Fto Plays an Oncogenic Role in Acute Myeloid Leukemia As a N6-Methyladenosine RNA Demethylase-猫眼课题宝

DOI：

10.1182/blood.v128.22.2706.2706

发表时间：

2016-12

期刊：

Blood

影响因子：

20.3

通讯作者：

中科院分区：

医学1区

文献类型：

作者：

R. Su;Zejuan Li;Hengyou Weng;X. Weng;Zhixiang Zuo;Chenying Li;Huilin Huang;Chao Hu;Xi Qin;L. Tang;Yungui Wang;Gia-ming Hong;Hao Huang;Xiao Wang;Ping Chen;S. Gurbuxani;Stephen Arnovitz;Yuanyuan Li;Shenglai Li;J. Strong;M. E. Neilly;R. Larson;Xi Jiang;Pumin Zhang;Jie Jin;Chuan He;Jianjun Chen

简介 N6-甲基腺苷 (m6A) 是信使 RNA (mRNA) 中最丰富的内部修饰，主要发生在 G[G>A]m6AC[U>A>C] 的共有基序上。尽管 m6A 修饰在各种基本生物过程中具有重要的功能，但 m6A 修饰在癌症（尤其是白血病）中的研究在很大程度上受到限制。脂肪量和肥胖相关蛋白 (FTO)（第一个 RNA 去甲基酶）已知与人类体重增加和肥胖密切相关。然而，FTO，特别是作为 RNA 去甲基化酶，在癌症发生和进展中的影响仍有待研究。急性髓系白血病 (AML) 是最常见和致命的造血系统恶性肿瘤之一，具有明显的遗传异常和对治疗的不同反应。在这里，我们的目标是定义 FTO 作为 AML 中 m6A 去甲基化酶的作用。方法为了了解 FTO 的潜在作用，我们分析了其在具有不同基因突变的 AML 患者中的表达。为了确定FTO对转化能力/细胞活力和白血病发生的影响，进行了集落形成/再接种测定（CFA）、MTT测定、细胞凋亡和骨髓移植（BMT）。为了确定 FTO 的潜在靶标，进行了全转录组 m6A-seq 和 RNA-seq。为了评估 FTO 对 m6A 修饰和 mRNA 代谢的功能，进行了 m6A 斑点印迹、基因特异性 m6A qPCR 测定和 RNA 稳定性测定。为了阐明 FTO 介导的对其靶标的调节是否取决于其去甲基化酶活性，进行了基因特异性 m6A qPCR 测定以及荧光素酶报告基因和诱变测定。为了研究 FTO 及其靶标在造血中的潜在作用，使用 ATRA 诱导的 APL 细胞分化。结果在对 AML 数据集的分析中，我们发现 FTO 在具有 t(11q23)/MLL 重排、t(15;17)/PML-RARA、FLT3-ITD 和/或 NPM1 突变的 AML 中高表达。慢病毒诱导野生型 FTO 的表达，而非突变型 FTO（携带两个点突变 H231A 和 D233A，破坏其酶活性），在体外显着增强集落形成活性，促进细胞增殖/转化，限制细胞凋亡并降低总体 mRNA m6A 水平。 Fto 显着强制表达 (p) 通过在 MONOMAC-6 AML 细胞中进行全转录组 m6A 测序和 RNA 测序 (RNA-Seq) 测定，无论是否过度表达或敲低 FTO，我们鉴定了 FTO 的两个功能关键靶标：ASB2 和 RARA。强制表达野生型 FTO（而非突变型 FTO）会降低 RARA 和 ASB2 的表达。 ASB2 或 RARA 在很大程度上概括了 FTO 敲低引起的表型。此外，FTO过表达或敲低的作用在很大程度上可以被RARA或ASB2的作用所挽救，表明它们是FTO功能上的重要靶点。 FTO的强制表达和敲低分别降低和增加了ASB2和RARA mRNA转录本上的m6A水平，并分别缩短和延长了重要的是，FTO 降低了具有完整 m6A 位点的 ASB239UTR、RARA39UTR 或 RARA59UTR 构建体的荧光素酶活性，而 m6A 位点的突变消除了这种抑制，表明 FTO 介导的基因调控依赖于其去甲基酶活性。 ATRA 治疗后，FTO 显着 NB4 APL 细胞中 RARA 和 ASB2 下调，而 RARA 和 ASB2 上调。 FTO 的强制表达明显受到抑制，而 FTO 的缺失则增强了 ATRA 诱导的细胞分化。RARA 或 ASB2 的强制表达也可以显着增强 NB4 细胞的分化。结论总之，我们提供了令人信服的体外和体内证据，证明 FTO（一种 m6A）去甲基酶通过降低其关键靶基因（如 ASB2 和 RARA）的 mRNA 转录本中的 m6A 水平，从而触发相应的信号级联反应，在细胞转化和白血病发生以及 ATRA 介导的白血病细胞分化中发挥关键的致癌作用。我们的研究强调了 m6A 修饰机制在白血病中的功能重要性。披露无相关冲突有兴趣申报。

