LSD1 Inhibitor Activates Retinoic Acid Pathway in MLL Fusion Positive Acute Myeloid Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1345-1345
Author(s):  
Mio Yano ◽  
Toshihiko Imamura ◽  
Kenichi Sakamoto ◽  
Hideki Yoshida ◽  
Atsushi Fujiki ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Morphological Changes ◽  
Pcr Analysis ◽  
Monocytic Differentiation ◽  
Differentiation Therapy ◽  
Cd11b Expression ◽  
Nbt Reduction ◽  
Acid Pathway

Abstract Abstract 1345 Background: Among the subtypes of acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) responds dramatically to differentiation therapy with all-trans retinoic acid (ATRA). However, ATRA is not sufficient to induce differentiation in non-APL AML. Although the molecular basis for the poor response of non-APL AML to ATRA was poorly understood, Lysine-specific demethylase 1 (LSD1), the histone demetylase, was found to inhibit the retinoic acid pathway by chromatin modification through H3K4 demethylation, resulting in silencing of gene expression targeted by retinoic acid. Herein, we first evaluated whether MLL fusion partners, such as MLL-AF9 and MLL-AF4/AF5q31, affect the sensitivity of ATRA in human and murine MLL fusion positive AML cells, which is one of the most aggressive pediatric AML. In addition, we also assess whether the LSD1 inhibitor affects the ATRA sensitivity in MLL fusion positive AML cells. Methods: Three human AML cell lines with MLL fusion (THP-1 and MOLM-13 expressing MLL-AF9, and KOCL48 expressing MLL-AF4) and two murine leukemic cell lines derived from murine Lin- hematopoietic progenitors transduced by retroviral vector expressing MLL fusion genes, such as MLL-AF9 and MLL-AF5q31 were used in this study. To test the sensitivity of ATRA, all cell lines were treated with 1 μM ATRA for three days. Cell growth was analyzed by counting nuclei using a Coulter counter. Monocytic differentiation was assessed by morphological analysis, NBT reduction test and flow cytometric analysis (FCM) of CD11b expression. FCM analysis was also carried out to evaluate cell cycle and annexin V assay. Quantitative RT-PCR (qRT-PCR) analysis and western blotting was carried out to measure the RARα, C/EBPα, C/EBPε, and PU.1 expressions. To determine whether Tranylcypromine (TCP), which is a nonreversible LSD1 inhibitor, could decrease the IC50 of ATRA in MLL-AF4/AF5q31 positive cells, KOCL48 and murine MLL-AF5q31 expressing cells were treated with 0μM or 10μM TCP and titrating doses of ATRA (ranging from 0μM to 10μM). After three days, cell count was analyzed by counting nuclei using a Coulter counter to evaluate IC50 of ATRA in each cell lines. Results: We first determined that morphological changes characteristic of monocytic differentiation, CD11b expression and NBT reduction are more readily induced by ATRA in human and murine MLL-AF9 positive cells than human and murine MLL-AF4/AF5q31 positive cells The NBT reduction percentage was 17.6±1.69 in THP-1, but 2.7±1.2 in KOCL48 cells (p<0.01). The ATRA treatment also induced growth inhibition accompanied with G0/G1 arrest and apoptosis more efficiency in MLL-AF9 positive cells than MLL-AF4/AF5q31 cells. The IC50 of ATRA for THP-1 cells was 0.21±0.04 μM, but 5.31±1.50 μM for KOCL48 cells (p<0.01) The percentage of cells arrested in G0/G1 phase and Annexin/PI positive cells were 84% and 17.8% in THP-1 but 40% and 4.8% in KOCL48, respectively. Furthermore, qRT-PCR analysis and western blot analysis revealed that ATRA increased expression level of RARα, C/EBPα, C/EBPε, and PU.1, which is involved in monocytic differentiation through retinoic acid pathway, in MLL-AF9 positive cells, but not in MLL-AF4/AF5q31 positive cells. Collectively, retinoic acid pathway is more impaired in MLL-AF4/AF5q31 positive cells than MLL-AF9 positive cells. Next, we also determined that ATRA and TCP combination treatment suppressed cell growth and decreased the IC50 of ATRA in KOCL48 and murine MLL-AF5q31 expressing cells (IC50 of ATRA: 0.20±0.10 μM and 0.20±0.09 μM with TCP, vs 5.5±3.2 μM and over 10 μM without TCP, p<0.05), accompanied with morphological changes and CD11b expression, suggesting that inhibition of LSD1 restores ATRA sensitivity in both cell lines. Conclusions: Our data demonstrate that retinoic acid pathway was more profoundly impaired in MLL-AF4/AF5q31 positive cell than MLL-AF9 positive cells, suggesting MLL-AF4/AF5q31 contributes inactivation of retinoic acid pathway. Our data also demonstrate TCP restore the sensitivity of ATRA in ATRA-resistant MLL-AF4/AF5q31 positive cell lines, suggesting LSD1 plays a major role in inactivation of retinoic acid pathway in MLL-AF4/AF5q31 positive cells. Therefore, LSD1 inhibitor might be important novel therapeutic option for differentiation therapy of MLL-fusion positive AML, especially for ATRA resistant MLL-AF4/AF5q31 positive cells. Disclosures: No relevant conflicts of interest to declare.

