Post-translational regulation of MLL in leukemogenesis

描述（由申请人提供）：MLL相关白血病占婴儿髓性白血病的80%，约占成人白血病的10%，表明需要彻底了解导致转化的机制。MLL是组蛋白H3赖氨酸4甲基转移酶转录因子，其调节MLL诱导的白血病所需的HOX基因的表达。MLL基因的易位将MLL的N-末端融合到60多种不同易位伴侣中的一种，从而产生有效的致癌融合蛋白。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;在MLL相关白血病中暗示野生型MLL。拟议研究的长期目标是确定和表征野生型MLL和MLL融合蛋白的调节机制，这些机制可能会被破坏，以实现骨髓白血病的治疗价值。目前的研究集中在MLL和聚合酶相关因子复合物（PAFc）之间的一种新的物理相互作用。PAFc是一种转录激活复合物，与RNA聚合酶II结合并促进组蛋白H2 B遍在蛋白化（H3赖氨酸4和79甲基化的先决条件）。MLL-PAFc相互作用对于MLL融合蛋白的白血病发生是必不可少的。从机制上讲，PAFc与MLL或MLL融合蛋白协同作用，通过帮助将MLL募集到靶基因座来增强转录。拟议的研究重点是使用肽和小化合物破坏MLL-PAFc相互作用。将通过核磁共振（NMR）成像获得MLL-PAFc相互作用表面的详细结构。将采用化学文库筛选来鉴定MLL-PAFc相互作用的抑制剂。将通过荧光共振能量转移（FRET）监测MLL-PAFc结合，并通过NMR结构分析验证候选化合物的相互作用。将使用小鼠模型中的体内骨髓移植测定来评估肽或化合物介导的MLL-PAFc相互作用的破坏在减轻MLL融合诱导的白血病中的功效。目前的另一个焦点是确定MLL中高度保守的PHD指的功能。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MLL的PHD指，并促进蛋白体依赖性降解。这是令人感兴趣的，因为PHD指总是从MLL融合蛋白中缺失，并且PHD包含对转化有害。拟议的研究调节ASB 2表达，以确定对HOX依赖性和HOX非依赖性人类细胞系的MLL稳定性和生长的影响。将进行造血发育期间ASB家族的表达分析，以测试ASB蛋白是否降解成熟血细胞中的MLL蛋白。转化试验将确定ASB 2介导的MLL降解是否与MLL融合白血病不相容。这些研究集中在MLL-PAFc相互作用和MLL的泛素化，这与MLL和HOX依赖性白血病直接相关，并可能被证明是有效的治疗靶点。

DESCRIPTION (provided by applicant): 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.