GenomeRNAi - a database for RNAi phenotypes and reagents

Phenotype information for gene 5599 (MAPK8)

Screen TitleGene IDGene SymbolReagent IDScorePhenotypeComment
Hepatitis C virus replication (1)
5599
MAPK8
PL-50002
0
Decreased Hepatitis C virus replication

Reference

A functional genomic screen identifies cellular cofactors of hepatitis C virus replication. Tai et al., 2009

Hepatitis C virus (HCV) chronically infects 3% of the world''s population, and complications from HCV are the leading indication for liver transplantation. Given the need for better anti-HCV therapies, one strategy is to identify and target cellular cofactors of the virus lifecycle. Using a genome-wide siRNA library, we identified 96 human genes that support HCV replication, with a significant number of them being involved in vesicle organization and biogenesis. Phosphatidylinositol 4-kinase PI4KA and multiple subunits of the COPI vesicle coat complex were among the genes identified. Consistent with this, pharmacologic inhibitors of COPI and PI4KA blocked HCV replication. Targeting hepcidin, a peptide critical for iron homeostasis, also affected HCV replication, which may explain the known dysregulation of iron homeostasis in HCV infection. The host cofactors for HCV replication identified in this study should serve as a useful resource in delineating new targets for anti-HCV therapies.

Screen Details

Stable ID: GR00180-A-1
Screen Title: Hepatitis C virus replication (1)
Assay: HCV replicon RNA copy number
Method: Luminescence
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: Huh7/Rep-Feo
Library: Dharmacon, siARRAY Human Genome siRNA Library
Reagent Type: siRNA
Score Type: q-value
Cutoff: Complex criteria
Notes:

Human papillomavirus oncogene expression regulation (1)
5599
MAPK8
-0.1
none

Reference

Genome-wide siRNA screen identifies SMCX, EP400, and Brd4 as E2-dependent regulators of human papillomavirus oncogene expression. Smith et al., 2010

An essential step in the pathogenesis of human papillomavirus (HPV)-associated cancers is the dysregulated expression of the viral oncogenes. The papillomavirus E2 protein can silence the long control region (LCR) promoter that controls viral E6 and E7 oncogene expression. The mechanisms by which E2 represses oncogene expression and the cellular factors through which E2 mediates this silencing are largely unknown. We conducted an unbiased, genome-wide siRNA screen and series of secondary screens that identified 96 cellular genes that contribute to the repression of the HPV LCR. In addition to confirming a role for the E2-binding bromodomain protein Brd4 in E2-mediated silencing, we identified a number of genes that have not previously been implicated in E2 repression, including the demethylase JARID1C/SMCX as well as EP400, a component of the NuA4/TIP60 histone acetyltransferase complex. Each of these genes contributes independently and additively to E2-mediated silencing, indicating that E2 functions through several distinct cellular complexes to repress E6 and E7 expression.

Screen Details

Stable ID: GR00197-A-1
Screen Title: Human papillomavirus oncogene expression regulation (1)
Assay: HPV18 LCR reporter activity
Method: Luminescence
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: C33A/BE2/18LCR c4
Library: Dharmacon, Human siGENOME SMARTpool library
Reagent Type: siRNA
Score Type: Z-score
Cutoff: >= 2
Notes: Author-submitted data. Phenotype strength according to Z-scores: weak: 2 - 3; moderate: 3 - 5; strong: > 5

Circadian clock regulation
NM_002750
MAPK8
np
np
Increased circadian period length double hit

Reference

A genome-wide RNAi screen for modifiers of the circadian clock in human cells. Zhang et al., 2009

Two decades of research identified more than a dozen clock genes and defined a biochemical feedback mechanism of circadian oscillator function. To identify additional clock genes and modifiers, we conducted a genome-wide small interfering RNA screen in a human cellular clock model. Knockdown of nearly 1000 genes reduced rhythm amplitude. Potent effects on period length or increased amplitude were less frequent; we found hundreds of these and confirmed them in secondary screens. Characterization of a subset of these genes demonstrated a dosage-dependent effect on oscillator function. Protein interaction network analysis showed that dozens of gene products directly or indirectly associate with known clock components. Pathway analysis revealed these genes are overrepresented for components of insulin and hedgehog signaling, the cell cycle, and the folate metabolism. Coupled with data showing many of these pathways are clock regulated, we conclude the clock is interconnected with many aspects of cellular function.

Screen Details

Stable ID: GR00213-A
Screen Title: Circadian clock regulation
Assay: Circadian clock Bmal1 reporter
Method: Luminescence
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: U2OS
Library: Qiagen, np
Reagent Type: siRNA
Score Type: Complex, sp
Cutoff: Complex criteria
Notes: Additional information about a secondary screen

Vaccinia virus (VACV) infection
5599
MAPK8
1.04
Increased vaccinia virus (VACV) infection number of cells compared to control (%): 88.73

Reference

Human genome-wide RNAi screen reveals a role for nuclear pore proteins in poxvirus morphogenesis. Sivan et al., 2013

Poxviruses are considered less dependent on host functions than other DNA viruses because of their cytoplasmic site of replication and large genomes, which encode enzymes for DNA and mRNA synthesis. Nevertheless, RNAi screens with two independent human genome-scale libraries have identified more than 500 candidate genes that significantly inhibited and a similar number that enhanced replication and spread of infectious vaccinia virus (VACV). Translational, ubiquitin-proteosome, and endoplasmic reticulum-to-Golgi transport functions, known to be important for VACV, were enriched in the siRNA-inhibiting group, and RNA polymerase II and associated functions were enriched in the siRNA-enhancing group. Additional findings, notably the inhibition of VACV spread by siRNAs to several nuclear pore genes, were unanticipated. Knockdown of nucleoporin 62 strongly inhibited viral morphogenesis, with only a modest effect on viral gene expression, recapitulating and providing insight into previous studies with enucleated cells.

Screen Details

Stable ID: GR00249-S
Screen Title: Vaccinia virus (VACV) infection
Assay: Vaccinia virus VACV IHD-J/GFP protein expression and DNA content
Method: Fluorescence
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HeLa
Library: Ambion and Dharmacon, Silencer Select Version 4, siGENOME SMARTpool and OnTargetPlus
Reagent Type: siRNA
Score Type: Z-score
Cutoff: >= 1 OR <= -1.5
Notes: Author-submitted data. Primary screen. Decreased viability phenotype if number of cells compared to control < 50 %.

Wnt/beta-catenin pathway regulation (1)
NM_002750
MAPK8
-0.83
none

Reference

A genome-wide RNAi screen for Wnt/beta-catenin pathway components identifies unexpected roles for TCF transcription factors in cancer. Tang et al., 2008

The Wnt family of secreted proteins coordinate cell fate decision-making in a broad range of developmental and homeostatic contexts. Corruption of Wnt signal transduction pathways frequently results in degenerative diseases and cancer. We have used an iterative genome-wide screening strategy that employs multiple nonredundant RNAi reagents to identify mammalian genes that participate in Wnt/beta-catenin pathway response. Among the genes that were assigned high confidence scores are two members of the TCF/LEF family of DNA-binding proteins that control the transcriptional output of the pathway. Surprisingly, we found that the presumed cancer-promoting gene TCF7L2 functions instead as a transcriptional repressor that restricts colorectal cancer (CRC) cell growth. Mutations in TCF7L2 identified from cancer genome sequencing efforts abolish its ability to function as a transcriptional regulator and result in increased CRC cell growth. We describe a growth-promoting transcriptional program that is likely activated in CRC tumors with compromised TCF7L2 function. Taken together, the results from our screen and studies focused on members of the TCF/LEF gene family refine our understanding of how aberrant Wnt pathway activation sustains CRC growth.

Screen Details

Stable ID: GR00057-A-1
Screen Title: Wnt/beta-catenin pathway regulation (1)
Assay: Wnt pathway reporter
Method: Luminescence
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HeLa
Library: Dharmacon, Human siArray siRNA library
Reagent Type: siRNA
Score Type: Z-score
Cutoff: > 4
Notes: Screen without Wnt3A stimulation. Additional information about secondary screens (Dharmacon and Qiagen libraries).

Homologous recombination DNA double-strand break repair (HR-DSBR) (1)
5599
MAPK8
2.08
Increased homologous recombination repair frequency

Reference

A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response. Adamson et al., 2012

Repair of DNA double-strand breaks is critical to genomic stability and the prevention of developmental disorders and cancer. A central pathway for this repair is homologous recombination (HR). Most knowledge of HR is derived from work in prokaryotic and eukaryotic model organisms. We carried out a genome-wide siRNA-based screen in human cells. Among positive regulators of HR we identified networks of DNA-damage-response and pre-mRNA-processing proteins, and among negative regulators we identified a phosphatase network. Three candidate proteins localized to DNA lesions, including RBMX, a heterogeneous nuclear ribonucleoprotein that has a role in alternative splicing. RBMX accumulated at DNA lesions through multiple domains in a poly(ADP-ribose) polymerase 1-dependent manner and promoted HR by facilitating proper BRCA2 expression. Our screen also revealed that off-target depletion of RAD51 is a common source of RNAi false positives, raising a cautionary note for siRNA screens and RNAi-based studies of HR.

Screen Details

Stable ID: GR00236-A-1
Screen Title: Homologous recombination DNA double-strand break repair (HR-DSBR) (1)
Assay: (HR-DSBR) DR-GFP reporter and DNA content
Method: Fluorescence
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: DR-U2OS
Library: Dharmacon, Human siGENOME siRNA (G-005000-05)
Reagent Type: siRNA
Score Type: Relative HR ratio
Cutoff: < ~0.4 OR > 1.88
Notes: Cutoff values correspond 2 standard deviations from the screen-wide mean

Proliferation of cells with active beta-catenin (4)
MAPK8
-0.41
none

Reference

CK1epsilon is required for breast cancers dependent on beta-catenin activity. Kim et al., 2010

Aberrant beta-catenin signaling plays a key role in several cancer types, notably colon, liver and breast cancer. However approaches to modulate beta-catenin activity for therapeutic purposes have proven elusive to date.

Screen Details

Stable ID: GR00221-A-4
Screen Title: Proliferation of cells with active beta-catenin (4)
Assay: Viability
Method: Luminescence
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: T47D
Library: The RNAi Consortium (TRC), TRC shRNA Library
Reagent Type: shRNA
Score Type: B-score
Cutoff: < -1
Notes: Essential gene: gene with B-score < -1 for >= 2 shRNAs

Combinatorial effect with MLN4924, a NAE inhibitor (2)
5599
MAPK8
sp
Increased viability with MLN4924 (a NAE inhibitor) sextuplicate screen

Reference

Novel DNA damage checkpoints mediating cell death induced by the NEDD8-activating enzyme inhibitor MLN4924. Blank et al., 2013

MLN4924 is an investigational small-molecule inhibitor of the NEDD8-activating enzyme (NAE) in phase I clinical trials. NAE inhibition prevents the ubiquitination and proteasomal degradation of substrates for cullin-RING ubiquitin E3 ligases that support cancer pathophysiology, but the genetic determinants conferring sensitivity to NAE inhibition are unknown. To address this gap in knowledge, we conducted a genome-wide siRNA screen to identify genes and pathways that affect the lethality of MLN4924 in melanoma cells. Of the 154 genes identified, approximately one-half interfered with components of the cell cycle, apoptotic machinery, ubiquitin system, and DNA damage response pathways. In particular, genes involved in DNA replication, p53, BRCA1/BRCA2, transcription-coupled repair, and base excision repair seemed to be important for MLN4924 lethality. In contrast, genes within the G(2)-M checkpoint affected sensitivity to MLN4924 in colon cancer cells. Cell-cycle analysis in melanoma cells by flow cytometry following RNAi-mediated silencing showed that MLN4924 prevented the transition of cells from S-G(2) phase after induction of rereplication stress. Our analysis suggested an important role for the p21-dependent intra-S-phase checkpoint and extensive rereplication, whereas the ATR-dependent intra-S-phase checkpoint seemed to play a less dominant role. Unexpectedly, induction of the p21-dependent intra-S-phase checkpoint seemed to be independent of both Cdt1 stabilization and ATR signaling. Collectively, these data enhance our understanding of the mechanisms by which inhibition of NEDD8-dependent ubiquitination causes cell death, informing clinical development of MLN4924.

Screen Details

Stable ID: GR00250-A-2
Screen Title: Combinatorial effect with MLN4924, a NAE inhibitor (2)
Assay: Viability (synthetic lethal)
Method: Luminescence
Scope: Selected genes
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: A375
Library: Dharmacon, np
Reagent Type: siRNA
Score Type: Complex, sp
Cutoff: Complex criteria
Notes: Phenotypes for 650 nmol/L MLN4924. Additional information about screens with 250 nmol/L MLN4924.

Proliferation of cells with active beta-catenin (1)
MAPK8
-1.03
Decreased viability essential gene

Reference

CK1epsilon is required for breast cancers dependent on beta-catenin activity. Kim et al., 2010

Aberrant beta-catenin signaling plays a key role in several cancer types, notably colon, liver and breast cancer. However approaches to modulate beta-catenin activity for therapeutic purposes have proven elusive to date.

Screen Details

Stable ID: GR00221-A-1
Screen Title: Proliferation of cells with active beta-catenin (1)
Assay: Viability
Method: Luminescence
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: MCF7
Library: The RNAi Consortium (TRC), TRC shRNA Library
Reagent Type: shRNA
Score Type: B-score
Cutoff: < -1
Notes: Essential gene: gene with B-score < -1 for >= 2 shRNAs

Ciliogenesis and cilium length (1)
5599
MAPK8
1225
107.45
none

Reference

Functional genomic screen for modulators of ciliogenesis and cilium length. Kim et al., 2010

Primary cilia are evolutionarily conserved cellular organelles that organize diverse signalling pathways. Defects in the formation or function of primary cilia are associated with a spectrum of human diseases and developmental abnormalities. Genetic screens in model organisms have discovered core machineries of cilium assembly and maintenance. However, regulatory molecules that coordinate the biogenesis of primary cilia with other cellular processes, including cytoskeletal organization, vesicle trafficking and cell-cell adhesion, remain to be identified. Here we report the results of a functional genomic screen using RNA interference (RNAi) to identify human genes involved in ciliogenesis control. The screen identified 36 positive and 13 negative ciliogenesis modulators, which include molecules involved in actin dynamics and vesicle trafficking. Further investigation demonstrated that blocking actin assembly facilitates ciliogenesis by stabilizing the pericentrosomal preciliary compartment (PPC), a previously uncharacterized compact vesiculotubular structure storing transmembrane proteins destined for cilia during the early phase of ciliogenesis. The PPC was labelled by recycling endosome markers. Moreover, knockdown of modulators that are involved in the endocytic recycling pathway affected the formation of the PPC as well as ciliogenesis. Our results uncover a critical regulatory step that couples actin dynamics and endocytic recycling with ciliogenesis, and also provides potential target molecules for future study.

Screen Details

Stable ID: GR00149-A-1
Screen Title: Ciliogenesis and cilium length (1)
Assay: Smoothed protein expression
Method: Fluorescence
Scope: Druggable genes
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: htRPE
Library: Ambion, Human druggable genome siRNA library V3.1
Reagent Type: siRNA
Score Type: Normalized percent inhibition
Cutoff: > 1.5 OR < -1.5 standard deviations from mean
Notes:

tau phosphorylation
MAPK8
MAPK8 s1
sp
none

Reference

High-content siRNA screening of the kinome identifies kinases involved in Alzheimer's disease-related tau hyperphosphorylation. Azorsa et al., 2010

Neurofibrillary tangles (NFT), a cardinal neuropathological feature of Alzheimer''s disease (AD) that is highly correlated with synaptic loss and dementia severity, appear to be partly attributable to increased phosphorylation of the microtubule stabilizing protein tau at certain AD-related residues. Identifying the kinases involved in the pathologic phosphorylation of tau may provide targets at which to aim new AD-modifying treatments.

Screen Details

Stable ID: GR00143-A
Screen Title: tau phosphorylation
Assay: Total tau and 12E8 tau protein expression
Method: Fluorescence
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: H4 overexpressing 4RON tau
Library: Qiagen, Validated human kinase siRNA Set 2.0
Reagent Type: siRNA
Score Type: p-value
Cutoff: Complex criteria
Notes:

Proliferation of cells with active beta-catenin (2)
MAPK8
-1.13
Decreased viability

Reference

CK1epsilon is required for breast cancers dependent on beta-catenin activity. Kim et al., 2010

Aberrant beta-catenin signaling plays a key role in several cancer types, notably colon, liver and breast cancer. However approaches to modulate beta-catenin activity for therapeutic purposes have proven elusive to date.

Screen Details

Stable ID: GR00221-A-2
Screen Title: Proliferation of cells with active beta-catenin (2)
Assay: Viability
Method: Luminescence
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: MDA-MB-231
Library: The RNAi Consortium (TRC), TRC shRNA Library
Reagent Type: shRNA
Score Type: B-score
Cutoff: < -1
Notes: Essential gene: gene with B-score < -1 for >= 2 shRNAs

Ciliogenesis and cilium length (1)
5599
MAPK8
37184
-6.59
none

Reference

Functional genomic screen for modulators of ciliogenesis and cilium length. Kim et al., 2010

Primary cilia are evolutionarily conserved cellular organelles that organize diverse signalling pathways. Defects in the formation or function of primary cilia are associated with a spectrum of human diseases and developmental abnormalities. Genetic screens in model organisms have discovered core machineries of cilium assembly and maintenance. However, regulatory molecules that coordinate the biogenesis of primary cilia with other cellular processes, including cytoskeletal organization, vesicle trafficking and cell-cell adhesion, remain to be identified. Here we report the results of a functional genomic screen using RNA interference (RNAi) to identify human genes involved in ciliogenesis control. The screen identified 36 positive and 13 negative ciliogenesis modulators, which include molecules involved in actin dynamics and vesicle trafficking. Further investigation demonstrated that blocking actin assembly facilitates ciliogenesis by stabilizing the pericentrosomal preciliary compartment (PPC), a previously uncharacterized compact vesiculotubular structure storing transmembrane proteins destined for cilia during the early phase of ciliogenesis. The PPC was labelled by recycling endosome markers. Moreover, knockdown of modulators that are involved in the endocytic recycling pathway affected the formation of the PPC as well as ciliogenesis. Our results uncover a critical regulatory step that couples actin dynamics and endocytic recycling with ciliogenesis, and also provides potential target molecules for future study.

