The pursuit of novel therapeutic agents in cancer relies on the identification and validation of molecular targets. Hallmarks of cancer include self-sufficiency in growth signals and evasion from apoptosis1; genes that regulate these processes may be optimal for therapeutic attack. Here we describe a loss-of-function screen for genes required for the proliferation and survival of cancer cells using an RNA interference library. We used a doxycycline-inducible retroviral vector for the expression of small hairpin RNAs (shRNAs) to construct a library targeting 2,500 human genes. We used retroviral pools from this library to infect cell lines representing two distinct molecular subgroups of diffuse large B-cell lymphoma (DLBCL), termed activated B-cell-like DLBCL and germinal centre B-cell-like DLBCL. Each vector was engineered to contain a unique 60-base-pair ‘bar code’, allowing the abundance of an individual shRNA vector within a population of transduced cells to be measured using microarrays of the bar-code sequences. We observed that a subset of shRNA vectors was depleted from the transduced cells after three weeks in culture only if shRNA expression was induced. In activated B-cell-like DLBCL cells, but not germinal centre B-cell-like DLBCL cells, shRNAs targeting the NF-κB pathway were depleted, in keeping with the essential role of this pathway in the survival of activated B-cell-like DLBCL. This screen uncovered CARD11 as a key upstream signalling component responsible for the constitutive IκB kinase activity in activated B-cell-like DLBCL. The methodology that we describe can be used to establish a functional taxonomy of cancer and help reveal new classes of therapeutic targets distinct from known oncogenes.
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Hanahan, D. & Weinberg, R. A. The hallmarks of cancer. Cell 100, 57–70 (2000)
Paddison, P. J. et al. A resource for large-scale RNA-interference-based screens in mammals. Nature 428, 427–431 (2004)
Berns, K. et al. A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428, 431–437 (2004)
Alizadeh, A. A. et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 403, 503–511 (2000)
Rosenwald, A. et al. The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma. N. Engl. J. Med. 346, 1937–1947 (2002)
Davis, R. E., Brown, K. D., Siebenlist, U. & Staudt, L. M. Constitutive nuclear factor κB activity is required for survival of activated B cell-like diffuse large B cell lymphoma cells. J. Exp. Med. 194, 1861–1874 (2001)
Lam, L. T. et al. Small molecule inhibitors of IκB-kinase are selectively toxic for subgroups of diffuse large B cell lymphoma defined by gene expression profiling. Clin. Cancer Res. 11, 28–40 (2005)
Thome, M. CARMA1, BCL-10 and MALT1 in lymphocyte development and activation. Nature Rev. Immunol. 4, 348–359 (2004)
Rosenwald, A. et al. Molecular diagnosis of primary mediastinal B cell lymphoma identifies a clinically favorable subgroup of diffuse large B cell lymphoma related to Hodgkin lymphoma. J. Exp. Med. 198, 851–862 (2003)
Savage, K. J. et al. The molecular signature of mediastinal large B-cell lymphoma differs from that of other diffuse large B-cell lymphomas and shares features with classical Hodgkin lymphoma. Blood 102, 3871–3879 (2003)
Ruland, J., Duncan, G. S., Wakeham, A. & Mak, T. W. Differential requirement for Malt1 in T and B cell antigen receptor signaling. Immunity 19, 749–758 (2003)
Isaacson, P. G. & Du, M. Q. MALT lymphoma: from morphology to molecules. Nature Rev. Cancer 4, 644–653 (2004)
Zhang, Q. et al. Inactivating mutations and overexpression of BCL10, a caspase recruitment domain-containing gene, in MALT lymphoma with t(1;14)(p22;q32). Nature Genet. 22, 63–68 (1999)
Ruefli-Brasse, A. A., French, D. M. & Dixit, V. M. Regulation of NF-κB-dependent lymphocyte activation and development by paracaspase. Science 302, 1581–1584 (2003)
