OCI/AML2 ATRA AND VPA GENE EXPRESSION
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE2668 | GEO | 2005/05/17
SECONDARY ACCESSION(S): PRJNA92103
REPOSITORIES: GEO
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Project description:Purpose: Treatment of acute promyelocytic leukemia (APL) with the retinoid, all trans retinoic acid (ATRA), along with standard chemotherapy has significantly improved survival compared to chemotherapy alone1. ATRA mediates its benefit in APL by overcoming the transcriptional block mediated by PML-RARï¡ fusion oncoprotein, thereby, restoring expression of retinoid response genes2. The therapeutic benefits observed with ATRA treatment in APL have not been achieved in the other more common sub-types of acute myeloblastic leukemia (AML)3. This likely reflects the recruitment of histone deacetylase complexes by other recurrent chromosomal translocations in AML cells4. In this experiment, we evaluated if treatment of the AML cell line, OCI/AML-2, with the histone deacetylase inhibitor, valproic acid (VPA), would alter the expression of sub-sets of genes by itself or in conjunction with ATRA that have been shown to be modulated by ATRA in APL2. Experiment Overall Design: Methods: OCI/AML-2 cells were cultured at 37°C and 5% CO2 in ï¡MEM growth media supplemented with antibiotics and 10% fetal calf serum (HyClone, UT, USA). The drugs, ATRA and VPA, were obtained from Sigma-Aldrich Canada Ltd. (ON, CAN) and were re-suspended in 100% ethanol. OCI/AML-2 cells were treated with ATRA as a single 10-6 M dose and VPA was dosed at 0.6mM every 8 hours over 24 hours. The following four treatment combinations were evaluated: i) controls (solvent only) ii) ATRA iii) VPA iv) ATRA + VPA. Experiment Overall Design: Suspension cells were pelleted at 300 g 24 h after initial drug exposure. Total RNA was isolated using an RNeasy kit (Qiagen Inc., Canada) and stored at â70°C in DEPC-H2O. Total RNA was then further concentrated to 2.5µg/ïL by ethanol precipitation overnight at â20°C in 0.3M sodium acetate pH 5.2 (Sigma-Aldrich Canada Ltd.). Experiment Overall Design: GeneChip experiments were carried out by the Microarray Facility, The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, ON. A total of 20µg of total RNA was used to generate biotin labeled cRNA probes according to Affymetrixâs recommended protocol using a labeling kit from EnzoBiochem, Inc. (NY, USA). A total of 15µg of the biotin labeled cRNA was hybridized onto the human U133A Affymetrix GeneChip. The GeneChip was washed as per the Affymetrix EukGE-ws2v4 protocol available at (https://www.affymetrix.com/site/login/login.affx). The GeneChip was scanned using a GeneArray 2500 scanner (Agilent, CA, USA) and hybridization intensity was obtained and detection p value and signal were calculated using MAS 5.0 software. Target signal was set to 150 for scaling for all of the experiments. The output chp file was exported to text file and used for further analysis with the Multiple experiment viewer (MeV) version 2.2 available from the Institute for Genomic Research at http://www.tigr.org/. Signal intensities obtained from the OCI/AML-2 cells treated with ATRA, VPA, or ATRA + VPA were compared to untreated OCI/AML-2 controls. Only genes that were identified as present (P) and displayed more than two fold change in signal intensity compared to untreated cells (controls) were carried forward for further analysis. Gene expression experiments were normalized using total intensity, median centered and log(2) transformed to give equal weight to expression values relative to the median for analysis. Unsupervised hierarchical clustering was performed using average linkage and Pearson correlation as a measure of distance. Experiment Overall Design: Results: Relatively few genes were regulated by ATRA treatment in OCI/AML-2 cells. However, treatment of OCI/AML-2 cells with VPA altered the expression of genes that encode for proteins involved with regulation of cell cycle, interferon response, apoptosis and differentiation. Examples of genes depressed by VPA, but not ATRA, involved regulators of cell cycle, cyclin A2 and MYC. The combination of ATRA + VPA further suppressed expression of these genes. Genes such as IRF1, neutrophilic cytosolic factor, ICAM3 and C/EBPï¢ increased in response to ATRA in OCI/AML-2 cells whereas VPA did not alter their expression over untreated OCI/AML-2 cells. However the combination of ATRA + VPA further enhanced the expression of these genes. Examples of genes induced