Screen Details

Stable ID: GR00149-A-1
Screen Title: Ciliogenesis and cilium length (1)
Assay: Smoothed protein expression
Method: Fluorescence
Scope: Druggable genes
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: htRPE
Library: Ambion, Human druggable genome siRNA library V3.1
Reagent Type: siRNA
Score Type: Normalized percent inhibition
Cutoff: > 1.5 OR < -1.5 standard deviations from mean
Notes:

Wnt/beta-catenin pathway regulation
5599
MAPK8
np
1.55
none

Reference

Bruton's tyrosine kinase revealed as a negative regulator of Wnt-beta-catenin signaling. James et al., 2009

Wnts are secreted ligands that activate several receptor-mediated signal transduction cascades. Homeostatic Wnt signaling through beta-catenin is required in adults, because either elevation or attenuation of beta-catenin function has been linked to diverse diseases. To contribute to the identification of both protein and pharmacological regulators of this pathway, we describe a combinatorial screen that merged data from a high-throughput screen of known bioactive compounds with an independent focused small interfering RNA screen. Each screen independently revealed Bruton''s tyrosine kinase (BTK) as an inhibitor of Wnt-beta-catenin signaling. Loss of BTK function in human colorectal cancer cells, human B cells, zebrafish embryos, and cells derived from X-linked agammaglobulinemia patients with a mutant BTK gene resulted in elevated Wnt-beta-catenin signaling, confirming that BTK acts as a negative regulator of this pathway. From affinity purification-mass spectrometry and biochemical binding studies, we found that BTK directly interacts with a nuclear component of Wnt-beta-catenin signaling, CDC73. Further, we show that BTK increased the abundance of CDC73 in the absence of stimulation and that CDC73 acted as a repressor of beta-catenin-mediated transcription in human colorectal cancer cells and B cells.

Screen Details

Stable ID: GR00016-A
Screen Title: Wnt/beta-catenin pathway regulation
Assay: Wnt/beta-catenin pathway reporter
Method: Luminescence
Scope: Selected genes
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: RKO
Library: rp, rp
Reagent Type: siRNA
Score Type: Z-score
Cutoff: > 2
Notes:

Proliferation of cells with active beta-catenin (3)
MAPK8
1.41
none

Reference

CK1epsilon is required for breast cancers dependent on beta-catenin activity. Kim et al., 2010

Aberrant beta-catenin signaling plays a key role in several cancer types, notably colon, liver and breast cancer. However approaches to modulate beta-catenin activity for therapeutic purposes have proven elusive to date.

Screen Details

Stable ID: GR00221-A-3
Screen Title: Proliferation of cells with active beta-catenin (3)
Assay: Viability
Method: Luminescence
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: MDA-MB-453
Library: The RNAi Consortium (TRC), TRC shRNA Library
Reagent Type: shRNA
Score Type: B-score
Cutoff: < -1
Notes: Essential gene: gene with B-score < -1 for >= 2 shRNAs

Combinatorial effect with Poly (ADP‐ribose)‐polymerase‐1 (PARP)
MAPK8
np
0.27
none 109%

Reference

A synthetic lethal siRNA screen identifying genes mediating sensitivity to a PARP inhibitor. Turner et al., 2008

Inhibitors of poly (ADP-ribose)-polymerase-1 (PARP) are highly lethal to cells with deficiencies in BRCA1, BRCA2 or other components of the homologous recombination pathway. This has led to PARP inhibitors entering clinical trials as a potential therapy for cancer in carriers of BRCA1 and BRCA2 mutations. To discover new determinants of sensitivity to these drugs, we performed a PARP-inhibitor synthetic lethal short interfering RNA (siRNA) screen. We identified a number of kinases whose silencing strongly sensitised to PARP inhibitor, including cyclin-dependent kinase 5 (CDK5), MAPK12, PLK3, PNKP, STK22c and STK36. How CDK5 silencing mediates sensitivity was investigated. Previously, CDK5 has been suggested to be active only in a neuronal context, but here we show that CDK5 is required in non-neuronal cells for the DNA-damage response and, in particular, intra-S and G(2)/M cell-cycle checkpoints. These results highlight the potential of synthetic lethal siRNA screens with chemical inhibitors to define new determinants of sensitivity and potential therapeutic targets.

Screen Details

Stable ID: GR00114-A
Screen Title: Combinatorial effect with Poly (ADP‐ribose)‐polymerase‐1 (PARP)
Assay: Viability
Method: Luminescence
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: CAL51
Library: Dharmacon, siARRAY SMARTpool
Reagent Type: siRNA
Score Type: Z-score
Cutoff: <= -3
Notes: Growth rates (%) for vehicle-alone experiments are given in the comment field.

Proliferation of cells with active beta-catenin (3)
MAPK8
1.85
none

Reference

CK1epsilon is required for breast cancers dependent on beta-catenin activity. Kim et al., 2010

Aberrant beta-catenin signaling plays a key role in several cancer types, notably colon, liver and breast cancer. However approaches to modulate beta-catenin activity for therapeutic purposes have proven elusive to date.

Screen Details

Stable ID: GR00221-A-3
Screen Title: Proliferation of cells with active beta-catenin (3)
Assay: Viability
Method: Luminescence
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: MDA-MB-453
Library: The RNAi Consortium (TRC), TRC shRNA Library
Reagent Type: shRNA
Score Type: B-score
Cutoff: < -1
Notes: Essential gene: gene with B-score < -1 for >= 2 shRNAs

Homologous recombination DNA double-strand break repair (HR-DSBR) (2)
5599
MAPK8
1.68
none

Reference

A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response. Adamson et al., 2012

Repair of DNA double-strand breaks is critical to genomic stability and the prevention of developmental disorders and cancer. A central pathway for this repair is homologous recombination (HR). Most knowledge of HR is derived from work in prokaryotic and eukaryotic model organisms. We carried out a genome-wide siRNA-based screen in human cells. Among positive regulators of HR we identified networks of DNA-damage-response and pre-mRNA-processing proteins, and among negative regulators we identified a phosphatase network. Three candidate proteins localized to DNA lesions, including RBMX, a heterogeneous nuclear ribonucleoprotein that has a role in alternative splicing. RBMX accumulated at DNA lesions through multiple domains in a poly(ADP-ribose) polymerase 1-dependent manner and promoted HR by facilitating proper BRCA2 expression. Our screen also revealed that off-target depletion of RAD51 is a common source of RNAi false positives, raising a cautionary note for siRNA screens and RNAi-based studies of HR.

Screen Details

Stable ID: GR00236-A-2
Screen Title: Homologous recombination DNA double-strand break repair (HR-DSBR) (2)
Assay: (HR-DSBR) DR-GFP reporter and DNA content
Method: Fluorescence
Scope: Selected genes
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: DR-U2OS
Library: Dharmacon, np
Reagent Type: siRNA
Score Type: Relative HR ratio
Cutoff: Strong: < ~0.4, weak: < ~0.59 (for mediators); strong: > 1.88, weak: > 1.69 (for suppressors)
Notes: Cutoff values correspond 2 standard deviations from the screen-wide mean (strong) or 1.5 SD (weak)

Cdk9 T-loop phosphorylation
5599
MAPK8
1225
143169
np
none

Reference

Cdk9 T-loop phosphorylation is regulated by the calcium signaling pathway. Ramakrishnan and Rice, 2012

Eukaryotic RNA polymerase II transcriptional elongation is a tightly regulated process and is dependent upon positive transcription elongation factor-b (P-TEFb). The core P-TEFb complex is composed of Cdk9 and Cyclin T and is essential for the expression of most protein coding genes. Cdk9 kinase function is dependent upon phosphorylation of Thr186 in its T-loop. In this study, we examined kinases and signaling pathways that influence Cdk9 T-loop phosphorylation. Using an RNAi screen in HeLa cells, we found that Cdk9 T-loop phosphorylation is regulated by Ca(2+)/calmodulin-dependent kinase 1D (CaMK1D). Using small molecules inhibitors in HeLa cells and primary CD4(+) T lymphocytes, we found that the Ca(2+) signaling pathway is required for Cdk9 T-loop phosphorylation. Inhibition of Ca(2+) signaling led to dephosphorylation of Thr186 on Cdk9. In reporter plasmid assays, inhibition of the Ca(2+) signaling pathway repressed the PCNA promoter and HIV-1 Tat transactivation of the HIV-1 LTR, but not HTLV-1 Tax transactivation of the HTLV-1 LTR, suggesting that perturbation of the Ca(2+) pathway and reduction of Cdk9 T-loop phosphorylation inhibits transcription units that have a rigorous requirement for P-TEFb function.

Screen Details

Stable ID: GR00243-A
Screen Title: Cdk9 T-loop phosphorylation
Assay: Cdk9 T-loop phosphorylation and beta-Actin protein expression
Method: Fluorescence
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HeLa
Library: Ambion, np
Reagent Type: siRNA
Score Type: np
Cutoff: >= 2 of 3 siRNAs decreasing phosphorylation
Notes:

Homologous recombination DNA double-strand break repair (HR-DSBR) (2)
5599
MAPK8
1.02
none

Reference

A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response. Adamson et al., 2012

Repair of DNA double-strand breaks is critical to genomic stability and the prevention of developmental disorders and cancer. A central pathway for this repair is homologous recombination (HR). Most knowledge of HR is derived from work in prokaryotic and eukaryotic model organisms. We carried out a genome-wide siRNA-based screen in human cells. Among positive regulators of HR we identified networks of DNA-damage-response and pre-mRNA-processing proteins, and among negative regulators we identified a phosphatase network. Three candidate proteins localized to DNA lesions, including RBMX, a heterogeneous nuclear ribonucleoprotein that has a role in alternative splicing. RBMX accumulated at DNA lesions through multiple domains in a poly(ADP-ribose) polymerase 1-dependent manner and promoted HR by facilitating proper BRCA2 expression. Our screen also revealed that off-target depletion of RAD51 is a common source of RNAi false positives, raising a cautionary note for siRNA screens and RNAi-based studies of HR.

Screen Details

Stable ID: GR00236-A-2
Screen Title: Homologous recombination DNA double-strand break repair (HR-DSBR) (2)
Assay: (HR-DSBR) DR-GFP reporter and DNA content
Method: Fluorescence
Scope: Selected genes
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: DR-U2OS
Library: Dharmacon, np
Reagent Type: siRNA
Score Type: Relative HR ratio
Cutoff: Strong: < ~0.4, weak: < ~0.59 (for mediators); strong: > 1.88, weak: > 1.69 (for suppressors)
Notes: Cutoff values correspond 2 standard deviations from the screen-wide mean (strong) or 1.5 SD (weak)

Combinatorial effect with MLN4924, a NAE inhibitor (2)
5599
MAPK8
sp
Synthetic lethal with MLN4924 (a NAE inhibitor) sextuplicate screen

Reference

Novel DNA damage checkpoints mediating cell death induced by the NEDD8-activating enzyme inhibitor MLN4924. Blank et al., 2013

MLN4924 is an investigational small-molecule inhibitor of the NEDD8-activating enzyme (NAE) in phase I clinical trials. NAE inhibition prevents the ubiquitination and proteasomal degradation of substrates for cullin-RING ubiquitin E3 ligases that support cancer pathophysiology, but the genetic determinants conferring sensitivity to NAE inhibition are unknown. To address this gap in knowledge, we conducted a genome-wide siRNA screen to identify genes and pathways that affect the lethality of MLN4924 in melanoma cells. Of the 154 genes identified, approximately one-half interfered with components of the cell cycle, apoptotic machinery, ubiquitin system, and DNA damage response pathways. In particular, genes involved in DNA replication, p53, BRCA1/BRCA2, transcription-coupled repair, and base excision repair seemed to be important for MLN4924 lethality. In contrast, genes within the G(2)-M checkpoint affected sensitivity to MLN4924 in colon cancer cells. Cell-cycle analysis in melanoma cells by flow cytometry following RNAi-mediated silencing showed that MLN4924 prevented the transition of cells from S-G(2) phase after induction of rereplication stress. Our analysis suggested an important role for the p21-dependent intra-S-phase checkpoint and extensive rereplication, whereas the ATR-dependent intra-S-phase checkpoint seemed to play a less dominant role. Unexpectedly, induction of the p21-dependent intra-S-phase checkpoint seemed to be independent of both Cdt1 stabilization and ATR signaling. Collectively, these data enhance our understanding of the mechanisms by which inhibition of NEDD8-dependent ubiquitination causes cell death, informing clinical development of MLN4924.

Screen Details

Stable ID: GR00250-A-2
Screen Title: Combinatorial effect with MLN4924, a NAE inhibitor (2)
Assay: Viability (synthetic lethal)
Method: Luminescence
Scope: Selected genes
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: A375
Library: Dharmacon, np
Reagent Type: siRNA
Score Type: Complex, sp
Cutoff: Complex criteria
Notes: Phenotypes for 650 nmol/L MLN4924. Additional information about screens with 250 nmol/L MLN4924.

tau phosphorylation
MAPK8
MAPK8 s2
sp
none

Reference

High-content siRNA screening of the kinome identifies kinases involved in Alzheimer's disease-related tau hyperphosphorylation. Azorsa et al., 2010

Neurofibrillary tangles (NFT), a cardinal neuropathological feature of Alzheimer''s disease (AD) that is highly correlated with synaptic loss and dementia severity, appear to be partly attributable to increased phosphorylation of the microtubule stabilizing protein tau at certain AD-related residues. Identifying the kinases involved in the pathologic phosphorylation of tau may provide targets at which to aim new AD-modifying treatments.

Screen Details

Stable ID: GR00143-A
Screen Title: tau phosphorylation
Assay: Total tau and 12E8 tau protein expression
Method: Fluorescence
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: H4 overexpressing 4RON tau
Library: Qiagen, Validated human kinase siRNA Set 2.0
Reagent Type: siRNA
Score Type: p-value
Cutoff: Complex criteria
Notes:

Human cytomegalovirus (HCMV) strain AD169 replication
NM_002750
MAPK8
MAPK8_siRNA3
0.93
none

Reference

Human kinome profiling identifies a requirement for AMP-activated protein kinase during human cytomegalovirus infection. Terry et al., 2012

Human cytomegalovirus (HCMV) modulates numerous cellular signaling pathways. Alterations in signaling are evident from the broad changes in cellular phosphorylation that occur during HCMV infection and from the altered activity of multiple kinases. Here we report a comprehensive RNAi screen, which predicts that 106 cellular kinases influence growth of the virus, most of which were not previously linked to HCMV replication. Multiple elements of the AMP-activated protein kinase (AMPK) pathway scored in the screen. As a regulator of carbon and nucleotide metabolism, AMPK is poised to activate many of the metabolic pathways induced by HCMV infection. An AMPK inhibitor, compound C, blocked a substantial portion of HCMV-induced metabolic changes, inhibited the accumulation of all HCMV proteins tested, and markedly reduced the production of infectious progeny. We propose that HCMV requires AMPK or related activity for viral replication and reprogramming of cellular metabolism.

Screen Details

Stable ID: GR00248-A
Screen Title: Human cytomegalovirus (HCMV) strain AD169 replication
Assay: Human cytomegalovirus (HCMV) IE1 protein expression
Method: Fluorescence
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: MRC5
Library: Sigma-Aldrich, Mission siRNA Human Kinase Panel
Reagent Type: siRNA
Score Type: Z-score
Cutoff: >=2 or <=-2 for >= 2 of 3 siRNA
Notes:

Melanogenesis
NM_002750
MAPK8
np
0.8
none

Reference

Genome-wide siRNA-based functional genomics of pigmentation identifies novel genes and pathways that impact melanogenesis in human cells. Ganesan et al., 2008

Melanin protects the skin and eyes from the harmful effects of UV irradiation, protects neural cells from toxic insults, and is required for sound conduction in the inner ear. Aberrant regulation of melanogenesis underlies skin disorders (melasma and vitiligo), neurologic disorders (Parkinson''s disease), auditory disorders (Waardenburg''s syndrome), and opthalmologic disorders (age related macular degeneration). Much of the core synthetic machinery driving melanin production has been identified; however, the spectrum of gene products participating in melanogenesis in different physiological niches is poorly understood. Functional genomics based on RNA-mediated interference (RNAi) provides the opportunity to derive unbiased comprehensive collections of pharmaceutically tractable single gene targets supporting melanin production. In this study, we have combined a high-throughput, cell-based, one-well/one-gene screening platform with a genome-wide arrayed synthetic library of chemically synthesized, small interfering RNAs to identify novel biological pathways that govern melanin biogenesis in human melanocytes. Ninety-two novel genes that support pigment production were identified with a low false discovery rate. Secondary validation and preliminary mechanistic studies identified a large panel of targets that converge on tyrosinase expression and stability. Small molecule inhibition of a family of gene products in this class was sufficient to impair chronic tyrosinase expression in pigmented melanoma cells and UV-induced tyrosinase expression in primary melanocytes. Isolation of molecular machinery known to support autophagosome biosynthesis from this screen, together with in vitro and in vivo validation, exposed a close functional relationship between melanogenesis and autophagy. In summary, these studies illustrate the power of RNAi-based functional genomics to identify novel genes, pathways, and pharmacologic agents that impact a biological phenotype and operate outside of preconceived mechanistic relationships.

Screen Details

Stable ID: GR00056-A
Screen Title: Melanogenesis
Assay: Melanin protein expression and viability
Method: Absorbance and luminescence
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: MNT-1
Library: Dharmacon, rp
Reagent Type: siRNA
Score Type: Normalized absorbance ratio
Cutoff: > 2 standard deviations below mean
Notes: Additional information about a secondary screen (retest to determine false-positive rate)

Proliferation of cells with active beta-catenin (1)
MAPK8
-1.72
Decreased viability essential gene

Reference

CK1epsilon is required for breast cancers dependent on beta-catenin activity. Kim et al., 2010

Aberrant beta-catenin signaling plays a key role in several cancer types, notably colon, liver and breast cancer. However approaches to modulate beta-catenin activity for therapeutic purposes have proven elusive to date.

Screen Details

Stable ID: GR00221-A-1
Screen Title: Proliferation of cells with active beta-catenin (1)
Assay: Viability
Method: Luminescence
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: MCF7
Library: The RNAi Consortium (TRC), TRC shRNA Library
Reagent Type: shRNA
Score Type: B-score
Cutoff: < -1
Notes: Essential gene: gene with B-score < -1 for >= 2 shRNAs

Human cytomegalovirus (HCMV) strain AD169 replication
NM_002750
MAPK8
MAPK8_siRNA1
0.26
none

Reference

Human kinome profiling identifies a requirement for AMP-activated protein kinase during human cytomegalovirus infection. Terry et al., 2012

Human cytomegalovirus (HCMV) modulates numerous cellular signaling pathways. Alterations in signaling are evident from the broad changes in cellular phosphorylation that occur during HCMV infection and from the altered activity of multiple kinases. Here we report a comprehensive RNAi screen, which predicts that 106 cellular kinases influence growth of the virus, most of which were not previously linked to HCMV replication. Multiple elements of the AMP-activated protein kinase (AMPK) pathway scored in the screen. As a regulator of carbon and nucleotide metabolism, AMPK is poised to activate many of the metabolic pathways induced by HCMV infection. An AMPK inhibitor, compound C, blocked a substantial portion of HCMV-induced metabolic changes, inhibited the accumulation of all HCMV proteins tested, and markedly reduced the production of infectious progeny. We propose that HCMV requires AMPK or related activity for viral replication and reprogramming of cellular metabolism.

