MLL associated leukemias account for up to 80% of infant myeloid leukemias and about 10% of adult leukemias demonstrating a need for a thorough understanding of the mechanisms that lead to transformation. MLL is a histone H3 lysine 4 methyltransferase transcription factor that regulates the expression of HOX genes, which are required for MLL induced leukemia. Translocations of the MLL gene fuse the N-terminus of MLL to one of more than 60 different translocation partners resulting in a potent oncogenic fusion protein. Importantly, MLL fusion induced leukemias require expression of the non-mutated wild type MLL allele; implicating wild type MLL in MLL associated leukemias. The long term goal of the proposed research is to identify and characterize regulatory mechanisms for both wild type MLL and MLL fusion proteins which may be disrupted for therapeutic value in myeloid leukemia. Current research focuses on a novel physical interaction between MLL and the Polymerase Associated Factor complex (PAFc). PAFc is a transcription activation complex that associates with RNA polymerase II and promotes histone H2B ubiquitination (a prerequisite for H3 lysine 4 and 79 methylation). The MLL-PAFc interaction is essential for leukemogenesis by MLL fusion proteins. Mechanistically, PAFc synergizes with MLL or MLL fusion proteins to augment transcription by aiding in the recruitment of MLL to target loci. The proposed research focuses on disruption of the MLL-PAFc interaction with the use of peptides and small chemical compounds. A detailed structure of the MLL-PAFc interaction surface will be obtained by nuclear magnetic resonance (NMR) imaging. Chemical library screening will be employed to identify inhibitors of the MLL-PAFc interaction. MLL-PAFc binding will be monitored by fluorescence resonance energy transfer (FRET) and interaction of candidate chemical compounds will be verified by NMR structural analysis. In vivo bone marrow transplantation assays in mouse models will be used to assess the efficacy of peptide or chemical compound mediated disruption of the MLL-PAFc interaction in mitigating MLL fusion induced leukemia. Another current focus is determining the function of highly conserved PHD fingers in MLL. Current research identified ASB2 and an associated E3 ubiquitin ligase complex binds to the PHD fingers of MLL and promotes proteosomal dependent degradation. This is intriguing since the PHD fingers are invariably deleted from MLL fusion proteins and PHD inclusion is deleterious to transformation. The proposed research modulates ASB2 expression to determine the effects on both MLL stability and growth of HOX dependent and HOX independent human cell lines. An expression analysis of the ASB family during hematopoietic development will be performed to test whether ASB proteins degrade MLL protein in mature blood cells. Transformation assays will determine whether ASB2 mediated MLL degradation is incompatible with MLL fusion leukemia. These studies focus on the MLL-PAFc interaction and ubiquitination of MLL which are directly relevant to MLL and HOX dependent leukemias and may prove as effective therapeutic targets.
Mutations in the MLL gene, which lead to deregulated protein function, are one of the most common abnormalities found in human leukemia. Using biochemical and cell biology approaches, protein-protein interactions and regulatory mechanisms were identified that are critical for the onset of MLL associated leukemia. The proposed research will focus on understanding these regulatory molecular mechanisms and the development of therapeutic compounds for the treatment of leukemia.
|Muntean, Andrew G; Chen, Wei; Jones, Morgan et al. (2013) MLL fusion protein-driven AML is selectively inhibited by targeted disruption of the MLL-PAFc interaction. Blood 122:1914-22|