Erythroleukemia Cell Line K562 Research Articles

TET2 Exon 2 Skipping Confers Sensitivity to AraC and Is an Independent Favorable Prognostic Factor in AML Patients Treated with Intensive Chemotherapy

The nucleoside analogue cytarabine (AraC) has served as the backbone of acute myeloid leukemia (AML) treatment for nearly forty years. About one-third of expressed genes are abnormally spliced in AML yet alternative exon usage (AEU) plays a role in the plasticity of tumor cells and may influence the response to treatment. Here the exon expression profiles of the erythroleukemia K562 cell line were compared to that of its AraC-resistant variant K562/AraC through Affymetrix HTA2 exon arrays. 5140 exon events harbored by 2583 genes distinguished the 2 cell lines. Among these, the skipping of TET2 exon 2 was identified in K562 cells sensitive to AraC whereas TET2 gene expression remained unchanged at the whole transcript level. The results were confirmed by exon-specific RTPCR (ESPCR). Microarray analysis did not evidenced any significant change in mRNA splicing for the 10 remaining exons of the TET2 gene.TET2 is a dioxygenase that catalyzes the conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) and promote DNA demethylation. TET2 somatic mutations occur in about 25% AML, distributing across the whole coding sequence without obvious hot spots. These mutations decrease TET2 enzymatic activity by truncating the protein or affecting its catalytic activity. TET2 exon 2 is spliced in a mutually exclusive manner with exon 1 yet it is used as an alternative promoter (https://fasterdb.lyon.unicancer.fr/). However, TET2 exon 2 is not translated into protein and its role in TET2 regulation is still unknown. Having found that AraC sensitive cells harbor the spliced TET2 isoform, we investigated whether or not skipping of TET2 exon 2 correlate with disease outcome in AML patients treated with AraC-based intensive chemotherapy (AraC-IC).The discovery cohort included 106 consecutive AML patients treated with AraC-IC (median age 57.91, 64 males). RNA was extracted from bone marrow MNCs and assayed for TET2 exon 2 skipping through real-time quantitative ESRTPCR (qESRTPCR) amplification of E1E3 (spliced) and E2E3 (unspliced) TET2 isoforms. TET2 exon 2 skipping was quantified by calculating the ratio E1E3/E2E3. For statistical analysis, the ratio E1E3/E2E3 was dichotomized using median value as the cutoff value. Skipping of TET2 exon 2 was associated with a significantly lower response rate: 65% vs 92%, p = 0.001 but with a significantly lower relapse rate: 39% vs 85%: p<10-4. Univariate analysis (27 months median follow-up) showed that in addition to age and cytogenetic risk, TET2 exon 2 skipping significantly influenced DFS; the higher the level of E1E3 isoform expression, the better the outcome [HR=0.27 (95% confidence interval (CI):0.13-0.53), p<10-4]. The favorable effect of TET2 exon 2 skipping on DFS remained in the 49 patients with normal karyotype: HR=0.31 (CI: 0.12-0.84), p=0.022. Multivariate analysis showed that age [HR=1.83 (CI:1.03-3.24), p=0.039], cytogenetics [HR=3.03 (CI:1.55-5.96), p=0.001], and TET2 exon 2 skipping [HR=0.38 (CI:0.19-0.77), p=0.007] were independent prognostic factors for DFS. The validation cohort included 103 patients with normal karyotype treated with AraC-IC (median age 61.12, 63 males). Skipping of TET2 exon 2 was associated with a lower response rate: 87.5% vs 91.1% (NS) and a significantly lower relapse rate: 3% vs 66%: p<10-4. Univariate analysis showed that in addition to age, FLT3 internal tandem duplication (FLT3-ITD), nucleophosmin (NPM1) exon-12 gene mutation, TET2 exon 2 skipping significantly influenced DFS, EFS, and OS. In multivariate analysis, TET2 exon 2 skipping was the sole prognostic factor for DFS [HR=0.07 (CI: 0.02-0.3), p<10-4], EFS [HR=0.32 (CI: 0.14-0.74), p=0.008], and OS [HR=0.42 (CI: 0.19-0.90), p=0.025]. As an additional control, the prognostic effect of TET2 exon 2 skipping was evaluated in a series of 36 AML unfit for IC and treated with hydroxyurea, azacitidine, decitabine, low dose AraC, or supportive care only. TET2 exon 2 skipping possessed no significant impact on the response rate, the relapse rate, OS, DFS, and EFS in this population.In conclusion TET2 exon 2 skipping is associated with a low relapse rate and possesses a strong favorable prognostic impact in AML treated with AraC-CTI. The mechanism linking this alternative exon usage with resistances to AraC are currently investigated while our results suggest that the determination of TET2 exon 2 splicing status might assist risk stratification. DisclosuresNicolini:Novartis: Consultancy.

Genomic and proteomic analysis of transcription factor TFII-I reveals insight into the response to cellular stress.

The ubiquitously expressed transcription factor TFII-I exerts both positive and negative effects on transcription. Using biotinylation tagging technology and high-throughput sequencing, we determined sites of chromatin interactions for TFII-I in the human erythroleukemia cell line K562. This analysis revealed that TFII-I binds upstream of the transcription start site of expressed genes, both upstream and downstream of the transcription start site of repressed genes, and downstream of RNA polymerase II peaks at the ATF3 and other stress responsive genes. At the ATF3 gene, TFII-I binds immediately downstream of a Pol II peak located 5 kb upstream of exon 1. Induction of ATF3 expression increases transcription throughout the ATF3 gene locus which requires TFII-I and correlates with increased association of Pol II and Elongin A. Pull-down assays demonstrated that TFII-I interacts with Elongin A. Partial depletion of TFII-I expression caused a reduction in the association of Elongin A with and transcription of the DNMT1 and EFR3A genes without a decrease in Pol II recruitment. The data reveal different interaction patterns of TFII-I at active, repressed, or inducible genes, identify novel TFII-I interacting proteins, implicate TFII-I in the regulation of transcription elongation and provide insight into the role of TFII-I during the response to cellular stress.

