APX3330 inhibition of the redox function of ape-1/ref-1 (Ref-1) in promyelocytic leukemia cells enhances retinoic acid (ATRA) induced myeloid differentiation and limits cell proliferation as an approach to the prevention of the retinoic acid syndrome (RAS)

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e14613-e14613
Author(s):  
K. A. Robertson ◽  
E. S. Colvin ◽  
M. R. Kelley ◽  
M. L. Fishel
Keyword(s):  
Flow Cytometry ◽  
Retinoic Acid ◽  
Cell Growth ◽  
Real Time ◽  
Real Time Pcr ◽  
Mapk Pathway ◽  
Promyelocytic Leukemia ◽  
Leukemia Cells ◽  
Myeloid Differentiation ◽  
Granulocytic Differentiation

e14613 Background: ATRA + chemotherapy has improved the treatment of promyelocytic leukemia(APL). However, 25% of ATRA treated APL patients experience toxicities that comprise the RAS (life-threatening respiratory distress, edema, renal failure, hypotension, coagulopathy and rising blast count). One approach to prevent RAS is to limit blast proliferation and enhance myeloid differentiation. Ref-1 is a DNA repair protein that functions in redox regulation of cellular proteins, such as Fos, Jun, p53, and NFkB. HL60 myeloid leukemia cells are promyeloblasts that respond to ATRA with granulocytic differentiation/growth arrest. Prior studies suggest Ref-1 redox control is integral to ATRA-induced differentiation. To define the role of the redox function of Ref-1, we used the Ref-1 specific drug, APX3330, to block Ref-1 redox function and examined the response of HL60 cells to ATRA. Methods: Cell growth assessed using trypan blue. Differentiation was evaluated by morphology and expression of CD11b by flow cytometry. Apoptosis was assayed by annexin-PI staining on flow cytometry and cell cycle analysis assayed with propidium iodide flow cytometry. To assess activation of the MAPK pathway, BLR-1 expression was determined by real time PCR. Results: 1) APX3330 blockade of Ref-1 redox function resulted in limited cell growth yet a profound increase in differentiation and a moderate increase in apoptosis. 2) dose dependent studies with ATRA showed a similar degree of differentiation in cells treated with 10 μM ATRA to cells treated with APX3330 + 0.01 μM ATRA; allowing HL60 cells + APX3330 to give a similar response to a 1000 fold lower dose of ATRA. APX3330 alone did not induce differentiation and induced only minimal apoptosis but in combination with ATRA, increased the number of cells in G1/G0 phase significantly. 3) APX3330 + ATRA increased BLR-1 expression significantly by real time PCR suggesting enhanced activation of the MAPK pathway. Conclusions: APX3330 + ATRA limits HL60 growth and dramatically enhances terminal granulocytic differentiation. These finding may provide a therapeutic approach for prevention of the RAS. No significant financial relationships to disclose.

Impact of Protein Phosphatase PP2A on ATRA-Induced Activation of FoxO3a In Acute Promyelocytic Leukemia Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2492-2492
Author(s):  
Yasuhiko Sakoe ◽  
Kumi Sakoe ◽  
Haruo Shimazaki ◽  
Keita Kirito ◽  
Norio Komatsu
Keyword(s):  
Retinoic Acid ◽  
Cell Growth ◽  
Fusion Protein ◽  
Acute Promyelocytic Leukemia ◽  
Cellular Responses ◽  
Promyelocytic Leukemia ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Atra Treatment ◽  
Granulocytic Differentiation

