Disrupting the AF4-AF9 protein complex in MLL leukemias.

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DESCRIPTION (provided by applicant): The ultimate goal of this project is to develop promising new agents for the treatment of acute lymphoblastic leukemia (ALL) characterized by a specific t(4;11) chromosome translocation. t(4;11) leukemia is a relatively common subtype of ALL and is unusually resistant to cytotoxic chemotherapy. The isolation, validation, and optimization of new compounds to treat t(4;11) leukemia is important given the limitations of currently available treatments. We have identified a protein:protein interaction that appears to be critical for the survival of t(4;11) leukemia. More important, small peptides that disrupt the interaction of these two proteins, AF4 and AF9, induce programmed cell death in t(4;11) leukemia cells. Additionally, these peptides do not inhibit the growth and differentiation of normal hematopoietic progenitor cells. These data indicate that the AF4-AF9 protein complex is a promising target for drug development. The NIH Molecular Libraries Screening Center Network offers a unique opportunity to screen an extensive molecular library to identify additional compounds that block AF4-AF9 binding. Here, we propose to develop an assay system that is well suited for high throughput screening of inhibitors of AF4-AF9 binding. Fluorescence polarization (FP) is a powerful technique to measure protein binding in a homogeneous solution. The technique is particularly well suited to measuring AF4-AF9 binding as the molecular complex can be mimicked by a peptide "probe" bound to the C-terminal domain of AF9. Furthermore, FP assays can be easily modified to analyze inhibitors of protein binding. FP competition assays have been used successfully for high throughput screening of inhibitors of other protein:protein interactions. Once optimized, the assay is simple, rapid, and inexpensive. In addition to a primary FP screening assay, we describe secondary assays to analyze "hits". These secondary assays include analyses of the anti-leukemic activity of lead compounds and have already been validated. It is anticipated that, following refinement, compounds identified and verified by this screening process will provide useful new anti-leukemic therapies. Despite improvements in the treatment of leukemia, some forms of the disease remain resistant to therapy. Here, we describe a system to rapidly identify chemicals that may serve as new drugs for the treatment of one of the most resistant forms of acute leukemia. We propose to develop and refine this testing system so that it may be used to isolate promising drug candidates from a large collection of chemicals developed by NIH.