Niflumic acid affects store-operated Ca(2+)-permeable (SOC) and Ca (2+)-dependent K (+) and Cl (-) ion channels and induces apoptosis in K562 cells.

Non-steroidal anti-inflammatory drugs (NSAIDs) are known to induce apoptosis in a variety of cancer cells. However, the precise mechanisms by which NSAIDs facilitate apoptosis in tumor cells are not clear. In the present study, we show that niflumic acid (NA), a member of the fenamates group of NSAIDs and Cl(-) and Ca(2+)-activated Cl(-) (CAC) channels blocker, induced apoptosis (by ~8 %, 24 h treatment) and potentiated (by 8-10 %) apoptotic effect of endoplasmic reticulum Ca(2+) mobilizer thapsigargin (Tg) in human erythroleukemic K562 cell line. The whole-cell patch clamp and Fluo-3 flow cytometric experiments confirmed an inhibitory effect of NA (100 and 300 µM) on store-operated (SOC) channels. We also found that NA-blocked CAC channels were activated by acute application of Tg (2 µM) in K562 cells. NA blockage of CAC channels was accompanied by activation of Ca(2+)-activated K(+) (SK4) channels. The observed effects of NA were not connected with COX-2 inhibition since 100-nM NA (IC50 for COX-2 inhibition) did not induce either apoptosis or affect the channels activity. We conclude that inhibition of SOC channels plays a major role in NA-induced apoptosis. Increased apoptotic levels in Tg-treated K562 cells in the presence of NA may be due to the blockage of CAC and stimulation of SK4 channels in addition to SOC channels inhibition.

All-trans retinoic acid enhances the effect of 5-aza-2'-deoxycytidine on p16INK4a demethylation, and the two drugs synergistically activate retinoic acid receptor β gene expression in the human erythroleukemia K562 cell line.

The aim of the current study was to investigate the antineoplastic activities of 5-aza-2′-deoxycytidine (also known as decitabine; DAC) and all-trans retinoic acid (ATRA), administered alone or in combination, in K562 cells in vitro, as well as the effects on the expression of the tumor suppressor genes, p16INK4a (p16) and retinoic acid receptor β (RAR-β). Cell growth inhibition, differentiation and apoptosis in K562 cells treated with DAC and/or ATRA were detected. The methylation of the p16 and RAR-β genes in the K562 cells was detected using the methylation-specific polymerase chain reaction (PCR) method. Quantitative PCR was used for the detection of the mRNA expression of the p16 and RAR-β genes, and western blot analysis was used to detect protein expression. DAC and ATRA, alone or in combination, had no effect on the growth inhibition, differentiation and apoptosis of the K562 cells. DAC alone induced the demethylation of the p16 gene, and combination of DAC and ATRA demonstrated more evident demethylation of the p16 gene, however, ATRA alone had no effect on methylation. The RAR-β promoter region was not methylated in the K562 cells. DAC in combination with ATRA appeared to produce a greater activation of the RAR-β gene, which led to the upregulation of the RAR-β expression level. ATRA enhanced the effect of DAC on p16 demethylation, and the combination of the two drugs was found to activate RAR-β expression, which indicated that DAC used in combination with ATRA has clinical potential in the treatment of human erythroleukemia.

HLA-G1 increases the radiosensitivity of human tumoral cells

Different molecules regulate the response of tumoral tissues to ionizing radiation. The objective of this work was to determine if HLA-G1 expression modulates the radiosensitivity of human tumoral cell lines. To this end, human melanoma M8 and human erythroleukemia K562 cell lines, with their correspondent HLA-G1 negative and positive variants, were gamma irradiated and the survival frequency was determined by clonogenic assay. The survival fraction of HLA-G1 expressing cells was around 60% of HLA-G1 negative cells. The generation of acidic vesicular organelles was higher in HLA-G1 positive cells. Apoptosis levels showed statistically significant differences only in K562 cells, whereas the variation in G2/M cycle progression was only significant in M8 cells. In addition, irradiation diminished cell-surface HLA-G1 and increased soluble HLA-G1 levels. Soluble HLA-G1 has no influence on cell survival in any cell line. In summary, we could demonstrate that HLA-G1 confers higher radiosensitivity to HLA-G1 expressing cells.

PiggyBac Transposon plus Insulators Overcome Epigenetic Silencing to Provide for Stable Signaling Pathway Reporter Cell Lines

Genetically modified hematopoietic progenitors represent an important testing platform for a variety of cell-based therapies, pharmaceuticals, diagnostics and other applications. Stable expression of a transfected gene of interest in the cells is often obstructed by its silencing. DNA transposons offer an attractive non-viral alternative of transgene integration into the host genome, but their broad applicability to leukocytes and other “transgene unfriendly” cells has not been fully demonstrated. Here we assess stability of piggyBac transposon-based reporter expression in murine prostate adenocarcinoma TRAMP-C2, human monocyte THP-1 and erythroleukemia K562 cell lines, along with macrophages and dendritic cells (DCs) that have differentiated from the THP-1 transfects. The most efficient and stable reporter activity was observed for combinations of the transposon inverted terminal repeats and one 5’- or two cHS4 core insulators flanking a green fluorescent protein reporter construct, with no detectable silencing over 10 months of continuous cell culture in absence of any selective pressure. In monocytic THP-1 cells, the functional activity of luciferase reporters for NF-κB, Nrf2, or HIF-1α has not decreased over time and was retained following differentiation into macrophages and DCs, as well. These results imply pB as a versatile tool for gene integration in monocytic cells in general, and as a convenient access route to DC-based signaling pathway reporters suitable for high-throughput assays, in particular.

