An expanded universe of cancer targets.

The characterization of cancer genomes has provided insight into somatically altered genes across tumors, transformed our understanding of cancer biology, and enabled tailoring of therapeutic strategies. However, the function of most cancer alleles remains mysterious, and many cancer features transcend their genomes. Consequently, tumor genomic characterization does not influence therapy for most patients. Approaches to understand the function and circuitry of cancer genes provide complementary approaches to elucidate both oncogene and non-oncogene dependencies. Emerging work indicates that the diversity of therapeutic targets engendered by non-oncogene dependencies is much larger than the list of recurrently mutated genes. Here we describe a framework for this expanded list of cancer targets, providing novel opportunities for clinical translation.

Compendium of Immune Signatures Identifies Conserved and Species-Specific Biology in Response to Inflammation.

Gene-expression profiling has become a mainstay in immunology, but subtle changes in gene networks related to biological processes are hard to discern when comparing various datasets. For instance, conservation of the transcriptional response to sepsis in mouse models and human disease remains controversial. To improve transcriptional analysis in immunology, we created ImmuneSigDB: a manually annotated compendium of ∼5,000 gene-sets from diverse cell states, experimental manipulations, and genetic perturbations in immunology. Analysis using ImmuneSigDB identified signatures induced in activated myeloid cells and differentiating lymphocytes that were highly conserved between humans and mice. Sepsis triggered conserved patterns of gene expression in humans and mouse models. However, we also identified species-specific biological processes in the sepsis transcriptional response: although both species upregulated phagocytosis-related genes, a mitosis signature was specific to humans. ImmuneSigDB enables granular analysis of transcriptomic data to improve biological understanding of immune processes of the human and mouse immune systems.

Gαs is dispensable for ß-arrestin coupling but dictates GRK selectivity and is predominant for gene expression regulation by ß2-adrenergic receptor.

ß-arrestins play a key role in G protein-coupled receptor (GPCR) internalization, trafficking, and signaling. Whether ß-arrestins act independently of G protein-mediated signaling has not been fully elucidated. Studies using genome-editing approaches revealed that whereas G proteins are essential for mitogen-activated protein kinase activation by GPCRs., ß-arrestins play a more prominent role in signal compartmentalization. However, in the absence of G proteins, GPCRs may not activate ß-arrestins, thereby limiting the ability to distinguish G protein from ß-arrestin-mediated signaling events. We used ß2-adrenergic receptor (ß2AR) and its ß2AR-C tail mutant expressed in human embryonic kidney 293 cells wildtype or CRISPR-Cas9 gene edited for Gαs, ß-arrestin1/2, or GPCR kinases 2/3/5/6 in combination with arrestin conformational sensors to elucidate the interplay between Gαs and ß-arrestins in controlling gene expression. We found that Gαs is not required for ß2AR and ß-arrestin conformational changes, ß-arrestin recruitment, and receptor internalization, but that Gαs dictates the GPCR kinase isoforms involved in ß-arrestin recruitment. By RNA-Seq analysis, we found that protein kinase A and mitogen-activated protein kinase gene signatures were activated by stimulation of ß2AR in wildtype and ß-arrestin1/2-KO cells but absent in Gαs-KO cells. These results were validated by re-expressing Gαs in the corresponding KO cells and silencing ß-arrestins in wildtype cells. These findings were extended to cellular systems expressing endogenous levels of ß2AR. Overall, our results support that Gs is essential for ß2AR-promoted protein kinase A and mitogen-activated protein kinase gene expression signatures, whereas ß-arrestins initiate signaling events modulating Gαs-driven nuclear transcriptional activity.

Synthetic lethal interaction between oncogenic KRAS dependency and STK33 suppression in human cancer cells.

An alternative to therapeutic targeting of oncogenes is to perform "synthetic lethality" screens for genes that are essential only in the context of specific cancer-causing mutations. We used high-throughput RNA interference (RNAi) to identify synthetic lethal interactions in cancer cells harboring mutant KRAS, the most commonly mutated human oncogene. We find that cells that are dependent on mutant KRAS exhibit sensitivity to suppression of the serine/threonine kinase STK33 irrespective of tissue origin, whereas STK33 is not required by KRAS-independent cells. STK33 promotes cancer cell viability in a kinase activity-dependent manner by regulating the suppression of mitochondrial apoptosis mediated through S6K1-induced inactivation of the death agonist BAD selectively in mutant KRAS-dependent cells. These observations identify STK33 as a target for treatment of mutant KRAS-driven cancers and demonstrate the potential of RNAi screens for discovering functional dependencies created by oncogenic mutations that may enable therapeutic intervention for cancers with "undruggable" genetic alterations.

