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Different roles of E proteins in t(8;21) leukemia: E2-2 compromises the function of AETFC and negatively regulates leukemogenesis.

ABSTRACT: The AML1-ETO fusion protein, generated by the t(8;21) chromosomal translocation, is causally involved in nearly 20% of acute myeloid leukemia (AML) cases. In leukemic cells, AML1-ETO resides in and functions through a stable protein complex, AML1-ETO-containing transcription factor complex (AETFC), that contains multiple transcription (co)factors. Among these AETFC components, HEB and E2A, two members of the ubiquitously expressed E proteins, directly interact with AML1-ETO, confer new DNA-binding capacity to AETFC, and are essential for leukemogenesis. However, the third E protein, E2-2, is specifically silenced in AML1-ETO-expressing leukemic cells, suggesting E2-2 as a negative factor of leukemogenesis. Indeed, ectopic expression of E2-2 selectively inhibits the growth of AML1-ETO-expressing leukemic cells, and this inhibition requires the bHLH DNA-binding domain. RNA-seq and ChIP-seq analyses reveal that, despite some overlap, the three E proteins differentially regulate many target genes. In particular, studies show that E2-2 both redistributes AETFC to, and activates, some genes associated with dendritic cell differentiation and represses MYC target genes. In AML patients, the expression of E2-2 is relatively lower in the t(8;21) subtype, and an E2-2 target gene, THPO, is identified as a potential predictor of relapse. In a mouse model of human t(8;21) leukemia, E2-2 suppression accelerates leukemogenesis. Taken together, these results reveal that, in contrast to HEB and E2A, which facilitate AML1-ETO-mediated leukemogenesis, E2-2 compromises the function of AETFC and negatively regulates leukemogenesis. The three E proteins thus define a heterogeneity of AETFC, which improves our understanding of the precise mechanism of leukemogenesis and assists development of diagnostic/therapeutic strategies.

SUBMITTER: Liu N

PROVIDER: S-EPMC6338862 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Different roles of E proteins in t(8;21) leukemia: E2-2 compromises the function of AETFC and negatively regulates leukemogenesis.

Liu Na N Song Junhong J Xie Yangyang Y Wang Xiao-Lin XL Rong Bowen B Man Na N Zhang Meng-Meng MM Zhang Qunling Q Gao Fei-Fei FF Du Mei-Rong MR Zhang Ying Y Shen Jian J Xu Chun-Hui CH Hu Cheng-Long CL Wu Ji-Chuan JC Liu Ping P Zhang Yuan-Liang YL Xie Yin-Yin YY Liu Ping P Huang Jin-Yan JY Huang Qiu-Hua QH Lan Fei F Shen Shuhong S Nimer Stephen D SD Chen Zhu Z Chen Sai-Juan SJ Roeder Robert G RG Wang Lan L Sun Xiao-Jian XJ

Proceedings of the National Academy of Sciences of the United States of America 20181228 3

The AML1-ETO fusion protein, generated by the t(8;21) chromosomal translocation, is causally involved in nearly 20% of acute myeloid leukemia (AML) cases. In leukemic cells, AML1-ETO resides in and functions through a stable protein complex, AML1-ETO-containing transcription factor complex (AETFC), that contains multiple transcription (co)factors. Among these AETFC components, HEB and E2A, two members of the ubiquitously expressed E proteins, directly interact with AML1-ETO, confer new DNA-bindi ...[more]

PMID: 30593567

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Project description:BackgroundThe 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.MethodsQualitative 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.ResultsEYA4 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.ConclusionsOur 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.

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

Different roles of E proteins in t(8;21) leukemia: E2-2 compromises the function of AETFC and negatively regulates leukemogenesis.

Publications

Different roles of E proteins in t(8;21) leukemia: E2-2 compromises the function of AETFC and negatively regulates leukemogenesis.

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