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Estrogen receptor alpha (ER) is the first biomarker to have been clinically validated as a predictor of cancer therapy response. Measurements of tumor ER expression were based on radiolabeled ligand binding to receptor present in tumor lysates. Despite substantial tissue requirement, lack of specificity for ER isoforms (alpha versus beta) or receptor integrity, quantitative determination of tumor ER content (fmol/mg total protein) was possible. Introduction of anti-ER antibodies later permitted immunohistochemical (***lHC) evaluation of ER expression. IHC determination of ER status in newly diagnosed breast cancer is now a standard of care. While these IHC assays have been shown to be as predictive of endocrine responsiveness as ligand binding assays, they remain semi-quantitative at best reporting tumor ER status either categorically (e.g. + or -) or as a numeric score which is subjective and lacks a linear relationship with endocrine responsiveness. Thus the lack of precision for quantifying ER as a predictive biomarker is one of the most important unresolved issues in breast cancer. We are working to develop a proteomic liquid chromatography-mass spectrometry (LC-MS) assay to help resolve this issue. Samples included recombinant ER (rER), immunoprecipitated (IP) rER, and IP ER from MCF7 cells. ER was digested with trypsin, lyophilised and solubilised in 5 femto-mol/microliter (100 μl) heavy peptide internal standard mix. ER peptides were resolved by LC (100 μl/minute) and detected by selected reaction monitoring MS. The area under the total ion chromatogram for each peptide were used to quantify the amount of analyte present in each sample as a single point reference to the signal of the heavy peptide spike. An 11 point calibration curve (0.1-1000 fmol on column (o/c)) of light peptides with each point in the curve spiked with 100 fmol heavy peptide was also produced to determine assay characteristics such as limits of detection (LOD), limits of quantification (LOQ), linearity, accuracy and precision. Three ER peptides were selected for quantification as they gave the greatest LOD, LOQ, linearity as well as reasonable intra- and inter-assay precision following multiple digestions of rER (intra = 3 digestions in 1 day; inter = 9 digestions over 3 weeks). Following IP of ER from four replicate MCF7 cell lysates (1mg/ml total protein) and measurement of ion intensities of the three ER peptides the mean concentration of ER was calculated to be 52 fmol (S.D of 7.5 fmol; n=4) per mg of total cell lysate after normalising for IP efficiency. We continue to develop the method to improve sensitivity and normalise for variability in IP and digestion. With the inclusion of reference peptides to known ER phosphorylation sites we are also in the process of quantifying ER phosphorylation. We aim to accurately determine ER concentration and phosphorylation status in tumor lysates and assess how these correlate with responsiveness to antiestrogen therapies. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-23.

Background: HER2 is an important target for antitumor therapy in several cancers, and while currently available HER2-targeting drugs provide a great treatment improvement they rarely achieve full disease control. We have developed a new HER2-targeting molecule with a unique pro-apoptotic mode of action that may provide additional benefit to patients. The DARPin® molecule MP0274 shows potency in several HER2-expressing PDX models and has been shown to directly induce apoptosis in cells that are addicted to HER2. Here we show through phospho-proteome analyses that MP0274 not only potently inhibits HER2/HER3 downstream signaling, but also shows a very distinct changes in the phospho-proteome pattern compared to approved HER2-targeting drugs. This provides mechanistic support to the unique mode of action of MP0274 that results in direct tumor cell killing without the need of effector-functions like ADCC. Methods: The effect of MP0274 on HER2 signaling was compared to trastuzumab, pertuzumab and a combination of both in the HER2-addicted cancer cell lines. Briefly, cells were incubated with drugs for 18 hours and then lysed. Lysates were analyzed for changes in the total proteome and phospho-proteome by Proteome Sciences’ proprietary SysQuant® Global Phosphoproteomics workflow. Results: Heat-maps of genes of interest indicate that MP0274 has a differential mode of action compared to trastuzumab, pertuzumab or a combination of both. On the total peptide and phospho-peptide level, the samples cluster specifically, based on the cancer cells used as well as drug treatment. Twenty nine unique global