Post-translational regulation of MLL in leukemogenesis

描述(申请人提供)：MLL相关性白血病占婴儿髓系白血病的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是一种组蛋白H3赖氨酸4甲基转移酶转录因子，它调节HOX基因的表达，而HOX基因是MLL诱导白血病所必需的。MLL基因的易位将MLL的N端融合到60多个不同的易位伙伴中的一个，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表达未突变的野生型MLL等位基因；这意味着MLL相关性白血病中存在野生型MLL。这项拟议研究的长期目标是识别和表征野生型MLL和MLL融合蛋白的调节机制，这些融合蛋白可能在髓系白血病的治疗价值方面受到干扰。目前的研究集中在MLL和聚合酶相关因子复合体(PAFC)之间的一种新的物理相互作用。PAFC是一种转录激活复合体，与RNA聚合酶II结合，促进组蛋白H2 B泛素化(H3赖氨酸4和79甲基化的先决条件)。MLL-PAFC相互作用是MLL融合蛋白致白血病所必需的。从机制上讲，PAFC与MLL或MLL融合蛋白协同作用，通过帮助MLL向靶基因位点募集来增强转录。拟议的研究重点是使用多肽和小化合物来破坏MLL-PAFC的相互作用。通过核磁共振成像将获得MLL-PAFC相互作用表面的详细结构。将利用化学文库筛选来鉴定MLL-PAFC相互作用的抑制剂。MLL-PAFC的结合将通过荧光共振能量转移(FRET)来监测，候选化合物之间的相互作用将通过核磁共振结构分析来验证。在小鼠体内的骨髓移植试验将被用来评估多肽或化合物介导的破坏MLL-PAFC相互作用在减轻MLL融合诱导的白血病方面的效果。目前的另一个焦点是确定高度保守的PhD手指在MLL中的功能。目前的研究发现，ASB2和相关的E3泛素连接酶复合体与MLL的PhD手指结合，促进蛋白酶体依赖的降解。这很耐人寻味，因为PhD指总是从MLL融合蛋白中删除，而PHD的包含对转化是有害的。这项拟议的研究通过调节ASB2的表达来确定对HOX依赖和非HOX依赖的人类细胞系MLL稳定性和生长的影响。将对ASB家族在造血发育过程中的表达进行分析，以测试ASB蛋白是否能降解成熟血细胞中的MLL蛋白。转化试验将确定ASB2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的相互作用和MLL的泛素化，这些与MLL和HOX依赖的白血病直接相关，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.