Transcriptomic profiling and genomic rearrangement landscape of Nigerian prostate cancer.

BACKGROUND: Men of African ancestry have disproportionately high incidence rates of prostate cancer (PCa) and have high mortality rates. While there is evidence for a higher genetic predisposition for incidence of PCa in men of African ancestry compared to men of European ancestry, there have been few transcriptomic studies on PCa in men of African ancestry in the African continent. OBJECTIVE: We performed transcriptomic profiling and fusion analysis on bulk RNA sequencing (RNA-seq) samples from 24 Nigerian PCa patients to investigate the transcriptomic and genomic rearrangement landscape of PCa in Nigerian men. DESIGN: Bulk RNA-seq was performed on 24 formalin-fixed paraffin-embeded (FFPE) prostatectomy specimens of Nigerian men. Transcriptomic analysis was performed on 11 high-quality samples. Arriba Fusion and STAR Fusion were used for fusion detection. RESULTS: 4/11 (36%) of the samples harbored an erythroblast transformation-specific (ETS) fusion event; 1/11 (9%) had a TMPRSS2-ERG fusion; 2/11 had a TMPRSS2-ETV5 fusion, and 1/11 had a SLC45A3-SKIL fusion. Hierarchical clustering of normalized and mean-centered gene expression showed clustering of fusion positive samples. Furthermore, we developed gene set signatures for Nigerian PCa based on fusion events. By projecting the cancer genome atlas prostate adenocarcinoma (TCGA-PRAD) bulk RNA-seq data set onto the transcriptional space defined by these signatures derived from Nigerian PCa patients, we identified a positive correlation between the Nigerian fusion signature and fusion positive samples in the TCGA-PRAD data set. CONCLUSIONS: Less frequent ETS fusion events other than TMPRSS2-ERG such as TMPRSS2-ETV5 and non-ETS fusion events such as SLC45A3-SKIL may be more common in PCa in Nigerian men. This study provides useful working transcriptomic signatures that characterize oncogenic states representative of specific gene fusion events in PCa from Nigerian men.

Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway.

Plasticity of the cell state has been proposed to drive resistance to multiple classes of cancer therapies, thereby limiting their effectiveness. A high-mesenchymal cell state observed in human tumours and cancer cell lines has been associated with resistance to multiple treatment modalities across diverse cancer lineages, but the mechanistic underpinning for this state has remained incompletely understood. Here we molecularly characterize this therapy-resistant high-mesenchymal cell state in human cancer cell lines and organoids and show that it depends on a druggable lipid-peroxidase pathway that protects against ferroptosis, a non-apoptotic form of cell death induced by the build-up of toxic lipid peroxides. We show that this cell state is characterized by activity of enzymes that promote the synthesis of polyunsaturated lipids. These lipids are the substrates for lipid peroxidation by lipoxygenase enzymes. This lipid metabolism creates a dependency on pathways converging on the phospholipid glutathione peroxidase (GPX4), a selenocysteine-containing enzyme that dissipates lipid peroxides and thereby prevents the iron-mediated reactions of peroxides that induce ferroptotic cell death. Dependency on GPX4 was found to exist across diverse therapy-resistant states characterized by high expression of ZEB1, including epithelial-mesenchymal transition in epithelial-derived carcinomas, TGFß-mediated therapy-resistance in melanoma, treatment-induced neuroendocrine transdifferentiation in prostate cancer, and sarcomas, which are fixed in a mesenchymal state owing to their cells of origin. We identify vulnerability to ferroptic cell death induced by inhibition of a lipid peroxidase pathway as a feature of therapy-resistant cancer cells across diverse mesenchymal cell-state contexts.

Metabolic Rewiring by Oncogenic BRAF V600E Links Ketogenesis Pathway to BRAF-MEK1 Signaling.

