Mariette Labots

Strategies for kinome profiling in cancer and potential clinical applications: chemical proteomics and array-based methods

AbstractKinases are key enzymes involved in deregulated signal transduction associated with cancer development and progression. The advent of personalized medicine drives the development of new diagnostic tools for patient stratification and therapy selection Ginsburg and Willard (Transl Res 154:277-287, 2009). Since deregulation of kinase-mediated signal transduction is implied in tumorigenesis, the analysis of all kinases (the kinome) active in a particular tumor may yield tumor-specific information on aberrant cell signalling pathways. Tumor tissue kinase activity profiles may correlate with response to therapy and therefore may be used for future therapy selection. In this Trend paper we describe peptide array and mass spectrometry-based technologies and new developments for kinome profiling, and we present an outlook towards future implementation of therapy selection based on kinome profiling in clinical practice.n FigureProtein kinases and interacting proteins are enriched by immobilized kinase inhibitor affinity capture, and their identity and phosphorylation sites are identified by mass spectrometry. Kinases that are differential between responders to therapy and non-responders can be used to design custom peptide substrate arrays to detect kinase-mediated aberrant signalling in cancer tissues. These custom peptide substrate arrays can be used, when validated, for therapy selection in the clinic.

Evaluation of different phospho-tyrosine antibodies for label-free phosphoproteomics☆

BACKGROUNDnMass spectrometry based phosphoproteomics emerged as advantageous approach for the analysis of tyrosine phosphorylation on proteins and tyrosine kinase signaling. Immunoaffinity purification is required for comprehensive analysis. Here we compared the performance of two antibodies for label-free phosphotyrosine-based phosphoproteomics.nnnMETHODSnPhosphopeptide immunoprecipitation of six technical replicates corresponding to 10mg protein from HCT116 cells was performed using agarose bead-coupled phosphotyrosine antibodies P-Tyr-1000 (N=3) and 4G10 (N=3). NanoLC-MS/MS was performed using a Q Exactive mass spectrometer. For relative quantitation of protein phosphorylation, spectral counts of phosphoproteins and ion intensities of phosphopeptides were determined using MaxQuant.nnnRESULTSnFrom the 3 samples incubated with P-Tyr-1000 a total of 689 phosphopeptides were identified with 60% ID reproducibility. The phosphopeptide capture using 4G10 resulted in a total of 421 at 46% ID reproducibility. The P-Tyr-1000 was applied to EGFR mutated U87 cells. Erlotinib reduced EGFR phosphorylation with 59% at y978, y1125, y1138, y1172, and y1197. EGFR inhibition was accompanied by enhanced phosphorylation of FYN, MET, PTK2, DYRK1A, MAPK1 and EPHA2.nnnCONCLUSIONnThe P-Tyr-1000 phosphotyrosine antibody performs superiorly when compared to 4G10 antibody for label-free phosphotyrosine-based phosphoproteomics. This workflow allows evaluation of drug target phosphorylation and may give insights in the pharmacodynamic effects of tyrosine kinase inhibitors.nnnCLINICAL SIGNIFICANCEnIn the past decade multiple tyrosine kinase inhibitors (TKIs) have been implemented in standard treatment regimens for patients with cancer. Unfortunately the majority of patients develops resistance to these drugs. Reliable tools for analysis of pharmacodynamic effects and drug resistance mechanisms are therefore warranted. Phosphoproteomic analyses have meanwhile emerged as a sophisticated approach for the determination of protein phosphorylation. These analyses rely on antibodies for enrichment of tyrosine-phosphorylated peptides. Here we compared two commercially available phosphotyrosine antibodies and show that P-Tyr-1000 yields 64% more phosphopeptides than 4G10 antibody, while including almost all 4G10 captured phosphopeptides. The workflow can be reproducibly performed at intermediate protein input levels of 10mg. Furthermore, application of the P-Tyr-1000 antibody in a standardized phosphoproteomics workflow allows relative quantitation of drug target inhibition and provides insights in alternative signaling pathways in cancer cells. This article is part of a Special Issue entitled: HUPO 2014.