Screen Details

Stable ID: GR00248-A
Screen Title: Human cytomegalovirus (HCMV) strain AD169 replication
Assay: Human cytomegalovirus (HCMV) IE1 protein expression
Method: Fluorescence
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: MRC5
Library: Sigma-Aldrich, Mission siRNA Human Kinase Panel
Reagent Type: siRNA
Score Type: Z-score
Cutoff: >=2 or <=-2 for >= 2 of 3 siRNA
Notes:

pRB-induced cell cycle regulation (1)
NM_139049
MAPK8
-4.11
Decreased cell viability after pRB stimulation

Reference

A kinase shRNA screen links LATS2 and the pRB tumor suppressor. Tschöp et al., 2011

pRB-mediated inhibition of cell proliferation is a complex process that depends on the action of many proteins. However, little is known about the specific pathways that cooperate with the Retinoblastoma protein (pRB) and the variables that influence pRB''s ability to arrest tumor cells. Here we describe two shRNA screens that identify kinases that are important for pRB to suppress cell proliferation and pRB-mediated induction of senescence markers. The results reveal an unexpected effect of LATS2, a component of the Hippo pathway, on pRB-induced phenotypes. Partial knockdown of LATS2 strongly suppresses some pRB-induced senescence markers. Further analysis shows that LATS2 cooperates with pRB to promote the silencing of E2F target genes, and that reduced levels of LATS2 lead to defects in the assembly of DREAM (DP, RB [retinoblastoma], E2F, and MuvB) repressor complexes at E2F-regulated promoters. Kinase assays show that LATS2 can phosphorylate DYRK1A, and that it enhances the ability of DYRK1A to phosphorylate the DREAM subunit LIN52. Intriguingly, the LATS2 locus is physically linked with RB1 on 13q, and this region frequently displays loss of heterozygosity in human cancers. Our results reveal a functional connection between the pRB and Hippo tumor suppressor pathways, and suggest that low levels of LATS2 may undermine the ability of pRB to induce a permanent cell cycle arrest in tumor cells.

Screen Details

Stable ID: GR00230-A-1
Screen Title: pRB-induced cell cycle regulation (1)
Assay: Cell number and viability
Method: Colorimetrics
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: SaOS2-TR-pRB
Library: TRC, Lentiviral shRNA library
Reagent Type: shRNA
Score Type: SD from the trendline
Cutoff: > 3.0 OR < -3.0 for >= 2 shRNAs
Notes:

Combinatorial effect with gemcitabine
MAPK8
MAPK8_A
-0.71
none

Reference

Synthetic lethal RNAi screening identifies sensitizing targets for gemcitabine therapy in pancreatic cancer. Azorsa et al., 2009

Pancreatic cancer retains a poor prognosis among the gastrointestinal cancers. It affects 230,000 individuals worldwide, has a very high mortality rate, and remains one of the most challenging malignancies to treat successfully. Treatment with gemcitabine, the most widely used chemotherapeutic against pancreatic cancer, is not curative and resistance may occur. Combinations of gemcitabine with other chemotherapeutic drugs or biological agents have resulted in limited improvement.

Screen Details

Stable ID: GR00225-A
Screen Title: Combinatorial effect with gemcitabine
Assay: Viability (synthetic lethal)
Method: Luminescence
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: MIA PaCa-2
Library: Qiagen, Validated kinase siRNA library version 1.0
Reagent Type: siRNA
Score Type: log2 ratio
Cutoff: 1.65 SD below mean ratio level
Notes:

Negative genetic interactions (5)
5599
MAPK8
0.07
none

Reference

A negative genetic interaction map in isogenic cancer cell lines reveals cancer cell vulnerabilities. Vizeacoumar et al., 2013

Improved efforts are necessary to define the functional product of cancer mutations currently being revealed through large-scale sequencing efforts. Using genome-scale pooled shRNA screening technology, we mapped negative genetic interactions across a set of isogenic cancer cell lines and confirmed hundreds of these interactions in orthogonal co-culture competition assays to generate a high-confidence genetic interaction network of differentially essential or differential essentiality (DiE) genes. The network uncovered examples of conserved genetic interactions, densely connected functional modules derived from comparative genomics with model systems data, functions for uncharacterized genes in the human genome and targetable vulnerabilities. Finally, we demonstrate a general applicability of DiE gene signatures in determining genetic dependencies of other non-isogenic cancer cell lines. For example, the PTEN(-/-) DiE genes reveal a signature that can preferentially classify PTEN-dependent genotypes across a series of non-isogenic cell lines derived from the breast, pancreas and ovarian cancers. Our reference network suggests that many cancer vulnerabilities remain to be discovered through systematic derivation of a network of differentially essential genes in an isogenic cancer cell model.

Screen Details

Stable ID: GR00255-A-5
Screen Title: Negative genetic interactions (5)
Assay: shRNA abundance
Method: Microarray
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HCT116
Library: TRC lentiviral library, np
Reagent Type: shRNA
Score Type: differential Gene Activity Ranking Profile (dGARP)
Cutoff: < -0.8
Notes: HCT116 KRASG13D/- and HCT116 KRAS+/- cells used. Cutoff corresponds to p-value < 0.05. Additional information about a secondary screen (genetic interactions with Cetuximab/Erbitux in LIM1215 cells)

Telomerase activity regulation
MAPK8
np
199.03
Increased telomerase activity

Reference

High-throughput RNAi screening reveals novel regulators of telomerase. Cerone et al., 2011

Telomerase is considered an attractive anticancer target on the basis of its common and specific activation in most human cancers. While direct telomerase inhibition is being explored as a therapeutic strategy, alternative strategies to target regulators of telomerase that could disrupt telomere maintenance and cancer cell proliferation are not yet available. Here, we report the findings of a high-throughput functional RNA interference screen to globally profile the contribution of kinases to telomerase activity (TA). This analysis identified a number of novel telomerase modulators, including ERK8 kinase, whose inhibition reduces TA and elicited characteristics of telomere dysfunction. Given that kinases represent attractive drug targets, we addressed the therapeutic implications of our findings, such as demonstrating how limiting TA via kinase blockade could sensitize cells to inhibition of the telomere-associated protein tankyrase. Taken together, our findings suggest novel combinatorial approaches to targeting telomere maintenance as a strategy for cancer therapy.

Screen Details

Stable ID: GR00156-A
Screen Title: Telomerase activity regulation
Assay: Telomerase activity
Method: Real-time qTRAP
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HeLa
Library: Dharmacon, SMARTpool siRNA kinase library
Reagent Type: siRNA
Score Type: Percentage
Cutoff: < 35 % OR > 150 %
Notes:

Non-small cell lung cancer (NSCLC) cytotoxicity (2)
MAPK8
np
-1
Decreased shRNA abundance

Reference

Proteasome inhibitors block DNA repair and radiosensitize non-small cell lung cancer. Cron et al., 2013

Despite optimal radiation therapy (RT), chemotherapy and/or surgery, a majority of patients with locally advanced non-small cell lung cancer (NSCLC) fail treatment. To identify novel gene targets for improved tumor control, we performed whole genome RNAi screens to identify knockdowns that most reproducibly increase NSCLC cytotoxicity. These screens identified several proteasome subunits among top hits, including the topmost hit PSMA1, a component of the core 20 S proteasome. Radiation and proteasome inhibition showed synergistic effects. Proteasome inhibition resulted in an 80-90% decrease in homologous recombination (HR), a 50% decrease in expression of NF-κB-inducible HR genes BRCA1 and FANCD2, and a reduction of BRCA1, FANCD2 and RAD51 ionizing radiation-induced foci. IκBα RNAi knockdown rescued NSCLC radioresistance. Irradiation of mice with NCI-H460 xenografts after inducible PSMA1 shRNA knockdown markedly increased murine survival compared to either treatment alone. Proteasome inhibition is a promising strategy for NSCLC radiosensitization via inhibition of NF-κB-mediated expression of Fanconi Anemia/HR DNA repair genes.

Screen Details

Stable ID: GR00251-A-2
Screen Title: Non-small cell lung cancer (NSCLC) cytotoxicity (2)
Assay: shRNA abundance
Method: Microarray
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: NCI-H460
Library: Hannon-Elledge whole genome pooled shRNA, np
Reagent Type: shRNA
Score Type: Complex, sp
Cutoff: Complex criteria
Notes: All listed genes are final hits. Final hit: >= 1 shRNA with >= 2-fold abundance decrease in both cell lines (A549 and NCI-H460)

Synthetic lethal interaction with Ras
MAPK8
-0.98 (0.06)
Synthetic lethal with Ras

Reference

A genome-wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene. Luo et al., 2009

Oncogenic mutations in the small GTPase Ras are highly prevalent in cancer, but an understanding of the vulnerabilities of these cancers is lacking. We undertook a genome-wide RNAi screen to identify synthetic lethal interactions with the KRAS oncogene. We discovered a diverse set of proteins whose depletion selectively impaired the viability of Ras mutant cells. Among these we observed a strong enrichment for genes with mitotic functions. We describe a pathway involving the mitotic kinase PLK1, the anaphase-promoting complex/cyclosome, and the proteasome that, when inhibited, results in prometaphase accumulation and the subsequent death of Ras mutant cells. Gene expression analysis indicates that reduced expression of genes in this pathway correlates with increased survival of patients bearing tumors with a Ras transcriptional signature. Our results suggest a previously underappreciated role for Ras in mitotic progression and demonstrate a pharmacologically tractable pathway for the potential treatment of cancers harboring Ras mutations.

Screen Details

Stable ID: GR00018-A-0
Screen Title: Synthetic lethal interaction with Ras
Assay: Synthetic lethal interaction with Ras
Method: Micoarray hybridization
Scope:
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: DLD1 colon adenocarcinoma cells (Ras mutant and wildtype)
Library: , shRNA-mir (G. Hannon)
Reagent Type: shRNA
Score Type: Log2 diff MUT-WT (and P-value)
Cutoff: -0.7 (0.3)
Notes:

Therapeutic kinase targets in neuroblastoma (3)
5599
MAPK8
MAPK8
0.92
none Illumina gene expression above basal level (p < 0.05)

Reference

RNAi screen of the protein kinome identifies checkpoint kinase 1 (CHK1) as a therapeutic target in neuroblastoma. Cole et al., 2011

Neuroblastoma is a childhood cancer that is often fatal despite intense multimodality therapy. In an effort to identify therapeutic targets for this disease, we performed a comprehensive loss-of-function screen of the protein kinome. Thirty kinases showed significant cellular cytotoxicity when depleted, with loss of the cell cycle checkpoint kinase 1 (CHK1/CHEK1) being the most potent. CHK1 mRNA expression was higher in MYC-Neuroblastoma-related (MYCN)-amplified (P < 0.0001) and high-risk (P = 0.03) tumors. Western blotting revealed that CHK1 was constitutively phosphorylated at the ataxia telangiectasia response kinase target site Ser345 and the autophosphorylation site Ser296 in neuroblastoma cell lines. This pattern was also seen in six of eight high-risk primary tumors but not in control nonneuroblastoma cell lines or in seven of eight low-risk primary tumors. Neuroblastoma cells were sensitive to the two CHK1 inhibitors SB21807 and TCS2312, with median IC(50) values of 564 nM and 548 nM, respectively. In contrast, the control lines had high micromolar IC(50) values, indicating a strong correlation between CHK1 phosphorylation and CHK1 inhibitor sensitivity (P = 0.0004). Furthermore, cell cycle analysis revealed that CHK1 inhibition in neuroblastoma cells caused apoptosis during S-phase, consistent with its role in replication fork progression. CHK1 inhibitor sensitivity correlated with total MYC(N) protein levels, and inducing MYCN in retinal pigmented epithelial cells resulted in CHK1 phosphorylation, which caused growth inhibition when inhibited. These data show the power of a functional RNAi screen to identify tractable therapeutical targets in neuroblastoma and support CHK1 inhibition strategies in this disease.

Screen Details

Stable ID: GR00193-A-3
Screen Title: Therapeutic kinase targets in neuroblastoma (3)
Assay: Substrate adherent cell growth
Method: rp
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: SKNAS
Library: Thermo Scientific, Kinase siGenome library
Reagent Type: siRNA
Score Type: Relative growth
Cutoff: 0.5 standard deviations below mean
Notes:

TRAIL-induced apoptosis (1)
NM_002750
MAPK8
-0.17
none

Reference

A synthetic lethal screen identifies FAT1 as an antagonist of caspase-8 in extrinsic apoptosis. Kranz and Boutros, 2014

The extrinsic apoptosis pathway is initiated by binding of death ligands to death receptors resulting in the formation of the death-inducing signaling complex (DISC). Activation of procaspase-8 within the DISC and its release from the signaling complex is required for processing executor caspases and commiting cell death. Here, we report that the atypical cadherin FAT1 interacts with caspase-8 preventing the association of caspase-8 with the DISC. We identified FAT1 in a genome-wide siRNA screen for synthetic lethal interactions with death receptor-mediated apoptosis. Knockdown of FAT1 sensitized established and patient-derived glioblastoma cell lines for apoptosis transduced by cell death ligands. Depletion of FAT1 resulted in enhanced procaspase-8 recruitment to the DISC and increased formation of caspase-8 containing secondary signaling complexes. In addition, FAT1 knockout cell lines generated by CRISPR/Cas9-mediated genome engineering were more susceptible for death receptor-mediated apoptosis. Our findings provide evidence for a mechanism to control caspase-8-dependent cell death by the atypical cadherin FAT1. These results contribute towards the understanding of effector caspase regulation in physiological conditions.

Screen Details

Stable ID: GR00240-S-1
Screen Title: TRAIL-induced apoptosis (1)
Assay: Viability
Method: Luminescence
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: U251MG
Library: Dharmacon, SMART-pool siRNA
Reagent Type: siRNA
Score Type: Z-score
Cutoff: > 4
Notes: Author-submitted data

Apoptosis regulation after Chlamydia trachomatis serovar L2 infection
5599
MAPK8
np
np
none

Reference

HIF-1α is involved in mediating apoptosis resistance to Chlamydia trachomatis-infected cells. Sharma et al., 2011

Chlamydiae are obligate intracellular Gram-negative bacteria that cause widespread diseases in humans. Due to the intimate association between bacterium and host, Chlamydia evolved various strategies to protect their host cell against death-inducing stimuli, allowing the bacterium to complete its development cycle. An RNA interference (RNAi)-based screen was used to identify host cell factors required for apoptosis resistance of human epithelial cells infected with Chlamydia trachomatis serovar L2. Among the 32 validated hits, the anti-apoptotic Bcl-2 family member Mcl-1 was identified as a target. Protein network analyses implicated the transcription factor hypoxia-induced factor 1 alpha (HIF-1α) to be central to the regulation of many of the identified targets. Further mechanistic investigations showed that HIF-1α was stabilized within the host cell cytoplasm during early infection time points, followed by its translocation to the nucleus and eventual transcriptional activation of Mcl-1. siRNA-mediated depletion of HIF-1α led to a drastic decrease in Mcl-1, rendering the cell sensitive to apoptosis induction. Taken together, our findings identify HIF-1α as responsible for upregulation of Mcl-1 and the maintenance of apoptosis resistance during Chlamydia infection.

Screen Details

Stable ID: GR00206-A
Screen Title: Apoptosis regulation after Chlamydia trachomatis serovar L2 infection
Assay: Cleaved cytokeratin-18 protein expression
Method: Fluorescence
Scope: Apoptosis, cellular trafficking and cell signalling genes
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HeLa
Library: Custom-made, Custom-made
Reagent Type: siRNA
Score Type: p-value
Cutoff: <= 0.05
Notes: Author-reviewed data

Telomerase activity regulation
MAPK8
np
169.77
Increased telomerase activity

Reference

High-throughput RNAi screening reveals novel regulators of telomerase. Cerone et al., 2011

Telomerase is considered an attractive anticancer target on the basis of its common and specific activation in most human cancers. While direct telomerase inhibition is being explored as a therapeutic strategy, alternative strategies to target regulators of telomerase that could disrupt telomere maintenance and cancer cell proliferation are not yet available. Here, we report the findings of a high-throughput functional RNA interference screen to globally profile the contribution of kinases to telomerase activity (TA). This analysis identified a number of novel telomerase modulators, including ERK8 kinase, whose inhibition reduces TA and elicited characteristics of telomere dysfunction. Given that kinases represent attractive drug targets, we addressed the therapeutic implications of our findings, such as demonstrating how limiting TA via kinase blockade could sensitize cells to inhibition of the telomere-associated protein tankyrase. Taken together, our findings suggest novel combinatorial approaches to targeting telomere maintenance as a strategy for cancer therapy.

Screen Details

Stable ID: GR00156-A
Screen Title: Telomerase activity regulation
Assay: Telomerase activity
Method: Real-time qTRAP
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HeLa
Library: Dharmacon, SMARTpool siRNA kinase library
Reagent Type: siRNA
Score Type: Percentage
Cutoff: < 35 % OR > 150 %
Notes:

Cell viability
MAPK8
v2HS_170501
-1.7
Increased cell death HMECs cells

Reference

Cancer proliferation gene discovery through functional genomics. Schlabach et al., 2008

Retroviral short hairpin RNA (shRNA)-mediated genetic screens in mammalian cells are powerful tools for discovering loss-of-function phenotypes. We describe a highly parallel multiplex methodology for screening large pools of shRNAs using half-hairpin barcodes for microarray deconvolution. We carried out dropout screens for shRNAs that affect cell proliferation and viability in cancer cells and normal cells. We identified many shRNAs to be antiproliferative that target core cellular processes, such as the cell cycle and protein translation, in all cells examined. Moreover, we identified genes that are selectively required for proliferation and survival in different cell lines. Our platform enables rapid and cost-effective genome-wide screens to identify cancer proliferation and survival genes for target discovery. Such efforts are complementary to the Cancer Genome Atlas and provide an alternative functional view of cancer cells.

Screen Details

Stable ID: GR00103-A-0
Screen Title: Cell viability
Assay: Cell viability
Method: Micoarray hybridization
Scope:
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: DLD-1, HCT116 (colon cancer); HCC1954 (breast cancer); HMECs (mammary epithelia cells)
Library: , shRNA-mir (G. Hannon)
Reagent Type: shRNA
Score Type: log ratio
Cutoff: <= -1 (>= 50% cell death)
Notes:

Focal adhesion formation
5599
MAPK8
np
sp
Decreased focal adhesion (FA) area, decreased FA length, decreased FA mean intensity, increased number of small and round FAs clusters: MC4, DC-NE, CC-NE

Reference

Multiparametric analysis of focal adhesion formation by RNAi-mediated gene knockdown. Winograd-Katz et al., 2009

Cell adhesion to the extracellular matrix is mediated by elaborate networks of multiprotein complexes consisting of adhesion receptors, cytoskeletal components, signaling molecules, and diverse adaptor proteins. To explore how specific molecular pathways function in the assembly of focal adhesions (FAs), we performed a high-throughput, high-resolution, microscopy-based screen. We used small interfering RNAs (siRNAs) to target human kinases, phosphatases, and migration- and adhesion-related genes. Multiparametric image analysis of control and of siRNA-treated cells revealed major correlations between distinct morphological FA features. Clustering analysis identified different gene families whose perturbation induced similar effects, some of which uncoupled the interfeature correlations. Based on these findings, we propose a model for the molecular hierarchy of FA formation, and tested its validity by dynamic analysis of FA formation and turnover. This study provides a comprehensive information resource on the molecular regulation of multiple cell adhesion features, and sheds light on signaling mechanisms regulating the formation of integrin adhesions.

