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A High-Throughput siRNA Screening Platform to Identify MYC-Synthetic Lethal Genes as Candidate Therapeutic Targets

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The Myc Gene

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1012))

Abstract

Targeted therapeutics toward specific genes and pathways represent the future of oncological treatments. However, several commonly activated oncogenes, such as MYC, have proven difficult to target by pharmacological agents. To broaden the menu of potentially druggable therapeutic targets, we describe a method to detect genes essential for the survival of MYC overexpressing cells, which we will refer to as MYC-synthetic lethal genes (MYC-SL) (Toyoshima et al., Proc Natl Acad Sci USA 109:9545–9550, 2012). These genes represent candidate targets for drug development to be utilized for MYC-driven cancers as well as probes to further our understanding of the biology of MYC-driven tumorigenesis. The discovery platform includes the following components: (1) an isogenic cell system that enables overexpression of MYC without oncogene-induced senescence (OIS) response (Benanti and Galloway, Mol Cell Biol 24:2842–2852, 2004; Benanti et al., Mol Cancer Res 5:1181–1189, 2007); (2) arrayed siRNA libraries targeting individual genes; (3) automated laboratory equipment for dispensing of cells, siRNAs, and readout assays; and (4) bioinformatics and software for data mining and visualization. This flexible platform can be readily applied to other oncogenes or tumor suppressor genes.

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References

  1. Soucek L, Whitfield J, Martins CP, Finch AJ, Murphy DJ, Sodir NM, Karnezis AN, Swigart LB, Nasi S, Evan GI (2008) Modelling Myc inhibition as a cancer therapy. Nature 455:679–683

    Article  PubMed  CAS  Google Scholar 

  2. Felsher DW, Bishop JM (1999) Reversible tumorigenesis by MYC in hematopoietic lineages. Mol Cell 4:199–207

    Article  PubMed  CAS  Google Scholar 

  3. Jain M, Arvanitis C, Chu K, Dewey W, Leonhardt E, Trinh M, Sundberg CD, Bishop JM, Felsher DW (2002) Sustained loss of a neoplastic phenotype by brief inactivation of MYC. Science 297:102–104

    Article  PubMed  CAS  Google Scholar 

  4. Evan GI, Wyllie AH, Gilbert CS, Littlewood TD, Land H, Brooks M, Waters CM, Penn LZ, Hancock DC (1992) Induction of apoptosis in fibroblasts by c-myc protein. Cell 69:119–128

    Article  PubMed  CAS  Google Scholar 

  5. Juin P, Hueber AO, Littlewood T, Evan G (1999) c-Myc-induced sensitization to apoptosis is mediated through cytochrome c release. Genes Dev 13:1367–1381

    Article  PubMed  CAS  Google Scholar 

  6. Morrish F, Isern N, Sadilek M, Jeffrey M, Hockenbery DM (2009) c-Myc activates multiple metabolic networks to generate substrates for cell-cycle entry. Oncogene 28:2485–2491

    Article  PubMed  CAS  Google Scholar 

  7. Dang CV (2011) Therapeutic targeting of Myc-reprogrammed cancer cell metabolism. Cold Spring Harb Symp Quant Biol 76:369–374

    Article  PubMed  CAS  Google Scholar 

  8. Barna M, Pusic A, Zollo O, Costa M, Kondrashov N, Rego E, Rao PH, Ruggero D (2008) Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency. Nature 456:971–975

    Article  PubMed  CAS  Google Scholar 

  9. Mai S, Fluri M, Siwarski D, Huppi K (1996) Genomic instability in MycER-activated Rat1A-MycER cells. Chromosome Res 4:365–371

    Article  PubMed  CAS  Google Scholar 

  10. Felsher DW, Zetterberg A, Zhu J, Tlsty T, Bishop JM (2000) Overexpression of MYC causes p53-dependent G2 arrest of normal fibroblasts. Proc Natl Acad Sci USA 97:10544–10548

    Article  PubMed  CAS  Google Scholar 

  11. Wade M, Wahl GM (2006) c-Myc, genome instability, and tumorigenesis: the devil is in the details. Curr Top Microbiol Immunol 302:169–203

    Article  PubMed  CAS  Google Scholar 

  12. Goga A, Yang D, Tward AD, Morgan DO, Bishop JM (2007) Inhibition of CDK1 as a potential therapy for tumors over-expressing MYC. Nat Med 13:820–827

    Article  PubMed  CAS  Google Scholar 

  13. Dominguez-Sola D, Ying CY, Grandori C, Ruggiero L, Chen B, Li M, Galloway DA, Gu W, Gautier J, Dalla-Favera R (2007) Non-transcriptional control of DNA replication by c-Myc. Nature 448:445–451

