines, 3-azabicyclo[3.1.0]hexanes, and pyrrolo[3,4-a]pyrrolizines spiro-fused with a benzo[4,5]imidazo[1,2-a]indole fragment were synthesized. Spirocyclic compounds were obtained in moderate to good yields, albeit with poor diastereoselectivity. Density functional theory calculations were performed to obtain an insight into the mechanism of the 1,3-dipolar cycloaddition of 11H-benzo[4,5]imidazo[1,2-a]indol-11-one-derived azomethine ylides to cyclopropenes. The cytotoxic activity of some of the obtained cycloadducts against the human erythroleukemia (K562) cell line was evaluated in vitro by MTS-assay.

Discovery of JAK2/3 Inhibitors from Quinoxalinone-Containing Compounds.

Janus kinases (JAKs) are involved in a wide variety of cell signaling associated with T-cell and B-cell mediated diseases. The pathogenesis of common lymphoid-derived diseases and leukemia cancer has been implicated in JAK2 and JAK3. Therefore, to decrease the risk of these diseases, targeting this pathway using JAK2/3 inhibitors could serve as a valuable research tool. Herein, we used a combination of the computational and biological approaches to identify the quinoxalinone-based dual inhibitors of JAK2/3. First, an in-house library of 49 quinoxalinones was screened by molecular docking. Then, the inhibitory activities of 17 screened compounds against both JAKs as well as against two human erythroleukemia cell lines, TF1 and HEL were examined. The obtained results revealed that several quinoxalinones could potentially inhibit JAK2/3, and among them, ST4j showed strong inhibition against JAKs with the IC50 values of 13.00 ± 1.31 nM for JAK2 and 14.86 ± 1.29 nM for JAK3, which are better than ruxolitinib and tofacitinib. In addition, ST4j potentially inhibited TF1 cells (IC50 of 15.53 ± 0.82 μM) and HEL cells (IC50 of 17.90 ± 1.36 μM), similar to both tofacitinib ruxolitinib. Mechanistically, ST4j inhibited JAK2 autophosphorylation and induced cell apoptosis in dose- and time-dependent manners. From molecular dynamics simulations, ST4j was mainly stabilized by van der Waals interactions, and its hydroxyl group could form hydrogen bonds in the hinge region at residues S936 and R938 of JAK2. This research highlights the potential of ST4j to be a novel therapeutic agent for the treatment of lymphoid-derived diseases and leukemia cancer.

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.

Abstract 3849: Exosomes in polycythemia vera: "mini platelets" with oncogenic and thrombogenic potential

Abstract Polycythemia vera (PV) is a classical "myeloproliferative neoplasm" characterized by clonal proliferation of myeloid progenitor cells caused by their acquisition of a mutation in the JAK2 gene. Its common complications are thrombosis and second primary malignancies. Secreted from virtually all cell types, exosomes are extracellular vesicles that carry bioactive cargo derived from their cells of origin. When engulfed, their cargo may alter the phenotype of the recipient cells. For example, neoplastic exosomes modulate the cellular makeup of bystander cells. Therefore, since mutated JAK2 is present in virtually all PV-patients, we hypothesize that PV-derived exosomes (hereafter PV-exo) carry the mutated JAK2 oncogene and are taken up by non-clonal cells, thus contributing to the thrombotic manifestations and oncogenic potential of patients with PV. We isolated and characterized exosomes from PV-patients and from the Human erythroleukemia (HEL) cell line (hereafter HEL-exo). We exposed normal cells implicated in thrombosis and skin cells to these exosomes and examined the cells' phenotypic changes and activation after exposure. Using Sanger sequencing we discovered that both PV-exo and HEL-exo, like their parental cells, carry the mutated JAK2 transcripts, allowing clonal cells to export oncogenes to distant, non-clonal sites. Our preliminary findings suggest that these exosomes promote pro-coagulant and malignant phenotypes in multiple ways. Thrombin generation assay pointed to a significantly higher generation of thrombin in the presence of PV-exo than those derived from healthy donors. Similarly, flow cytometry analysis revealed that PV-exo increased the expression of platelet activation markers. Moreover, Trans Endothelial Electrical Resistance (TEER) assay results suggest that HEL-exo induces endothelial dysfunction. Furthermore, using the Sulforhodamine B assay, we found that exposure to HEL-exo increased the proliferation of normal keratinocytes. In summary, we developed the concept of exosomes as “mini metastasis” that might promote disease manifestations either directly or indirectly. Since unlike solid tumors, hematological malignancies do not metastasize, exosomes derived from neoplastic hematological cells may fulfill this function. Such exosomes may spread oncogenes (e.g., mutated JAK2), damage non-clonal tissues (e.g., endothelial dysfunction) or act as a miniature reflection of their cells of origin (e.g., exosomes as mini platelets) in a parallel way to "real" metastasis; this concept may affect treatment related decisions. For example, by reducing clonal burden, early treatment even in “low risk” patients may prevent exosomal spread and exosomal-dependent damage to distant tissues. The results of our study highlight PV-exo as promoters of PV-complications, thrombosis and malignant transformation. Citation Format: Orit Uziel, Adi Shacham-Abulafia, Galia Spectre, Ester Ziv, Karyn Revital Geiger, Zinab Sarsor, Neria Ron, Einat Beery, Pia Raanani, Shoshana Revel-Vilk, Mira Naamad, Uri Rozovski. Exosomes in polycythemia vera: "mini platelets" with oncogenic and thrombogenic potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3849.