Abstract Abstract 2492 Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia caused by reciprocal translocations of the long arms of chromosomes 15 and 17, which prevent cellular differentiation into mature neutrophils. The translocation of the promyelocytic leukemia (PML) gene on chromosome 15 and a retinoic acid receptor α (RARα) gene on chromosome 17 generates a PML-RARα fusion protein that inhibits PML-dependent apoptotic pathways in a dominant negative fashion. This fusion protein also blocks granulocytic differentiation by direct transcriptional inhibition of retinoic acid target genes. All-trans retinoic acid (ATRA) arrests cell growth, granulocytic differentiation, and apoptosis in APL cells via proteasome-dependent degradation of PML-RARα fusion protein and subsequent PML-nuclear body (NB) formation. Although PML is the essential component of PML-NBs and functions as a tumor suppressor, disruption of PML-NBs by the PML-RARα fusion protein inhibits endogenous PML tumor-suppressive functions in APL cells. Therefore, degradation of PML-RARα fusion protein and reorganization of PML-NBs during ATRA treatment are regarded as critical cellular responses, similar to the cell growth arrest and apoptosis of leukemia cells. Recently we demonstrated that FoxO3a (also named FKHRL1), a member of the Forkhead family of transcription factors, is a key molecule for the ATRA-induced cellular responses in APL cells (Blood 2010; 115: 3787–3795). In this study, we investigated the mechanism by which FoxO3a is activated by ATRA treatment in a human promyelocytic leukemia cell line NB4. Okadaic acid, a potent PP2A inhibitor, cancelled ATRA-induced dephosphorylation of AKT and its downstream molecule FoxO3a in NB4 cells. Knockdown of endogenous PP2A by siRNA significantly enhanced phosphorylation of both AKT and FoxO3a. These results suggested that PP2A is involved in ATRA-induced dephosphorylation of AKT and FoxO3a. Concomitantly, PP2AC, a catalytic subunit of PP2A, was dephoshorylated at tyrosine 307, and phosphatase activity of PP2A increased after ATRA treatment. Co-immunoprecipitation assay revealed that PP2A constitutively and directly binds to FoxO3a. Using artificial oligopeptides, we demonstrated that enhanced PP2A activity by ATRA directly dephosphorylates phosphothreonine 32 on FoxO3a. In addition, we found that 14-3-3 epsilon binded to phosphorylated FoxO3a in the cytoplasm in the absence of ATRA. After ATRA treatment, however, dephosphorylated FoxO3a dissociated from 14-3-3 epsilon and moved into the nucleus. Confocal microscopic analysis revealed that PP2A-FoxO3a complex partially co-localized with PML-NBs in the nucleus after ATRA treatment. Together, PML orchestrates nuclear networking with PP2A and FoxO3a for ATRA-induced granulocytic differentiation and apoptosis of APL cells. Disclosures: No relevant conflicts of interest to declare.

Inhibition of the Redox Function of Ape-1/ref-1 in Myeloid Leukemia Cells Results in Enhanced Sensitivity to Retinoic Acid Induced Differentiation and Apoptosis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4217-4217
Author(s):  
Kent A. Robertson ◽  
Edwin S. Colvin ◽  
Mark R. Kelley
Keyword(s):  
Flow Cytometry ◽  
Retinoic Acid ◽  
Myeloid Leukemia ◽  
Leukemia Cells ◽  
Minimal Amount ◽  
Morphologic Change ◽  
Similar Response ◽  
Moderate Increase ◽  
Granulocytic Differentiation ◽  
Cell Counts

