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 ...

Drug resistance remains an elusive problem in cancer therapy, particularly for novel targeted therapies. Much work is focused upon the development of an arsenal of targeted therapies, towards oncogenic driver genes such as ALK-EML4, to overcome the inevitable resistance that develops over time. Currently, after failure of first line ALK TKI therapy, another ALK TKI is administered, though collateral sensitivity is not considered. To address this, we evolved resistance in an ALK rearranged non-small cell lung cancer line (H3122) to a panel of 4 ALK TKIs, and performed a collateral sensitivity analysis. All ALK inhibitor resistant cell lines displayed significant cross-resistance to all other ALK inhibitors. We then evaluated ALK-inhibitor sensitivities after drug holidays of varying length (1–21 days), and observed dynamic patterns of resistance. This unpredictability led us to an expanded search for treatment options, where we tested 6 further anti-cancer agents for collateral sensitivity among resistant cells, uncovering possibilities for further treatment, including cross-sensitivity to standard cytotoxic therapies, as well as Hsp90 inhibitors. Taken together, these results imply that resistance to targeted therapy in non-small cell lung cancer is highly dynamic, and also one where there are many opportunities to re-establish sensitivities where there was once resistance. Drug resistance in cancer inevitably emerges during treatment; particularly with novel targeted therapies, designed to inhibit specific molecules. A clinically-relevant example of this phenomenon occurs in ALK-positive non-small cell lung cancer, where targeted therapies are used to inhibit the ALK-EML4 fusion protein. A potential solution to this may lie in finding drug sensitivities in the resistant population, termed collateral sensitivities, and then using these as second-line agents. This study shows how the evolution of resistance in ALK-positive lung cancer is a dynamic process through time, one in which patterns of drug resistance and collateral sensitivity change substantially, and therefore one where temporal regimens, such as drug cycling and drug holidays may have great benefit.

Purpose: The clinical use of MEK inhibitors in uveal melanoma is limited by the rapid acquisition of resistance. This study has used multiomics approaches and drug screens to identify the pan-HDAC inhibitor panobinostat as an effective strategy to limit MEK inhibitor resistance. Experimental Design: Mass spectrometry-based proteomics and RNA-Seq were used to identify the signaling pathways involved in the escape of uveal melanoma cells from MEK inhibitor therapy. Mechanistic studies were performed to evaluate the escape pathways identified, and the efficacy of the MEK-HDAC inhibitor combination was demonstrated in multiple in vivo models of uveal melanoma. Results: We identified a number of putative escape pathways that were upregulated following MEK inhibition, including the PI3K/AKT pathway, ROR1/2, and IGF-1R signaling. MEK inhibition was also associated with increased GPCR expression, particularly the endothelin B receptor, and this contributed to therapeutic escape through ET-3-mediated YAP signaling. A screen of 289 clinical grade compounds identified HDAC inhibitors as potential candidates that suppressed the adaptive YAP and AKT signaling that followed MEK inhibition. In vivo, the MEK-HDAC inhibitor combination outperformed either agent alone, leading to a long-term decrease of tumor growth in both subcutaneous and liver metastasis models and the suppression of adaptive PI3K/AKT and YAP signaling. Conclusions: Together, our studies have identified GPCRmediated YAP activation and RTK-driven AKT signaling as key pathways involved in the escape of uveal melanoma cells from MEK inhibition. We further demonstrate that HDAC inhibition is a promising combination partner for MEK inhibitors in advanced uveal melanoma.

Purpose: Covalent inhibitors of KRASG12C specifically target tumors driven by this form of mutant KRAS, yet early studies show that bypass signaling drives adaptive resistance. Although several combination strategies have been shown to improve efficacy of KRASG12C inhibitors (KRASi), underlying mechanisms and predictive strategies for patient enrichment are less clear. Experimental Design: We performed mass spectrometry–based phosphoproteomics analysis in KRASG12C cell lines after short-term treatment with ARS-1620. To understand signaling diversity and cell type–specific markers, we compared proteome and phosphoproteomes of KRASG12C cells. Gene expression patterns of KRASG12C cell lines and lung tumor tissues were examined. Results: Our analysis suggests cell type–specific perturbation to ERBB2/3 signaling compensates for repressed ERK and AKT signaling following ARS-1620 treatment in epithelial cell type, and this subtype was also more responsive to coinhibition of SHP2 and SOS1. Conversely, both high basal and feedback activation of FGFR or AXL signaling were identified in mesenchymal cells. Inhibition of FGFR signaling suppressed feedback activation of ERK and mTOR, while AXL inhibition suppressed PI3K pathway. In both cell lines and human lung cancer tissues with KRASG12C, we observed high basal ERBB2/3 associated with epithelial gene signatures, while higher basal FGFR1 and AXL were observed in cells/tumors with mesenchymal gene signatures. Conclusions: Our phosphoproteomic study identified cell type–adaptive responses to KRASi. Markers and targets associated with ERBB2/3 signaling in epithelial subtype and with FGFR1/AXL signaling in mesenchymal subtype should be considered in patient enrichment schemes with KRASi.

Epithelial-mesenchymal transition (EMT) is one mechanism of acquired resistance to inhibitors of the epidermal growth factor receptor-tyrosine kinases (EGFR-TKIs) in non-small cell lung cancer (NSCLC). The precise mechanisms of EMT-related acquired resistance to EGFR-TKIs in NSCLC remain unclear. We generated erlotinib-resistant HCC4006 cells (HCC4006ER) by chronic exposure of EGFR-mutant HCC4006 cells to increasing concentrations of erlotinib. HCC4006ER cells acquired an EMT phenotype and activation of the TGF-β/SMAD pathway, while lacking both T790M secondary EGFR mutation and MET gene amplification. We employed gene expression microarrays in HCC4006 and HCC4006ER cells to better understand the mechanism of acquired EGFR-TKI resistance with EMT. At the mRNA level, ZEB1 (TCF8), a known regulator of EMT, was >20-fold higher in HCC4006ER cells than in HCC4006 cells, and increased ZEB1 protein level was also detected. Furthermore, numerous ZEB1 responsive genes, such as CDH1 (E-cadherin), ST14, and vimentin, were coordinately regulated along with increased ZEB1 in HCC4006ER cells. We also identified ZEB1 overexpression and an EMT phenotype in several NSCLC cells and human NSCLC samples with acquired EGFR-TKI resistance. Short-interfering RNA against ZEB1 reversed the EMT phenotype and, importantly, restored erlotinib sensitivity in HCC4006ER cells. The level of micro-RNA-200c, which can negatively regulate ZEB1, was significantly reduced in HCC4006ER cells. Our results suggest that increased ZEB1 can drive EMT-related acquired resistance to EGFR-TKIs in NSCLC. Attempts should be made to explore targeting ZEB1 to resensitize TKI-resistant tumors.