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

• 批准号: 8049797
• 负责人: ANDREW NAPPER
• 金额: $ 12.75万
• 依托单位: ALFRED I. DU PONT HOSP FOR CHILDREN
• 依托单位国家: 美国
• 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/8049797
• 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; Gene Rearrangement; Generations; Genes; Genetic Transcription; Goals; HOXA9 gene; Histones; Homeobox; Infant; Inhibitory Concentration 50; Institutes; Lasers; Lead; Length; Letters; Leukemic Cell; Libraries; Lysine; MLL 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; research study; response; self-renewal; small molecule; tool; transcription factor

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）：儿童白血病是在混合谱系白血病（MLL）基因重排产生MLL融合蛋白后发生的，具有侵袭性，非常难以治疗。目前使用的化疗剂量几乎没有增加的余地，因为这是为治疗成人癌症而开发的，对儿童毒性很大。许多MLL融合导致婴儿白血病，他们特别容易受到毒性作用的影响。因此，迫切需要专门设计用于治疗婴儿和幼儿的靶向药物。MLL和转录因子AF 4（来自4号染色体的ALL 1融合基因）的融合导致特别严峻的预后，特别是在婴儿急性淋巴细胞白血病（ALL）中，在所有病例中有一半被发现，并且导致五年无事件生存率仅为34%。MLL-AF 4募集激活HOX（同源异型盒）癌基因HOXA 9转录的蛋白质复合物，并且通过组蛋白甲基转移酶DOT 1 L的异常组蛋白甲基化维持高水平的HOXA 9表达。这种表观遗传控制的破坏导致异常的基因表达，阻断了祖细胞的正常分化，而不是赋予干细胞样的自我更新能力，导致白血病。MLL-AF 4与转录因子AF 9的直接相互作用已被证明是白血病发生过程的关键。该提案的目的是开发MLL-AF 4与AF 9结合的小分子抑制剂的高通量筛选（HTS）测定，并在中试筛选中验证该测定。HTS之后将进行一组二次和反筛选试验，包括化合物结合动力学的生化评价和化合物对白血病细胞系和正常细胞的基于细胞的检测。将使用AlphaScreen技术测量AF 4衍生肽与AF 9蛋白的结合，AlphaScreen技术是一种非常适合HTS的基于均相邻近的方法。供体珠的激光激发导致单线态氧的产生，单线态氧遇到通过AF 4肽与AF 9结合而紧密接近的受体珠，从而产生放大的发光信号。将在微孔板生物传感器中评价降低HTS中发光信号的抑制剂，以确认与AF 9的直接结合。最近的一项研究表明，含有源自AF 4中AF 9结合位点的少至10个氨基酸的肽以低纳摩尔效力结合AF 9，并选择性杀死含有MLL-AF 4的白血病细胞系。将类似地测试结合至AF 9的小分子对携带MLL-AF 4融合蛋白的白血病细胞系的选择性杀伤。最终，将在小鼠白血病模型中测试具有合适药代动力学特性的化合物，并将其优化为用于靶向携带MLL-AF 4的儿科白血病的新型疗法的先导化合物，既作为独立药剂又作为现有化疗剂的协同增效剂。 公共卫生关系：在美国每年发生的2,500例儿童急性淋巴细胞白血病（ALL）中，至少有10%仍然很难治疗，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，对儿童有很高的毒性。由基因MLL（混合谱系白血病）重排引发的白血病（其导致基因表达的正常控制的破坏）具有特别严峻的预后，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。