Transfection of chimeric anti-CD138 gene enhances natural killer cell activation and killing of multiple myeloma cells

Reprogramming of NK cells with a chimeric antigen receptor (CAR) proved an effective strategy to increase NK cell reactivity and recognition specificity toward tumor cells. To enhance the cytotoxicity of NK cells against CD138-positive multiple myeloma (MM) cells, we generated genetically modified NK-92MI cells carrying a CAR that consists of an anti-CD138 single-chain variable fragment (scFv) fused to the CD3ζ chain as a signaling moiety. The genetic modification through a lentiviral vector did not affect the intrinsic cytolytic activity of NK-92MI toward human erythroleukemic cell line K562 cells or CD138-negative targets. However, these retargeted NK-92MI (NK-92MI-scFv) displayed markedly enhanced cytotoxicity against CD138-positive human MM cell lines (RPMI8226, U266 and NCI-H929) and primary MM cells at various effector-to-target ratios (E:T) as compared to the empty vector-transfected NK-92MI (NK-92MI-mock). In line with the enhanced cytotoxicity of NK-92MI-scFv, significant elevations in the secretion of granzyme B, interferon-γ and proportion of CD107a expression were also found in NK-92MI-scFv in response to CD138-positive targets compared with NK-92MI-mock. Most importantly, the enhancement in the cytotoxicity of NK-92MI-scFv did not attenuate with 10Gy-irradiation that sufficiently blocked cell proliferation. Moreover, the irradiated NK-92MI-scFv exerted definitely intensified anti-tumor activity toward CD138-positive MM cells than NK-92MI-mock in the xenograft NOD-SCID mouse model. This study provides the rationale and feasibility for adoptive immunotherapy with CD138-specific CAR-modified NK cells in CD138-positive plasmacytic malignancies, which potentially further improves remission quality and prolongs the remission duration of patients with MM after upfront chemotherapy.

Expression Profiling Of Nuclear Hormone Receptors In Myeloid Leukemia Reveals Potential Novel Drug Targets For Combination Therapy

Nuclear Hormone Receptors (NHRs) are a large superfamily of ligand dependent transcription factors regulating a plethora of genes involved in metabolism, growth and differentiation. Recent studies identified the overwhelming role played by NHR in determining hematopoiesis as well as HSC function and fate, suggesting that defective proliferation and maturation of progenitors in hematopoietic malignancies could be attributed to altered expression of these transcriptional regulators. NHRs are attractive but relatively unexplored drug targets and there is limited data on their expression profile in hematological malignancies. Our preliminary screening of AML cell lines and primary cells revealed differential expression of PPARg and RXRα among AML cell lines and decreased expression of these targets in resistant cell lines vs. sensitive ones (Fig.1a). We extended this study aiming to identify the expression pattern of all NHRs in myeloid malignancies, compare with ex-vivo chemoresistance and prognostic markers. [Display omitted] Myeloid leukemia cell lines AML(n=12) carrying AML-ETO, Inv-16, PML-RAR fusion transcript, NPM1 or FLT3 mutation and CML(n=8), pooled primary samples harboring only FLT3 ITD mutation and only NPM1 mutation, CD34+ cells and total cellular RNA samples from normal donors were included in the study. Bone marrow samples from AML patients and peripheral blood samples from healthy volunteers were collected after informed consent. PBSC from normal donors (n=7) were enriched for CD34+ cells by magnetic enrichment. RNA extraction followed by purification and cDNA synthesis were done as per manufacturer's recommendation. The expression of 42 NHRs and 42 co-regulators was profiled by qRT PCR using RT2 PCR Profiler array (SABiosciences, Germany). Expression of each gene was normalized to the average of 5 housekeeping genes (GAPDH, b-actin, b2-microglobulin, HGPRT & RPLP0) and differential expression was calculated by normalizing this with normal total RNA samples to determine fold change. Data normalization and differential gene expression were computed using SABiosciences web-based analysis software. Ex-vivo cytotoxicity to cytarabine and daunorubicin was analyzed using MTT assay and IC50 was calculated using Adapt software. Based on Ara-C and Dnr IC50, AML samples were categorized as sensitive or resistant (IC50<6.25µM and >6.25µM for Ara-C;<0.5µM and >0.5µM for Dnr). FLT3-ITD and NPM1 mutations were screened using PCR followed by GeneScan methods.Upon analysis we observed steroid receptors except PPARG, NR5A1, RXRG, NR1D2, glucocorticoid receptors, androgen receptors and their co-regulatory molecules were expressed across all cell lines. In contrast, expression of certain NHRs such as NR2E3, NR1I2, NR1H4, NR0B1, NR0B2, ESRRG, ESRRB, NR1D1 and the co-regulator HDAC7 were less or at undetectable levels, while estrogen receptors and its co-regulatory molecules except PPARGC1B, steroid receptors HNF4A, NR2F1, NR2F2, RARG and NR3C2 were moderately expressed across the cell lines (Fig.1b). Analysis also revealed significantly reduced expression of ESR2, ESRRG, NR2F2, NR2F6 and THRB and increased PPARG in AML compared to CML cell lines. Comparison between CD34+ progenitor population and well differentiated hematopoietic cells demonstrated >6 fold change in NR4A1 expression. Likewise, expression of several differentiation markers NR1H3, NR1D2, RORA, RORB, RARA and VDR were elevated in cells with increased degree of maturation. The receptor inducing terminal differentiation of erythrocytes THRA, had lesser expression (<60 Fold) in erythroleukemic cell lines K562, HEL and LAMA-84 compared to normal (Fig.1c). In Ara-C resistant cell lines, expression of NR1I3, AHR, and HNF4A were up-regulated while PPARG and RXRA were down-regulated. NPM1 positive cell lines and patient samples had increased expression of PPARG, PPARGC1A, NOTCH2, NR1H3 and NR2F2 than the FLT3 mutated group (Fig.1d) and similar expression of PPARG and RXRA was validated in NPM mutated primary AML blasts (Fig.1e).Our comprehensive analysis of NHRs and its co regulatory molecules provides us with insights on NHR expression on differentiation and drug resistance across myeloid leukemia cell lines with various genetic and molecular characteristics. This could be further explored to identify potential novel drug targets to be used as combination therapy in myeloid leukemias to overcome drug resistance. Disclosures:No relevant conflicts of interest to declare.

