The following hypothesis is to be tested. """"""""Transient activation of apoptotic nucleases initiate leukemogenic translocations of MLL through DNA cleavage and error prone repair"""""""". It is proposed that apoptotic execution may be arrested, after targeted DNA damage has occurred, in cells that survive. Preliminary data shows that apoptotic triggers initiate cleavage of MLL, which is subsequently translocated to AF9, creating the leukemogenic MLL-AF9 fusion gene that is transcribed in cells capable of division. The relevance to health care is that this process would represent a novel pathway for the generation of fusion genes implicated in leukemogenesis that would be open to therapeutic intervention.
Three Aims are proposed that sequentially address the mechanism controlling cleavage within MLL, the growth of cells that survive and validation of these in-vitro data with a clinical model of early stage leukemogenesis.
Aim 1 Mechanism of site-specific cleavage in MLL. It is proposed that apoptotic cleavage of MLL is regulated by adjacent sequence motifs, including ATC tracts or topoisomerase II binding sites. These possibilities will be tested by modifying each motif-using site directed mutagenesis within an MLL containing episome (pREP4MLL) that reproduces genomic MLL cleavage in ceils undergoing apoptosis.
Aim 2. Selection of cells that survive apoptotic activation. Cell lines will be selected using the pREP4 episome containing the minimal MLL apoptotic cleavage motif intemal to one of two corrupt antibiotic resistance genes. Apoptotic cleavage within pREP4MLL min will be used to stimulate selection of surviving cells containing a stable copy ofpREP4MLL min by gene conversion repair of the resistance gene.
Aim 3. Detection ofMLL translocations in patients at risk of therapy related leukemia. Blood taken from patients after treatment for non-Hodgkins lymphoma will be analyzed for the presence of MLL-AF9 message by RT-PCR and MLL rearrangements at the apoptotic cleavage site using inverse PCR. In each case the ability of cells containing such aberrations to divide will be determined by the presence of duplicated genomic breakpoint junctions within each patients sample.
|Betti, C J; Villalobos, M J; Jiang, Q et al. (2005) Cleavage of the MLL gene by activators of apoptosis is independent of topoisomerase II activity. Leukemia 19:2289-95|
|Vaughan, Andrew T; Betti, Christopher J; Villalobos, Michael J et al. (2005) Surviving apoptosis: a possible mechanism of benzene-induced leukemia. Chem Biol Interact 153-154:179-85|