TANK-binding kinase 1 (TBK1) has emerged as a novel therapeutic target for unspecified subset of lung cancers. TBK1 reportedly mediates prosurvival signaling by activating NF-κB and AKT. However, we observed that TBK1 knockdown also decreased viability of cells expressing constitutively active NF-κB and interferon regulatory factor 3. Basal phospho-AKT level was not reduced after TBK1 knockdown in TBK1-sensitive lung cancer cells, implicating that TBK1 mediates unknown survival mechanisms. To gain better insight into TBK1 survival signaling, we searched for altered phosphoproteins using mass spectrometry following RNAi-mediated TBK1 knockdown. In total, we identified 2,080 phosphoproteins (4,621 peptides), of which 385 proteins (477 peptides) were affected after TBK1 knockdown. A view of the altered network identified a central role of Polo-like kinase 1 (PLK1) and known PLK1 targets. We found that TBK1 directly phosphorylated PLK1 in vitro. TBK1 phosphorylation was induced at mitosis, and loss of TBK1 impaired mitotic phosphorylation of PLK1 in TBK1-sensitive lung cancer cells. Furthermore, lung cancer cell sensitivity to TBK1 was highly correlated with sensitivity to pharmacological PLK inhibition. We additionally found that TBK1 knockdown decreased metadherin phosphorylation at Ser-568. Metadherin was associated with poor outcome in lung cancer, and loss of metadherin caused growth inhibition and apoptosis in TBK1-sensitive lung cancer cells. These results collectively revealed TBK1 as a mitosis regulator through activation of PLK1 and also suggested metadherin as a putative TBK1 downstream effector involved in lung cancer cell survival.non-canonical IκB kinase | stable isotope labeling by amino acids (SILAC) | astrocyte elevated gene-1 (AEG-1) T ANK-binding kinase 1 (TBK1) was originally identified as an NF-κB-activating kinase (1). TBK1 activates the innate immune response through phosphorylation of two transcription factors, NF-κB and interferon regulatory factor 3 (IRF3), in response to proinflammatory cytokines and Toll-like receptor activation (2, 3). Besides its pivotal role in the innate immune response, increasing evidence indicates that aberrant activation of TBK1 and its closest homolog, IκB kinase e (IKKe), is associated with development of human cancers. IKKe renders cells tumorigenic and is required for breast cancer cell proliferation and survival (4). IKKe has also been found to be amplified or activated in cancers, including lung cancer, ovarian cancer, glioma, and breast cancer (4-7).Mechanisms that activate TBK1 and the downstream proteins and pathways regulated by TBK1 in cancer remain incompletely understood. Some studies have placed TBK1 downstream of RAS signaling, increasing the potential for TBK1-targeting therapeutics in cancer. The RalB GTPase engages TBK1 with oncogenic RAS to support cancer cell survival, suggesting that TBK1 is an unappreciated RAS downstream protein involved in cancer cell survival (8). A systemic RNAi screening study revealed that TBK1 is specifically required ...

Little is known about mammalian preRC stoichiometry, the number of preRCs on chromosomes, and how this relates to replicon size and usage. We show here that, on average, each 100-kb of the mammalian genome contains a preRC composed of approximately one ORC hexamer, 4–5 MCM hexamers, and 2 Cdc6. Relative to these subunits, ∼0.35 total molecules of the pre-Initiation Complex factor Cdc45 are present. Thus, based on ORC availability, somatic cells contain ∼70,000 preRCs of this average total stoichiometry, although subunits may not be juxtaposed with each other. Except for ORC, the chromatin-bound complement of preRC subunits is even lower. Cdc45 is present at very low levels relative to the preRC subunits, but is highly stable, and the same limited number of stable Cdc45 molecules are present from the beginning of S-phase to its completion. Efforts to artificially increase Cdc45 levels through ectopic expression block cell growth. However, microinjection of excess purified Cdc45 into S-phase nuclei activates additional replication foci by three-fold, indicating that Cdc45 functions to activate dormant preRCs and is rate-limiting for somatic replicon usage. Paradoxically, although Cdc45 colocalizes in vivo with some MCM sites and is rate-limiting for DNA replication to occur, neither Cdc45 nor MCMs colocalize with active replication sites. Embryonic metazoan chromatin consists of small replicons that are used efficiently via an excess of preRC subunits. In contrast, somatic mammalian cells contain a low density of preRCs, each containing only a few MCMs that compete for limiting amounts of Cdc45. This provides a molecular explanation why, relative to embryonic replicon dynamics, somatic replicons are, on average, larger and origin efficiency tends to be lower. The stable, continuous, and rate-limiting nature of Cdc45 suggests that Cdc45 contributes to the staggering of replicon usage throughout S-phase, and that replicon activation requires reutilization of existing Cdc45 during S-phase.