G-Quadruplex Forming Sequences Found In The Beta-Globin Gene Cluster Regulate Erythroid Differentiation Of K562 Cells

IntroductionGuanine-rich DNA sequences can form stable four stranded structures by folding of the DNA strands, forming stacks of G-tetrads called G-quadruplex. G-quadruplex forming sequences are found in eukaryotic telomeres, in promoter regions and in noncoding regions of the genome. Although the function of these structures has not yet been identified it has been suggested that they play a negative regulatory role at the transcription level. The most studied G-quadruplex sequences (AS1411 and PU27) show activity in reducing growth and invasiveness of malignant cells, as well as increasing cell death of a large array of tumors with promising therapeutic applications. We identified two G-quadruplex forming sequences in the human beta globin cluster (Hbd and Hbg2). We confirmed quadruplex formation by these sequences using circular dichroism spectroscopy. We also observed in Southern blot assay that these oligo-sequences bind to specific β-globin gene fragments after EcoRI digestion. Finally we demonstrate that neither Hbd nor Hbg2 (5 or 10µM) were growth inhibitory to four different leukemia cell lines after 6 days culture measured by MTT. ResultsThe β-globin cluster contains 5 functional globin-like genes and long range regulator elements, the expression of each of these genes is tightly regulated during development (from embryo to fetus to adult), in cell type and in the course of differentiation/maturation of the erythroid lineage. Recent studies have demonstrated the importance of folding and looping of the DNA in the control of globin genes expression. We hypothesized that the G-quadruplex forming sequences within the β-globin gene cluster participate in regulation and splicing of the different globin genes during erythroid maturation. The erythroleukemia cell line K562 was used in this study because of its ability to differentiate into erythrocytes under Hemin stimulation. K562 cells were divided into 6 groups of treatment and cultured for 3 days in the presence or absence of Hemin (30µM) with or without either Hbd or Hbg2 oligonucleotides (5µM). Erythroid differentiation was evaluated by quantitative QRT-PCT for the expression of β-, γ- and ε-globin genes as well as α-globin and by flow-cytometry for the presence of β-, γ- and ε-globin; α-globin and CD235a (Glycophorin A) were used as controls. The data reveal a difference in gene expression for all globin genes when K562 were exposed to Hbd or Hbg2 only, interestingly, a significant up-regulation of β-globin was observed only in Hbg2 treated cells. Similarly in K562 induced to differentiate with Hemin show up-regulation of γ-, ε- and α- globin, the addition of Hbd or Hbg2 oligonucleotides to Hemin at the beginning of the culture slightly improve the expression of γ- and ε- however there was no difference for the expression of β- globin in the hemin +/- Hb oligonucleotides treated cells compared to control untreated cells. Flow-cytometry experiments confirmed the increase in the expression of each globin gene in all treatments compared to control. Altogether the data suggest a role for Hbd and Hbg2 in the regulation of β-globin gene transcription and therefore in the differentiation of K562 in erythroid lineage. ConclusionsThe implications of our findings are multiple. 1) the presence of G-quadruplex sequences in a gene complex not involved in growth regulation or oncogenesis; 2) to our knowledge this is the first time that G-quadruplex sequences are shown to specifically alter the expression of the gene in which they are located; and 3) the potential use of globin G-quadruplex to modify the globin gene expression therapeutically. Disclosures:No relevant conflicts of interest to declare.

The Leukemia-Associated Rho Guanine Nucleotide Exchange Factor (LARG) /ARHGEF12 Is Important For Erythroid Differentiation

ARHGEF12,which was initially identified as a fusion partner of MLL in acute myeloid leukemia, is a regulatory protein involved in the GDP/GTP exchange reaction of the Rho A and activates a Rho-GTPase-dependent signaling pathway. However, the role of ARHGEF12 in hematopoiesis remains unknown. Here, we first report that ARHGEF12 takes part in erythroid differentiation not only in erythroleukemia cell line K562 but also in zebra fish based on knockdown technology. Although primitive hematopoiesis including erythroid differentiation progressed normally, double silencing of both orthologues arhgef12a and arhgef12b in Danio rerio with morpholino caused erythropenia during definitive hematopoiesis. Meanwhile, cytology assay reveal less mature red blood cells with large and loose nuclear in morpholino injected embryos. The phenotype of blocked erythroid differentiation can be rescured by mRNA overexpression of RhoA active mutant. On the other hand, injection of mRNA transcripted from the dominant negative mutant of RhoA could replicate the phenotype of arhgef12 silencing. Antibody array screen of kinase phosphorylation suggested STAT1 and MAPK pathways downstream of Rho A may be important for erythroid differentiation in our modles. Disclosures:No relevant conflicts of interest to declare.

