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.

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.

Specific MLL Fusion Partners Affect the Sensitivity of Human and Murine Leukemic Cell Lines to Demethylating Agents

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2158-2158
Author(s):  
Atsushi Fujiki ◽  
Toshihiko Imamura ◽  
Hideki Yoshida ◽  
Yoshifumi Hirashima ◽  
Mitsuru Miyachi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Growth Inhibition ◽  
Cell Lines ◽  
Fusion Proteins ◽  
Leukemic Cells ◽  
Pcr Analysis ◽  
Inhibition Rate ◽  
Monocytic Differentiation ◽  
Murine Cell ◽  
Qrt Pcr

Abstract Abstract 2158 Background and Introduction: The CCAAT/enhancer binding protein (C/EBP)α is a transcriptional factor that plays a key role in granulocytic/monocytic differentiation. We have demonstrated that ATRA and 5-Aza-2′-deoxycytidine (5-Aza) efficiently induce over-expression of C/EBPα, leading to monocytic differentiation, cell cycle arrest and apoptosis in various AML cell lines (Fujiki A, et al. The 51st Annual ASH Meeting). In addition, we also found that THP-1 cells, which express MLL-AF9, are more sensitive to treatment with a combination of ATRA and 5-Aza than KOCL48 cells, which express MLL-AF4. To explore whether the expression of MLL fusion partners affects the degree of monocytic differentiation induced by combination treatment with ATRA and 5-Aza, we compared the expression levels of various transcriptional factors and cell surface markers in human and murine leukemic cells expressing variousMLL fusion proteins. Materials and Methods: THP-1 and KOCL48, two human AML cell lines containing MLL rearrangements, and MLL-AF9, MLL-ENL and MLL-AF5q31, three murine leukemic cell lines expressing MLL fusion proteins, were used in this study. Thee murine cell lines were derived from murine Lin-hematopoietic progenitors transduced by retroviral vectors expressing MLL fusion proteins. To test the effect of Aza and ATRA on cell growth, all of the cell lines were treated with 50 nM 5-Aza for two days, followed by 1 μM ATRA for an additional three or five days. Cell growth was then analyzed by nuclei counting using a Coulter counter. Cell cycle analysis was also performed by flow cytometry (FCM). In addition, an Annexin V assay was performed to measure the level of apoptosis. To assess whether monocytic differentiation was induced, the level of CD11b/Mac 1 expression was evaluated by FCM. In addition, the level of expression of different transcription factors, including C/EBPa, C/EBPe and PU.1/ Sfpi1, was analyzed by quantitative PCR (qRT-PCR) analysis. Results: Although both THP-1 and KOCL-48 cells were moderately resistant to ATRA (growth inhibition rate of 40–50%), the addition of 5-Aza efficiently suppressed the growth in these two cell lines (growth inhibition rate of 80%). Cell cycle analysis revealed that G1 arrest occurred at almost the same level in both cell lines. However, an Annexin V assay revealed that treatment with ATRA and 5-Aza induced 1.5 times more Annexin positive THP-1 cells than KOCL-48 cells. Morphological studies of treated THP-1 and KOCL-48 cells revealed characteristic features of apoptosis, such as an extended cytoplasm containing vacuoles, the presence of fine granules, and irregular shaped nuclei. However, FCM analysis revealed that, following treatment, CD11b was expressed at higher levels in THP-1 cells than in KOCL-48 cells. Similarly, qRT-PCR analysis demonstrated that PU.1 expression was induced to higher levels in the THP-1 cell line than in KOCL-48 cells following ATRA/5-Aza treatment (p<0.05), suggesting that these two cell lines are differentially sensitive to combination therapy. In addition, the murine cell lines expressing either MLL-AF5q31 or MLL-ENL were resistant to treatment with ATRA /5-Aza (growth inhibition rate: 10–30%). However, the murine cell line expressing MLL-AF9 was sensitive to ATRA/5-Aza treatment (growth inhibition rate: 90%). Using qRT-PCR analysis, it was found that C/EBPα and Sfpi1 expression increased in MLL-AF9-expressing murine leukemic cells but not in MLL-ENL or MLL-AF5q31-expressing murine leukemic cells. Conclusions: Monocytic differentiation is more readily induced by combination treatment with ATRA and 5-Aza in MLL-AF9-expressing cells than in MLL-ENL- or MLL-AF4/AF5q31-expressing cells. The presence of specific MLL fusion partners might affect the sensitivity of cell lines to demethylating agents through inducing different degrees of DNA methylation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Increased FLYWCH1 Expression is Negatively Correlated with Wnt/β-catenin Target Gene Expression in Acute Myeloid Leukemia Cells

