A Genome-Wide Rnai Screen Identifies Multiple Synthetic Lethal

A Genome-wide RNAi Screen Identifies Multiple Synthetic Lethal Interactions with the Ras Oncogene Ji Luo,1 Michael J. Emanuele,1 Danan Li,2 Chad J. Creighton,3 Michael R. Schlabach,1 Thomas F. Westbrook,4 Kwok-Kin Wong,2 and Stephen J. Elledge1,* 1Howard Hughes Medical Institute and Department of Genetics, Harvard Medical School, Division of Genetics, Brigham and Women’s Hospital 2Department of Medicine, Harvard Medical School and Department of Medical Oncology, Dana Farber Cancer Center, Ludwig Center at Dana-Farber/Harvard Cancer Center Boston, MA 02115, USA 3Dan L. Duncan Cancer Center Division of Biostatistics 4Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Department of Molecular and Human Genetics, Dan L. Duncan Cancer Center Baylor College of Medicine, Houston, TX 77030, USA *Correspondence: [email protected] DOI 10.1016/j.cell.2009.05.006 SUMMARY (Ngo et al., 2006; Schlabach et al., 2008; Silva et al., 2008). We have developed barcoded, retroviral/lentiviral-based short Oncogenic mutations in the small GTPase Ras are hairpin RNA (shRNA) libraries targeting the entire human genome highly prevalent in cancer, but an understanding of to enable genome-wide loss-of-function analysis through stable the vulnerabilities of these cancers is lacking. We gene knockdown (Silva et al., 2005). Our design also allowed undertook a genome-wide RNAi screen to identify us to use microarray deconvolution to develop a multiplex synthetic lethal interactions with the KRAS onco- screening platform that enables the highly parallel screening gene. We discovered a diverse set of proteins whose of >10,000 shRNAs in a pool-based format (Schlabach et al., 2008; Silva et al., 2008). These technological breakthroughs depletion selectively impaired the viability of Ras have made it possible to rapidly interrogate the genome for func- mutant cells. Among these we observed a strong tional vulnerability of cancer cells and here we apply these to the enrichment for genes with mitotic functions. We Ras oncogene. describe a pathway involving the mitotic kinase The Ras family of small GTPases are frequently mutated in PLK1, the anaphase-promoting complex/cyclosome, human cancers (reviewed in Karnoub and Weinberg, 2008). Ras and the proteasome that, when inhibited, results in is a membrane-bound signaling molecule that cycles between prometaphase accumulation and the subsequent the inactive, GDP-bound state and the active, GTP-bound state. death of Ras mutant cells. Gene expression analysis Growth factor receptor signaling promotes GTP loading and indicates that reduced expression of genes in this activation of Ras, which in turn activates an array of downstream pathway correlates with increased survival of pathways to promote cell proliferation and survival. Among patients bearing tumors with a Ras transcriptional the major Ras effector pathways is the MAP kinase pathway, the PI3-kinase (PI3K) pathway, RalGDS proteins, phospholi- signature. Our results suggest a previously underap- pase-C3, and Rac. Each of these has been implicated in media- preciated role for Ras in mitotic progression and tion of Ras oncogenesis. Ras GAPs (GTPase-activating proteins) demonstrate a pharmacologically tractable pathway inactivate Ras by stimulating GTP hydrolysis. Oncogenic muta- for the potential treatment of cancers harboring Ras tions in Ras are invariably point mutations that either interfere mutations. with Ras GAP binding or directly disrupt Ras GTPase activity, locking Ras in a constitutively active, GTP-bound state. Onco- INTRODUCTION genic mutations have been found in all three members of the Ras gene family with KRAS being the most frequently mutated. A major challenge in cancer therapeutics is the identification of KRAS mutations are found at high frequencies in pancreatic, cellular drug targets whose inhibition leads to the selective killing thyroid, colon, lung, and liver cancers and in myelodyspastic of cancer cells while sparing normal cells. Recent advances in syndrome and are correlated with poor prognosis (Karnoub and mammalian RNA interference (RNAi) technologies have made it Weinberg, 2008). possible to systematically interrogate the human genome for Despite its prominent status as a cancer drug target, thera- genes whose loss of function constitutes synthetic lethality either peutics aimed at disrupting the Ras pathway have proven chal- with the oncogenic state or with particular oncogenic mutations lenging thus far. Inhibitors of farnesyl transferase, the enzyme Cell 137, 835–848, May 29, 2009 ª2009 Elsevier Inc. 835 that prenylates Ras for its membrane localization, have met with We screened the parental KRAS WT/G13D (Ras Mut) DLD-1 cells only limited success (Karnoub and Weinberg, 2008). Chemical and the isogenic