Critical functions specified by the MLL CXXC domain determine leukemogenic capaci

描述(申请人提供)：混合血统白血病(MLL)基因涉及染色体易位，产生一种融合蛋白，导致侵袭性急性白血病，预后不良。得到的嵌合蛋白保留了MLL的氨基末端部分，包括CXXC DNA结合域，而癌蛋白的羧基末端部分由融合伙伴组成。CXXC结构域在较大的阻遏结构域中被发现，并与非甲基化的CpG DNA结合。这个CXXC DNA结合域包含一个由八个半胱氨酸组成的特定簇，这些半胱氨酸与两个锌离子配位，对于表达MLL融合蛋白的细胞的转化能力是必不可少的。MLL最接近的同源物MLL2(也被命名为MLL4)含有类似的CXXC结构域，但结合DNA的亲和力较低。有趣的是，人工mll2-enl在体外不能转化造血祖细胞。CXXC结构域或MLL和MLL2较大的阻遏结构域之间的氨基酸差异肯定是导致这些功能差异的原因。DNA接触界面上的特定氨基酸可能在不同的DNA结合亲和力中发挥作用。此外，我们之前已经证明MLL抑制域(包含CXXC结构域)与共抑制蛋白结合。这些阻遏蛋白与MLL和MLL2的不同结合可能会影响整体功能，但也可能有助于CXXC DNA结合的亲和力。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，与锌配位相关的保守半胱氨酸相邻。当在MLL-AF9的背景下引入Cys1188到天冬氨酸的突变时，完全取消了CXXC结构域与DNA结合的能力，并防止了体外永生化和小鼠白血病的发展。相反，Cys1188突变为丙氨酸保留了MLL与CpG DNA结合的能力，以及在MLL-AF9融合的情况下在体外和体内永生化的能力。由于Cys1188在CXXC结构域的DNA结合表面上有一个关键位置，并且硫醇基团容易被修饰，我们假设Cys1188的侧链可能被生理上改变来调节DNA结合亲和力，使这个残基能够作为一个分子开关来调节MLL与其非甲基化的CpG DNA靶标的结合。在这项申请中提出了两个特定的目标，将探索MLL和M112CXXC结构域的特定氨基酸残基如何功能改变DNA结合、共抑制物结合和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确定特征。关键词：MLL、MLL2、白血病、造血 公共卫生相关性：混合血统白血病(MLL)基因参与染色体重排，产生MLL融合蛋白，最终导致急性白血病，预后不良。目前还不完全清楚MLL融合蛋白的功能如何导致MLL相关性白血病。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，以最终允许MLL融合蛋白的治疗靶向，并改善这种毁灭性的急性白血病的临床结果。

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.