Abstract Ape-1/ref-1 is a multifunctional base excision DNA repair protein that is involved in the repair of abasic sites in DNA. However, it also has a distinct role in the redox regulation of a variety of cellular proteins, such as Fos, Jun, p53, NFκB, PAX, HIF-1α, HLF, and others. Ape-1/ref-1 maintains these proteins in a reduced state thereby facilitating their DNA binding and transcriptional activation capability. HL-60 cells are known to respond to retinoic acid (RA) with terminal granulocytic differentiation and apoptosis, which is mediated through the RA receptors. Previous experiments suggested that elevated Ape-1/ref-1 expression is related to differentiation and apoptosis. To further define the role of the redox function of Ape-1/ref-1 in this relationship, redox function was blocked using two techniques. First, we used retroviral gene transduction to over-express a redox-inactive C-65 mutant of Ape1/ref-1 in HL-60 myeloid leukemia cells and examined the response to retinoic acid. In a second set of experiments we used an Ape-1/ref-1 specific small molecule inhibitor to pharmacologically block the redox function and again examined the response to retinoic acid. Differentiation was evaluated by morphologic change in differential cell counts and expression of CD11b by flow cytometry. Apoptosis was assayed by annexin-PI staining on flow cytometry and cell cycle analysis was examined with propidium iodide flow cytometry. Results: HL-60 cells expressing high levels of C-65 Ape-1/ref-1 responded to retinoic acid with a significantly higher level of differentiation and a moderate increase in apoptosis. Pharmacologic blockade of Ape-1/ref-1 redox function resulted in a profound increase in differentiation and a moderate increase in apoptosis compared to controls. dose dependent studies with retinoic acid demonstrated a similar degree of differentiation (CD11b expression) in cells treated with 10 μmolar retinoic acid and those treated with the redox inhibitor + 0.1 μmolar retinoic acid; alllowing HL-60 cells in the presence of the redox inhibitor to give a similar response to a 100 fold lower dose of retinoic acid. The redox inhibitor alone did not induce differentiation and induced only a minimal amount of apoptosis but did increase the number of cells in S phase significantly. In conclusion, our data supports the contention that redox function of Ape-1/ref-1 may be important for controlling RA-induced myeloid differentiation and programmed cell death. The implication of these findings is that myeloid leukemia cells may be sensitized to retinoids by manipulation of the redox status of Ape-1/ref-1.

scholarly journals PML-RAR Binds to the +7kb Enhancer of CEBPE and Inhibits Its Expression

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 43-43
Author(s):  
Pavithra Shyamsunder ◽  
Shree Pooja Sridharan ◽  
Pushkar Dakle ◽  
Zeya Cao ◽  
Vikas Madan ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Regulatory Element ◽  
Promyelocytic Leukemia ◽  
Luciferase Reporter ◽  
Myeloid Differentiation ◽  
Atra Treatment ◽  
Granulocytic Differentiation ◽  
Nb4 Cells ◽  
Acute Myeloid

Acute promyelocytic leukemia (APL) is a unique subtype of acute myeloid leukemia (AML). The disease is identified by distinctive morphology and is distinguished by a balanced reciprocal translocation between chromosomes 15 and 17. This aberration leads to the fusion between promyelocytic leukemia (PML) gene located on chromosome 15q21, and retinoic acid receptor α (RARA) gene from chromosome 17q21, leading to the resultant chimeric onco-fusion protein PML-RARA, which is detectable in more than 95% patients and disturbs proper promyelocytic differentiation. All-trans retinoic acid (ATRA) can induce granulocytic differentiation in APL and is used to treat APL patients. Genes containing PML-RARA-targeted promoters are transcriptionally suppressed in APL and most likely constitute a major mechanism of transcriptional repression occurring in APL. A growing body of evidence points to the role of distal regulatory elements, including enhancers, in the control of gene expression. In order to understand the unique sets of enhancers that might be under the control of PML-RAR and crucial for granulocytic differentiation of NB4 cells, we analysed the enhancer landscape of control and ATRA treated NB4 cells. H3K9Ac mapping identified a repertoire of enhancers that were gained in NB4 cells treated with ATRA. Closer investigation of these enhancer elements revealed enrichment of H3K9Ac signals around major drivers of myeloid differentiation. Of note, we identified a gain in enhancer signature for a region about 7kb downstream of the CEBPE gene. Our previous studies identified a novel enhancer for CEBPE in murine hematopoietic cells, which was 6 downstream of CEBPE core promoter. It appears that the +7kb region we identified in human APL cells may be analogous to the murine enhancer. We also observed that PML-RAR binds this +7kb region and ATRA treatment of NB4 cells displaced binding of PML-RAR from the + 7kb region, suggestive of a transcriptional repressive effect of PML-RAR at such enhancer elements. To test the transcription regulating potential of this +7kb region, we used catalytically inactive Cas9 fused to Krüppel associated box (KRAB) domain (dCas9-KRAB). We designed three guide RNAs covering this regulatory region. The sgRNAs effectively repressed expression of CEBPE accompanied by lowered granulocytic differentiation of these guide RNA targeted NB4 cells after ATRA treatment. To explore transcription factor (TF) occupancy at this +7 kb region, we analysed public available ChIP-seq datasets for hematopoiesis-specific factors. Analysis revealed that the +7kb region was marked by an open chromatin signature, accompanied by binding of a majority of hematopoietic TFs around this putative regulatory element with concurrent binding of EP300. Strikingly we noticed binding of CEBPA, CEBPB and CEBPE at this regulatory element. To assess whether binding of these members of the CEBP family of TFs is functionally relevant, luciferase reporter and electrophoretic mobility shift assays (EMSA) were performed. Co expression of the CEBP TFs led to significant induction of luciferase expression, and this data was further confirmed using EMSA assays. Based on these observations, we propose that PML-RAR blocks granulocytic differentiation by occupying this +7kb enhancer of CEBPE, hinders binding of other cell type/lineage specific TFs, and blocks CEBPE expression. When cells are stimulated with ATRA, PML-RAR is displaced from the CEBPE enhancer, allowing for efficient binding of myeloid-specific TFs. This results in increased CEBPE expression, which in turn promotes efficient granulocytic differentiation. The findings from our study expands our current understanding of the mechanism of differentiation therapy, the role of onco-fusion proteins in inhibiting myeloid differentiation, and may provide new therapeutic approaches to many acute myeloid leukemias. Disclosures Ong: National University of Singapore: Other: Royalties.

