R-loop-mediated genome instability in mRNA cleavage and polyadenylation mutants.

Genome instability via RNA:DNA hybrid-mediated R loops has been observed in mutants involved in various aspects of transcription and RNA processing. The prevalence of this mechanism among essential chromosome instability (CIN) genes remains unclear. In a secondary screen for increased Rad52 foci in CIN mutants, representing ∼25% of essential genes, we identified seven essential subunits of the mRNA cleavage and polyadenylation (mCP) machinery. Genome-wide analysis of fragile sites by chromatin immunoprecipitation (ChIP) and microarray (ChIP-chip) of phosphorylated H2A in these mutants supported a transcription-dependent mechanism of DNA damage characteristic of R loops. In parallel, we directly detected increased RNA:DNA hybrid formation in mCP mutants and demonstrated that CIN is suppressed by expression of the R-loop-degrading enzyme RNaseH. To investigate the conservation of CIN in mCP mutants, we focused on FIP1L1, the human ortholog of yeast FIP1, a conserved mCP component that is part of an oncogenic fusion in eosinophilic leukemia. We found that truncation fusions of yeast FIP1 analogous to those in cancer cause loss of function and that siRNA knockdown of FIP1L1 in human cells increases DNA damage and chromosome breakage. Our findings illuminate how mCP maintains genome integrity by suppressing R-loop formation and suggest that this function may be relevant to certain human cancers.

An evolutionarily conserved synthetic lethal interaction network identifies FEN1 as a broad-spectrum target for anticancer therapeutic development.

Harnessing genetic differences between cancerous and noncancerous cells offers a strategy for the development of new therapies. Extrapolating from yeast genetic interaction data, we used cultured human cells and siRNA to construct and evaluate a synthetic lethal interaction network comprised of chromosome instability (CIN) genes that are frequently mutated in colorectal cancer. A small number of genes in this network were found to have synthetic lethal interactions with a large number of cancer CIN genes; these genes are thus attractive targets for anticancer therapeutic development. The protein product of one highly connected gene, the flap endonuclease FEN1, was used as a target for small-molecule inhibitor screening using a newly developed fluorescence-based assay for enzyme activity. Thirteen initial hits identified through in vitro biochemical screening were tested in cells, and it was found that two compounds could selectively inhibit the proliferation of cultured cancer cells carrying inactivating mutations in CDC4, a gene frequently mutated in a variety of cancers. Inhibition of flap endonuclease activity was also found to recapitulate a genetic interaction between FEN1 and MRE11A, another gene frequently mutated in colorectal cancers, and to lead to increased endogenous DNA damage. These chemical-genetic interactions in mammalian cells validate evolutionarily conserved synthetic lethal interactions and demonstrate that a cross-species candidate gene approach is successful in identifying small-molecule inhibitors that prove effective in a cell-based cancer model.

Synthetic lethality of cohesins with PARPs and replication fork mediators.

Synthetic lethality has been proposed as a way to leverage the genetic differences found in tumor cells to affect their selective killing. Cohesins, which tether sister chromatids together until anaphase onset, are mutated in a variety of tumor types. The elucidation of synthetic lethal interactions with cohesin mutants therefore identifies potential therapeutic targets. We used a cross-species approach to identify robust negative genetic interactions with cohesin mutants. Utilizing essential and non-essential mutant synthetic genetic arrays in Saccharomyces cerevisiae, we screened genome-wide for genetic interactions with hypomorphic mutations in cohesin genes. A somatic cell proliferation assay in Caenorhabditis elegans demonstrated that the majority of interactions were conserved. Analysis of the interactions found that cohesin mutants require the function of genes that mediate replication fork progression. Conservation of these interactions between replication fork mediators and cohesin in both yeast and C. elegans prompted us to test whether other replication fork mediators not found in the yeast were required for viability in cohesin mutants. PARP1 has roles in the DNA damage response but also in the restart of stalled replication forks. We found that a hypomorphic allele of the C. elegans SMC1 orthologue, him-1(e879), genetically interacted with mutations in the orthologues of PAR metabolism genes resulting in a reduced brood size and somatic cell defects. We then demonstrated that this interaction is conserved in human cells by showing that PARP inhibitors reduce the viability of cultured human cells depleted for cohesin components. This work demonstrates that large-scale genetic interaction screening in yeast can identify clinically relevant genetic interactions and suggests that PARP inhibitors, which are currently undergoing clinical trials as a treatment of homologous recombination-deficient cancers, may be effective in treating cancers that harbor cohesin mutations.