Green Synthesis of Gold, Iron and Selenium Nanoparticles Using Phytoconstituents: Preliminary Evaluation of Antioxidant and Biocompatibility Potential.

This study aimed at fabricating gold (Au), iron (Fe) and selenium (Se) nanoparticles (NPs) using various natural plant extracts from the Fertile Crescent area and evaluating their potential application as antioxidant and biocompatible agents to be used in the pharmaceutical field, especially in drug delivery. The Au-NPs were synthesized using Ephedra alata and Pistacia lentiscus extracts, whereas the Fe-NPs and Se-NPs were synthesized using peel, fruit and seed extracts of Punica granatum. The phytofabricated NPs were characterized by the UV-visible spectroscopy, scanning electron microscope, Fourier transform infrared spectroscopy, X-ray diffraction (XRD) and energy-dispersive X-ray (EDS) spectroscopy. Scanning electron microscope technique showed that the synthesized NPs surface was spherical, and the particle size analysis confirmed a particle size of 50 nm. The crystalline nature of the NPs was confirmed by the XRD analysis. All synthesized NPs were found to be biocompatible in the fibroblast and human erythroleukemic cell lines. Se-NPs showed a dose-dependent antitumor activity as evidenced from the experimental results with breast cancer (MCF-7) cells. A dose-dependent, free-radical scavenging effect of the Au-NPs and Se-NPs was observed in the DPPH (2,2-Diphenyl-1-picrylhydrazyl) assay, with the highest effect recorded for Au-NPs.

N-acetylcysteine potentiates the tumor cytotoxicity of cytokine-induced killer cells.

Cytokine-induced killer (CIK) cells are an ex vivo expanded heterogeneous population of natural killer (NK)-like T cells that can exert potent MHC-unrestricted antitumor activity. A number of pre-clinical and clinical studies have demonstrated that CIK cells can serve as a safe and potent immunotherapy of malignant tumors. N-acetylcysteine (NAC) has been demonstrated to enhance the T-cell functions by increasing their proliferation and cytokine production. To investigate whether the incorporation of NAC to CIK cell culture could enhance the antitumor activity of CIK cells. The phenotypes of human CIK cells, including CD3+CD56+, IFN-γ, granzyme B, and perforin, were determined by flow cytometry. The cytotoxic activity against the human erythroleukemic cell line (K562) and cholangiocarcinoma cell line (CL6) prelabeled with CFSE was investigated by flow cytometry. The mRNA expression levels of IFNG, PRF1, and GZMB were measured by real-time PCR. By adding NAC into CIK cell culture, the percentage of CD3+CD56+ cells along with the expression of Th1 cytokines and cytolytic granules increased significantly, resulting in an improvement of cytotoxicity against the cancer cell lines CL6 and K562. The incorporation of NAC into CIK culture can markedly improve the cytotoxicity against cancer cells due to the significant increase in the major effector population of CIK cells expressing Th1 cytokines and cytolytic granules.

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.

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.

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.

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.

Aberrant Repression of GFI1 Super-Enhancer By LSD1 Plays an Important Role in Leukemia Programs in Acute Myeloid Leukemia