MLL-AF9 Positive Acute Myeloid Leukemia Cells Are Sensitive To All-Trans Retinoic Acid

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3827-3827
Author(s):  
Kenichi Sakamoto ◽  
Toshihiko Imamura ◽  
Mio Yano ◽  
Hideki Yoshida ◽  
Atsushi Fujiki ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Morphological Changes ◽  
Monocytic Differentiation ◽  
Cd11b Expression ◽  
Qrt Pcr ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Nbt Reduction

Abstract Background Among the subtypes of acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) responds dramatically to differentiation therapy with all-trans retinoic acid (ATRA). But, ATRA is not sufficient to induce differentiation in non-APL AML. Herein, we first evaluated whether MLL fusion partners, such as MLL-AF9 and MLL-AF4/AF5q31, affect the sensitivity of ATRA in human and murine MLL fusion positive AML cell lines. In addition, we also assessed the level of H3K4me2 modification for the RARα gene in human AML cell lines, and whether the LSD1 inhibitor affected the ATRA-resistant MLLfusion positive AML cell lines. Methods Three human AML cell lines with MLL fusion (THP1 and MOLM13 expressing MLL-AF9, and KOCL48 expressing MLL-AF4) and two murine leukemic cell lines derived from murine Lin- hematopoietic progenitors transduced by retroviral vector expressing MLL fusion genes, such as MLL-AF9 and MLL-AF5q31 were used in this study. To test the sensitivity of ATRA, all cell lines were treated with 1 μM ATRA for three days. Monocytic differentiation was assessed by morphological analysis, NBT reduction test and flow cytometric analysis (FCM) of CD11b expression. Quantitative RT-PCR (qRT-PCR) analysis and western blotting was carried out to measure the RARα, C/EBPα, C/EBPε, and PU.1 expressions. Cytotoxicity assay was performed to determine the IC50 of ATRA in these cell lines and whether ATRA could decrease the IC50 of cytarabine in MLL-AF9positive cells by using WST assays. Chromatin immunoprecipitation (ChIP) assay was performed to determine the value of H3K4me2 status using RARα-specific primers. To determine whether tranylcypromine (TCP), which is a nonreversible LSD1 inhibitor, could reactivate ATRA sensitivity, we treated KOCL48 with 10 μM TCP and 1μM ATRA. Results We first determined that morphological changes characteristic of monocytic differentiation, CD11b expression and NBT reduction are more readily induced by ATRA in human and murine MLL-AF9 positive cells than MLL-AF4/AF5q31 positive cells. The NBT reduction percentage was 12.5±3.77 in THP1, 13.1±2.02 in MOLM13, but 7.00±2.64 in KOCL48 cells (p<0.05). The ATRA treatment also induced growth inhibition and increased gene expression related to monocytic differentiation through retinoic acid (RA) pathway, more efficiency in MLL-AF9 positive cells than MLL-AF4/AF5q31 cells. The IC50 of ATRA for THP1, MOLM13 and murine MLL-AF9 cells was 3.85, 1.24 and 1.95 μM, but over 10 μM for KOCL48 and murine MLL-AF5q31 cells. Furthermore, qRT-PCR and western blot revealed that ATRA increased expression level of RARα, C/EBPα, C/EBPε, and PU.1 in MLL-AF9 positive cells, but not in MLL-AF4/AF5q31 positive cells. Collectively, RA pathway is more impaired in MLL-AF4/AF5q31 positive cells than MLL-AF9 positive cells. In addition, the increase in RARα, C/EBPα, C/EBPε, and PU.1 mRNA expressions were observed in two primary MLL-AF9 positive AML cells treated with ATRA. Next, we also carried out ChIP assay and the H3K4me2/ H3 on the RARα promoter in MLL-AF9 positive cells were higher than MLL-AF4 positive cell. Furthermore, ATRA and TCP combination treatment in KOCL48 induced morphological changes, CD11b expression, and increased the expression level of RARα, C/EBPα, C/EBPε, and PU.1, suggesting that inhibition of LSD1 restores ATRA sensitivity. Finally, ATRA in combination with cytarabine treatment in MLL-AF9 positive cells enhanced cytarabine sensitivity: the IC50 of cytarabine in THP1, MOLM13, and murine MLL-AF9cells was 4.18, 0.04, and0.065 μM without ATRA and 0.13, 0.0005, and 0.015 μM with ATRA, respectively. Conclusions Our data demonstrated that RA pathway was more profoundly impaired in MLL-AF4/AF5q31 positive cells than MLL-AF9 positive cells, suggesting type of MLL fusion protein contributes inactivation of RA pathway. Our data also identified the sensitivity of ATRA was correlated with the ratio of H3K4me2/ H3 on the RARα promoter, and TCP restore the sensitivity of ATRA in KOCL48, suggesting the decrease of the H3K4me2/H3 plays a role in inactivation of RA pathway. Additionally, we revealed that synergistic antileukemic activity of ATRA in combination with cytarabine in MLL-AF9 positive cells. Therefore, ATRA in combination with cytarabine might be novel therapeutic option for the ATRA sensitive AML cells, especially for MLL-AF9 positive cells. Disclosures: No relevant conflicts of interest to declare.