Targeting The PRMT5 Enzyme To Induce γ-Globin Gene Derepression In Sickle Cell Anemia

Sickle Cell Disease (SCD) is an autosomal recessive disorder that affects thousands of people across the United States. Although it is not a disease exclusive to the African American population, it is most common among persons from this ethnic group with an estimated 1 out of every 500 individuals affected. The disease is caused by a point mutation in the sixth codon of the beta-globin gene which is found on chromosome 11. This missense mutation involves the replacement of the hydrophilic glutamic acid with a hydrophobic valine that affects the structure of the b-GLOBIN protein leading to altered erythrocyte elasticity and oxygen carrying capacity. As a result, less oxygen is delivered to the tissue and the deformed, rigid cells often become trapped in the microvasculature causing local tissue ischemia, inflammation and the pain crisis that is characteristic of the disease. Early research into the understanding of this devastating disease has shown that a modulator of SCD severity is the persistence of fetal hemoglobin (HbF or γ-GLOBIN). Indeed SCD patients carrying a genetic mutation leading to hereditary persistence of HbF are able to maintain a near crisis-free life. This is so because HbF is made up of 2 alpha and 2 gamma subunits and lacks the beta subunits found in adult hemoglobin. As a result, HbF lacks the ability to sickle when there are significant changes in the oxygen levels. Several genetic, biochemical and clinical observations indicate that even modest induction of γ-GLOBIN may be sufficient to ameliorate the SCD phenotype. In the past decade efforts have focused on identifying regulatory complexes that modulate fetal-γ-GLOBIN gene expression that could be targeted for pharmacological intervention to improve and to monitor the outcome of this incurable disease. Physiologic transcriptional silencing of the γ-GLOBIN locus during development could be reversed for therapeutic benefit. To date, compounds such as 5-aza-2-deoxycytidine, hydroxyurea, short-chain fatty acids, and histone deacetylase (HDAC) inhibitors, have been used to increase HbF, however, effects are variable and mechanism of action is poorly understood. Thus, it is imperative that additional therapies be developed to enhance HbF expression in patients not responding well to the drugs available on the market. Recent work has identified key components of the repressor complex that silences fetal-γ-GLOBIN including protein arginine methyltransferase 5 enzyme (PRMT5) and histone deacetylase protein 1 and 2 (HDAC1/2). Studies are ongoing to characterize the regulatory complex that is recruited to the γ-GLOBIN gene promoter and develop a lead optimized PRMT5 inhibitor drug for clinical translation. Pretreatment of the erythroleukemia cell line K562 with three of our newly developed PRMT5 inhibitors (CMP5, HLCL7 and CMP12) leads to re-expression of g-GLOBIN gene product HbF at mRNA and protein levels in a time and dose dependent fashion. As PRMT5, LSD1 and HDAC1/2 have been shown to be all involved in the regulation of expression of HbF gene we further evaluated the effect of specific inhibitors targeting each of these proteins alone or in combination. Our data strongly indicate that while each agent alone led to re-expression of HbF, combination treatments led to an enhanced protein expression. Studies are ongoing to caracterize the regulatory complex that is recruited to the γ-GLOBIN gene promoter and develop a lead optimized PRMT5 inhibitor drug for clinical translation. We believe that our results will be the key for developing of more effective and specific therapies for this incurable disease. Disclosures:No relevant conflicts of interest to declare.

