Post-translational regulation of MLL in leukemogenesis

项目摘要/摘要 MLL相关性白血病占婴儿髓系白血病的80%，约占成人的10% 白血病表明需要彻底了解导致转化的机制。 MLL是一种组蛋白H3赖氨酸4甲基转移酶转录因子，调节HOX的表达 MLL诱导白血病所必需的基因。MLL基因易位融合N端 MLL与60多个不同易位伙伴中的一个结合，产生一种有效的致癌融合蛋白。 重要的是，MLL融合诱导的白血病需要表达未突变的野生型MLL等位基因； MLL相关性白血病中涉及野生型MLL。拟议研究的长期目标是 鉴定和表征野生型MLL和MLL融合蛋白的调节机制，可能是 对髓系白血病的治疗价值被打乱。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体(PAFC)之间的关系。PAFC是一种转录激活 与RNA聚合酶II结合并促进组蛋白H2B泛素化的复合体(这是 H3赖氨酸4和79甲基化)。MLL-PAFC相互作用在MLL融合致白血病中的作用 蛋白质。在机制上，PAFC与MLL或MLL融合蛋白协同作用，通过辅助转录增强 在以MLL为靶点的招募中。拟议的研究重点是MLL-PAFC的中断 与使用多肽和小化合物的相互作用。MLL-PAFC的详细结构 相互作用表面将通过核磁共振成像获得。化学文库筛选 将被用来确定MLL-PAFC相互作用的抑制剂。MLL-PAFC绑定将由 荧光共振能量转移(FRET)和候选化合物的相互作用 经核磁共振结构分析验证。将使用小鼠模型的体内骨髓移植试验 评价多肽或化合物介导的阻断MLL-PAFC相互作用的有效性。 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 MLL中的博士手指。目前的研究发现ASB2和相关的E3泛素连接酶复合体与 PHD指的MLL和促进蛋白酶体依赖的降解。这很耐人寻味，因为博士 Finger总是从MLL融合蛋白中删除，而PHD包涵体不利于转化。这个 拟议的研究通过调节ASB2的表达来确定对MLL稳定性和生长的影响 HOX依赖和非HOX依赖的人类细胞系。ASB家族在不同时期的表达分析 将进行造血发育，以测试ASB蛋白是否在成熟时降解MLL蛋白 血细胞。转化试验将确定ASB2介导的MLL降解是否不相容 患有MLL融合白血病。这些研究主要集中在MLL-PAFC的相互作用和MLL的泛素化 与MLL和HOX依赖的白血病直接相关，并可能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.