Monocytic Differentiation of Myeloid Leukemia Cell Lines Induced by ATRA and 5-Aza-2'-Deoxycitidine.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3111-3111
Author(s):  
Atsushi Fujiki ◽  
Toshihiko Imamura ◽  
Yoshifumi Hirashima ◽  
Mitsuru Miyachi ◽  
Shigeki Yagyu ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Annexin V ◽  
G1 Arrest ◽  
Inhibition Rate ◽  
Monocytic Differentiation ◽  
Differentiation Therapy ◽  
Specific Pcr ◽  
Annexin V Assay

Abstract Abstract 3111 Poster Board III-48 Backgrounds and Introduction The CCAAT/enhancer binding protein (C/EBP)αa is a transcriptional factor of hematopoietic system and plays a key role in monocytic differentiation. Recently, several studies have reported that C/EBPαa expression is down-regulated in acute myeloid leukemia (AML), leading to block of granulocytic and monocytic differentiation. Differentiation therapy of ATRA is highly effective for acute promyelocytic leukemia (APL). The mechanism of induction of differentiation in the treatment of APL is induction of a set of transcriptional factors which are responsible for myeloid differentiation. However, ATRA alone is not sufficient to treat another type of AML. Thus, it is worth to explore the agents which intensify the efficacy of ATRA. To assess the possibility of differentiation therapy in AML, except for APL, we evaluated the efficacy of demethylation agent combined with ATRA for various AML cell lines. Materials and Methods The five AML cell lines (K562, U-937, HL-60, THP-1, and KOCL48 expressing MLL-AF4) were treated with 50nM 5-Aza-2'-deoxycytidine (5-Aza) for two days and 1 mM ATRA for additional five days. Then, we analyzed cell growth with counting nuclei using Coulter counter. The cell cycle analysis was also performed by flow cytometry (FCM). In addition, Annexin V assay was performed to determine whether apoptosis occurred or not. To assess whether monocytic differentiation was induced or not, the expression of CD11b was evaluated by FCM. In addition, the expression of transcriptional factors, such as C/EBPαa, PU.1 and c-myc ) were analyzed by real time PCR analysis. Methylation specific PCR was also performed to evaluate the methylation status of promoter region of C/EBPαa. Results HL-60 was highly sensitive to ATRA (growth inhibition rate: 80%). THP-1 and KOCL-48 were moderately sensitive to ATRA (growth inhibition rate: 50-60%). Addition of 5-Aza induced suppression of the growth in these two cell lines efficiently (growth inhibition rate: 80%). K562 and U937 were resistant to ATRA (growth inhibition rate: 10-20%). Addition of 5-Aza induced suppression of cell-growth in U937 (growth inhibition rate: 70%). However, 5-Aza did not show the effect in K562. Morphological studies revealed the characteristic features, such as extended cytoplasm with vacuoles, fine granules and irregular shaped nucleus, were evident in four cell lines which was sensitive to 5-Aza. FCM analysis revealed intensification of CD11b expression. In addition, real time PCR determined the increased expression of C/EBPαa and PU.1 (Fold change: 2-3.0) in the four cell lines except for K562. On the other hand, the expression level of c-myc was decreased under treatment with ATRA and 5-Aza (Fold change: 0.2). Cell cycle analysis revealed G1 arrest was occurred. Annexin V assay also revealed that combination therapy of ATRA and 5-Aza induced apoptosis in theses cell lines except for K562. Methylation specific PCR did not identified hypermetylation of pormorter region of C/EBPαa in these cell lines except for K562. Conclusion Addition of 5-Aza to ATRA induced further expression of C/EBPαa and PU.1 efficiently, leading to monocytic differentiation in AML cell lines. Monocytic differentiation was accompanied with G1 arrest through down-regulation of c-myc, and apoptosis was induced finally. Combination of ATRA and 5-Aza might be effective therapeutic option even for AML which is resistant to differentiation therapy with ATRA. Disclosures No relevant conflicts of interest to declare.

