TBK1 is a non-canonical IκB protein kinase that phosphorylates and activates downstream targets such as IRF3 and cRel and, reportedly mediates NFkB activation in cancer. We used both genetic and pharmacological approaches to evaluate TBK1 function in cancer cells. In oncogenic KRAS mutant lung cancer cell lines, the TBK1 knockdown did not correlate with reduction in a 2D viability assay. Using TBK1 small molecule inhibitors, we found that the inhibition of cell growth in 2D viability assays did not correlate with Kras dependency or RAS genetic signature. Since the pIRF3(S386) levels in lung lines were too low to assess TBK1 target engagement, we identified cell lines with high pIRF3(S386) levels. We discovered a subset of pancreatic and colorectal cell lines that have high constitutive pIRF3(S386) levels and this correlated with high levels of pTBK1(S172). TBK1 inhibitors were able to dose-dependently inhibit pIRF3S386 in pancreatic & colon lines. These observations indicate that pIRF3(S386) could be used as a biomarker for direct target engagement of TBK1 in cancer cell lines with activated TBK1 pathway. pTBK1(S172), a phosphorylation site within the TBK1 kinase domain activation loop, does not decrease upon inhibition with TBK1 inhibitors but, instead increases. This suggests stimulation of a feedback loop resulting in the activation of an upstream kinase(s) responsible for activating TBK1 in cancer cell lines. Although several pancreatic and colon cell lines were identified to have low μM sensitivity to TBK1 inhibitors, TBK1 shRNA knockdown did not impact proliferation in these cell lines even though pIRF3(S386) levels were reduced. Further studies using knockdown of the related IKKe homolog in cell lines with activated pathway are also being pursued to determine if IKKe knockdown alone or in combination with TBK1 knockdown would result in more substantial growth reduction. It is likely that inhibition of additional nodes in the NF-kB pathway and other pathways are needed to achieve robust anti-tumor efficacy.

Citation Format: Asli Muvaffak, Qi Pan, Richard Middleton, Erick Morris, Rafael Fernandez, Jonwong Lim, Brian Dolinski, Thi T. Nguyen, Peter Strack, Haiyan Yan, Rossana Chung, Stephen Wu, Weiqun Zhang, Chris Hulton, Heather Hirsch, Kumiko Nagashimo, Yan Wang. Evaluating TBK1 as a cancer therapeutic target in cancer cell lines with activated IRF3. [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 4372. doi:10.1158/1538-7445.AM2013-4372