Introduction N6-methyladenosine (m6A) is the most abundant internal modification in messenger RNA (mRNA) mainly occurring at consensus motif of G[G>A]m6AC[U>A>C]. Despite the functional importance of m6A modification in various fundamental bioprocesses, the studies of m6A modification in cancer, especially in leukemia have largely been limited.Fat mass and obesity-associated protein (FTO), the first RNA demethylase,was known to be robustly associated with increased body mass and obesity in humans. However, the impact of FTO, especially as a RNA demethylase, in cancer development and progression has yet to be investigated. Acute myeloid leukemia (AML) is one of the most common and fatal forms of hematopoietic malignancies with distinct geneticabnormalities and variable response to treatment.Here, we aim to definethe roleof FTO as an m6A demethylase in AML. Methods To access the potential effect of FTO, we analyzed its expression in AML patients with distinct genetic mutations. To determine the influence of FTO on transformation capacity/ cell viability and leukemogenesis, colony-forming/replating assay (CFA), MTT assays, cell apoptosis and bone marrow transplantation (BMT) were carried out. To identify potential targets of FTO, transcriptome-wide m6A-seq and RNA-seq were performed. To evaluate the function of FTO on m6A modification and mRNA metabolism,m6A dot blot, gene-specific m6A qPCR assays and RNA stability assays were conducted. To elucidate whether FTO-mediated regulation of its targets depends on its demethylase activity, gene-specific m6A qPCR assays and luciferase reporter and mutagenesis assays were carried out. To investigate the potential roles of FTO and its targets in hematopoiesis, ATRA-induced APL cell differentiation was used. Results In analysis of AML datasets, we found FTO is highly expressed in AMLs with t(11q23)/MLL-rearrangements, t(15;17)/PML-RARA, FLT3-ITD and/or NPM1 mutations. Lentivirus-induced expression of wild-type FTO, but not mutant FTO (carrying two point mutations, H231A and D233A , which disrupt its enzymatic activity), significantly enhanced colony forming activities, promoted cell proliferation/transformation, restricted cell apoptosis and decreased global mRNA m6A levelin vitro. Forced expression of Fto significantly (p Via transcriptome-wide m6A-sequencingand RNA-sequencing (RNA-Seq) assays in MONOMAC-6 AML cells with or without overexpression or knockdown of FTO, we identified two functionally critical targets of FTO, ASB2 and RARA.Forced expression of wild-type FTO, but not mutant FTO, reduced expression of RARA and ASB2. Forced expression of either ASB2 or RARA largely recapitulated the phenotypes caused by FTO knockdown. Moreover, the effects of overexpression or knockdownof FTO can be largely rescued by that of RARA or ASB2, indicating that they are functional important targets of FTO. Forced expression and knockdown of FTO reduced and increased, respectively, the m6A levels on ASB2 and RARA mRNA transcripts,and shortened and prolonged, respectively, the half-life of ASB2 and RARAmRNA transcripts in AML cells.Importantly, FTO reduced luciferase activity ofASB239UTR, RARA39UTR or RARA59UTR constructs with intact m6A sites, while mutations in the m6A sites abrogated the inhibition, demonstrating that FTO-mediated gene regulation relies on its demethylase activity. Upon ATRA treatment, FTO was significantly down-regulated, while RARA and ASB2were up-regulated in NB4 APL cells. Forced expression of FTO noticeably suppressed, while depletion of FTO enhanced, ATRA-induced cell differentiation.Forced expression of either RARA or ASB2 could also substantially enhance NB4 cell differentiation. Conclusions In summary, we provide compelling in vitro and in vivo evidence demonstrating that FTO, an m6A demethylase, plays a critical oncogenic role in cell transformation and leukemogenesis as well as in ATRA-mediated differentiation of leukemic cells, through reducing m6A levels in mRNA transcripts of its critical target genes such as ASB2 and RARA and thereby triggering corresponding signaling cascades. Our study highlights the functional importance of the m6A modification machinery in leukemia. Disclosures No relevant conflicts of interest to declare.

Fto Plays an Oncogenic Role in Acute Myeloid Leukemia As a N6-Methyladenosine RNA Demethylase

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