Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1

Abstract

The proto-oncogene KRAS is mutated in a wide array of human cancers, most of which are aggressive and respond poorly to standard therapies. Although the identification of specific oncogenes has led to the development of clinically effective, molecularly targeted therapies in some cases, KRAS has remained refractory to this approach. A complementary strategy for targeting KRAS is to identify gene products that, when inhibited, result in cell death only in the presence of an oncogenic allele. Here we have used systematic RNA interference to detect synthetic lethal partners of oncogenic KRAS and found that the non-canonical IkappaB kinase TBK1 was selectively essential in cells that contain mutant KRAS. Suppression of TBK1 induced apoptosis specifically in human cancer cell lines that depend on oncogenic KRAS expression. In these cells, TBK1 activated NF-kappaB anti-apoptotic signals involving c-Rel and BCL-XL (also known as BCL2L1) that were essential for survival, providing mechanistic insights into this synthetic lethal interaction. These observations indicate that TBK1 and NF-kappaB signalling are essential in KRAS mutant tumours, and establish a general approach for the rational identification of co-dependent pathways in cancer.

Fig. 1

Meta-analysis of RNAi screens identifying KRAS synthetic lethals. (a) Supervised analysis of viability data (B-score) identified 250 shRNAs that distinguished mutant KRAS from wild-type (WT) cells, including genes targeted by multiple shRNAs. (b) Hairpin set analysis (RIGER). Genes were assigned NES (red lines) based on the KRAS mutant/WT differential survival scores (blue lines) for each shRNA. Negative values represent mutant KRAS-selectivity. (c) Union of 17 genes identified in (a) and 40 genes identified in (b). (d) Secondary screening data normalized using percent of control (POC) and analyzed using RIGER. FDR for KRAS and TBK1 was 0.04 and 0.18 respectively.

Fig. 2

TBK1 synthetic lethality with oncogenic KRAS. (a) Top-scoring TBK1-specific shRNAs (*) induced lethality and TBK1 suppression (immunoblot) in NCI-H23 cells (mutant KRAS). (b) Suppression of KRAS or TBK1 in NSCLC cell lines. HCC-1359 and HCC-193 cells expressed RAS and NF-κB signatures. (c) KRAS and TBK1 dependence of lung epithelial cells expressing oncogenic KRAS (AALE-K) or vector (AALE-V). (d) Tumor formation following TBK1 suppression. Mean and SEM of at least 11 replicates shown. (e) Immunoblot of cleaved PARP following TBK1 or KRAS suppression. (f) Percentage of TUNEL positive nuclei following TBK1 or KRAS suppression. Mean and SD shown. (g) Differential cell viability following KRAS, TBK1, CRAF, BRAF, AKT1 or RALB suppression in KRAS mutant vs. WT cell lines (t-test for comparisons). SEM of triplicate samples normalized to shGFP control vector shown.

Fig. 3

Oncogenic KRAS-induced NF-κB signaling involves TBK1 (a) GSEA of AALE-V (vector), AALE-K (KRAS G12V) or AALE-WT-K (KRAS WT) cells (positive NES). A RAS oncogenic signature (black) and NF-κB signatures (red) showed significant enrichment (FDR<0.25) in AALE-K cells. N.S. = non-significant. (b) RAS signatures in mutant KRAS lung adenocarcinomas correlate with NF-κB but not IRF3 signatures (red=activation, blue=inactivation). (c) RAS and NF-κB signature expression in WT KRAS lung adenocarcinomas and normal lung tissue. (d) AALE-K signature enrichment plot following shTBK1 or shGFP expression in triplicate samples. Heatmap shows top KRAS-induced genes with negative enrichment in AALE-K-shTBK1 samples. Immunoblot of IKBα, p105, TBK1, and KRAS in AALE-K and AALE-V cells (e) or NCI-H23 cells (f) following KRAS or TBK1 suppression. (g) Cell viability after expression of control vector (pBP) or IκBα-super-repressor (pBP-IKB-SR) in mutant or WT KRAS cells. Mean and SEM of triplicate samples shown, t-test for comparisons.

Fig. 4

TBK1 regulates c-REL and BCL-XL in KRAS mutant cells. (a) Differential cell viability following IRF3 or cREL suppression in KRAS mutant vs. WT cell lines. (b) Immunoblot of p105, c-REL, p50, BCL-XL and BIRC2 in KRAS mutant cell lines following TBK1 suppression. (c) Cell viability following KRAS or TBK1 suppression in NCI-H23 cells expressing a control protein (LACZ) or V5-tagged BCL-XL. SEM of triplicate samples normalized to shGFP control vector shown. (d) Immunoblot showing over-expression of V5-tagged BCL-XL and inhibition of PARP cleavage.