RAD001, An Inhibitor of mTOR, Enhances Monocytic Differentiation Induced by ATRA through Phosphorylation of C/EBPα In AML Cell Lines

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2152-2152
Author(s):  
Hideki Yoshida ◽  
Toshihiko Imamura ◽  
Atsushi Fujiki ◽  
Yoshifumi Hirashima ◽  
Mitsuru Miyachi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Microarray Analysis ◽  
Combination Treatment ◽  
Annexin V ◽  
Resistant Cell ◽  
Pcr Analysis ◽  
Monocytic Differentiation ◽  
Cd11b Expression ◽  
Qrt Pcr ◽  
Induced Apoptosis

Abstract Abstract 2152 Background: CCAAT/ enhancer binding protein alpha (C/EBPα) is a critical transcription factor that controls monocytic and granulocytic differentiation. Several recent studies have reported that C/EBPα expression is down-regulated in acute myeloid leukemia (AML), leading to suppression of monocytic differentiation. All-trans-retinoic acid (ATRA) induces numerous transcriptional factors, including C/EBPα; however, ATRA alone is not sufficient to induce monocytic differentiation in AML. The purpose of this study was to identify agents that increase the efficacy of ATRA. RAD001 (Everolimus; provided by Novartis), a rapamycin analog, is a relatively new drug that inhibits the Akt/ PI3K/ mTOR pathway. To assess the utility of differentiation therapy as a treatment for types of AML other than acute promyelocytic leukemia, we evaluated the effects of RAD001 and ATRA combination treatment in several AML cell lines. Methods: Three AML cell lines (U-937, THP-1, and KOCL48) and two primary AML samples were treated with 2.5–5.0 nM RAD001 and 1 μM ATRA for five days. Cell growth was analyzed by counting nuclei using a Coulter counter. Monocytic differentiation was assessed by morphological analysis and flow cytometric analysis (FCM) of CD11b expression. An Annexin V assay was carried out to measure apoptosis. Microarray analysis using an Agilent expression array was employed to determine changes in gene expression associated with ATRA and RAD001 combination treatment. Quantitative RT-PCR (qRT-PCR) analysis was performed to validate the microarray results. Western blotting was carried out to measure the phosphorylation of C/EBPα at Ser 21. Results: We determined that ATRA and RAD001 treatment induced morphological changes characteristic of monocytic differentiation. Microarray analysis of THP-1 revealed that ATRA and RAD001 induced expression of a set of genes associated with monocytic differentiation, including MPEG1, CD11b, CD115 and CD14. FCM analysis confirmed that ATRA and RAD001 intensified CD11b expression in the three cell lines tested, especially in the two ATRA-resistant cell lines (KOCL48 and U937). qRT-PCR analysis also revealed that ATRA and RAD001 treatment increased expression of C/EBPα and C/EBPε, which is involved in the terminal stages of monocytic differentiation, in all three cell lines and two primary samples compared to treatment with ATRA only. Expression of PU.1 was also increased by combination treatment in all cells tested except the U937 cell line. Western blot analysis revealed that ATRA and RAD001 decreased phosphorylation of C/EBPα at serine 21. ATRA and RAD001 combination treatment also suppressed cell growth in two ATRA-resistant cell lines (growth inhibition rate: 70–80%). The Annexin V assay demonstrated that ATRA and RAD001 combination treatment strongly induced apoptosis in the three cell lines tested. Microarray analysis revealed that FasL,FADD, and caspase 8, which are associated with apoptotic pathways, showed the greatest degree of up-regulation in THP-1 cells treated with ATRA and RAD001. qRT-PCR analysis confirmed up-regulation of these genes in all three cell lines and in both primary AML samples, indicating that ATRA and RAD001 induce apoptosis in AML cells through the extrinsic cell death signaling pathway. Conclusions: RAD001 induced monocytic differentiation through induction of a set of genes associated with monocytic differentiation and phosphorylation of C/EBPα at Ser 21 when combined with ATRA. This combination therapy also induced apoptosis in AML cells through activation of the extrinsic cell death signaling pathway. Disclosures: No relevant conflicts of interest to declare.