2019 ◽  
Vol 20 (11) ◽  
pp. 2739
Author(s):  
Amany Almars ◽  
Panagiota S. Chondrou ◽  
Emenike K. Onyido ◽  
Sheema Almozyan ◽  
Claire Seedhouse ◽  
...  
Keyword(s):  
Cell Lines ◽  
Myeloid Leukemia ◽  
Target Gene ◽  
Negative Regulator ◽  
G1 Arrest ◽  
Dividing Cells ◽  
Acute Myeloid Leukemia Cells ◽  
G0 Phase ◽  
New Protein ◽  
Acute Myeloid

Acute myeloid leukaemia (AML) is a heterogeneous clonal malignancy of hematopoietic progenitor cells. The Wnt pathway and its downstream targets are tightly regulated by β-catenin. We recently discovered a new protein, FLYWCH1, which can directly bind nuclear β-catenin. Herein, we studied the FLYWCH1/β-catenin pathway in AML cells using qRT-PCR, Western blot, and immunofluorescence assays. In addition, the stemness activity and cell cycle were analysed by the colony-forming unit (CFU) using methylcellulose-based and Propidium iodide/flow cytometry assays. We found that FLYWCH1 mRNA and protein were differentially expressed in the AML cell lines. C-Myc, cyclin D1, and c-Jun expression decreased in the presence of higher FLYWCH1 expression, and vice versa. There appeared to be the loss of FLYWCH1 expression in dividing cells. The sub-G0 phase was prolonged and shortened in the low and high FLYWCH1 expression cell lines, respectively. The G0/G1 arrest correlated with FLYWCH1-expression, and these cell lines also formed colonies, whereas the low FLYWCH1 expression cell lines could not. Thus, FLYWCH1 functions as a negative regulator of the Wnt/β-catenin pathway in AML.

Identification of Sensitizing Targets to the Hypomethylating Agent 5-Azacytidine in Acute Myeloid Leukemia Cells Using RNAi-Based Functional Genomics Screening

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2665-2665
Author(s):  
Raoul Tibes ◽  
Ashish Choudhary ◽  
Amanda Henrichs ◽  
Sadia Guled ◽  
Irma Monzon ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Growth Inhibition ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Statistical Significance ◽  
Molecular Targets ◽  
Leukemia Cells ◽  
Hypomethylating Agents ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

Abstract • Hypomethylating agents like 5-Azacytidine (5Aza) have become an effective therapy for myelodysplastic syndromes (MDS) and show promise in acute myeloid leukemia (AML). In AML, complimentary mechanisms including epigenetic silencing of growth controlling genes, i.e. tumor suppressors, and activation of kinases contribute to malignant transformation. In order to enhance the therapeutic potential of epigenetic therapies, we developed a high-throughput RNA interference (HT-RNAi) platform for large-scale transient gene silencing in acute myeloid leukemia cells. This assay allows for the first time to individually silence hundreds or thousands of genes in combination with 5Aza to identify molecular targets whose inhibition enhances the anti-leukemic effect of hypomethylating agents. As part of assay development for HT-RNAi, ten AML cell lines were used to determine the median inhibitory concentration (IC50) of 5Aza for each AML cell lines. Furthermore, the ten cell lines were tested with a panel of cationic lipid transfection reagents at varying weight to volume (wt:vol) ratios to determine the optimal siRNA transfection conditions. Results from these studies identified two AML cell lines TF1 and ML4, which were advanced into kinome-epigenetic RNAi screens. Using a lipid-based method, cells were reverse transfected for 48hrs with 2 different siRNA sequences per gene targeting a total of 572 kinases. After 48hrs, 5Aza at the calculated IC25 was added for an additional 72 hrs and cell proliferation was measured using a luminescence-based assay. Data was background corrected and analyzed using the B-score method to report the strength and statistical significance of growth inhibition compared to controls. A B-score of &lt;−2 indicates statistical significance with p&lt;0.05 (&gt;95% confidence); a B-score &lt;−1.5 provides &gt;87% confidence and was used as lowest cutoff given that screens are focused and contain validated siRNA to kinases. Analysis of two independent RNAi kinome screens, one in TF1 and the other in ML4, in combination with 5Aza, identified six and eleven kinases respectively whose silencing by two different siRNA sequences (2× coverage) potentiated the effects of 5Aza at B-score &lt;−1.5. In ML4 cells 2 kinases were highly significant with a B-score for both siRNA &lt;−2. Six kinases were common targets in both cell lines with significant growth inhibition at a B-score for both siRNA of at least &lt;−1.5 making these kinases potential important modifiers of response to 5Aza. In summary, initial kinome RNAi screens in myeloid cells identified specific kinases as potential sensitizing targets to hypomethylating agents. Moreover, functional genomic RNAi screens provide a fast and attractive approach to identify molecular targets in AML for the rational development of combination therapies with hypomethylating agents as well as other drug classes.

