Chromosomal translocations that affect the proto-oncogene MLL (Mixed Lineage Leukemia) occur in aggressive human acute leukemia's, both in children and adults. The normal MLL protein plays a key role in regulation of HOX gene expression, which is required for proper hematopoiesis (blood cell development). In leukemia's, this function is destroyed by a fusion of MLL with one of 60 alternative partner genes to form a chimeric oncogenes. MLL fusion proteins upregulate HOX expression resulting in a blockage of blood cell differentiation that ultimately leads to acute leukemia. Patients with leukemia harboring MLL translocations have a very poor prognosis (20 % event free survival at 3 years) and it is clear that there is a need for new therapies to treat these leukemia's. Despite the heterogeneity of fusion partner, the N-terminus of MLL translocations is always invariant and contains the conserved CXXC domain. Deletion of this domain eliminates transactivation properties of oncogenic MLL fusions. The CXXC domain functions as a DNA binding motif that specifically recognizes unmethylated CpG sequences. Importantly, the interaction of the CXXC domain with DNA is absolutely required for leukemogenic function of MLL translocations. To inhibit the oncogenic potential of MLL-translocations we plan to develop small molecules that could specifically inhibit interaction of the CXXC domain with DNA. Here, we propose to use high throughput screening (HTS) to identify inhibitors of this protein-DNA interaction. We propose to use a well-validated fluorescence polarization (FP) assay to screen the MLPCN library of compounds and TR-FRET (time resolved fluorescence resonance energy transfer) as a secondary assay to validate hits obtained by initial screening. To address selectivity, active compounds will be tested by FP with the methyl-CpG DNA binding domain of the MBD2 protein, which binds specifically to methylated CpG DNA elements and not to unmethylated CpG elements. Direct binding of the most active compounds to the CXXC domain will be verified by NMR spectroscopy. The compounds will be subsequently optimized to increase binding affinity to the CXXC domain by structure-based drug design approaches combined with standard medicinal chemistry. The most potent compounds will be tested in appropriate leukemia cell-lines harboring MLL-translocations and, if feasible, in appropriate mouse models of leukemia's.
We are proposing to use high throughput screening (HTS) methods to try to identify chemicals which can inhibit specific proteins that cause leukemia. These chemicals have the potential to be developed into so-called targeted drugs which very specifically attack the proteins causing specific types of leukemia while resulting in limited side-effects, unlike conventional chemotherapy.
