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Inhibition of the MLL-AF4-AF9 Interaction in Pediatric Leukemia

抑制小儿白血病中的 MLL-AF4-AF9 相互作用

基本信息

批准号：
8413724
负责人：
ANDREW NAPPER
金额：
$ 3.31万
依托单位：
ALFRED I. DU PONT HOSP FOR CHILDREN
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-27 至 2012-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8413724
关键词：
AF-9 protein Acute Lymphocytic Leukemia Adult Amino Acids Binding Binding Sites Biochemical Biological Assay Biosensor Blood Cells Cell Cycle Kinetics Cell Line Cells Chemical Agents Chemicals Child Childhood Childhood Acute Lymphocytic Leukemia Childhood Leukemia Chimeric Proteins Chromosomes, Human, Pair 4 Complement DNA Sequence Rearrangement Detection Development Dimethyl Sulfoxide Disease-Free Survival Dose Drug Design Drug Kinetics Employee Strikes Epigenetic Process Evaluation Event Gene Expression Generations Genes Genetic Transcription Goals HOXA9 gene Histones Homeobox Infant Inhibitory Concentration 50 Institutes Lasers Lead Length Letters Leukemic Cell Libraries Lysine MLL gene MLLT2 gene MLLT3 gene Malignant Neoplasms Measures Mediating Methods Methylation Miniaturization Modeling Molecular Molecular Target Morphologic artifacts Mus Myeloid-Lymphoid Leukemia Protein Natural Product Drug Normal Cell Oncogenes Patients Peptides Phenotype Process Property Proteins Protocols documentation Reagent Recruitment Activity Reporting Role Scientist Screening procedure Signal Transduction Singlet Oxygen Stem cells Survival Rate Technology Testing Therapeutic Agents Toxic effect United States Variant base cancer stem cell carcinogenesis cell killing chemotherapeutic agent chemotherapy counterscreen design dosage drug candidate high throughput screening histone methyltransferase inhibitor/antagonist killings leukemia leukemogenesis metaplastic cell transformation novel outcome forecast prevent protein complex public health relevance research study response self-renewal small molecule tool transcription factor

项目摘要

DESCRIPTION (provided by applicant): Childhood leukemia that develops following rearrangement of the mixed lineage leukemia (MLL) gene to give MLL fusion proteins is aggressive and very difficult to treat. There is little scope to increase the dosage of currently used chemotherapy, as this was developed to treat adult cancer and is highly toxic to children. Many of the MLL-fusions give rise to leukemia in infants, who are especially vulnerable to toxic effects. Thus targeted drugs designed specifically to treat infants and young children are urgently needed. The fusion of MLL and the transcription factor AF4 (ALL1 fused gene from chromosome 4) leads to a particularly grim prognosis, especially in infant acute lymphocytic leukemia (ALL), where it is found in half of all cases and results in a five-year event-free survival rate of only 34%. MLL-AF4 recruits a complex of proteins that activate transcription of the HOX (homeobox) oncogene HOXA9, and aberrant histone methylation by the histone methyltransferase DOT1L maintains a high level of HOXA9 expression. This breakdown of epigenetic control resulting in abnormal gene expression blocks normal differentiation of progenitor cells, instead conferring stem cell-like capacity for self-renewal that leads to leukemia. Direct interaction of MLL-AF4 with the transcription factor AF9 has been shown to be critical to this process of leukemogenesis. The aim of this proposal is to develop a high-throughput screening (HTS) assay for small-molecule inhibitors of the binding of MLL-AF4 to AF9 and to validate the assay in a pilot screen. HTS will be followed by a panel of secondary and counterscreen assays consisting of biochemical evaluation of compound binding kinetics and cell-based testing of compounds against leukemia cell lines and normal cells. Binding of an AF4-derived peptide to AF9 protein will be measured using AlphaScreen technology, a homogeneous proximity-based method well-suited to HTS. Laser excitation of donor beads results in the generation of singlet oxygen, which encounters acceptor beads brought into close proximity by the binding of AF4 peptide to AF9, giving an amplified luminescent signal. Inhibitors that reduce the luminescent signal in HTS will be evaluated in a microplate biosensor to confirm direct binding to AF9. A recent study showed that peptides containing as few as 10 amino acids derived from the AF9 binding site in AF4 bind to AF9 with low nanomolar potency and selectively kill an MLL-AF4-containing leukemia cell line. Small molecules binding to AF9 will similarly be tested for selective killing of leukemia cell lines that harbor the MLL-AF4 fusion protein. Ultimately, compounds with suitable pharmacokinetic properties will be tested in a mouse leukemia model and optimized into lead compounds for novel therapies targeted to pediatric leukemia harboring MLL-AF4, both as stand-alone agents and as synergistic potentiators of existing chemotherapeutic agents. PUBLIC HEALTH RELEVANCE: At least 10% of the 2,500 cases of pediatric acute lymphocytic leukemia (ALL) that strike children in the United States each year remain very difficult to treat, and there is little scope to increase the dosage of chemotherapy, which was developed to treat adult cancer and is highly toxic to children. Leukemias triggered by rearrangement of the gene MLL (mixed lineage leukemia), which leads to disruption of the normal control of gene expression, have a particularly grim prognosis, especially in infants. The ultimate goal of this project is to discover targeted molecular therapies that treat these infant leukemias.