phosphorylation sites specific to HER2, including C-terminal tyrosines which are reported to recruit adaptor proteins starting signaling processes after auto-phosphorylation, were identified for MP0274. Several proteins were identified which were differentially expressed and phosphorylated after MP0274 treatment and which are involved in three key downstream signaling pathways activated by HER2/HER3 heterodimers: RAF/MAP kinase cascade, PI3K-induced AKT signaling, and signaling by PLCG1. Conclusions: MP0274 shows a unique and distinct inhibition of the HER2 signaling cascade, different from trastuzumab, pertuzumab and a combination of both. It induces a more profound inhibition of downstream signaling which provides mechanistic support to the finding that MP0274 direct cell killing by induction of apoptosis in HER2-addicted tumor cells. * DARPins are small repeat proteins, designed to bind targets with high affinity and specificity, and which can be combined in a modular fashion to produce multi-functional agents. Citation Format: Vikram Mitra, Ulrike Fiedler, Dan Snell, Keith M. Dawson, Stephan Jung, Ian Pike, Elmar vom Baur. Phospho-proteome analyses confirm the unique mode of action of MP0274, an apoptosis inducing, biparatopic HER2-targeting DARPin® drug candidate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4966. doi:10.1158/1538-7445.AM2017-4966

Targeted kinase inhibition is a promising treatment modality for melanoma, which has significantly increased available clinical strategies and improved survival outcomes for patients, whose tumors harbor the BRAF V600E mutation. However, responses are transient and multiple mechanisms for drug resistance can contribute to therapeutic escape. Therefore, a novel multiplexed proteomics approach provides an optimal choice for comprehensively dissecting multiple mechanisms of resistance to BRAFi and combination therapy within one experiment. A large-scale quantitative expression proteomics and phosphoproteomics analysis (SysQuant) was carried out on BRAF V600E mutant melanoma cell lines treated with two different clinically relevant kinase inhibitor combinations: 1) BRAFi/MEKi and 2) BRAFi/PI3Ki. Changes in protein expression and phosphorylation in response to each treatment are determined by comparison to vehicle controls. Cell line models all harbor BRAF V600E mutations, but differ in PTEN status (A375 is WT, while WM793 is PTEN null). Briefly, cells were treated with either drug combination (as above) and harvested at 1hr, 6hrs, 24hrs and 48hrs post-treatment. Control cells were treated with DMSO. Samples were lysed, reduced, alkylated and digested with trypsin. Tryptic peptides from each sample were chemically labeled or “barcoded” with TMT-10plex reagents (4 BRAFi/MEKi time points, 4 BRAFi/PI3Ki time points, 1 DMSO control and a pooled reference sample) and combined for LC-MS/MS. After peptide fractionation with strong cation exchange chromatography and phosphopeptide enrichment, LC-MS/MS peptide sequencing and relative quantification was performed using an Orbitrap Fusion mass spectrometer (Thermo). Raw MS data was searched by Proteome Discoverer and analyzed by in-house R-scripts and Perseus statistical software package. Pathway analysis was done in GeneGO (Metacore). The SysQuant workflow identified >9,000 protein groups and >17,000 unique phosphosites per cell line across different treatments. Principal component analysis of the phosphoproteomics data revealed signaling differences across different treatment conditions and drug combinations are mainly driven by BRAFi and the time post-treatment. K-means clustering was also used to examine trends in the data; as an example, this technique could be used to track signaling changes that correlate with reduction and recovery in ERK signaling. The SysQuant approach provides a systems view of global signaling changes occurring in response to drug treatment. The ability to multiplex samples with TMT allows quantitative deduction of protein expression and phosphorylation patterns that are common or unique in different cell lines, time post-treatment and the effect of combination treatments. This proteomics approach ties protein expression and phosphorylation status in response to combination therapy and generates several hypotheses for further testing with the goal of developing novel combination therapy strategies. Citation Format: Ritin Sharma, Inna Fedorenko, Sasa Koncarevic, Vikram Mitra, Stefan Selzer, Gitte Boehm, Ian Pike, Keiran Smalley, John M. Koomen. Functional proteomics elucidates signaling adaptation driven by combination therapy in BRAF mutant melanoma cell line models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C12.

