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

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.

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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)

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.e14613 ◽

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.

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Terminal differentiation of human promyelocytic leukemic cells in primary culture in response to retinoic acid

Blood ◽

10.1182/blood.v57.6.1000.bloodjournal5761000 ◽

1981 ◽

Vol 57 (6) ◽

pp. 1000-1004 ◽

Cited By ~ 14

Author(s):

TR Breitman ◽

SJ Collins ◽

BR Keene

Keyword(s):

Retinoic Acid ◽

Acute Promyelocytic Leukemia ◽

Leukemia Cell ◽

Promyelocytic Leukemia ◽

Leukemic Cells ◽

Leukemia Cell Line ◽

Granulocytic Differentiation ◽

Myelocytic Leukemia ◽

Functional Maturation ◽

Promyelocytic Leukemia Cell Line

The recent finding that retinoic acid induces terminal granulocytic differentiation of the human promyelocytic leukemia cell line, HL-60, prompted an investigation of the sensitivity to this inducer of human myelocytic leukemia cells in primary suspension culture. Of the 21 leukemic specimens, only cells from the two patients with acute promyelocytic leukemia differentiated in response to retinoic acid. After an incubation period of 5--7 days in 1 microM retinoic acid, the cells from these two patients showed extensive morphological and functional maturation. Thus, because it appears that retinoic acid specifically induces granulocytic differentiation of leukemic promyelocytes, this compound may have therapeutic utility in the treatment of acute promyelocytic leukemia.

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Retinoic acid is required for and potentiates differentiation of acute promyelocytic leukemia cells by nonretinoid agents

Blood ◽

10.1182/blood.v84.7.2122.2122 ◽

1994 ◽

Vol 84 (7) ◽

pp. 2122-2129 ◽

Cited By ~ 33

Author(s):

A Chen ◽

JD Licht ◽

Y Wu ◽

N Hellinger ◽

W Scher ◽

...

Keyword(s):

Retinoic Acid ◽

Cell Line ◽

Acute Promyelocytic Leukemia ◽

Drug Exposure ◽

Primary Cultures ◽

Leukemia Cell ◽

Promyelocytic Leukemia ◽

Leukemia Cell Line ◽

Granulocytic Differentiation ◽

Nb4 Cells

Abstract Patients with acute promyelocytic leukemia (APL) associated with the t(15;17) translocation and fusion of the promyelocytic leukemia (PML) and retinoic acid receptor-alpha (RAR-alpha) genes achieve complete remission but not cure with all-trans retinoic acid (RA), NB4, a cell line derived from a patient with t(15;17) APL that undergoes granulocytic differentiation when treated with pharmacologic doses of RA, was used as a model for differentiation therapy of APL. We found that NB4 cells are resistant to differentiation by nonretinoid inducers such as hexamethylene bisacetamide (HMBA), butyrates, vitamin D3, or hypoxanthine, all of which can induce differentiation in the commonly used HL60 leukemia cell line. Preexposure of NB4 cells to low concentrations of RA for a period as short as 30 minutes abolished resistance to nonretinoids and potentiated differentiation. Sequential RA and HMBA treatment yielded maximal differentiation by 3 days of drug exposure, whereas the effect of RA alone peaked after 6 days and yielded a smaller percentage of differentiated cells. RA also reversed NB4 cell resistance to butyrates and allowed for synergistic differentiation by these agents. Pretreatment with HMBA before exposure to RA failed to stimulate differentiation. Sequential RA/HMBA treatment also markedly increased the extent of differentiation of primary cultures of bone marrow and peripheral blood mononuclear cells from three APL patients. In one case RA/HMBA treatment overcame resistance to RA in vitro. Together, these results suggest that intermittent low doses of RA followed by either HMBA or butyrates may be a useful combination in the treatment of APL. This clinical strategy may help prevent or overcome RA resistance in APL.

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

Blood ◽

10.1182/blood-2004-01-0023 ◽

2004 ◽

Vol 104 (10) ◽

pp. 3335-3342 ◽

Cited By ~ 16

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)

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Retinoic acid is required for and potentiates differentiation of acute promyelocytic leukemia cells by nonretinoid agents

Blood ◽

10.1182/blood.v84.7.2122.bloodjournal8472122 ◽

1994 ◽

Vol 84 (7) ◽

pp. 2122-2129 ◽

Cited By ~ 3

Author(s):

A Chen ◽

JD Licht ◽

Y Wu ◽

N Hellinger ◽

W Scher ◽

...

