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

描述（由申请人提供）：混合谱系白血病（MLL）基因重排后发展为MLL融合蛋白的儿童白血病具有侵袭性，非常难以治疗。目前使用的化疗药物几乎没有增加剂量的余地，因为这是为了治疗成人癌症而开发的，对儿童有很高的毒性。许多mll融合在婴儿中引起白血病，婴儿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）的融合导致了特别糟糕的预后，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发现了MLL，并导致5年无事件生存率仅为34%。MLL-AF4募集一组蛋白复合物，激活HOX（同源盒）癌基因HOXA9的转录，而组蛋白甲基转移酶DOT1L的异常组蛋白甲基化维持了高水平的HOXA9表达。这种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，而不是赋予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这一白血病发生过程至关重要。本提案的目的是开发MLL-AF4与AF9结合的小分子抑制剂的高通量筛选（HTS）试验，并在中试筛选中验证该试验。HTS之后将进行一组二级和反筛分析，包括化合物结合动力学的生化评估和基于细胞的化合物对白血病细胞系和正常细胞的测试。af4衍生肽与AF9蛋白的结合将使用alphasgreen技术进行测量，该技术是一种非常适合HTS的基于均质近似的方法。激光激发供体小珠产生单线态氧，与因AF4肽与AF9结合而靠近的受体小珠相遇，发出放大的发光信号。减少HTS中发光信号的抑制剂将在微孔板生物传感器上进行评估，以确认与AF9的直接结合。最近的一项研究表明，从AF4的AF9结合位点衍生的含有10个氨基酸的肽以低纳摩尔效价与AF9结合，选择性地杀死含有mll -AF4的白血病细胞系。与AF9结合的小分子也将被用于选择性杀死含有MLL-AF4融合蛋白的白血病细胞系。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，并优化为针对含有MLL-AF4的儿童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。

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.