EoL-1 Cell Research Articles

Uncovering the Role of CD59 As a Novel Therapeutic Target Against High-Risk Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a hematologic malignancy driven by acquired genetic mutations in hematopoietic stem and progenitor cells. Continued disease progression leads to the unregulated growth of immature cells of the myeloid lineage, resulting in impaired hematopoiesis, organ failure, and death. High-risk AML is a disease subtype, classified by cytogenetic abnormalities, TP53 mutations, chemotherapy resistance, and dismal overall survival. To uncover the driving factors of poor outcomes in high-risk AML, we performed a multivariable analysis of the BEAT-AML-2.0 patient cohort. We identified that overexpression of the membrane regulatory protein, CD59, serves as a prognostic marker for an increased risk of death. Furthermore, we uncovered that CD59 is overexpressed in high-risk AML patients, including samples with TP53 mutations and refractory disease. CD59 prevents the formation of the membrane attack complex of the complement system and its overexpression is linked with resistance to cancer therapies that activate the complement cascade. However, the intracellular role of CD59 is poorly understood and it is unexplored in AML. Given that the standard of care for AML treatment, which consists of a combination therapy of cytarabine and an anthracycline, does not activate the complement system, we hypothesize that CD59 confers a survival advantage to AML cells in a complement-independent manner. To study the role of CD59 in AML, we constructed doxycycline-inducible short-hairpin RNA (shRNA) expression vectors against CD59 (shCD59) and a non-targeting control (shNT) and transduced them into SKM-1, HL-60, EOL-1, and OCI-M1 AML cell lines. First, we examined the impact of CD59 knockdown on cell growth through a cell competition assay between shCD59 or shNT cells and untransduced cells by culturing them at a 1:1 ratio. We observed that upon the addition of doxycycline, shCD59 cells are outcompeted by untransduced cells whereas the percentage of shNT cells remains unchanged. Cell viability analysis revealed an increased level of cell death in shCD59 cells in comparison to shNT cells. Moreover, we quantified the level of cellular proliferation in shCD59 and shNT cells through 5-ethyl-2'-deoxyuridine (EdU) staining. We observed that shCD59 cells have a signification reduction in cellular proliferation in comparison to shNT cells, highlighting changes in both cellular division and survival pathways upon CD59 knockdown. Importantly, to rule out the impact of alternative complement activation on the observed phenotype, we repeated the previously described experiments in heat-inactivated fetal bovine serum supplement cell culture medium and observed an identical response, confirming a complement-independent role of CD59. Based on our findings, we investigated the mechanism through which CD59 exerts pro-survival signals by performing RNA-sequencing on shCD59 and shNT cells. Through differential expression and pathway analyses, we discovered shCD59 cells exhibit increased inflammatory signalling and apoptotic pathway activation. Conversely, mTOR and MYC signaling pathways are significantly downregulated upon CD59 knockdown, including a reduction in mitotic spindle activity, DNA repair machinery, and DNA replication checkpoints. These results indicate that upon CD59 knockdown, AML cells undergo mitotic catastrophe, driven by deficient cell-cycle checkpoints, DNA damage, and ultimately, cell death. We further examined changes in the signaling landscape of shCD59 and shNT cells by analyzing the cellular phospho-proteome through mass spectrometry. In agreement with our RNA-sequencing analysis, shCD59 cells have increased activation of DNA damage and inflammation markers, such as BCLAF1 and IKBKG, respectively. In contrast, shNT cells display elevated activation of mTOR pathway markers, including EIF4B, and cell cycle drivers, such as CDC20. In summary, we present CD59 overexpression as a driver of AML progression and poor clinical outcomes. Through functional and computational studies, we have shown CD59 expression is critical for cell growth and cell survival, and as such, identifying it as a novel therapeutic target. The survival outlook for high-risk AML patients has remained unchanged for the past 25 years. Our findings offer valuable insights that can significantly alter the current treatment landscape of AML.

IgE Immune Complexes Mitigate Eosinophilic Immune Responses through NLRC4 Inflammasome.

Immune complexes (ICs) skew immune responses toward either a pro- or anti-inflammatory direction based on the type of stimulation. Immunoglobulin E (IgE) is associated with Th2 immune responses and known to activate innate immune cells. However, roles of antigen (Ag)-specific-IgE ICs in regulating human eosinophil responses remain elusive; therefore, this study builts upon the mechanism of which ovalbumin (Ova)-IgE ICs affects eosinophilic responses utilizing human EoL-1 cell line as a model. Eosinophils are granulocytes functioning through pattern recognition receptors (PRRs) and destructive granule contents in allergic inflammation and parasitic infections. One of the PRRs that eosinophils express is NLRC4, a member of the CARD domain containing nucleotide-binding oligomerization (NOD)-like receptor (NLR) family. Upon recognition of its specific ligand flagellin, NLRC4 inflammasome is formed and leads to the release of interleukin-1β (IL-1β). We exhibited that Ova-IgE ICs induced the NLRC4-inflammasome components, including NLRC4, caspase-1, intracellular IL-1β, and secretion of IL-1β, as well as the granule contents MMP9, TIMP1, and TIMP2 proteins via TLR2 signaling; these responses were suppressed, when NLRC4 inflammasome got actived in the presence of ICs. Furthermore, Ova-IgE ICs induced mRNA expressions of MMP9, TIMP2, and ECP and protein expressions of MMP9 and TIMP2 in EoL-1 through FcɛRII. Interestingly, TLR2 ligand and Ova-IgE ICs costimulation elevated the number of CD63+ cells, a degranulation marker, as compared to the native IgE. Collectively, our findings provide a mechanism for the impacts of Ova-IgE ICs on eosinophilic responses via NLRC4-inflammasome and may help understand eosinophil-associated diseases, including chronic eosinophilic pneumonia, eosinophilic esophagitis, eosinophilic granulomatosis, parasitic infections, allergy, and asthma.