Mass Spectrometry-Based Serum and Plasma Peptidome Profiling for Prediction of Treatment Outcome in Patients With Solid Malignancies

INTRODUCTIONnTreatment selection tools are needed to enhance the efficacy of targeted treatment in patients with solid malignancies. Providing a readout of aberrant signaling pathways and proteolytic events, mass spectrometry-based (MS-based) peptidomics enables identification of predictive biomarkers, whereas the serum or plasma peptidome may provide easily accessible signatures associated with response to treatment. In this systematic review, we evaluate MS-based peptide profiling in blood for prompt clinical implementation.nnnMETHODSnPubMed and Embase were searched for studies using a syntax based on the following hierarchy: (a) blood-based matrix-assisted or surface-enhanced laser desorption/ionization time-of-flight MS peptide profiling (b) in patients with solid malignancies (c) prior to initiation of any treatment modality, (d) with availability of outcome data.nnnRESULTSnThirty-eight studies were eligible for review; the majority were performed in patients with non-small cell lung cancer (NSCLC). Median classification prediction accuracy was 80% (range: 66%-93%) in 11 models from 14 studies reporting an MS-based classification model. A pooled analysis of 9 NSCLC studies revealed clinically significant median progression-free survival in patients classified as poor outcome and good outcome of 2.0 ± 1.06 months and 4.6 ± 1.60 months, respectively; median overall survival was also clinically significant at 4.01 ± 1.60 months and 10.52 ± 3.49 months, respectively.nnnCONCLUSIONnPretreatment MS-based serum and plasma peptidomics have shown promising results for prediction of treatment outcome in patients with solid tumors. Limited sample sizes and absence of signature validation in many studies have prohibited clinical implementation thus far. Our pooled analysis and recent results from the PROSE study indicate that this profiling approach enables treatment selection, but additional prospective studies are warranted.

Feasibility of label-free phosphoproteomics and application to base-line signaling of colorectal cancer cell lines.

UNLABELLEDnRobust phosphopeptide enrichment methods with minimal fractionation are required to profile signaling network analysis in cancer cell lines and tissues. We assessed performance of single-shot LC-MS/MS label-free phosphoproteomics using TiOx-based phosphopeptide enrichment and report phosphopeptide identification reproducibility (75.8%), depth of identification (6014-6150 phosphopeptides) and reproducibility of label-free quantification (CV 17.8%). Subsequently, we have profiled the baseline global phosphorylation of 8 colorectal cancer (CRC) cell lines representing different CRC prognostic subtypes. Global single-shot phosphoproteomics can distinguish CRC subtypes previously identified by transcriptomics and identifies signaling proteins and processes associated with the CCS3 poor prognosis subtype. Data are available via ProteomeXchange with identifiers PXD001546 and PXD001550.nnnBIOLOGICAL SIGNIFICANCEnLabel-free single-shot phosphoproteomics is a mature workflow that can be used for global quantitative profiling of biological cell lines and tissues to map signaling networks in comparative analyses. Here we show the feasibility of label-free profiling of CRC cell lines at sample input levels compatible with clinical samples such as tumor biopsies. This article is part of a Special Issue entitled: HUPO 2014.

High-level copy number gains of established and potential drug target genes in gastric cancer as a lead for treatment development and selection

PurposeThe overall survival rate of patients with advanced gastric cancer is poor. Therefore, there is an urgent need for new treatment options for these patients. The identification of drug target genes located on DNA regions exhibiting high-level copy number gains (CNG) may be an effective approach, as has e.g. previously been shown for HER2. The aim of the present study was to identify putative drug targets in patients with gastric cancer by applying this strategy.MethodsGenome-wide array comparative genomic hybridization (array CGH) data available from 183 primary gastric cancer samples were analyzed through Ingenuity Pathway Analysis (IPA) to assess whether any established or potential anticancer drug target genes showed high-level CNG, including focal amplifications.ResultsA total of 147 high-level gained regions were identified in the gastric cancer samples, harboring 167 genes that had previously been annotated as drug target genes. Thirty (18xa0%) of these genes showed high-level gains in at least 2xa0% of the tumors. The identified drug target genes included those for drugs known to be active in advanced (gastric) cancer, targets for targeted therapies in clinical development, as well as targets for drugs currently used for other indications but of potential interest for anticancer treatment. In addition, 12 potential drug target genes were identified, including genes involved in growth factor signaling and cell cycle regulation.ConclusionThe majority of gastric cancers carried one or more high-level CNGs or focal amplifications encompassing putative drug target genes. A number of the associated drugs are currently not being considered for treatment of gastric cancer. Based on these results we hypothesize that DNA copy number profiling may be a useful tool to identify new drug targets and to guide individualized treatment strategies in patients with gastric cancer.

