Critical functions specified by the MLL CXXC domain determine leukemogenic capaci

MLL CXXC 结构域指定的关键功能决定白血病发生能力

• 批准号: 7912640
• 负责人: Noah Warren Birch
• 金额: $ 2.75万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-02 至 2013-10-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7912640
• 关键词: Acute leukemia; Affect; Affinity; Alanine; Amino Acids; Aspartate; Binding; Cells; Chimeric Proteins; Chromosomal Rearrangement; Chromosomal translocation; Clinical; Cysteine; DNA; DNA Binding; DNA Binding Domain; Development; Hematopoiesis; Hematopoietic stem cells; Homologous Gene; In Vitro; Ions; Location; MLL gene; MLL2 gene; Modification; Molecular; Mus; Mutation; Names; Oncogene Proteins; Outcome; Play; Positioning Attribute; Post-Translational Protein Processing; Predisposition; Protein Binding; Repression; Repressor Proteins; Research; Role; Side; Specific qualifier value; Sulfhydryl Compounds; Surface; Zinc; in vivo; leukemia; outcome forecast; prevent; public health relevance; therapeutic target

DESCRIPTION (provided by applicant): The Mixed Lineage Leukemia (MLL) gene is involved in chromosomal translocations which generate a fusion protein causing aggressive, acute leukemia with poor prognosis. The resulting chimeric protein retains the amino-terminal portion of MLL including the CXXC DNA-binding domain while the carboxy-terminal portion of the oncoprotein is comprised of the fusion partner. The CXXC domain is found within a larger repression domain and binds to non- methylated CpG DNA. This CXXC DNA-binding domain contains a specific clustering of eight cysteines which coordinate two zinc ions and is essential for the transformation capacity of cells expressing an MLL fusion protein. The closest homolog of MLL, MLL2 (alternatively named MLL4), contains a similar CXXC domain but binds with lower DNA-binding affinity. Interestingly, artificial MLL2-ENL does not transform hematopoietic progenitor cells in vitro. Amino acid differences between the CXXC domain or the larger repression domain of MLL and MLL2 must contribute to these functional differences. Specific amino acids along the DNA contact interface may play a role in differential DNA binding affinity. In addition, we have previously shown that the MLL repression domain (containing the CXXC domain) binds to co-repressor proteins. Differential binding of these repressor proteins to MLL and MLL2 may affect overall function, but might also contribute to CXXC DNA-binding affinity. Cysteine 1188, the only non-zinc-coordinating cysteine residue within the CXXC domain, is critically positioned on the DNA-binding surface and adjacent to one of the conserved cysteines involved in zinc coordination. Mutation of Cys1188 to aspartate completely abrogated the ability of the CXXC domain to bind DNA and prevented both in vitro immortalization and development of leukemia in mice when introduced in the context of MLL-AF9. In contrast, mutation of Cys1188 to alanine retained MLL's ability to bind CpG DNA, as well as to immortalize in vitro and in vivo in the context of the MLL-AF9 fusion. With a critical location on the DNA-binding surface of the CXXC domain and a thiol group susceptible to modification, we hypothesize that the side chain of Cys1188 may be physiologically altered to regulate DNA-binding affinity, allowing this residue to function as a molecular switch that regulates the binding of MLL to its non-methylated CpG DNA targets. Two specific aims are proposed in this application which will explore how specific amino acid residues of the MLL and MLL2 CXXC domains function to alter DNA binding, co-repressor binding, and susceptibility to post-translational modification. We hypothesize that these are critical determination features for MLL's role in acute leukemia. Keywords: MLL, MLL2, leukemia, hematopoiesis PUBLIC HEALTH RELEVANCE: The Mixed Lineage Leukemia (MLL) gene is involved in chromosomal rearrangements that produce MLL fusion proteins and ultimately cause acute leukemia with poor prognosis. It is not yet fully understood how the functions of MLL fusion proteins cause MLL-associated leukemia. The research proposed in this application aims to understand how the MLL functions of DNA binding and repressor protein binding are regulated to ultimately allow for therapeutic targeting of MLL fusion proteins and better clinical outcomes for this devastating form of acute leukemia.

描述（由申请人提供）：混合谱系白血病（MLL）基因参与染色体易位，产生融合蛋白，导致预后不良的侵袭性急性白血病。所得嵌合蛋白保留了MLL的氨基末端部分，包括CXXC DNA结合结构域，而癌蛋白的羧基末端部分由融合伴侣组成。CXXC结构域存在于较大的阻遏结构域中，并与非甲基化的CpG DNA结合。该CXXC DNA结合结构域包含八个半胱氨酸的特定簇，其与两个锌离子配位，并且对于表达MLL融合蛋白的细胞的转化能力是必需的。MLL的最接近的同源物MLL 2（或者称为MLL 4）含有类似的CXXC结构域，但以较低的DNA结合亲和力结合。有趣的是，人工MLL 2-ENL在体外不转化造血祖细胞。MLL和MLL 2的CXXC结构域或更大的抑制结构域之间的氨基酸差异必须有助于这些功能差异。沿着DNA接触界面的特定氨基酸沿着可能在差异DNA结合亲和力中起作用。此外，我们以前已经表明，MLL阻遏结构域（包含CXXC结构域）结合到辅阻遏蛋白。这些阻遏蛋白与MLL和MLL 2的差异结合可能影响整体功能，但也可能有助于CXXC DNA结合亲和力。半胱氨酸1188是CXXC结构域中唯一的非锌配位半胱氨酸残基，位于DNA结合表面的关键位置，与锌配位中的保守半胱氨酸之一相邻。Cys 1188突变为天冬氨酸完全消除了CXXC结构域结合DNA的能力，并且当在MLL-AF 9的背景下引入时，防止了体外永生化和小鼠白血病的发展。相比之下，Cys 1188突变为丙氨酸保留了MLL结合CpG DNA的能力，以及在MLL-AF 9融合的情况下在体外和体内永生化的能力。由于CXXC结构域的DNA结合表面上的关键位置和易于修饰的巯基，我们假设Cys 1188的侧链可能在生理上改变以调节DNA结合亲和力，从而使该残基作为分子开关发挥作用，调节MLL与其非甲基化CpG DNA靶点的结合。在本申请中提出了两个具体目标，其将探索MLL和MLL 2 CXXC结构域的特定氨基酸残基如何起作用以改变DNA结合、共阻遏物结合和对翻译后修饰的敏感性。我们假设这些是MLL在急性白血病中作用的关键决定特征。关键词：MLL，MLL 2，白血病，造血 公共卫生相关性：混合谱系白血病（MLL）基因参与染色体重排，产生MLL融合蛋白，并最终导致预后不良的急性白血病。MLL融合蛋白的功能如何导致MLL相关白血病尚未完全了解。本申请中提出的研究旨在了解如何调节DNA结合和阻遏蛋白结合的MLL功能，以最终实现MLL融合蛋白的治疗靶向，并为这种毁灭性的急性白血病形式提供更好的临床结局。