Mapping genetic interactions in cancer: a road to rational combination therapies.

The discovery of synthetic lethal interactions between poly (ADP-ribose) polymerase (PARP) inhibitors and BRCA genes, which are involved in homologous recombination, led to the approval of PARP inhibition as a monotherapy for patients with BRCA1/2-mutated breast or ovarian cancer. Studies following the initial observation of synthetic lethality demonstrated that the reach of PARP inhibitors is well beyond just BRCA1/2 mutants. Insights into the mechanisms of action of anticancer drugs are fundamental for the development of targeted monotherapies or rational combination treatments that will synergize to promote cancer cell death and overcome mechanisms of resistance. The development of targeted therapeutic agents is premised on mapping the physical and functional dependencies of mutated genes in cancer. An important part of this effort is the systematic screening of genetic interactions in a variety of cancer types. Until recently, genetic-interaction screens have relied either on the pairwise perturbations of two genes or on the perturbation of genes of interest combined with inhibition by commonly used anticancer drugs. Here, we summarize recent advances in mapping genetic interactions using targeted, genome-wide, and high-throughput genetic screens, and we discuss the therapeutic insights obtained through such screens. We further focus on factors that should be considered in order to develop a robust analysis pipeline. Finally, we discuss the integration of functional interaction data with orthogonal methods and suggest that such approaches will increase the reach of genetic-interaction screens for the development of rational combination therapies

Incorporating genome-scale tools for studying energy homeostasis

Mammals have evolved complex regulatory systems that enable them to maintain energy homeostasis despite constant environmental challenges that limit the availability of energy inputs and their composition. Biological control relies upon intricate systems composed of multiple organs and specialized cell types that regulate energy up-take, storage, and expenditure. Because these systems simultaneously perform diverse functions and are highly integrated, they are extremely difficult to understand in terms of their individual component contributions to energy homeostasis. In order to provide improved treatments and clinical options, it is important to identify the principle genetic and molecular components, as well as the systemic features of regulation. To begin, many of these features can be discovered by integrating experimental technologies with advanced methods of analysis. This review focuses on the analysis of transcriptional data derived from microarrays and how it can complement other experimental techniques to study energy homeostasis

Functional analysis of the Drosophila embryonic germ cell transcriptome by RNA interference

In Drosophila melanogaster, primordial germ cells are specified at the posterior pole of the very early embryo. This process is regulated by the posterior localized germ plasm that contains a large number of RNAs of maternal origin. Transcription in the primordial germ cells is actively down-regulated until germ cell fate is established. Bulk expression of the zygotic genes commences concomitantly with the degradation of the maternal transcripts. Thus, during embryogenesis, maternally provided and zygotically transcribed mRNAs determine germ cell development collectively. In an effort to identify novel genes involved in the regulation of germ cell behavior, we carried out a large-scale RNAi screen targeting both maternal and zygotic components of the embryonic germ line transcriptome. We identified 48 genes necessary for distinct stages in germ cell development. We found pebble and fascetto to be essential for germ cell migration and germ cell division, respectively. Our data uncover a previously unanticipated role of mei-P26 in maintenance of embryonic germ cell fate. We also performed systematic co-RNAi experiments, through which we found a low rate of functional redundancy among homologous gene pairs. As our data indicate a high degree of evolutionary conservation in genetic regulation of germ cell development, they are likely to provide valuable insights into the biology of the germ line in general

VAMP3/Syb and YKT6 are required for the fusion of constitutive secretory carriers with the plasma membrane

The cellular machinery required for the fusion of constitutive secretory vesicles with the plasma membrane in metazoans remains poorly defined. To address this problem we have developed a powerful, quantitative assay for measuring secretion and used it in combination with combinatorial gene depletion studies in Drosophila cells. This has allowed us to identify at least three SNARE complexes mediating Golgi to PM transport (STX1, SNAP24/29 and Syb; STX1, SNAP24/29 and YKT6; STX4, SNAP24 and Syb). RNAi mediated depletion of YKT6 and VAMP3 in mammalian cells also blocks constitutive secretion suggesting that YKT6 has an evolutionarily conserved role in this process. The unexpected role of YKT6 in plasma membrane fusion may in part explain why RNAi and gene disruption studies have failed to produce the expected phenotypes in higher eukaryotes

Systems analysis of early endosome motility through identification of molecular motors

Endocytosis is an evolutionary conserved process of internalization of cargo from the extracellular environment, be they ligands, nutritional and signaling or pathogens into cells. Following their entry, cargo is received into vesiculo-tubular network of early endosomal compartments from where they are sorted and routed to appropriate cellular destinations through transport along the endocytic network. Recycling cargo is sorted away from other cargo resident in early endosomes through tubulation resulting in fission of recycling vesicles, while those to be degraded are progressively concentrated in early endosomes to be degraded in lysosomes. Early endosomes are dynamic organelles that have been shown to move centripetally following the internalization of cargo into at the cell periphery. Their motility from the cell periphery to the juxtanuclear location of the cell involves convoluted trajectories that include directed motility, bi-directional switches, saltatory behavior and stalls. This complex motility presumably contributes toward the cargo sorting, duration of cargo residence and spatio-temporal signaling by early endosomes. How the different regimes of motility, and nature and number of molecular motors involved in early endosome motility contribute toward endosome function is not understood. The aim of this study was to probe into the regulation of endosome motility and understand how transport organizes early endosome network. Towards this end, live cell time-lapse movies of Rab5 endosomes were analyzed to derive motility properties contributing to organization of early endosomes. Consistent and significant bias toward the cell centre (minus end motility) in kinetic parameters such as speed, displacement and duration of motility contribute to centripetal flux of Rab5 early endosomes. A phenomenological property of early endosome motility is its saltatory behavior that produces saturation curves in Mean Square Displacement (MSD) plots. This phase of motility is descriptive, with no understanding of its mechanism or function. Live cell candidate RNAi screen and cytoskeletal perturbation analysis were performed to identify molecules regulating saltatory motility. To this end, cellular microtubule perturbation and RNAi knock down of several Kinesin motor candidates showed a loss in saturation behavior. Potential candidates identified have to be tested for their effect on endosome function through cargo sorting and kinetic assays to gain insights into the role of saltatory motility in endosome function. Molecular motors mediate Rab5 motility. Therefore, understanding regulation of motility requires identifying number and nature of molecular motors involved in their transport. Towards this end, a functional cargo (LDL) degradation RNAi screen targeting molecular motors was performed. The Ambion Select technology was used with 3 siRNAs targeting every gene in the library. Analysis of screen produced by lack of phenotype consistency between the multiple siRNAs targeting the same gene. Hence, a search for technology with better target specificity was initiated. Technologies tested were Ambion Select, Ambion Silencer Select, Dharmacon ON-TARGET Plus, esiRNA and Invitrogen Stealth. Invitrogen Stealth technology was found to produce the least off-targets and was most specific in terms of consistency of phenotypes produced by multiple siRNAs silencing the same target gene. Assay conditions were also found to influence the silencing specificities to a significant extent. Hence, a systematic assay optimization exercise was performed in terms of the concentration of siRNA used for transfection and time window of assay to maximize specificity of siRNA silencing. Insights obtained from methodologies developed herein not only provide invaluable guidelines in choosing RNAi commercial libraries for screens, but also underscore the importance of establishing optimal assay conditions to minimize off-targets and improve specificity of silencing target genes. The motor screen was repeated with RNAi library from Invitrogen Stealth. Several potentially interesting candidates have been identified. Also, correlation analyses of phenotypes produced in the screen have indicated toward potential regulatory motor complexes, all of which await biochemical validation