Combination of retinoic acid and tumor necrosis factor overcomes the maturation block in a variety of retinoic acid-resistant acute promyelocytic leukemia cells

Blood ◽  
2004 ◽  
Vol 104 (10) ◽  
pp. 3335-3342 ◽  
Author(s):  
Michael Witcher ◽  
Hoi Ying Shiu ◽  
Qi Guo ◽  
Wilson H. Miller
Keyword(s):  
Retinoic Acid ◽  
Tumor Necrosis Factor ◽  
Cell Line ◽  
Acute Promyelocytic Leukemia ◽  
Tumor Necrosis ◽  
Promyelocytic Leukemia ◽  
Leukemia Cells ◽  
Monocytic Differentiation ◽  
Granulocytic Differentiation ◽  
Necrosis Factor

Abstract Retinoic acid (RA) overcomes the maturation block in t(15:17) acute promyelocytic leukemia (APL), leading to granulocytic differentiation. Patients receiving RA alone invariably develop RA resistance. RA-resistant cells can serve as useful models for the development of treatments for both APL and other leukemias. Previously, we showed that RA and tumor necrosis factor (TNF) promote monocytic differentiation of the APL cell line NB4 and U937 monoblastic cells. Here, we report that combining TNF with RA leads to maturation of several RA-resistant APL cells along a monocytic pathway, whereas UF-1, a patient-derived RA-resistant cell line, showed characteristics of granulocytic differentiation. We found distinct differences in gene regulation between UF-1 cells and cells showing monocytic differentiation. Although IRF-7 was up-regulated by TNF and RA in all cells tested, expression of c-jun and PU.1 correlated with monocytic differentiation. Furthermore, synergistic induction of PU.1 DNA binding and macrophage colony-stimulating factor receptor (m-CSF-1R) mRNA was observed only in cells differentiating into monocytes. Using neutralizing antibodies against m-CSF-1R or its ligand, we found that inhibiting this pathway strongly reduced CD14 expression in response to RA and TNF, suggesting that this pathway is essential for their synergy in RA-resistant leukemia cells. (Blood. 2004;104:3335-3342)

