Systematic investigation of genetic vulnerabilities across cancer cell lines reveals lineage-specific dependencies in ovarian cancer.

A comprehensive understanding of the molecular vulnerabilities of every type of cancer will provide a powerful roadmap to guide therapeutic approaches. Efforts such as The Cancer Genome Atlas Project will identify genes with aberrant copy number, sequence, or expression in various cancer types, providing a survey of the genes that may have a causal role in cancer. A complementary approach is to perform systematic loss-of-function studies to identify essential genes in particular cancer cell types. We have begun a systematic effort, termed Project Achilles, aimed at identifying genetic vulnerabilities across large numbers of cancer cell lines. Here, we report the assessment of the essentiality of 11,194 genes in 102 human cancer cell lines. We show that the integration of these functional data with information derived from surveying cancer genomes pinpoints known and previously undescribed lineage-specific dependencies across a wide spectrum of cancers. In particular, we found 54 genes that are specifically essential for the proliferation and viability of ovarian cancer cells and also amplified in primary tumors or differentially overexpressed in ovarian cancer cell lines. One such gene, PAX8, is focally amplified in 16% of high-grade serous ovarian cancers and expressed at higher levels in ovarian tumors. Suppression of PAX8 selectively induces apoptotic cell death of ovarian cancer cells. These results identify PAX8 as an ovarian lineage-specific dependency. More generally, these observations demonstrate that the integration of genome-scale functional and structural studies provides an efficient path to identify dependencies of specific cancer types on particular genes and pathways.

Polar N-terminal Residues Conserved in Type 2 Secretion Pseudopilins Determine Subunit Targeting and Membrane Extraction Steps during Fibre Assembly.

Bacterial type 2 secretion systems (T2SS), type 4 pili, and archaeal flagella assemble fibres from initially membrane-embedded pseudopilin and pilin subunits. Fibre subunits are made as precursors with positively charged N-terminal anchors, whose cleavage via the prepilin peptidase, essential for pilin membrane extraction and assembly, is followed by N-methylation of the mature (pseudo)pilin N terminus. The conserved Glu residue at position 5 (E5) of mature (pseudo)pilins is essential for assembly. Unlike T4 pilins, where E5 residue substitutions also abolish N-methylation, the E5A variant of T2SS pseudopilin PulG remains N-methylated but is affected in interaction with the T2SS component PulM. Here, biochemical and functional analyses showed that the PulM interaction defect only partly accounts for the PulGE5A assembly defect. First, PulGT2A variant, equally defective in PulM interaction, remained partially functional. Furthermore, pseudopilus assembly defect of pulG(E5A) mutant was stronger than that of the pulM deletion mutant. To understand the dominant effect of E5A mutation, we used molecular dynamics simulations of PulGE5A, methylated PulGWT (MePulGWT), and MePulGE5A variant in a model membrane. These simulations pointed to a key role for an intramolecular interaction between the pseudopilin N-terminal amine and E5 to limit polar interactions with membrane phospholipids. N-methylation of the N-terminal amine further limited its interactions with phospholipid head-groups to facilitate pseudopilin membrane escape. By binding to polar residues in the conserved N-terminal region of PulG, we propose that PulM acts as chaperone to promote pseudopilin recruitment and coordinate its membrane extraction with subsequent steps of the fibre assembly process.

Structural Basis of Pullulanase Membrane Binding and Secretion Revealed by X-Ray Crystallography, Molecular Dynamics and Biochemical Analysis.

The Klebsiella lipoprotein pullulanase (PulA) is exported to the periplasm, triacylated, and anchored via lipids in the inner membrane (IM) prior to its transport to the bacterial surface through a type II secretion system (T2SS). X-Ray crystallography and atomistic molecular dynamics (MD) simulations of PulA in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) model membrane provided an unprecedented molecular view of an N-terminal unstructured tether and the IM lipoprotein retention signal, and revealed novel interactions with the IM via N-terminal immunoglobulin-like domains in PulA. An efficiently secreted nonacylated variant (PulANA) showed similar peripheral membrane association during MD simulations, consistent with the binding of purified PulANA to liposomes. Remarkably, combined X-ray, MD, and functional studies identified a novel subdomain, Ins, inserted in the α-amylase domain, which is required for PulA secretion. Available data support a model in which PulA binding to the IM promotes interactions with the T2SS, possibly via the Ins subdomain.

Efficient Characterization of Protein Cavities within Molecular Simulation Trajectories: trj_cavity.

Protein cavities and tunnels are critical in determining phenomena such as ligand binding, molecular transport, and enzyme catalysis. Molecular dynamics (MD) simulations enable the exploration of the flexibility and conformational plasticity of protein cavities, extending the information available from static experimental structures relevant to, for example, drug design. Here, we present a new tool (trj_cavity) implemented within the GROMACS ( www.gromacs.org ) framework for the rapid identification and characterization of cavities detected within MD trajectories. trj_cavity is optimized for usability and computational efficiency and is applicable to the time-dependent analysis of any cavity topology, and optional specialized descriptors can be used to characterize, for example, protein channels. Its novel grid-based algorithm performs an efficient neighbor search whose calculation time is linear with system size, and a comparison of performance with other widely used cavity analysis programs reveals an orders-of-magnitude improvement in the computational cost. To demonstrate its potential for revealing novel mechanistic insights, trj_cavity has been used to analyze long-time scale simulation trajectories for three diverse protein cavity systems. This has helped to reveal, respectively, the lipid binding mechanism in the deep hydrophobic cavity of a soluble mite-allergen protein, Der p 2; a means for shuttling carbohydrates between the surface-exposed substrate-binding and catalytic pockets of a multidomain, membrane-proximal pullulanase, PulA; and the structural basis for selectivity in the transmembrane pore of a voltage-gated sodium channel (NavMs), embedded within a lipid bilayer environment. trj_cavity is available for download under an open-source license ( http://sourceforge.net/projects/trjcavity ). A simplified, GROMACS-independent version may also be compiled.

RNA-programmed genome editing in human cells.

Type II CRISPR immune systems in bacteria use a dual RNA-guided DNA endonuclease, Cas9, to cleave foreign DNA at specific sites. We show here that Cas9 assembles with hybrid guide RNAs in human cells and can induce the formation of double-strand DNA breaks (DSBs) at a site complementary to the guide RNA sequence in genomic DNA. This cleavage activity requires both Cas9 and the complementary binding of the guide RNA. Experiments using extracts from transfected cells show that RNA expression and/or assembly into Cas9 is the limiting factor for Cas9-mediated DNA cleavage. In addition, we find that extension of the RNA sequence at the 3' end enhances DNA targeting activity in vivo. These results show that RNA-programmed genome editing is a facile strategy for introducing site-specific genetic changes in human cells.DOI:http://dx.doi.org/10.7554/eLife.00471.001.