Eosinophil-derived TGFβ1 controls the new bone formation in chronic rhinosinusitis with nasal polyps

Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by chronic eosinophilic inflammation and new bone formation (NBF). These processes may be associated with each other in the pathogenesis and influence the severity and prognosis of the disease. However, it is still unclear how eosinophilic inflammation is involved in the NBF. Sinus bone cells were isolated from ethmoid bone tissues of patients with CRSwNP and controls. Transforming growth factor beta 1 (TGFβ1) and alkaline phosphatase (ALP) expression in sinus bone cells was determined using quantitative RT-PCR, immunoblotting, and immunohistochemistry. The co-localization of TGFβ1 with eosinophils was assessed by immunofluorescence staining. Sinus bone cells were co-cultured with eosinophils (Eol-1 cell line), which were differentiated with butyrate, to measure the osteoblast differentiation activity of sinus bone cells. TGFβ1 expression was increased in sinus bone tissues and correlated with CT scores in CRSwNP. TGFβ1 was also increased in the submucosa of CRSwNP and co-localized predominantly with eosinophils compared with neutrophils Differentiated Eol-1 cells-derived TGFβ1 increased ALP expression in sinus bone cells. Treatment with a TGFβ inhibitor attenuated TGFβ1-induced ALP expression and staining in sinus bone cells of CRSwNP, leading to loss of bone formation. Eosinophil-derived TGFβ1 was enriched in the submucosa of CRSwNP, which induced ALP expression in sinus bone cells and NBF. Therefore, eosinophil-derived TGFβ1 may mediate aberrant bone remodeling in CRSwNP.

Methyl P-Coumarate Ameliorates the Inflammatory Response in Activated-Airway Epithelial Cells and Mice with Allergic Asthma

Methyl p-coumarate (methyl p-hydroxycinnamate) (MH) is a natural compound found in a variety of plants. In the present study, we evaluated the ameliorative effects of MH on airway inflammation in an experimental model of allergic asthma (AA). In this in vitro study, MH was found to exert anti-inflammatory activity on PMA-stimulated A549 airway epithelial cells by suppressing the secretion of IL-6, IL-8, MCP-1, and ICAM-1. In addition, MH exerted an inhibitory effect not only on NF-κB (p-NF-κB and p-IκB) and AP-1 (p-c-Fos and p-c-Jun) activation but also on A549 cell and EOL-1 cell (eosinophil cell lines) adhesion. In LPS-stimulated RAW264.7 macrophages, MH had an inhibitory effect on TNF-α, IL-1β, IL-6, and MCP-1. The results from in vivo study revealed that the increases in eosinophils/Th2 cytokines/MCP-1 in the bronchoalveolar lavage fluid (BALF) and IgE in the serum of OVA-induced mice with AA were effectively inhibited by MH administration. MH also exerted a reductive effect on the immune cell influx, mucus secretion, and iNOS/COX-2 expression in the lungs of mice with AA. The effects of MH were accompanied by the inactivation of NF-κB. Collectively, the findings of the present study indicated that MH attenuates airway inflammation in mice with AA, suggesting its potential as an adjuvant in asthma therapy.

A pyridinesulfonamide derivative FD268 suppresses cell proliferation and induces apoptosis via inhibiting PI3K pathway in acute myeloid leukemia.

Aberration of PI3K signaling pathway has been confirmed to be associated with several hematological malignancies including acute myeloid leukemia (AML). FD268, a pyridinesulfonamide derivative characterized by the conjugation of 7-azaindole group, is a newly identified PI3K inhibitor showing high potent enzyme activity at nanomole concentration. In this study, we demonstrated that FD268 dose-dependently inhibits survival of AML cells with the efficacy superior to that of PI-103 (pan-PI3K inhibitor) and CAL-101 (selective PI3Kδ inhibitor) in the tested HL-60, MOLM-16, Mv-4-11, EOL-1 and KG-1 cell lines. Further mechanistic studies focused on HL-60 revealed that FD268 significantly inhibits the PI3K/Akt/mTOR signaling pathway, promotes the activation of pro-apoptotic protein Bad and downregulates the expression of anti-apoptotic protein Mcl-1, thus suppressing the cell proliferation and inducing caspase-3-dependent apoptosis. The bioinformatics analysis of the transcriptome sequencing data also indicated a potential involvement of the PI3K/Akt/mTOR pathway. These studies indicated that FD268 possesses high potent activity toward AML cells via inhibition of PI3K/Akt/mTOR signaling pathway, which sheds some light on the pyridinesulfonamide scaffold for further optimization and investigation.