C/EBP Is Deregulated by PML-RAR in Acute Promyelocytic Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3015-3015
Author(s):  
Florence Guibal ◽  
Hanna S. Radomska ◽  
Lisa M. Johansen ◽  
Daniel G. Tenen
Keyword(s):  
Retinoic Acid ◽  
Fusion Protein ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia ◽  
Myeloid Differentiation ◽  
Computer Search ◽  
Granulocytic Differentiation ◽  
Transient Transfection Assay

Abstract Acute promyelocytic leukemia (APL) cells are blocked at the promyelocyte stage of myeloid differentiation. The majority of APL cells display the t(15;17) reciprocal chromosomal translocation leading to the expression of the fusion protein promyelocytic leukemia-retinoic acid receptor alpha (PML-RARa). Cells harboring this reciprocal translocation can be induced to differentiate after treatment with all-trans retinoic acid (at-RA) both in vivo and in vitro. During normal hematopoiesis, differentiation is regulated by several key transcription factors. One of them, CCAAT/enhancer binding protein alpha (C/EBPa), controls expression of genes regulating normal myeloid differentiation. Its disruption leads to a block of granulocytic differentiation. We thus hypothesize that C/EBPa could be deregulated in APL and therefore participate in the pathogenesis of APL. Using the U937PR9 cell line, which expresses an inducible PML-RARa, we observed that expression of PML-RARa induced a decrease of both C/EBPa mRNA and protein, leading to decreased C/EBPa DNA binding activity. Using a transient transfection assay with a C/EBPa promoter construct in presence or absence of PML-RARa, we are able to demonstrate that PML-RARa can repress C/EBPa promoter activity. This repression is specific to the fusion protein, as both PML and RARa have no effect upon the C/EBPa promoter. A computer search of the C/EBPa promoter sequence did not exhibit any evident RARE binding site, and therefore we are currently mapping the site(s) responsible for this repression. In conclusion, PML-RARa down regulates C/EBPa expression; this down regulation could participate in the pathogenesis of APL. This hypothesis is also supported by the observation that at-RA treatment of APL cell lines (NB4 and HT93) induces a rapid restoration of both C/EBPa RNA and protein. Thus, a decrease in both C/EBPa expression and activity could contribute to the differentiation block of APL cells by deregulating the normal myeloid differentiation program.

scholarly journals PML-Retinoic Acid Receptor α Inhibits PML IV Enhancement of PU.1-Induced C/EBPε Expression in Myeloid Differentiation

2007 ◽  
Vol 27 (16) ◽  
pp. 5819-5834 ◽  
Author(s):  
Hitoshi Yoshida ◽  
Hitoshi Ichikawa ◽  
Yusuke Tagata ◽  
Takuo Katsumoto ◽  
Kazunori Ohnishi ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Fusion Protein ◽  
Retinoic Acid Receptor ◽  
Promyelocytic Leukemia ◽  
Myeloid Differentiation ◽  
Granulocytic Differentiation ◽  
Acid Receptor ◽  
Type Iv ◽  
Retinoic Acid Receptor Α ◽  
Deficient Mice

ABSTRACT PML and PU.1 play important roles in myeloid differentiation. PML-deficient mice have an impaired capacity for terminal maturation of their myeloid precursor cells. This finding has been explained, at least in part, by the lack of PML action to modulate retinoic acid-differentiating activities. In this study, we found that C/EBPε expression is reduced in PML-deficient mice. We showed that PU.1 directly activates the transcription of the C/EBPε gene that is essential for granulocytic differentiation. The type IV isoform of PML interacted with PU.1, promoted its association with p300, and then enhanced PU.1-induced transcription and granulocytic differentiation. In contrast to PML IV, the leukemia-associated PML-retinoic acid receptor α fusion protein dissociated the PU.1/PML IV/p300 complex and inhibited PU.1-induced transcription. These results suggest a novel pathogenic mechanism of the PML-retinoic acid receptor α fusion protein in acute promyelocytic leukemia.

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