Fumi Kinose

Dissection of TBK1 signaling via phosphoproteomics in lung cancer cells

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.

Mass Spectrometry Mapping of Epidermal Growth Factor Receptor Phosphorylation Related to Oncogenic Mutations and Tyrosine Kinase Inhibitor Sensitivity

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) correlated with activating mutations and three sites (pY1110, pY1172, pY1197) correlated with erlotinib sensitivity. Five sites (pT693, pY1092, pY1110, pY1172, and pY1197) were inhibited by erlotinib in concentration-dependent 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.

GSK3 Alpha and Beta Are New Functionally Relevant Targets of Tivantinib in Lung Cancer Cells

Tivantinib has been described as a potent and highly selective inhibitor of the receptor tyrosine kinase c-MET and is currently in advanced clinical development for several cancers including non-small cell lung cancer (NSCLC). However, recent studies suggest that tivantinibs anticancer properties are unrelated to c-MET inhibition. Consistently, in determining tivantinibs activity profile in a broad panel of NSCLC cell lines, we found that, in contrast to several more potent c-MET inhibitors, tivantinib reduces cell viability across most of these cell lines. Applying an unbiased, mass-spectrometry-based, chemical proteomics approach, we identified glycogen synthase kinase 3 (GSK3) alpha and beta as novel tivantinib targets. Subsequent validation showed that tivantinib displayed higher potency for GSK3α than for GSK3β and that pharmacological inhibition or simultaneous siRNA-mediated loss of GSK3α and GSK3β caused apoptosis. In summary, GSK3α and GSK3β are new kinase targets of tivantinib that play an important role in its cellular mechanism-of-action in NSCLC.

ZEB1 Mediates Acquired Resistance to the Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors in Non-Small Cell Lung Cancer.

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.

Adaptive Responses to Dasatinib-Treated Lung Squamous Cell Cancer Cells Harboring DDR2 Mutations

DDR2 mutations occur in approximately 4% of lung squamous cell cancer (SCC) where the tyrosine kinase inhibitor dasatinib has emerged as a new therapeutic option. We found that ERK and AKT phosphorylation was weakly inhibited by dasatinib in DDR2-mutant lung SCC cells, suggesting that dasatinib inhibits survival signals distinct from other oncogenic receptor tyrosine kinases (RTK) and/or compensatory signals exist that dampen dasatinib activity. To gain better insight into dasatinibs action in these cells, we assessed altered global tyrosine phosphorylation (pY) after dasatinib exposure using a mass spectrometry-based quantitative phosphoproteomics approach. Overlaying protein-protein interaction relationships upon this dasatinib-regulated pY network revealed decreased phosphorylation of Src family kinases and their targets. Conversely, dasatinib enhanced tyrosine phosphorylation in a panel of RTK and their signaling adaptor complexes, including EGFR, MET/GAB1, and IGF1R/IRS2, implicating a RTK-driven adaptive response associated with dasatinib. To address the significance of this observation, these results were further integrated with results from a small-molecule chemical library screen. We found that dasatinib combined with MET and insulin-like growth factor receptor (IGF1R) inhibitors had a synergistic effect, and ligand stimulation of EGFR and MET rescued DDR2-mutant lung SCC cells from dasatinib-induced loss of cell viability. Importantly, we observed high levels of tyrosine-phosphorylated EGFR and MET in a panel of human lung SCC tissues harboring DDR2 mutations. Our results highlight potential RTK-driven adaptive-resistant mechanisms upon DDR2 targeting, and they suggest new, rationale cotargeting strategies for DDR2-mutant lung SCC.

Phosphoproteomics Reveals MAPK Inhibitors Enhance MET- and EGFR-Driven AKT Signaling in KRAS-Mutant Lung Cancer

Pathway inhibition of the RAS-driven MAPK pathway using small-molecule kinase inhibitors has been a key focus for treating cancers driven by oncogenic RAS, yet significant clinical responses are lacking. Feedback reactivation of ERK driven by drug-induced RAF activity has been suggested as one of the major drug resistance mechanisms, especially in the context of oncogenic RAS. To determine whether additional adaptive resistance mechanisms may coexist, we characterized global phosphoproteomic changes after MEK inhibitor selumetinib (AZD6244) treatment in KRAS-mutant A427 and A549 lung adenocarcinoma cell lines employing mass spectrometry–based phosphoproteomics. We identified 9,075 quantifiable unique phosphosites (corresponding to 3,346 unique phosphoproteins), of which 567 phosphosites were more abundant and 512 phosphosites were less abundant after MEK inhibition. Selumetinib increased phosphorylation of KSR-1, a scaffolding protein required for assembly of MAPK signaling complex, as well as altered phosphorylation of GEF-H1, a novel regulator of KSR-1 and implicated in RAS-driven MAPK activation. Moreover, selumetinib reduced inhibitory serine phosphorylation of MET at Ser985 and potentiated HGF- and EGF-induced AKT phosphorylation. These results were recapitulated by pan-RAF (LY3009120), MEK (GDC0623), and ERK (SCH772984) inhibitors, which are currently under early-phase clinical development against RAS-mutant cancers. Our results highlight the unique adaptive changes in MAPK scaffolding proteins (KSR-1, GEF-H1) and in RTK signaling, leading to enhanced PI3K–AKT signaling when the MAPK pathway is inhibited. Implications: This study highlights the unique adaptive changes in MAPK scaffolding proteins (KSR-1, GEF-H1) and in RTK signaling, leading to enhanced PI3K/AKT signaling when the MAPK pathway is inhibited. Mol Cancer Res; 14(10); 1019–29. ©2016 AACR.