Ruland, J. et al. Bcl10 is a positive regulator of antigen receptor-induced activation of NF-κB and neural tube closure. Cell 104, 33–42 (2001)
Xue, L. et al. Defective development and function of Bcl10-deficient follicular, marginal zone and B1 B cells. Nature Immunol. 4, 857–865 (2003)
Newton, K. & Dixit, V. M. Mice lacking the CARD of CARMA1 exhibit defective B lymphocyte development and impaired proliferation of their B and T lymphocytes. Curr. Biol. 13, 1247–1251 (2003)
Egawa, T. et al. Requirement for CARMA1 in antigen receptor-induced NF-κB activation and lymphocyte proliferation. Curr. Biol. 13, 1252–1258 (2003)
Hara, H. et al. The MAGUK family protein CARD11 is essential for lymphocyte activation. Immunity 18, 763–775 (2003)
Jun, J. E. et al. Identifying the MAGUK protein Carma-1 as a central regulator of humoral immune responses and atopy by genome-wide mouse mutagenesis. Immunity 18, 751–762 (2003)
McAllister-Lucas, L. M. et al. Bimp1, a MAGUK family member linking protein kinase C activation to Bcl10-mediated NF-κB induction. J. Biol. Chem. 276, 30589–30597 (2001)
Gaide, O. et al. Carma1, a CARD-containing binding partner of Bcl10, induces Bcl10 phosphorylation and NF-κB activation. FEBS Lett. 496, 121–127 (2001)
Bertin, J. et al. CARD11 and CARD14 are novel caspase recruitment domain (CARD)/membrane-associated guanylate kinase (MAGUK) family members that interact with BCL10 and activate NF-κB. J. Biol. Chem. 276, 11877–11882 (2001)
Zhou, H. et al. Bcl10 activates the NF-κB pathway through ubiquitination of NEMO. Nature 427, 167–171 (2004)
Sun, L., Deng, L., Ea, C. K., Xia, Z. P. & Chen, Z. J. The TRAF6 ubiquitin ligase and TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T lymphocytes. Mol. Cell 14, 289–301 (2004)
Kolfschoten, I. G. et al. A genetic screen identifies PITX1 as a suppressor of RAS activity and tumorigenicity. Cell 121, 849–858 (2005)
Westbrook, T. F. et al. A genetic screen for candidate tumor suppressors identifies REST. Cell 121, 837–848 (2005)
Brummelkamp, T. R., Bernards, R. & Agami, R. Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2, 243–247 (2002)
van de Wetering, M. et al. Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector. EMBO Rep. 4, 609–615 (2003)
Reynolds, A. et al. Rational siRNA design for RNA interference. Nature Biotechnol. 22, 326–330 (2004)
This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. V.N.N. was also supported by a Damon Runyon-Walter Winchell Cancer Research Foundation Fellowship.
The microarray data discussed in this publication have been deposited in the Gene Expression Omnibus of NCBI (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO series accession number GSE3896. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.
Knockdown of gene expression depends upon induction of shRNA expression by doxycycline. This figure shows Q-PCR and Western blot analysis of shRNA-mediated knockdown of target gene expression in DLBCL cell lines. (PDF 978 kb)
Identification of shRNAs that block the proliferation or survival of lymphoma cell lines. (PDF 194 kb)
Gene expression profiles and NF-κB pathway activity in DLBCL cell lines. (PDF 302 kb)
CARD11 mRNA expression in ABC DLBCL, GCB DLBCL, and PMBL tumor biopsies. (PDF 91 kb)
Sequence of effective shRNAs and position within the targeted Refseq mRNA sequence. (DOC 31 kb)
More detailed methods are described here for preparing doxycycline-inducible cell lines; performing barcode DNA microarrays; cell-based IKK assay; cytokine measurement; and survival assay. (DOC 52 kb)
Text to accompany the above Supplementary Figures. (DOC 28 kb)
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Ngo, V., Davis, R., Lamy, L. et al. A loss-of-function RNA interference screen for molecular targets in cancer. Nature 441, 106–110 (2006). https://doi.org/10.1038/nature04687
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