Abstract 700: Investigating KRAS synthetic lethal/co-dependency interactions using siRNA and CRISPR
Authors: Scrace Simon F. , Tsonou Elpida , Russell Paul , Wickenden Julie A. , Lawo Steffen , Scales Tim M. , Wiggins Ceri M. , Moore Jonathan D. 
Published: August 2015

Abstract
No molecularly targeted therapy has yet been identified for KRAS mutant cancers. As oncogenic mutations reduce RAS enzymatic activity, classic small molecule approaches are ineffective, hence most work has focussed on drugging RAS-effector pathways. Multiple inhibitors of MEK, RAF and PI3K have been identified but toxicity issues and pathway adaptation have stymied their success against KRAS-driven cancers. An alternative approach is to exploit “non-oncogene addiction” by identifying targets with synthetic lethal or co-dependence interactions with KRAS.
A number of siRNA and shRNA screens have identified targets that exhibit differential dependencies between KRAS mutant and KRAS wild-type tumours, but there is poor overlap between the different published studies. This discordance may arise from (1) the noise inherent in using cell line panels differing in much more than their KRAS mutant/wild-type status and (2) the use of RNA interference methodologies driving incomplete knockdown and associated with substantial off-target effects. Next generation screens that exploit both isogenic cell lines and cell line panels, and use a combination of knockdown and knock-out (i.e. CRISPR/Cas9-sgRNA) methodologies, may be better suited for identifying novel targets that withstand validation. However, if we are to detect co-dependence as well as synthetic lethal interactions, screens must be performed under conditions where mutant KRAS alleles are essential for growth.
A library of siRNAs targeting proposed KRAS synthetic lethal targets was assembled and screened under conditions where proliferation is dependent on KRAS status. DLD1 cells harbour an activating KRASG13D mutation dispensable for proliferation in 2D, but essential for proliferation under 3D (soft agar) conditions. Knockdown of several targets including KRAS itself, PLK1, TBK1, BCL-XL & RAF1 proved more anti-proliferative under 3D conditions. This screen was extended to a panel of KRAS-mutant colon lines, with varying levels of KRAS sensitivity, where we found the requirement for RAF1 highly correlated with the requirement for KRAS.
With the advent of CRISPR we are now able to design sgRNA libraries capable of probing the effect of ‘knocking out’ rather than ‘knocking down’ targets, providing a potentially superior alternative to RNA interference. Data from mouse models indicates RAF1 is required for the initiation of lung cancer by oncogenic KRAS. Although we found good correlation between sensitivity to KRAS and RAF1 depletion, we were unable to unambiguously validate RAF1 as a target in human lung cancer cells using RNA interference methodologies. However, using CRISPR-Cas9, we found complete loss of RAF1 expression was anti-proliferative in A549 cells, supporting the concept of targeting RAF1 in a KRAS mutant lung cancers. These results demonstrate that a more penetrant sgRNA based screening approach may identify novel KRAS synthetic lethal or co-dependent interactions.
Citation Format: Simon F. Scrace, Elpida Tsonou, Paul Russell, Julie A. Wickenden, Steffen Lawo, Tim M. Scales, Ceri M. Wiggins, Jonathan D. Moore. Investigating KRAS synthetic lethal/co-dependency interactions using siRNA and CRISPR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 700. doi:10.1158/1538-7445.AM2015-700