O-Glcnacylation Regulates γ-Globin Transcription

Patients with sickle cell disease (SCD), caused by mutation of the adult β-globin gene, are phenotypically normal if they carry compensatory mutations that result in continued expression of the fetal γ-globin genes, a condition termed hereditary persistence of fetal hemoglobin (HPFH). Thus, a logical clinical goal for treatment of SCD is to up-regulate γ-globin synthesis using compounds that are specific for increasing fetal hemoglobin (HbF) without pleiotropic effects on cellular homeostasis. Developmental regulation of the γ-globin genes is complex and normal silencing during the adult stage of erythropoiesis likely results from a combination of the loss of transcriptional activators and the gain of transcriptional repressor complexes. One mode of γ-globin silencing occurs at the GATA binding sites located at -566 or -567 relative to the Aγ-globin or Gγ-globin CAP sites respectively, and is mediated through the DNA binding moiety of GATA-1 and its recruitment of co-repressor partners, FOG-1 and Mi-2 (NuRD complex). Modifications of repressor complexes can regulate gene transcription; one such modification is O-GlcNAcylation. The O-GlcNAc post-translational modification is the attachment of a single N-acetyl-glucosamine moiety to either a serine or threonine residue on nuclear and cytoplasmic proteins. O-GlcNAc is added to proteins by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA) in response to changes in extracellular signals and nutrients. A dynamic balance in protein levels also exists between these two enzymes; an increase or decrease of one results in a like compensatory change in the other. Thus, the rate of O-GlcNAc addition and removal is a dynamic cycling event that is exquisitely controlled for a given target molecule, which may offer a point of intervention in the turning off or on of gene expression. O-GlcNAcylation is involved in the regulation of many cellular processes such as stress response, cell cycle progression, and transcription. Potentially, O-GlcNAc plays a pivotal role in regulating transcription of the human γ-globin genes. We induced human erythroleukemia cell line K562 with sodium butyrate to differentiate toward the erythroid lineage and observed the expected increase of γ-globin gene expression. A robust increase of γ-globin gene expression was measured after pharmacological inhibition of OGA using Thiamet-G (TMG). Using chromatin immunoprecipitation (ChIP), we demonstrated that OGT and OGA are recruited to the -566 region of the Aγ-globin promoter, the same region occupied by the GATA-1-FOG-1-Mi-2 (NuRD) repressor complex. However, OGT recruitment to this region was decreased when O-GlcNAc levels were artificially elevated by OGA inhibition with TMG. When γ-globin expression was not induced, Mi-2 was modified with O-GlcNAc and interacted with both OGT and OGA. After induction, O-GlcNAcylation of Mi-2 was reduced and Mi2 no longer interacted with OGT. Stable K562 cells were generated in which OGA was knocked down using shRNA. Following induction of these cells with sodium butyrate, γ-globin gene expression was higher compared to control cells. These data suggest that the dynamic cycling of O-GlcNAc on the Mi-2 (NuRD) moiety contributes towards regulation of γ-globin transcription. Concurrent ChIP experiments in human β-globin locus yeast artificial chromosome (β-YAC) transgenic mice demonstrated that GATA-1, Mi2 and OGT were recruited to the -566 Aγ-globin GATA silencer site in day E18 fetal liver when γ-globin is repressed, but not in day E12 fetal liver when γ-globin is expressed. These data demonstrate that O-GlcNAc cycling is a novel mechanism regulating γ-globin gene expression and will provide new avenues to explore in how alterations in gene regulation lead to the onset, progression, and severity of hematological disease. Disclosures:No relevant conflicts of interest to declare.

Glycophorin C ligation: another biochemical pathway in red blood cell senescence?

In this issue of TRANSFUSION two articles describe the effects on erythroid cells of antibody ligation to the red blood cell (RBC) membrane glycoprotein glycophorin C (GPC).1, 2 GPC is a monotopic high-abundance RBC membrane protein that forms a structural linkage to the membrane skeleton via the proteins 4.1R and p55. GPC derives from the GYPC gene, which additionally expresses GPC in a truncated form, glycophorin D (GPD). Glycophorins are extensively sialyated and contribute significantly to the zeta potential (net negative charge) that prevents systemic aggregation of RBCs by mutual repulsion. RBCs lacking both GPC and GPD are known as the Leach phenotype and have irregular ellipsoid shape. GPC and GPD express the 11 Gerbich blood group antigens,3 and three Gerbich-negative phenotypes are known, Gerbich (Ge:–2,–3,4), Yus (Ge:–2,3,4), and the aforementioned Leach phenotype (Ge:–2,–3,–4). Anti-Gerbich (notably anti-Ge3) has previously been noted as a cause of hemolytic disease of the fetus and newborn (HDFN; and further described here by Pate and colleagues2), so knowledge regarding the immune destruction of the fetal RBCs is of relevance to the clinical management of these cases. The study of Wang and coworkers1 reveals that ligation using anti-GPC induces cell death in the erythroleukemic cell line K562 and erythroid progenitors derived from CD34+ umbilical cord blood stem cells. This process remains only partially characterized biochemically, but involves the exposure of phosphatidylserine (PS) at the extracellular leaflet of the membrane (it is normally polarized to the intracellular face of the membrane). These findings confirm our earlier study,4 which interestingly also found that Gerbich and Yus phenotype cells lacked this pathway, despite binding anti-GPC on their surface. Thus, anti-Ge3 causing the HDFN described in previous studies, most likely induces PS exposure on fetal RBCs and induces their death and is clearly a different mechanism to that induced by anti-D which involves monocyte binding to the Fc region of RBC bound antibody. The GPC pathway induced by anti-Ge3 (and other anti-GPC) most likely is mimicking binding to the protein by an as yet unknown ligand(s), possibly on macrophages or other white blood cells. The mechanism of PS exposure induced by GPC ligation is also intriguing; the previously mentioned GPC-p4.1R-p55 interaction clearly must trigger the PS exposure in some manner. Indeed, p4.1R is known to bind PS and is regulated by calmodulin.5 It is therefore important that this pathway is unraveled as it may play a fundamental part in RBC turnover. It is somewhat puzzling that despite its huge physiologic significance, mechanisms of RBC turnover (dubbed eryptosis) remain relatively poorly characterized; this deficiency no doubt will be rectified based on the identification of the key RBC membrane proteins that appear to be involved. GPC is now among the candidates. The author is a member of the Transfusion Medicine Advisory Board (TMAB) for Grifols SA.