Specific synthetic lethal killing of RAD54B-deficient human colorectal cancer cells by FEN1 silencing.

Mutations that cause chromosome instability (CIN) in cancer cells produce "sublethal" deficiencies in an essential process (chromosome segregation) and, therefore, may represent a major untapped resource that could be exploited for therapeutic benefit in the treatment of cancer. If second-site unlinked genes can be identified, that when knocked down, cause a synthetic lethal (SL) phenotype in combination with a somatic mutation in a CIN gene, novel candidate therapeutic targets will be identified. To test this idea, we took a cross species SL candidate gene approach by recapitulating a SL interaction observed between rad54 and rad27 mutations in yeast, via knockdown of the highly sequence- and functionally-related proteins RAD54B and FEN1 in a cancer cell line. We show that knockdown of RAD54B, a gene known to be somatically mutated in cancer, causes CIN in mammalian cells. Using high-content microscopy techniques, we demonstrate that RAD54B-deficient human colorectal cancer cells are sensitive to SL killing by reduced FEN1 expression, while isogenic RAD54B proficient cells are not. This conserved SL interaction suggests that extrapolating SL interactions observed in model organisms for homologous genes mutated in human cancers will aid in the identification of novel therapeutic targets for specific killing of cancerous cells exhibiting CIN.

Chromatid cohesion defects may underlie chromosome instability in human colorectal cancers.

Although the majority of colorectal cancers exhibit chromosome instability (CIN), only a few genes that might cause this phenotype have been identified and no general mechanism underlying their function has emerged. To systematically identify somatic mutations in potential CIN genes in colorectal cancers, we determined the sequence of 102 human homologues of 96 yeast CIN genes known to function in various aspects of chromosome transmission fidelity. We identified 11 somatic mutations distributed among five genes in a panel that included 132 colorectal cancers. Remarkably, all but one of these 11 mutations were in the homologs of yeast genes that regulate sister chromatid cohesion. We then demonstrated that down-regulation of such homologs resulted in chromosomal instability and chromatid cohesion defects in human cells. Finally, we showed that down-regulation or genetic disruption of the two major candidate CIN genes identified in previous studies (MRE11A and CDC4) also resulted in abnormal sister chromatid cohesion in human cells. These results suggest that defective sister chromatid cohesion as a result of somatic mutations may represent a major cause of chromosome instability in human cancers.

Lead identification of conformationally restricted benzoxepin type combretastatin analogs: synthesis, antiproliferative activity, and tubulin effects.

We have synthesized a series of polymethoxylated rigid analogs of combretastatin A-4 which contain a benzoxepin ring in place of the usual ethylene bridge present in the natural combretastatin products. The compounds display antiproliferative activity when evaluated against the MCF-7 and MDA human breast carcinoma cell lines. 5-(3-Hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihydro-benzoxepine (11g) was found to be the most potent product when evaluated against the MCF-7 breast cancer cell line. A brief computational study of the structure-activity relationship for the synthesized compounds is presented. These 4,5-diarylbenzoxepins are identified as potentially useful scaffolds for the further development of antitumor agents which target tubulin.

Placental weight in pregnancies with trisomy confined to the placenta.

OBJECTIVE: Mosaicism with trisomy confined to the placenta is present in ~1% of ongoing pregnancies at the time of chorionic villus sampling. Some studies have found reduced fetal growth in confined placental trisomy. The objective of this study was to assess placental weight and feto-placental weight ratio in pregnancies with trisomy confined to the placenta, and to correlate them with the level of trisomy in the three major placental lineages. METHODS: We conducted a retrospective study of 69 pregnancies with prenatally diagnosed mosaic trisomy in which the trisomic cells were confined to the placenta. Placental weight and feto-placental weight ratio were compared to those of matched controls, and placental weight was also analyzed for associations with the type and level of trisomy. Placental pathology was also reviewed. RESULTS: The pregnancies with mosaic trisomy were found to have lower placental weights than matched controls, but normal feto-placental weight ratios. Placental weight was not associated with the type or level of trisomic cells in the three placental lineages at term (chorionic plate, chorionic villus mesenchyme, and trophoblast). There were no pathognomonic findings on routine placental pathology of the trisomic placentas. CONCLUSION: Although placental weight was reduced (with normal feto-placental weight ratio) in pregnancies with trisomy confined to the placenta, the level of placental trisomy was not correlated with placental weight. Thus, trisomy may alter placental function rather than have a direct hypoplastic effect on placental growth. More in-depth studies beyond routine pathology are required to identify how trisomy affects placental function.