Melanoma, the most lethal form of skin cancer, is marked by numerous genetic modifications, including point mutations as well as overexpression and deletion of genes. Intrinsic and acquired resistance to BRAF V600E targeted therapies (BRAFi) in metastatic melanoma patients further underscores the need for global profiling of melanoma circuitry at the functional level. Therefore, activity-based protein profiling (ABPP) and phosphoproteomics was carried out to decipher steady state differences in global signaling mechanisms in naïve and BRAFi resistant melanoma cell lines with BRAF V600E mutations. Four cell lines (A375, 1205Lu, WM164 and WM793) were selected to evaluate different molecular backgrounds of BRAF mutation based on their PTEN status (either WT or null). For each cell type, both the naïve and BRAFi resistant lines were analyzed via ABPP as well as chemical labeling with tandem mass tags (TMT) prior to discovery phosphoproteomics. While the ABPP approach mined for kinases, phosphoproteomics identified STY phosphorylated peptides providing information on signaling via kinase substrates. LC-MS/MS discovery proteomics (RSLC and Q Exactive, Thermo) identified and relatively quantified all peptides observed in ABPP and TMT phosphoproteomics experiments. MaxQuant was used for data evaluation; preliminary statistical analyses were performed in Perseus to select significant differences for pathway mapping (GeneGO, Metacore) and follow up experiments using siRNA or pharmacological inhibition. Adaptive responses to combination treatment were also explored using the SysQuant workflow for quantitative expression analysis and phosphoproteomics. These experiments served as a basis for comparison for a pilot project of 12 metastatic tumors from BRAF mutant melanoma patients selected for comparison of good and poor survival outcomes. ABPP measurements on different cell line models (A375, 1205Lu, WM793 and WM164) reveal significant differences in ATP uptake of proteins in the resistant cell line model compared to its naïve counterpart. For example, in the 1205Lu cell line several proteins including EGFR, p38alpha, DNA-PK formed an interconnected pathway. Overall we identified between 2,000-2,800 proteins in each cell line with ∼150 kinases. Isobaric labeling coupled to phosphoproteomics identified ∼1,600 quantifiable proteins with ∼4,000 phosphorylation sites. Phosphoproteomics revealed concomitant increase in phosphorylation levels of the substrates acted upon by kinases showing higher ATP uptake in ABPP measurements. For example, in 1205Lu BRAFi resistant cells, CDK1, CDK2 and DNA-PK showed higher ATP uptake and their substrates SSK1, DPYSL3, and vimentin showed higher phosphorylation levels. The complementary nature of the two functional proteomics approaches provided holistic overview of signalling network in melanoma and enabled selection of targets for follow-up studies. Citation Format: Ritin Sharma, Manali Phadke, David Britton, Ian Pike, Keiran Smalley, John M. Koomen. Kinases and adaptive signaling contribute to drug resistance in BRAF mutant melanoma. [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 2006. doi:10.1158/1538-7445.AM2015-2006

Introduction: Quantitative mass spectrometry assays using the Tandem Mass Tag (TMT) labeling strategy have been developed for measuring biologically relevant phosphorylation and mutation sites, which are not easily amenable to quantification in standard proteomics workflows. Quantification of the c-terminal peptide from HER2, which can be phosphorylated at Tyrosine 1248, reports on potential binding of Shc and activation of Ras signaling. In melanoma, N-Ras mutations at Q61 can occur de novo or develop as a resistance mechanism to B-Raf inhibition. However, tryptic peptides containing these specific sites have poor or no signal for liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) quantification. Different proteolysis strategies and TMT labeling enhanced the signal intensity of those specific peptides and enabled quantification of these candidate biomarkers. The quantification of target peptides with specific phosphorylation and mutation sites will help to understand cancer biology and may predict drug response to support