Vikram Mitra

Quantification of pancreatic cancer proteome and phosphorylome: Indicates molecular events likely contributing to cancer and activity of drug targets

Objective LC-MS/MS phospho-proteomics is an essential technology to help unravel the complex molecular events that lead to and propagate cancer. We have developed a global phospho-proteomic workflow to determine activity of signaling pathways and drug targets in pancreatic cancer tissue for clinical application. 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 IMAC and TiO2, prior to analysis. In-house, commercial and freeware bioinformatic platforms were used to identify relevant biological events from the complex dataset. Results Of 2,101 proteins identified, 152 demonstrated significant difference in abundance 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 HIPK1 & MLCK. 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 guanine nucleotide exchange factors & MRCKα) and formation of focal adhesions. Activator phosphorylation sites on FYN, AKT1, ERK2, HDAC1 and other drug targets were found to be highly modulated (≥2 fold) in different cases highlighting their predictive power. Conclusion Here we provided critical information enabling us to identify the common and unique molecular events likely contributing to cancer in each case. Such information may be used to help predict more bespoke therapy suitable for an individual case.

Glycosylation of Human Plasma Clusterin Yields a Novel Candidate Biomarker of Alzheimer's Disease.

Specific glycosylated peptides of clusterin are found associated with hippocampal atrophy. The glycosylation of clusterin from human plasma was comprehensively analyzed and characterized using mass spectrometry (MS)-based glycoproteomics analysis. All six known N-glycosylation sites are covered, three in the alpha subunit (α64N, α81N and α123N) and three in the beta subunit (β64N, β127N, and β147N). More detailed structural characterization of clusterin glycopeptides was also performed, demonstrating the presence of glycosylated peptides and their corresponding glycans. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we have determined the differences in the glycoforms associated at each of the different glycosylation sites in plasma clusterin obtained from subjects of low hippocampal atrophy (n = 13) and high hippocampal atrophy (n = 14). In our pilot study, the β64N site shows the most significant regulations between clinical groups. Eight β64N glycoforms are significantly reduced in patients with high atrophy compared with those with low atrophy, which demonstrates the utility of clusterin isoforms as diagnostic and prognostic Alzheimers disease (AD) markers. These results provide a novel and robust workflow suitable for rapid verification of specific clusterin glycoforms with utility as AD biomarkers.

A global proteomic study identifies distinct pathological features of IgG4-related and primary sclerosing cholangitis.

This combined proteomic and histopathological study was aimed to compare tissue characteristics of immunoglobulin (Ig)G4‐related sclerosing cholangitis (ISC) and primary sclerosing cholangitis (PSC) in a global, non‐biased manner.

Tubulin β-III: a novel immunohistochemical marker for intrahepatic peripheral cholangiocarcinoma

Our recent proteomic study identified tubulin β‐III (TUBB3) as a potential tissue marker for intrahepatic cholangiocarcinomas (CCs). This validation study was conducted to see whether or not TUBB3 can serve as a novel immunohistochemical marker for peripheral CCs, using a large cohort (n = 197) covering various liver tumours and premalignant conditions.

Low-pH Solid-Phase Amino Labeling of Complex Peptide Digests with TMTs Improves Peptide Identification Rates for Multiplexed Global Phosphopeptide Analysis

We present a novel tandem mass tag solid-phase amino labeling (TMT-SPAL) protocol using reversible immobilization of peptides onto octadecyl-derivatized (C18) solid supports. This method can reduce the number of steps required in complex protocols, saving time and potentially reducing sample loss. In our global phosphopeptide profiling workflow (SysQuant), we can cut 24 h from the protocol while increasing peptide identifications (20%) and reducing side reactions. Solid-phase labeling with TMTs does require some modification to typical labeling conditions, particularly pH. It has been found that complete labeling equivalent to standard basic pH solution-phase labeling for small and large samples can be achieved on C18 resins under slightly acidic buffer conditions. Improved labeling behavior on C18 compared to that with standard basic pH solution-phase labeling is demonstrated. We analyzed our samples for histidine, serine, threonine, and tyrosine labeling to determine the degree of overlabeling and observed higher than expected levels (25% of all peptide spectral matches (PSMs)) of overlabeling at all of these amino acids (predominantly at tyrosine and serine) in our standard solution-phase labeling protocol. Overlabeling at all of these sites is greatly reduced (4-fold, to 7% of all PSMs) by the low-pH conditions used in the TMT-SPAL protocol. Overlabeling seems to represent a so-far overlooked mechanism causing reductions in peptide identification rates with NHS-activated TMT labeling compared to that with label-free methods. Our results also highlight the importance of searching data for overlabeling when labeling methods are used.