by VPA that were not affected by ATRA in OCI/AML-2 cells include cell surface markers CD9, and MHC class II, promoters/enhancers of apoptosis such as MAPK kinase, interferon responsive genes, and caspase 7, and negative regulators of cell cycle like p21 and p19. The addition of ATRA to VPA increased the expression of some, but not all of these genes. It would appear that VPA dramatically affects the gene expression pattern of OCI/AML-2 cells, and modulates the expression of a set of genes, similar to those modulated by ATRA in APL cells2. Experiment Overall Design: References Experiment Overall Design: 1. Tallman MS, Andersen JW, Schiffer CA, Appelbaum FR, Feusner JH, Ogden A, Shepherd L, Experiment Overall Design: Willman C, Bloomfield CD, Rowe JM, Wiernik PH. All-trans-retinoic acid in acute Experiment Overall Design: promyelocytic leukemia. N Engl J Med 1997; 337(15):1021-1028. Experiment Overall Design: 2. Tamayo P, Slonim D, Mesirov J, Zhu Q, Kitareewan S, Dmitrovsky E, Lander ES, Experiment Overall Design: Golub TR. Interpreting patterns of gene expression with self-organizing maps: methods Experiment Overall Design: and application to hematopoietic differentiation. Proc Natl Acad Sci U S A 1999; Experiment Overall Design: 96(6):2907-2912. Experiment Overall Design: 3. Estey EH, Thall PF, Pierce S, Cortes J, Beran M, Kantarjian H, Keating MJ, Andreeff Experiment Overall Design: M, Freireich E. Randomized phase II study of fludarabine + cytosine arabinoside + Experiment Overall Design: idarubicin +/- all-trans retinoic acid +/- granulocyte colony- stimulating factor in poor Experiment Overall Design: prognosis newly diagnosed acute myeloid leukemia and myelodysplastic syndrome. Experiment Overall Design: Blood 1999; 93(8):2478-2484 Experiment Overall Design: 4. Gelmetti V, Zhang J, Fanelli M, Minucci S, Pelicci PG, Lazar MA. Aberrant Experiment Overall Design: recruitment of the nuclear receptor corepressor-histone deacetylase complex by the acute Experiment Overall Design: myeloid leukemia fusion partner ETO. Mol Cell Biol 1998; 18(12):7185-7191.
2008-06-12 | E-GEOD-2668 | biostudies-arrayexpress
Project description:Searching for new strategies of acute myeloid leukemia (AML) treatment is of particular interest. Cell lines, e. g. HL-60 and NB4, represent model systems to study molecular features of leukemic cells. The all-trans-retinoic acid (ATRA) has proven itself to be an effective treatment for one of AML subtypes, i.e., acute promyelocytic leukemia (APL). At the same time, ATRA causes granulocytic differentiation of non-APL leukemic cells in vitro. Combination of new therapeutics with ATRA could improve efficiency of treatment. Studying the proteome perturbation in leukemic cells under the ATRA treatment allows to determine potential regulatory molecules that could be affected pharmacologically. Thus, the TMT-based proteomic profiles of HL-60, NB4, and K562 cell lines under the ATRA treatment were obtained at 0, 3, 12, 24, and 72 h after the ATRA treatment.
2024-06-23 | PXD050317 | Pride
Project description:All-trans-retinoic acid (ATRA) has been successfully used in therapy of acute promyelocytic leukemia (APL), a cytogenetically distinct subtype of acute myeloid leukemia (AML) but the response of non-APL AML cases to ATRA-based treatment has been poor. Here we show that, via epigenetic reprogramming, inhibitors of LSD1/KDM1 demethylase including tranylcypromine (TCP) unlocked the ATRA-driven therapeutic response in non-APL AML. LSD1 inhibition did not lead to an increase in genome-wide H3 lysine4 dimethylation (H3K4me2) but did increase H3K4me2 and expression of myeloid differentiation-associated genes. Importantly, treatment with ATRA plus TCP dramatically diminished engraftment of primary human AML cells in vivo in NOD.SCID mice, suggesting that ATRA in combination with TCP may target leukemia-initiating cells. Furthermore, initiation of ATRA plus TCP co-treatment 15 days post-engraftment of human AML cells in NOD.SCID gamma mice also revealed the ATRA plus TCP drug combination to have a potent anti-leukemic effect, which was superior to treatment with either drug alone. These data identify LSD1 as a therapeutic target and strongly suggest that it may contribute to AML pathogenesis by inhibiting the normal pro-differentiative function of ATRA, paving the way for novel combinatorial therapies of AML. Overall, 30 specimens derived from HL-60 or TEX cell line were treated with drugs and hybridized to Illumina HumanHT-12 gene expression arrays.