Inhibition Of LSD1 As a Therapeutic Strategy For The Treatment Of Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3964-3964 ◽  
Author(s):  
Ryan G. Kruger ◽  
Helai Mohammad ◽  
Kimberly Smitheman ◽  
Monica Cusan ◽  
Yan Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Cell Surface ◽  
Growth Inhibition ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Stem Cell Differentiation ◽  
Ethical Review Process ◽  
Acute Myeloid

Abstract Lysine specific demethylase 1 (LSD1) is a histone H3K4me1/2 demethylase found in various transcriptional co-repressor complexes. These complexes include Histone Deacetylases (HDAC1/2) and Co-Repressor for Element-1-Silencing Transcription factor (CoREST). LSD1 mediated H3K4 demethylation can result in a repressive chromatin environment that silences gene expression. LSD1 has been shown to play a role in development in various contexts. LSD1 can interact with pluripotency factors in human embryonic stem cells and is important for decommissioning enhancers in stem cell differentiation. Beyond embryonic settings, LSD1 is also critical for hematopoietic differentiation. LSD1 is overexpressed in multiple cancer types and recent studies suggest inhibition of LSD1 reactivates the all-trans retinoic acid receptor pathway in acute myeloid leukemia (AML). These studies implicate LSD1 as a key regulator of the epigenome that modulates gene expression through post-translational modification of histones and through its presence in transcriptional complexes. The current study describes the anti-tumor effects of a novel LSD1 inhibitor (GSK2879552) in AML. GSK2879552 is a potent, selective, mechanism-based, irreversible inhibitor of LSD1. Screening of over 150 cancer cell lines revealed that AML cells have a unique requirement for LSD1. While LSD1 inhibition did not affect the global levels of H3K4me1 or H3K4me2, local changes in these histone marks were observed near transcriptional start sites of putative LSD1 target genes. This increase in the transcriptionally activating histone modification correlated with a dose dependent increase in gene expression. Treatment with GSK2879552 promoted the expression of cell surface markers, including CD11b and CD86, associated with a differentiated immunophenotype in 12 of 13 AML cell lines. For example, in SKM-1 cells, increases in cell surface expression of CD86 and CD11b occurred after as early as one day of treatment with EC50 values of 13 and 7 nM respectively. In a separate study using an MV-4-11 engraftment model, increases in CD86 and CD11b were observed as early as 8 hours post dosing. GSK2879552 treatment resulted in a potent anti-proliferative growth effect in 19 of 25 AML cell lines (average EC50 = 38 nM), representing a range of AML subtypes. Potent growth inhibition was also observed on AML blast colony forming ability in 4 out of 5 bone marrow samples derived from primary AML patient samples (average EC50 = 205 nM). The effects of LSD1 inhibition were further characterized in an in vivo mouse model of AML induced by transduction of mouse hematopoietic progenitor cells with a retrovirus encoding MLL-AF9 and GFP. Primary AML cells were transplanted into a cohort of secondary recipient mice and upon engraftment, the mice were treated for 17 days. After 17 days of treatment, control treated mice had 80% GFP+ cells in the bone marrow whereas treated mice possessed 2.8% GFP positive cells (p<0.012). The percentage of GFP+ cells continued to decrease to 1.8% by 1-week post therapy. Remarkably, in a preliminary assessment for survival, control-treated mice succumbed to AML by 28 days post transplant, while treated mice showed prolonged survival. Together, these data demonstrate that pharmacological inhibition of LSD1 may provide a promising treatment for AML by promoting differentiation and subsequent growth inhibition of AML blasts. GSK2879552 is currently in late preclinical development and clinical trials are anticipated to start in 2014. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed. Disclosures: Kruger: GlaxoSmithKline Pharmaceuticals: Employment. Mohammad:GlaxoSmithKline Pharmaceuticals: Employment. Smitheman:GlaxoSmithKline Pharmaceuticals: Employment. Liu:GlaxoSmithKline Pharmaceuticals: Employment. Pappalardi:GlaxoSmithKline Pharmaceuticals: Employment. Federowicz:GlaxoSmithKline Pharmaceuticals: Employment. Van Aller:GlaxoSmithKline Pharmaceuticals: Employment. Kasparec:GlaxoSmithKline Pharmaceuticals: Employment. Tian:GlaxoSmithKline Pharmaceuticals: Employment. Suarez:GlaxoSmithKline Pharmaceuticals: Employment. Rouse:GlaxoSmithKline Pharmaceuticals: Employment. Schneck:GlaxoSmithKline Pharmaceuticals: Employment. Carson:GlaxoSmithKline Pharmaceuticals: Employment. McDevitt:GlaxoSmithKline Pharmaceuticals: Employment. Ho:GlaxoSmithKline Pharmaceuticals: Employment. McHugh:GlaxoSmithKline Pharmaceuticals: Employment. Miller:GlaxoSmithKline Pharmaceuticals: Employment. Johnson:GlaxoSmithKline Pharmaceuticals: Employment. Armstrong:Epizyme Inc.: Has consulted for Epizyme Inc. Other. Tummino:GlaxoSmithKline Pharmaceuticals: Employment.