Keyword(s):

Retinoic Acid ◽

Cell Line ◽

Acute Promyelocytic Leukemia ◽

Drug Exposure ◽

Primary Cultures ◽

Leukemia Cell ◽

Promyelocytic Leukemia ◽

Leukemia Cell Line ◽

Granulocytic Differentiation ◽

Nb4 Cells

Patients with acute promyelocytic leukemia (APL) associated with the t(15;17) translocation and fusion of the promyelocytic leukemia (PML) and retinoic acid receptor-alpha (RAR-alpha) genes achieve complete remission but not cure with all-trans retinoic acid (RA), NB4, a cell line derived from a patient with t(15;17) APL that undergoes granulocytic differentiation when treated with pharmacologic doses of RA, was used as a model for differentiation therapy of APL. We found that NB4 cells are resistant to differentiation by nonretinoid inducers such as hexamethylene bisacetamide (HMBA), butyrates, vitamin D3, or hypoxanthine, all of which can induce differentiation in the commonly used HL60 leukemia cell line. Preexposure of NB4 cells to low concentrations of RA for a period as short as 30 minutes abolished resistance to nonretinoids and potentiated differentiation. Sequential RA and HMBA treatment yielded maximal differentiation by 3 days of drug exposure, whereas the effect of RA alone peaked after 6 days and yielded a smaller percentage of differentiated cells. RA also reversed NB4 cell resistance to butyrates and allowed for synergistic differentiation by these agents. Pretreatment with HMBA before exposure to RA failed to stimulate differentiation. Sequential RA/HMBA treatment also markedly increased the extent of differentiation of primary cultures of bone marrow and peripheral blood mononuclear cells from three APL patients. In one case RA/HMBA treatment overcame resistance to RA in vitro. Together, these results suggest that intermittent low doses of RA followed by either HMBA or butyrates may be a useful combination in the treatment of APL. This clinical strategy may help prevent or overcome RA resistance in APL.

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A Chimeric Gene, FIP1L1-RARα, Is Isolated from t(4;17)-Positive Acute Promyelocytic Leukemia; a Report of Cloning and Functional Analysis.

Blood ◽

10.1182/blood.v110.11.1820.1820 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1820-1820

Author(s):

Takeshi Kondo ◽

Akio Mori ◽

Masahiro Onozawa ◽

Kaoru Kahata ◽

Satoshi Hashino ◽

...

Keyword(s):

Retinoic Acid ◽

Acute Promyelocytic Leukemia ◽

Luciferase Activity ◽

Promyelocytic Leukemia ◽

Leukemia Cells ◽

Atra Treatment ◽

Partner Gene ◽

Vitro Analysis ◽

In Vitro Analysis

Abstract Acute promyelocytic leukemia (APL) comprises 5 to 10% of acute myelogenous leukemia, the leukemia cells of which is characterized by the differentiation block at the stage of promyelocyte. In APL cells, a reciprocal translocation in chromosome 17 is usually observed, and consequently, a retinoic acid receptor alpha (RARα) gene is rearranged. The rearranged RARα gene is fused to another gene, and as a result, the product of newly composed fusion gene provokes the onset of APL. So far, there are five RARα partner genes reported; PML gene in 15q22, PLZF gene in 11q23, NPM gene in 5q35, NuMA gene in 11q13 Stat5b in 17q21. We recently experienced APL case, in which a chromosomal translocation, t(4;17) was observed. FISH analysis of leukemia cells indicated that RARα gene was translocated. To identify the RARα-partner gene, we performed 5′-rapid amplification of cDNA end (RACE) and identified FIP1L1 gene as RARα-partner gene. It is known that FIP1L1 gene is fused to PGDFRα gene in the hypereosinophilic syndrome. DNA sequence revealed that exon 15 of FIP1L1 gene was fused to exon 3 of RARα gene. In previous reports, five X-RARα have the common character; X-RARα manifests homodimerization. X-RARα suppresses retinoic acid-induced transcription. We examined that FIP1L1-RARα has these two characters. Transient transfection analysis showed that FIP1L1-RARα has the ability to form homodimer. And luciferase assay suggested that FIP1L1-RARα suppressed the retinoic acid-induced transcriptional activity at the physiological concentration of all-trans retinoic acid (ATRA). And the level of luciferase activity suppressed by FIP1L1-RARα was similar to that suppressed by PML-RARα. And the therapeutic concentration of ATRA activated the significant level of luciferase activity. Thus, in vitro analysis suggested that ATRA treatment could be effective to FIP1L1-RARα-positive APL patients. Consistent with the results of in vitro analysis, ATRA treatment was clinically effective, and the patient achieved complete remission after five weeks of ATRA treatment. Collectively, we suggest that FIP1L1-RAR? is the sixth pathogenic gene of APL.