Synthesis, biological evaluation, structural-activity relationship, and docking study for a series of benzoxepin-derived estrogen receptor modulators.

The estrogen receptors ERalpha and ERbeta are recognized as important pharmaceutical targets for a variety of diseases including osteoporosis and breast cancer. A series of novel benzoxepin-derived compounds are described as potent selective modulators of the human estrogen receptor modulators (SERMs). We report the antiproliferative effects of these compounds on human MCF-7 breast tumor cells. These heterocyclic compounds contain the triarylethylene arrangement as exemplified by tamoxifen, conformationally restrained through the incorporation of the benzoxepin ring system. The compounds demonstrate potency at nanomolar concentrations in antiproliferative assays against an MCF-7 human breast cancer cell line with low cytotoxicity together with low nanomolar binding affinity for the estrogen receptor. The compounds also demonstrate potent antiestrogenic properties in the human uterine Ishikawa cell line. The effect of a number of functional group substitutions on the ER binding properties of the benzoxepin molecular scaffold is examined through a detailed docking and 2D-QSAR computational investigation. The best QSAR model developed for ERalphabeta selectivity yielded R(2) of 0.84 with an RMSE for the training set of 0.30. The predictive quality of the model was Q(2) of 0.72 and RMSE of 0.18 for the test set. One particular compound bearing a 4-fluoro substituent, exhibits 15-fold selectivity for ERbeta and both our docking and QSAR studies converge on the correlation between enhanced lipophilicity and enhanced ERbeta binding for this benzoxepin ring scaffold.

Lead Optimization of Benzoxepin-Type Selective Estrogen Receptor (ER) Modulators and Downregulators with Subtype-Specific ERα and ERß Activity.

Estrogen receptor α (ERα) is an important target for the design of drugs such as tamoxifen (2a) and fulvestrant (5). Three series of ER-ligands based on the benzoxepin scaffold structure were synthesized: series I containing an acrylic acid, series II with an acrylamide, and series III with a saturated carboxylic acid substituent. These compounds were shown to be high affinity ligands for the ER with nanomolar IC50 binding values. Series I acrylic acid ligands were generally ERα selective. In particular, compound 13e featuring a phenylpenta-2,4-dienoic acid substituent was shown to be antiproliferative and downregulated ERα and ERß expression in MCF-7 breast cancer cells. Interestingly, from series III, the phenoxybutyric acid derivative compound 22 was not antiproliferative and selectively downregulated ERß. A docking study of the benzoxepin ligands was undertaken. Compound 13e is a promising lead for development as a clinically relevant SERD, while compound 22 will be a useful experimental probe for helping to elucidate the role of ERß in cancer cells.

Genome-wide synthetic lethal screens identify an interaction between the nuclear envelope protein, Apq12p, and the kinetochore in Saccharomyces cerevisiae.

The maintenance of genome stability is a fundamental requirement for normal cell cycle progression. The budding yeast Saccharomyces cerevisiae is an excellent model to study chromosome maintenance due to its well-defined centromere and kinetochore, the region of the chromosome and associated protein complex, respectively, that link chromosomes to microtubules. To identify genes that are linked to chromosome stability, we performed genome-wide synthetic lethal screens using a series of novel temperature-sensitive mutations in genes encoding a central and outer kinetochore protein. By performing the screens using different mutant alleles of each gene, we aimed to identify genetic interactions that revealed diverse pathways affecting chromosome stability. Our study, which is the first example of genome-wide synthetic lethal screening with multiple alleles of a single gene, demonstrates that functionally distinct mutants uncover different cellular processes required for chromosome maintenance. Two of our screens identified APQ12, which encodes a nuclear envelope protein that is required for proper nucleocytoplasmic transport of mRNA. We find that apq12 mutants are delayed in anaphase, rereplicate their DNA, and rebud prior to completion of cytokinesis, suggesting a defect in controlling mitotic progression. Our analysis reveals a novel relationship between nucleocytoplasmic transport and chromosome stability.