scholarly journals Voruciclib, an Oral, Selective CDK9 Inhibitor, Enhances Cell Death Induced By the Bcl-2 Selective Inhibitor Venetoclax in Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1361-1361 ◽  
Author(s):  
Daniel A Luedtke ◽  
Yongwei Su ◽  
Holly Edwards ◽  
Lisa Polin ◽  
Juiwanna Kushner ◽  
...  
Keyword(s):  
Cell Lines ◽  
Myeloid Leukemia ◽  
Caspase 3 ◽  
Combination Index ◽  
Survival Rates ◽  
Annexin V ◽  
Antileukemic Activity ◽  
Induction Of Apoptosis ◽  
Induced Apoptosis

Abstract Introduction: Patients with acute myeloid leukemia (AML) face overall 5-year survival rates of 65% and 27% for children and adults, respectively, leaving significant room for improvement. Relapse remains a major contributor to such low overall survival rates, and leukemic stem cells (LSCs) that survive treatment are believed to be responsible for AML relapse. The anti-apoptotic protein Bcl-2 is overexpressed in bulk AML cells and LSCs and is associated with poor clinical outcomes. Thus, Bcl-2 represents a promising therapeutic target for the treatment of AML. Venetoclax (ABT-199) is a selective Bcl-2 inhibitor that has shown great potential for treating a number of malignancies, including AML. Venetoclax inhibits Bcl-2, preventing it from sequestering pro-apoptotic Bcl-2 family protein Bim, leading to Bim activated Bax/Bak, resulting in apoptosis. However, Mcl-1 can also sequester Bim and prevent apoptosis. We previously showed that directly targeting Mcl-1 can enhance the antileukemic activity of venetoclax (Luedtke DA, et al. Signal Transduct Target Ther. Apr 2017). Alternatively, we proposed that indirect targeting of Mcl-1 may preserve or enhance the antileukemic activity of venetoclax, and prevent resistance resulting from Mcl-1. It has been reported that inhibition of CDK9 can downregulate cell survival genes regulated by superenhancers, including Mcl-1, MYC, and Cyclin D1. One CDK9 inhibitor in clinical development, flavopiridol (alvocidib), has progressed to phase II clinical trials in AML. However, off target effects and dose-limiting toxicities remain a concern. Voruciclib is an oral, selective CDK inhibitor differentiated by its potent inhibition of CDK9 as compared to other CDK inhibitors. This selectivity may potentially circumvent toxicities resulting from inhibition of non-CDK targets like MAK and ICK that are inhibited by flavopiridol. Voruciclib has been shown in vitro to promote apoptosis and decrease Mcl-1 expression levels in chronic lymphocytic leukemia (CLL) cells (Paiva C, et al. PLOS One. Nov 2015) and inhibit tumor growth in mouse xenograft models of diffuse large B-cell lymphoma (DLBCL) in combination with venetoclax (Dey J. et al Scientific Reports. Dec 2017). Based on these data, voruciclib may downregulate Mcl-1 in AML cells and therefore synergistically enhance the antileukemic activity of venetoclax. Methods/Results: Culturing AML cell lines (THP-1, U937, MOLM-13, MV4-11, and OCI-AML3) and primary patient samples with various concentrations of voruciclib resulted in a concentration-dependent increase in Annexin V+ cells (2 μM voruciclib induced 13.8-55.8% Annexin V+ cells) along with increased levels of cleaved caspase 3 and PARP, demonstrating that voruciclib induces apoptosis in AML cells. Next, we tested the combination of voruciclib and venetoclax in AML cell lines and primary AML patient samples at clinically achievable concentrations. Annexin V/PI staining, flow cytometry analysis, and combination index calculation (using CalcuSyn software) revealed synergistic induction of apoptosis by voruciclib and venetoclax combination (combination index values for MV4-11, U937, THP-1, and MOLM-13 cells were <0.73; treatment with 2 µM voruciclib and venetoclax for 24 h resulted in >80% apoptosis). Importantly, synergy was observed in both venetoclax sensitive and resistant cell lines. This was accompanied by increased cleavage of caspase 3 and PARP. Lentiviral shRNA knockdown of Bak and Bax partially rescued AML cells from voruciclib-induced apoptosis, showing that voruciclib induces apoptosis at least partially through the intrinsic apoptosis pathway. However, Bak and Bax knockdown had little to no effect on induction of apoptosis by the combination treatment, indicating that there might be other molecular mechanisms underlying the synergistic interaction between the two agents. Treatment with the pan-caspase inhibitor Z-VAD-FMK partially rescued cells from combination treatment induced-apoptosis. Discussion: Collectively, these results demonstrate that voruciclib and venetoclax synergistically induce apoptosis in AML cells in vitro and reverse venetoclax resistance. Further studies to determine the mechanism of action and in vivo efficacy of this promising combination in AML xenografts and PDX models are underway. Disclosures Ge: MEI Pharma: Research Funding.