Curcumin Enhances Differentiation of All-Trans Retinoic Acid (ATRA)-Sensitive and ATRA-Resistant Acute Promyelocytic (APL) Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4456-4456 ◽  
Author(s):  
Ameet R. Kini ◽  
Moolky Nagabhushan ◽  
Martin S. Tallman ◽  
Shantanu Roychowdhury
Keyword(s):  
Retinoic Acid ◽  
Therapeutic Agent ◽  
Atra Treatment ◽  
Differentiation Therapy ◽  
Cd11b Expression ◽  
Nb4 Cells ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Nbt Reduction ◽  
Therapeutic Doses

Abstract The introduction of ATRA-based differentiation therapy has significantly enhanced outcomes in patients with APL. However, retinoic acid syndrome and ATRA-resistance remain significant concerns. It would therefore be useful to develop drugs that reduce the therapeutic doses of ATRA needed, and would be effective in ATRA-resistant cases. We have shown previously that curcumin, the yellow compound isolated from spice turmeric, suppresses the initiation and promotion stages of cancer development. In the present study we evaluated whether curcumin affects differentiation of NB4 APL cells. The NB4 cells were derived from a patient with APL, and differentiate in response to ATRA, while NB4-R1 cells are resistant to ATRA. Treatment of NB4 cells with 5 μM curcumin enhanced ATRA-mediated differentiation. Differentiation was assessed by evaluating CD11b expression, nitroblue tetrazolium (NBT) reduction and by morphologic examination. This curumin-mediated enhanced differentiation was apparent at 1 μM as well as 0.1 μM of ATRA. Curcumin alone did not cause differentiation of the NB4 cells, although higher concentrations of curcumin caused apoptosis. We then examined the effect of curcumin on the ATRA-resistant NB-R1 cells. Addition of ATRA and curcumin together induced differentiation of the NB4-R1 cells, whereas either agent alone did not cause differentiation. The differentiation was characterized by increased CD11b expression, NBT reduction and the typical morphologic changes. In addition, differentiation of the NB4-R1 cells was accompanied by restoration of the PML-oncogenic domains (PODs). These results indicate that curcumin may be another unconventional therapeutic agent in APL, following the successful use of ATRA and arsenic trioxide.

Restoration of CCAAT Enhancer Binding Protein Alpha P42 Overcomes Myeloid Differentiation Block and All-Trans Retinoic Acid Resistance in Human Acute Promyelocytic Leukemia NB4-R1 Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1238-1238 ◽  
Author(s):  
Limengmeng Wang ◽  
Haowen Xiao ◽  
Xing Zhang ◽  
Weichao Liao ◽  
Shan Fu ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Acute Promyelocytic Leukemia ◽  
Morphological Changes ◽  
Promyelocytic Leukemia ◽  
Mtor Pathway ◽  
Myeloid Differentiation ◽  
Differentiation Therapy ◽  
Cd11b Expression ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid

Abstract All-trans retinoic acid (ATRA) is one of the first line agents in differentiation therapy for acute promyelocytic leukemia (APL). However, drug resistance becomes a major problem which influences the efficacy of ATRA. Mechanisms of ATRA resistance are ugly needed to be identified. Here we found that expression of C/EBPα, an important transcription factor for myeloid differentiation, was significantly suppressed in ATRA resistant APL cell line NB4-R1 compared with ATRA sensitive NB4 cells. Moreover, two forms of C/EBPα were unequally suppressed in NB4-R1 cells. Suppression of the full-length form P42 was more pronounced than the truncated form P30. Inhibition of PI3K/Akt/mTOR pathway was also observed in NB4-R1 cells. Meanwhile, C/EBPα expression was reduced by PI3K inhibitor LY294002 and mTOR inhibitor RAD001 in NB4 cell, suggesting that inactivation of the PI3K/Akt/mTOR pathway was responsible for C/EBPα suppression in APL cells. Then, we restored C/EBPα P42 and P30 by lentivirus vectors in NB4-R1 cells respectively, and found C/EBPα P42, but not P30, could increase CD11b, CD14, G-CSFR and GM-CSFR expression, which indicated the occurrence myeloid differentiation. Moreover, further upregulating of CD11b expression and differential morphological changes were found in NB4-R1 cells with restored C/EBPα P42 after ATRA treatment. However, CD11b expression and differential morphological changes could not be induced by ATRA in NB4-R1 cells infected with P30 expressing or control vector. Thus, we inferred that ATRA sensibility of NB4-R1 cells was enhanced by restoration of C/EBPα P42. In addition, we used histone deacetylase inhibitor trichostatin (TSA) to restore C/EBPα expression in NB4-R1 cells. Similar enhancement of myeloid differentiation and cell growth arrest were detected. Together, this study demonstrated that suppression of C/EBPα P42 induced by PI3K/Akt/mTOR inhibition impaired the differentiation and ATRA sensibility of APL cells. Restoring C/EBPα P42 is an attractive approach for differentiation therapy in ATRA resistant APL. Disclosures No relevant conflicts of interest to declare.

scholarly journals All-trans retinoic acid enhances, and a pan-RAR antagonist counteracts, the stem cell promoting activity of EVI1 in acute myeloid leukemia

2019 ◽  
Vol 10 (12) ◽  
Author(s):  
Chi Huu Nguyen ◽  
Katharina Bauer ◽  
Hubert Hackl ◽  
Angela Schlerka ◽  
Elisabeth Koller ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Retinoic Acid ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Acute Myeloid

AbstractEcotropic virus integration site 1 (EVI1), whose overexpression characterizes a particularly aggressive subtype of acute myeloid leukemia (AML), enhanced anti-leukemic activities of all-trans retinoic acid (atRA) in cell lines and patient samples. However, the drivers of leukemia formation, therapy resistance, and relapse are leukemic stem cells (LSCs), whose properties were hardly reflected in these experimental setups. The present study was designed to address the effects of, and interactions between, EVI1 and retinoids in AML LSCs. We report that Evi1 reduced the maturation of leukemic cells and promoted the abundance, quiescence, and activity of LSCs in an MLL-AF9-driven mouse model of AML. atRA further augmented these effects in an Evi1 dependent manner. EVI1 also strongly enhanced atRA regulated gene transcription in LSC enriched cells. One of their jointly regulated targets, Notch4, was an important mediator of their effects on leukemic stemness. In vitro exposure of leukemic cells to a pan-RAR antagonist caused effects opposite to those of atRA. In vivo antagonist treatment delayed leukemogenesis and reduced LSC abundance, quiescence, and activity in Evi1high AML. Key results were confirmed in human myeloid cell lines retaining some stem cell characteristics as well as in primary human AML samples. In summary, our study is the first to report the importance of EVI1 for key properties of AML LSCs. Furthermore, it shows that atRA enhances, and a pan-RAR antagonist counteracts, the effects of EVI1 on AML stemness, thus raising the possibility of using RAR antagonists in the therapy of EVI1high AML.

