Kevin R. Brown

High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities

The ability to perturb genes in human cells is crucial for elucidating gene function and holds great potential for finding therapeutic targets for diseases such as cancer. To extend the catalog of human core and context-dependent fitness genes, we have developed a high-complexity second-generation genome-scale CRISPR-Cas9 gRNA library and applied it to fitness screens in five human cell lines. Using an improved Bayesian analytical approach, we consistently discover 5-fold more fitness genes than were previously observed. We present a list of 1,580 human core fitness genes and describe their general properties. Moreover, we demonstrate that context-dependent fitness genes accurately recapitulate pathway-specific genetic vulnerabilities induced by known oncogenes and reveal cell-type-specific dependencies for specific receptor tyrosine kinases, even in oncogenic KRAS backgrounds. Thus, rigorous identification of human cell line fitness genes using a high-complexity CRISPR-Cas9 library affords a high-resolution view of the genetic vulnerabilities of a cell.

Unequal evolutionary conservation of human protein interactions in interologous networks

BackgroundProtein-protein interaction (PPI) networks have been transferred between organisms using interologs, allowing model organisms to supplement the interactomes of higher eukaryotes. However, the conservation of various network components has not been fully explored. Unequal conservation of certain network components may limit the ability to fully expand the target interactomes using interologs.ResultsIn this study, we transfer high quality human interactions to lower eukaryotes, and examine the evolutionary conservation of individual network components. When human proteins are mapped to yeast, we find a strong positive correlation (r = 0.50, P = 3.9 × 10-4) between evolutionary conservation and the number of interacting proteins, which is also found when mapped to other model organisms. Examining overlapping PPI networks, Gene Ontology (GO) terms, and gene expression data, we are able to demonstrate that protein complexes are conserved preferentially, compared to transient interactions in the network. Despite the preferential conservation of complexes, and the fact that the human interactome comprises an abundance of transient interactions, we demonstrate how transferring human PPIs to yeast augments this well-studied protein interaction network, using the coatomer complex and replisome as examples.ConclusionHuman proteins, like yeast proteins, show a correlation between the number of interacting partners and evolutionary conservation. The preferential conservation of proteins with higher degree leads to enrichment in protein complexes when interactions are transferred between organisms using interologs.

NAViGaTOR: Network Analysis, Visualization and Graphing Toronto

Summary: NAViGaTOR is a powerful graphing application for the 2D and 3D visualization of biological networks. NAViGaTOR includes a rich suite of visual mark-up tools for manual and automated annotation, fast and scalable layout algorithms and OpenGL hardware acceleration to facilitate the visualization of large graphs. Publication-quality images can be rendered through SVG graphics export. NAViGaTOR supports community-developed data formats (PSI-XML, BioPax and GML), is platform-independent and is extensible through a plug-in architecture. Availability: NAViGaTOR is freely available to the research community from http://ophid.utoronto.ca/navigator/. Installers and documentation are provided for 32- and 64-bit Windows, Mac, Linux and Unix. Contact: juris@ai.utoronto.ca Supplementary information: Supplementary data are available at Bioinformatics online.

Regulation of epidermal growth factor receptor trafficking by lysine deacetylase hdac6

HDAC6 sets a brake that slows down the delivery of activated epidermal growth factor receptors to the degradative compartment. Setting a Speed Limit on EGFR Traffic Receptor tyrosine kinases interact with ligands at the cell surface to trigger intracellular signaling cascades. In some cases, these receptors are internalized, a process that can either enable them to initiate signaling cascades from intracellular membranes or target them for lysosomal degradation. Lissanu Deribe et al. connect acetylation of the microtubule cytoskeleton to regulation of delivery of epidermal growth factor receptors (EGFRs) to lysosomes. HDAC6, a cytoplasmic lysine deacetylase, was identified as binding to the inactive EGFR, stimulating deacetylation of α-tubulin, and decreasing the rate of delivery of EGFR from the early endosome to late endosomes or lysosomes. Phosphorylation of HDAC6, which decreased its activity, by activated EGFR created a negative feedback loop, leading to increased degradation of activated EGFRs. Binding of epidermal growth factor (EGF) to its receptor leads to receptor dimerization, assembly of protein complexes, and activation of signaling networks that control key cellular responses. Despite their fundamental role in cell biology, little is known about protein complexes associated with the EGF receptor (EGFR) before growth factor stimulation. We used a modified membrane yeast two-hybrid system together with bioinformatics to identify 87 candidate proteins interacting with the ligand-unoccupied EGFR. Among them was histone deacetylase 6 (HDAC6), a cytoplasmic lysine deacetylase, which we found negatively regulated EGFR endocytosis and degradation by controlling the acetylation status of α-tubulin and, subsequently, receptor trafficking along microtubules. A negative feedback loop consisting of EGFR-mediated phosphorylation of HDAC6 Tyr570 resulted in reduced deacetylase activity and increased acetylation of α-tubulin. This study illustrates the complexity of the EGFR-associated interactome and identifies protein acetylation as a previously unknown regulator of receptor endocytosis and degradation.