scholarly journals Biological Evaluation of Arylsemicarbazone Derivatives as Potential Anticancer Agents

2019 ◽  
Vol 12 (4) ◽  
pp. 169
Author(s):  
Anne Cecília Nascimento da Cruz ◽  
Dalci José Brondani ◽  
Temístocles I´talo de Santana ◽  
Lucas Oliveira da Silva ◽  
Elizabeth Fernanda da Oliveira Borba ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Anticancer Agents ◽  
Cell Cycle Progression ◽  
Annexin V ◽  
Biological Evaluation ◽  
G1 Phase ◽  
Cellular Functions ◽  
Ic50 Values ◽  
Annexin V Assay

Fourteen arylsemicarbazone derivatives were synthesized and evaluated in order to find agents with potential anticancer activity. Cytotoxic screening was performed against K562, HL-60, MOLT-4, HEp-2, NCI-H292, HT-29 and MCF-7 tumor cell lines. Compounds 3c and 4a were active against the tested cancer cell lines, being more cytotoxic for the HL-60 cell line with IC50 values of 13.08 μM and 11.38 μM, respectively. Regarding the protein kinase inhibition assay, 3c inhibited seven different kinases and 4a strongly inhibited the CK1δ/ε kinase. The studied kinases are involved in several cellular functions such as proliferation, migration, cell death and cell cycle progression. Additional analysis by flow cytometry revealed that 3c and 4a caused depolarization of the mitochondrial membrane, suggesting apoptosis mediated by the intrinsic pathway. Compound 3c induced arrest in G1 phase of the cell cycle on HL-60 cells, and in the annexin V assay approximately 50% of cells were in apoptosis at the highest concentration tested (26 μM). Compound 4a inhibited cell cycle by accumulation of abnormal postmitotic cells at G1 phase and induced DNA fragmentation at the highest concentration (22 μM).