Synthesis and biological activity evaluation of novel 2,6,9-trisubstituted purine conjugates as potential protein kinases inhibitors

Deregulation of protein kinases is often associated with uncontrolled cell proliferation in various tumours and the inhibition of kinase activity remains an important target for anti-tumour drug development. Here, we report a novel series of 2-aminocyclohexylamino-6-(substituted benzylamino/anilino)-9-cyclopentylpurine derivatives conjugated with putrescine, spermidine or spermine moiety in an effort to expand library of highly potent 2,6,9-trisubstituted purine kinase inhibitors. Presented aniline-type conjugates exhibit significant cytotoxic activity in MV4-11 and EOL-1 cell lines which correlates with FLT3-ITD and PDGFRα inhibition. Furthermore, 6-anilinopurines affected MAPK and STAT pathways in the treated MV4-11 cells and induced cell cycle arrest in the G1 phase. 6-Benzylaminopurines showed comparable CDK2 inhibitory activity to 6-anilinopurines, however, the PDGFRα and FLT3-ITD inhibition was strongly suppressed. Our results show that novel compounds containing aniline in the structure can be involved in the development of potent tyrosine kinase inhibitors with strong activity toward acute myeloid leukemia or chronic eosinophilic leukemia.

Docosahexaenoic Acid Inhibits Proliferation of EoL-1 Leukemia Cells and Induces Cell Cycle Arrest and Cell Differentiation.

Τhe effect of docosahexaenoic acid (DHA, an omega-3 polyunsaturated fatty acid) upon the proliferation of EoL-1 (Eosinophilic leukemia) cell line was assessed, while additional cellular events during the antiproliferative action were recorded. DHA inhibited EoL-1 cells growth dose-dependently by inducing growth arrest at G0/1 phase of the cell cycle. After DHA addition to the cells, the expression of MYC oncogene was decreased, PTAFR-mRNA overexpression was observed which was used as a marker of differentiation, and PLA2G4A-mRNA increase was recorded. The enzymatic activities of phospholipase A2 (PLA2), a group of hydrolytic enzymes, whose action precedes and leads to PAF biosynthesis through the remodeling pathway, as well as platelet activating factor acetylhydrolase (PAFAH) which hydrolyses and deactivates PAF, were also measured. DHA had an effect on the levels of both the intracellular and secreted activities of PLA2 and PAFAH. The inflammatory cytokines IL-6 and TNF-α were also detected in high levels. In conclusion, DHA-induced EoL-1 cells differentiation was correlated with downregulation of MYC oncogene, overexpression of PTAFR and PLA2G4A-mRNAs, increase of the inflammatory cytokines production, and alteration of the enzymatic activities that regulate PAF levels. DHA is a natural substance and the understanding of its action on EoL-1 cells on molecular level could be useful in further investigation as a future therapeutic tool against F/P + hypereosinophilic syndrome.

FLT3, a prognostic biomarker for acute myeloid leukemia (AML): Quantitative monitoring with a simple anti‐FLT3 interaction and flow cytometric method

BackgroundOverexpression of fms‐like tyrosine kinase 3 (FLT3) protein in leukemia is highly related to poor prognosis and reduced survival rate in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients. Simple but efficient quantification of FLT3 protein levels on the leukemic cell surface using flow cytometry had been developed for rapid determination of FLT3 on intact cell surface.MethodsQuantitation protocol for FLT3 biomarker in clinical samples was developed and validated. Cell model selection for calibration curve construction was identified and evaluated. Selected antibody concentrations, cell density, and incubation time were evaluated for most appropriate conditions. Comparison of the developed FLT3 determination protocol with the conventional Western blot analysis was performed.ResultsEoL‐1 cell line was selected for using as positive control cells. Calibration curve (20%‐120% of FLT3 positive cells) and quality control (QC) levels were constructed and evaluated. The results demonstrated good linearity (r 2 > 0.99). The intra‐ and inter‐day precision and accuracy, expressed as the coefficient of variation (%CV) and % recovery, were <20% and fell in 80%‐120% in all cases. When compared with Western blotting results, FLT3 protein expression levels in leukemia patient's bone marrow samples were demonstrated in the same trend.ConclusionsThe effective, reliable, rapid, and economical analytical technique using the developed flow cytometric method was demonstrated for FLT3 protein determination on leukemic cell surface. This method provided a practical analysis of FLT‐3 biomarker levels which is valuable for physician decision in acute leukemia treatment.

