Abstract 3135: In vitro and in vivo synthetic lethal screens to identify novel targets in the context of PTEN deficiency.
Authors: Zhang Jing-xin , Murtie Joshua , Iartchouk Oleg , Cao Hui , Shapiro Gary , Li Zhifang , Wang Hongyun , Ding Zhihu , Zhang Yu-an , Light Madelyn , Hoffman Dietmar , Cindhuchao May , Jani Saurin , Newcombe Richard , Bric-Furlong Eva , Wu Bin , Virone-Oddos Angela , Reiling Stephan , Theihaber Joachim , Lengauer Christoph , Pollard Jack , Watters James , Silver Serena , Reddy Venkat 
Published: August 2014

Functional genomic screens have been employed by many groups to identify novel targets for cancer therapeutics. However, translation of these data sets into new drug discovery programs has proved challenging, in part due to difficulties in obtaining strong on-target knockdown, coupled with misleading off-target effects. Most commercially available pooled shRNA screening libraries provide 5-6 shRNAs per gene, with little knowledge of knock-down efficiency. In an effort to overcome these issues, we designed a custom shRNA library targeting 580 human genes, including key nodes in signal transduction pathways and genes of interest for oncology drug development. This pathway-centric design allows us to determine critical pathways for cancer cell growth in addition to individual gene phenotypes. For each gene, we have total of 11 shRNAs, a subset of which have been previously shown high efficiency knock-down. In particular, we emphasized genes where tool or lead compounds were available for rapid follow-up. Both in size and scope, this library was designed to quickly identify important signaling pathways that are essential on their own or when perturbed in the presence of small molecule drugs in our pipeline.

With this focused library, we performed functional genomic shRNA screens in PTEN mutant or null lines across cancer types in vitro, including four cell lines that are PTEN deficient (UACC62, MDA-MB-468, U87-MG and PC3), and one cell line, (HCT-116) that is PTEN wild type. Meanwhile, we performed in vivo screens with PC3 cells. To identify hits from our screens, we used an internally developed algorithm, c-FOLD, to calculate p-value and fold changes for each shRNA. As confirmation that our library and methods can identify essential genes in a context specific manner, we observe BRAF as the top essential gene hit in the cell line which contains an activated BRAF allele, UACC62, but not in other lines tested. We then examined the concordance of hits in our in vitro and in vivo experiments with PC3 cells. In general, individual shRNAs were highly correlated between in vitro and in vivo assays, but a subset is specific to one setting, and may present particularly interesting targets.

To discover synthetic lethal partners to PTEN deficiency, we required that a gene is essential in all four PTEN deficient lines in vitro, as well as in PC3 in vivo, but not essential in HCT-116. Among the top hits are multiple shRNAs against ENTPD5, an ER enzyme hydrolyzing UDP to UMP. In addition, using our pathway approach, we identified components in JNK pathways as synthetic lethal partners to PTEN deficiency. The results obtained from these studies established a strong platform for in vivo and in vitro identification of novel targets and novel combination partners. These approaches can also be integrated with other Omics data, such as mutation and expression, to ultimately find promising targets to develop novel cancer therapeutics.

Citation Format: Jing-xin Zhang, Joshua Murtie, Oleg Iartchouk, Hui Cao, Gary Shapiro, Zhifang Li, Hongyun Wang, Zhihu Ding, Yu-an Zhang, Madelyn Light, Dietmar Hoffman, May Cindhuchao, Saurin Jani, Richard Newcombe, Eva Bric-Furlong, Bin Wu, Angela Virone-Oddos, Stephan Reiling, Joachim Theihaber, Christoph Lengauer, Jack Pollard, James Watters, Serena Silver, Venkat Reddy. In vitro and in vivo synthetic lethal screens to identify novel targets in the context of PTEN deficiency. [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 3135. doi:10.1158/1538-7445.AM2013-3135