Many human cancers share similar metabolic alterations, including the Warburg effect. However, it remains unclear whether oncogene-specific metabolic alterations are required for tumor development. Here we demonstrate a "synthetic lethal" interaction between oncogenic BRAF V600E and a ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL). HMGCL expression is upregulated in BRAF V600E-expressing human primary melanoma and hairy cell leukemia cells. Suppression of HMGCL specifically attenuates proliferation and tumor growth potential of human melanoma cells expressing BRAF V600E. Mechanistically, active BRAF upregulates HMGCL through an octamer transcription factor Oct-1, leading to increased intracellular levels of HMGCL product, acetoacetate, which selectively enhances binding of BRAF V600E but not BRAF wild-type to MEK1 in V600E-positive cancer cells to promote activation of MEK-ERK signaling. These findings reveal a mutation-specific mechanism by which oncogenic BRAF V600E "rewires" metabolic and cell signaling networks and signals through the Oct-1-HMGCL-acetoacetate axis to selectively promote BRAF V600E-dependent tumor development.

Differential regulation of TNFα and IL-6 expression contributes to immune evasion in prostate cancer.

BACKGROUND: The role of the inflammatory milieu in prostate cancer progression is not well understood. Differences in inflammatory signaling between localized and metastatic disease may point to opportunities for early intervention. METHODS: We modeled PCa disease progression by analyzing RNA-seq of localized vs. metastatic patient samples, followed by CIBERSORTx to assess their immune cell populations. The VHA CDW registry of PCa patients was analyzed for anti-TNF clinical outcomes. RESULTS: We observed statistically significant opposing patterns of IL-6 and TNFα expression between localized and metastatic disease. IL-6 was robustly expressed in localized disease and downregulated in metastatic disease. The reverse was observed with TNFα expression. Metastatic disease was also characterized by downregulation of adhesion molecule E-selectin, matrix metalloproteinase ADAMTS-4 and a shift to M2 macrophages whereas localized disease demonstrated a preponderance of M1 macrophages. Treatment with anti-TNF agents was associated with earlier stage disease at diagnosis. CONCLUSIONS: Our data points to clearly different inflammatory contexts between localized and metastatic prostate cancer. Primary localized disease demonstrates local inflammation and adaptive immunity, whereas metastases are characterized by immune cold microenvironments and a shift towards resolution of inflammation and tissue repair. Therapies that interfere with these inflammatory networks may offer opportunities for early intervention in monotherapy or in combination with immunotherapies and anti-angiogenic approaches.

Human induced pluripotent stem cell-derived macrophages ameliorate liver fibrosis.

With an increasing number of patients with degenerative hepatic diseases, such as liver fibrosis, and a limited supply of donor organs, there is an unmet need for therapies that can repair or regenerate damaged liver tissue. Treatment with macrophages that are capable of phagocytosis and anti-inflammatory activities such as secretion of matrix metalloproteinases (MMPs) provide an attractive cellular therapy approach. Human induced pluripotent stem cells (iPSCs) are capable of efficiently generating a large-scale, homogenous population of human macrophages using fully defined feeder- and serum-free differentiation protocol. Human iPSC-macrophages exhibit classical surface cell markers and phagocytic activity similar to peripheral blood-derived macrophages. Moreover, gene and cytokine expression analysis reveal that these macrophages can be efficiently polarized to pro-inflammatory M1 or anti-inflammatory M2 phenotypes in presence of LPS + IFN-γ and IL-4 + IL-13, respectively. M1 macrophages express high level of CD80, TNF-α, and IL-6 while M2 macrophages show elevated expression of CD206, CCL17, and CCL22. Here, we demonstrate that treatment of liver fibrosis with both human iPSC-derived macrophage populations and especially M2 subtype significantly reduces fibrogenic gene expression and disease associated histological markers including Sirius Red, αSMA and desmin in immunodeficient Rag2-/- γc-/- mice model, making this approach a promising cell-based avenue to ameliorate fibrosis.

Tissue- and development-stage-specific mRNA and heterogeneous CNV signatures of human ribosomal proteins in normal and cancer samples.

We give results from a detailed analysis of human Ribosomal Protein (RP) levels in normal and cancer samples and cell lines from large mRNA, copy number variation and ribosome profiling datasets. After normalizing total RP mRNA levels per sample, we find highly consistent tissue specific RP mRNA signatures in normal and tumor samples. Multiple RP mRNA-subtypes exist in several cancers, with significant survival and genomic differences. Some RP mRNA variations among subtypes correlate with copy number loss of RP genes. In kidney cancer, RP subtypes map to molecular subtypes related to cell-of-origin. Pan-cancer analysis of TCGA data showed widespread single/double copy loss of RP genes, without significantly affecting survival. In several cancer cell lines, CRISPR-Cas9 knockout of RP genes did not affect cell viability. Matched RP ribosome profiling and mRNA data in humans and rodents stratified by tissue and development stage and were strongly correlated, showing that RP translation rates were proportional to mRNA levels. In a small dataset of human adult and fetal tissues, RP protein levels showed development stage and tissue specific heterogeneity of RP levels. Our results suggest that heterogeneous RP levels play a significant functional role in cellular physiology, in both normal and disease states.