Screen Details

Stable ID: GR00210-A
Screen Title: Focal adhesion formation
Assay: paxillin protein expression
Method: Fluorescence
Scope: Kinases, phosphatases and selected genes
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HeLa
Library: Thermo Fisher Scientific, SMARTpool siARRAY siRNA Libraries
Reagent Type: siRNA
Score Type: Z-score
Cutoff: > 3.5 OR < -3.5
Notes:

Vaccinia virus (VACV) infection
5599
MAPK8
J-003514-17
1.04
Increased vaccinia virus (VACV) infection number of cells compared to control (%): 87.19

Reference

Human genome-wide RNAi screen reveals a role for nuclear pore proteins in poxvirus morphogenesis. Sivan et al., 2013

Poxviruses are considered less dependent on host functions than other DNA viruses because of their cytoplasmic site of replication and large genomes, which encode enzymes for DNA and mRNA synthesis. Nevertheless, RNAi screens with two independent human genome-scale libraries have identified more than 500 candidate genes that significantly inhibited and a similar number that enhanced replication and spread of infectious vaccinia virus (VACV). Translational, ubiquitin-proteosome, and endoplasmic reticulum-to-Golgi transport functions, known to be important for VACV, were enriched in the siRNA-inhibiting group, and RNA polymerase II and associated functions were enriched in the siRNA-enhancing group. Additional findings, notably the inhibition of VACV spread by siRNAs to several nuclear pore genes, were unanticipated. Knockdown of nucleoporin 62 strongly inhibited viral morphogenesis, with only a modest effect on viral gene expression, recapitulating and providing insight into previous studies with enucleated cells.

Screen Details

Stable ID: GR00249-S
Screen Title: Vaccinia virus (VACV) infection
Assay: Vaccinia virus VACV IHD-J/GFP protein expression and DNA content
Method: Fluorescence
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HeLa
Library: Ambion and Dharmacon, Silencer Select Version 4, siGENOME SMARTpool and OnTargetPlus
Reagent Type: siRNA
Score Type: Z-score
Cutoff: >= 1 OR <= -1.5
Notes: Author-submitted data. Primary screen. Decreased viability phenotype if number of cells compared to control < 50 %.

Ciliogenesis and cilium length (1)
5599
MAPK8
1415
93.9
none

Reference

Functional genomic screen for modulators of ciliogenesis and cilium length. Kim et al., 2010

Primary cilia are evolutionarily conserved cellular organelles that organize diverse signalling pathways. Defects in the formation or function of primary cilia are associated with a spectrum of human diseases and developmental abnormalities. Genetic screens in model organisms have discovered core machineries of cilium assembly and maintenance. However, regulatory molecules that coordinate the biogenesis of primary cilia with other cellular processes, including cytoskeletal organization, vesicle trafficking and cell-cell adhesion, remain to be identified. Here we report the results of a functional genomic screen using RNA interference (RNAi) to identify human genes involved in ciliogenesis control. The screen identified 36 positive and 13 negative ciliogenesis modulators, which include molecules involved in actin dynamics and vesicle trafficking. Further investigation demonstrated that blocking actin assembly facilitates ciliogenesis by stabilizing the pericentrosomal preciliary compartment (PPC), a previously uncharacterized compact vesiculotubular structure storing transmembrane proteins destined for cilia during the early phase of ciliogenesis. The PPC was labelled by recycling endosome markers. Moreover, knockdown of modulators that are involved in the endocytic recycling pathway affected the formation of the PPC as well as ciliogenesis. Our results uncover a critical regulatory step that couples actin dynamics and endocytic recycling with ciliogenesis, and also provides potential target molecules for future study.

Screen Details

Stable ID: GR00149-A-1
Screen Title: Ciliogenesis and cilium length (1)
Assay: Smoothed protein expression
Method: Fluorescence
Scope: Druggable genes
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: htRPE
Library: Ambion, Human druggable genome siRNA library V3.1
Reagent Type: siRNA
Score Type: Normalized percent inhibition
Cutoff: > 1.5 OR < -1.5 standard deviations from mean
Notes:

Cell proliferation
NM_002750
MAPK8
0.82
none

Reference

Time-resolved human kinome RNAi screen identifies a network regulating mitotic-events as early regulators of cell proliferation. Zhang et al., 2011

Analysis of biological processes is frequently performed with the help of phenotypic assays where data is mostly acquired in single end-point analysis. Alternative phenotypic profiling techniques are desired where time-series information is essential to the biological question, for instance to differentiate early and late regulators of cell proliferation in loss-of-function studies. So far there is no study addressing this question despite of high unmet interests, mostly due to the limitation of conventional end-point assaying technologies. We present the first human kinome screen with a real-time cell analysis system (RTCA) to capture dynamic RNAi phenotypes, employing time-resolved monitoring of cell proliferation via electrical impedance. RTCA allowed us to investigate the dynamics of phenotypes of cell proliferation instead of using conventional end-point analysis. By introducing data transformation with first-order derivative, i.e. the cell-index growth rate, we demonstrate this system suitable for high-throughput screenings (HTS). The screen validated previously identified inhibitor genes and, additionally, identified activators of cell proliferation. With the information of time kinetics available, we could establish a network of mitotic-event related genes to be among the first displaying inhibiting effects after RNAi knockdown. The time-resolved screen captured kinetics of cell proliferation caused by RNAi targeting human kinome, serving as a resource for researchers. Our work establishes RTCA technology as a novel robust tool with biological and pharmacological relevance amenable for high-throughput screening.

Screen Details

Stable ID: GR00231-A
Screen Title: Cell proliferation
Assay: Viability
Method: Electrical impedance
Scope: Kinases and selected genes
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HeLa
Library: Dharmacon, Human siARRAY - Protein Kinase and Cell Cycle libraries
Reagent Type: siRNA
Score Type: Z-score
Cutoff: < -1.96 OR > 1.96
Notes: Additional information about cell titer blue cell viability screen

hepcidin regulation
NM_002750
MAPK8
np
0.05
none

Reference

Unbiased RNAi screen for hepcidin regulators links hepcidin suppression to proliferative Ras/RAF and nutrient-dependent mTOR signaling. Mleczko-Sanecka et al., 2014

The hepatic hormone hepcidin is a key regulator of systemic iron metabolism. Its expression is largely regulated by 2 signaling pathways: the "iron-regulated" bone morphogenetic protein (BMP) and the inflammatory JAK-STAT pathways. To obtain broader insights into cellular processes that modulate hepcidin transcription and to provide a resource to identify novel genetic modifiers of systemic iron homeostasis, we designed an RNA interference (RNAi) screen that monitors hepcidin promoter activity after the knockdown of 19 599 genes in hepatocarcinoma cells. Interestingly, many of the putative hepcidin activators play roles in signal transduction, inflammation, or transcription, and affect hepcidin transcription through BMP-responsive elements. Furthermore, our work sheds light on new components of the transcriptional machinery that maintain steady-state levels of hepcidin expression and its responses to the BMP- and interleukin-6-triggered signals. Notably, we discover hepcidin suppression mediated via components of Ras/RAF MAPK and mTOR signaling, linking hepcidin transcriptional control to the pathways that respond to mitogen stimulation and nutrient status. Thus using a combination of RNAi screening, reverse phase protein arrays, and small molecules testing, we identify links between the control of systemic iron homeostasis and critical liver processes such as regeneration, response to injury, carcinogenesis, and nutrient metabolism.

Screen Details

Stable ID: GR00253-A
Screen Title: hepcidin regulation
Assay: hepcidin::fluc mRNA expression
Method: Luminescence
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: Huh7
Library: ThermoFisher, siGenome siARRAY SMARTpool
Reagent Type: siRNA
Score Type: Z-score
Cutoff: >= 1.755 OR <= -1.389
Notes: Cutoff <= -1.389 defined based on hepcidin promotor activity. Additional information about secondary siRNA screens.

TNF-induced apoptosis
NM_139049
MAPK8
np
0.66
Increased viability after TNF/CHX stimulation 95% confidence interval

Reference

A functional RNAi screen identifies hexokinase 1 as a modifier of type II apoptosis. Schindler and Foley, 2010

Tumor necrosis factor alpha (TNF-alpha) signals through NF-kappaB, JNK, and caspase modules to drive physiological responses that range from inflammation to apoptosis. The balance between the individual modules determines the nature of the response, and deregulated TNF signaling has been implicated in numerous pathological conditions. We used a quantitative high-throughput RNA interference assay to probe the entire complement of human kinases and phosphatases for gene products that tilt the balance of TNF signal transduction in favor of cell death or cell viability. Of all gene products tested, loss of hexokinase 1 resulted in the greatest elevations in TNF-dependent death. In secondary assays, we demonstrated that hexokinase 1 does not alter TNF-dependent activation of NF-kappaB or JNK modules. Instead, hexokinase 1 modifies the induction of caspase-driven cell death. Specifically, we showed that hexokinase 1 inhibits the formation of active, pro-apoptotic caspases in response to extrinsic inducers of apoptosis. These data are the first loss-of-function reports to examine the involvement of hexokinase 1 in the transduction of cell death signals and indicate that hexokinases are critical determinants of the viability of cells in response to extrinsic apoptotic cues.

Screen Details

Stable ID: GR00228-A
Screen Title: TNF-induced apoptosis
Assay: Viability (synthetic lethal)
Method: Fluorescence
Scope: Kinases and phosphatases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HeLa
Library: Applied Biosystems, Human kinase siRNA library V3 and Human phosphatase siRNA library V3
Reagent Type: siRNA
Score Type: Death index
Cutoff: 99% confidence interval: > 2.58 SD; 95% confidence interval: > 1.96 SD
Notes:

Combinatorial effect with paclitaxel
NM_002750
MAPK8
np
1.1
none

Reference

Synthetic lethal screen identification of chemosensitizer loci in cancer cells. Whitehurst et al., 2007

Abundant evidence suggests that a unifying principle governing the molecular pathology of cancer is the co-dependent aberrant regulation of core machinery driving proliferation and suppressing apoptosis. Anomalous proteins engaged in support of this tumorigenic regulatory environment most probably represent optimal intervention targets in a heterogeneous population of cancer cells. The advent of RNA-mediated interference (RNAi)-based functional genomics provides the opportunity to derive unbiased comprehensive collections of validated gene targets supporting critical biological systems outside the framework of preconceived notions of mechanistic relationships. We have combined a high-throughput cell-based one-well/one-gene screening platform with a genome-wide synthetic library of chemically synthesized small interfering RNAs for systematic interrogation of the molecular underpinnings of cancer cell chemoresponsiveness. NCI-H1155, a human non-small-cell lung cancer line, was employed in a paclitaxel-dependent synthetic lethal screen designed to identify gene targets that specifically reduce cell viability in the presence of otherwise sublethal concentrations of paclitaxel. Using a stringent objective statistical algorithm to reduce false discovery rates below 5%, we isolated a panel of 87 genes that represent major focal points of the autonomous response of cancer cells to the abrogation of microtubule dynamics. Here we show that several of these targets sensitize lung cancer cells to paclitaxel concentrations 1,000-fold lower than otherwise required for a significant response, and we identify mechanistic relationships between cancer-associated aberrant gene expression programmes and the basic cellular machinery required for robust mitotic progression.

Screen Details

Stable ID: GR00054-A
Screen Title: Combinatorial effect with paclitaxel
Assay: Viability (synthetic lethal)
Method: ATP level
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: NCI-H1155
Library: Dharmacon, # G-005000-01
Reagent Type: siRNA
Score Type: Paclitaxel/control ratio
Cutoff: Complex criteria
Notes: Additional information about 87 high-confidence hits

Aryl hydrocarbon receptor (AhR) transduction pathway regulation
5599
MAPK8
MAPK8_1
MAPK8_3
MAPK8_2
np
none

Reference

RNAi-based screening identifies kinases interfering with dioxin-mediated up-regulation of CYP1A1 activity. Gilot et al., 2011

The aryl hydrocarbon receptor (AhR) is a transcription factor activated by several environmental pollutants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and involved in carcinogenesis and various physiological processes, including immune response and endocrine functions. Characterization of kinases-related AhR transduction pathway remains an important purpose.

Screen Details

Stable ID: GR00155-A
Screen Title: Aryl hydrocarbon receptor (AhR) transduction pathway regulation
Assay: TCDD-induced CYP1A1-related EROD activity and cell viability
Method: Fluorescence and methylene blue
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: MCF-7
Library: Sigma-Genesys, MISSION siRNA Human Kinase Panel library
Reagent Type: siRNA
Score Type: Z-score
Cutoff: Top 150 for >= 2 of 3 siRNAs AND no effect on cell proliferation
Notes:

Proliferation of cells with active beta-catenin (4)
MAPK8
0.24
none

Reference

CK1epsilon is required for breast cancers dependent on beta-catenin activity. Kim et al., 2010

Aberrant beta-catenin signaling plays a key role in several cancer types, notably colon, liver and breast cancer. However approaches to modulate beta-catenin activity for therapeutic purposes have proven elusive to date.

Screen Details

Stable ID: GR00221-A-4
Screen Title: Proliferation of cells with active beta-catenin (4)
Assay: Viability
Method: Luminescence
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: T47D
Library: The RNAi Consortium (TRC), TRC shRNA Library
Reagent Type: shRNA
Score Type: B-score
Cutoff: < -1
Notes: Essential gene: gene with B-score < -1 for >= 2 shRNAs

Genome stability
NM_002750
MAPK8
np
sp
none

Reference

A genome-wide siRNA screen reveals diverse cellular processes and pathways that mediate genome stability. Paulsen et al., 2009

Signaling pathways that respond to DNA damage are essential for the maintenance of genome stability and are linked to many diseases, including cancer. Here, a genome-wide siRNA screen was employed to identify additional genes involved in genome stabilization by monitoring phosphorylation of the histone variant H2AX, an early mark of DNA damage. We identified hundreds of genes whose downregulation led to elevated levels of H2AX phosphorylation (gammaH2AX) and revealed links to cellular complexes and to genes with unclassified functions. We demonstrate a widespread role for mRNA-processing factors in preventing DNA damage, which in some cases is caused by aberrant RNA-DNA structures. Furthermore, we connect increased gammaH2AX levels to the neurological disorder Charcot-Marie-Tooth (CMT) syndrome, and we find a role for several CMT proteins in the DNA-damage response. These data indicate that preservation of genome stability is mediated by a larger network of biological processes than previously appreciated.

Screen Details

Stable ID: GR00053-A
Screen Title: Genome stability
Assay: gamma-H2AX phosphorylation and DNA content
Method: Fluorescence
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HeLa
Library: ThermoFisher Scientific, siARRAY human genome siRNA library
Reagent Type: siRNA
Score Type: p-value
Cutoff: Complex criteria
Notes: Confidence groupings from 4 to 1 (highest level of confidence in group 4)

Homologous recombination DNA double-strand break repair (HR-DSBR) (2)
5599
MAPK8
1.17
none

Reference

A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response. Adamson et al., 2012

Repair of DNA double-strand breaks is critical to genomic stability and the prevention of developmental disorders and cancer. A central pathway for this repair is homologous recombination (HR). Most knowledge of HR is derived from work in prokaryotic and eukaryotic model organisms. We carried out a genome-wide siRNA-based screen in human cells. Among positive regulators of HR we identified networks of DNA-damage-response and pre-mRNA-processing proteins, and among negative regulators we identified a phosphatase network. Three candidate proteins localized to DNA lesions, including RBMX, a heterogeneous nuclear ribonucleoprotein that has a role in alternative splicing. RBMX accumulated at DNA lesions through multiple domains in a poly(ADP-ribose) polymerase 1-dependent manner and promoted HR by facilitating proper BRCA2 expression. Our screen also revealed that off-target depletion of RAD51 is a common source of RNAi false positives, raising a cautionary note for siRNA screens and RNAi-based studies of HR.

Screen Details

Stable ID: GR00236-A-2
Screen Title: Homologous recombination DNA double-strand break repair (HR-DSBR) (2)
Assay: (HR-DSBR) DR-GFP reporter and DNA content
Method: Fluorescence
Scope: Selected genes
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: DR-U2OS
Library: Dharmacon, np
Reagent Type: siRNA
Score Type: Relative HR ratio
Cutoff: Strong: < ~0.4, weak: < ~0.59 (for mediators); strong: > 1.88, weak: > 1.69 (for suppressors)
Notes: Cutoff values correspond 2 standard deviations from the screen-wide mean (strong) or 1.5 SD (weak)

Proliferation of cells with active beta-catenin (2)
MAPK8
-0.78
none

Reference

CK1epsilon is required for breast cancers dependent on beta-catenin activity. Kim et al., 2010

Aberrant beta-catenin signaling plays a key role in several cancer types, notably colon, liver and breast cancer. However approaches to modulate beta-catenin activity for therapeutic purposes have proven elusive to date.

Screen Details

Stable ID: GR00221-A-2
Screen Title: Proliferation of cells with active beta-catenin (2)
Assay: Viability
Method: Luminescence
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: MDA-MB-231
Library: The RNAi Consortium (TRC), TRC shRNA Library
Reagent Type: shRNA
Score Type: B-score
Cutoff: < -1
Notes: Essential gene: gene with B-score < -1 for >= 2 shRNAs

Wnt/beta-catenin pathway regulation (2)
NM_002750
MAPK8
sp
none

Reference

A genome-wide RNAi screen for Wnt/beta-catenin pathway components identifies unexpected roles for TCF transcription factors in cancer. Tang et al., 2008

The Wnt family of secreted proteins coordinate cell fate decision-making in a broad range of developmental and homeostatic contexts. Corruption of Wnt signal transduction pathways frequently results in degenerative diseases and cancer. We have used an iterative genome-wide screening strategy that employs multiple nonredundant RNAi reagents to identify mammalian genes that participate in Wnt/beta-catenin pathway response. Among the genes that were assigned high confidence scores are two members of the TCF/LEF family of DNA-binding proteins that control the transcriptional output of the pathway. Surprisingly, we found that the presumed cancer-promoting gene TCF7L2 functions instead as a transcriptional repressor that restricts colorectal cancer (CRC) cell growth. Mutations in TCF7L2 identified from cancer genome sequencing efforts abolish its ability to function as a transcriptional regulator and result in increased CRC cell growth. We describe a growth-promoting transcriptional program that is likely activated in CRC tumors with compromised TCF7L2 function. Taken together, the results from our screen and studies focused on members of the TCF/LEF gene family refine our understanding of how aberrant Wnt pathway activation sustains CRC growth.