Bortezomib Sensitizes Non-Hodgkin’s Lymphoma Cells to Apoptosis Induced by Antibodies to TRAIL Receptors R1 and R2.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4614-4614
Author(s):  
Mitchell Reed Smith ◽  
Fang Jin ◽  
Indira Joshi
Keyword(s):  
Growth Inhibition ◽  
Cell Lines ◽  
Annexin V ◽  
Hematologic Malignancies ◽  
Caspase Inhibitor ◽  
Apoptosis Induction ◽  
Minimal Growth ◽  
Trail Receptors ◽  
Independent Mechanism ◽  
Induction Of Apoptosis

Abstract TRAIL-R1 and -R2 signaling induces apoptosis via a pathway that activates caspase 8. The proteosome inhibitor bortezomib may act via several pathways. Agonistic antibodies to TRAIL-R1 and -R2 and bortezomib are in clinical trials in solid tumors and hematologic malignancies. To develop rational combinations for future clinical studies, we investigated the actions of these agents on non-Hodgkin s lymphoma (NHL) cell lines. The t(14;18)+, EBV- NHL cell lines DoHH2 and WSU-FSCCL were treated with agonistic monoclonal antibodies to TRAIL-R1 (HGS-ETR1) and -R2 (HGS-ETR2) (Human Genome Sciences, Rockville, MD) and/or bortezomib. While HGS-ETR 1 and HGS-ETR 2 are effective inducers of apoptosis in FSCCL, DoHH2, which expresses dim TRAIL-R1 (DR4, HGS-ETR1 target) and TRAIL-R2 (DR5, HGS-ETR2 target), shows minimal growth inhibition or apoptosis induction by HGS-ETR1 or HGS-ETR2. Bortezomib has modest effects on DoHH2 cells in growth inhibition and apoptosis assays. HGS-ETR1 and HGS-ETR2 induction of apoptosis in WSU-FSCCL is efficiently blocked by the caspase inhibitor ZVAD. In contrast, bortezomib effects are not blocked by ZVAD, indicating an independent mechanism of action. To determine if these separate pathways would provide enhanced combination activity, DoHH2 cells were pre-treated with bortezomib for 30 min, followed by incubation with HGS-ETR1 or HGS-ETR2. This led to supra-additive induction of apoptosis (annexin V staining). We conclude that bortezomib sensitizes DoHH2 cells to the action of HGS-ETR1 and HGS-ETR2. Further, bortezomib induces apoptosis in DoHH2 cells by an independent mechanism, and the combination of TRAIL-receptor signaling and bortezomib may be a useful combination to explore.

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.

Evaluation of a-Catenin(CTNNA1) As a Tumor Suppressor Gene in Acute Myeloid Leukemis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5240-5240
Author(s):  
Xianwei Zeng ◽  
Ya Liu ◽  
Cui Lv ◽  
Zhenbo Hu
Keyword(s):  
Tumor Suppressor ◽  
Tumor Suppressor Gene ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Suppressor Gene ◽  
Leukemia Cell Line ◽  
G1 Arrest ◽  
Endogenous Expression ◽  
Leukemia Cell Lines

Abstract Abstract 5240 Human a-catenin (CTNNA1) is a 102-kDa cadherin-binding protein that has been recognized as a tumor suppressor gene in solid tumors. Recent studies suggested that a-catenin functional as a tumor suppressor gene in leukemia with 5q deletion as well. In the present study, we speculate that a-catenin may play an important role as a tumor suppressor gene in AML and MDS cases with del(5q), since it is located on chromosome 5, band q31, and within the interval that is consistently deleted in these malignancies. Here, we report the evaluation of a-catenin as a tumor suppressor in myeloid disorders by mutational and functional analysis. Northern blot analysis revealed a very low level of expression of a-catenin mRNA in a panel of leukemia cell lines, whereas it was slightly higher in breast, colon, and prostate cancer lines. Protein truncation analysis in 11 myeloid leukemia cell lines revealed no mutations. By genomic sequencing selected exons in 23 clinical samples of AML/MDS patients with del(5q), we found that 1 of the samples carries a mismatch mutation on exon 5 of the gene. Using pcDNA-catenin expression vector for gene transfection analysis, we found that constitutive expression of a-catenin in the del(5q) leukemia cell line MUTZ-8 inhibited colony formation by 38.7%±6.3%(mea±sd) in the catenin-expressing cells when compared to those with empty vector and caused G1 arrest in the catenin-expressing cells (85.2%±7.3% vs 65.1%±4.6% in control cells) by cell cycle analysis. We conclude that a-catenin causes growth suppression in myeloid leukemia cells, which is consistent with a tumor suppressor gene, and endogenous expression of a-catenin is decreased in most of these cells; However, the finding a-catenin mutation in 1 out of 23 leukemia samples provides limited evidence that it is mutationally inactivated in human leukemias. Disclosures: No relevant conflicts of interest to declare.

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