Mechanisms of Leukemogenesis in AMKL

项目摘要/摘要 需要对白血病发生机制进行基础分析，才能开发出最优化的 治疗方法正在接近。N6-甲基腺苷(M6A)是最丰富的RNA修饰，在 RNA代谢、转录本稳定性和翻译效率。M6A写入器和擦除器的异常调节 有助于白血病的发生，尽管甲基转移酶机制改变的确切机制 在白血病中的作用仍有待证实。到目前为止，甲基转移酶编写者的突变还没有被确定， 一个例外：在急性巨核细胞白血病(AMKL)复发的t(1；22)易位中 在m6A编写器中，RBM15与血清反应因子的转录辅助因子MKL1融合 (SRF)。了解RBM15-MKL1(RM)融合蛋白导致白血病的机制将会脱落 M6A RNA甲基化紊乱在白血病发生中的作用，特别是AMKL和AML和TO 一般都是癌症。我们假设，为了发展与RM相关的AMKL，MKL1结构域异常地靶向 RBM15对SRF结合位点的活性以及RBM15将M6A编写器复合体招募到相关RNA， 改变m6A mRNA甲基化和转化所需和/或 对巨核细胞的命运承诺和成熟很重要。我们建议在全基因组范围内聚焦、不偏不倚 研究确定m6A表位转录组和SRF转录网络如何在RBM15- MKL1 AMKL.基因组方法共有的候选靶基因(m6A、RNA)的功能效应 免疫沉淀、染色质作图、RNA稳定性分析和翻译组作图)以及 在RM介导的白血病发生中的关键区域的相关性将通过生长、分化的分析来测试 Rm诱导的小鼠AMKL和原发人AMKL患者的癌基因依赖性 异种移植(PDX)。这些研究具有高度的临床相关性，因为它们解决了一种独特的致病机制 通过全面分析AMKL的基本生物学机制，将揭示以前未知的 表位转录组的调节，这可能是AML和其他癌症的一种新的共同致癌机制。 建议的方法是多方面的，使用细胞系和基因工程动物模型， 以及原发的人类和小鼠白血病样本。这些研究将有助于我们对 阐明m6A RNA甲基酶复合体的一个组分在白血病中的直接作用 揭示了m6A mRNA修饰在AML中的更广泛作用。

Project Summary/Abstract Fundamental analysis of the mechanisms of leukemogenesis is required in order to develop optimized treatment approaches. N6-methyladenosine (m6A), the most abundant RNA modification, plays key roles in RNA metabolism, transcript stability, and translation efficiency. Aberrant regulation of m6A writers and erasers contributes to leukemogenesis, though the exact mechanisms how the methyltransferase machinery is altered in leukemia remains to be shown. Mutations in methyltransferase writers have not been identified to date, with one exception: in the recurrent t(1;22) translocation in acute megakaryoblastic leukemia (AMKL) a component of the m6A writer machinery, RBM15, is fused with MKL1, a transcriptional cofactor of serum response factor (SRF). Understanding the mechanism by which the RBM15-MKL1 (RM) fusion protein causes leukemia will shed light on the role of disordered m6A RNA methylation in leukemogenesis, specifically AMKL but also AML and to cancer in general. We hypothesize that for RM-associated AMKL to develop, the MKL1 domain aberrantly targets RBM15 activity to sites of SRF binding and that RBM15 recruits the m6A writer complex to associated RNAs, altering m6A mRNA methylation and expression of genes that are required for transformation and/or are important for megakaryocyte fate commitment and maturation. We propose focused, unbiased genome-wide studies to determine how the m6A epitranscriptome and the SRF transcriptional networks are coopted in RBM15- MKL1 AMKL. The functional effects of candidate target genes common to the genomic approaches (m6A RNA immunoprecipitation, chromatin mapping, RNA stability assays, and mapping of the translatome) as well as the relevance of critical domains in RM-mediated leukemogenesis will be tested with assays of growth, differentiation and oncogene dependence in RM-induced murine AMKL as well as primary human AMKL patient derived xenotransplants (PDX). The studies are highly clinically relevant as they address a unique mechanism causative of AMKL via comprehensive analysis of fundamental biologic mechanisms, and will reveal previously unidentified regulation of the epitranscriptome that may be a novel shared oncogenic mechanism in AML and other cancers. The proposed approaches are multifaceted, using cell lines as well as genetically engineered animal models, and primary human and murine leukemia samples. The studies will contribute to our understanding of leukemogenesis by elucidating the direct role of a component of the m6A RNA methylase complex in leukemia shedding light on the broader role of m6A mRNA modifications in AML.