KRAS WT/À (Ras WT) DLD-1 control cells with screens in isogenic Ras mutant and wild-type cell lines have our library of 74,905 retroviral shRNAs targeting 32,293 unique identified compounds that exhibit preferential toxicity toward human transcripts (including 19,542 RefSeq transcripts). The Ras mutant cells (Torrance et al., 2001; Dolma et al., 2003). library was screened in 6 pools of 13,000 shRNAs per pool However, the translation of these chemical screens into clinical using a protocol described previously (Figure 1A) (Schlabach practice has been impeded by the challenge in identifying the et al., 2008). We analyzed the change in relative abundance of protein targets of these chemical entities and subsequent drug each shRNA over time by microarray hybridization to identify development. Inhibitors targeting various Ras effecter pathways those that are antiproliferative and are thus depleted from the could also prove efficacious in treating tumors with Ras muta- population. We compared the lethality signature of the Ras tions, as it was recently shown that a combined application of Mut and WT cells to identify those shRNAs showing selective MEK and PI3K/mTOR inhibitors can reduce tumor burden in depletion in the Ras Mut but not Ras WT cells. Such shRNAs a mouse model of Ras-driven lung cancer (Engelman et al., are potential Ras synthetic lethal (RSL) candidates. Relaxed 2008). However, the prevalence of de novo and acquired drug statistical criteria identified 1741 RSL shRNAs targeting 1613 resistance to other targeted therapies suggests that combina- genes, whereas a more stringent cutoff identified a subset 379 tions of multiple therapeutic agents will be required to effectively RSL shRNAs targeting 368 genes (Tables S1 and S2 available inhibit malignant progression. online). In principle, tumors can be attacked either by reversing the We devised a multicolor competition assay (MCA) to test the effects of oncoproteins through inhibition (i.e., exploiting onco- reproducibility of candidate RSL shRNAs from the primary gene addiction) or by attacking tumor-specific vulnerabilities screen (Smogorzewska et al., 2007). Individual shRNAs were caused by the oncogenic state, often by inhibiting proteins that packaged into retroviruses and infected into a 50:50 mixture of are not oncoproteins themselves (i.e., exploiting nononcogene GFP+ DLD-1 Ras Mut cells and GFPÀ (colorless) DLD-1 Ras addiction) (Solimini et al., 2007; Luo et al., 2009). The inappro- WT cells. The relative ratio of Ras Mut versus WT cells at priate rewiring of cellular signaling through oncogene activation 7 days post-shRNA infection was analyzed by FACS and should result in vulnerabilities that could be exploited for cancer compared to that of the same cells infected with a negative therapies in theory. Since these vulnerabilities are not obvious control shRNA targeting luciferase (Figure 1E). Using this assay and cannot be predicted, the most direct approach to their we tested 320 candidate RSL shRNAs from the primary screen discovery is through genetic exploration. The systematic identi- and found 83 shRNA (26%) targeting 77 genes to preferentially fication of genes and pathways necessary for the Ras-driven decrease the fitness of Ras Mut cells compared to Ras WT cells oncogenic state would provide additional drug targets for thera- (Table S3). To rule out cell-line-specific effects, we performed peutic exploration, shed new light on Ras’ mechanisms of MCAs in a second isogenic pair of colorectal cancer cell lines: action, and potentially provide new biomarkers for patient strat- HCT116 KRAS WT/G13D and HCT116 KRAS WT/À, which were ification. To this end, we screened our shRNA library for genes derived in the same manner as the DLD-1 isogenic pair (Shira- whose inhibition constitutes synthetic lethality with the KRAS sawa et al., 1993; Torrance et al., 2001). Many shRNAs that oncogene. We identified a functionally diverse set of genes scored in the DLD-1 cells also showed synthetic lethality in the including a number of mitotic proteins and demonstrate that HCT116 cells (50 of 68 tested, 73.5%, Table S3), indicating pharmacological inhibitors targeting these mitotic proteins can that the majority of candidate RSL shRNAs are likely to interact selectively impair the viability of Ras mutant cells. These findings genetically with KRAS. point to a previously underappreciated role of Ras in mitotic progression and demonstrate that mitotic stress induced by Functional Diversity of Candidate RSL Genes the Ras oncogene might be exploited for therapeutic purposes. We recovered shRNAs against KRAS itself from the screen. In accordance with the phenotype of the Ras WT isogenic controls, shRNA-mediated knockdown of KRAS expression (both Mut RESULTS

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