2012-03-14 | E-GEOD-34672 | biostudies-arrayexpress
Project description:Acute promyelocytic leukaemia (APL) can be cured by the co-administration of arsenic trioxide (ATO) and all-trans retinoic acid (ATRA). These small molecules relieve the differentiation blockade of the cancerous promyelocytes and trigger their maturation into functional neutrophils, which are physiologically primed for apoptosis. This normalization therapy uses low dose treatments and represents a compelling alternative to cytotoxic anticancer chemotherapy. However, the serendipitous discovery of this combination treatment prevented the establishment of methodologies to screen for novel combination treatments consisting of inducers of differentiation and other metallopharmaceuticals. Here, we present an experimental framework that enables interrogating the suitability, directionality and extent of normalization of novel combination treatments. Since the endpoint of this anticancer treatment is remodelling of cancer phenotypes, this approach requires a mechanism-driven understanding of the drug-induced cellular adaptions along the path to mature neutrophils. For this purpose, label-free quantification proteomics is used to probe the proteome changes upon differentiation and metal(-loid) treatment on the molecular level in a panel of ATRA-responsive and ATRA-non-responsive acute myeloid leukaemia (AML) cell lines, including APL. The induction of differentiation was confirmed by the up-regulation of canonical surface markers (e.g. CD11β) and validated by flow cytometry. The regulation of key transcription factors in this panel of AML cell lines reflects their position in the myeloid differentiation cascade (e.g. SPI1, GFI-1 and CEBPE). The additive/synergistic contributions of ATO to the combination treatment were thoroughly characterized and include changes in metabolic activities, as well as protein signatures related to management of reactive oxygen species and an integrated stress response. The latter two are also characteristic for the organoruthenium-based drug candidate plecstatin-1, which was recently shown to modulate the scaffold protein plectin. The combination of differentiation-induction and plecstatin-1 treatment featured a striking similarity in the responsiveness of the AML cancer cells compared to ATO+ATRA. It turned out to be at least equivalent in the regulation of key processes underlying normalization therapy in AML with granulocytic maturation. By exploiting response patterns of AML cancer cells induced by the ATO+ATRA combination treatment, this approach may allow identifying novel small molecule combinations, which may have the potential to achieve normalization and thus therapeutic benefit beyond APL.
2022-02-22 | PXD019619 | Pride
Project description:All-trans-retinoic acid (ATRA) has been successfully used in therapy of acute promyelocytic leukemia (APL), a cytogenetically distinct subtype of acute myeloid leukemia (AML) but the response of non-APL AML cases to ATRA-based treatment has been poor. Here we show that, via epigenetic reprogramming, inhibitors of LSD1/KDM1 demethylase including tranylcypromine (TCP) unlocked the ATRA-driven therapeutic response in non-APL AML. LSD1 inhibition did not lead to an increase in genome-wide H3 lysine4 dimethylation (H3K4me2) but did increase H3K4me2 and expression of myeloid differentiation-associated genes. Importantly, treatment with ATRA plus TCP dramatically diminished engraftment of primary human AML cells in vivo in NOD.SCID mice, suggesting that ATRA in combination with TCP may target leukemia-initiating cells. Furthermore, initiation of ATRA plus TCP co-treatment 15 days post-engraftment of human AML cells in NOD.SCID gamma mice also revealed the ATRA plus TCP drug combination to have a potent anti-leukemic effect, which was superior to treatment with either drug alone. These data identify LSD1 as a therapeutic target and strongly suggest that it may contribute to AML pathogenesis by inhibiting the normal pro-differentiative function of ATRA, paving the way for novel combinatorial therapies of AML. ChIP-seq was used to study the effects of ATRA, TCP and ATRA/TCP treatment on H3K4 dimethylation. In addition to the three treatment samples, two reference samples were processed: (i) An untreated sample using the same anti-H3K4me2 antibody and an untreated sample using IgG. These five sequencing experiments were conducted using HL-60 cells and TEX cells, leading to 10 ChIP-seq samples in total.
2012-03-14 | E-GEOD-34725 | biostudies-arrayexpress
Project description:All-trans-retinoic acid (ATRA) has been successfully used in therapy of acute promyelocytic leukemia (APL), a cytogenetically distinct subtype of acute myeloid leukemia (AML) but the response of non-APL AML cases to ATRA-based treatment has been poor. Here we show that, via epigenetic reprogramming, inhibitors of LSD1/KDM1 demethylase including tranylcypromine (TCP) unlocked the ATRA-driven therapeutic response in non-APL AML. LSD1 inhibition did not lead to an increase in genome-wide H3 lysine4 dimethylation (H3K4me2) but did increase H3K4me2 and expression of myeloid differentiation-associated genes. Importantly, treatment with ATRA plus TCP dramatically diminished engraftment of primary human AML cells in vivo in NOD.SCID mice, suggesting that ATRA in combination with TCP may target leukemia-initiating cells. Furthermore, initiation of ATRA plus TCP co-treatment 15 days post-engraftment of human AML cells in NOD.SCID gamma mice also revealed the ATRA plus TCP drug combination to have a potent anti-leukemic effect, which was superior to treatment with either drug alone. These data identify LSD1 as a therapeutic target and strongly suggest that it may contribute to AML pathogenesis by inhibiting the normal pro-differentiative function of ATRA, paving the way for novel combinatorial therapies of AML.
