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 allele1,2. Here we have used systematic RNA interference (RNAi) to detect synthetic lethal partners of oncogenic KRAS and found that the non-canonical IκB kinase, TBK1, was selectively essential in cells that harbor 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-κB anti-apoptotic signals involving cREL and BCL-XL that were essential for survival, providing mechanistic insights into this synthetic lethal interaction. These observations identify TBK1 and NF-κB signaling as essential in KRAS mutant tumors and establish a general approach for the rational identification of co-dependent pathways in cancer.

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.

Oncogenic mutations in the serine/threonine kinase B-RAF are found in 50–70% of malignant melanomas1. Pre-clinical studies have demonstrated that the B-RAFV600E mutation predicts a dependency on the mitogen activated protein kinase (MAPK) signaling cascade in melanoma1–5—an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials6–8. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance9–11. Identification of resistance mechanisms in a manner that elucidates alternative ‘druggable’ targets may inform effective long-term treatment strategies12. Here, we expressed ~600 kinase and kinase-related open reading frames (ORFs) in parallel to functionally interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (COT/TPL2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAFV600E cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signaling. Moreover, COT expression is associated with de novo resistance in B-RAFV600E cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibition. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.

SUMMARY Diffuse large B cell lymphoma (DLBCL) is the most common form of blood cancer and is characterized by a striking degree of genetic and clinical heterogeneity. This heterogeneity poses a major barrier to understanding the genetic basis of the disease and its response to therapy. Here, we performed an integrative analysis of whole exome sequencing and transcriptome sequencing in a cohort of 1001 DLBCL patients to comprehensively define the landscape of 150 genetic drivers of the disease. We characterized the functional impact of these genes using an unbiased CRISPR screen of DLBCL cell lines to define oncogenes that promote cell growth. A prognostic model comprising these genetic alterations outperformed current established methods: cell of origin, the International Prognostic Index comprising clinical variables, and dual MYC and BCL2 expression. These results comprehensively define the genetic drivers and their functional roles in DLBCL to identify new therapeutic opportunities in the disease.

Lineage survival oncogenes are activated by somatic DNA alterations in cancers arising from the cell lineages in which these genes play a role in normal development.1,2 Here we show that a peak of genomic amplification on chromosome 3q26.33, found in squamous cell carcinomas (SCCs) of the lung and esophagus, contains the transcription factor gene SOX2—which is mutated in hereditary human esophageal malformations3 and necessary for normal esophageal squamous development4, promotes differentiation and proliferation of basal tracheal cells5 and co-operates in induction of pluripotent stem cells.6,7,8 SOX2 expression is required for proliferation and anchorage-independent growth of lung and esophageal cell lines, as shown by RNA interference experiments. Furthermore, ectopic expression of SOX2 cooperated with FOXE1 or FGFR2 to transform immortalized tracheobronchial epithelial cells. SOX2-driven tumors show expression of markers of both squamous differentiation and pluripotency. These observations identify SOX2 as a novel lineage survival oncogene in lung and esophageal SCC.

Intratumor heterogeneity is a major clinical problem because tumor cell subtypes display variable sensitivity to therapeutics and may play different roles in progression. We previously characterized 2 cell populations in human breast tumors with distinct properties: CD44 + CD24 -cells that have stem cell-like characteristics, and CD44 -CD24 + cells that resemble more differentiated breast cancer cells. Here we identified 15 genes required for cell growth or proliferation in CD44 + CD24 -human breast cancer cells in a large-scale loss-of-function screen and found that inhibition of several of these (IL6, PTGIS, HAS1, CXCL3, and PFKFB3) reduced Stat3 activation. We found that the IL-6/JAK2/Stat3 pathway was preferentially active in CD44 + CD24 -breast cancer cells compared with other tumor cell types, and inhibition of JAK2 decreased their number and blocked growth of xenografts. Our results highlight the differences between distinct breast cancer cell types and identify targets such as JAK2 and Stat3 that may lead to more specific and effective breast cancer therapies.

