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High-grade serous ovarian cancers (HGSCs) are characterized by a high frequency of TP53 mutations, BRCA1/2 inactivation, homologous recombination dysfunction, and widespread copy number changes. Cyclin E1 (CCNE1) gene amplification has been reported to occur independently of BRCA1/2 mutation, and it is associated with primary treatment failure and reduced patient survival. Insensitivity of CCNE1-amplified tumors to platinum cross-linking agents may be partly because of an intact BRCA1/2 pathway. Both BRCA1/2 dysfunction and CCNE1 amplification are known to promote genomic instability and tumor progression. These events may be mutually exclusive, because either change provides a path to tumor development, with no selective advantage to having both mutations. Using data from a genome-wide shRNA synthetic lethal screen, we show that BRCA1 and members of the ubiquitin pathway are selectively required in cancers that harbor CCNE1 amplification. Furthermore, we show specific sensitivity of CCNE1-amplified tumor cells to the proteasome inhibitor bortezomib. These findings provide an explanation for the observed mutual exclusivity of CCNE1 amplification and BRCA1/2 loss in HGSC and suggest a unique therapeutic approach for treatment-resistant CCNE1-amplified tumors.

Histone deacetylase inhibitors (HDACis) inhibit tumor cell growth and survival, possibly through their ability to regulate the expression of specific proliferative and/or apoptotic genes. However, the HDACi-regulated genes necessary and/or sufficient for their biological effects remain undefined. We demonstrate that the HDACis suberoylanilide hydroxamic acid (SAHA) and depsipeptide regulate a highly overlapping gene set with at least 22% of genes showing altered expression over a 16-h culture period. SAHA and depsipeptide coordinately regulated the expression of several genes within distinct apoptosis and cell cycle pathways. Multiple genes within the Myc, type β TGF, cyclin/cyclin-dependent kinase, TNF, Bcl-2, and caspase pathways were regulated in a manner that favored induction of apoptosis and decreased cellular proliferation. APAF - 1 , a gene central to the intrinsic apoptotic pathway, was induced by SAHA and depsipeptide and shown to be important, but not essential, for HDACi-induced cell death. Overexpression of p16 INK4A and arrest of cells in G 1 can suppress HDACi-mediated apoptosis. Although p16 INK4A did not affect the genome-wide transcription changes mediated by SAHA, a small number of apoptotic genes, including BCLXL and B - MYB , were differentially regulated in a manner consistent with attenuated HDACi-mediated apoptosis in arrested cells. We demonstrate that different HDACi alter transcription of a large and common set of genes that control diverse molecular pathways important for cell survival and proliferation. The ability of HDACi to target multiple apoptotic and cell proliferation pathways may provide a competitive advantage over other chemotherapeutic agents because suppression/loss of a single pathway may not confer resistance to these agents.

Induction of wild-type p53 in mouse fibroblasts causes cell cycle arrest at the G 1 phase, whereas coexpression of p53 and the protooncogene c- myc induces apoptosis. Although p53 transcriptional activity generally is required for both pathways, the molecular components mediating p53-dependent apoptosis are not well understood. To identify factors that could mediate p53-induced cell death, we used a comparative RNA differential display procedure. We have identified Pw1/Peg3 as a gene product induced during p53/c- myc -mediated apoptosis. Pw1/Peg3 is not induced during p53-mediated G 1 growth arrest nor by c- myc alone. Although it is not clear whether the induction of Pw1/Peg3 depends on p53 activity, we show that Pw1/Peg3 interacts with a p53-inducible gene product Siah1a. We demonstrate that coexpression of Pw1/Peg3 with Siah1a induces apoptosis independently of p53 whereas expression of Pw1/Peg3 or Siah1a separately has no effect on cell death. These data suggest that Siah1a and Pw1/Peg3 cooperate in the p53-mediated cell death pathway. Furthermore, we show that inhibiting Pw1/Peg3 activity blocks p53-induced apoptosis. The observation that Pw1/Peg3 is necessary for the p53 apoptotic response suggests a pivotal role for this gene in determining cell death versus survival.

Several findings suggest that signals from tyrosine kinases are transduced, at least in part, through ras proteins. These findings include (i) blockage of the transforming activity of constitutively active tyrosine kinases by inhibiting ras function and (ii) genetic screens in Caenorhabditis elegans and in Drosophila that identified ras genes as downstream effectors of tyrosine kinases. The recently isolated Drosophila gene Son of sevenless (Sos) is postulated to act as a positive regulatory link between tyrosine kinase and ras proteins by catalyzing exchange of GDP for GTP on ras protein. Such exchange proteins have been reported in extracts of mammalian cells but have not been previously characterized at a molecular level. As Sos appears to function in this role in Drosophila, we sought to isolate a vertebrate counterpart(s). We have characterized two widely expressed murine genes with a high degree of homology to Sos. Hybridization with human DNA and RNA indicates a high degree of conservation of these genes in other vertebrates.

The sevenless gene encodes a protein-tyrosine kinase receptor expressed in a complex pattern during the development of the Drosophila melanogaster eye. We have previously shown that this pattern is regulated transcriptionally by an enhancer located in the body of the sevenless gene. Here we extend our analysis of the sevenless enhancer, defining a 475-base-pair fragment that contains elements necessary for the correct qualitative and quantitative expression of the sevenless gene. Within this fragment are sequence elements conserved in the sevenless gene of a distantly related Drosophila species and protected from DNase I digestion by nuclear extracts isolated from adult heads and imaginal discs. Partial deletions of the 475-base-pair fragment result in preferential loss of expression in different subsets of cells. These results suggest that the normal pattern of expression is generated by the combined action of separate cell-specific regulatory elements.

The sevenless gene encodes a putative protein tyrosine kinase receptor that is required for the proper differentiation of the R7 photoreceptor cells of the Drosophila eye. We have expressed the sevenless protein in Drosophila tissue culture cells and studied its synthesis, processing, and activity. Our results show that the sevenless protein possesses protein tyrosine kinase activity. The protein is first synthesized as a 280-kDa glycoprotein precursor that is subsequently cleaved into 220-kDa amino-terminal and 58-kDa carboxyl-terminal subunits that remain associated by noncovalent interactions. The 220-kDa subunit is glycosylated and contains most of the extracellular portion of the protein, and the 58-kDa subunit is composed of a small portion of the extracellular sequences and the intracellular protein tyrosine kinase domain. This complex is subsequently cleaved into either 49- or 48-kDa carboxyl-terminal fragments with concomitant degradation of the rest of the protein.

The sevenless gene encodes a cell surface receptor that has protein-tyrosine kinase activity and is expressed in a highly specific and complex pattern in the developing Drosophila eye. We have coupled the sevenless promoter to the reporter gene lacZ and have examined the pattern of beta-galactosidase expression in the developing eyes of transgenic larvae. Our results indicate that the dynamic pattern of sevenless protein expression is regulated transcriptionally. Promoter sequences located 5' of the coding region are insufficient for the wild-type level of gene expression but appear to be able to confer the correct pattern of expression. In contrast, enhancer sequences within the body of the gene can confer both the correct pattern and a normal level of expression on either the sevenless promoter or heterologous promoters. Thus the complex pattern of sevenless expression is redundantly encoded within proximal promoter sequences and enhancer elements internal to the gene but relies on these enhancer sequences for correct quantitative expression.