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Structure-Guided Mechanistic Studies of Dot1L in Mixed Lineage Leukemia

Dot1L 在混合谱系白血病中的结构引导机制研究

基本信息

批准号：
10224919
负责人：
Catherine Jeanette Spangler
金额：
$ 3.74万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2022-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10224919
关键词：
Acute leukemia Automobile Driving Cell Differentiation process Cell Line Cell Survival Cells Chimeric Proteins Chromosomal translocation Clinical Clinical Treatment Complex Cryoelectron Microscopy Deposition Development Disease Drug Targeting Enzymes Epigenetic Process Foundations Gene Expression Genes Genetic Genetic Transcription Histone H2B Histone H3 Histones Homeobox Genes In Vitro Knowledge Lead Leukemic Cell Lysine MLL gene Maintenance Malignant Neoplasms Mammalian Cell Methylation Methyltransferase Mixed-Lineage Leukemia Modification Molecular Mutate Mutation Normal Cell Nucleosomes Oncogene Activation Oncogenes Oncogenic Outcome Pathway interactions Phase Post-Translational Protein Processing Postdoctoral Fellow Protein Biochemistry Protein Chemistry Proteins RNA Polymerase II Regulator Genes Reporting Research Resolution Role Site Site-Directed Mutagenesis Structure Transcription Elongation Transcription Regulatory Protein Ubiquitin Up-Regulation Variant Work combat epigenetic regulation genetic regulatory protein histone methylation histone methyltransferase histone modification improved insight leukemia leukemogenesis member mixed lineage leukemia cell new therapeutic target novel novel strategies organizational structure overexpression pre-clinical promoter protein complex recruit structural biology targeted treatment therapeutic development therapeutic target tool tumorigenesis

项目摘要

PROJECT SUMMARY Mixed lineage leukemia is an aggressive subset of acute leukemias with poor clinical outcome and a severe need for improved treatment options. A genetic hallmark of mixed lineage leukemia is chromosomal translocation of the MLL gene, resulting in the formation of MLL-fusion proteins that drive leukemogenesis by mislocalizing essential cellular epigenetic machinery. Many MLL-fusion partner proteins interact with the histone methyltransferase Dot1L, mistargeting its activating methylation mark to developmental regulatory genes that are native targets of MLL during development. Dot1L catalytic activity is essential for both leukemogenic transformation and maintenance, making Dot1L inhibition one of the major strategies underlying current therapeutic development. Defining the molecular basis of Dot1L activity is an essential step towards rational development of Dot1L-targeted therapeutics, but our understanding of the structural and mechanistic basis for Dot1L activity on its native nucleosome substrate is still incomplete. Dot1L methylation of its target histone H3 lysine 79 residue is dependent on prior ubiquitylation of its nucleosome substrate on histone H2B at lysine 120. However, the structural basis for this trans-histone crosstalk has not been established. To elucidate the mechanistic basis for Dot1L activity on its nucleosome substrate and identify novel strategies for Dot1L inhibition, we solved a 3.9Å cryo-EM structure of Dot1L bound to a site-specifically ubiquitylated nucleosome, providing the first high-resolution insight into how Dot1L engages with its nucleosome substrate and is regulated by ubiquitin. Guided by this structure, we identified residues in Dot1L essential for both its nucleosome-specific activity and upregulation by ubiquitin. In Aim 1, we will mutate these Dot1L residues to observe their effect on leukemia cell viability and Dot1L activity at known oncogenes. This work will define the nucleosome-specific and ubiquitin-dependent activities of Dot1L in leukemogenesis and probe the potential of the Dot1L-nucleosome and Dot1L-ubiquitin interfaces as therapeutic targets. Many MLL-fusion partner proteins are also components of the super elongation complex (SEC), a large transcriptional regulatory protein complex that promotes the elongation phase of transcription by releasing RNA polymerase II from promoter-proximal pausing. The SEC is also essential for leukemogenic transformation in mixed lineage leukemia, further promoting misregulation of gene expression at MLL-fusion target genes through overactivation of transcriptional elongation. Still, the structural organization of the SEC and the influence of MLL- fusions on SEC structure and function has not been explored. In Aim 2, I propose a complete structural and functional characterization of the SEC to determine the mechanistic basis for SEC-driven leukemogenic progression in the context of MLL-fusion proteins. Together, these aims will identify novel targets along the molecular pathways driving leukemogenesis to form the pre-clinical foundation for rational therapeutic development.

项目总结混合血统白血病是急性白血病的一种侵袭性亚型，临床转归较差，严重需要改进治疗方案。混合血统白血病的一个遗传特征是染色体易位导致MLL融合蛋白的形成，该融合蛋白通过错误定位而驱动白血病的发生基本的细胞表观遗传机制。许多MLL融合伙伴蛋白与组蛋白相互作用甲基转移酶DOT1L，错误地将其激活的甲基化标记定位为发育调控基因是MLL在开发期间的本地目标。DOT1L催化活性对两种白血病的发生都是必不可少的改造和维护，使DOT1L抑制成为电流的主要策略之一治疗方面的发展。确定DOT1L活性的分子基础是迈向理性的重要一步 DOT1L靶向疗法的发展，但我们对DOT1L靶向治疗的结构和机制基础的理解 DOT1L在其天然核小体底物上的活性仍然不完整。其靶向组蛋白H3赖氨酸79残基的DOT1L甲基化依赖于其核小体先前的泛素化赖氨酸为120的组蛋白H_2B的底物。然而，这种跨组蛋白串扰的结构基础还没有已经建立了。阐明DOT1L在其核小体底物上活性的机制并鉴定针对DOT1L抑制的新策略，我们解决了与位点特异性结合的DOT1L的3.9Cryo-EM结构泛素化的核小体，为DOT1L如何与其核小体接合提供了第一个高分辨率洞察力底物，受泛素调节。在这种结构的指导下，我们确定了DOT1L中对它的核小体特异性活性和泛素的上调。在目标1中，我们将突变这些DOT1L残基观察它们对白血病细胞存活率和已知癌基因DOT1L活性的影响。这项工作将定义 DOT1L在白血病发生中的核小体特异性和泛素依赖活性，并探讨其潜在的作用 DOT1L-核小体和DOT1L-泛素作为治疗靶点。许多MLL融合伙伴蛋白也是超延长复合体(SEC)的组成部分，SEC是一种通过释放RNA促进转录延伸阶段的转录调节蛋白复合体来自启动子-近端停顿的聚合酶II。SEC对白血病的转化也是必不可少的。混合血统白血病，通过进一步促进MLL融合靶基因的基因表达失调转录延伸过度激活。尽管如此，SEC的结构组织和MLL的影响- 关于SEC结构和功能的融合还没有被探索。在目标2中，我提出了一个完整的结构和 SEC的功能特征以确定SEC驱动白血病的机制基础 MLL融合蛋白的研究进展。总之，这些目标将沿着驱动白血病形成的分子途径识别新的靶点。合理开发治疗方法的临床前基础。