Aminopeptidase Inhibitor Bestatin Potentiates the Induced-Differentiation Activity of All-trans-Retinoic Acid in NB4 Cells Possibly through Down-Regulating c-myc Expression.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4443-4443
Author(s):  
Mao-fang Lin ◽  
Xi-jun Qian
Keyword(s):  
Retinoic Acid ◽  
Tumor Therapy ◽  
Expression Level ◽  
Induced Differentiation ◽  
Cd11b Expression ◽  
Nb4 Cells ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Pharmacologic Activity ◽  
Nbt Reduction

Abstract All-trans-retinoic acid (ATRA) represents the sole example of clinically useful cyto-differentiating agent. ATRA treatment alone results in complete remission of nearly 80% patients with acute promyelocytic leukemia (APL). However, the therapeutic use of this compound is limited by a number of problems, including the systemic toxicity and ATRA resistant leukemia. One way to circumvent these problems is to identify the agents capable of enhancing the pharmacologic activity of ATRA. As we know, an aminopeptidase inhibitor, bestatin, had been used as an immunomodulator in anti-tumor therapy. Recently, we have reported bestatin can induce apoptosis in HL-60 and K562 cells. In the present study, we investigated whether bestatin can potentiate the ATRA induced-differentiation of APL cell line NB4 cells and whether changes of transcription factors expression are involved in this course. The cellular morphology observed by optical microscopy, the expression level of CD11b measured by flow cytometry and the nitroblue-tetrazolium (NBT) reduction assay was performed to determine the cyto-differentiation in NB4 cells. The mRNA expression levels of c-myc and c-EBPε in NB4 cells were detected by RT-PCR. NB4 cells incubated with 10nM ATRA plus 100μg/ml bestatin showed more morphologic character of metamyelocyte and band neutrophil than that of the cells treated by ATRA alone. Compared with 10nM ATRA used alone, after treating NB4 cells for 72 hours, the addition of various concentration of bestatin (50μg/ml, 75μg/ml, 100μg/ml) dose-dependently enhancesd NBT reduction of NB4 cells (17.6±2.5 vs. 12.0±2.2, p<0.05; 23.5±3.2 vs. 12.0±2.2, p<0.01; 36.0±8.3 vs. 12.0±2.2, p<0.01, respectively). 100μg/ml bestatin time-dependently increased 10nM ATRA induced NBT reduction of NB4 cells from 24 to 72 hours (p<0.01). The effect of various concentration of ATRA in combination with 100μg/ml bestatin was statistically different with the sum of the effects of individual drugs after subtracting the value of background (31.2±9.1 vs. 12.7±4.3, p<0.01; 39.5±5.0 vs.16.0±1.8, p<0.001; 49.6±5.3 vs. 22.1±1.6, p<0.001, respectively). Moreover, 10nM ATRA plus 100μg/ml bestatin could prominently elevate CD11b expression in NB4 cells compared with ATRA alone treated NB4 cells group(60.58±9.18% vs. 31.95±5.52%, p<0.01), while 100μg/ml bestatin could not induced significant changes in the expression level of CD11b in NB4 cells after 72 hours incubation. The various concentration (50μg/ml, 75μg/ml, 100μg/ml) of bestatin synergizes with 10nM ATRA to down-regulate the expression level of c-myc mRNA (p<0.01), which was inversely correlated with the NBT reduction activity of NB4 cells induced by 10nM ATRA plus various concentration bestatin (r=−0.917, p=0.028). However, 100μg/ml bestatin plus 10nM ATRA could not induce any significant changes in the expression level of c-EBPε mRNA compared with ATRA treated alone group. In conclusion, an aminopeptidase inhibitor bestatin can potentiate ATRA-induced differentiation of NB4 cells, which may be through down-regulating the expression of c-myc in concert with ATRA. Bestatin would be useful in anti-APL therapy by enhancing the pharmacologic activity of ATRA.

scholarly journals Enzasataurin Enhances ATRA-induced Differentiation of Acute Myeloid Leukemia Cells

2020 ◽  
Author(s):  
Ze-yi Li ◽  
Cui Liang ◽  
Ming Ding ◽  
Xiang-qin Weng ◽  
yan Sheng ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Specific Inhibitor ◽  
Akt Inhibitor ◽  
Induced Differentiation ◽  
Cd11b Expression ◽  
Akt Activation ◽  
Protein Levels ◽  
Acute Myeloid

