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

Making the most of RNAi Two papers this week highlight the impact of RNAi (RNA interference) in clinical medicine. Ngo et al . have developed a novel ‘Achilles heel' screen to identify genes that, if silenced, cause cancer cells to stop dividing. The novelty lies in a successful ‘negative’ screen that can reveal potential therapeutic targets that do not necessarily contain mutations or other alterations. Use of the screen on 2,500 genes in B-cell lymphoma cells identified three genes that were essential for cancer cell survival and growth of one particular B-cell lymphoma subtype. In particular the protein CARD11 looks a prime target. Zimmermann et al . report a significant step towards harnessing RNAi as a new class of drug treatment. They used systemic administration of small interfering RNA (siRNA) to silence a disease-causing gene in a non-human primate: it had previously been demonstrated in mice. Specifically, siRNA targeted against the gene for apolipoprotein B (ApoB) in cynomolgus monkeys successfully reduced in ApoB protein, serum cholesterol and low-density lipoprotein levels. This has implications for diseases associated with high cholesterol levels, such as coronary heart disease, and more broadly demonstrates that potential therapies may be developed against historically ‘non-druggable’ targets. 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 apoptosis 1 ; 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 expressio