A Curated Resource for Phosphosite-specific Signature Analysis.

Signaling pathways are orchestrated by post-translational modifications (PTMs) such as phosphorylation. However, pathway analysis of PTM data sets generated by mass spectrometry (MS)-based proteomics is typically performed at a gene-centric level because of the lack of appropriately curated PTM signature databases and bioinformatic tools that leverage PTM site-specific information. Here we present the first version of PTMsigDB, a database of modification site-specific signatures of perturbations, kinase activities and signaling pathways curated from more than 2,500 publications. We adapted the widely used single sample Gene Set Enrichment Analysis approach to utilize PTMsigDB, enabling PTMSignature Enrichment Analysis (PTM-SEA) of quantitative MS data. We used a well-characterized data set of epidermal growth factor (EGF)-perturbed cancer cells to evaluate our approach and demonstrated better representation of signaling events compared with gene-centric methods. We then applied PTM-SEA to analyze the phosphoproteomes of cancer cells treated with cell-cycle inhibitors and detected mechanism-of-action specific signatures of cell cycle kinases. We also applied our methods to analyze the phosphoproteomes of PI3K-inhibited human breast cancer cells and detected signatures of compounds inhibiting PI3K as well as targets downstream of PI3K (AKT, MAPK/ERK) covering a substantial fraction of the PI3K pathway. PTMsigDB and PTM-SEA can be freely accessed at https://github.com/broadinstitute/ssGSEA2.0.

Illuminating the Onco-GPCRome: Novel G protein-coupled receptor-driven oncocrine networks and targets for cancer immunotherapy.

G protein-coupled receptors (GPCRs) are the largest gene family of cell membrane-associated molecules mediating signal transmission, and their involvement in key physiological functions is well-established. The ability of GPCRs to regulate a vast array of fundamental biological processes, such as cardiovascular functions, immune responses, hormone and enzyme release from endocrine and exocrine glands, neurotransmission, and sensory perception (e.g. vision, odor, and taste), is largely due to the diversity of these receptors and the layers of their downstream signaling circuits. Dysregulated expression and aberrant functions of GPCRs have been linked to some of the most prevalent human diseases, which renders GPCRs one of the top targets for pharmaceutical drug development. However, the study of the role of GPCRs in tumor biology has only just begun to make headway. Recent studies have shown that GPCRs can contribute to the many facets of tumorigenesis, including proliferation, survival, angiogenesis, invasion, metastasis, therapy resistance, and immune evasion. Indeed, GPCRs are widely dysregulated in cancer and yet are underexploited in oncology. We present here a comprehensive analysis of GPCR gene expression, copy number variation, and mutational signatures in 33 cancer types. We also highlight the emerging role of GPCRs as part of oncocrine networks promoting tumor growth, dissemination, and immune evasion, and we stress the potential benefits of targeting GPCRs and their signaling circuits in the new era of precision medicine and cancer immunotherapies.

Modelling bistable tumour population dynamics to design effective treatment strategies.

Despite recent advances in targeted drugs and immunotherapy, cancer remains "the emperor of all maladies" due to almost inevitable emergence of resistance. Drug resistance is thought to be driven by genetic alterations and/or dynamic plasticity that deregulate pathway activities and regulatory programs of a highly heterogeneous tumour. In this study, we propose a modelling framework to simulate population dynamics of heterogeneous tumour cells with reversible drug resistance. Drug sensitivity of a tumour cell is determined by its internal states, which are demarcated by coordinated activities of multiple interconnected oncogenic pathways. Transitions between cellular states depend on the effects of targeted drugs and regulatory relations between the pathways. Under this framework, we build a simple model to capture drug resistance characteristics of BRAF-mutant melanoma, where two cell states are determined by two mutually inhibitory - main and alternative - pathways. We assume that cells with an activated main pathway are proliferative yet sensitive to the BRAF inhibitor, and cells with an activated alternative pathway are quiescent but resistant to the drug. We describe a dynamical process of tumour growth under various drug regimens using the explicit solutions of mean-field equations. Based on these solutions, we compare efficacy of three treatment strategies from simulated data: static treatments with continuous and constant dosages, periodic treatments with regular intermittent active phases and drug holidays, and treatments derived from optimal control theory (OCT). Periodic treatments outperform static treatments with a considerable margin, while treatments based on OCT outperform the best periodic treatment. Our results provide insights regarding optimal cancer treatment modalities for heterogeneous tumours, and may guide the development of optimal therapeutic strategies to circumvent plastic drug resistance. They can also be used to evaluate the efficacy of suboptimal treatments that may account for side effects of the treatment and the cost of its application.