Phosphotyrosine-based-phosphoproteomics scaled-down to biopsy level for analysis of individual tumor biology and treatment selection

Mass spectrometry-based phosphoproteomics of cancer cell and tissue lysates provides insight in aberrantly activated signaling pathways and potential drug targets. For improved understanding of individual patients tumor biology and to allow selection of tyrosine kinase inhibitors in individual patients, phosphoproteomics of small clinical samples should be feasible and reproducible. We aimed to scale down a pTyr-phosphopeptide enrichment protocol to biopsy-level protein input and assess reproducibility and applicability to tumor needle biopsies. To this end, phosphopeptide immunoprecipitation using anti-phosphotyrosine beads was performed using 10, 5 and 1mg protein input from lysates of colorectal cancer (CRC) cell line HCT116. Multiple needle biopsies from 7 human CRC resection specimens were analyzed at the 1mg-level. The total number of phosphopeptides captured and detected by LC-MS/MS ranged from 681 at 10mg input to 471 at 1mg HCT116 protein. ID-reproducibility ranged from 60.5% at 10mg to 43.9% at 1mg. Per 1mg-level biopsy sample, >200 phosphopeptides were identified with 57% ID-reproducibility between paired tumor biopsies. Unsupervised analysis clustered biopsies from individual patients together and revealed known and potential therapeutic targets.nnnSIGNIFICANCEnThis study demonstrates the feasibility of label-free pTyr-phosphoproteomics at the tumor biopsy level based on reproducible analyses using 1mg of protein input. The considerable number of identified phosphopeptides at this level is attributed to an effective down-scaled immuno-affinity protocol as well as to the application of ID propagation in the data processing and analysis steps. Unsupervised cluster analysis reveals patient-specific profiles. Together, these findings pave the way for clinical trials in which pTyr-phosphoproteomics will be performed on pre- and on-treatment biopsies. Such studies will improve our understanding of individual tumor biology and may enable future pTyr-phosphoproteomics-based personalized medicine.

INKA, an integrative data analysis pipeline for phosphoproteomic inference of active phosphokinases

Identifying (hyper)active kinases in cancer patient tumors is crucial to enable individualized treatment with specific inhibitors. Conceptually, kinase activity can be gleaned from global protein phosphorylation profiles obtained with mass spectrometry-based phosphoproteomics. A major challenge is to relate such profiles to specific kinases to identify (hyper)active kinases that may fuel growth/progression of individual tumors. Approaches have hitherto focused on phosphorylation of either kinases or their substrates. Here, we combine kinase-centric and substrate-centric information in an Integrative Inferred Kinase Activity (INKA) analysis. INKA utilizes label-free quantification of phosphopeptides derived from kinases, kinase activation loops, kinase substrates deduced from prior experimental knowledge, and kinase substrates predicted from sequence motifs, yielding a single score. This multipronged, stringent analysis enables ranking of kinase activity and visualization of kinase-substrate relation networks in a biological sample. As a proof of concept, INKA scoring of phosphoproteomic data for different oncogene-driven cancer cell lines inferred top activity of implicated driver kinases, and relevant quantitative changes upon perturbation. These analyses show the ability of INKA scoring to identify (hyper)active kinases, with potential clinical significance.

Selection of Protein Kinase Inhibitors Based on Tumor Tissue Kinase Activity Profiles in Patients with Refractory Solid Malignancies: An Interventional Molecular Profiling Study

Abstract Lessons Learned. Clinically applicable tools are needed for treatment selection and repurposing of available protein kinase inhibitors (PKIs) in patients with advanced solid tumors refractory to standard treatment. Using a tyrosine kinase peptide substrate microarray, observed inhibitory activity in vitro could not sufficiently predict clinical benefit of treatment with the selected PKI. Background. This exploratory molecular profiling study determined the feasibility and benefit of the selection of protein kinase inhibitors (PKIs) based on kinase activity profiling in patients with refractory solid malignancies. Methods. Adult patients with biopsy‐accessible refractory solid tumors were eligible. Per patient, the inhibitory potency of sunitinib, dasatinib, erlotinib, sorafenib, everolimus, and lapatinib was determined in tumor lysates from fresh biopsies using a tyrosine kinase peptide substrate microarray. The most active PKI in this in vitro assay was selected for treatment. Results. Thirteen patients were enrolled in the feasibility part and underwent tumor biopsy. Of 12 patients in whom kinase activity profiling was performed, 11 started treatment with a selected PKI: dasatinib in 8, sunitinib in 2, and erlotinib in 1 patient(s). Eight patients were evaluable for response. One patient had stable disease (SD) >4 months on sunitinib; one patient had SD at 6 weeks but progressive disease (PD) at 12 weeks. The remaining patients had PD after 6 weeks of treatment. Conclusion. Kinase inhibition profiles of multiple PKIs can be reliably determined using fresh tumor biopsies from patients with refractory solid tumors. However, the current in vitro microarray selection approach insufficiently predicted clinical benefit of PKI treatment in these patients.

Abstract 1621: Short-time exposure to high concentrations of sunitinib causes tumor cell death.

