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Epigenetic Silencing of Eyes Absent 4 Gene by Acute Myeloid Leukemia 1-Eight-twenty-one Oncoprotein Contributes to Leukemogenesis in t(8;21) Acute Myeloid Leukemia.

ABSTRACT: The acute myeloid leukemia 1 (AML1)-eight-twenty-one (ETO) fusion protein generated by the t(8;21)(q22;q22) translocation is considered to display a crucial role in leukemogenesis in AML. By focusing on the anti-leukemia effects of eyes absent 4 (EYA4) gene on AML cells, we investigated the biologic and molecular mechanism associated with AML1-ETO expressed in t(8;21) AML.Qualitative polymerase chain reaction (PCR), quantitative reverse transcription PCR (RT-PCR), and Western blotting analysis were used to observe the mRNA and protein expression levels of EYA4 in cell lines. Different plasmids (including mutant plasmids) of dual luciferase reporter vector were built to study the binding status of AML1-ETO to the promoter region of EYA4. Chromatin immunoprecipitation assay was used to study the epigenetic silencing mechanism of EYA4. Bisulfite sequencing was applied to detect the methylation status in EYA4 promoter region. The influence of EYA4 gene in the cell proliferation, apoptosis, and cell clone-forming ability was detected by the technique of Cell Counting Kit-8, flow cytometry, and clonogenic assay.EYA4 gene was hypermethylated in AML1-ETO+ patients and its expression was down-regulated by 6-fold in Kasumi-1 and SKNO-1 cells, compared to HL-60 and SKNO-1-siA/E cells, respectively. We demonstrated that AML1-ETO triggered the epigenetic silencing of EYA4 gene by binding at AML1-binding sites and recruiting histone deacetylase 1 and DNA methyltransferases. Enhanced EYA4 expression levels inhibited cellular proliferation and suppressed cell colony formation in AML1-ETO+ cell lines. We also found EYA4 transfection increased apoptosis of Kasumi-1 and SKNO-1 cells by 1.6-fold and 1.4-fold compared to negative control, respectively.Our study identified EYA4 gene as targets for AML1-ETO and indicated it as a novel tumor suppressor gene. In addition, we provided evidence that EYA4 gene might be a novel therapeutic target and a potential candidate for treating AML1-ETO+ t (8;21) AML.

SUBMITTER: Huang S

PROVIDER: S-EPMC4894048 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Epigenetic Silencing of Eyes Absent 4 Gene by Acute Myeloid Leukemia 1-Eight-twenty-one Oncoprotein Contributes to Leukemogenesis in t(8;21) Acute Myeloid Leukemia.

Huang Sai S Jiang Meng-Meng MM Chen Guo-Feng GF Qian Kun K Gao Hong-Hao HH Guan Wei W Shi Jin-Long JL Liu An-Qi AQ Liu Jing J Wang Bian-Hong BH Li Yong-Hui YH Yu Li L

Chinese medical journal 20160601 11

<h4>Background</h4>The acute myeloid leukemia 1 (AML1)-eight-twenty-one (ETO) fusion protein generated by the t(8;21)(q22;q22) translocation is considered to display a crucial role in leukemogenesis in AML. By focusing on the anti-leukemia effects of eyes absent 4 (EYA4) gene on AML cells, we investigated the biologic and molecular mechanism associated with AML1-ETO expressed in t(8;21) AML.<h4>Methods</h4>Qualitative polymerase chain reaction (PCR), quantitative reverse transcription PCR (RT-PC ...[more]

PMID: 27231175

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Project description:Backgroundt(8;21)(q22;q22) is one of the most frequent chromosomal abnormalities in acute myeloid leukemia (AML), leading to the generation of the fusion protein AML1-ETO. Despite t(8;21) AML being considered as a subtype with a favorable prognosis, approximately 30-50% of patients experience drug resistance and subsequent relapse. N6-methyladenosine (m6A) is demonstrated to be involved in the development of AML. However, the regulatory mechanisms between AML1-ETO and m6A-related enzymes and the roles of dysregulated m6A modifications in the t(8;21)-leukemogenesis and chemoresistance remain elusive.MethodsChromatin immunoprecipitation, dual-luciferase reporter assay, m6A-qPCR, RNA immunoprecipitation, and RNA stability assay were used to investigate a regulatory loop between AML1-ETO and FTO, an m6A demethylase. Gain- and loss-of-function experiments both in vitro and in vivo were further performed. Transcriptome-wide RNA sequencing and m6A sequencing were conducted to identify the potential targets of FTO.ResultsHere we show that FTO is highly expressed in t(8;21) AML, especially in patients with primary refractory disease. The expression of FTO is positively correlated with AML1-ETO, which is attributed to a positive regulatory loop between the AML1-ETO and FTO. Mechanistically, AML1-ETO upregulates FTO expression through inhibiting the transcriptional repression of FTO mediated by PU.1. Meanwhile, FTO promotes the expression of AML1-ETO by inhibiting YTHDF2-mediated AML1-ETO mRNA decay. Inactivation of FTO significantly suppresses cell proliferation, promotes cell differentiation and renders resistant t(8;21) AML cells sensitive to Ara-C. FTO exerts functions by regulating its mRNA targets, especially IGFBP2, in an m6A-dependent manner. Regain of Ara-C tolerance is observed when IGFBP2 is overexpressed in FTO-knockdown t(8;21) AML cells.ConclusionOur work reveals a therapeutic potential of targeting AML1-ETO/FTO/IGFBP2 minicircuitry in the treatment for t(8;21) patients with resistance to Ara-C.

Dataset Information

Epigenetic Silencing of Eyes Absent 4 Gene by Acute Myeloid Leukemia 1-Eight-twenty-one Oncoprotein Contributes to Leukemogenesis in t(8;21) Acute Myeloid Leukemia.

Publications

Epigenetic Silencing of Eyes Absent 4 Gene by Acute Myeloid Leukemia 1-Eight-twenty-one Oncoprotein Contributes to Leukemogenesis in t(8;21) Acute Myeloid Leukemia.

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