Catecholamine Exposure Accelerates In Vivo Leukemogenesis in Acute Myeloid Leukemia Patient Derived Xenografts

Mouse xenografts are routinely used for in vivo studies on human acute myeloid leukemia (AML) cells. However, a significant proportion of primary AML samples (~50-60% of cases) are not able to repopulate mice, or respectively require a particularly long time (up to 9 months) to show detectable engraftment in such models. Here we report that changing the transplantation time-point from day (5:00 pm) to night (4:00 am) strongly improved the engraftment of human AML cells in NOD/SCID/IL2Rgnull (NSG) mice. Sublethally irradiated gender-matched NSG mice of 6-8 weeks of age (n=40) were transplanted with primary AML cells (n=5 patients). For each sample, equal numbers of freshly thawed AML cells were injected via the tail vein at both time-points; transplanted mice were afterwards monitored for human leukemic engraftment in peripheral blood (PB) and bone marrow (BM) using multicolor flow cytometry and immunohistochemistry. In all n=5 analyzed AML patient samples, transplantation at night strongly promoted leukemogenesis, with 2/5 samples showing engraftment only in the night transplant condition and 3/5 samples showing engraftment in both but significantly higher leukemic burden in night vs. day transplanted mice (Fig. 1a), although similar leukemia-specific mutations were retrieved in mice of the two conditions by next generation sequencing analysis (Fig. 1b). Limiting dilution experiments (n=2 AML samples) further confirmed these findings and showed that in fact lower numbers of AML cells were required to initiate leukemia when cells were transplanted at night versus day times.Given that the immediate process that happens after intravenous injection of AML cells into mice involves homing to BM niches, we hypothesized that the time-point of transplantation influences engraftment by modulating homing. Therefore, we performed homing assays as described for healthy hematopoietic stem/progenitor cells (HSPCs); we injected equally treated human CFSE labeled AML cells via tail vein injection at day versus night time points and analyzed murine BMs 8 hours later for the presence of human leukemic cells. Indeed, significantly higher BM colonization by leukemic cells was observed after night vs. day time injections (0.31±0.09 vs. 0.13±0.18, p<0.001, n= 7 AML patient samples).Since mice transplanted at night showed stress-related behavioral abnormalities, we hypothesized that this transplantation method might induce catecholamine release. ELISAs indeed revealed enhanced epinephrine levels in PB (318.00±12.96 vs. 122.20±47.11 ng/mL, p<0.001) as well as BM (21.08±1.04 vs. 13.50±3.57 ng/mL, p<0.05) of night vs. day transplanted animals. The role of catecholamines influencing leukemia initiation is underinvestigated; b-adrenergic receptors are expressed on several cell types in the BM including HSCs, osteoblasts and mesenchymal stem cells and catecholamine release was shown to regulate HSC egress from BM niches. We hypothesized that enhanced adrenergic signaling may promote leukemogenesis by enhancing leukemic cell homing to BM niches, which in this setting are more readily available due to enhanced healthy HSC egress. To explore this hypothesis, we treated mice with the b1/2-blocker propranolol prior to night transplantation (10mg/kg, intraperitoneal (i.p) injection, 1h pre-transplant, to reverse the “night effect”) and respectively with epinephrine prior to day transplantation (2mg/kg, i.p injection, 1h pre-transplant, to mimic the “night effect”). Indeed, propranolol treatment reduced homing and delayed leukemogenesis in night transplanted mice. Vice-versa, epinephrine enhanced AML cell homing and accelerated long-term leukemia induction from patient derived AML cells and cells of the EOL-1 cell line, and these effects could be counteracted by co-treatment with propranolol. Interestingly, in vitro co-cultures with epinephrine versus empty carrier control enhanced AML cell but not healthy HSPC adhesion to MS5 stroma cells (shown for n=3 cell lines and n=8 primary AML), suggesting that enhanced catecholamine levels may specifically induce AML cell adhesion.We conclude that enhanced catecholamine levels can accelerate leukemia development from AML cells transplanted in xenograft models by facilitating BM niche colonization by AML cells. Catecholamines might in general promote leukemia progression by favoring the retention of leukemic versus healthy HSCs in BM niches. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Wogonin attenuates nasal polyp formation by inducing eosinophil apoptosis through HIF-1\u03b1 and survivin suppression

Chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) is an inflammatory sinonasal disorder characterized by eosinophilic inflammation and T-helper 2 skewing. Eosinophil accumulation in sinonasal mucosa comprises a major feature of CRSwNP. The study aimed to investigate the effect of the flavone wogonin in nasal polyposis by assessing its ability to induce eosinophil apoptosis in vitro and attenuate eosinophilic CRSwNP in mice. Double immunofluorescence, immunohistochemistry, flow cytometry, and immunoblotting were performed to evaluate hypoxia-inducible factor (HIF)-1α, survivin, and apoptotic markers in the human eosinophilic EoL-1 cell line or sinonasal tissues from patients with CRS with or without NPs. In sinonasal specimens from patients with CRS, HIF-1α and survivin were up-regulated in eosinophils from patients with NPs compared with levels in patients without NPs. Under hypoxia, HIF-1α and survivin expression was up-regulated in EoL-1 cells. Wogonin down-regulated both HIF-1α and survivin in EoL-1 cells. In addition, overexpression of survivin protected EoL-1 cells against apoptosis in response to wogonin. Moreover, wogonin attenuated nasal polyp formation in a murine model. Our findings suggest that wogonin could induce caspase-3 activation by suppressing HIF-1α and survivin expression in EoL-1 cells. Further studies regarding novel therapeutic options for CRSwNP targeting eosinophil apoptosis are needed.