Screen Details

Stable ID: GR00057-A-2
Screen Title: Wnt/beta-catenin pathway regulation (2)
Assay: Wnt pathway reporter
Method: Luminescence
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HeLa
Library: Dharmacon, Human siArray siRNA library
Reagent Type: siRNA
Score Type: Complex, SP
Cutoff: Complex criteria
Notes: Screen with Wnt3A stimulation. Additional information about secondary screens (Dharmacon and Qiagen libraries).

Homologous recombination DNA double-strand break repair (HR-DSBR) (1)
ENSG00000107643
MAPK8
np
1.15
none

Reference

A genome-scale DNA repair RNAi screen identifies SPG48 as a novel gene associated with hereditary spastic paraplegia. Słabicki et al., 2010

DNA repair is essential to maintain genome integrity, and genes with roles in DNA repair are frequently mutated in a variety of human diseases. Repair via homologous recombination typically restores the original DNA sequence without introducing mutations, and a number of genes that are required for homologous recombination DNA double-strand break repair (HR-DSBR) have been identified. However, a systematic analysis of this important DNA repair pathway in mammalian cells has not been reported. Here, we describe a genome-scale endoribonuclease-prepared short interfering RNA (esiRNA) screen for genes involved in DNA double strand break repair. We report 61 genes that influenced the frequency of HR-DSBR and characterize in detail one of the genes that decreased the frequency of HR-DSBR. We show that the gene KIAA0415 encodes a putative helicase that interacts with SPG11 and SPG15, two proteins mutated in hereditary spastic paraplegia (HSP). We identify mutations in HSP patients, discovering KIAA0415/SPG48 as a novel HSP-associated gene, and show that a KIAA0415/SPG48 mutant cell line is more sensitive to DNA damaging drugs. We present the first genome-scale survey of HR-DSBR in mammalian cells providing a dataset that should accelerate the discovery of novel genes with roles in DNA repair and associated medical conditions. The discovery that proteins forming a novel protein complex are required for efficient HR-DSBR and are mutated in patients suffering from HSP suggests a link between HSP and DNA repair.

Screen Details

Stable ID: GR00151-A-1
Screen Title: Homologous recombination DNA double-strand break repair (HR-DSBR) (1)
Assay: (HR-DSBR) DR-GFP reporter
Method: Flow cytometry
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HeLa
Library: Custom-made, Custom-made
Reagent Type: esiRNA
Score Type: Z-score
Cutoff: < -2 OR > 2
Notes:

Combinatorial effect with gemcitabine
MAPK8
MAPK8_B
-0.59
none

Reference

Synthetic lethal RNAi screening identifies sensitizing targets for gemcitabine therapy in pancreatic cancer. Azorsa et al., 2009

Pancreatic cancer retains a poor prognosis among the gastrointestinal cancers. It affects 230,000 individuals worldwide, has a very high mortality rate, and remains one of the most challenging malignancies to treat successfully. Treatment with gemcitabine, the most widely used chemotherapeutic against pancreatic cancer, is not curative and resistance may occur. Combinations of gemcitabine with other chemotherapeutic drugs or biological agents have resulted in limited improvement.

Screen Details

Stable ID: GR00225-A
Screen Title: Combinatorial effect with gemcitabine
Assay: Viability (synthetic lethal)
Method: Luminescence
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: MIA PaCa-2
Library: Qiagen, Validated kinase siRNA library version 1.0
Reagent Type: siRNA
Score Type: log2 ratio
Cutoff: 1.65 SD below mean ratio level
Notes:

Negative genetic interactions (2)
5599
MAPK8
0.01
none

Reference

A negative genetic interaction map in isogenic cancer cell lines reveals cancer cell vulnerabilities. Vizeacoumar et al., 2013

Improved efforts are necessary to define the functional product of cancer mutations currently being revealed through large-scale sequencing efforts. Using genome-scale pooled shRNA screening technology, we mapped negative genetic interactions across a set of isogenic cancer cell lines and confirmed hundreds of these interactions in orthogonal co-culture competition assays to generate a high-confidence genetic interaction network of differentially essential or differential essentiality (DiE) genes. The network uncovered examples of conserved genetic interactions, densely connected functional modules derived from comparative genomics with model systems data, functions for uncharacterized genes in the human genome and targetable vulnerabilities. Finally, we demonstrate a general applicability of DiE gene signatures in determining genetic dependencies of other non-isogenic cancer cell lines. For example, the PTEN(-/-) DiE genes reveal a signature that can preferentially classify PTEN-dependent genotypes across a series of non-isogenic cell lines derived from the breast, pancreas and ovarian cancers. Our reference network suggests that many cancer vulnerabilities remain to be discovered through systematic derivation of a network of differentially essential genes in an isogenic cancer cell model.

Screen Details

Stable ID: GR00255-A-2
Screen Title: Negative genetic interactions (2)
Assay: shRNA abundance
Method: Microarray
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HCT116
Library: TRC lentiviral library, np
Reagent Type: shRNA
Score Type: differential Gene Activity Ranking Profile (dGARP)
Cutoff: < -1.0
Notes: HCT116 MUS81-/- and HCT116 MUS81+/+ cells used. Cutoff corresponds to p-value < 0.05. Additional information about a secondary screen (genetic interactions with Cetuximab/Erbitux in LIM1215 cells)

Combinatorial effect with gemcitabine
MAPK8
MAPK8_B
-0.17
none

Reference

Synthetic lethal RNAi screening identifies sensitizing targets for gemcitabine therapy in pancreatic cancer. Azorsa et al., 2009

Pancreatic cancer retains a poor prognosis among the gastrointestinal cancers. It affects 230,000 individuals worldwide, has a very high mortality rate, and remains one of the most challenging malignancies to treat successfully. Treatment with gemcitabine, the most widely used chemotherapeutic against pancreatic cancer, is not curative and resistance may occur. Combinations of gemcitabine with other chemotherapeutic drugs or biological agents have resulted in limited improvement.

Screen Details

Stable ID: GR00225-A
Screen Title: Combinatorial effect with gemcitabine
Assay: Viability (synthetic lethal)
Method: Luminescence
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: MIA PaCa-2
Library: Qiagen, Validated kinase siRNA library version 1.0
Reagent Type: siRNA
Score Type: log2 ratio
Cutoff: 1.65 SD below mean ratio level
Notes:

Human cytomegalovirus (HCMV) strain AD169 replication
NM_002750
MAPK8
MAPK8_siRNA2
1.45
none

Reference

Human kinome profiling identifies a requirement for AMP-activated protein kinase during human cytomegalovirus infection. Terry et al., 2012

Human cytomegalovirus (HCMV) modulates numerous cellular signaling pathways. Alterations in signaling are evident from the broad changes in cellular phosphorylation that occur during HCMV infection and from the altered activity of multiple kinases. Here we report a comprehensive RNAi screen, which predicts that 106 cellular kinases influence growth of the virus, most of which were not previously linked to HCMV replication. Multiple elements of the AMP-activated protein kinase (AMPK) pathway scored in the screen. As a regulator of carbon and nucleotide metabolism, AMPK is poised to activate many of the metabolic pathways induced by HCMV infection. An AMPK inhibitor, compound C, blocked a substantial portion of HCMV-induced metabolic changes, inhibited the accumulation of all HCMV proteins tested, and markedly reduced the production of infectious progeny. We propose that HCMV requires AMPK or related activity for viral replication and reprogramming of cellular metabolism.

Screen Details

Stable ID: GR00248-A
Screen Title: Human cytomegalovirus (HCMV) strain AD169 replication
Assay: Human cytomegalovirus (HCMV) IE1 protein expression
Method: Fluorescence
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: MRC5
Library: Sigma-Aldrich, Mission siRNA Human Kinase Panel
Reagent Type: siRNA
Score Type: Z-score
Cutoff: >=2 or <=-2 for >= 2 of 3 siRNA
Notes:

Cytokine regulation
5599
MAPK8
AAA13.E11 NM_139049.x-984s1c1
1.2
Increased IL-13 protein expression

Reference

RNA interference screen in primary human T cells reveals FLT3 as a modulator of IL-10 levels. Astier et al., 2010

Functional studies of human primary immune cells have been hampered by the lack of tools to silence gene functions. In this study, we report the application of a lentiviral RNA interference library in primary human T cells. Using a subgenomic short hair RNA library targeting approximately 1000 signaling genes, we identified novel genes that control the levels of IL-10 produced. IL-10 is a potent anti-inflammatory cytokine secreted by several cell types, including T regulatory type 1 cells, a subset of T regulatory cells that exert their suppressive activity through IL-10 secretion. FLT3, a known hematopoeitic growth factor, was found to be a negative regulator of IL-10 levels in activated T cells. This was based on several observations. First, FLT3 and its ligand (FL) were both induced by T cell activation. Second, silencing of FLT3 led to increased IL-10 levels, whereas addition of FL suppressed IL-10 secretion and increased FLT3 surface levels. Third, engagement of CD46, a known inducer of T regulatory type 1 cells, upregulated surface FLT3, and secreted FL, which then inhibited IL-10 production by T cells. Hence, FL and FLT3 form a novel regulatory feedback loop that limits IL-10 production in T cells. Our results identified FLT3 as a new regulator of T cell function and offer a strategy to genetically dissect specific pathways in T cells.

Screen Details

Stable ID: GR00219-A
Screen Title: Cytokine regulation
Assay: IL-10, IL-13 and IFN-gamma protein expression
Method: Fluorescence
Scope: Kinases and phosphatases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Primary cells
Biomodel: primary CD4+ T cells
Library: The RNAi Consortium (TRC), TRC shRNA Library
Reagent Type: shRNA
Score Type: Z-score
Cutoff: Complex criteria
Notes:

Ciliogenesis and cilium length (1)
5599
MAPK8
1225
86.95
none

Reference

Functional genomic screen for modulators of ciliogenesis and cilium length. Kim et al., 2010

Primary cilia are evolutionarily conserved cellular organelles that organize diverse signalling pathways. Defects in the formation or function of primary cilia are associated with a spectrum of human diseases and developmental abnormalities. Genetic screens in model organisms have discovered core machineries of cilium assembly and maintenance. However, regulatory molecules that coordinate the biogenesis of primary cilia with other cellular processes, including cytoskeletal organization, vesicle trafficking and cell-cell adhesion, remain to be identified. Here we report the results of a functional genomic screen using RNA interference (RNAi) to identify human genes involved in ciliogenesis control. The screen identified 36 positive and 13 negative ciliogenesis modulators, which include molecules involved in actin dynamics and vesicle trafficking. Further investigation demonstrated that blocking actin assembly facilitates ciliogenesis by stabilizing the pericentrosomal preciliary compartment (PPC), a previously uncharacterized compact vesiculotubular structure storing transmembrane proteins destined for cilia during the early phase of ciliogenesis. The PPC was labelled by recycling endosome markers. Moreover, knockdown of modulators that are involved in the endocytic recycling pathway affected the formation of the PPC as well as ciliogenesis. Our results uncover a critical regulatory step that couples actin dynamics and endocytic recycling with ciliogenesis, and also provides potential target molecules for future study.

Screen Details

Stable ID: GR00149-A-1
Screen Title: Ciliogenesis and cilium length (1)
Assay: Smoothed protein expression
Method: Fluorescence
Scope: Druggable genes
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: htRPE
Library: Ambion, Human druggable genome siRNA library V3.1
Reagent Type: siRNA
Score Type: Normalized percent inhibition
Cutoff: > 1.5 OR < -1.5 standard deviations from mean
Notes:

Vaccinia virus (VACV) infection
5599
MAPK8
M-003514-04
1.76
Increased vaccinia virus (VACV) infection number of cells compared to control (%): 84.44

Reference

Human genome-wide RNAi screen reveals a role for nuclear pore proteins in poxvirus morphogenesis. Sivan et al., 2013

Poxviruses are considered less dependent on host functions than other DNA viruses because of their cytoplasmic site of replication and large genomes, which encode enzymes for DNA and mRNA synthesis. Nevertheless, RNAi screens with two independent human genome-scale libraries have identified more than 500 candidate genes that significantly inhibited and a similar number that enhanced replication and spread of infectious vaccinia virus (VACV). Translational, ubiquitin-proteosome, and endoplasmic reticulum-to-Golgi transport functions, known to be important for VACV, were enriched in the siRNA-inhibiting group, and RNA polymerase II and associated functions were enriched in the siRNA-enhancing group. Additional findings, notably the inhibition of VACV spread by siRNAs to several nuclear pore genes, were unanticipated. Knockdown of nucleoporin 62 strongly inhibited viral morphogenesis, with only a modest effect on viral gene expression, recapitulating and providing insight into previous studies with enucleated cells.

Screen Details

Stable ID: GR00249-S
Screen Title: Vaccinia virus (VACV) infection
Assay: Vaccinia virus VACV IHD-J/GFP protein expression and DNA content
Method: Fluorescence
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HeLa
Library: Ambion and Dharmacon, Silencer Select Version 4, siGENOME SMARTpool and OnTargetPlus
Reagent Type: siRNA
Score Type: Z-score
Cutoff: >= 1 OR <= -1.5
Notes: Author-submitted data. Primary screen. Decreased viability phenotype if number of cells compared to control < 50 %.

Proliferation of cells with active beta-catenin (2)
MAPK8
1.41
none

Reference

CK1epsilon is required for breast cancers dependent on beta-catenin activity. Kim et al., 2010

Aberrant beta-catenin signaling plays a key role in several cancer types, notably colon, liver and breast cancer. However approaches to modulate beta-catenin activity for therapeutic purposes have proven elusive to date.

Screen Details

Stable ID: GR00221-A-2
Screen Title: Proliferation of cells with active beta-catenin (2)
Assay: Viability
Method: Luminescence
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: MDA-MB-231
Library: The RNAi Consortium (TRC), TRC shRNA Library
Reagent Type: shRNA
Score Type: B-score
Cutoff: < -1
Notes: Essential gene: gene with B-score < -1 for >= 2 shRNAs

Therapeutic kinase targets in neuroblastoma (4)
5599
MAPK8
MAPK8
0.99
none Illumina gene expression above basal level (p < 0.05)

Reference

RNAi screen of the protein kinome identifies checkpoint kinase 1 (CHK1) as a therapeutic target in neuroblastoma. Cole et al., 2011

Neuroblastoma is a childhood cancer that is often fatal despite intense multimodality therapy. In an effort to identify therapeutic targets for this disease, we performed a comprehensive loss-of-function screen of the protein kinome. Thirty kinases showed significant cellular cytotoxicity when depleted, with loss of the cell cycle checkpoint kinase 1 (CHK1/CHEK1) being the most potent. CHK1 mRNA expression was higher in MYC-Neuroblastoma-related (MYCN)-amplified (P < 0.0001) and high-risk (P = 0.03) tumors. Western blotting revealed that CHK1 was constitutively phosphorylated at the ataxia telangiectasia response kinase target site Ser345 and the autophosphorylation site Ser296 in neuroblastoma cell lines. This pattern was also seen in six of eight high-risk primary tumors but not in control nonneuroblastoma cell lines or in seven of eight low-risk primary tumors. Neuroblastoma cells were sensitive to the two CHK1 inhibitors SB21807 and TCS2312, with median IC(50) values of 564 nM and 548 nM, respectively. In contrast, the control lines had high micromolar IC(50) values, indicating a strong correlation between CHK1 phosphorylation and CHK1 inhibitor sensitivity (P = 0.0004). Furthermore, cell cycle analysis revealed that CHK1 inhibition in neuroblastoma cells caused apoptosis during S-phase, consistent with its role in replication fork progression. CHK1 inhibitor sensitivity correlated with total MYC(N) protein levels, and inducing MYCN in retinal pigmented epithelial cells resulted in CHK1 phosphorylation, which caused growth inhibition when inhibited. These data show the power of a functional RNAi screen to identify tractable therapeutical targets in neuroblastoma and support CHK1 inhibition strategies in this disease.

Screen Details

Stable ID: GR00193-A-4
Screen Title: Therapeutic kinase targets in neuroblastoma (4)
Assay: Substrate adherent cell growth
Method: rp
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: NLF
Library: Thermo Scientific, Kinase siGenome library
Reagent Type: siRNA
Score Type: Relative growth
Cutoff: 0.5 standard deviations below mean
Notes:

Cell viability
MAPK8
v2HS_170503
-1.19
Increased cell death HMECs cells

Reference

Cancer proliferation gene discovery through functional genomics. Schlabach et al., 2008

Retroviral short hairpin RNA (shRNA)-mediated genetic screens in mammalian cells are powerful tools for discovering loss-of-function phenotypes. We describe a highly parallel multiplex methodology for screening large pools of shRNAs using half-hairpin barcodes for microarray deconvolution. We carried out dropout screens for shRNAs that affect cell proliferation and viability in cancer cells and normal cells. We identified many shRNAs to be antiproliferative that target core cellular processes, such as the cell cycle and protein translation, in all cells examined. Moreover, we identified genes that are selectively required for proliferation and survival in different cell lines. Our platform enables rapid and cost-effective genome-wide screens to identify cancer proliferation and survival genes for target discovery. Such efforts are complementary to the Cancer Genome Atlas and provide an alternative functional view of cancer cells.

Screen Details

Stable ID: GR00103-A-0
Screen Title: Cell viability
Assay: Cell viability
Method: Micoarray hybridization
Scope:
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: DLD-1, HCT116 (colon cancer); HCC1954 (breast cancer); HMECs (mammary epithelia cells)
Library: , shRNA-mir (G. Hannon)
Reagent Type: shRNA
Score Type: log ratio
Cutoff: <= -1 (>= 50% cell death)
Notes:

Cell division (1)
ENSG00000107643
MAPK8
ENSG00000107643
sp
Increased S DNA content

Reference

Genome-scale RNAi profiling of cell division in human tissue culture cells. Kittler et al., 2007

Cell division is fundamental for all organisms. Here we report a genome-scale RNA-mediated interference screen in HeLa cells designed to identify human genes that are important for cell division. We have used a library of endoribonuclease-prepared short interfering RNAs for gene silencing and have used DNA content analysis to identify genes that induced cell cycle arrest or altered ploidy on silencing. Validation and secondary assays were performed to generate a nine-parameter loss-of-function phenoprint for each of the genes. These phenotypic signatures allowed the assignment of genes to specific functional classes by combining hierarchical clustering, cross-species analysis and proteomic data mining. We highlight the richness of our dataset by ascribing novel functions to genes in mitosis and cytokinesis. In particular, we identify two evolutionarily conserved transcriptional regulatory networks that govern cytokinesis. Our work provides an experimental framework from which the systematic analysis of novel genes necessary for cell division in human cells can begin.

