Myelodysplasia (MDS), a premalignant disorder of hematopoietic stem cells, is usually fatal unless treated with allogeneic bone marrow transplantation, an option not generally available for persons over age 60, who are most often affected by this disease. An area of great urgency is the development of molecularly targeted therapy for a subset of MDS patients defined by a high frequency of complex aberrant karyotypes and p53 mutations (MDS-CAK, ~20% of all MDS cases). In MDS-CAK, P53 mutations block apoptosis in response to DNA damage induced by genotoxic drugs and total-body irradiation, rendering the MDS stem cells resistant to these agents. Ideally, one would like to identify endogenous proteins whose inhibition by antibodies or small-molecules is incompatible with the survival of p53 mutant cells but spares cells expressing the wild- type protein. Our proposal seeks to fill this gap by exploiting the zebrafish experimental system, which affords a faithful model of human myelopoiesis and is uniquely suited to identify critical pathways, using 1) large-scale forward genetic screens and 2) rapid (morpholino-based) candidate-gene approaches. Our underlying hypothesis is that identification of genes 'synthetically lethal'to p53 mutants [s/(p53) genes] will implicate novel pathways through which p53-defective cells evade apoptosis;hence, the wild-type products of such genes should provide useful new therapeutic targets in MDS-CAK and other pathologic conditions associated with p53 mutations. The feasibility of this approach is demonstrated by our recent discovery of chk1 as an s/(p53) gene whose morpholino knockdown suppresses mutant p53e7/e7 -associated resistance to DNA damage-induced apoptosis in myeloid progenitors, while not affecting non-irradiated p53+/+ embryos.
In Aim1 we propose to elucidate the mechanistic basis of the synthetic-lethal relationship between p53 and chk1, by applying in-vivo genetic epistasis and cell biological techniques available for zebrafish research. Specifically, morpholino-based gene targeting in p53 mutant zebrafish will be used to define mechanisms of gene action and compensatory apoptotic pathways, in Aim 2 we have developed the required methodology and expertise to conduct an unbiased, forward-genetic, synthetic-lethal screen in zebrafish using ethylnitrosourea (END) mutagenesis and an acridine-orange embryonic assay. The sl(p53) morphants/mutants identified in Aims 1 and 2 will be tested for synthetic lethality in the zebrafish myeloid lineage by in situ hybridization with a variety of myeloid specific probes, as well as in vivo using our recently generated pu. 1 gfp/p53e7/e7 line. Successful completion of Aims 1 and 2 will yield a group of fully validated sl(p53) genes, with the long-range goal that their wild-type protein products will embody exquisite targets eligible for human phase I clinical trials against MDS-CAK. Lay summary: MDS is a disorder of blood stem cells with a very poor prognosis. This proposal seeks to improve therapy for MDS carrying a particular type of gene defect. The planned research uses the zebrafish animal model system to identify genes whose protein products can be inhibited with antibodies or small molecules, thus causing the MDS cells to be selectively killed, sparing normal cells in the body.
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