Identification of Anticancer Drug Targets

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There is increasing evidence that genetic instability underlies the pathogenesis of many cancers, particularly solid tumors. It was recently demonstrated that chromosomal instability (CIN), which occurs in most sporadic colorectal cancers, can be caused by mutations in mitotic-spindle-checkpoint genes. Microsatellite instability (MIN) occurs in most hereditary non-polyposis colorectal cancers (HNPCCs), and is caused by mutations in mismatch-repair genes. Mutations causing CIN and MIN increase tumor heterogeneity, which is thought to drive tumor progression and complicate anticancer drug therapies. However, because these mutations also distinguish tumor cells from normal cells, they may provide critical avenues for the identification of anticancer drug targets. The S. cerevisiae genes BUB1 and MSH2 are homologous to genes known to cause CIN and MIN in human tumors. The existence of yeast homologs of genes with fundamental roles in cancer pathogenesis should allow the identification of anticancer drug targets using synthetic lethal analysis. Synthetic lethal analysis is a technique used by yeast geneticists to identify genes that, when mutated, result in lethality to the cell in the context of mutations in previously characterized genes. Yeast with mutations in BUB1 or MSH2 are known to be viable. Using synthetic lethal analysis, one can identify genes that, when mutated, result in synthetic lethality with BUB1 or MSH2 mutations. The proteins encoded by the human homologs of genes that bring about synthetic lethality represent potential drug targets for cancers with mutations in hBUB1 or hMSH2. The Specific Aims are: 1) To identify genes by synthetic lethal analysis that are required for the viability of S. cerevisiae strains lacking BUB1 or MSH2. Synthetic lethal analysis will be performed using appropriately constructed bub1 and msh2 strains rescued by BUB1-ADE3 and MSH2 ADE3 plasmids, respectively. Synthetic lethality of mutated genes with bub1 and msh2 will be confirmed and the wild-type versions will be cloned by complementation. 2) To test the synthetic lethality of mutations in the human homologs of genes identified in Aim 1 in cells with CIN and MIN due to mutations in hBUB1 or hMSH2. The human homologs of genes identified in Aim 1 will be cloned, and the consequences of their mutation in appropriate cell lines will be determined.