Screen Details

Stable ID: GR00098-A-1
Screen Title: Cell division (1)
Assay: Cell number and DNA content
Method: Laser scanning cytometry
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HeLa
Library: Custom-made, rp
Reagent Type: esiRNA
Score Type: Complex, sp
Cutoff: Complex criteria
Notes:

Epithelial cell migration (1)
5599
MAPK8
np
np
none

Reference

Identification of genes that regulate epithelial cell migration using an siRNA screening approach. Simpson et al., 2008

To provide a systematic analysis of genes that regulate epithelial cell migration, we performed a high throughput wound healing screen with MCF-10A breast epithelial cells, using siRNAs targeting 1,081 human genes encoding phosphatases, kinases and proteins predicted to influence cell migration and adhesion. The primary screen identified three categories of hits: those that accelerate, those that inhibit and those that impair migration with associated effects on cell proliferation or metabolism. Extensive validation of all the hits yielded 66 high confidence genes that, when downregulated, either accelerated or impaired migration; 42 of these high confidence genes have not been previously associated with motility or adhesion. Time-lapse video microscopy revealed a broad spectrum of phenotypic changes involving alterations in the extent and nature of disruption of cell-cell adhesion, directionality of motility, cell polarity and shape, and protrusion dynamics. Informatics analysis highlighted three major signalling nodes, beta-catenin, beta1-integrin and actin, and a large proportion of the genes that accelerated migration impaired cell-cell adhesion.

Screen Details

Stable ID: GR00055-A-1
Screen Title: Epithelial cell migration (1)
Assay: Cell migration and viability
Method: Microscopy and fluorescence
Scope: Kinases, phosphatases and migration and adhesion related (MAR) genes
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: MCF-10A
Library: Dharmacon and custom-made, SMARTpool siRNA library and MAR library
Reagent Type: siRNA
Score Type: Area score and Alamar score
Cutoff: Complex criteria
Notes:

Hepatitis C virus replication (2)
5599
MAPK8
np
np
none

Reference

A functional genomic screen identifies cellular cofactors of hepatitis C virus replication. Tai et al., 2009

Hepatitis C virus (HCV) chronically infects 3% of the world''s population, and complications from HCV are the leading indication for liver transplantation. Given the need for better anti-HCV therapies, one strategy is to identify and target cellular cofactors of the virus lifecycle. Using a genome-wide siRNA library, we identified 96 human genes that support HCV replication, with a significant number of them being involved in vesicle organization and biogenesis. Phosphatidylinositol 4-kinase PI4KA and multiple subunits of the COPI vesicle coat complex were among the genes identified. Consistent with this, pharmacologic inhibitors of COPI and PI4KA blocked HCV replication. Targeting hepcidin, a peptide critical for iron homeostasis, also affected HCV replication, which may explain the known dysregulation of iron homeostasis in HCV infection. The host cofactors for HCV replication identified in this study should serve as a useful resource in delineating new targets for anti-HCV therapies.

Screen Details

Stable ID: GR00180-A-2
Screen Title: Hepatitis C virus replication (2)
Assay: HCV replicon RNA copy number
Method: Luminescence
Scope: Selected genes
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: Huh7/Rep-Feo
Library: Dharmacon, siARRAY Human Genome siRNA Library
Reagent Type: siRNA
Score Type: Number of positive siRNA
Cutoff: >= 2 of 4 siRNAs with standard deviation of decreased HCV replication >= 2
Notes:

Vaccinia virus (VACV) infection
5599
MAPK8
-0.73
none number of cells compared to control (%): 95.77

Reference

Human genome-wide RNAi screen reveals a role for nuclear pore proteins in poxvirus morphogenesis. Sivan et al., 2013

Poxviruses are considered less dependent on host functions than other DNA viruses because of their cytoplasmic site of replication and large genomes, which encode enzymes for DNA and mRNA synthesis. Nevertheless, RNAi screens with two independent human genome-scale libraries have identified more than 500 candidate genes that significantly inhibited and a similar number that enhanced replication and spread of infectious vaccinia virus (VACV). Translational, ubiquitin-proteosome, and endoplasmic reticulum-to-Golgi transport functions, known to be important for VACV, were enriched in the siRNA-inhibiting group, and RNA polymerase II and associated functions were enriched in the siRNA-enhancing group. Additional findings, notably the inhibition of VACV spread by siRNAs to several nuclear pore genes, were unanticipated. Knockdown of nucleoporin 62 strongly inhibited viral morphogenesis, with only a modest effect on viral gene expression, recapitulating and providing insight into previous studies with enucleated cells.

Screen Details

Stable ID: GR00249-S
Screen Title: Vaccinia virus (VACV) infection
Assay: Vaccinia virus VACV IHD-J/GFP protein expression and DNA content
Method: Fluorescence
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HeLa
Library: Ambion and Dharmacon, Silencer Select Version 4, siGENOME SMARTpool and OnTargetPlus
Reagent Type: siRNA
Score Type: Z-score
Cutoff: >= 1 OR <= -1.5
Notes: Author-submitted data. Primary screen. Decreased viability phenotype if number of cells compared to control < 50 %.

Non-small cell lung cancer (NSCLC) cytotoxicity (1)
MAPK8
np
-1.5
Decreased shRNA abundance

Reference

Proteasome inhibitors block DNA repair and radiosensitize non-small cell lung cancer. Cron et al., 2013

Despite optimal radiation therapy (RT), chemotherapy and/or surgery, a majority of patients with locally advanced non-small cell lung cancer (NSCLC) fail treatment. To identify novel gene targets for improved tumor control, we performed whole genome RNAi screens to identify knockdowns that most reproducibly increase NSCLC cytotoxicity. These screens identified several proteasome subunits among top hits, including the topmost hit PSMA1, a component of the core 20 S proteasome. Radiation and proteasome inhibition showed synergistic effects. Proteasome inhibition resulted in an 80-90% decrease in homologous recombination (HR), a 50% decrease in expression of NF-κB-inducible HR genes BRCA1 and FANCD2, and a reduction of BRCA1, FANCD2 and RAD51 ionizing radiation-induced foci. IκBα RNAi knockdown rescued NSCLC radioresistance. Irradiation of mice with NCI-H460 xenografts after inducible PSMA1 shRNA knockdown markedly increased murine survival compared to either treatment alone. Proteasome inhibition is a promising strategy for NSCLC radiosensitization via inhibition of NF-κB-mediated expression of Fanconi Anemia/HR DNA repair genes.

Screen Details

Stable ID: GR00251-A-1
Screen Title: Non-small cell lung cancer (NSCLC) cytotoxicity (1)
Assay: shRNA abundance
Method: Microarray
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: A549
Library: Hannon-Elledge whole genome pooled shRNA, np
Reagent Type: shRNA
Score Type: Complex, sp
Cutoff: Complex criteria
Notes: All listed genes are final hits. Final hit: >= 1 shRNA with >= 2-fold abundance decrease in both cell lines (A549 and NCI-H460)

Cell cycle
MAPK8
shMAPK8-864
15.4
Increased mitotic index

Reference

A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Moffat et al., 2006

To enable arrayed or pooled loss-of-function screens in a wide range of mammalian cell types, including primary and nondividing cells, we are developing lentiviral short hairpin RNA (shRNA) libraries targeting the human and murine genomes. The libraries currently contain 104,000 vectors, targeting each of 22,000 human and mouse genes with multiple sequence-verified constructs. To test the utility of the library for arrayed screens, we developed a screen based on high-content imaging to identify genes required for mitotic progression in human cancer cells and applied it to an arrayed set of 5,000 unique shRNA-expressing lentiviruses that target 1,028 human genes. The screen identified several known and approximately 100 candidate regulators of mitotic progression and proliferation; the availability of multiple shRNAs targeting the same gene facilitated functional validation of putative hits. This work provides a widely applicable resource for loss-of-function screens, as well as a roadmap for its application to biological discovery.

Screen Details

Stable ID: GR00110-A-0
Screen Title: Cell cycle
Assay: Mitotic index
Method: High content (microscopy)
Scope:
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HT29 (colon cancer)
Library: , TRC shRNA library
Reagent Type: shRNA
Score Type: Mitotic index
Cutoff: >=14 / <= 0.3
Notes:

Combinatorial effect with gemcitabine
MAPK8
MAPK8_A
-0.37
none

Reference

Synthetic lethal RNAi screening identifies sensitizing targets for gemcitabine therapy in pancreatic cancer. Azorsa et al., 2009

Pancreatic cancer retains a poor prognosis among the gastrointestinal cancers. It affects 230,000 individuals worldwide, has a very high mortality rate, and remains one of the most challenging malignancies to treat successfully. Treatment with gemcitabine, the most widely used chemotherapeutic against pancreatic cancer, is not curative and resistance may occur. Combinations of gemcitabine with other chemotherapeutic drugs or biological agents have resulted in limited improvement.

Screen Details

Stable ID: GR00225-A
Screen Title: Combinatorial effect with gemcitabine
Assay: Viability (synthetic lethal)
Method: Luminescence
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: MIA PaCa-2
Library: Qiagen, Validated kinase siRNA library version 1.0
Reagent Type: siRNA
Score Type: log2 ratio
Cutoff: 1.65 SD below mean ratio level
Notes:

Ciliogenesis and cilium length (1)
5599
MAPK8
37184
-38.41
none

Reference

Functional genomic screen for modulators of ciliogenesis and cilium length. Kim et al., 2010

Primary cilia are evolutionarily conserved cellular organelles that organize diverse signalling pathways. Defects in the formation or function of primary cilia are associated with a spectrum of human diseases and developmental abnormalities. Genetic screens in model organisms have discovered core machineries of cilium assembly and maintenance. However, regulatory molecules that coordinate the biogenesis of primary cilia with other cellular processes, including cytoskeletal organization, vesicle trafficking and cell-cell adhesion, remain to be identified. Here we report the results of a functional genomic screen using RNA interference (RNAi) to identify human genes involved in ciliogenesis control. The screen identified 36 positive and 13 negative ciliogenesis modulators, which include molecules involved in actin dynamics and vesicle trafficking. Further investigation demonstrated that blocking actin assembly facilitates ciliogenesis by stabilizing the pericentrosomal preciliary compartment (PPC), a previously uncharacterized compact vesiculotubular structure storing transmembrane proteins destined for cilia during the early phase of ciliogenesis. The PPC was labelled by recycling endosome markers. Moreover, knockdown of modulators that are involved in the endocytic recycling pathway affected the formation of the PPC as well as ciliogenesis. Our results uncover a critical regulatory step that couples actin dynamics and endocytic recycling with ciliogenesis, and also provides potential target molecules for future study.

Screen Details

Stable ID: GR00149-A-1
Screen Title: Ciliogenesis and cilium length (1)
Assay: Smoothed protein expression
Method: Fluorescence
Scope: Druggable genes
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: htRPE
Library: Ambion, Human druggable genome siRNA library V3.1
Reagent Type: siRNA
Score Type: Normalized percent inhibition
Cutoff: > 1.5 OR < -1.5 standard deviations from mean
Notes:

TRAIL-induced apoptosis (2)
NM_002750
MAPK8
0.28
none Z-score 0.134

Reference

A synthetic lethal screen identifies FAT1 as an antagonist of caspase-8 in extrinsic apoptosis. Kranz and Boutros, 2014

The extrinsic apoptosis pathway is initiated by binding of death ligands to death receptors resulting in the formation of the death-inducing signaling complex (DISC). Activation of procaspase-8 within the DISC and its release from the signaling complex is required for processing executor caspases and commiting cell death. Here, we report that the atypical cadherin FAT1 interacts with caspase-8 preventing the association of caspase-8 with the DISC. We identified FAT1 in a genome-wide siRNA screen for synthetic lethal interactions with death receptor-mediated apoptosis. Knockdown of FAT1 sensitized established and patient-derived glioblastoma cell lines for apoptosis transduced by cell death ligands. Depletion of FAT1 resulted in enhanced procaspase-8 recruitment to the DISC and increased formation of caspase-8 containing secondary signaling complexes. In addition, FAT1 knockout cell lines generated by CRISPR/Cas9-mediated genome engineering were more susceptible for death receptor-mediated apoptosis. Our findings provide evidence for a mechanism to control caspase-8-dependent cell death by the atypical cadherin FAT1. These results contribute towards the understanding of effector caspase regulation in physiological conditions.

Screen Details

Stable ID: GR00240-S-2
Screen Title: TRAIL-induced apoptosis (2)
Assay: Viability (synthetic lethal)
Method: Luminescence
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: U251MG
Library: Dharmacon, SMART-pool siRNA
Reagent Type: siRNA
Score Type: Differential score
Cutoff: > 3.6 AND viability Z-score < 4
Notes: Author-submitted data. Z-scores from viability screen (1) are considered in score interpretation for this screen.

Selective autophagy regulation (1)
NM_002750
MAPK8
np
sp
none

Reference

Image-based genome-wide siRNA screen identifies selective autophagy factors. Orvedahl et al., 2011

Selective autophagy involves the recognition and targeting of specific cargo, such as damaged organelles, misfolded proteins, or invading pathogens for lysosomal destruction. Yeast genetic screens have identified proteins required for different forms of selective autophagy, including cytoplasm-to-vacuole targeting, pexophagy and mitophagy, and mammalian genetic screens have identified proteins required for autophagy regulation. However, there have been no systematic approaches to identify molecular determinants of selective autophagy in mammalian cells. Here, to identify mammalian genes required for selective autophagy, we performed a high-content, image-based, genome-wide small interfering RNA screen to detect genes required for the colocalization of Sindbis virus capsid protein with autophagolysosomes. We identified 141 candidate genes required for viral autophagy, which were enriched for cellular pathways related to messenger RNA processing, interferon signalling, vesicle trafficking, cytoskeletal motor function and metabolism. Ninety-six of these genes were also required for Parkin-mediated mitophagy, indicating that common molecular determinants may be involved in autophagic targeting of viral nucleocapsids and autophagic targeting of damaged mitochondria. Murine embryonic fibroblasts lacking one of these gene products, the C2-domain containing protein, SMURF1, are deficient in the autophagosomal targeting of Sindbis and herpes simplex viruses and in the clearance of damaged mitochondria. Moreover, SMURF1-deficient mice accumulate damaged mitochondria in the heart, brain and liver. Thus, our study identifies candidate determinants of selective autophagy, and defines SMURF1 as a newly recognized mediator of both viral autophagy and mitophagy.

Screen Details

Stable ID: GR00242-A-1
Screen Title: Selective autophagy regulation (1)
Assay: Sindbis virus (SIN) capsid SIN-mCherry.capsid and autophagosome GFP–LC3 protein expression
Method: Fluorescence
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HeLa/GFP-LC3
Library: Dharmacon, siGenome
Reagent Type: siRNA
Score Type: Z-score
Cutoff: Complex criteria
Notes:

Vaccinia virus (VACV) infection
5599
MAPK8
-0.7
none number of cells compared to control (%): 79.01

Reference

Human genome-wide RNAi screen reveals a role for nuclear pore proteins in poxvirus morphogenesis. Sivan et al., 2013

Poxviruses are considered less dependent on host functions than other DNA viruses because of their cytoplasmic site of replication and large genomes, which encode enzymes for DNA and mRNA synthesis. Nevertheless, RNAi screens with two independent human genome-scale libraries have identified more than 500 candidate genes that significantly inhibited and a similar number that enhanced replication and spread of infectious vaccinia virus (VACV). Translational, ubiquitin-proteosome, and endoplasmic reticulum-to-Golgi transport functions, known to be important for VACV, were enriched in the siRNA-inhibiting group, and RNA polymerase II and associated functions were enriched in the siRNA-enhancing group. Additional findings, notably the inhibition of VACV spread by siRNAs to several nuclear pore genes, were unanticipated. Knockdown of nucleoporin 62 strongly inhibited viral morphogenesis, with only a modest effect on viral gene expression, recapitulating and providing insight into previous studies with enucleated cells.

Screen Details

Stable ID: GR00249-S
Screen Title: Vaccinia virus (VACV) infection
Assay: Vaccinia virus VACV IHD-J/GFP protein expression and DNA content
Method: Fluorescence
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HeLa
Library: Ambion and Dharmacon, Silencer Select Version 4, siGENOME SMARTpool and OnTargetPlus
Reagent Type: siRNA
Score Type: Z-score
Cutoff: >= 1 OR <= -1.5
Notes: Author-submitted data. Primary screen. Decreased viability phenotype if number of cells compared to control < 50 %.

Combinatorial effect with MLN4924, a NAE inhibitor (1)
5599
MAPK8
sp
Synthetic lethal with MLN4924 (a NAE inhibitor) duplicate screen

Reference

Novel DNA damage checkpoints mediating cell death induced by the NEDD8-activating enzyme inhibitor MLN4924. Blank et al., 2013

MLN4924 is an investigational small-molecule inhibitor of the NEDD8-activating enzyme (NAE) in phase I clinical trials. NAE inhibition prevents the ubiquitination and proteasomal degradation of substrates for cullin-RING ubiquitin E3 ligases that support cancer pathophysiology, but the genetic determinants conferring sensitivity to NAE inhibition are unknown. To address this gap in knowledge, we conducted a genome-wide siRNA screen to identify genes and pathways that affect the lethality of MLN4924 in melanoma cells. Of the 154 genes identified, approximately one-half interfered with components of the cell cycle, apoptotic machinery, ubiquitin system, and DNA damage response pathways. In particular, genes involved in DNA replication, p53, BRCA1/BRCA2, transcription-coupled repair, and base excision repair seemed to be important for MLN4924 lethality. In contrast, genes within the G(2)-M checkpoint affected sensitivity to MLN4924 in colon cancer cells. Cell-cycle analysis in melanoma cells by flow cytometry following RNAi-mediated silencing showed that MLN4924 prevented the transition of cells from S-G(2) phase after induction of rereplication stress. Our analysis suggested an important role for the p21-dependent intra-S-phase checkpoint and extensive rereplication, whereas the ATR-dependent intra-S-phase checkpoint seemed to play a less dominant role. Unexpectedly, induction of the p21-dependent intra-S-phase checkpoint seemed to be independent of both Cdt1 stabilization and ATR signaling. Collectively, these data enhance our understanding of the mechanisms by which inhibition of NEDD8-dependent ubiquitination causes cell death, informing clinical development of MLN4924.

Screen Details

Stable ID: GR00250-A-1
Screen Title: Combinatorial effect with MLN4924, a NAE inhibitor (1)
Assay: Viability (synthetic lethal)
Method: Luminescence
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: A375
Library: Dharmacon siGENOME SMARTpool, np
Reagent Type: siRNA
Score Type: Complex, sp
Cutoff: Complex criteria
Notes: Phenotypes for 650 nmol/L MLN4924. Additional information about screens with 250 nmol/L MLN4924.

Proliferation of cells with active beta-catenin (1)
MAPK8
-0.31
none essential gene

Reference

CK1epsilon is required for breast cancers dependent on beta-catenin activity. Kim et al., 2010

Aberrant beta-catenin signaling plays a key role in several cancer types, notably colon, liver and breast cancer. However approaches to modulate beta-catenin activity for therapeutic purposes have proven elusive to date.