individualized selection of therapy. Experimental Procedures: Model systems included breast cancer (MCF7, BT474) and melanoma (N-Ras WT M257, N-Ras Q61K M245, N-Ras Q61R 2032, N-Ras Q61L M318) cell lines inter al. Peptide-based quantitative assays were developed for HER2 expression and phosphorylation as well as N-Ras expression and mutation using TMT labeling prior to LC-MRM analysis. Endogenous peptides including wild type N-Ras and all known Q61 mutations have been synthesized and tested for TMT labeling efficiency with LC-MRM. Then, proteins were fractionated by SDS-PAGE from whole cell lysates. Different proteolytic strategies including chymotrypsin or Glu-C digestion were optimized for biomarker quantification. The assay characteristics were evaluated using serial dilutions of cell lysate before analysis of clinical specimens. Data Summary: LC-MRM assays for both unmodified and phosphorylated HER2 as well as wild type and mutant N-Ras have been developed and characterized using cell line models. TMT labeling efficiency has been evaluated under different conditions to accommodate different digestion buffers. Assays have been implemented to study these biomarkers. Conclusions: Proteomic quantification of cancer biomarkers defined by known biology, including expression, phosphorylation, and mutation status, has the potential to improve the selection of targeted therapeutics and prediction of drug response. Citation Format: Yi Chen, David J. Britton, Kim Paraiso, Inna Fedorenko, Elizabeth R. Wood, Anthony Magliocco, Vernon K. Sondak, Keiran Smalley, Ian Pike, John M. Koomen. Quantification of biomarker expression, phosphorylation, and mutation in cancer using TMT labeling prior to liquid chromatography-multiple reaction monitoring mass spectrometry. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2491. doi:10.1158/1538-7445.AM2014-2491

INTRODUCTION: LC-MS/MS proteomics is an essential technology to help unravel the complex molecular events that lead to and propagate cancer. We have developed an analytical workflow to quantify the expression and phosphorylation status of thousands of proteins simultaneously in frozen resected human pancreatic tumor (T), relative to matched non-tumor (NT) tissue. These measurements enabled us to determine activity of anti-neoplastic drug targets and other signaling proteins. METHODS: Peptides resulting from tryptic digestion of proteins extracted from frozen tissue of pancreatic ductal adenocarcinoma and background pancreas (n=12), were labelled with tandem mass tags (TMT 8-plex), separated by strong cation exchange chromatography, then were analysed by LC-MS/MS directly or first enriched for phosphopeptides using Fe3+ and TiO2, prior to analysis. In-house, commercial and freeware bio-informatics platforms were used to identify relevant biological events from the complex dataset. RESULTS: Of 2,101 proteins identified, 152 demonstrated significant difference in expression between tumor and non-tumor tissue. They included proteins that are known to be up-regulated in pancreatic cancer (e.g. Mucin-1), but the majority were new candidate markers such as homeodomain interacting protein kinase 1 & Myosin light chain kinase. Of the 6,543 unique phosphopeptides identified (6,284 unique phosphorylation sites), 635 showed significant regulation, particularly those from proteins involved in cell migration (Rho GTPase signaling proteins such as Rho guanine nucleotide exchange factors & Serine/threonine-protein kinase MRCKα) as well as proteins involved in disassembly of cell-cell junctions (tight junction, adherens junction) and formation of cell-extracellular matrix (ECM) junctions (focal adhesions). We quantified activator & inhibitory phosphorylation sites on FYN, AKT1, HDAC1&2, GSK3α&β, RAF kinases, MAPKs (p38, ERK1&2), PKCs, Casein Kinases and >20 others, as well as their downstream substrates some of which were often found to be highly modulated (≥ 2 fold) in T versus NT in different cases. CONCLUSION: Application of our LC-MS/MS proteomic workflow to frozen resected human pancreatic T versus NT tissue elucidated molecular events likely contributing to pancreatic cancer in each case, particularly those contributing to cell migration, and in future may help clinicians predict the best targeted anti-cancer therapy bespoke for an individual patient. Citation Format: David Britton, Yoh Zen, Stefan Selzer, Vikram Mitra, Alberto Quaglia, Debashis Sarker, Leandro Castellano, Justin Stebbing, Julia Gee, Rob Nicholson, Nigel Heaton, Ian Pike. Quantification of pancreatic cancer proteome & phosphorylome: Indicates molecular events likely contributing to cancer & activation status of drug targets. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1617. doi:10.1158/1538-7445.AM2014-1617

