Post-translational regulation of MLL in leukemogenesis

项目概要/摘要 MLL相关白血病占婴儿髓性白血病的高达80%和成人髓性白血病的约10%。 白血病表明需要彻底了解导致转化的机制。 MLL是一种调节HOX表达的组蛋白H3赖氨酸4甲基转移酶转录因子 MLL诱导的白血病所需的基因。MLL基因的易位融合了 MLL与60多种不同的易位伴侣之一结合，产生一种强致癌融合蛋白。 重要的是，MLL融合诱导的白血病需要非突变的野生型MLL等位基因的表达; 表明野生型MLL与MLL相关的白血病有关。拟议研究的长期目标是 鉴定和表征野生型MLL和MLL融合蛋白调节机制， 在髓性白血病中的治疗价值被破坏。目前的研究集中在一种新的物理相互作用上 MLL和聚合酶相关因子复合物（PAFc）之间的相互作用。PAFc是一种转录激活因子， 一种与RNA聚合酶II结合并促进组蛋白H2 B泛素化的复合物（ H3赖氨酸4和79甲基化）。MLL-PAFc相互作用是MLL融合致白血病的关键 proteins.从机制上讲，PAFc与MLL或MLL融合蛋白协同作用，通过帮助MLL或MLL融合蛋白增强转录。 在MLL向靶位点的募集中。拟议的研究重点是破坏MLL-PAFc 与使用肽和小的化学化合物的相互作用。MLL-PAFc的详细结构 相互作用表面将通过核磁共振（NMR）成像获得。化学文库筛选 将用于鉴定MLL-PAFc相互作用的抑制剂。MLL-PAFc结合将通过以下方法监测： 荧光共振能量转移（FRET）和候选化合物的相互作用将是 通过NMR结构分析证实。将使用小鼠模型中的体内骨髓移植试验 为了评估肽或化合物介导的MLL-PAFc相互作用的破坏在 减轻MLL融合诱导的白血病。目前的另一个焦点是确定高度保守的 手指在MLL。目前的研究确定了ASB 2和相关的E3泛素连接酶复合物结合到 PHD指MLL并促进蛋白体依赖性降解。这是耐人寻味的，因为博士 指状物总是从MLL融合蛋白中缺失，而PHD的包含对转化有害。的 拟议的研究调节ASB 2表达，以确定对MLL稳定性和生长的影响。 HOX依赖性和HOX非依赖性人细胞系。ASB家族在人乳腺癌中的表达分析 将进行造血发育以测试ASB蛋白是否降解成熟造血细胞中的MLL蛋白。 血细胞转化试验将确定ASB 2介导的MLL降解是否不相容 MLL融合白血病这些研究集中在MLL-PAFc相互作用和MLL的泛素化， 与MLL和HOX依赖性白血病直接相关，并可被证明是有效的治疗靶点。

Project Summary/Abstract MLL associated leukemias account for up to 80% of infant myeloid leukemias and about 10% of adult leukemias demonstrating a need for a thorough understanding of the mechanisms that lead to transformation. MLL is a histone H3 lysine 4 methyltransferase transcription factor that regulates the expression of HOX genes, which are required for MLL induced leukemia. Translocations of the MLL gene fuse the N-terminus of MLL to one of more than 60 different translocation partners resulting in a potent oncogenic fusion protein. Importantly, MLL fusion induced leukemias require expression of the non-mutated wild type MLL allele; implicating wild type MLL in MLL associated leukemias. The long term goal of the proposed research is to identify and characterize regulatory mechanisms for both wild type MLL and MLL fusion proteins which may be disrupted for therapeutic value in myeloid leukemia. Current research focuses on a novel physical interaction between MLL and the Polymerase Associated Factor complex (PAFc). PAFc is a transcription activation complex that associates with RNA polymerase II and promotes histone H2B ubiquitination (a prerequisite for H3 lysine 4 and 79 methylation). The MLL-PAFc interaction is essential for leukemogenesis by MLL fusion proteins. Mechanistically, PAFc synergizes with MLL or MLL fusion proteins to augment transcription by aiding in the recruitment of MLL to target loci. The proposed research focuses on disruption of the MLL-PAFc interaction with the use of peptides and small chemical compounds. A detailed structure of the MLL-PAFc interaction surface will be obtained by nuclear magnetic resonance (NMR) imaging. Chemical library screening will be employed to identify inhibitors of the MLL-PAFc interaction. MLL-PAFc binding will be monitored by fluorescence resonance energy transfer (FRET) and interaction of candidate chemical compounds will be verified by NMR structural analysis. In vivo bone marrow transplantation assays in mouse models will be used to assess the efficacy of peptide or chemical compound mediated disruption of the MLL-PAFc interaction in mitigating MLL fusion induced leukemia. Another current focus is determining the function of highly conserved PHD fingers in MLL. Current research identified ASB2 and an associated E3 ubiquitin ligase complex binds to the PHD fingers of MLL and promotes proteosomal dependent degradation. This is intriguing since the PHD fingers are invariably deleted from MLL fusion proteins and PHD inclusion is deleterious to transformation. The proposed research modulates ASB2 expression to determine the effects on both MLL stability and growth of HOX dependent and HOX independent human cell lines. An expression analysis of the ASB family during hematopoietic development will be performed to test whether ASB proteins degrade MLL protein in mature blood cells. Transformation assays will determine whether ASB2 mediated MLL degradation is incompatible with MLL fusion leukemia. These studies focus on the MLL-PAFc interaction and ubiquitination of MLL which are directly relevant to MLL and HOX dependent leukemias and may prove as effective therapeutic targets.