Abstract 4289: 3-Dimensional growth reveals KRAS dependency.
Authors: Foster Rebecca , Mudd Clare , Wiggins Ceri , Torrance Chris 
Published: August 2014

Advances in the development of clinically effective therapeutic strategies for targeting KRAS mutant tumors have been hampered by the lack of suitable in vitro tools and the inability to correlate findings from current in vitro KRAS dependency models with clinical results. Studies to evaluate targeted agents in KRAS dependent and independent cell systems have traditionally utilized panels of cell lines grown in 2D, which differ not only in KRAS status but also exhibit multiple genetic differences. An additional confounding element is that traditional 2D assays do not accurately mimic tumor architecture or growth. Our study focused on addressing two essential questions: (1) Can the consequence of a KRAS mutation be studied without the influence of other genetic factors? (2) Is there a requirement for more in vivo-like conditions to reveal KRAS dependency in vitro? To overcome confounding genetic factors, Horizon Discovery's proprietary gene editing technology GENESIS™ was used to knock-out an endogenous KRAS G13D mutation from DLD-1 colon carcinoma cells in order to create a pair of isogenic cell lines that differ only in terms of their KRAS status. Extensive profiling of the KRAS mutant and wild type cells under a range of growth formats was then performed to identify conditions which reveal dependency on KRAS. In standard 2D formats, cell growth was independent of KRAS with both cell lines demonstrating identical growth rates. However, knock-out of the KRAS mutation severely compromised cell growth under 3D conditions which more closely mimic true tumor biology. Interestingly, growth of the wild type cells was compromised when grown in two distinct 3D formats; soft agar or grown without a supporting matrix under low anchorage conditions. These results demonstrate the essential nature of 3D conditions for revealing KRAS dependency. In order to profile anti-cancer compounds using a truly KRAS dependent system, cellular responses to a series of targeted agents were compared in DLD-1 cells in 2D and 3D assays. Multiple MEK inhibitors demonstrated clear sensitivity under KRAS dependent conditions, with increased potency seen in the 3D assay compared to the 2D assay. Targeted agents unrelated to KRAS such as JAK and SYK inhibitors and non-targeted agents such as Paclitaxel exhibited similar potency in both 2D and 3D assays, further validating this KRAS dependency model. The increasing evidence that 3D conditions are critical for revealing KRAS dependency suggests that previous studies performed under standard 2D conditions may have underestimated levels of dependency. To address this, a study to systematically evaluate KRAS dependency across a panel of more than thirty KRAS mutant and wild type cancer cell lines under 2D and 3D conditions is now underway. Greater knowledge surrounding the biology and an understanding of how best to model KRAS dependency will be key to advancing effective KRAS pathway targeted agents into the clinic.

Citation Format: Rebecca Foster, Clare Mudd, Ceri Wiggins, Chris Torrance. 3-Dimensional growth reveals KRAS dependency. [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 4289. doi:10.1158/1538-7445.AM2013-4289