A mammalian functional-genetic approach to characterizing cancer therapeutics.

Identifying mechanisms of drug action remains a fundamental impediment to the development and effective use of chemotherapeutics. Here we describe an RNA interference (RNAi)-based strategy to characterize small-molecule function in mammalian cells. By examining the response of cells expressing short hairpin RNAs (shRNAs) to a diverse selection of chemotherapeutics, we could generate a functional shRNA signature that was able to accurately group drugs into established biochemical modes of action. This, in turn, provided a diversely sampled reference set for high-resolution prediction of mechanisms of action for poorly characterized small molecules. We could further reduce the predictive shRNA target set to as few as eight genes and, by using a newly derived probability-based nearest-neighbors approach, could extend the predictive power of this shRNA set to characterize additional drug categories. Thus, a focused shRNA phenotypic signature can provide a highly sensitive and tractable approach for characterizing new anticancer drugs.

[1]  M. N. Gupta,et al.  Affinity-based strategies for protein purification. , 2006, Analytical chemistry.

[2]  K. Cimprich,et al.  ATR: an essential regulator of genome integrity , 2008, Nature Reviews Molecular Cell Biology.

[3]  Mark J. Smyth,et al.  Analysis of the apoptotic and therapeutic activities of histone deacetylase inhibitors by using a mouse model of B cell lymphoma , 2007, Proceedings of the National Academy of Sciences.

[4]  A. Strasser,et al.  The BCL-2 protein family: opposing activities that mediate cell death , 2008, Nature Reviews Molecular Cell Biology.

[5]  J. C. Hinshaw,et al.  Discovering Modes of Action for Therapeutic Compounds Using a Genome-Wide Screen of Yeast Heterozygotes , 2004, Cell.

[6]  S. Lowe,et al.  Probing tumor phenotypes using stable and regulated synthetic microRNA precursors , 2005, Nature Genetics.

[7]  Inmar E. Givoni,et al.  Exploring the Mode-of-Action of Bioactive Compounds by Chemical-Genetic Profiling in Yeast , 2006, Cell.

[8]  D. Housman,et al.  p53 status and the efficacy of cancer therapy in vivo. , 1994, Science.

[9]  Grant W. Brown,et al.  Integration of chemical-genetic and genetic interaction data links bioactive compounds to cellular target pathways , 2004, Nature Biotechnology.

[10]  Yudong D. He,et al.  Functional Discovery via a Compendium of Expression Profiles , 2000, Cell.

[11]  Dirk Eick,et al.  TFIIH kinase places bivalent marks on the carboxy-terminal domain of RNA polymerase II. , 2009, Molecular cell.

[12]  L. Maquat,et al.  The mRNA surveillance protein hSMG-1 functions in genotoxic stress response pathways in mammalian cells. , 2004, Molecular cell.

[13]  Daphne Koller,et al.  Systematic analysis of genome-wide fitness data in yeast reveals novel gene function and drug action , 2010, Genome Biology.

[14]  Elizabeth A. Winzeler,et al.  Genomic profiling of drug sensitivities via induced haploinsufficiency , 1999, Nature Genetics.

[15]  M. Lavin.  Ataxia-telangiectasia: from a rare disorder to a paradigm for cell signalling and cancer , 2008, Nature Reviews Molecular Cell Biology.

[16]  M. Ljungman,et al.  The cyclin-dependent kinase inhibitor roscovitine inhibits RNA synthesis and triggers nuclear accumulation of p53 that is unmodified at Ser15 and Lys382. , 2001, Molecular pharmacology.

[17]  W. Richard McCombie,et al.  Topoisomerase levels determine chemotherapy response in vitro and in vivo , 2008, Proceedings of the National Academy of Sciences.

[18]  S. Lowe,et al.  INK4a/ARF mutations accelerate lymphomagenesis and promote chemoresistance by disabling p53. , 1999, Genes & development.

[19]  Qinguo Zheng,et al.  Role of Postreplicative DNA Mismatch Repair in the Cytotoxic Action of Thioguanine , 1996, Science.

[20]  Jiri Bartek,et al.  Chk1 and Chk2 kinases in checkpoint control and cancer. , 2003, Cancer cell.

[21]  Shin-ichi Sato,et al.  Biochemical target isolation for novices: affinity-based strategies. , 2010, Chemistry & biology.

[22]  D. Housman,et al.  p53-dependent apoptosis modulates the cytotoxicity of anticancer agents , 1993, Cell.

[23]  Paul A Clemons,et al.  The Connectivity Map: Using Gene-Expression Signatures to Connect Small Molecules, Genes, and Disease , 2006, Science.

[24]  R. Shoemaker.  The NCI60 human tumour cell line anticancer drug screen , 2006, Nature Reviews Cancer.

[25]  T. Humphrey,et al.  Integrating stress-response and cell-cycle checkpoint pathways. , 2001, Trends in cell biology.

[26]  R. Palmiter,et al.  The c-myc oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice , 1985, Nature.

[27]  M. Roussel,et al.  Arf gene loss enhances oncogenicity and limits imatinib response in mouse models of Bcr-Abl-induced acute lymphoblastic leukemia. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[28]  W. El-Deiry,et al.  Targeting p53 for enhanced radio- and chemo-sensitivity , 2009, Apoptosis.

[29]  Zigang Dong,et al.  Post-translational modification of p53 in tumorigenesis , 2004, Nature Reviews Cancer.

[30]  T. Golub,et al.  Gene expression signature-based chemical genomic prediction identifies a novel class of HSP90 pathway modulators. , 2006, Cancer cell.

[31]  Robert P. St.Onge,et al.  The Chemical Genomic Portrait of Yeast: Uncovering a Phenotype for All Genes , 2008, Science.

[32]  M. Lopes,et al.  Mismatch repair-dependent processing of methylation damage gives rise to persistent single-stranded gaps in newly replicated DNA. , 2007, Genes & development.

[33]  M. Yaffe,et al.  The combined status of ATM and p53 link tumor development with therapeutic response. , 2009, Genes & development.

[34]  D. Lane,et al.  Seliciclib (CYC202, R-Roscovitine) induces cell death in multiple myeloma cells by inhibition of RNA polymerase II-dependent transcription and down-regulation of Mcl-1. , 2005, Cancer research.

[35]  Michael I. Jordan,et al.  Chemogenomic profiling: identifying the functional interactions of small molecules in yeast. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[36]  J. Collins,et al.  Inferring Genetic Networks and Identifying Compound Mode of Action via Expression Profiling , 2003, Science.

[37]  Michael B Yaffe,et al.  p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage. , 2007, Cancer cell.

[38]  D. Lauffenburger,et al.  Three-kinase inhibitor combination recreates multipathway effects of a geldanamycin analogue on hepatocellular carcinoma cell death , 2009, Molecular Cancer Therapeutics.