SiRNA Delivery Improvement by Co-formulation of Different Modified Polymers in Erythroleukemic Cell Line K562.

siRNA may be a very promising tool for treatment of various diseases especially in cancer therapy due to high specificity. One of the main hurdles applications of siRNAs in vivo is optimization of the delivery strategy, especially the carrier systems. The aim of this study was to optimize siRNA delivery into suspended erythroleukemic cell line K562. We applied polyethyleneimine (PEI) and oligoethyleneimine (OEI) derivatives alone or their co-formulation with different agents such as chloroquine (a drug known to alter lysosomal pH and thus to inhibit lysosomal degradation of macromolecules), DOPE (lipophilic agent), succinic acid (introduction of negatively charged to polymer) and transferrin (the ligand of transferring receptor which is over-expressed in many types of tumors and hematopoietic cells). In this study it was shown that utilizing a combination of 70% OEI-HA10 (ten hexyl acrylate residues per one OEI chain) plus 30% of transferin-PEI with Luc-siRNA was highly effective for transfecting K562 cell. This co-formulation silenced luciferase activity up to 70% after short time without any significant inhibition in the luciferase activity in siCONTROL wells. In conclusion, the combination of modified PEI with transferrin and OEI by hexyl acrylate may increase siRNA delivery and reduce toxicity in hematopoietic suspended cells.

Antioxidants May Protect Cancer Cells from Apoptosis Signals and Enhance Cell Viability

Quercetin is one of the most abundant dietary flavonoids widely present in many fruits and vegetables. Previous in vitro studies has shown that quercetin acts as an antioxidant and anti-inflammatory agent and it has potent anticarcinogenic properties as an apoptosis inducer. In this study we examined apoptotic effects of quercetin on the K562 erythroleukemia cell line. K562 cells were induced to undergo apoptosis by hydrogen peroxide. Cell viability and apoptosis level were assessed by annexin V and PI staining methods using flow cytometry. Viability of K562 cells was increased by low dose of quercetin (5-100 μM) for 3 hours. High doses of quercetin proved toxic (100-500 μM, 24 hours) and resulted in decrease of K562 cell viability as expected (P<0.01). As to results, 100 μM quercetin was defined as a protective dose. Also, K562 cell apoptosis due to hydrogen peroxide was decreased in a dose dependent manner. As indicated in previous studies, reduction of superoxides by free radical scavengers like quercetin could be beneficial for prevention of cancer but consumption of such flavonoids during cancer treatment may weaken effects of chemotherapeutics and radiotherapy. Especially cancer patients should be carefully considered for traditional phytotherapy during cancer treatment, which can lead to controversial results.

Enhanced erythroid cell differentiation in hypoxic condition is in part contributed by miR-210

Erythropoiesis, a process of erythroid production, is controlled by several factors including oxygen level. In this study, the effect of oxygen tension on erythropoiesis was investigated in K562 erythroleukemic cell line and erythroid progenitor cells derived from normal and β-thalassemia/hemoglobin (Hb) E individuals. The enhanced erythroid differentiation specific markers including increased levels of α-, β- and γ-globin gene expressions, numbers of HbF positive cells and the presence of glycophorin A surface marker were observed during cell culture under hypoxic atmosphere. The result also showed that miR-210, one of the hypoxia-induced miRNAs, was up-regulated in K562 and β-thalassemia/HbE progenitor cells cultured under hypoxic condition. Inhibition of miR-210 expression leads to reduction of the globin gene expression and delayed maturation in K562 and erythroid progenitor cells. This indicated that miR-210 contributes to hypoxia-induced erythroid differentiation in both K562 cells and β-thalassemia/HbE erythroid progenitor cells.

The role of Lysophosphatidic acid in erythropoiesis in K562 human erythroleukemia cell line

Lysophosphatidic acid (LPA) is an essential factor in the regulation of cellular responses through activating different LPA receptors (LPARs), which belong to a family of G‐protein‐coupled receptors. While LPA has been widely studied for its effects on cardiovascular system development and cancer metastasis, the relationship between LPARs and erythropoiesis remains unclear. The aim of this study is to use human erythroleukemia cell line K562 as a model to investigate the role of LPA and LPARs in erythorocyte development. Our data demonstrated that exogenous addition of LPA had no significant effect on hemin‐induced erythropoiesis by the detection of γ‐globin mRNA expression. However, treatment with Ki16425, a specific antagonist for LPAR1 and LPAR3, significantly decreased γ‐globin mRNA levels. Moreover, treatment with LPAR2 specific agonist, mono‐dodecylphosphate (MDP), also resulted in the reduction of γ‐globin mRNA levels. These results suggested that LPAR1/3 and LPAR2 reciprocally regulate erythropoiesis in K562 cells.

Identification of promoter targets of enhancers by epigenetic knockdown using TAL DNA binding proteins

A fundamental question in biology is how an organism’s genome controls cellular differentiation and identity to produce numerous distinct cell types. At the epigenome level, this is partially influenced by cell type specific regulatory regions marked by accessible chromatin. Recent genome-wide maps of chromatin modifications and DNA associated proteins have identified over a million candidate regulatory elements in the human genome. These DNA sequence elements are proposed to drive the activation or repression of nearby genes, however new tools are needed to do a thorough and systematic functional analysis of these elements in an endogenous context. Materials and methods To directly test distal elements in a loss of function manner, we have fused the chromatin modifying enzyme lysine specific demethylase 1 (LSD1, also known as KDM1A) to transcription activator-like (TAL) effector DNA binding proteins. The TAL:LSD1 fusions are designed to specifically bind target DNA sequences and alter the nearby chromatin state, specifically demethylation of histone H3 lysine 4 di and mono-methylation (H3K4me2/1), a mark of distal enhancers. We have designed TALs to target over 50 putative distal enhancers. These constructs were individually transfected into the erythroleukemic cell line K562, after which we examined changes in H3K4me2 and H3K27acetylation by ChIP, or examined gene expression changes by RNA-seq. Additionally we designed TALs fused to a 3X FLAG epitope to do ChIP-seq analysis and determine the specificity of TAL targeting. Results We show using TAL:3XFLAG ChIP-seq that binding of TALs is virtually exclusive to their target sequence. We show the TAL:LSD1 fusions have the ability to specifically decrease H3K4me2, as well as H3K27acetyl, another mark of distal enhancers, at their intended target sites. We find roughly 50% of TALs significantly reduce the chromatin modifications at the target enhancer, but not at non-target control enhancers. For a subset of these TAL:LSD1 fusions, we used RNA-seq to identify a nearby gene that is downregulated upon loss of the chromatin state at the enhancer. These nearby target genes show a range of decreased expression, allowing us to relatively quantitate the contribution on transcriptional activation of the target enhancer. Conclusions