Collateral sensitivity networks reveal evolutionary instability and novel treatment strategies in ALK mutated non-small cell lung cancer

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.

Target Identification in Small Cell Lung Cancer via Integrated Phenotypic Screening and Activity-Based Protein Profiling

To overcome hurdles in identifying key kinases in small cell lung cancer (SCLC), we integrated a target-agnostic phenotypic screen of kinase inhibitors with target identification using activity-based protein profiling (ABPP) in which a desthiobiotin-ATP probe was used. We screened 21 SCLC cell lines with known c-MYC amplification status for alterations in viability using a chemical library of 235 small-molecule kinase inhibitors. One screen hit compound was interrogated with ABPP, and, through this approach, we reidentified Aurora kinase B as a critical kinase in MYC-amplified SCLC cells. We next extended the platform to a second compound that had activity in SCLC cell lines lacking c-MYC amplification and identified TANK-binding kinase 1, a kinase that affects cell viability, polo-like kinase-1 signaling, G2–M arrest, and apoptosis in SCLC cells lacking MYC amplification. These results demonstrate that phenotypic screening combined with ABPP can identify key disease drivers, suggesting that this approach, which combines new chemical probes and disease cell screens, has the potential to identify other important targets in other cancer types. Mol Cancer Ther; 15(2); 334–42. ©2016 AACR.

PAXIP1 Potentiates the Combination of WEE1 Inhibitor AZD1775 and Platinum Agents in Lung Cancer

The DNA damage response (DDR) involves a complex network of signaling events mediated by modular protein domains such as the BRCA1 C-terminal (BRCT) domain. Thus, proteins that interact with BRCT domains and are a part of the DDR constitute potential targets for sensitization to DNA-damaging chemotherapy agents. We performed a pharmacologic screen to evaluate 17 kinases, identified in a BRCT-mediated interaction network as targets to enhance platinum-based chemotherapy in lung cancer. Inhibition of mitotic kinase WEE1 was found to have the most effective response in combination with platinum compounds in lung cancer cell lines. In the BRCT-mediated interaction network, WEE1 was found in complex with PAXIP1, a protein containing six BRCT domains involved in transcription and in the cellular response to DNA damage. We show that PAXIP1 BRCT domains regulate WEE1-mediated phosphorylation of CDK1. Furthermore, ectopic expression of PAXIP1 promotes enhanced caspase-3–mediated apoptosis in cells treated with WEE1 inhibitor AZD1775 (formerly, MK-1775) and cisplatin compared with cells treated with AZD1775 alone. Cell lines and patient-derived xenograft models expressing both PAXIP1 and WEE1 exhibited synergistic effects of AZD1775 and cisplatin. In summary, PAXIP1 is involved in sensitizing lung cancer cells to the WEE1 inhibitor AZD1775 in combination with platinum-based treatment. We propose that WEE1 and PAXIP1 levels may be used as mechanism-based biomarkers of response when WEE1 inhibitor AZD1775 is combined with DNA-damaging agents. Mol Cancer Ther; 15(7); 1669–81. ©2016 AACR.

Polypharmacology-based ceritinib repurposing using integrated functional proteomics

Targeted drugs are effective when they directly inhibit strong disease drivers, but only a small fraction of diseases feature defined actionable drivers. Alternatively, network-based approaches can uncover new therapeutic opportunities. Applying an integrated phenotypic screening, chemical and phosphoproteomics strategy, here we describe the anaplastic lymphoma kinase (ALK) inhibitor ceritinib as having activity across several ALK-negative lung cancer cell lines and identify new targets and network-wide signaling effects. Combining pharmacological inhibitors and RNA interference revealed a polypharmacology mechanism involving the noncanonical targets IGF1R, FAK1, RSK1 and RSK2. Mutating the downstream signaling hub YB1 protected cells from ceritinib. Consistent with YB1 signaling being known to cause taxol resistance, combination of ceritinib with paclitaxel displayed strong synergy, particularly in cells expressing high FAK autophosphorylation, which we show to be prevalent in lung cancer. Together, we present a systems chemical biology platform for elucidating multikinase inhibitor polypharmacology mechanisms, subsequent design of synergistic drug combinations, and identification of mechanistic biomarker candidates.