2012-03-14 | GSE34672 | GEO
Project description:All-trans-retinoic acid (ATRA) has been successfully used in therapy of acute promyelocytic leukemia (APL), a cytogenetically distinct subtype of acute myeloid leukemia (AML) but the response of non-APL AML cases to ATRA-based treatment has been poor. Here we show that, via epigenetic reprogramming, inhibitors of LSD1/KDM1 demethylase including tranylcypromine (TCP) unlocked the ATRA-driven therapeutic response in non-APL AML. LSD1 inhibition did not lead to an increase in genome-wide H3 lysine4 dimethylation (H3K4me2) but did increase H3K4me2 and expression of myeloid differentiation-associated genes. Importantly, treatment with ATRA plus TCP dramatically diminished engraftment of primary human AML cells in vivo in NOD.SCID mice, suggesting that ATRA in combination with TCP may target leukemia-initiating cells. Furthermore, initiation of ATRA plus TCP co-treatment 15 days post-engraftment of human AML cells in NOD.SCID gamma mice also revealed the ATRA plus TCP drug combination to have a potent anti-leukemic effect, which was superior to treatment with either drug alone. These data identify LSD1 as a therapeutic target and strongly suggest that it may contribute to AML pathogenesis by inhibiting the normal pro-differentiative function of ATRA, paving the way for novel combinatorial therapies of AML.
2012-03-14 | GSE34725 | GEO
Project description:All-trans retinoic acid (ATRA)-based differentiation therapy has achieved success with the treatment of acute promyelocytic leukemia (APL), a unique subtype of acute myeloid leukemia (AML). However, other subtypes of AML display resistance to ATRA-based treatment. Here, we demonstrate that a novel natural vibsane-type diterpenoid vibsanin A promotes the differentiation of myeloid leukemia cell lines and primary AML blasts. To reveal how vibsanin A function on promoting myeloid leukemia cell differentiation, we analyzed and compared the gene expression profiles in myeloid leukemia HL-60 cells treated with vibsanin A, PMA, and ATRA.
2016-02-28 | GSE78734 | GEO
Project description:All-trans retinoic acid (ATRA)-based differentiation therapy has achieved success with the treatment of acute promyelocytic leukemia (APL), a unique subtype of acute myeloid leukemia (AML). However, other subtypes of AML display resistance to ATRA-based treatment. Here, we demonstrate that a novel natural vibsane-type diterpenoid vibsanine A promotes the differentiation of myeloid leukemia cell lines and primary AML blasts. To reveal how vibsanine A function on promoting myeloid leukemia cell differentiation, we analyzed and compared the gene expression profiles in myeloid leukemia HL-60 cells treated with vibsanine A, PMA, and ATRA. HL-60 cells were treated with vibsanine A, PMA and ATRA for 6 hours or longer up to 24 hours. Gene expression profiling was conducted
2016-02-28 | E-GEOD-78734 | biostudies-arrayexpress
Project description:To identify the top 20 up-regulated genes in CD34+ cells from AML patients in comparison with healthy donors, we examined the microarray gene expression profile of CD34+ blasts from patients with newly diagnosed AML vs CD34+ normal cells from healthy donors. Despite the fact that combined therapy of all-trans retinoic acid (ATRA) with arsenic trioxide (ATO) or chemotherapy dramatically improves the prognosis of patients with APL, these regimens can cause systemic infections and secondary leukemias. Here we report that expression of the pseudokinase Tribble 3 (TRIB3) associates positively with APL progression and therapeutic resistance. The elevated TRIB3 expression promotes APL by interacting with PML-RARa and suppressing its sumoylation, ubiquitylation and degradation. This represses PML nuclear body assembly, p53-mediated senescence, cell differentiation, and supports cellular self-renewal. Genetically inhibiting Trib3 expression or disturbing the TRIB3/PML-RARa interaction produces potent therapeutic efficacy against APL and has synergic anti-APL effects when used in combination with ATRA or ATO by promoting PML-RARa degradation and PML expression. Our study provides new insight into APL pathogenesis and a new therapeutic option against APL.
2017-05-12 | GSE90062 | GEO