Measuring error rates in genomic perturbation screens: gold standards for human functional genomics

Technological advancement has opened the door to systematic genetics in mammalian cells. Genome‐scale loss‐of‐function screens can assay fitness defects induced by partial gene knockdown, using RNA interference, or complete gene knockout, using new CRISPR techniques. These screens can reveal the basic blueprint required for cellular proliferation. Moreover, comparing healthy to cancerous tissue can uncover genes that are essential only in the tumor; these genes are targets for the development of specific anticancer therapies. Unfortunately, progress in this field has been hampered by off‐target effects of perturbation reagents and poorly quantified error rates in large‐scale screens. To improve the quality of information derived from these screens, and to provide a framework for understanding the capabilities and limitations of CRISPR technology, we derive gold‐standard reference sets of essential and nonessential genes, and provide a Bayesian classifier of gene essentiality that outperforms current methods on both RNAi and CRISPR screens. Our results indicate that CRISPR technology is more sensitive than RNAi and that both techniques have nontrivial false discovery rates that can be mitigated by rigorous analytical methods.

Integrated proteomic and transcriptomic profiling of mouse lung development and Nmyc target genes.

Although microarray analysis has provided information regarding the dynamics of gene expression during development of the mouse lung, no extensive correlations have been made to the levels of corresponding protein products. Here, we present a global survey of protein expression during mouse lung organogenesis from embryonic day E13.5 until adulthood using gel‐free two‐dimensional liquid chromatography coupled to shotgun tandem mass spectrometry (MudPIT). Mathematical modeling of the proteomic profiles with parallel DNA microarray data identified large groups of gene products with statistically significant correlation or divergence in coregulation of protein and transcript levels during lung development. We also present an integrative analysis of mRNA and protein expression in Nmyc loss‐ and gain‐of‐function mutants. This revealed a set of 90 positively and negatively regulated putative target genes. These targets are evidence that Nmyc is a regulator of genes involved in mRNA processing and a repressor of the imprinted gene Igf2r in the developing lung.

Transcriptional targets of hepatocyte growth factor signaling and Ki- ras oncogene activation in colorectal cancer

Both Ki-ras mutation and hepatocyte growth factor (HGF) receptor Met overexpression occur at high frequency in colon cancer. This study investigates the transcriptional changes induced by Ki-ras oncogene and HGF/Met signaling activation in colon cancer cell lines in vitro and in vivo. The model system used in these studies included the DLD-1 colon cancer cell line with a mutated Ki-ras allele, and the DKO-4 cell line generated from DLD-1, with its mutant Ki-ras allele inactivated by targeted disruption. These cell lines were transduced with cDNAs of full-length Met receptor. Microarray transcriptional profiling was conducted on cell lines stimulated with HGF, as well as on tumor xenograft tissues. Overlapping genes between in vitro and in vivo microarray data sets were selected as a subset of HGF/Met and Ki-ras oncogene-regulated targets. Using the Online Predicted Human Interaction Database, novel HGF/Met and Ki-ras regulated proteins with putative functional linkage were identified. Novel proteins identified included histone acetyltransferase 1, phosphoribosyl pyrophosphate synthetase 2, chaperonin containing TCP1, subunit 8, CSE1 chromosome segregation 1-like (yeast)/cellular apoptosis susceptibility (mammals), CCR4–NOT transcription complex, subunit 8, and cyclin H. Transcript levels for these Met-signaling targets were correlated with Met expression levels, and were significantly elevated in both primary and metastatic human colorectal cancer samples compared to normal colorectal mucosa. These genes represent novel Met and/or Ki-ras transcriptionally coregulated genes with a high degree of validation in human colorectal cancers.

Genome-wide CRISPR screens reveal a Wnt-FZD5 signaling circuit as a druggable vulnerability of RNF43-mutant pancreatic tumors

Forward genetic screens with CRISPR–Cas9 genome editing enable high-resolution detection of genetic vulnerabilities in cancer cells. We conducted genome-wide CRISPR–Cas9 screens in RNF43-mutant pancreatic ductal adenocarcinoma (PDAC) cells, which rely on Wnt signaling for proliferation. Through these screens, we discovered a unique requirement for a Wnt signaling circuit: engaging FZD5, one of the ten Frizzled receptors encoded in the human genome. Our results uncover an underappreciated level of context-dependent specificity at the Wnt receptor level. We further derived a panel of recombinant antibodies that reports the expression of nine FZD proteins and confirms that FZD5 functional specificity cannot be explained by protein expression patterns. Additionally, antibodies that specifically bind FZD5 and FZD8 robustly inhibited the growth of RNF43-mutant PDAC cells grown in vitro and as xenografts in vivo, providing orthogonal support for the functional specificity observed genetically. Proliferation of a patient-derived PDAC cell line harboring an RNF43 variant was also selectively inhibited by the FZD5 antibodies, further demonstrating their use as a potential targeted therapy. Tumor organoid cultures from colorectal carcinoma patients that carried RNF43 mutations were also sensitive to the FZD5 antibodies, highlighting the potential generalizability of these findings beyond PDAC. Our results show that CRIPSR-based genetic screens can be leveraged to identify and validate cell surface targets for antibody development and therapy.

