The role and mechanism of METTL3/METTL14-mediated RNA modification in the pathogenesis and drug-resistance of AML

项目摘要(摘要)： 背景：N6-甲基腺苷(M6A)修饰是最丰富的内部修饰 真核生物信使RNAs(MRNAs)，在各种正常的生物过程中起着关键作用。证据是 研究发现，m6A及其相关机制也在癌症中发挥作用。急性髓系白血病(AML) 一种最常见、最致命的血液系统恶性肿瘤，具有明显的细胞遗传学特征(如MLL重排) 以及分子(例如，Flt3和NPM1的突变)异常。超过70%的AML患者无法通过 由于耐药和复发而导致的当代治疗。白血病干细胞/起始细胞(LSCs/LICs)是 被认为是急性髓细胞白血病治疗失败和复发的根本原因。因此，更好地理解 AML发病、LSC/LIC自我更新和耐药的分子机制可能导致 开发有效的治疗急性髓系白血病的新治疗策略。RNAm6A修饰由 METTL3-METTL14甲基转移酶复合体(MTC；即编写者)，并可能影响稳定性和/或翻译 靶向mRNA的效率。我们和其他人已经报道了METTL3和METTL14都发挥了致癌作用 在MLL重排的AML中，但它们在其他(即非MLL-AML)的发展和维持中起决定性作用 AML的亚型，尤其是LSC/LIC的自我更新仍然难以捉摸。我们未出版的 初步数据表明，METTL3和METTL14也可能在急性髓系白血病的耐药性中发挥作用，这是有根据的 进一步的系统研究。此外，它们在AML中的关键靶点还没有得到很好的研究。 假设：METTL3和METTL14在AML发病机制、LSC/LIC中起重要致癌作用 通过依赖m6A调节关键靶基因的表达来维持和耐药。 具体目标：1)确定METTL3和METTL14在发育和维护中的作用 不同亚型的AML，以及相关LSC/LIC和正常HSC的自我更新；2)至 确定METTL3和METTL14在急性髓系白血病耐药中的作用；3)破译分子 METTL3和METTL14在AML发生/维持中的病理作用机制， LSC/LIC自我更新与耐药 研究设计：1)我们将使用可诱导的METTL3和Mettl14基因敲除小鼠模型 AML模型以及患者来源的异种移植(PDX)模型，以研究METTL3的作用 和METTL14在不同亚型AML的开发/维护以及LSC/LIC的自我更新和 正常造血干细胞(HSCs)。2)我们将进行体内功能获得和丧失的研究，以 确定METTL3和METTL14是否在AML耐药中起关键作用。3)我们将执行PAR- Clip-seq、m6A-seq、rna-seq、rna寿命分析-seq和核糖体分析-seq以识别所有直接靶标 METTL3和METTL14在急性髓系白血病中的表达及其调控机制 靶标RNA，随后对一组顶级候选靶标进行验证和功能研究。

PROJECT SUMMARY (ABSTRACT): Background: N6-methyladenosine (m6A) modification is the most abundant internal modification in eukaryotic messenger RNAs (mRNAs) and plays critical roles in various normal bioprocesses. Evidence is emerging that m6A and the associated machinery also play roles in cancers. Acute myeloid leukemia (AML) is one of the most common and fatal forms of hematopoietic malignancies with distinct cytogenetic (e.g., MLL-rearranged) and molecular (e.g., mutations in FLT3 and NPM1) abnormalities. Over 70% of AML patients cannot be cured by contemporary therapies due to drug resistance and relapse. The leukemia stem/initiating cells (LSCs/LICs) are considered to be the root cause for the treatment failure and relapse of AML. Thus, it is critical to better understand the molecular mechanisms underlying AML pathogenesis, LSC/LIC self-renewal and drug resistance, which may lead to the development of effective novel therapeutic strategies to treat AML. RNA m6A modification is deposited by the METTL3-METTL14 methyltransferase complex (MTC; i.e., writer), and may affect the stability and/or translation efficiency of target mRNAs. We and others have reported that both METTL3 and METTL14 play oncogenic roles in MLL-rearranged AML, but their definitive roles in the development and maintenance of other (i.e., non-MLL- rearranged) subtypes of AMLs and especially in LSC/LIC self-renewal remain elusive. Our unpublished preliminary data imply that METTL3 and METTL14 may also play roles in drug resistance in AML, which warrants further systematic studies. Furthermore, their critical targets in AMLs have yet to be well investigated. Hypothesis: METTL3 and METTL14 play essential oncogenic roles in AML pathogenesis, LSC/LIC maintenance and drug resistance through m6A-dependent regulation of expression of critical target genes. Specific Aims: 1) To determine the roles of METTL3 and METTL14 in the development and maintenance of different subtypes of AMLs, as well as in the self-renewal of relevant LSCs/LICs and normal HSCs; 2) To determine the roles of METTL3 and METTL14 in drug resistance of AMLs; and 3) To decipher the molecular mechanisms underlying the pathological roles of METTL3 and METTL14 in AML development/maintenance, LSC/LIC self-renewal and drug resistance. Study Design: 1) We will use inducible Mettl3 and Mettl14 knockout mouse models coupled with mouse AML models, as well as patient-derived xeno-transplantation (PDX) models, to investigate the roles of METTL3 and METTL14 in development/maintenance of different subtypes of AMLs and self-renewal of LSCs/ LICs and normal hematopoietic stem cells (HSCs). 2) We will conduct both gain- and loss-of-function in vivo studies to determine whether METTL3 and METTL14 play critical roles in drug resistance of AMLs. 3) We will perform PAR- CLIP-seq, m6A-seq, RNA-seq, RNA life-time profiling-seq, and ribosome profiling-seq to identify all direct targets of METTL3 and METTL14 in AMLs and to understand how METTL3 and METTL14 regulate expression of their target RNAs, followed by validation and functional studies of a set of top candidate targets.