(S)-(+)-Decursin and its Analogues as Inhibitors of Cytokines Secretion Induced by DpE in THP-1/EoL-1 Cell Line

(S)-(+)-Decursin and its analogues were synthesized and evaluated for their inhibitory effects on monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), and IL-6 secretion induced by Dermatophagoides pteronyssinus extract (DpE) in human acute monocytic leukemia (THP-1) cells and eosinophilic leukemia (EoL-1) cell lines. (S)-(+)- Decursin analogues with alkyl acryloyl groups, apart from (S)-(+)-decursin (1) and (S)-(+)-decursinol angelate (4a), did not influence the regulation of cytokine secretion. (S)-(+)-Decursin analogues (8d, 8f, 8g) with cinnamoyl groups carrying hydroxyl- and acetoxy-substitutes potently inhibited the secretion of MCP-1 and IL-6. The structure-activity relationship study revealed that increasing π-rich environment at the extensions from the eastern alcohol of (S)-(+)-decursin is a key element in building the pharmacophore for achieving the desired biological activity.

The Role of S100A8 and S100A9 in Differentiation of Human Eosinophilic Leukemia Cells, EoL-1

S100A8 and S100A9 are associated with myeloid cell differentiation, chemotactic activities, adhesion of neutrophils, and apoptosis. In this study, we investigated the contribution of S100A8 and S100A9 to differentiation of the human eosinophilic leukemia cell line, EoL-1. S100A8 and S100A9 increased the number of vacuole per one cell and the protein expression of EPO and MBP. Rottlerin, an inhibitor of protein kinase C delta (PKCδ), inhibited the EoL-1 cell differentiation induced by S100A8 and S100A9. These results suggest that S100A8 and S100A9 may regulate the differentiation of eosinophilic progenitors. Moreover, these findings may shed light on elucidation of eosinophil differentiation due to S100 proteins.

Inhibitory effect of Panax ginseng and Pleurotus osteratus complex on expression of cytokine genes induced by extract of Dermatophagoides pteronissinus in human monocytic THP-1 and EoL-1 cells

A recent study reported that Pleurotus ostreatus has the potential to be used as a β-glucan- based cream for supportive complementary therapy of atopic dermatitis. KH054 is a new herbal prescription consisting of P. ostreatus and Panax ginseng. The effects of atopic dermatitis-induced materials on the expression of cytokine genes in human monocytes (THP-1, EoL- 1) have been examined. Some reports demonstrated that P. ginseng augments the activity of natural killer cells, which plays an important role in innate immunity against infection and tumor development. Monocyte chemotactic protein 1 (MCP-1), interleukin (IL)-6, and IL-8 have important roles in mediating the infiltration of various cells into the skin of atopic dermatitis and psoriasis. The present study investigated whether KH054 on induced IL-6, IL-8, and MCP-1 secretion by house dust mite (Dermatophagoides pteronissinus) in THP-1 (human acute monocytic leukemia) and EoL-1 (Human eosinophilic leukemia) cell. D. pteronissinus functions in the pathogenesis of allergic diseases, including atopic dermatitis and asthma. The inhibitory effect of KH054 on the induction of IL-6, IL-8, and MCP-1 secretion by D. pteronissinus extract in THP-1 and EoL-1 cells was examined. KH054 potently suppressed the elevated production of IL-6 and IL-8 induced by D. pteronissinus treatment in THP-1 and EoL-1 cells. Based on the present results, KH054 may be useful for developing functional foods to treat atopic dermatitis.

Chromatin Conformation Signatures Associated with Epigenetic Deregulation of the FIP1L1 and PDGFRA Genes

