Abstract 1375: A homogeneous high-throughput screening assay for inhibitors of the interaction of MLL-AF4 and AF9 in pediatric leukemia

Abstract

Abstract Chromosomal translocations of the mixed lineage leukemia (MLL) gene occur in 5-10% of pediatric acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), including up to 80% of infant ALL and most treatment-related secondary AML. These MLL rearrangements result in a portion of the MLL protein being fused to one of a number of transcription factors. The prognosis for MLL-rearranged (MLL-R) leukemia is much poorer than for leukemia devoid of MLL fusions; five-year survival of <40% in the case of infant ALL. MLL fusions are potent inducers of leukemia, through recruitment of a protein complex that activates transcription of the HOX (homeobox) oncogene HOXA9, which in turn blocks normal differentiation of progenitor cells and confers a stem cell-like phenotype. The transcription factor AF4 is the most common MLL fusion partner, and MLL-AF4 is associated with a particularly poor prognosis. Direct interaction of MLL-AF4 with the transcription factor AF9 has been shown to be critical to leukemic transformation. Here we describe the development of a homogeneous high-throughput screening (HTS) assay for small-molecule inhibitors of the binding of MLL-AF4 to AF9 and the validation of this assay in pilot HTS. 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 MLL-AF4-containing leukemia cell lines. We have developed an assay that measures binding of an AF4-derived peptide to AF9 protein using AlphaScreen technology, a homogeneous proximity-based method well suited to HTS in 384- and 1536-well plates. 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 are identified by reduction of the luminescent signal. We have validated the assay by demonstrating low nanomolar inhibition by several AF4-derived peptides and by screening a library of 2000 drugs and natural products. The next step is transfer of the assay to the Broad Institute (Cambridge, MA) for HTS against the 400,000-compound NIH library. The ultimate goal is the optimization of lead compounds as novel therapies targeted to pediatric leukemia harboring MLL-AF4, both as stand-alone agents and as synergistic potentiators of existing chemotherapeutic agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1375. doi:10.1158/1538-7445.AM2011-1375