描述（由申请人提供）：混合谱系白血病（MLL）基因重排产生 MLL 融合蛋白后发生的儿童白血病具有侵袭性且非常难以治疗。目前使用的化疗剂量几乎没有增加的余地，因为它是为治疗成人癌症而开发的，对儿童具有剧毒。许多 MLL 融合会导致婴儿患白血病，而婴儿特别容易受到毒性作用。因此，迫切需要专门用于治疗婴幼儿的靶向药物。 MLL 和转录因子 AF4（来自 4 号染色体的 ALL1 融合基因）的融合导致了特别严峻的预后，尤其是在婴儿急性淋巴细胞白血病 (ALL) 中，该病占所有病例的一半，导致五年无事件生存率仅为 34%。 MLL-AF4 招募一种蛋白质复合物，激活 HOX（同源盒）癌基因 HOXA9 的转录，组蛋白甲基转移酶 DOT1L 引起的异常组蛋白甲基化维持 HOXA9 的高水平表达。表观遗传控制的破坏导致基因表达异常，阻碍了祖细胞的正常分化，反而赋予了干细胞样的自我更新能力，从而导致白血病。 MLL-AF4 与转录因子 AF9 的直接相互作用已被证明对于白血病发生过程至关重要。该提案的目的是开发一种针对 MLL-AF4 与 AF9 结合的小分子抑制剂的高通量筛选 (HTS) 测定方法，并在中试筛选中验证该测定方法。 HTS 之后将进行一系列二次和反筛选测定，包括化合物结合动力学的生化评估以及针对白血病细胞系和正常细胞的化合物的细胞测试。 AF4 衍生肽与 AF9 蛋白的结合将使用 AlphaScreen 技术进行测量，这是一种非常适合 HTS 的均质邻近方法。供体珠的激光激发会产生单线态氧，单线态氧遇到通过 AF4 肽与 AF9 结合而紧密靠近的受体珠，从而产生放大的发光信号。减少 HTS 中发光信号的抑制剂将在微孔板生物传感器中进行评估，以确认与 AF9 的直接结合。最近的一项研究表明，源自 AF4 中 AF9 结合位点的肽含有少至 10 个氨基酸，以低纳摩尔效力与 AF9 结合，并选择性杀死含有 MLL-AF4 的白血病细胞系。类似地，将测试与 AF9 结合的小分子对含有 MLL-AF4 融合蛋白的白血病细胞系的选择性杀伤。最终，具有合适药代动力学特性的化合物将在小鼠白血病模型中进行测试，并优化为针对携带 MLL-AF4 的儿科白血病的新疗法的先导化合物，既可以作为独立药物，也可以作为现有化疗药物的协同增强剂。公共卫生相关性：美国每年发生 2,500 例小儿急性淋巴细胞白血病 (ALL) 病例，其中至少有 10% 仍然非常难以治疗，而且几乎没有增加化疗剂量的余地，化疗是为治疗成人癌症而开发的，对儿童具有剧毒。由基因MLL（混合谱系白血病）重排引发的白血病会导致基因表达正常控制的破坏，其预后尤其严峻，尤其是在婴儿中。该项目的最终目标是发现治疗这些婴儿白血病的靶向分子疗法。