The human genome is organized into topological domains with discrete structural and regulatory units that influence gene expression epigenetically (Crutchley et al., 2010). In glioma patients with IDH1 (R132H) mutations, researchers detected aberrant methylation of transcriptional CTCF-enhancer regions between the FIPL1L1 and PDGFRA genes. Conformational changes to the chromosomal arrangement of these regions have been shown to upregulate PDGFRA expression (Flavahan et al; 2016). Flavahan et al also demonstrated the down regulation of cell growth and proliferation in the glioma cells with the addition of tyrosine kinase inhibitors that target the interaction.We explored this epigenetic interaction and its utility in the stratification of patients for treatment with tyrosine kinase inhibitors. Using a high-resolution chromosome-conformation capture or 3C analysis platform known as EpiSwitchTMand quantitative PCR, we mapped, evaluated, and quantified the conformational juxtaposition between FIP1L1 and PDGFRA in glioma-cell lines with and without IDH mutations. Deregulation of PDGFRA by interstitial deletion at 4q12 and fusion to FIP1L1 associated with chronic esosinophic leukemias prompted our group to also investigate whether the same chromosome-conformation interactions are present in EOL-1 and other leukemic cell lines.EpiSwitchTM templates were produced using a modified process to purify the 3C DNA. Unlike previous protocols, only one set of primers is required. Chromosome conformation capture analysis with a probe, single step PCR and gel purification was used to identify and sequence 3C interactions in the AML cell lines EOL-1 and HL-60. qPCR templates, adjusted to 20 ng of 3C library DNA, were used with concentration-matched negative controls (ie, 3C libraries derived from adipose biopies and normal blood). A 3C interaction with MMP-1 was used as a internal control for the EpiSwitchTM library. A dual label hydrolysis probe was used to detect the sequenced interaction. Five novel interactions were identified between FIP1L1 and PDGFRA by single step PCR and confirmed by sequencing. We quantified one interaction with a dual-labeled hydrolysis probe based qPCR assay to determine copy number in eight cell lines: BT-412 (anaplastic oligoastrocytoma), DBTRG-05MG (glioblastoma multiforme), U-3T3 (glioblastoma astrocytoma), U-87 (glioblastoma astrocytoma), GDM-1 (acute myelomonocytic leukemia), EOL-1 (acute myeloid leukemia, AML), HL-60 (AML) and KG-1 (AML).Using EpiSwitchTM and ISO standard MIQE-compliant qPCR design, five specific epigenetic interactions were detected in three types of cancerous cell lines including EOL-1 and HL-60. qPCR was used to detect one specific difference between normal blood (n = 4) and leukemic cell lines (n = 4). By qPCR the interaction was detected in two AML cell lines known to be highly sensitive to the tyrosine kinase inhibitor Glivec (EOL-1 and GDM-1), as well as HL-60 cells, but not in KG-1. The FIP1L1 PDGFRA 3C interaction was also found in DBTRG-05MG and U-373 at a similar copy number compared to the AML cell lines. A glioma patient biopsy tissue also tested positive for the conformational-chromosome interaction by qPCR compared with an adipose biopsy control. An interaction in the MMP-1 gene was used as an internal positive control in all samples tested.Emerging epigenetic approaches, such as chromosome conformation signatures as detected by EpiSwitchTM, are providing sensitive and accurate methods for improving patient treatment stratification. Detection of interactions between PDGFRA and FIP1L1 based on chromosomal conformational changes have the potential to improve upon the available methods for identifying patients who may benefit from treatment with PDGFRA-targeted tyrosine kinase inhibitors. Further research is needed to validate the method described using blood and biopsy samples in patients with glioma, AML and other myeloproliferative neoplasms associated with the FIP1L1-PDGFRA fusion gene. DisclosuresGrand:Oxford BioDynamics Limited: Employment, Equity Ownership. Bird:Oxford BioDynamics Limited: Employment. Corfield:Oxford BioDynamics Limited: Employment. Dezfouli:Oxford BioDynamics Limited: Employment. Warren:Oxford BioDynamics Limited: Employment. Foulkes:Oxford BioDynamics Limited: Employment. Khudari:Oxford BioDynamics Limited: Employment. Salter:Oxford BioDynamics Limited: Employment. Mahecha:Oxford BioDynamics Limited: Employment. Ssentongo:Oxford BioDynamics Limited: Employment. Green:Oxford BioDynamics Limited: Employment, Equity Ownership. Womersley:Oxford BioDynamics Limited: Employment, Patents & Royalties. Hunter:Oxford BioDynamics Limited: Employment. Ramadass:Oxford BioDynamics Limited: Employment. Akoulitchev:Oxford BioDynamics Limited: Employment, Equity Ownership.

CDX2 regulates Human Leukemic Cell Clonogenicity and in Vivo Repopulation Capacity

Objectives: The caudal-type homeobox (CDX) gene family has been mainly studied during early development for its role in axial elongation and antero-posterior patterning. More recently, CDX genes were shown to regulate embryonic hematopoiesis via downstream HOX genes and interactions with the WNT signaling pathway. The role of CDX genes in adult hematopoiesis is poorly understood and almost no data exists on human cells. Healthy bone marrow (BM) derived hematopoietic cells express low levels of CDX1 and CDX4 but lack CDX2 expression. However, CDX2 expression is found in >80% of human acute myeloid (AML) and lymphoid leukemia (ALL) and its induction in murine BM cells results in myeloid leukemia. Here, we explore the role of CDX2 in human healthy hematopoietic and leukemic cells.Methods: CDX2 expression was modulated via lentiviral treatment in human BM CD34+ and SKM-1, EOL-1 and NALM16 human leukemic cell lines. CDX2 knockdown experiments were performed using two different shRNAs against CDX2 to control for potential off-target effects. To generate cultures displaying a very strong knockdown, individual clones were generated from single transduced cells and analyzed also separately. Efficient modulation of gene and protein CDX2 expression was analyzed by qRT-PCR and respectively immunoblot analyses. CDX2-modified (overexpressing or knockdown) or control cells were subjected to growth, colony forming (CFU), cell cycle, flow cytometry and qRT-PCR assays and analyzed in vivo upon xenotransplantation in NOD/SCID/IL2Rγnull (NSG) mice. Human recombinant DKK-1 protein was supplemented in CFU assays to CDX2 knockdown and respectively control cells.Results: shRNA-mediated CDX2 knockdown performed on SKM-1 as well as EOL-1 cells strongly reduced clonogenic capacity in CFU assays while only slightly reducing growth. Consistently, proliferation, apoptosis sensitivity and cell cycle were not influenced by CDX2 downregulation in any of the three analyzed leukemic cell lines. Importantly, CDX2 knockdown SKM-1 as well as NALM16 cells transplanted into immunopermissive NSG mice showed profoundly suppressed in vivo leukemogenic properties compared to control cells. However, overexpression of the human CDX2 gene using an SFFV-promotor driven lentiviral vector in human healthy CD34+ bone marrow-derived and also in leukemic cells resulted in a G0/G1 cell cycle arrest, reducing in vitro growth and CFU formation. Consistent with these results and in contrast to previous results reported in mice, CDX2 overexpression did not confer in vitro serial replating capacity or in vivo leukemogenic properties to healthy human CD34+ cells.To further investigate the molecular mechanisms underlying these CDX2-mediated effects in human leukemia, we analyzed the expression of HOX and Wnt-pathway associated genes via qRT-PCR. Modulation of HOX gene expression was indeed observed upon CDX2 knockdown or overexpression. Previously reported repressive effects of CDX2 on Klf4 gene expression were confirmed. Surprisingly however, we found that CDX2 expression positively regulates the expression of the WNT-inhibitory molecule DKK-1 in both leukemic and healthy CD34+ stem/progenitor cells. Supplementation of DKK-1 was able to rescue the clonogenic capacity of CDX2 knockdown leukemic cells in CFU-assays while opposite effects were noted in control cells.Conclusion: Our data suggest that CDX2 regulates in vivo leukemogenesis by inducing clonogenic properties in human leukemic cells. Its downstream molecular pathways in human leukemic cells may include, next to Klf4, HOX gene family members and the Wnt-inhibitor DKK-1. Leukemic cells might use DKK-1 expression to fine-tune their Wnt-signaling activity to an optimal dosage required for leukemia initiation and growth. Work underway in our laboratory is further investigating this hypothesis. DisclosuresNo relevant conflicts of interest to declare.