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Neutrophil Secondary-Granule Deficiency as a Hallmark of All-Trans Retinoic Acid–Induced Differentiation of Acute Promyelocytic Leukemia Cells

Blood ◽

10.1182/blood.v90.2.803.803_803_813 ◽

1997 ◽

Vol 90 (2) ◽

pp. 803-813 ◽

Cited By ~ 2

Author(s):

Jun Miyauchi ◽

Kazuma Ohyashiki ◽

Yuka Inatomi ◽

Keisuke Toyama

Keyword(s):

Retinoic Acid ◽

Acute Promyelocytic Leukemia ◽

Chromatin Condensation ◽

Leukemia Cell ◽

Promyelocytic Leukemia ◽

Leukemia Cells ◽

Atra Treatment ◽

Trans Retinoic Acid ◽

All Trans Retinoic Acid ◽

Secondary Granule

Acute promyelocytic leukemia (APL) is a neoplasm with the unique chromosomal translocation t(15; 17), which involves the retinoic acid receptor α gene. All-trans retinoic acid (ATRA) has been used for APL patients as a potent therapeutic agent to induce differentiation of leukemia cells. Although polymorphonuclear leukocytes (PMNs) appearing in the blood and bone marrow during ATRA treatment often possess Auer rods, indicating their neoplastic origin, other morphological abnormalities of PMNs have not been elucidated. We studied the morphological changes of APL cells during ATRA treatment at the ultrastructural level. Although most aberrant primary granules, including Auer rods, became morphologically normal in response to ATRA therapy and the nuclei showed chromatin condensation and lobulation, resulting in the emergence of PMNs, the lobulated nuclei often had nuclear filamentous connections and/or nuclear blebs, indicating some pathological process. Furthermore, PMNs, particularly early in ATRA treatment, lacked neutrophil secondary granules as did the PMNs appearing in a culture of APL cells incubated with ATRA, findings consistent with previously reported data that acute myeloid leukemia cell lines do not produce secondary granule proteins even after induction of differentiation towards mature neutrophils. The present data indicate that ATRA is incapable of inducing complete morphological maturation of APL cells and that secondary-granule deficiency may be a hallmark of aberrantly differentiated leukemic cells.

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

Blood ◽

10.1182/blood.v106.11.3015.3015 ◽

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.

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Neutrophil Secondary-Granule Deficiency as a Hallmark of All-Trans Retinoic Acid–Induced Differentiation of Acute Promyelocytic Leukemia Cells

Blood ◽

10.1182/blood.v90.2.803 ◽

1997 ◽

Vol 90 (2) ◽

pp. 803-813 ◽

Cited By ~ 19

Author(s):

Jun Miyauchi ◽

Kazuma Ohyashiki ◽

Yuka Inatomi ◽

Keisuke Toyama

Keyword(s):

Retinoic Acid ◽

Acute Promyelocytic Leukemia ◽

Chromatin Condensation ◽

Leukemia Cell ◽

Promyelocytic Leukemia ◽

Leukemia Cells ◽

Atra Treatment ◽

Trans Retinoic Acid ◽

All Trans Retinoic Acid ◽

Secondary Granule

Abstract Acute promyelocytic leukemia (APL) is a neoplasm with the unique chromosomal translocation t(15; 17), which involves the retinoic acid receptor α gene. All-trans retinoic acid (ATRA) has been used for APL patients as a potent therapeutic agent to induce differentiation of leukemia cells. Although polymorphonuclear leukocytes (PMNs) appearing in the blood and bone marrow during ATRA treatment often possess Auer rods, indicating their neoplastic origin, other morphological abnormalities of PMNs have not been elucidated. We studied the morphological changes of APL cells during ATRA treatment at the ultrastructural level. Although most aberrant primary granules, including Auer rods, became morphologically normal in response to ATRA therapy and the nuclei showed chromatin condensation and lobulation, resulting in the emergence of PMNs, the lobulated nuclei often had nuclear filamentous connections and/or nuclear blebs, indicating some pathological process. Furthermore, PMNs, particularly early in ATRA treatment, lacked neutrophil secondary granules as did the PMNs appearing in a culture of APL cells incubated with ATRA, findings consistent with previously reported data that acute myeloid leukemia cell lines do not produce secondary granule proteins even after induction of differentiation towards mature neutrophils. The present data indicate that ATRA is incapable of inducing complete morphological maturation of APL cells and that secondary-granule deficiency may be a hallmark of aberrantly differentiated leukemic cells.

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