Abstract 4966: Phospho-proteome analyses confirm the unique mode of action of MP0274, an apoptosis inducing, biparatopic HER2-targeting DARPin®drug candidate

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

Correction to “Glycosylation of Human Plasma Clusterin Yields a Novel Candidate Biomarker of Alzheimer’s Disease”

■ ACKNOWLEDGMENTS We formally acknowledge the scientific contribution of our colleagues at Proteome Science plc and collaborators at the Institute of Psychiatry, King’s College London. We are grateful for grant funding from the Alzheimer’s Research U.K. and the NIHR Biomedical Research Centre for Mental Health and Biomedical Research Unit for Dementia at the South London and Maudsley NHS Foundation Trust and Kings College London. AddNeuroMed was funded through the EU FP6 program.

Abstract LB-C12: Functional proteomics elucidates signaling adaptation driven by combination therapy in BRAF mutant melanoma cell line models

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.

Discovery of multiple phosphorylated tau biomarkers in CSF using a novel mass spectrometry method

mutations (n1⁄418, CDR 1⁄4 0.5 in 12) or at 50% risk for inheriting such mutations (n1⁄425). Among the 31 mutation carriers (MCs), 26 had PSEN1 mutations and 5 had the V717I APP mutation. CSF was depleted of 14 high abundance proteins, digested with trypsin and the resulting peptides were analyzed by liquid chromatography-mass spectrometry (LCMS). Peptides were identified by shotgun sequencing and clustered into their parent proteins. Peptide peaks were aligned across all samples and peak intensities were measured and compared to determine relative peptide and protein abundance in each sample. Protein levels were separately compared between mutation non-carriers (NCs, n1⁄412) versus all ADADMCs and versus all presymptomaticMCs (n1⁄413).Results: 1,018 proteins were differentially quantified and identified. Using an intensity difference of 2-fold, and pand q-values (false discovery rate) of 0.05 as cut-offs for significance, we identified 12 proteins that were differentially expressed between ADAD MCs and NCs. Thrombospondin-2, LRP 11, NPDC1, Complement factor H, Fibronectin, Complement component C7, Transcription factor SOX-13, Clusterin, Fatty acid-binding protein, and APP were increased and Folate receptor alpha and CRP were decreased in ADAD MCs. An additional 22 proteins were differentially expressed with significant but smaller differences. When only presymptomatic ADAD mutation carriers were included, no protein was expressed with a 2-fold difference and only one (LRP1) was elevated with pand q-values 0.05. Conclusions: Proteins associated with the cell matrix, inflammation, vascular remodeling, and Abeta transport are elevated in ADAD MCs Only LRP1 was elevated in presymptomatic MCs, suggesting that its overexpression represents an adaptive response to Abeta overproduction.

Microglial-derived proteins in CSF are candidate biomarkers for early diagnosis of Alzheimer’s disease

and insula) and in temporal lobe. Volume of WMHs was distinctive of dementia status in YO but not in OO. Conclusions:The emphasis on decline in medial temporal and posterior brain regions in the non-demented OO is different from the documented “fronto-temporal” pattern in young elders and the temporo-parietal pattern in typical Alzheimer’s disease. Absence of dementia in the OO correlates with relatively preserved anterior cortex and the temporal lobe, which could inform mechanisms of cognitive maintenance in late life.