IFN-γ and TNF-α potentiate prostaglandin D2-induced human eosinophil chemotaxis through up-regulation of CRTH2 surface receptor

Prostaglandin D2 (PGD2) receptor CRTH2, is a pro-inflammatory molecule involved in eosinophil recruitment to the allergic airway. We investigated the expression of CRTH2 in eosinophil from allergic rhinitis patients (AR) and tested the modulatory role of several TH1 and TH2 cytokines closely related to the allergic immunological response, on the expression of CRTH2 receptor, utilizing human eosinophil cell line (Eol-1).The expression of CRTH2 was tested by immunohistochemistry and flow cytometry (FACS). Chemotaxis was performed in micro-chemotaxis chambers. It is shown that the expression of CRTH2 by eosinophils was significantly higher in the nasal tissue and peripheral blood of AR patients, when compared to control subjects. PGD2 exhibited a typical bell shape dose response in attracting eosinophil from AR patients with optimal activity at 10−7M. Eol-1 cell surface expression of CRTH2 was significantly up-regulated by 10ng/ml IFN-γ and TNF-α. The percentage of Eol-1 cells expressing the receptor increased by IFN-γ and TNF-α from 12.74%±2.66 to 55%±8 and 33.8%±9.4, respectively. PGD2-induced Eol-1 chemotaxis was not blocked by SB203580, H-89 Dihydrochloride, Bisindo-lylmaleimide, or Genistein. PGD2-induced Eol-1 chemotaxis was potentiated by IFN-γ and TNF-α without changing the signal transduction pathway. Correlation of our results to peripheral blood eosinophils from allergic rhinitis patients confirmed that 3hour pretreatment of eosinophils by 10ng/ml IFN-γ and TNF-α, increased the mean fluorescence intensity (MFI) of CRTH2 from 8.23 to 9.68 and 9.38, respectively, and potentiated PGD2-induced eosinophil chemotaxis. Our results demonstrate a novel synergism between PGD2, IFN-γ and TNF-α, in eosinophil chemotaxis.

Effect of Benzo[a]pyrene on the Production of Vascular Endothelial Growth Factor by Human Eosinophilic Leukemia EoL-1 Cells

Benzo[a]pyrene (BaP) has been shown to affect both the development and response of T and B cells in the immune system. However, the effect of BaP on other immune cells, such as eosionophils, is unknown. In this study, we investigated the effect of BaP on the production of vascular endothelial growth factor (VEGF) using an in vitro eosinophilic EoL-1 cell and human umbilical vein endothelial cell (HUVEC) co-culture system. EoL-1-conditioned medium was found to promote the growth of HUVEC in a time-dependent manner. The growth stimulating activity was due to the production of VEGF by the EoL-1 cells. The production of VEGF was correlated with the enhanced expression of the phosphorylated form of extracellular signal-regulated kinases (p-ERKs) and the upregulated expression of VEGF mRNA. Furthermore, BaP-induced expression of VEGF mRNA was reduced by the ERK inhibitor PD98059. Results from this study suggested that BaP might affect the growth of endothelial cells through the modulation of VEGF production by eosinophils.