Backgrounds: Precise gene regulation by cell-type-specific enhancers is fundamental to cell fate decision in hematopoietic differentiation. Super-enhancers (SEs) consist of multiple enhancer elements containing a number of transcription factor-binding motifs. Dysregulation of SEs has been recognized to be one of the major causes of leukemogenesis. Recent studies reported that SEs near to oncogenes such as MYC and EVI1 are often aberrantly activated in leukemia cells, while little is known about whether aberrant silencing of SEs near to onco-suppressor genes is involved in leukemia development.Lysine specific demethylase 1 (LSD1) is a histone modifier that catalyze mono- and di-methylated lysine 4 of histone 3 (H3K4me1/2) directly and acetylated lysine 27 of histone 3 (H3K27ac) via recruiting complex with histone deacetylases 1 (HDAC1) and histone deacetylase 2 (HDAC2). Because both H3K4me1/2 and H3K27ac are activated markers of enhancers, LSD1 is supposed to act as an enhancer repressor. We recently demonstrated that a novel LSD1 inhibitor, NCD38, exerts anti-leukemia effects through inducing myeloid differentiation and activates approximately 500 SEs in leukemia cells. Growth factor independent 1 (GFI1) is an essential regulator for neutrophil development and is strongly upregulated by LSD1 inhibitors. Moreover, the GFI1-SE is included in the list of activated SEs by NCD38. Here we investigate the mechanism and role of the GFI1-SE dysregulation in leukemia cells.Results: Previously we performed ChIP-seq of histone modifications (H3K4me2/3 and H3K27ac) in a human erythroleukemia cell line (HEL) with or without NCD38, and identified the GFI1-SE regulated by LSD1. To better know if status of the GFI1-SE could influence anti-leukemic effect of LSD1 inhibitors, we compared the H3K27ac level of the GFI1-SE to the GFI1 transcript level, the CD11b level, and cell viability, after treatment with LSD1 inhibitors (NCD38, NCD25 and ORY86) in 4 leukemia cell lines. Activation of the GFI1-SE, which is defined as significant elevation of H3K27ac level, followed by induction of the GFI1 transcript and CD11b was observed in 2 cell lines (HEL and CMK11-5) but not in the remaining 2 cell lines (K562 and UT7-EPO). In conjunction with this response, the anti-leukemic effect was exerted only in the former 2 cell lines. These data suggest that activation of the GFI1-SE seems necessary for anti-leukemic effect of LSD1 inhibitors.The GFI1-SE has an evolutionarily conserved region which contains several DNA binding motifs of key transcription factors including GATA1 and RUNX1. Thus, to better understand an underlying mechanism of the GFI1-SE regulation, we performed luciferase reporter assay with several deletion mutants. The reporter activity was significantly elevated by LSD1 inhibitors in the vector cloned with the entire region of the GFI1-SE compared with the empty control vector. However, the elevation of the reporter activity was hardly observed with deletion of the conserved region and attenuated with mutation of both GATA1 and RUNX1 motifs. These results suggest that the conserved region is a core regulatory part of the GFI1-SE and is regulated by the coordination of LSD1 and several key transcriptional factors.Finally, in order to investigate to what extent the GFI1-SE can directly affect leukemia programs by itself alone, we knocked out the GFI1-SE locus using the CRISPR/Cas9-mediated gene editing system in HEL cells. In all 3 GFI1-SE knockout (KO) clones we finally established, GFI1 induction by LSD1 inhibitors was significantly attenuated and consequently CD11b was hardly induced. Next, we compared gene expression profiling after treatment with NCD38 between GFI1-SE KO HEL cells and control HEL cells. Gene set enrichment analysis revealed that gene-sets associated with leukemogenesis and stemness are enriched in GFI1-SE KO cells. These data indicate that deletion of the GFI1-SE suppresses the attenuation of leukemia program by NCD38.Conclusions: Our study disclosed that the newly identified GFI1-SE is essential for sufficient GFI1 induction and is regulated by the coordination of LSD1 and several key transcriptional factors. Aberrant repression or deletion of the GFI1-SE is closely associated with leukemia programs. DisclosuresTakaori:Celgene: Research Funding; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Janssen Pharma: Honoraria; Pfizer​: Honoraria.

Relation between ABCB1 overexpression and COX2 and ALOX5 genes in human erythroleukemia cell lines

This study aimed to characterize the relationship between the COX2 and ALOX5 genes, as well as their link with the multidrug resistance (MDR) phenotype in sensitive (K562) and MDR (K562-Lucena and FEPS) erythroleukemia cells. For this, the inhibitors of 5-LOX (zileuton) and COX-2 (acetylsalicylic acid-ASA) and cells with the silenced ABCB1 gene were used. The treatment with ASA caused an increase in the gene expression of COX2 and ABCB1 in both MDR cell lines, and a decrease in the expression of ALOX5 in the FEPS cells. Silencing the ABCB1 gene induced a decrease in COX2 expression and an increase in the ALOX5 gene. Treatment with zileuton did not alter the expression of COX2 and ABCB1. Cytometry data showed that there was an increase in ABCB1 protein expression after exposure to ASA. In addition, the increased activity of ABCB1 in the K562-Lucena cell line indicates that ASA may be a substrate for this efflux pump, corroborating the molecular docking that showed that ASA can bind to ABCB1. Regardless of the genetic alteration in COX2 and ABCB1, the direct relationship between these genes and the inverse relationship with ALOX5 remained in the MDR cell lines. We assume that ABCB1 can play a regulatory role in COX2 and ALOX5 during the transformation of the parental cell line K562, explaining the increased gene expression of COX2 and decreased ALOX5 in the MDR cell lines.

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.