Abstract 3205: In vivo oncology target screening using a lentiviral inducible- shRNA knockdown system.

Background: Small RNAs are able to suppress expression of any gene through the endogenous RNA interference pathway and have been used to identify therapeutic targets in a variety of pathological conditions. In this project, we aimed to optimise a lentiviral inducible-knockdown small hairpin RNA (shRNA) system for efficient in vivo cancer target validation, using polo-like kinase 1 (PLK1), an essential cell-cycle protein, as a proof-of-concept target. Methods: An inducible shRNA construct targeting PLK1 or a control construct was transduced into the colon carcinoma cell line SW620. Inducible shRNA expression (marked by turbo-RFP) was monitored by fluorescence microscopy and flow cytometry in vitro and by spectrum CT in vivo. PLK1 gene expression was quantified by real-time PCR and protein expression was quantified by Western blotting in vitro or IHC ex vivo. Cell viability was assessed by MTT assays. Xenografts were established by subcutaneous injection of PLK1 inducible-knockdown cells into nude mice. Growth inhibitory effect was assessed by calliper measurement of tumour size and correlated with ex vivo assessment of gene and protein knockdown, and of cell proliferation as measured by Ki67 staining. Results: Cell viability assays demonstrated that concentrations of 2ug/ml of doxycycline or less did not result in cell death over an assay period of 7 days in both wild-type cells and cells transduced with a control shRNA construct. Following doxycycline treatment, PLK1 inducible-knockdown cells showed dose- and time-dependent shRNA induction with an increase in the level of shRNA expression and the percentage of positive cells. PLK1 down-regulation correlated with induction of shRNA expression reaching 80% gene knock-down and 60% protein knock-down 72 hours post-induction. The optimal delivery route of doxycycline via measurement of turbo-RFP induction and PK/PD measurements was assessed in vivo. In tumour-bearing mice treated with doxycycline, there was a decline in tumour growth rate in the PLK1 knockdown group compared with the control group. Ex vivo analysis showed significantly lower PLK1 gene and protein expression in the doxycycline-treated group compared with the control group which correlated with lower rates of cell proliferation in the treated group. Conclusion: The results support the anti-tumour effects of PLK1 down-regulation and confirm an efficient target validation methodology for cancer target screening using a lentiviral inducible-knockdown shRNA system. Citation Format: Yinfei Yin, Rajendra Kumari, Siddharth Subramaniam, Peter King, Sue Watson, Philip Mallinder, Anna Grabowska. In vivo oncology target screening using a lentiviral inducible- shRNA knockdown system. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3205. doi:10.1158/1538-7445.AM2013-3205