Hypomethylation of Tumor Suppressor Genes in Acute Myeloid Leukemia: Characteristic of Cell Lines with MLL Abnormalities?.

Abstract Abstract 365 Background: Translocations of the Mixed Lineage Leukemia (MLL) gene occur in a subset (5%) of acute myeloid leukemia (AML) and in mixed phenotype acute leukemia in infancy, a disease with extremely poor prognosis. Animal model systems show that MLL gain of function mutations may contribute to leukemogenesis. Wild-type MLL carries histone methyltransferase activity and affects specific target genes, such us HOXA cluster genes. While the more than three dozen MLL fusion proteins known today exert different specific functions, they finally induce transcription of individual target genes. Consequently, acute lymphoblastic leukemias (ALL) with MLL mutations (MLLmu) exhibit typical gene expression profiles including high-level expression of HOXA cluster genes. Aim of this study was to find a correlation between the MLL mutational status and tumor suppressor gene methylation/expression in acute leukemia cell lines. Results: Using MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification assay), methylation of 24 different TSG was analyzed in 28 MLLmu and MLLwt acute leukemia cell lines. 1.8/24 TSG were methylated in MLLmu AML cells, 6.2/24 TSG were methylated in MLLwt AML cells. Hypomethylation and expression of the tumor suppressor genes (TSG) BEX2, IGSF4 and TIMP3 turned out to be characteristic of MLLmu acute myeloid leukemia (AML) cell lines. MLL wild-type (MLLwt) AML cell lines displayed hypermethylated TSG promoters resulting in transcriptional silencing. Demethylating agents and inhibitors of histone deacetylases restored expression of BEX2, IGSF4 and TIMP3 confirming epigenetic silencing of these genes in MLLwt cells. The positive correlation between MLL translocation, TSG hypomethylation and expression suggested that MLL fusion proteins were responsible for dysregulation of TSG expression in MLLmu cells. This concept was supported by our observation that Bex2 mRNA levels in MLL-ENL transgenic mouse cell lines required expression of the MLL fusion gene. Conclusion: These results suggest that the conspicuous expression of the TSG BEX2, IGSF4 and TIMP3 in MLLmu AML cell lines is the consequence of altered epigenetic properties of MLL fusion proteins. Disclosures: No relevant conflicts of interest to declare.

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Mircrorna-550 Functions As a Critical Tumor Suppressor in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v126.23.1240.1240 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1240-1240

Author(s):

Chao Hu ◽

Xi Jiang ◽

Bryan Ulrich ◽

Yungui Wang ◽

Rui Su ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Tumor Suppressor ◽

Progenitor Cells ◽

Cell Lines ◽

Myeloid Leukemia ◽

Molecular Mechanisms ◽

Target Genes ◽

The Future ◽

Acute Myeloid

Abstract Background: Acute Myeloid leukemia (AML) is one of the most common and fatal form of hematologic malignancies. Recurring chromosomal aberrations and gene mutations have been shown to contribute to AML pathogenesis and clinical outcomes. However, no effective therapy is available to selectively target the cytogenetic and molecular abnormalities, except for PML-RARA in acute promyelocytic leukemia (APL) which can be targeted by all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). As a result, the majority of AML patients are suffering from unsatisfied treatment of standard chemotherapy, associated with high rate of relapse and inferior survival. Thus, better understanding of the molecular mechanisms underlying the pathogenesis and drug resistance of AML, and more effective treatments based on such understanding, are urgently needed. MiRNAs are a class of non-coding RNAs which post-transcriptionally regulate targeted gene expression. They usually consist of 20~24 nucleotides. MiRNAs are closely involved in almost all physiological and pathological processes. The widespread dysregulation of miRNA expressions have been shown to be correlated with various types of malignancies, including AML. In our recent publication, we show through Exiqon miRNA microarray analysis that several miRNAs are significantly down-regulated in most subtypes of de novo AML; miR-550 is one of them (Jiang et al., Cancer Cell, 2012). However, its role and regulatory mechanism in AML have been poorly elucidated. The present study is to investigate the biological functions and molecular mechanisms of miR-550 in AML. Methods: The expression levels of miR-550 were analyzed in multiple AML cell lines, AML patients' bone marrow (BM) mononuclear cells (MNC) and normal MNC control samples by using Taqman miRNA assay qPCR kit. Cell viability and proliferation assays, i.e., MTT assays, were performed in human AML cell lines with stable ectopic expression of miR-550 or control plasmids induced by retrovirus. Cell apoptosis and cell cycle were assessed via flow cytometry analysis. To determine the influence of miR-550 on the transformation capacity of mouse BM progenitor cells transduced with leukemic fusion genes, e.g. MLL-AF9 and AML-ETO9a (AE9a), colony-forming/replating assay (CFA) was carried out. To evaluate the effect of restoration of miR-550 expression/function in AML progression in vivo, we retrovirally infected leukemic blast cells carrying MLL-AF9 with miR-550 or empty vector, and performed secondary BM transplantation by i.v. injecting recipient mice with these donor cells. To identify potential target genes of miR-550, two independent AML patient datasets were analyzed and the correlation patterns between miR-550 and the candidate targets were shown. Results: Consistent with the results of our previous miRNA array, the expression level of miR-550 was significantly down-regulated in most AML patient samples and AML cell lines as compared with normal controls. In AML cell lines, retrovirus induced enforced expression of miR-550 resulted in G1-phase arrest, increased apoptosis, and inhibited cell growth and viability. In mouse BM progenitor cells, forced expression of miR-550 dramatically attenuated colony-forming capacity driven by MLL-AF9 or AE9a. Overexpression of miR-550 significantly inhibited progression of AML induced by MLL-AF9 (MLL-AF9+miR-550, with medium survival of 33 days; MLL-AF9, with medium survival of 27 days; P=0.01) in vivo. We further analyzed in two independent AML patient datasets the expressional correlation between miR-550 and its potential gene targets predicted by miRanda, miRWalk, PITA and Targetscan, etc. 77 candidate target genes inversely correlated with miR-550 in expression. Amongst these genes, FOXE1, IGFBP5 and KSR2 etc. have been shown to be oncogenes and are closely related with AML pathology. Therefore, these genes are candidate oncogenic targets that we will focus on in the future. Conclusions: The above results suggest that miR-550 is an important tumor suppressor in AML. Through targeting a series of oncogenes, miR-550 represses the viability and proliferation of leukemic cells, promotes apoptosis and differentiation, and inhibits cell transformation. Our study indicates that down-regulation of miR-550 likely plays a critical role in AML pathogenesis and restoration of miR-550 might hold great potential in treating AML in the future. Disclosures No relevant conflicts of interest to declare.