Hospital Ward Antibiotic Prescribing and the Risks of Clostridium difficile Infection

IMPORTANCE Only a portion of hospital-acquired Clostridium difficile infections can be traced back to source patients identified as having symptomatic disease. Antibiotic exposure is the main risk factor for C difficile infection for individual patients and is also associated with increased asymptomatic shedding. Contact with patients taking antibiotics within the same hospital ward may be a transmission risk factor for C difficile infection, but this hypothesis has never been tested. OBJECTIVES To obtain a complete portrait of inpatient risk that incorporates innate patient risk factors and transmission risk factors measured at the hospital ward level and to investigate ward-level rates of antibiotic use and C difficile infection risk. DESIGN, SETTING, AND PATIENTS A 46-month (June 1, 2010, through March 31, 2014) retrospective cohort study of inpatients 18 years or older in a large, acute care teaching hospital composed of 16 wards, including 5 intensive care units and 11 non-intensive care unit wards. EXPOSURES Patient-level risk factors (eg, age, comorbidities, hospitalization history, antibiotic exposure) and ward-level risk factors (eg, antibiotic therapy per 100 patient-days, hand hygiene adherence, mean patient age) were identified from hospital databases. MAIN OUTCOMES AND MEASURES Incidence of hospital-acquired C difficile infection as identified prospectively by hospital infection prevention and control staff. RESULTS A total of 255 of 34 298 patients developed C difficile (incidence rate, 5.95 per 10,000 patient-days; 95% CI, 5.26-6.73). Ward-level antibiotic exposure varied from 21.7 to 56.4 days of therapy per 100 patient-days. Each 10% increase in ward-level antibiotic exposure was associated with a 2.1 per 10,000 (P < .001) increase in C difficile incidence. The association between C difficile incidence and ward antibiotic exposure was the same among patients with and without recent antibiotic exposure, and C difficile risk persisted after multilevel, multivariate adjustment for differences in patient-risk factors among wards (relative risk, 1.34 per 10% increase in days of therapy; 95% CI, 1.16-1.57). CONCLUSIONS AND RELEVANCE Among hospital inpatients, ward-level antibiotic prescribing is associated with a statistically significant and clinically relevant increase in C difficile risk that persists after adjustment for differences in patient-level antibiotic use and other patient- and ward-level risk factors. These data strongly support the use of antibiotic stewardship as a means of preventing C difficile infection.

Identification of novel ATP13A2 interactors and their role in α-synuclein misfolding and toxicity

Lysosomes are responsible for degradation and recycling of bulky cell material, including accumulated misfolded proteins and dysfunctional organelles. Increasing evidence implicates lysosomal dysfunction in several neurodegenerative disorders, including Parkinsons disease and related synucleinopathies, which are characterized by the accumulation of α-synuclein (α-syn) in Lewy bodies. Studies of lysosomal proteins linked to neurodegenerative disorders present an opportunity to uncover specific molecular mechanisms and pathways that contribute to neurodegeneration. Loss-of-function mutations in a lysosomal protein, ATP13A2 (PARK9), cause Kufor-Rakeb syndrome that is characterized by early-onset parkinsonism, pyramidal degeneration and dementia. While loss of ATP13A2 function plays a role in α-syn misfolding and toxicity, the normal function of ATP13A2 in the brain remains largely unknown. Here, we performed a screen to identify ATP13A2 interacting partners, as a first step toward elucidating its function. Utilizing a split-ubiquitin membrane yeast two-hybrid system that was developed to identify interacting partners of full-length integral membrane proteins, we identified 43 novel interactors that primarily implicate ATP13A2 in cellular processes such as endoplasmic reticulum (ER) translocation, ER-to-Golgi trafficking and vesicular transport and fusion. We showed that a subset of these interactors modified α-syn aggregation and α-syn-mediated degeneration of dopaminergic neurons in Caenorhabditis elegans, further suggesting that ATP13A2 and α-syn are functionally linked in neurodegeneration. These results implicate ATP13A2 in vesicular trafficking and provide a platform for further studies of ATP13A2 in neurodegeneration.