Introduction: A quantitative mass spectrometry platform has been developed for measuring biomarkers of protein expression and phosphorylation in breast cancer. The quantification of numerous biomarkers from a single tissue specimen has the potential to impact patient care. Specifically, the focus of initial development was on the expression and phosphorylation of estrogen receptor alpha (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2). However, immunohistochemical assessments of Ki-67 and vimentin are also widely used for monitoring proliferation in breast cancer. The integration of tissue quality control measurements, quantification of proliferation biomarkers, and ER/PR/HER2 evaluation could improve patient assessment and open novel avenues for patient classification, prognosis, and selection of therapy in breast cancer. Experimental Procedures: Breast cancer cell lines MCF7, BT474, and T47D were used to develop peptide-based quantitative assays, including stable isotope-labeled standard peptides, (n = 31) for protein expression and phosphorylation biomarkers using liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) of digested whole cell lysates or SDS-PAGE fractionated proteins followed by immunoprecipitation to examine expression and phosphorylation of ER/PR/HER2 (IP-LC-MRM). Using 2 mg of total protein, the sample is divided into three parts. Tissue QC biomarkers and expression levels of Ki67, vimentin, and HER2 are measured in digests of whole cell lysate or tissue homogenate (1% of sample). Then, parallel IP strategies are used for HER2 (10% of sample) and ER/PR (90%) before LC-MRM analysis of expression and phosphorylation. Multiplex IP of the three proteins was not successful, because the amplification of HER2 could not be accommodated. The sensitivity of each assay was tested using serial dilutions of cell lysate to evaluate requirements for analysis of clinical specimens. Data Summary: LC-MRM assays for both unmodified and phosphorylated peptides have been developed and characterized for each protein using cell line models. Assays have been implemented in cell lines to study Estradiol/EGF stimulation as well as lapatinib inhibition of HER2. Data has been generated for pre-treatment frozen tissue specimens from breast cancer patients that are either HER2+ or ER+ (n = 12 per group). The analysis of a larger group of patients and in clinical scenarios (e.g. comparison of naïve and drug resistant tissues from the same patient) will establish the clinical utility of these assays. Conclusions: The ability to examine protein expression as well as phosphorylation status in these biomarkers with quantitative mass spectrometry has the potential to improve the selection of targeted therapeutics. Citation Format: Yi Chen, David J. Britton, Elizabeth R. Wood, Anthony M. Magliocco, Ian Pike, John M. Koomen. Liquid chromatography-multiple reaction monitoring mass spectrometry biomarker quantification for breast cancer patient assessment. [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 2494. doi:10.1158/1538-7445.AM2013-2494

