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A loss-of-function RNA interference screen for molecular targets in cancer

Abstract

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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Figure 1: Inducible shRNA library screen for genes controlling cancer cell proliferation and survival.
Figure 2: Identification of shRNAs that block the proliferation or survival of lymphoma cell lines.
Figure 3: Toxicity of CARD11 and MALT1 shRNAs for activated B-cell-like DLBCL cell lines.
Figure 4: Role of CARD11, MALT1 and BCL10 in NF-κB signalling in activated B-cell-like DLBCL.

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Acknowledgements

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.

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Correspondence to Louis M. Staudt.

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Competing interests

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.

Supplementary information

Supplementary Figure 1

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)

Supplementary Figure 2

Identification of shRNAs that block the proliferation or survival of lymphoma cell lines. (PDF 194 kb)

Supplementary Figure 3

Gene expression profiles and NF-κB pathway activity in DLBCL cell lines. (PDF 302 kb)

Supplementary Figure 4

CARD11 mRNA expression in ABC DLBCL, GCB DLBCL, and PMBL tumor biopsies. (PDF 91 kb)

Supplementary Table 1

Sequence of effective shRNAs and position within the targeted Refseq mRNA sequence. (DOC 31 kb)

Supplementary Methods

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)

Supplementary Figure Legends

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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