Genomic analyses are yielding a host of new information on the multiple genetic abnormalities associated with specific types of cancer. A comprehensive description of cancer-associated genetic abnormalities can improve our ability to classify tumors into clinically relevant subgroups, and, on occasion, identify mutant genes that drive the cancer phenotype (“drivers”). More often, though, the functional significance of cancer-associated mutations is difficult to discern. Genome-wide pooled shRNA screens enable global identification of the genes essential for cancer cell survival and proliferation, providing a “functional genomic” map of human cancer to complement genomic studies. Using a lentiviral shRNA library targeting ~16,000 genes and a newly developed, dynamic scoring approach, we identified essential gene profiles in 72 breast, pancreatic, and ovarian cancer cell lines. Integrating our results with current and future genomic data should facilitate the systematic identification of drivers, unanticipated synthetic lethal relationships, and functional vulnerabilities of these tumor types.

Synthetic lethal genetic interaction networks define genes that work together to control essential functions and have been studied extensively in Saccharomyces cerevisiae using the synthetic genetic array (SGA) analysis technique (ScSGA). The extent to which synthetic lethal or other genetic interaction networks are conserved between species remains uncertain. To address this question, we compared literature-curated and experimentally derived genetic interaction networks for two distantly related yeasts, Schizosaccharomyces pombe and S. cerevisiae. We find that 23% of interactions in a novel, high-quality S. pombe literature-curated network are conserved in the existing S. cerevisiae network. Next, we developed a method, called S. pombe SGA analysis (SpSGA), enabling rapid, high-throughput isolation of genetic interactions in this species. Direct comparison by SpSGA and ScSGA of ϳ220 genes involved in DNA replication, the DNA damage response, chromatin remodeling, intracellular transport, and other processes revealed that ϳ29% of genetic interactions are common to both species, with the remainder exhibiting unique, species-specific patterns of genetic connectivity. We define a conserved yeast network (CYN) composed of 106 genes and 144 interactions and suggest that this network may help understand the shared biology of diverse eukaryotic species.comparative genomics ͉ Saccharomyces cerevisiae ͉ Schizosaccharomyces pombe ͉ synthetic genetic array

This study defines a network of synthetic sick/lethal interactions with a set of query genes in a series of isogenic cancer cell lines. Analysis of differential essentiality reveals general properties in genetic interaction networks derived from studies on model organisms.

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Myogenesis is a tightly controlled process involving the transcriptional activation and repression of thousands of genes. Although many components of the transcriptional network regulating the later phases of myogenesis have been identified, relatively few studies have described the transcriptional landscape during the first 24 h, when myoblasts commit to differentiate. Through dense temporal profiling of differentiating C2C12 myoblasts, we identify 193 transcriptional regulators (TRs) whose expression is significantly altered within the first 24 h of myogenesis. A high-content shRNA screen of 77 TRs involving 427 stable lines identified 42 genes whose knockdown significantly inhibits differentiation of C2C12 myoblasts. Of the TRs that were differentially expressed within the first 24 h, over half inhibited differentiation when knocked down, including known regulators of myogenesis (Myod1, Myog, and Myf5), as well as 19 TRs not previously associated with this process. Surprisingly, a similar proportion (55%) of shRNAs targeting TRs whose expression did not change also inhibited C2C12 myogenesis. We further show that a subset of these TRs inhibits myogenesis by downregulating expression of known regulatory and structural proteins. Our findings clearly illustrate that several TRs critical for C2C12 myogenesis are not differentially regulated, suggesting that approaches that focus functional studies on differentially-expressed transcripts will fail to provide a comprehensive view of this complex process.

Using integrative genomics and functional screening we identified coiled-coil domain containing 68 (CCDC68) as a novel putative tumor suppressor (TSG) in pancreatic ductal adenocarcinoma (PDAC). CCDC68 allelic losses were documented in 48% of primary PDAC patient tumors, 50% of PDAC cell lines, and 30% of primary patient derived xenografts. We also discovered a SNP variant (SNP rs1344011) that leads to exon skipping and generation of an unstable protein isoform CCDC68Δ69–114 in 31% of PDAC patients. Overexpression of full length CCDC68 (CCDC68wt) in PANC-1 and Hs.766T PDAC cell lines lacking CDCC68 expression decreased proliferation and tumorigenicity in scid mice. In contrast, downregulation of endogenous CCDC68 in MIAPaca-2 cells increased tumor growth rate. These effects were not observed with the deletion-containing isoform, CCDC68Δ69–114. In conclusion, our results suggest that CCDC68 is a novel candidate TSG in PDAC.

The Wee1 kinase, which is activated in response to DNA damage, regulates exit from G2 through inhibitory phosphorylation of Cdk1/Cdc2, and is an attractive drug target. However, recent work has highlighted effects of Cdk2 phosphorylation by Wee1 on movement through S-phase, suggesting the potential to sensitize to S-phase specific agents by Wee1 inhibitors. In this paper we applied multiparametric flow cytometry to patient-derived pancreatic cancer xenograft tumor cells to study the cell cycle perturbations of Wee1 disruption via the small molecule inhibitor MK-1775, and genetic knockdown. We find that in vitro treatment with MK-1775, and to a lesser degree, Wee1 RNA transcript knockdown, results in the striking appearance of S-phase cells prematurely entering into mitosis. This effect was not seen in vivo in any of the models tested. Here, although we noted an increase of S-phase cells expressing the damage response marker gH2AX, treatment with MK-1775 did not significantly sensitize cells to the cytidine analog gemcitabine. Treatment with MK-1775 did result in a transient but large increase in cells expressing the mitotic marker phosphorylated H3S10 that reached a peak 4 hours after treatment. This suggests a role for Wee1 regulating the progression of genomically unstable cancer cells through G2 in the absence of extrinsically-applied DNA damage. A single dose of 8Gy ionizing radiation resulted in the time-dependent accumulation of Cyclin A2 positive/phosphorylated H3S10 negative cells at the 4N position, which was abrogated by treatment with MK-1775. Consistent with these findings, a genomescale pooled RNA interference screen revealed that toxic doses of MK-1775 are suppressed by CDK2 or Cyclin A2 knockdown. These findings support G2 exit as the more significant effect of Wee1 inhibition in pancreatic cancers.