PDF file - 1334KB, S1. IC50 values of TBK1 inhibitors Compound number 5, Compound number 4 and Compound number 3 from the selected PDAC, colorectal cancer and NSCLC cancer cell lines using are shown. Table 1S. List of shRNAs Targeting TBK1 Table 2S. RAS Gene Signature Score Correlation with Sensitivity to the TBK1i (Compound #1) and Meki (PD0325901) in NSCLC Cell Lines Table 3S. List of Cell Lines from 6-Cell Line Panels that were Used in TBK1 Inhibitor Screening in the 2-Dimensional Cell Viability Assay (NSCLC and Colorectal cancer panels) Table 4S. List of Cell Lines from 6-Cell Line Panels that were Used in TBK1 Inhibitor Screening in the 2-Dimensional Cell Viability Assay (Ovarian cancer, GBM and Lymphoma panels).

Abstract Gastric cancer is the second leading cause of cancer deaths in the world. The genomics of gastric cancers is unique in that they harbor significantly more copy number alterations compared to point mutations, yet the functional importance of these genetic alterations in tumor maintenance is not known. To better understand oncogenic drivers of gastric cancer and identify potential therapeutic targets we performed negative selection RNAi screens in ten well annotated gastric cancer cell lines. Screens were performed using two different but overlapping shRNA libraries. The first library was the Decipher Human Module I pool from Cellecta composed of 27500 shRNAs targeting 5043 genes. The second library was a custom designed focused pool with 6500 shRNAs targeting 608 genes. In addition to screening the two shRNA libraries in vitro, the focused pool was also screened in subcutaneous xenograft tumor models in eight of the gastric cancer cell lines. The screens revealed distinct genetic vulnerabilities that correlated with the corresponding genomic alteration in the specific cell lines. In particular we found that KRAS amplifications confer dependency to the same degree as activating KRAS mutations. This KRAS dependency was further validated with additional shRNAs in KRAS amplified and mutated cell lines. Furthermore, we identified AMPK which is focally amplified in 9% of gastric cancer as a critical oncogenic driver. Multiple subunits of the AMPK holoenzyme scored in the screen and dependency on AMPK alpha and beta subunits was demonstrated with independent shRNAs in two cell lines from the primary screen. Consistent with the screen results we find that LMSU, a gastric cancer cell line not part of the primary screen but annotated as amplified for the AMPK alpha subunit shows elevated expression levels and is sensitive to knockdown of AMPK. These observations have identified AMPK as a novel oncogenic driver in gastric cancer with therapeutic potential. Citation Format: Meghana M. Kulkarni, Sushma Gurumurthy, Oleg Schmidt-Kittler, Jason Berglund, Christopher H. Hulton, David J. Wilson, David Jakubosky, Daniel Michaud, Robert E. Jones, Nicole M. Sjoblom, Russell McSweeney, Hongwei Zhou, Annapurna Venkatakrishnan, Karin J. Jensen, Jingxin Zhang, Parminder K. Mankoo, Jack Pollard, Christopher Winter, Pasi A. Jänne, Kwok-Kin Wong, Victoria M. Richon, Jessie M. English, Mark A. Bittinger. Functional genomics reveals genetic dependencies in gastric cancer. [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 1110. doi:10.1158/1538-7445.AM2015-1110

Abstract TBK1 (TANK-binding kinase 1) is a noncanonical IκB protein kinase that phosphorylates and activates downstream targets such as IRF3 and c-Rel and, mediates NF-κB activation in cancer. Previous reports demonstrated synthetic lethality of TBK1 with mutant KRAS in non–small cell lung cancer (NSCLC); thus, TBK1 could be a novel target for treatment of KRAS-mutant NSCLC. Here, the effect of TBK1 on proliferation in a panel of cancer cells by both genetic and pharmacologic approaches was evaluated. In KRAS-mutant cancer cells, reduction of TBK1 activity by knockdown or treatment with TBK1 inhibitors did not correlate with reduced proliferation in a two-dimensional viability assay. Verification of target engagement via reduced phosphorylation of S386 of IRF3 (pIRF3S386) was difficult to assess in NSCLC cells due to low protein expression. However, several cell lines were identified with high pIRF3S386 levels after screening a large panel of cell lines, many of which also harbor KRAS mutations. Specifically, a large subset of KRAS-mutant pancreatic cancer cell lines was uncovered with high constitutive pIRF3S386 levels, which correlated with high levels of phosphorylated S172 of TBK1 (pTBK1S172). Finally, TBK1 inhibitors dose-dependently inhibited pIRF3S386 in these cell lines, but this did not correlate with inhibition of cell growth. Taken together, these data demonstrate that the regulation of pathways important for cell proliferation in some NSCLC, pancreatic, and colorectal cell lines is not solely dependent on TBK1 activity. Implications: TBK1 has therapeutic potential under certain contexts and phosphorylation of its downstream target IRF3 is a biomarker of TBK1 activity. Visual Overview: http://mcr.aacrjournals.org/content/12/7/1055/F1.large.jpg. Mol Cancer Res; 12(7); 1055–66. ©2014 AACR.

Abstract 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