Screen Details

Stable ID: GR00221-A-1
Screen Title: Proliferation of cells with active beta-catenin (1)
Assay: Viability
Method: Luminescence
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: MCF7
Library: The RNAi Consortium (TRC), TRC shRNA Library
Reagent Type: shRNA
Score Type: B-score
Cutoff: < -1
Notes: Essential gene: gene with B-score < -1 for >= 2 shRNAs

TP53 interactions (1)
ENSG00000107643
np
sp
none

Reference

A systematic RNAi synthetic interaction screen reveals a link between p53 and snoRNP assembly. Krastev et al., 2011

TP53 (tumour protein 53) is one of the most frequently mutated genes in human cancer and its role during cellular transformation has been studied extensively. However, the homeostatic functions of p53 are less well understood. Here, we explore the molecular dependency network of TP53 through an RNAi-mediated synthetic interaction screen employing two HCT116 isogenic cell lines and a genome-scale endoribonuclease-prepared short interfering RNA library. We identify a variety of TP53 synthetic interactions unmasking the complex connections of p53 to cellular physiology and growth control. Molecular dissection of the TP53 synthetic interaction with UNRIP indicates an enhanced dependency of TP53-negative cells on small nucleolar ribonucleoprotein (snoRNP) assembly. This dependency is mediated by the snoRNP chaperone gene NOLC1 (also known as NOPP140), which we identify as a physiological p53 target gene. This unanticipated function of TP53 in snoRNP assembly highlights the potential of RNAi-mediated synthetic interaction screens to dissect molecular pathways of tumour suppressor genes.

Screen Details

Stable ID: GR00196-A-1
Screen Title: TP53 interactions (1)
Assay: TP53 protein expression and viability
Method: Fluorescence
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HCT116 ( wildtype and TP53 knockout)
Library: Custom-made, rp
Reagent Type: esiRNA
Score Type: Complex, sp
Cutoff: Complex criteria
Notes:

Salmonella enterica subspecies 1 serovar Typhimurium invasion (1)
5599
MAPK8
np
0.04
none

Reference

RNAi screen of Salmonella invasion shows role of COPI in membrane targeting of cholesterol and Cdc42. Misselwitz et al., 2011

The pathogen Salmonella Typhimurium is a common cause of diarrhea and invades the gut tissue by injecting a cocktail of virulence factors into epithelial cells, triggering actin rearrangements, membrane ruffling and pathogen entry. One of these factors is SopE, a G-nucleotide exchange factor for the host cellular Rho GTPases Rac1 and Cdc42. How SopE mediates cellular invasion is incompletely understood. Using genome-scale RNAi screening we identified 72 known and novel host cell proteins affecting SopE-mediated entry. Follow-up assays assigned these ''hits'' to particular steps of the invasion process; i.e., binding, effector injection, membrane ruffling, membrane closure and maturation of the Salmonella-containing vacuole. Depletion of the COPI complex revealed a unique effect on virulence factor injection and membrane ruffling. Both effects are attributable to mislocalization of cholesterol, sphingolipids, Rac1 and Cdc42 away from the plasma membrane into a large intracellular compartment. Equivalent results were obtained with the vesicular stomatitis virus. Therefore, COPI-facilitated maintenance of lipids may represent a novel, unifying mechanism essential for a wide range of pathogens, offering opportunities for designing new drugs.

Screen Details

Stable ID: GR00133-A-1
Screen Title: Salmonella enterica subspecies 1 serovar Typhimurium invasion (1)
Assay: Gentamycin protection invasion assay
Method: Fluorescence
Scope: Druggable genes
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HeLa
Library: Qiagen, Druggable genome library V2.0
Reagent Type: siRNA
Score Type: log2 median
Cutoff: Complex criteria
Notes:

Negative genetic interactions (3)
5599
MAPK8
0.7
none

Reference

A negative genetic interaction map in isogenic cancer cell lines reveals cancer cell vulnerabilities. Vizeacoumar et al., 2013

Improved efforts are necessary to define the functional product of cancer mutations currently being revealed through large-scale sequencing efforts. Using genome-scale pooled shRNA screening technology, we mapped negative genetic interactions across a set of isogenic cancer cell lines and confirmed hundreds of these interactions in orthogonal co-culture competition assays to generate a high-confidence genetic interaction network of differentially essential or differential essentiality (DiE) genes. The network uncovered examples of conserved genetic interactions, densely connected functional modules derived from comparative genomics with model systems data, functions for uncharacterized genes in the human genome and targetable vulnerabilities. Finally, we demonstrate a general applicability of DiE gene signatures in determining genetic dependencies of other non-isogenic cancer cell lines. For example, the PTEN(-/-) DiE genes reveal a signature that can preferentially classify PTEN-dependent genotypes across a series of non-isogenic cell lines derived from the breast, pancreas and ovarian cancers. Our reference network suggests that many cancer vulnerabilities remain to be discovered through systematic derivation of a network of differentially essential genes in an isogenic cancer cell model.

Screen Details

Stable ID: GR00255-A-3
Screen Title: Negative genetic interactions (3)
Assay: shRNA abundance
Method: Microarray
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HCT116
Library: TRC lentiviral library, np
Reagent Type: shRNA
Score Type: differential Gene Activity Ranking Profile (dGARP)
Cutoff: < -1.2
Notes: HCT116 PTEN-/- and HCT116 PTEN+/+ cells used. Cutoff corresponds to p-value < 0.05. Additional information about a secondary screen (genetic interactions with Cetuximab/Erbitux in LIM1215 cells)

Kinase-mediated spindle orientation
NM_139047|NM_139049|NM_002750|NM_139046
MAPK8
np
sp
none

Reference

ABL1 regulates spindle orientation in adherent cells and mammalian skin. Matsumara et al., 2012

Despite the growing evidence for the regulated spindle orientation in mammals, a systematic approach for identifying the responsible genes in mammalian cells has not been established. Here we perform a kinase-targeting RNAi screen in HeLa cells and identify ABL1 as a novel regulator of spindle orientation. Knockdown of ABL1 causes the cortical accumulation of Leu-Gly-Asn repeat-enriched-protein (LGN), an evolutionarily conserved regulator of spindle orientation. This results in the LGN-dependent spindle rotation and spindle misorientation. In vivo inactivation of ABL1 by a pharmacological inhibitor or by ablation of the abl1 gene causes spindle misorientation and LGN mislocalization in mouse epidermis. Furthermore, ABL1 directly phosphorylates NuMA, a binding partner of LGN, on tyrosine 1774. This phosphorylation maintains the cortical localization of NuMA during metaphase, and ensures the LGN/NuMA-dependent spindle orientation control. This study provides a novel approach to identify genes regulating spindle orientation in mammals and uncovers new signalling pathways for this mechanism.

Screen Details

Stable ID: GR00256-A
Screen Title: Kinase-mediated spindle orientation
Assay: beta-tubulin protein expression and DNA content
Method: Fluorescence
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HeLa
Library: Ambion, Silencer Kinase siRNA library (AM80010V3)
Reagent Type: siRNA
Score Type: Complex, sp
Cutoff: Complex criteria
Notes: HeLa cells stably expressing GFP-H2B (HeLa-GH2B) used. Additional information about secondary screens.

Regulation of FOXO1 nuclear localization (1)
MAPK8
np
sp
none rank: 17093

Reference

Whole genome siRNA cell-based screen links mitochondria to Akt signaling network through uncoupling of electron transport chain. Senapedis et al., 2011

Forkhead transcription factors (FOXOs) alter a diverse array of cellular processes including the cell cycle, oxidative stress resistance, and aging. Insulin/Akt activation directs phosphorylation and cytoplasmic sequestration of FOXO away from its target genes and serves as an endpoint of a complex signaling network. Using a human genome small interfering RNA (siRNA) library in a cell-based assay, we identified an extensive network of proteins involved in nuclear export, focal adhesion, and mitochondrial respiration not previously implicated in FOXO localization. Furthermore, a detailed examination of mitochondrial factors revealed that loss of uncoupling protein 5 (UCP5) modifies the energy balance and increases free radicals through up-regulation of uncoupling protein 3 (UCP3). The increased superoxide content induces c-Jun N-terminal kinase 1 (JNK1) kinase activity, which in turn affects FOXO localization through a compensatory dephosphorylation of Akt. The resulting nuclear FOXO increases expression of target genes, including mitochondrial superoxide dismutase. By connecting free radical defense and mitochondrial uncoupling to Akt/FOXO signaling, these results have implications in obesity and type 2 diabetes development and the potential for therapeutic intervention.

Screen Details

Stable ID: GR00247-A-1
Screen Title: Regulation of FOXO1 nuclear localization (1)
Assay: EGFP-FOXO1a protein expression and DNA content
Method: Fluorescence
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: U2OS
Library: Dharmacon, Human Genome library
Reagent Type: siRNA
Score Type: Complex, sp
Cutoff: Complex criteria
Notes:

Negative genetic interactions (1)
5599
MAPK8
0.04
none

Reference

A negative genetic interaction map in isogenic cancer cell lines reveals cancer cell vulnerabilities. Vizeacoumar et al., 2013

Improved efforts are necessary to define the functional product of cancer mutations currently being revealed through large-scale sequencing efforts. Using genome-scale pooled shRNA screening technology, we mapped negative genetic interactions across a set of isogenic cancer cell lines and confirmed hundreds of these interactions in orthogonal co-culture competition assays to generate a high-confidence genetic interaction network of differentially essential or differential essentiality (DiE) genes. The network uncovered examples of conserved genetic interactions, densely connected functional modules derived from comparative genomics with model systems data, functions for uncharacterized genes in the human genome and targetable vulnerabilities. Finally, we demonstrate a general applicability of DiE gene signatures in determining genetic dependencies of other non-isogenic cancer cell lines. For example, the PTEN(-/-) DiE genes reveal a signature that can preferentially classify PTEN-dependent genotypes across a series of non-isogenic cell lines derived from the breast, pancreas and ovarian cancers. Our reference network suggests that many cancer vulnerabilities remain to be discovered through systematic derivation of a network of differentially essential genes in an isogenic cancer cell model.

Screen Details

Stable ID: GR00255-A-1
Screen Title: Negative genetic interactions (1)
Assay: shRNA abundance
Method: Microarray
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HCT116
Library: TRC lentiviral library, np
Reagent Type: shRNA
Score Type: differential Gene Activity Ranking Profile (dGARP)
Cutoff: < -1.0
Notes: HCT116 BLM-/- and HCT116 BLM+/+ cells used. Cutoff corresponds to p-value < 0.05. Additional information about a secondary screen (genetic interactions with Cetuximab/Erbitux in LIM1215 cells)

Negative genetic interactions (4)
5599
MAPK8
-0.64
none

Reference

A negative genetic interaction map in isogenic cancer cell lines reveals cancer cell vulnerabilities. Vizeacoumar et al., 2013

Improved efforts are necessary to define the functional product of cancer mutations currently being revealed through large-scale sequencing efforts. Using genome-scale pooled shRNA screening technology, we mapped negative genetic interactions across a set of isogenic cancer cell lines and confirmed hundreds of these interactions in orthogonal co-culture competition assays to generate a high-confidence genetic interaction network of differentially essential or differential essentiality (DiE) genes. The network uncovered examples of conserved genetic interactions, densely connected functional modules derived from comparative genomics with model systems data, functions for uncharacterized genes in the human genome and targetable vulnerabilities. Finally, we demonstrate a general applicability of DiE gene signatures in determining genetic dependencies of other non-isogenic cancer cell lines. For example, the PTEN(-/-) DiE genes reveal a signature that can preferentially classify PTEN-dependent genotypes across a series of non-isogenic cell lines derived from the breast, pancreas and ovarian cancers. Our reference network suggests that many cancer vulnerabilities remain to be discovered through systematic derivation of a network of differentially essential genes in an isogenic cancer cell model.

Screen Details

Stable ID: GR00255-A-4
Screen Title: Negative genetic interactions (4)
Assay: shRNA abundance
Method: Microarray
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: HCT116
Library: TRC lentiviral library, np
Reagent Type: shRNA
Score Type: differential Gene Activity Ranking Profile (dGARP)
Cutoff: < -1.2
Notes: HCT116 PTTG1-/- and HCT116 PTTG1+/+ cells used. Cutoff corresponds to p-value < 0.05. Additional information about a secondary screen (genetic interactions with Cetuximab/Erbitux in LIM1215 cells)

Ciliogenesis and cilium length (1)
5599
MAPK8
1415
60.43
none

Reference

Functional genomic screen for modulators of ciliogenesis and cilium length. Kim et al., 2010

Primary cilia are evolutionarily conserved cellular organelles that organize diverse signalling pathways. Defects in the formation or function of primary cilia are associated with a spectrum of human diseases and developmental abnormalities. Genetic screens in model organisms have discovered core machineries of cilium assembly and maintenance. However, regulatory molecules that coordinate the biogenesis of primary cilia with other cellular processes, including cytoskeletal organization, vesicle trafficking and cell-cell adhesion, remain to be identified. Here we report the results of a functional genomic screen using RNA interference (RNAi) to identify human genes involved in ciliogenesis control. The screen identified 36 positive and 13 negative ciliogenesis modulators, which include molecules involved in actin dynamics and vesicle trafficking. Further investigation demonstrated that blocking actin assembly facilitates ciliogenesis by stabilizing the pericentrosomal preciliary compartment (PPC), a previously uncharacterized compact vesiculotubular structure storing transmembrane proteins destined for cilia during the early phase of ciliogenesis. The PPC was labelled by recycling endosome markers. Moreover, knockdown of modulators that are involved in the endocytic recycling pathway affected the formation of the PPC as well as ciliogenesis. Our results uncover a critical regulatory step that couples actin dynamics and endocytic recycling with ciliogenesis, and also provides potential target molecules for future study.

Screen Details

Stable ID: GR00149-A-1
Screen Title: Ciliogenesis and cilium length (1)
Assay: Smoothed protein expression
Method: Fluorescence
Scope: Druggable genes
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: htRPE
Library: Ambion, Human druggable genome siRNA library V3.1
Reagent Type: siRNA
Score Type: Normalized percent inhibition
Cutoff: > 1.5 OR < -1.5 standard deviations from mean
Notes:

Homologous recombination DNA double-strand break repair (HR-DSBR) (2)
5599
MAPK8
0.86
none

Reference

A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response. Adamson et al., 2012

Repair of DNA double-strand breaks is critical to genomic stability and the prevention of developmental disorders and cancer. A central pathway for this repair is homologous recombination (HR). Most knowledge of HR is derived from work in prokaryotic and eukaryotic model organisms. We carried out a genome-wide siRNA-based screen in human cells. Among positive regulators of HR we identified networks of DNA-damage-response and pre-mRNA-processing proteins, and among negative regulators we identified a phosphatase network. Three candidate proteins localized to DNA lesions, including RBMX, a heterogeneous nuclear ribonucleoprotein that has a role in alternative splicing. RBMX accumulated at DNA lesions through multiple domains in a poly(ADP-ribose) polymerase 1-dependent manner and promoted HR by facilitating proper BRCA2 expression. Our screen also revealed that off-target depletion of RAD51 is a common source of RNAi false positives, raising a cautionary note for siRNA screens and RNAi-based studies of HR.

Screen Details

Stable ID: GR00236-A-2
Screen Title: Homologous recombination DNA double-strand break repair (HR-DSBR) (2)
Assay: (HR-DSBR) DR-GFP reporter and DNA content
Method: Fluorescence
Scope: Selected genes
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: DR-U2OS
Library: Dharmacon, np
Reagent Type: siRNA
Score Type: Relative HR ratio
Cutoff: Strong: < ~0.4, weak: < ~0.59 (for mediators); strong: > 1.88, weak: > 1.69 (for suppressors)
Notes: Cutoff values correspond 2 standard deviations from the screen-wide mean (strong) or 1.5 SD (weak)

Proliferation of cells with active beta-catenin (3)
MAPK8
0.37
none

Reference

CK1epsilon is required for breast cancers dependent on beta-catenin activity. Kim et al., 2010

Aberrant beta-catenin signaling plays a key role in several cancer types, notably colon, liver and breast cancer. However approaches to modulate beta-catenin activity for therapeutic purposes have proven elusive to date.

Screen Details

Stable ID: GR00221-A-3
Screen Title: Proliferation of cells with active beta-catenin (3)
Assay: Viability
Method: Luminescence
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: MDA-MB-453
Library: The RNAi Consortium (TRC), TRC shRNA Library
Reagent Type: shRNA
Score Type: B-score
Cutoff: < -1
Notes: Essential gene: gene with B-score < -1 for >= 2 shRNAs

Combinatorial effect with MLN4924, a NAE inhibitor (2)
5599
MAPK8
sp
Synthetic lethal with MLN4924 (a NAE inhibitor) sextuplicate screen

Reference

Novel DNA damage checkpoints mediating cell death induced by the NEDD8-activating enzyme inhibitor MLN4924. Blank et al., 2013

MLN4924 is an investigational small-molecule inhibitor of the NEDD8-activating enzyme (NAE) in phase I clinical trials. NAE inhibition prevents the ubiquitination and proteasomal degradation of substrates for cullin-RING ubiquitin E3 ligases that support cancer pathophysiology, but the genetic determinants conferring sensitivity to NAE inhibition are unknown. To address this gap in knowledge, we conducted a genome-wide siRNA screen to identify genes and pathways that affect the lethality of MLN4924 in melanoma cells. Of the 154 genes identified, approximately one-half interfered with components of the cell cycle, apoptotic machinery, ubiquitin system, and DNA damage response pathways. In particular, genes involved in DNA replication, p53, BRCA1/BRCA2, transcription-coupled repair, and base excision repair seemed to be important for MLN4924 lethality. In contrast, genes within the G(2)-M checkpoint affected sensitivity to MLN4924 in colon cancer cells. Cell-cycle analysis in melanoma cells by flow cytometry following RNAi-mediated silencing showed that MLN4924 prevented the transition of cells from S-G(2) phase after induction of rereplication stress. Our analysis suggested an important role for the p21-dependent intra-S-phase checkpoint and extensive rereplication, whereas the ATR-dependent intra-S-phase checkpoint seemed to play a less dominant role. Unexpectedly, induction of the p21-dependent intra-S-phase checkpoint seemed to be independent of both Cdt1 stabilization and ATR signaling. Collectively, these data enhance our understanding of the mechanisms by which inhibition of NEDD8-dependent ubiquitination causes cell death, informing clinical development of MLN4924.

Screen Details

Stable ID: GR00250-A-2
Screen Title: Combinatorial effect with MLN4924, a NAE inhibitor (2)
Assay: Viability (synthetic lethal)
Method: Luminescence
Scope: Selected genes
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: A375
Library: Dharmacon, np
Reagent Type: siRNA
Score Type: Complex, sp
Cutoff: Complex criteria
Notes: Phenotypes for 650 nmol/L MLN4924. Additional information about screens with 250 nmol/L MLN4924.