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Activation of C/EBPa Myeloid-Specific Transcription Factor By NAMPT/SIRT1-Triggered Deacetylation

Blood ◽

10.1182/blood.v126.23.2199.2199 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2199-2199

Author(s):

Bardia Samareh ◽

Masoud Nasri ◽

Inna Zimmer ◽

Olga Klimenkova ◽

Leonie Keller ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Transcription Factor ◽

Cell Lines ◽

Myeloid Leukemia ◽

Target Genes ◽

Leukemia Cell ◽

Myeloid Differentiation ◽

Granulocytic Differentiation ◽

Acute Myeloid ◽

Myeloid Leukemia Cell

Abstract Previously, we described new mechanism of G-CSF-triggered granulocytic differentiation of hematopoietic stem cells (HSCs) via activation of the enzyme Nicotinamide Phosphorybosyltransferase (NAMPT) leading to NAD+ production and activation of NAD+ -dependent protein deacetylase sirtuin 1 (SIRT1). We found, that upon stimulation of HSCs with NAMPT, SIRT1 bound to the key myeloid transcription factor C/EBPα followed by transcriptional induction of C/EBPα target genes G-CSFR and G-CSF and granulocytic differentiation. In the present work we investigated the mechanism of NAMPT/SIRT1-triggered deacetylation of C/EBPα. We found that C/EBPα is acetylated at the position Lys 161, which is evolutionarily conserved. Lys 161 is localized in the transactivation element III (TE-III) of the transactivation domain (TAD) of C/EBPα protein, which is responsible for recruitment of SWI/SNF and CDK2/CDK4. Western blot and DUOLINK analysis using rabbit polyclonal antibody specifically recognizing acetyl-Lys 161 of C/EBPα revealed predominantly nuclear localization of acetylated C/EBPα protein in acute myeloid leukemia cell lines NB4 and HL60 as well as in primary HSCs. Induction of myeloid differentiation of HSCs by treatment with G-CSF as well as ATRA-induced differentiation of NB4 cells resulted in the deacetylation of C/EBPα. NAMPT inhibition in NB4 and HL60 cell lines using specific inhibitor FK866 led to the dramatically elevated levels of acetylated C/EBPα and reduced amounts of total C/EBPα protein, which was in line with diminished mRNA expression of C/EBPα target genes (G-CSF, G-CSFR and ELANE). Interestingly, treatment of acute myeloid leukemia cell line HL60 with NAMPT or transduction of HL-60 cells with NAMPT-expressing lentiviral construct induced myeloid differentiation of these cells even without addition of ATRA. This was in line with time- and dose-dependent increase of total C/EBPα protein levels upon NAMPT treatment. Therefore, NAMPT overcomes transcriptional repression of C/EBPα in HL-60 cells by activation of positive CEBPA autoregulation. Taken together, we described a new mechanism of regulation of C/EBPα activities in hematopoiesis and leukemogenesis by its post-translational modification via NAMPT/SIRT1-triggered de-/acetylation. Disclosures No relevant conflicts of interest to declare.