Introduction: Estrogen receptor alpha (ER alpha), progesterone receptor B (PR B), and the receptor tyrosine kinase, Her2, are tumor markers classified by the American Society of Clinical Oncology (ASCO) as level of evidence I in breast cancer. The quantitative evaluation of protein expression and phosphorylation status of these targets in tumor tissues will enable subtype classification and support clinical decision-making and selection of targeted therapy in breast cancer. Experimental Procedures: Breast cancer cell lines were used to develop peptide-based quantitative assays for protein expression and phosphorylation using immunoprecipitation (IP) and liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM). Stable isotope-labeled standard peptides were synthesized and developed as internal standards. Multiplexed IP for ER alpha, PR B, and HER2 will be developed to enable the enrichment of the proteins to enable quantification of the expression and phosphorylation of these biomarkers in the same sample. The sensitivity of each assay will be tested using serial dilutions of cell lysate to evaluate requirements for analysis of clinical specimens. Data Summary: LC-MRM assays for both unmodified and phosphorylated peptides have been developed and characterized for each protein. Recovery using multiplexed IP for these proteins is similar to recovery of individual IP. Data have been generated for cell line models of stimulation and treatment. For example, LC-MRM quantification of HER2 in cell line models stimulated with EGF or Heregulin shows that protein expression is unchanged, but phosphorylation increases; on the other hand, LC-MRM shows clearly that phosphorylation of Her2 is inhibited with Lapatinib. Conclusions: The ability to examine the protein expression as well as the phosphorylation status in these biomarkers with quantitative mass spectrometry will produce improved understanding of their role in the development and progression of breast cancer and has the potential to improve the selection of targeted therapeutics. 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 4810. doi:1538-7445.AM2012-4810

Introduction: The translation of genomic pre-disposition into a disease phenotype is driven primarily by protein expression and a complex network of post-translational modifications (PTM). One such PTM, phosphorylation, affords exquisite regulation of protein function by modulating protein folding, substrate affinity, stability, and activity and is proven to modulate the activity of thousands of onco- and tumor suppressor proteins. Phosphorylation-induced dysregulation of key checkpoints in signaling pathways that drive cell division, proliferation, differentiation, and apoptosis contributes to the cancer phenotype. A priori knowledge of which phosphorylation events are driving a particular tumor will assist in drug target validation, compound selection and ultimately treatment optimization for individual patients. We have developed a liquid chromatography mass spectrometry proteomics approach to monitor the activity of these signaling pathways in cancer cells. Experimental procedure: Peptides resulting from tryptic digestion of proteins from a human breast cancer cell line (BT474 +/− lapatinib, heregulin, lapatinib+heregulin), were labeled with tandem mass tags (TMT®) and passed through phospho-peptide enrichment columns (IMAC or TiO2). The enriched phospho-peptides were then analyzed by LC-MS/MS to identify and quantify the level of protein phosphorylation in each sample relative to a spiked internal reference sample. Data summary: In total we identified 4046 phosphorylation sites on 1054 phospho-proteins in BT474 cells under the various treatment conditions stated above. Lapatinib and heregulin were shown to significantly modulate phosphorylation on key proteins involved in 32 KEGG signaling pathways, including: the pancreatic cancer pathway (11 phospho-proteins), cell cycle pathway (18 phospho-proteins) and the ERBB signaling pathway, where we observed modulation of phosphorylation sites on 11 phospho-proteins including ERBB2 (pT701, pS703, pY877, pS1050/1051, pS1054, pS1083, pS1107, pS1166, pY1248 and 4 additional novel sites), AKT1 (pS124, pS126, pS129, pS137), and GSK3B (pS9, pY216 and an additional novel site). Conclusion: The global analysis of site-specific phosphorylation on key signaling pathways relevant to a specific cancer phenotype has been developed. Our initial data provide further insight into the on- and off-target effects of the ERBB1/ERBB2 tyrosine kinase inhibitor lapatinib. These data may help define optimal combination treatments in ERBB1/ERBB2 positive tumors. The application of this method to other tumor types and therapeutic agents is widely recommended. 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 LB-8. doi:1538-7445.AM2012-LB-8