    Article  PubMed  CAS  Google Scholar 

  14. Robinson K, Asawachaicharn N, Galloway DA, Grandori C (2009) c-Myc accelerates S-phase and requires WRN to avoid replication stress. PLoS One 4:e5951

    Article  PubMed  Google Scholar 

  15. Yang D, Liu H, Goga A, Kim S, Yuneva M, Bishop JM (2010) Therapeutic potential of a synthetic lethal interaction between the MYC proto-oncogene and inhibition of aurora-B kinase. Proc Natl Acad Sci USA 107:13836–13841

    Article  PubMed  CAS  Google Scholar 

  16. Moser R, Toyoshima M, Robinson K, Gurley KE, Howie HL, Davison J, Morgan M, Kemp CJ, Grandori C (2012) MYC-driven tumorigenesis is inhibited by WRN syndrome gene deficiency. Mol Cancer Res 10:535–545

    Article  PubMed  CAS  Google Scholar 

  17. Hartwell LH, Szankasi P, Roberts CJ, Murray AW, Friend SH (1997) Integrating genetic approaches into the discovery of anticancer drugs. Science 278:1064–1068

    Article  PubMed  CAS  Google Scholar 

  18. Kaelin WG Jr (2005) The concept of synthetic lethality in the context of anticancer therapy. Nat Rev Cancer 5:689–698

    Article  PubMed  CAS  Google Scholar 

  19. Paddison PJ, Silva JM, Conklin DS, Schlabach M, Li M, Aruleba S, Balija V, O’Shaughnessy A, Gnoj L, Scobie K, Chang K, Westbrook T, Cleary M, Sachidanandam R, McCombie WR, Elledge SJ, Hannon GJ (2004) A resource for large-scale RNA-interference-based screens in mammals. Nature 428:427–431

    Article  PubMed  CAS  Google Scholar 

  20. Hu G, Kim J, Xu Q, Leng Y, Orkin SH, Elledge SJ (2009) A genome-wide RNAi screen identifies a new transcriptional module required for self-renewal. Genes Dev 23:837–848

    Article  PubMed  CAS  Google Scholar 

  21. Blakely K, Ketela T, Moffat J (2011) Pooled lentiviral shRNA screening for functional genomics in mammalian cells. Methods Mol Biol 781:161–182

    Article  PubMed  CAS  Google Scholar 

  22. Toyoshima M, Howie HL, Imakura M, Walsh RM, Annis JE, Chang AN, Frazier J, Chau BN, Loboda A, Linsley PS, Cleary MA, Park JR, Grandori C (2012) Functional genomics identifies therapeutic targets for MYC-driven cancer. Proc Natl Acad Sci USA 109:9545–9550

    Article  PubMed  CAS  Google Scholar 

  23. Kessler JD, Kahle KT, Sun T, Meerbrey KL, Schlabach MR, Schmitt EM, Skinner SO, Xu Q, Li MZ, Hartman ZC, Rao M, Yu P, Dominguez-Vidana R, Liang AC, Solimini NL, Bernardi RJ, Yu B, Hsu T, Golding I, Luo J, Osborne CK, Creighton CJ, Hilsenbeck SG, Schiff R, Shaw CA, Elledge SJ, Westbrook TF (2012) A SUMOylation-dependent transcriptional subprogram is required for Myc-driven tumorigenesis. Science 335:348–353

    Article  PubMed  CAS  Google Scholar 

  24. Liu L, Ulbrich J, Muller J, Wustefeld T, Aeberhard L, Kress TR, Muthalagu N, Rycak L, Rudalska R, Moll R, Kempa S, Zender L, Eilers M, Murphy DJ (2012) Deregulated MYC expression induces dependence upon AMPK-related kinase 5. Nature 483:608–612

    Article  PubMed  CAS  Google Scholar 

  25. Benanti JA, Galloway DA (2004) Normal human fibroblasts are resistant to RAS-induced senescence. Mol Cell Biol 24:2842–2852

    Article  PubMed  CAS  Google Scholar 

  26. Benanti JA, Wang ML, Myers HE, Robinson KL, Grandori C, Galloway DA (2007) Epigenetic down-regulation of ARF expression is a selection step in immortalization of human fibroblasts by c-Myc. Mol Cancer Res 5:1181–1189

    Article  PubMed  CAS  Google Scholar 

  27. Wang ML, Walsh R, Robinson KL, Burchard J, Bartz SR, Cleary M, Galloway DA, Grandori C (2011) Gene expression signature of c-MYC-immortalized human fibroblasts reveals loss of growth inhibitory response to TGFbeta. Cell Cycle 10:2540–2548

    Article  PubMed  CAS  Google Scholar 

  28. Cerami EG, Gross BE, Demir E, Rodchenkov I, Babur O, Anwar N, Schultz N, Bader GD, Sander C (2011) Pathway commons, a web resource for biological pathway data. Nucleic Acids Res 39:D685–690