Background: Sunitinib, although initially developed as an antiangiogenic agent, has been shown to exert direct anti-tumor effects. Previously we demonstrated that sunitinib inhibits proliferation of various tumor cell lines in a dose-dependent manner. We here studied whether short exposure to high peak concentrations of sunitinib would induce tumor cell death. Methods: 786-O renal cancer cells were incubated with increasing concentrations of sunitinib (5, 10 and 20 μM) for various time intervals (1 hour, 3, 6, 9, 24, 48, 72 hours). At prespecified time points, sunitinib was removed and cells were subsequently allowed to grow in drug-free culture medium. Viability of these cells one week after short time sunitinib exposure and sensitivity of recovered cells after repetitive treatment with high sunitinib concentrations were determined by MTT assays. Flow cytometry with Propidium Iodide staining was employed to determine apoptosis. Results: Short-time exposure to high concentrations of sunitinib significantly induced cell death. Exposure to 20 μM of sunitinib for 9 hours provoked cell death in a manner identical to the prolonged exposure of 24, 48 or 72 hours. Flow cytometry confirmed the significant increase in cell death (60%) after 9-hour treatment with 20 μM sunitinib, compared to the untreated control (2%). Tumor cells that were repetitively treated with high concentrations of sunitinib retain their sensitivity to the drug identical to its parental cell line. Analysis of the key signaling pathways involved in sunitinib-induced cell death is ongoing. Conclusion: We here report that repetitive short-time exposure to high concentration sunitinib causes cell death without inducing resistance. Interestingly, single-dose sunitinib up to 300 mg was previously reported to be well tolerated by patients. Based on these findings, administration of high dose intermittent sunitinib may result in cytotoxic antitumor activity irrespective of angiogenesis inhibition. A phase I trial to test this hypothesis in patients with cancer will start shortly. Citation Format: Maria Rovithi, Dennis Poel, Jens Voortman, Mariette Labots, Henk Dekker, Kristy J. Gotink, Henk J. Broxterman, Henk M.W. Verheul. Short-time exposure to high concentrations of sunitinib causes tumor cell death. [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 1621. doi:10.1158/1538-7445.AM2013-1621

Abstract 3608: Measurement of kinase activity in cancer cell lines and tumor tissue using a tyrosine kinase peptide substrate array

Introduction Tyrosine kinases play an important role in tumor biology. Their activity can be measured using a kinase peptide substrate array consisting of 144 Tyr residue-containing peptides (PamChip®, PamGene, Den Bosch, The Netherlands). We evaluated this platform for the measurement of kinase activity in tumor tissue and cancer cell lines under various experimental conditions. Methods Lysates of colorectal and renal cancer cell lines, HCT116 and 786-0 respectively, were made using both Mammalian and Tissue Protein Extraction Reagent (M-PER and T-PER, Thermo Scientific) and Radio-Immunoprecipitation Assay (RIPA, home made) buffer. Lysates from patient-derived tumor tissues were prepared by adding T-PER to several 10 μm cryoslides containing >50% tumor. After lysate incubation with reaction buffer containing a fluorescent labeled antibody against phospo-tyrosine and ATP, kinase activity profiles were determined on kinetics of recorded peptide substrate phosphorylation intensities. The effect of protein and ATP concentration, different lysis buffers and number of freeze-thaw cycles on basal kinase activity was studied. Sunitinib, sorafenib and dasatinib, clinically available tyrosine kinase inhibitors (TKIs), were used to differentially inhibit kinase activity in the lysates. Results Application of 2.5-15 µg protein in 40 µl sample mix per array revealed linearly increasing phosphorylation signal intensities and initial velocities (Vini) of the kinetic curve (R2 = 0.98). Increasing ATP concentrations induced phosphorylation signal intensities, but above 400 µM the curve deviated from linearity. Basal kinase activity profiles of cell lines and tumor tissues were reproducible with CV9s below 15%, with good signal-to-background ratios and low aspecific binding. Different lysis buffers resulted in a maximum variation of phosphorylation signal intensity of 47±5.7% in both cell lines without affecting the actual profile. Quadruple freeze-thawing of lysates did not affect signal intensities by more than 10%. Inhibition profiles of treated vs. control lysates were reproducible within and between experiments, showing a higher and differential number of inhibited peptides at increasing TKI concentrations. In contrast to the ATP-independent inhibition of dasatinib, ATP-dependent inhibition for sunitinib and sorafenib was demonstrated by combining a fixed drug concentration with increasing concentrations of ATP up to 800 µM. Conclusion Kinase activity in lysates from cancer cell lines and patient-derived tumor tissue can be reproducibly profiled with a tyrosine kinase peptide substrate array. In addition, TKIs show differential ATP-dependent inhibition profiles on this array. Taken together, we expect that array-based tumor kinase activity profiling may lead to specific TKI-phosphorylation fingerprints for personalized treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3608. doi:1538-7445.AM2012-3608