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The re-expression of the epigenetically silenced e-cadherin gene by a polyamine analogue lysine-specific demethylase-1 (LSD1) inhibitor in human acute myeloid leukemia cell lines

Amino Acids ◽

10.1007/s00726-013-1485-1 ◽

2013 ◽

Vol 46 (3) ◽

pp. 585-594 ◽

Cited By ~ 27

Author(s):

Tracy Murray-Stewart ◽

Patrick M. Woster ◽

Robert A. Casero

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

Leukemia Cell ◽

Lysine Specific Demethylase ◽

Leukemia Cell Lines ◽

Human Acute Myeloid Leukemia ◽

Polyamine Analogue ◽

Acute Myeloid ◽

Myeloid Leukemia Cell

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TheCDCREL1 gene fused toMLL in de novo acute myeloid leukemia with t(11;22)(q23;q11.2) and its frequent expression in myeloid leukemia cell lines

Genes Chromosomes and Cancer ◽

10.1002/1098-2264(2000)9999:9999<::aid-gcc1084>3.0.co;2-j ◽

2001 ◽

Vol 30 (3) ◽

pp. 230-235 ◽

Cited By ~ 13

Author(s):

Ken Tatsumi ◽

Tomohiko Taki ◽

Masafumi Taniwaki ◽

Hideo Nakamura ◽

Jun Taguchi ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

De Novo ◽

Leukemia Cell ◽

Leukemia Cell Lines ◽

Acute Myeloid ◽

Myeloid Leukemia Cell

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Anti-leukemia properties of cadmium nanoparticles in the in vitro and in vivo condition: A chemobiological study

Archives of Medical Science ◽

10.5114/aoms/142465 ◽

2021 ◽

Author(s):

Yudi Miao ◽

Behnam Mahdavi ◽

Mohammad Zangeneh

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Lines ◽

Aqueous Extract ◽

Myeloid Leukemia ◽

Antioxidant Properties ◽

Leukemia Cell ◽

Sem Images ◽

Acute Myeloid

IntroductionThe present study investigated the anti-acute myeloid leukemia effects of Ziziphora clinopodides Lam leaf aqueous extract conjugated cadmium nanoparticles.Material and methodsTo synthesize CdNPs, Z. clinopodides aqueous extract was mixed with Cd(NO3)2 .4H2O. The characterization of the biosynthesized cadmium nanoparticles was carried out using many various techniques such as UV-Vis. and FT-IR spectroscopy, XRD, FE-SEM, and EDS.ResultsThe uniform spherical morphology of NPs was proved by FE-SEM images with NPs the average size of 26.78cnm. For investigating the antioxidant properties of Cd(NO3)2, Z. clinopodides, CdNPs, and Daunorubicin, the DPPH test was used. The cadmium nanoparticles inhibited half of the DPPH molecules in a concentration of 196 µg/mL. To survey the cytotoxicity and anti-acute myeloid leukemia effects of Cd(NO3)2, Z. clinopodides, CdNPs, and Daunorubicin, MTT assay was used on the human acute myeloid leukemia cell lines i.e., Murine C1498, 32D-FLT3-ITD, and Human HL-60/vcr. The IC50 of the cadmium nanoparticles was 168, 205, and 210 µg/mL against Murine C1498, 32D-FLT3-ITD, and Human HL-60/vcr cell lines, respectively. In the part of in vivo study, DMBA was used for inducing acute myeloid leukemia in mice. CdNPs similar to daunorubicin ameliorated significantly (p≤0.01) the biochemical, inflammatory, RBC, WBC, platelet, stereological, histopathological, and cellular-molecular parameters compared to the other groups.ConclusionsAs mentioned, the cadmium nanoparticles had significant anti-acute myeloid leukemia effects. After approving the above results in the clinical trial studies, these cadmium nanoparticles can be used as a chemotherapeutic drug to treat acute myeloid leukemia in humans.

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The Vitamin E Derivative Gamma Tocotrienol Promotes Anti-Tumor Effects in Acute Myeloid Leukemia Cell Lines

Nutrients ◽

10.3390/nu11112808 ◽

2019 ◽

Vol 11 (11) ◽

pp. 2808 ◽

Cited By ~ 6

Author(s):

Ghanem ◽

Zouein ◽

Mohamad ◽

Hodroj ◽

Haykal ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Vitamin E ◽

Cell Lines ◽

Myeloid Leukemia ◽

Apoptotic Cell ◽

Leukemia Cell ◽

Therapeutic Effects ◽

Apoptotic Pathway ◽

Acute Myeloid

Acute myeloid leukemia (AML) is a blood cancer characterized by the formation of faulty defective myelogenous cells with morphological heterogeneity and cytogenic aberrations leading to a loss of their function. In an attempt to find an effective and safe AML treatment, vitamin E derivatives, including tocopherols were considered as potential anti-tumor compounds. Recently, other isoforms of vitamin E, namely tocotrienols have been proposed as potential potent anti-cancerous agents, displaying promising therapeutic effects in different cancer types. In this study we evaluated the anti-cancerous effects of γ-tocotrienol, on AML cell lines in vitro. For this purpose, AML cell lines incubated with γ-tocotrienol were examined for their viability, cell cycle status, apoptotic cell death, DNA fragmentation, production of reactive oxygen species and expression of proapoptotic proteins. Our results showed that γ-tocotrienol exhibits time and dose-dependent anti-proliferative, pro-apoptotic and antioxidant effects on U937 and KG-1 cell lines, through the upregulation of proteins involved in the intrinsic apoptotic pathway.

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