Hepatitis C virus (HCV) infection (1)
NM_002750
MAPK8
np
sp
none

Reference

EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy. Lupberger et al., 2011

Hepatitis C virus (HCV) is a major cause of liver disease, but therapeutic options are limited and there are no prevention strategies. Viral entry is the first step of infection and requires the cooperative interaction of several host cell factors. Using a functional RNAi kinase screen, we identified epidermal growth factor receptor and ephrin receptor A2 as host cofactors for HCV entry. Blocking receptor kinase activity by approved inhibitors broadly impaired infection by all major HCV genotypes and viral escape variants in cell culture and in a human liver chimeric mouse model in vivo. The identified receptor tyrosine kinases (RTKs) mediate HCV entry by regulating CD81-claudin-1 co-receptor associations and viral glycoprotein-dependent membrane fusion. These results identify RTKs as previously unknown HCV entry cofactors and show that tyrosine kinase inhibitors have substantial antiviral activity. Inhibition of RTK function may constitute a new approach for prevention and treatment of HCV infection.

Screen Details

Stable ID: GR00234-A-1
Screen Title: Hepatitis C virus (HCV) infection (1)
Assay: Hepatitis C Virus pseudoparticles (HCVpp; H77; genotype 1a) protein expression
Method: Luminescence
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: Huh7
Library: Qiagen, Human Kinase RNAi Set V2.0
Reagent Type: siRNA
Score Type: Complex, sp
Cutoff: Complex criteria
Notes:

Cell viability
MAPK8
np
np
Increased cell death in breast cancer cell lines (MCF10A, MDA-MB-435)

Reference

Profiling essential genes in human mammary cells by multiplex RNAi screening. Silva et al., 2008

By virtue of their accumulated genetic alterations, tumor cells may acquire vulnerabilities that create opportunities for therapeutic intervention. We have devised a massively parallel strategy for screening short hairpin RNA (shRNA) collections for stable loss-of-function phenotypes. We assayed from 6000 to 20,000 shRNAs simultaneously to identify genes important for the proliferation and survival of five cell lines derived from human mammary tissue. Lethal shRNAs common to these cell lines targeted many known cell-cycle regulatory networks. Cell line-specific sensitivities to suppression of protein complexes and biological pathways also emerged, and these could be validated by RNA interference (RNAi) and pharmacologically. These studies establish a practical platform for genome-scale screening of complex phenotypes in mammalian cells and demonstrate that RNAi can be used to expose genotype-specific sensitivities.

Screen Details

Stable ID: GR00104-A-0
Screen Title: Cell viability
Assay: Cell viability
Method: Sequencing
Scope:
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: MCF-10A, MDA-MB-435 (breast cancer)
Library: , shRNA-mir (G. Hannon)
Reagent Type: shRNA
Score Type: log ratio
Cutoff: <= -1 (>= 50% cell death)
Notes:

Combinatorial effect with MLN4924, a NAE inhibitor (2)
5599
MAPK8
sp
Synthetic lethal with MLN4924 (a NAE inhibitor) sextuplicate screen

Reference

Novel DNA damage checkpoints mediating cell death induced by the NEDD8-activating enzyme inhibitor MLN4924. Blank et al., 2013

MLN4924 is an investigational small-molecule inhibitor of the NEDD8-activating enzyme (NAE) in phase I clinical trials. NAE inhibition prevents the ubiquitination and proteasomal degradation of substrates for cullin-RING ubiquitin E3 ligases that support cancer pathophysiology, but the genetic determinants conferring sensitivity to NAE inhibition are unknown. To address this gap in knowledge, we conducted a genome-wide siRNA screen to identify genes and pathways that affect the lethality of MLN4924 in melanoma cells. Of the 154 genes identified, approximately one-half interfered with components of the cell cycle, apoptotic machinery, ubiquitin system, and DNA damage response pathways. In particular, genes involved in DNA replication, p53, BRCA1/BRCA2, transcription-coupled repair, and base excision repair seemed to be important for MLN4924 lethality. In contrast, genes within the G(2)-M checkpoint affected sensitivity to MLN4924 in colon cancer cells. Cell-cycle analysis in melanoma cells by flow cytometry following RNAi-mediated silencing showed that MLN4924 prevented the transition of cells from S-G(2) phase after induction of rereplication stress. Our analysis suggested an important role for the p21-dependent intra-S-phase checkpoint and extensive rereplication, whereas the ATR-dependent intra-S-phase checkpoint seemed to play a less dominant role. Unexpectedly, induction of the p21-dependent intra-S-phase checkpoint seemed to be independent of both Cdt1 stabilization and ATR signaling. Collectively, these data enhance our understanding of the mechanisms by which inhibition of NEDD8-dependent ubiquitination causes cell death, informing clinical development of MLN4924.

Screen Details

Stable ID: GR00250-A-2
Screen Title: Combinatorial effect with MLN4924, a NAE inhibitor (2)
Assay: Viability (synthetic lethal)
Method: Luminescence
Scope: Selected genes
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: A375
Library: Dharmacon, np
Reagent Type: siRNA
Score Type: Complex, sp
Cutoff: Complex criteria
Notes: Phenotypes for 650 nmol/L MLN4924. Additional information about screens with 250 nmol/L MLN4924.

Cytokine regulation
5599
MAPK8
AAA13.E12 NM_139049.x-864s1c1
1.08
Increased IL-13 protein expression

Reference

RNA interference screen in primary human T cells reveals FLT3 as a modulator of IL-10 levels. Astier et al., 2010

Functional studies of human primary immune cells have been hampered by the lack of tools to silence gene functions. In this study, we report the application of a lentiviral RNA interference library in primary human T cells. Using a subgenomic short hair RNA library targeting approximately 1000 signaling genes, we identified novel genes that control the levels of IL-10 produced. IL-10 is a potent anti-inflammatory cytokine secreted by several cell types, including T regulatory type 1 cells, a subset of T regulatory cells that exert their suppressive activity through IL-10 secretion. FLT3, a known hematopoeitic growth factor, was found to be a negative regulator of IL-10 levels in activated T cells. This was based on several observations. First, FLT3 and its ligand (FL) were both induced by T cell activation. Second, silencing of FLT3 led to increased IL-10 levels, whereas addition of FL suppressed IL-10 secretion and increased FLT3 surface levels. Third, engagement of CD46, a known inducer of T regulatory type 1 cells, upregulated surface FLT3, and secreted FL, which then inhibited IL-10 production by T cells. Hence, FL and FLT3 form a novel regulatory feedback loop that limits IL-10 production in T cells. Our results identified FLT3 as a new regulator of T cell function and offer a strategy to genetically dissect specific pathways in T cells.

Screen Details

Stable ID: GR00219-A
Screen Title: Cytokine regulation
Assay: IL-10, IL-13 and IFN-gamma protein expression
Method: Fluorescence
Scope: Kinases and phosphatases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Primary cells
Biomodel: primary CD4+ T cells
Library: The RNAi Consortium (TRC), TRC shRNA Library
Reagent Type: shRNA
Score Type: Z-score
Cutoff: Complex criteria
Notes:

Proliferation of cells with active beta-catenin (4)
MAPK8
0.07
none

Reference

CK1epsilon is required for breast cancers dependent on beta-catenin activity. Kim et al., 2010

Aberrant beta-catenin signaling plays a key role in several cancer types, notably colon, liver and breast cancer. However approaches to modulate beta-catenin activity for therapeutic purposes have proven elusive to date.

Screen Details

Stable ID: GR00221-A-4
Screen Title: Proliferation of cells with active beta-catenin (4)
Assay: Viability
Method: Luminescence
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: T47D
Library: The RNAi Consortium (TRC), TRC shRNA Library
Reagent Type: shRNA
Score Type: B-score
Cutoff: < -1
Notes: Essential gene: gene with B-score < -1 for >= 2 shRNAs

Therapeutic kinase targets in neuroblastoma (2)
5599
MAPK8
MAPK8
1.12
none Illumina gene expression above basal level (p < 0.05)

Reference

RNAi screen of the protein kinome identifies checkpoint kinase 1 (CHK1) as a therapeutic target in neuroblastoma. Cole et al., 2011

Neuroblastoma is a childhood cancer that is often fatal despite intense multimodality therapy. In an effort to identify therapeutic targets for this disease, we performed a comprehensive loss-of-function screen of the protein kinome. Thirty kinases showed significant cellular cytotoxicity when depleted, with loss of the cell cycle checkpoint kinase 1 (CHK1/CHEK1) being the most potent. CHK1 mRNA expression was higher in MYC-Neuroblastoma-related (MYCN)-amplified (P < 0.0001) and high-risk (P = 0.03) tumors. Western blotting revealed that CHK1 was constitutively phosphorylated at the ataxia telangiectasia response kinase target site Ser345 and the autophosphorylation site Ser296 in neuroblastoma cell lines. This pattern was also seen in six of eight high-risk primary tumors but not in control nonneuroblastoma cell lines or in seven of eight low-risk primary tumors. Neuroblastoma cells were sensitive to the two CHK1 inhibitors SB21807 and TCS2312, with median IC(50) values of 564 nM and 548 nM, respectively. In contrast, the control lines had high micromolar IC(50) values, indicating a strong correlation between CHK1 phosphorylation and CHK1 inhibitor sensitivity (P = 0.0004). Furthermore, cell cycle analysis revealed that CHK1 inhibition in neuroblastoma cells caused apoptosis during S-phase, consistent with its role in replication fork progression. CHK1 inhibitor sensitivity correlated with total MYC(N) protein levels, and inducing MYCN in retinal pigmented epithelial cells resulted in CHK1 phosphorylation, which caused growth inhibition when inhibited. These data show the power of a functional RNAi screen to identify tractable therapeutical targets in neuroblastoma and support CHK1 inhibition strategies in this disease.

Screen Details

Stable ID: GR00193-A-2
Screen Title: Therapeutic kinase targets in neuroblastoma (2)
Assay: Substrate adherent cell growth
Method: rp
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: KELLY
Library: Thermo Scientific, Kinase siGenome library
Reagent Type: siRNA
Score Type: Relative growth
Cutoff: 0.5 standard deviations below mean
Notes:

Self-renewal and pluripotency in human embryonic stem cells (1)
NM_002750
MAPK8
M-003514-03
-0.32
none

Reference

A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity. Chia et al., 2010

The derivation of human ES cells (hESCs) from human blastocysts represents one of the milestones in stem cell biology. The full potential of hESCs in research and clinical applications requires a detailed understanding of the genetic network that governs the unique properties of hESCs. Here, we report a genome-wide RNA interference screen to identify genes which regulate self-renewal and pluripotency properties in hESCs. Interestingly, functionally distinct complexes involved in transcriptional regulation and chromatin remodelling are among the factors identified in the screen. To understand the roles of these potential regulators of hESCs, we studied transcription factor PRDM14 to gain new insights into its functional roles in the regulation of pluripotency. We showed that PRDM14 regulates directly the expression of key pluripotency gene POU5F1 through its proximal enhancer. Genome-wide location profiling experiments revealed that PRDM14 colocalized extensively with other key transcription factors such as OCT4, NANOG and SOX2, indicating that PRDM14 is integrated into the core transcriptional regulatory network. More importantly, in a gain-of-function assay, we showed that PRDM14 is able to enhance the efficiency of reprogramming of human fibroblasts in conjunction with OCT4, SOX2 and KLF4. Altogether, our study uncovers a wealth of novel hESC regulators wherein PRDM14 exemplifies a key transcription factor required for the maintenance of hESC identity and the reacquisition of pluripotency in human somatic cells.

Screen Details

Stable ID: GR00184-A-1
Screen Title: Self-renewal and pluripotency in human embryonic stem cells (1)
Assay: POU5F1 protein expression
Method: Fluorescence
Scope: Genome-wide
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: hESC H1
Library: Dharmacon, SMARTpool siRNA library
Reagent Type: siRNA
Score Type: Z-score
Cutoff: < -2
Notes:

Therapeutic kinase targets in neuroblastoma (1)
5599
MAPK8
MAPK8
0.83
none Illumina gene expression above basal level (p < 0.05)

Reference

RNAi screen of the protein kinome identifies checkpoint kinase 1 (CHK1) as a therapeutic target in neuroblastoma. Cole et al., 2011

Neuroblastoma is a childhood cancer that is often fatal despite intense multimodality therapy. In an effort to identify therapeutic targets for this disease, we performed a comprehensive loss-of-function screen of the protein kinome. Thirty kinases showed significant cellular cytotoxicity when depleted, with loss of the cell cycle checkpoint kinase 1 (CHK1/CHEK1) being the most potent. CHK1 mRNA expression was higher in MYC-Neuroblastoma-related (MYCN)-amplified (P < 0.0001) and high-risk (P = 0.03) tumors. Western blotting revealed that CHK1 was constitutively phosphorylated at the ataxia telangiectasia response kinase target site Ser345 and the autophosphorylation site Ser296 in neuroblastoma cell lines. This pattern was also seen in six of eight high-risk primary tumors but not in control nonneuroblastoma cell lines or in seven of eight low-risk primary tumors. Neuroblastoma cells were sensitive to the two CHK1 inhibitors SB21807 and TCS2312, with median IC(50) values of 564 nM and 548 nM, respectively. In contrast, the control lines had high micromolar IC(50) values, indicating a strong correlation between CHK1 phosphorylation and CHK1 inhibitor sensitivity (P = 0.0004). Furthermore, cell cycle analysis revealed that CHK1 inhibition in neuroblastoma cells caused apoptosis during S-phase, consistent with its role in replication fork progression. CHK1 inhibitor sensitivity correlated with total MYC(N) protein levels, and inducing MYCN in retinal pigmented epithelial cells resulted in CHK1 phosphorylation, which caused growth inhibition when inhibited. These data show the power of a functional RNAi screen to identify tractable therapeutical targets in neuroblastoma and support CHK1 inhibition strategies in this disease.

Screen Details

Stable ID: GR00193-A-1
Screen Title: Therapeutic kinase targets in neuroblastoma (1)
Assay: Substrate adherent cell growth
Method: rp
Scope: Kinases
Screen Type: Cell-based
Species: Homo sapiens
Biosource: Cell line
Biomodel: EBC1
Library: Thermo Scientific, Kinase siGenome library
Reagent Type: siRNA
Score Type: Relative growth
Cutoff: 0.5 standard deviations below mean
Notes:

Reagent information for gene 5599 (MAPK8)

Reagent IDTypeLibrary
D-003514-07 siRNA
siGENOME|Thermo Scientific Dharmacon|1|RefSeq release 5-7|84206 siRNAs in pools of four|siRNA|http://www.dharmacon.com/
D-003514-06 siRNA
siGENOME|Thermo Scientific Dharmacon|1|RefSeq release 5-7|84206 siRNAs in pools of four|siRNA|http://www.dharmacon.com/
M-003514-01 siRNA_Pool
siGENOME|Thermo Scientific Dharmacon|1|RefSeq release 5-7|84206 siRNAs in pools of four|siRNA|http://www.dharmacon.com/
D-003514-08 siRNA
siGENOME|Thermo Scientific Dharmacon|1|RefSeq release 5-7|84206 siRNAs in pools of four|siRNA|http://www.dharmacon.com/
D-003514-05 siRNA
siGENOME|Thermo Scientific Dharmacon|1|RefSeq release 5-7|84206 siRNAs in pools of four|siRNA|http://www.dharmacon.com/
SI02757209 siRNA
Druggable and whole genome supplement|Qiagen|1, 3|RefSeq|70308 siRNAs in pools of four|siRNA|http://www.qiagen.com/
SI02758651 siRNA
Druggable and whole genome supplement|Qiagen|1, 3|RefSeq|70308 siRNAs in pools of four|siRNA|http://www.qiagen.com/
SI02758637 siRNA
Druggable and whole genome supplement|Qiagen|1, 3|RefSeq|70308 siRNAs in pools of four|siRNA|http://www.qiagen.com/
SI02758644 siRNA
Druggable and whole genome supplement|Qiagen|1, 3|RefSeq|70308 siRNAs in pools of four|siRNA|http://www.qiagen.com/
SP00006522 siRNA_Pool
Druggable and whole genome supplement|Qiagen|1, 3|RefSeq|70308 siRNAs in pools of four|siRNA|http://www.qiagen.com/
TRCN0000010581 shRNA
TRC shRNA Library|The RNAi Consortium (TRC)|1|RefSeq|81054|shRNA|http://www.broadinstitute.org/rnai/public/
TRCN0000001055 shRNA
TRC shRNA Library|The RNAi Consortium (TRC)|1|RefSeq|81054|shRNA|http://www.broadinstitute.org/rnai/public/
TRCN0000001056 shRNA
TRC shRNA Library|The RNAi Consortium (TRC)|1|RefSeq|81054|shRNA|http://www.broadinstitute.org/rnai/public/
TRCN0000010580 shRNA
TRC shRNA Library|The RNAi Consortium (TRC)|1|RefSeq|81054|shRNA|http://www.broadinstitute.org/rnai/public/
TRCN0000196371 shRNA
TRC shRNA Library|The RNAi Consortium (TRC)|1|RefSeq|81054|shRNA|http://www.broadinstitute.org/rnai/public/
TRCN0000196304 shRNA
TRC shRNA Library|The RNAi Consortium (TRC)|1|RefSeq|81054|shRNA|http://www.broadinstitute.org/rnai/public/
TRCN0000001057 shRNA
TRC shRNA Library|The RNAi Consortium (TRC)|1|RefSeq|81054|shRNA|http://www.broadinstitute.org/rnai/public/
TRCN0000196850 shRNA
TRC shRNA Library|The RNAi Consortium (TRC)|1|RefSeq|81054|shRNA|http://www.broadinstitute.org/rnai/public/
TRCN0000196820 shRNA
TRC shRNA Library|The RNAi Consortium (TRC)|1|RefSeq|81054|shRNA|http://www.broadinstitute.org/rnai/public/
TRCN0000194860 shRNA
TRC shRNA Library|The RNAi Consortium (TRC)|1|RefSeq|81054|shRNA|http://www.broadinstitute.org/rnai/public/
v2HS_217930 siRNA
shRNAmir|OpenBiosystems|1|RefSeq|2120|shRNA|http://www.openbiosystems.com/
s11152 siRNA
Ambion Silencer Select|Ambion|1|RefSeq|64781|siRNA|http://www.invitrogen.com/site/us/en/home/brands/ambion.html?CID=fl-ambion
s11153 siRNA
Ambion Silencer Select|Ambion|1|RefSeq|64781|siRNA|http://www.invitrogen.com/site/us/en/home/brands/ambion.html?CID=fl-ambion
s11154 siRNA
Ambion Silencer Select|Ambion|1|RefSeq|64781|siRNA|http://www.invitrogen.com/site/us/en/home/brands/ambion.html?CID=fl-ambion

Gene information for gene 5599 (MAPK8)

Gene:
Alternate gene names:JNK, PRKM8, JNK1A2, JNK1, JNK21B1/2, SAPK1c, SAPK1, JNK-46
Description:mitogen-activated protein kinase 8
Chromosome:10
Start:48306638
Stop:48439359
Strand:positive
Locus:10q11.22
Biotype:protein-coding
Status:live
Entrez Gene:
GeneCards:
Ensembl:
Hgnc:
Hprd:
Mim:
Uniprot:
Vega:
RefSeq:

Homologs:

GeneChromosomeLocusOrganism
bsk2LDrosophila melanogaster

Genome browser for gene 5599 (MAPK8)

Homo sapiens GRCh38
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