Insertional mutagenesis in a haploid background can lead to complete disruption of gene function1. Here we generate a population of human cells that contain insertions in >98% of their expressed genes. We established Phenotypic Interrogation via Tag Sequencing (PhITSeq) as a method to examine millions of mutant alleles through selection and parallel sequencing. Analysis of pools of selected cells rather than individual clones provides a rapid assessment of the spectrum of genes involved in phenotypes under study. This facilitates comparative screens as illustrated here for the family of cytolethal distending toxins (CDTs). CDTs are virulence factors secreted by a variety of pathogenic gram-negative bacteria that cause tissue damage at distinct anatomical sites2. We identified 743 mutations distributed over 12 human genes important for intoxication by four different CDTs. While related CDTs may share host factors, they also exploit unique host factors yielding a characteristic profile for each CDT.

Linking the molecular aberrations of cancer to drug responses could guide treatment choice and identify new therapeutic applications. However, there has been no systematic approach for analyzing gene-drug interactions in human cells. We establish a multiplexed assay to study the cellular fitness of a panel of engineered isogenic cancer cells in response to a collection of drugs, enabling the systematic analysis of thousands of gene-drug interactions. Applying this approach to breast cancer revealed various synthetic-lethal interactions and drug resistance mechanisms, some of which were known, thereby validating the method. NOTCH pathway activation, which occurs frequently in breast cancer, unexpectedly conferred resistance to PI3K inhibitors, which are currently undergoing clinical trials in breast cancer patients. NOTCH1 and downstream induction of c-MYC overrode the dependency of cells on the PI3K/mTOR pathway for proliferation. These data reveal a novel mechanism of resistance to PI3K inhibitors with direct clinical implications.

H2S exhibits direct inhibitory action on ACE activity in HUVECs, obviously by interfering with the Zn in the active center of the enzyme. Thus, beside the known influence of H2S on SMC KATP-channels, the observed direct ACE inhibitory effect may add to the vasorelaxant effect of H2S in the vasculature by reducing angiotensin II production and inhibiting bradykinin degradation.

BackgroundCardiac glycosides are Na+/K+-pump inhibitors widely used to treat heart failure. They are also highly cytotoxic, and studies have suggested specific anti-tumor activity leading to current clinical trials in cancer patients. However, a definitive demonstration of this putative anti-cancer activity and the underlying molecular mechanism has remained elusive.Methodology/Principal FindingsUsing an unbiased transcriptomics approach, we found that cardiac glycosides inhibit general protein synthesis. Protein synthesis inhibition and cytotoxicity were not specific for cancer cells as they were observed in both primary and cancer cell lines. These effects were dependent on the Na+/K+-pump as they were rescued by expression of a cardiac glycoside-resistant Na+/K+-pump. Unlike human cells, rodent cells are largely resistant to cardiac glycosides in vitro and mice were found to tolerate extremely high levels.Conclusions/SignificanceThe physiological difference between human and mouse explains the previously observed sensitivity of human cancer cells in mouse xenograft experiments. Thus, published mouse xenograft models used to support anti-tumor activity for these drugs require reevaluation. Our finding that cardiac glycosides inhibit protein synthesis provides a mechanism for the cytotoxicity of CGs and raises concerns about ongoing clinical trials to test CGs as anti-cancer agents in humans.