Benzothiepin-derived molecular scaffolds for estrogen receptor modulators: synthesis and antagonistic effects in breast cancer cells.

A series of novel benzothiepin-derived compounds are described as potent selective modulators of the human estrogen receptor (SERMs). The objective of the study is to evaluate the antiproliferative effects of the compounds on human MCF-7 breast tumor cells. These heterocyclic compounds contain the traditional triarylethylene arrangement exemplified by tamoxifen, conformationally restrained through the incorporation of the benzothiepin ring system. The compounds demonstrated potency at nanomolar concentrations in antiproliferative assays against an MCF-7 human breast cancer cell line with low cytotoxicity. The compounds exhibited low nanomolar binding affinity for the estrogen receptor (ER) with some specificity for ERbeta, and also demonstrate potent antiestrogenic properties in the human uterine Ishikawa cell line. The effect of a number of functional group substitutions on the ER binding properties of the benzothiepin molecular scaffold is explored through a brief computational structure-activity relationship investigation with molecular simulation.

Postnatal follow-up of prenatally diagnosed trisomy 16 mosaicism.

OBJECTIVE: To determine the long-term outcome of pregnancies prenatally diagnosed with trisomy 16 and identify variables associated with the outcome. METHODS: We reviewed all published and our unpublished data from trisomy 16 pregnancies for which outcomes were available for children of greater than 1 year of age. RESULTS: Nineteen cases were diagnosed with trisomy 16 on chorionic villus sampling (CVS) and 17 cases at amniocentesis. Age at last follow-up ranges from 1 to 13 years. Among the CVS group, four out of five patients, with a birth weight and/or length below -2 SD and postnatal growth information, showed catch-up growth (80%). Among the amniotic fluid (AF) group, the birth weight was available in 13 cases. Eleven of the 13 cases had a birth weight less than -2 SD. In eight cases, the length was also below -2 SD (length data unavailable in one case). Nine out of ten cases (90%) and seven out of eight (87.5%) showed catch-up growth for weight and length, respectively. In terms of development, no cases of CVS mosaicism had global developmental delay. One child had a history of delay in speech development. Among the AF-detected cases, 4/17 cases had global developmental delay. All four children with global developmental delay had more than one major malformation compared to 6 out of 32 children in the group with normal development (p = 0.004). The finding of uniparental disomy (UPD) was not associated with developmental delay. CONCLUSIONS: The majority of prenatally diagnosed trisomy 16 mosaic cases have a good postnatal outcome. However, the finding of mosaicism on AF and the presence of major congenital anomalies are associated with an increased risk of developmental delay.

Origin of amnion and implications for evaluation of the fetal genotype in cases of mosaicism.

OBJECTIVE: To investigate presence of trisomy in amniotic epithelium (uncultured amnion) and mesenchyme (cultured amnion) from mosaic cases to understand the origins of these tissues and their relationship to pregnancy outcome. METHODS: Polymerase chain reaction (PCR) of microsatellite loci was used to determine the presence of trisomy (of meiotic origin only) in amnion samples from 33 placentas previously ascertained because of a prenatal diagnosis of trisomy mosaicism that was predominantly confined to the placental tissues. RESULTS: In 16 (48%) of 33 cases, trisomy was confirmed to be present by molecular analysis of uncultured amnion. In contrast, cytogenetic analysis of cultured amnion showed trisomy in only 2 of 20 informative cases. The molecular detection of trisomy in amnion was strongly associated with poor pregnancy outcome (intrauterine growth restriction, fetal anomalies and/or intrauterine/neonatal death) even when analysis was limited to cases negative for the trisomy on amniotic fluid (N = 22, p = 0.0005). CONCLUSIONS: We infer that amniotic mesenchyme (usually diploid) derives from early embryonic mesoderm of the primitive streak and not from the hypoblast as is commonly cited. Trisomy in amniotic epithelium suggests that high numbers of abnormal cells were present in the epiblast, and this correlates with poor outcome even when the subsequently derived fetus and amniotic mesenchyme appear to carry only diploid cells.