We are performing a uniform and unbiased imaging survey of the Large Magellanic Cloud (LMC; $7 ; 7) using the IRAC (3.6, 4.5, 5.8, and 8 m) and MIPS (24, 70, and 160 m) instruments on board the Spitzer Space Telescope in the Surveying the Agents of a Galaxy's Evolution (SAGE) survey, these agents being the interstellar medium (ISM) and stars in the LMC. This paper provides an overview of the SAGE Legacy project, including observing strategy, data processing, and initial results. Three key science goals determined the coverage and depth of the survey. The detection of diffuse ISM with column densities >1:2 ; 10 21 H cm À2 permits detailed studies of dust processes in the ISM. SAGE's point-source sensitivity enables a complete census of newly formed stars with masses >3 M that will determine the current star formation rate in the LMC. SAGE's detection of evolved stars with mass-loss rates >1 ; 10 À8 M yr À1 will quantify the rate at which evolved stars inject mass into the ISM of the LMC. The observing strategy includes two epochs in 2005, separated by 3 months, that both mitigate instrumental artifacts and constrain source variability. The SAGE data are nonproprietary. The data processing includes IRAC and MIPS pipelines and a database for mining the point-source catalogs, which will be released to the community in support of Spitzer proposal cycles 4 and 5. We present initial results on the epoch 1 data for a region near N79 and N83. The MIPS 70 and 160 m images of the diffuse dust emission of the N79/N83 region reveal a similar distribution to the gas emissions, especially the H i 21 cm emission. The measured point-source sensitivity for the epoch 1 data is consistent with expectations for the survey. The point-source counts are highest for the IRAC 3.6 m band and decrease dramatically toward longer wavelengths, A

Aberrant activation of the canonical Wnt/β-catenin pathway occurs in almost all colorectal cancers and contributes to their growth, invasion and survival. Although dysregulated β-catenin activity drives colon tumorigenesis, additional genetic perturbations are required to elaborate fully malignant disease. To identify genes that both modulate β-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. Suppression of CDK8 expression inhibited proliferation in colon cancer cells characterized by high levels of CDK8 and β-catenin hyperactivity. CDK8 kinase activity was necessary for β-catenin driven transformation and expression of several β-catenin transcriptional targets. Together these observations suggest that therapeutic interventions targeting CDK8 may confer clinical benefit in β-catenin-driven malignancies.Correspondence and Requests for materials should be addressed to W.C.H. (Email: william_hahn@dfci.harvard.edu).. The Wnt/β-catenin pathway is implicated in over 90% of colon cancers and in a fraction of other human malignancies. Loss of the tumor suppressor APC or activating CTNNB1 (β-catenin) mutations results in constitutive activity of the β-catenin-T cell factor (TCF) transcriptional complex, which drives adenoma formation 1,2 . Although mutations in TP53 or K-RAS cooperate with dysregulated β-catenin signaling to program a fully malignant phenotype 3 , these mutations are found in less than half of β-catenin-driven colon cancers 4 . NIH Public AccessTo identify oncogenes that modulate β-catenin-dependent transcription and regulate colon cancer cell proliferation, we conducted two RNAi-based loss-of-function screens. We engineered DLD1 colon cancer cells, which harbor APC deletions and depend on β-catenin for proliferation 5 , to stably express "TOPFLASH" β-catenin-luciferase and "FOPFLASH" mutant-Renilla reporter constructs 6,7 (DLD1 Rep ). Suppression of β-catenin expression in DLD1 Rep cells by three β-catenin-specific short hairpin RNAs (shRNA) markedly reduced the TOPFLASH/FOPFLASH ratio (Fig. 1a), confirming that reporter activity requires β-catenin expression. We then screened DLD1 Rep cells with a shRNA library containing 4849 shRNAs that target 1000 genes, including 95% of the human kinome 6 . We found 34 genes whose expression was necessary for β-catenin activity, including two known β-catenin regulators, CSNK1G3 8 and CSNK1E 9 ( Fig. 1b and Supplementary Table 1).In parallel, we performed an arrayed, kinase-enriched shRNA screen in another β-catenindependent colon cancer cell line, HCT116, to identify genes essential for cancer cell proliferation. We identified 166 candidate genes necessary for proliferatio...