Abstract Background All-trans retinoic acid (ATRA) is considered to be the sole clinically useful differentiating agent in the treatment of acute myeloid leukemia (AML). However, it has been effective only in acute promyelocytic leukemia (APL) but not other subtypes of AML. Therefore, finding strategies to sensitize cells to ATRA may develop ATRA-based therapy in the treatment of non-APL AML patients. Methods Cell proliferation was assessed by cell growth. Cell death was evaluated by cell viability and Annexin-V assay. Cell differentiation was analyzed by CD11b expression and morphology. To explore the underlying mechanisms, we studied the role of PKCβ, MEK, ERK, AKT, PU.1, C/EBPβ and C/EBPε by Western-blotting analysis. Results In this study, a clinically achievable concentration of enzastaurin enhanced ATRA-induced differentiation of AML cell lines, HL-60 and U937 as well as non-APL AML primary cells, while it also restored ATRA sensitivity in ATRA-resistant cell line, HL-60Res. Mechanistically, in all these cell lines, enzastaurin-ATRA (enz-ATRA) enhanced the protein levels of PU.1, CCAAT/enhancer binding protein β (C/EBPβ) and C/EBPε. The activity of protein kinase C β (PKCβ) was suppressed by enz-ATRA treatment in HL-60 and HL-60Res cells. However, another PKCβ-selective inhibitor mimicked the cellular and molecular effects of enzastaurin only in HL-60 cells. Only in U937 cells, enz-ATRA activated MEK and ERK, and a MEK specific inhibitor suppressed enz-ATRA-triggered differentiation and reduced the protein levels of PU.1, C/EBPβ and C/EBPε. Enz-ATRA activated Akt in HL-60 and HL-60Res cells. However, an Akt inhibitor blocked enz-ATRA-triggered differentiation and restored the protein levels of PU.1, C/EBPβ and C/EBPε only in HL-60Res cells. Therefore, PKCβ inhibition, MEK/ERK and Akt activation are involved in enz-ATRA-induced differentiation in HL-60, U937 and HL-60Res cells, respectively by modulation of the protein levels of C/EBPβ, C/EBPε and PU.1. Conclusions Enzastaurin, at the clinically achievable concentration, enhances ATRA-induced differentiation of AML cells by PKCβ inhibition, MEK/ERK and Akt activation. This study may provide a potential therapeutic strategy for AML patients.

scholarly journals Establishment of a human acute promyelocytic leukemia-ascites model in SCID mice

Blood ◽  
1996 ◽  
Vol 87 (8) ◽  
pp. 3404-3409 ◽  
Author(s):  
SY Zhang ◽  
J Zhu ◽  
GQ Chen ◽  
XX Du ◽  
LJ Lu ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Acute Promyelocytic Leukemia ◽  
Fusion Gene ◽  
Promyelocytic Leukemia ◽  
Scid Mice ◽  
Survival Times ◽  
Cd11b Expression ◽  
Nb4 Cells ◽  
Molecular Features ◽  
Nbt Reduction

Acute promyelocytic leukemia (APL) is an interesting model for cancer research because of the presence of the specific PML-RARalpha fusion gene associated with the clinical response to retinoic acid differentiation therapy. To better understand and improve differentiation induction with retinoic acid, we have established a human APL-ascites model in SCID mice using the NB4 human APL cell line. NB4 (1 x 10(6) cells) were transplanted into the peritoneum (IP) of SCID mice for 1 month. NB4 ascites cells (A-NB4) appeared, which were then engrafted in SCID mice periodically for 18 passages at an interval of 3 to 4 weeks with a 100% success rate of tumor induction. The mean survival times of SCID mice transplanted with 1 x 10(6) A-NB4 cells was 21.6 +/- 2.3 days. Analysis of the biologic characteristics of ninth passage NB4 ascitic cells was performed and they were found to have the morphologic, immunologic, cytogenetic, and molecular features of cultured NB4 cells. Furthermore, A-NB4 cells were capable of differentiating when treated with all-trans retinoic acid (ATRA), as manifested by enhanced NBT reduction and CD11b expression. In vivo treatment with ATRA in SCID mice for 4 days also increased NBT reduction by A-NB4 cells. ATRA treatment significantly prolonged survival time in the group after transplantation (28.1 +/- 6.8 to 29.1 +/- 8.4 days) compared with the control (P < .001). Furthermore, treatment with adriamycin, an effective chemotherapeutic drug in APL, had a strong growth suppressive effect on A-NB4 cells. These results demonstrate that this SCID-APL (NB4 ascites cells) model is a useful preclinical system for evaluating new or known drugs in the treatment of APL.

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