Abstract A24: Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) induces apoptosis via ectodomain shedding mediated by a disintegrin and metalloproteinase 9

Abstract Objectives: Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) is a type-II membrane protein that can induce apoptosis in immune cells including peripheral lymphocytes and natural killer cells after converted to a secreted type by proteolytic processing referred to as “ectodomain shedding.” So far, over 150 scientific reports have been published concerning the biological functions and clinical significance of RCAS1. Because RCAS1 not only helps tumor cells to evade immune surveillance but also induces cancer stromal tissue remodeling, RCAS1 is believed to exaggerate aggressive characteristics of human malignancies. Clinically, RCAS1 expression correlates with several pathological variables including tumor size, stage, invasion depth, and lymph node metastasis and is a negative predictor of overall survival in 15 different kinds of cancer derived from brain, oral cavity, lung, pleural mesothelium, esophagus, stomach, bile duct, gallbladder, pancreas, colon, gastrointestinal mesenchyme, kidney, prostate, uterine cervix, and endometrium. RCAS1 is secreted significantly higher in serum and pleural effusion in cancer patients than non-cancer individuals and its level is changed in response to treatment. We here studied regarding ectodomain shedding that affects the biological activity of membrane proteins such as growth factors, growth factor receptors, cell-adhesion molecules, extracellular matrix proteins by altering their localization and mode of action. The objective of this study is to investigate (1) a key protease involved in RCAS1 shedding and (2) an association between tumor protease expression and serum RCAS1 concentration in uterine cancer patients. Materials and Methods: (1) The key protease involved in RCAS1 shedding was investigated using two cell lines: a human cervical adenocarcinoma cell line SiSo and a breast adenocarcinoma cell line MCF-7. Both SiSo and MCF-7 express RCAS1, but RCAS1 secretion is undetectable in MCF-7. The protease expression was evaluated by microarray analysis and immnocytochemistry. The expression and secretion of RCAS1 were measured by using flow cytometry and ELISA after knockdown or introduction of protease expression. The induction of apoptosis in an erythroleukemia cell line K562, which express a putative RCAS1 receptor, was investigated. K562 cells were co-cultured with SiSo, MCF-7, and protease cDNA transfected MCF-7 to follow RCAS1 secretion in apoptosis initiation. (2) The association between tumor protease expression and serum RCAS1 concentration was studied in 50 cases of uterine cancer patients. The tumor protease expression was evaluated by immunohistochemistry and serum RCAS1 concentration was measured by ELISA. Results: (1) Microarray analysis revealed that SiSo expressed MMP-28, ADAM9, and ADAM21 at significantly higher levels than did MCF-7. The association of RCAS1 and ADAM9 was detected by immunocytochemistry in SiSo. ADAM9 cDNA transfection in MCF-7 induced RCAS1 secretion and reduced its expression, on the other hand, ADAM9 siRNA transfection in SiSo suppressed RCAS1 secretion and augmented its expression. Growth inhibition and apoptosis were induced by ADAM9 cDNA tansfected MCF-7 but not parent cell. Therefore, it was revealed that ADAM9 is a key protease for RCAS1 shedding. (2) There was a significant correlation between tumor ADAM9 expression and serum RCAS1 concentration in uterine cancer. Conclusions: Further exploration regarding the regulatory mechanisms involved in the conversion of membrane-anchored RCAS1 into its soluble form by ADAM9 should aid the development of novel therapeutic strategies against human malignancies by targeting RCAS1. Citation Format: Kenzo Sonoda. Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) induces apoptosis via ectodomain shedding mediated by a disintegrin and metalloproteinase 9. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A24.

Antibody‐mediated glycophorin C coligation on K562 cells induces phosphatidylserine exposure and cell death in an atypical apoptotic process

Glycophorin C (GPC) is necessary in the maintenance of red blood cell structure. Severe autoimmune hemolytic anemia and hemolytic disease of the fetus and newborn (HDFN) have been associated with Gerbich (Ge) blood group system antigens expressed on GPC. Previous in vitro studies with cord blood progenitor cells have shown that anti-Ge suppresses erythropoiesis. Here, we evaluated the K562 erythroleukemic cell line to study the cellular effects of a murine anti-GPC. Cell proliferation was evaluated after treatment with anti-GPC. Flow cytometry was used to evaluate exofacial phosphatidylserine (PS) expression and cell viability (propidium iodide binding). Cell morphology was evaluated under light microscopy with cytospin preparations stained with May-Grünwald Giemsa. Anti-GPC dramatically inhibited K562 proliferation and increased PS expression, consistent with cytoplasmic blebbing, suggesting evidence of apoptosis. Z-VAD-FMK, an inhibitor of classical apoptosis, was unable to reverse the suppressive effect of anti-GPC. However, hemin was able to attenuate growth suppression. Together, the data suggest that anti-GPC suppresses erythroid proliferation through the induction of nonclassical apoptosis.