    Article  PubMed  CAS  Google Scholar 

  29. Zhang JH, Chung TD, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4:67–73

    Article  PubMed  Google Scholar 

  30. Birmingham A, Selfors LM, Forster T, Wrobel D, Kennedy CJ, Shanks E, Santoyo-Lopez J, Dunican DJ, Long A, Kelleher D, Smith Q, Beijersbergen RL, Ghazal P, Shamu CE (2009) Statistical methods for analysis of high-throughput RNA interference screens. Nat Methods 6:569–575

    Article  PubMed  CAS  Google Scholar 

  31. Chung N, Zhang XD, Kreamer A, Locco L, Kuan PF, Bartz S, Linsley PS, Ferrer M, Strulovici B (2008) Median absolute deviation to improve hit selection for genome-scale RNAi screens. J Biomol Screen 13:149–158

    Article  PubMed  CAS  Google Scholar 

  32. Jackson AL, Linsley PS (2010) Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application. Nat Rev Drug Discov 9:57–67

    Article  PubMed  CAS  Google Scholar 

  33. Chau BN, Diaz RL, Saunders MA, Cheng C, Chang AN, Warrener P, Bradshaw J, Linsley PS, Cleary MA (2009) Identification of SULF2 as a novel transcriptional target of p53 by use of integrated genomic analyses. Cancer Res 69:1368–1374

    Article  PubMed  CAS  Google Scholar 

  34. Meerbrey KL, Hu G, Kessler JD, Roarty K, Li MZ, Fang JE, Herschkowitz JI, Burrows AE, Ciccia A, Sun T, Schmitt EM, Bernardi RJ, Fu X, Bland CS, Cooper TA, Schiff R, Rosen JM, Westbrook TF, Elledge SJ (2011) The pINDUCER lentiviral toolkit for inducible RNA interference in vitro and in vivo. Proc Natl Acad Sci USA 108:3665–3670

    Article  PubMed  CAS  Google Scholar 

  35. Zuber J, McJunkin K, Fellmann C, Dow LE, Taylor MJ, Hannon GJ, Lowe SW (2010) Toolkit for evaluating genes required for proliferation and survival using tetracycline-regulated RNAi. Nat Biotechnol 29:79–83

    Article  PubMed  Google Scholar 

  36. Scuoppo C, Miething C, Lindqvist L, Reyes J, Ruse C, Appelmann I, Yoon S, Krasnitz A, Teruya-Feldstein J, Pappin D, Pelletier J, Lowe SW (2012) A tumour suppressor network relying on the polyamine-hypusine axis. Nature 487:244–248

    Article  PubMed  CAS  Google Scholar 

  37. Schuhmacher M, Staege MS, Pajic A, Polack A, Weidle UH, Bornkamm GW, Eick D, Kohlhuber F (1999) Control of cell growth by c-Myc in the absence of cell division. Curr Biol 9:1255–1258

    Article  PubMed  CAS  Google Scholar 

  38. Lutz W, Stohr M, Schurmann J, Wenzel A, Lohr A, Schwab M (1996) Conditional expression of N-myc in human neuroblastoma cells increases expression of alpha-prothymosin and ornithine decarboxylase and accelerates progression into S-phase early after mitogenic stimulation of quiescent cells. Oncogene 13:803–812

    PubMed  CAS  Google Scholar 

  39. Mateyak MK, Obaya AJ, Adachi S, Sedivy JM (1997) Phenotypes of c-Myc-deficient rat fibroblasts isolated by targeted homologous recombination. Cell Growth Differ 8:1039–1048

    PubMed  CAS  Google Scholar 

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Acknowledgments

I am indebted to Maki Imakura and James Annis for sharing their expertise in high-throughput screening. I also thank Hamid Bolouri, Daniel Diolait-i, and Christopher Kemp for critical reading of this chapter and Daniel Diolait-i for figure design, Aaron Chang and Hamid Bolouri for discussion of bioinformatics tools, and Kristin Robinson in Denise Galloway’s laboratory for isolating HFFs cultures.

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Authors and Affiliations

  1. Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

    Carla Grandori

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  1. Carla Grandori
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Editors and Affiliations

  1. Fundació Vall d'Hebron Institute of Onco, Barcelona, Spain

    Laura Soucek

  2. Department of Biochemistry Sanger Building, University of Cambridge, Cambridge, United Kingdom

    Nicole M. Sodir

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Grandori, C. (2013). A High-Throughput siRNA Screening Platform to Identify MYC-Synthetic Lethal Genes as Candidate Therapeutic Targets. In: Soucek, L., Sodir, N. (eds) The Myc Gene. Methods in Molecular Biology, vol 1012. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-429-6_12

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  • DOI: https://doi.org/10.1007/978-1-62703-429-6_12

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-428-9

  • Online ISBN: 978-1-62703-429-6

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