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

Summary

Principal Investigator: Andrew Napper
Abstract: 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.
Funding Period: -------------------- - --------------------
more information: NIH RePORT

Top Publications

  1. pmc Development of a high-throughput screening-compatible assay for the discovery of inhibitors of the AF4-AF9 interaction using AlphaScreen technology
    VENITA GRESHAM WATSON
    High Throughput Screening and Drug Discovery Lab, Nemours Center for Childhood Cancer Research, A I duPont Hospital for Children, Wilmington, Delaware, USA
    Assay Drug Dev Technol 11:253-68. 2013

Detail Information

Publications1

  1. pmc Development of a high-throughput screening-compatible assay for the discovery of inhibitors of the AF4-AF9 interaction using AlphaScreen technology
    VENITA GRESHAM WATSON
    High Throughput Screening and Drug Discovery Lab, Nemours Center for Childhood Cancer Research, A I duPont Hospital for Children, Wilmington, Delaware, USA
    Assay Drug Dev Technol 11:253-68. 2013
    ....