The epidermal growth factor receptor (EGFR) plays an important role in cancer by activating downstream signals important in growth and survival. Inhibitors of EGFR are frequently selected as treatment for cancer including lung cancer. We performed an unbiased and comprehensive search for EGFR phosphorylation events related to somatic activating mutations and EGFR inhibitor (erlotinib) sensitivity. EGFR immunoprecipitation combined with high resolution liquid chromatography-mass spectrometry and label free quantitation characterized EGFR phosphorylation. Thirty (30) phosphorylation sites were identified including 12 tyrosine (pY), 12 serine (pS), and 6 threonine (pT). Site-specific phosphorylation was monitored by comparing ion signals from the corresponding unmodified peptide. Phosphorylation sites related to activating mutations in EGFR as well as sensitivity to erlotinib were identified using 31 lung cancer cell lines. We identified three sites (pY1092, pY1110, pY1172) correlating with activating mutations while three sites (pY1110, pY1172, pY1197) correlated with erlotinib sensitivity. Five sites (pT693, pY1092, pY1110, pY1172 and pY1197) were inhibited by erlotinib in concentrationdependent manner. Erlotinib sensitivity was confirmed using liquid chromatography coupled to multiple reaction monitoring (LC-MRM) and quantitative western blotting. This LC-MS/MS strategy can quantitatively assess site-specific EGFR phosphorylation and can identify relationships between somatic mutations or drug sensitivity and protein phosphorylation. § Correspondence should be addressed to: Eric B. Haura,

Introduction: Proteomic analyses using iTRAQ and reaction monitoring mass spectrometry are used to examine the mechanism of melphalan resistance and build an assessment platform to enable future selection of combination therapy in patients. Experimental Procedures: Drug IC50 values and interactions are assessed using cell viability measurements. Protein expression analysis using isobaric tags for relative and absolute quantification (iTRAQ) was used to compare melphalan resistant cell lines (8226/LR5 and U266/LR6) to their isogenic, naïve counterparts (RPMI-8226, U266). In this study, iTRAQ was used to identify proteins that have changed in expression level during the development of melphalan resistance in multiple myeloma (MM). In addition to manual selection of differentially expressed proteins, the resulting quantitative data were coalesced into pathway models using Metacore, GeneGO for further analysis. For targeted protein detection and quantification, liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM) assays are developed and implemented. Data Summary: Melphalan resistant cells are more susceptible to other chemotherapy agents, including steroids, proteasome inhibitors, and geldanamycin derivatives, than their isogenic, naïve counterparts. To investigate the differences, iTRAQ showed numerous changes in protein expression, including redox stress components, mitochondrial proteins, and heat shock proteins (HSPs). LC-MRM assays were developed for proteins detected in the iTRAQ experiments that are current chemotherapy targets, e.g. HSPs and proteasome components. Furthermore, assays have also been developed for additional protein targets of other chemotherapy agents, e.g. topoisomerases and the glucocorticoid receptor, that were not detected in the iTRAQ results. LC-MRM was then used to examine the expression of these proteins in the four model cell lines listed above and in the naïve cells following the onset of drug treatment. Endogenous proteins were quantified in less than 10,000 cells. These assays can be combined to develop an assessment tool to help elucidate the mechanisms of drug resistance and enable the assessment of myeloma cells from patients’ bone marrow aspirates. Conclusions: Quantification of the proteomes of myeloma cell lines using iTRAQ provided insights into the mechanism of melphalan resistance in multiple myeloma. Investigation of the expression of these proteins and other drug targets in myeloma cell lines has produced an LC-MRM platform that can be translated for patient assessment to assist in selection of chemotherapy combinations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5112. doi:10.1158/1538-7445.AM2011-5112

KRAS mutation accounts for development of approximately 30% of lung adenocarcinoma yet therapeutic options remain limited. We report our efforts to explore downstream signaling proteins driven by KRAS, including TANK-binding kinase 1 (TBK1), through a mass spectrometry based phosphoproteomics approach. We hypothesized that such a search would identify key survival proteins as well as uncover potential adaptive resistance mechanisms. We identified proteins whose phosphorylation is regulated TBK1 using SILAC and mass spectrometry following RNAi-mediated TBK1 knockdown. A cohesive network view of the underlying results centered major effects on Polo-like Kinase 1 (PLK1) and decreased phosphorylation of its targets implicating TBK1 as involved in mitosis. TBK1 activity was induced especially in late G1 and M phase, and loss of TBK1 sensitized lung cancer cells to a mitotic stressor. Surprisingly, we found that TBK1 knockdown increased phosphorylation of oncogenic kinases, including EGFR, Met, and ERK1/2. We screened multiple tyrosine kinase inhibitors in combination with TBK1 loss and found the pro-apoptotic effect of TBK1 loss was enhanced by dasatinib, a Src-family kinase inhibitor, providing a mechanistic basis for rational combinatorial therapies involving TBK1. Moving upstream, we analyzed global phosphoproteome change after KRAS knockdown and identified unexpected role of KRAS involved in CDK1 regulation as well as potential adaptive resistant mechanisms that can protect cells against KRAS loss. Finally, we have started exploring changes in the programmed kinome in KRAS mutated lung cancer cells following exposure to MEK inhibitors using activity based protein profiling. Initial experiments have characterized phenotypic effects of two MEK inhibitors in a subset of KRAS mutated lung cancer cells and preliminary studies have identified increased levels of some kinases following MEK inhibition. Collectively, we expect this kinome and phosphoproteomic network based approach will provide better insights into survival signaling mechanism maintaining survival of KRAS mutant lung cancer cell as well as novel therapeutic strategies for this cancer subtype. Updated work will be presented. Supported by the SPORE in Lung Cancer (P50-CA119997) Citation Format: Jae-Young Kim, Eric A. Welsh, Bin Fang, Umut Oguz, Jiannong Li, Fumi Kinose, Crystina Bronk, Amer A. Beg, Ann Chen, Steven Eschrich, John Koomen, Eric B. Haura. Dissection of KRAS-driven survival signaling networks via phosphoproteomics in lung cancer cells. [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 937. doi:10.1158/1538-7445.AM2013-937