The roles of MCP-1 and protein kinase Cδ activation in human eosinophilic leukemia EoL-1 cells

Idiopathic hypereosinophilc syndrome is a disorder associated with clonally eosinophilic proliferation. The importance of FIP1-like-1-platelet-derived growth factor receptor-α (FIP1L1-PDGFRA) in the pathogenesis and classification of HES has been recently reported. In this study, we investigated the contribution of monocyte chemoattractant protein-1 (MCP-1)/CCL2 to chemotactic activity and protein kinase C delta (PKCδ in the human eosinophilic leukemia cell line EoL-1. These cells express CCR2 protein among the CC chemokine receptors (CCR1-5). MCP-1 induces strong migration of EoL-1 cells and the chemotaxis signal in response to MCP-1 involves a G i/G o protein, phospholipase C (PLC), PKCδ, p38 MAPK and NF-κB. MCP-1 activates p38 MAPK via G i/G o protein, PLC and PKCδ cascade. MCP-1 also induces NF-κB translocation and the activation is inhibited by PKCδ activation. The increase in the basal expression and activity of PKCδ in EoL-1 cells, compared to normal eosinophils, inhibits apoptosis in EoL-1 cells. Anti-apoptotic mechanism of PKCδ is related to inhibition of caspase 3 and caspase 9, but not to FIP1L1-PDGFRA. PKCδ functions as an anti-apoptotic molecule, and is involved in EoL-1 cell movement stimulated by MCP-1. This study contributes to an understanding of MCP-1 in eosinophil biology and pathogenic mechanism of eosinophilic disorders.

Down Regulation of GATA-2 and the Inversely-Correlated Expression of Adipogenic Specific Gene-PPARγ in Mesenchymal Stem Cells from Patients with Aplastic Anemia.

(Introduction) Aplastic anemia (AA) is characterized by a reduced number of hematopoietic stem cells resulting in peripheral pancytopenia and fatty marrow replacement. Although immune-mediated suppression of hematopoiesis is considered to play an important role in most patients with AA, the mechanism of fatty marrow replacement remains unclear. Transcription factor GATA-2 is known to play important roles in hematopoiesis. Gene Chip analysis has implicated the decreased expression of GATA-2 in AA CD34 cells, a finding confirmed by quantitative real-time PCR analysis. In a mouse stromal preadipocyte cell line, the suppression of GATA-2 expression accelerated the differentiation of stromal preadipocytes. In order to explore the impact of GATA-2 expression on adipogenesis in AA, the expression of GATA-2, the adipogenic specific gene adipsin, CCAAT/enhancer binding protein alpha (C/EBPα), and peroxisome proliferator-activated receptor γ (PPARγ) was examined in mesenchymal stem cells (MSCs) derived from bone marrow from AA patients and normal individuals.(Patients and methods) Thirty-four AA patients and 15 healthy individuals were included in this study. The median patient age was 10 years, ranging from 2 to 21 years. Twelve patients were studied before immunosuppressive therapy (IST) or bone marrow transplantation. Twenty-two patients were studied after treatment, of whom 11 responded to IST and 11 did not respond. Mononuclear cells were separated from bone marrow aspirates and 1x107 cells used for MSCs culture. MesenCult (StemCell Technologies, USA) was used to culture and expand the MSCs in vitro, after which the cells retain their ability to differentiate along various lineages upon the addition of the appropriate stimulatory supplements. Total RNA was extracted from the third passage of MSCs and synthesis of cDNA was performed using the Thermoscript RT-PCR system. Quantitative real-time PCR was conducted using Taqman primers and probes. The K562 cell line for GATA-2, the EOL1 cell line for adipsin and the MOLM-13 cell line for C/EBPα and PPARγ were used as controls.(Results) GATA-2 expression by MSCs from patients with AA at diagnosis was significantly decreased compared with normal individuals (p=0.031). However, one of the adipogenic specific genes, PPARγ, was significantly increased in MSCs in AA at diagnosis (p=0.017). The expression of adipsin and C/EBPα in MSCs did not differ significantly between AA patients and healthy volunteers. Notably, in the 22 AA patients who received IST, the expression of GATA-2 and PPARγ did not significantly decrease if the patient had responded and hematopoiesis had improved (n=11). However, after the IST the 11 non-responders still had significantly lower GATA-2 and higher PPARγ expression compared to normal individuals.(Conclusion) The mechanism of fatty replacement in the bone marrow in AA may be explained by the down regulation of GATA-2 and the inversely-correlated expression of PPARγ in MSCs. The decreased expression of GATA-2 may be responsible for the pathogenesis and development of the clinical features of this disease.

All- trans-retinoic acid enhances apoptosis induction by tyrosine kinase inhibitors in the eosinophilic leukemia-derived EoL-1 cell line

Imatinib and retinoids induce apoptosis in FIP1L1/PDGFRα-positive EoL-1 leukemia cells. Although imatinib induces complete remission in most FIP1L1/PDGFRα-positive patients, response to imatinib is sometimes suboptimal. In order to enhance the potency of the molecularly targeted therapy of eosinophilic leukemia, we investigated the effect of retinoids combined with tyrosine kinase inhibitors on EoL-1 cells. We demonstrate that retinoids combined with tyrosine kinase inhibitors lead to enhanced apoptosis induction in EoL-1 cells. Our results suggest that tyrosine kinase inhibitors combined with retinoids may constitute a valuable therapeutic approach for sensitive neoplasias that may display enhanced anti-leukemic potency when compared to single drug treatments.