Medulloblastoma exome sequencing uncovers subtype-specific somatic mutations.

Medulloblastomas are the most common malignant brain tumours in children. Identifying and understanding the genetic events that drive these tumours is critical for the development of more effective diagnostic, prognostic and therapeutic strategies. Recently, our group and others described distinct molecular subtypes of medulloblastoma on the basis of transcriptional and copy number profiles. Here we use whole-exome hybrid capture and deep sequencing to identify somatic mutations across the coding regions of 92 primary medulloblastoma/normal pairs. Overall, medulloblastomas have low mutation rates consistent with other paediatric tumours, with a median of 0.35 non-silent mutations per megabase. We identified twelve genes mutated at statistically significant frequencies, including previously known mutated genes in medulloblastoma such as CTNNB1, PTCH1, MLL2, SMARCA4 and TP53. Recurrent somatic mutations were newly identified in an RNA helicase gene, DDX3X, often concurrent with CTNNB1 mutations, and in the nuclear co-repressor (N-CoR) complex genes GPS2, BCOR and LDB1. We show that mutant DDX3X potentiates transactivation of a TCF promoter and enhances cell viability in combination with mutant, but not wild-type, ß-catenin. Together, our study reveals the alteration of WNT, hedgehog, histone methyltransferase and now N-CoR pathways across medulloblastomas and within specific subtypes of this disease, and nominates the RNA helicase DDX3X as a component of pathogenic ß-catenin signalling in medulloblastoma.

ATARiS: computational quantification of gene suppression phenotypes from multisample RNAi screens.

Genome-scale RNAi libraries enable the systematic interrogation of gene function. However, the interpretation of RNAi screens is complicated by the observation that RNAi reagents designed to suppress the mRNA transcripts of the same gene often produce a spectrum of phenotypic outcomes due to differential on-target gene suppression or perturbation of off-target transcripts. Here we present a computational method, Analytic Technique for Assessment of RNAi by Similarity (ATARiS), that takes advantage of patterns in RNAi data across multiple samples in order to enrich for RNAi reagents whose phenotypic effects relate to suppression of their intended targets. By summarizing only such reagent effects for each gene, ATARiS produces quantitative, gene-level phenotype values, which provide an intuitive measure of the effect of gene suppression in each sample. This method is robust for data sets that contain as few as 10 samples and can be used to analyze screens of any number of targeted genes. We used this analytic approach to interrogate RNAi data derived from screening more than 100 human cancer cell lines and identified HNF1B as a transforming oncogene required for the survival of cancer cells that harbor HNF1B amplifications. ATARiS is publicly available at http://broadinstitute.org/ataris.

Gene signatures related to B-cell proliferation predict influenza vaccine-induced antibody response.

Vaccines are very effective at preventing infectious disease but not all recipients mount a protective immune response to vaccination. Recently, gene expression profiles of PBMC samples in vaccinated individuals have been used to predict the development of protective immunity. However, the magnitude of change in gene expression that separates vaccine responders and nonresponders is likely to be small and distributed across networks of genes, making the selection of predictive and biologically relevant genes difficult. Here we apply a new approach to predicting vaccine response based on coordinated upregulation of sets of biologically informative genes in postvaccination gene expression profiles. We found that enrichment of gene sets related to proliferation and immunoglobulin genes accurately segregated high responders to influenza vaccination from low responders and achieved a prediction accuracy of 88% in an independent clinical trial. Many of the genes in these gene sets would not have been identified using conventional, single-gene level approaches because of their subtle upregulation in vaccine responders. Our results demonstrate that gene set enrichment method can capture subtle transcriptional changes and may be a generally useful approach for developing and interpreting predictive models of the human immune response.

Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1.

