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系白血病的80%，约占成人白血病的10%，这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端与60多种不同的易位伙伴之一融合，从而产生一种强效的致癌融合蛋白。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；暗示野生型MLL与MLL相关白血病有关。该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，这些蛋白可能被破坏以用于髓性白血病的治疗价值。目前的研究重点是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相关，促进组蛋白H2B泛素化（H3赖氨酸4和79甲基化的先决条件）。MLL- pafc相互作用对MLL融合蛋白的白血病发生至关重要。从机制上讲，PAFc与MLL或MLL融合蛋白协同作用，通过帮助MLL招募到靶位点来增加转录。提出的研究重点是利用肽和小化合物破坏MLL-PAFc相互作用。核磁共振成像将获得mll - pacc相互作用表面的详细结构。化学文库筛选将用于鉴定MLL-PAFc相互作用的抑制剂。MLL-PAFc结合将通过荧光共振能量转移（FRET）监测，候选化合物的相互作用将通过核磁共振结构分析验证。小鼠模型的体内骨髓移植试验将用于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。另一个当前的焦点是确定MLL中高度保守的PHD指的功能。目前的研究发现ASB2和相关的E3泛素连接酶复合物与MLL的PHD指结合并促进蛋白体依赖性降解。这是有趣的，因为PHD手指总是从MLL融合蛋白中删除，PHD包含对转化有害。本研究通过调节ASB2的表达来确定对依赖和不依赖HOX的人细胞系的MLL稳定性和生长的影响。我们将对ASB家族在造血发育过程中的表达进行分析，以检测ASB蛋白是否会在成熟血细胞中降解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- pacc相互作用和MLL泛素化上，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. PUBLIC HEALTH RELEVANCE: Mutations in the MLL gene, which lead to deregulated protein function, are one of the most common abnormalities found in human leukemia. Using biochemical and cell biology approaches, protein-protein interactions and regulatory mechanisms were identified that are critical for the onset of MLL associated leukemia. The proposed research will focus on understanding these regulatory molecular mechanisms and the development of therapeutic compounds for the treatment of leukemia.