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

描述(申请人提供)：混合血统白血病(MLL)基因重排后发生的儿童白血病(MLL)融合蛋白具有侵袭性，非常难以治疗。目前使用的化疗剂量几乎没有增加的余地，因为这是为了治疗成人癌症而开发的，对儿童具有高度毒性。许多MLL融合会导致婴儿白血病，他们特别容易受到毒性影响。因此，迫切需要专门为治疗婴幼儿而设计的靶向药物。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，特别是在婴儿急性淋巴细胞白血病(ALL)，在所有病例中有一半被发现，导致五年无事件生存率只有34%。MLL-AF4招募一种激活HOX(同源盒)癌基因HOXA9转录的蛋白质复合体，组蛋白甲基转移酶DOT1L异常的组蛋白甲基化维持HOXA9的高水平表达。这种表观遗传控制的崩溃导致基因表达异常，阻止了祖细胞的正常分化，相反，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在这一白血病发生过程中起关键作用。这项建议的目的是建立一种高通量筛选(HTS)方法来检测MLL-AF4与AF9结合的小分子抑制剂，并在中试筛选中验证该方法。HTS之后将进行一系列二级和反筛选分析，包括化合物结合动力学的生化评估以及化合物对白血病细胞株和正常细胞的细胞测试。AF4衍生的多肽与AF9蛋白的结合将使用AlphaScreen技术进行测量，AlphaScreen技术是一种非常适合HTS的基于同质接近的方法。激光激发供体小球产生单线态氧，与AF4多肽与AF9结合而紧密结合的受体小球相遇，产生放大的发光信号。减少HTS中发光信号的抑制剂将在微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，AF4中AF9结合部位含有10个氨基酸的多肽与AF9结合，具有低纳摩尔效力，并选择性地杀伤含有MLL-AF4的白血病细胞系。与AF9结合的小分子也将进行类似的测试，以选择性杀伤含有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，并优化为先导化合物，用于针对携带MLL-AF4的儿童白血病的新疗法，既作为独立药物，也作为现有化疗药物的协同增强剂。 与公共卫生相关：在美国每年发作的2500例儿童急性淋巴细胞白血病(ALL)病例中，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，化疗是为了治疗成人癌症而开发的，对儿童具有高度毒性。由混合血统白血病(MLL)基因重排引发的白血病，导致基因表达的正常控制中断，预后尤其严重，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有针对性的分子疗法。

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.