The proto-oncogene KRAS is mutated in a wide array of human cancers, most of which are aggressive and respond poorly to standard therapies. Although the identification of specific oncogenes has led to the development of clinically effective, molecularly targeted therapies in some cases, KRAS has remained refractory to this approach. A complementary strategy for targeting KRAS is to identify gene products that, when inhibited, result in cell death only in the presence of an oncogenic allele. Here we have used systematic RNA interference to detect synthetic lethal partners of oncogenic KRAS and found that the non-canonical IkappaB kinase TBK1 was selectively essential in cells that contain mutant KRAS. Suppression of TBK1 induced apoptosis specifically in human cancer cell lines that depend on oncogenic KRAS expression. In these cells, TBK1 activated NF-kappaB anti-apoptotic signals involving c-Rel and BCL-XL (also known as BCL2L1) that were essential for survival, providing mechanistic insights into this synthetic lethal interaction. These observations indicate that TBK1 and NF-kappaB signalling are essential in KRAS mutant tumours, and establish a general approach for the rational identification of co-dependent pathways in cancer.

Twist1 suppresses senescence programs and thereby accelerates and maintains mutant Kras-induced lung tumorigenesis.

KRAS mutant lung cancers are generally refractory to chemotherapy as well targeted agents. To date, the identification of drugs to therapeutically inhibit K-RAS have been unsuccessful, suggesting that other approaches are required. We demonstrate in both a novel transgenic mutant Kras lung cancer mouse model and in human lung tumors that the inhibition of Twist1 restores a senescence program inducing the loss of a neoplastic phenotype. The Twist1 gene encodes for a transcription factor that is essential during embryogenesis. Twist1 has been suggested to play an important role during tumor progression. However, there is no in vivo evidence that Twist1 plays a role in autochthonous tumorigenesis. Through two novel transgenic mouse models, we show that Twist1 cooperates with Kras(G12D) to markedly accelerate lung tumorigenesis by abrogating cellular senescence programs and promoting the progression from benign adenomas to adenocarcinomas. Moreover, the suppression of Twist1 to physiological levels is sufficient to cause Kras mutant lung tumors to undergo senescence and lose their neoplastic features. Finally, we analyzed more than 500 human tumors to demonstrate that TWIST1 is frequently overexpressed in primary human lung tumors. The suppression of TWIST1 in human lung cancer cells also induced cellular senescence. Hence, TWIST1 is a critical regulator of cellular senescence programs, and the suppression of TWIST1 in human tumors may be an effective example of pro-senescence therapy.

Identification of RPS14 as a 5q- syndrome gene by RNA interference screen.

Somatic chromosomal deletions in cancer are thought to indicate the location of tumour suppressor genes, by which a complete loss of gene function occurs through biallelic deletion, point mutation or epigenetic silencing, thus fulfilling Knudson's two-hit hypothesis. In many recurrent deletions, however, such biallelic inactivation has not been found. One prominent example is the 5q- syndrome, a subtype of myelodysplastic syndrome characterized by a defect in erythroid differentiation. Here we describe an RNA-mediated interference (RNAi)-based approach to discovery of the 5q- disease gene. We found that partial loss of function of the ribosomal subunit protein RPS14 phenocopies the disease in normal haematopoietic progenitor cells, and also that forced expression of RPS14 rescues the disease phenotype in patient-derived bone marrow cells. In addition, we identified a block in the processing of pre-ribosomal RNA in RPS14-deficient cells that is functionally equivalent to the defect in Diamond-Blackfan anaemia, linking the molecular pathophysiology of the 5q- syndrome to a congenital syndrome causing bone marrow failure. These results indicate that the 5q- syndrome is caused by a defect in ribosomal protein function and suggest that RNAi screening is an effective strategy for identifying causal haploinsufficiency disease genes.

CDK8 is a colorectal cancer oncogene that regulates beta-catenin activity.

Aberrant activation of the canonical WNT/beta-catenin pathway occurs in almost all colorectal cancers and contributes to their growth, invasion and survival. Although dysregulated beta-catenin activity drives colon tumorigenesis, further genetic perturbations are required to elaborate full malignant transformation. To identify genes that both modulate beta-catenin activity and are essential for colon cancer cell proliferation, we conducted two loss-of-function screens in human colon cancer cells and compared genes identified in these screens with an analysis of copy number alterations in colon cancer specimens. One of these genes, CDK8, which encodes a member of the mediator complex, is located at 13q12.13, a region of recurrent copy number gain in a substantial fraction of colon cancers. Here we show that the suppression of CDK8 expression inhibits proliferation in colon cancer cells characterized by high levels of CDK8 and beta-catenin hyperactivity. CDK8 kinase activity was necessary for beta-catenin-driven transformation and for expression of several beta-catenin transcriptional targets. Together these observations suggest that therapeutic interventions targeting CDK8 may confer a clinical benefit in beta-catenin-driven malignancies.