Extracellular vesicles released by cancer-associated fibroblast-induced myeloid-derived suppressor cells inhibit T-cell function.

Myeloid cells are known to play a crucial role in creating a tumor-promoting and immune suppressive microenvironment. Our previous study demonstrated that primary human monocytes can be polarized into immunosuppressive myeloid-derived suppressor cells (MDSCs) by cancer-associated fibroblasts (CAFs) in a 3D co-culture system. However, the molecular mechanisms underlying the immunosuppressive function of MDSCs, especially CAF-induced MDSCs, remain poorly understood. Using mass spectrometry-based proteomics, we compared cell surface protein changes among monocytes, in vitro differentiated CAF-induced MDSCs, M1/M2 macrophages, and dendritic cells, and identified an extracellular vesicle (EV)-mediated secretory phenotype of MDSCs. Functional assays using an MDSC/T-cell co-culture system revealed that blocking EV generation in CAF-induced MDSCs reversed their ability to suppress T-cell proliferation, while EVs isolated from CAF-induced MDSCs directly inhibited T-cell function. Furthermore, we identified fructose bisphosphatase 1 (FBP1) as a cargo protein that is highly enriched in EVs isolated from CAF-induced MDSCs, and pharmacological inhibition of FBP1 partially reversed the suppressive phenotype of MDSCs. Our findings provide valuable insights into the cell surface proteome of different monocyte-derived myeloid subsets and uncover a novel mechanism underlying the interplay between CAFs and myeloid cells in shaping a tumor-permissive microenvironment.

Decrypting drug actions and protein modifications by dose- and time-resolved proteomics.

Although most cancer drugs modulate the activities of cellular pathways by changing posttranslational modifications (PTMs), little is known regarding the extent and the time- and dose-response characteristics of drug-regulated PTMs. In this work, we introduce a proteomic assay called decryptM that quantifies drug-PTM modulation for thousands of PTMs in cells to shed light on target engagement and drug mechanism of action. Examples range from detecting DNA damage by chemotherapeutics, to identifying drug-specific PTM signatures of kinase inhibitors, to demonstrating that rituximab kills CD20-positive B cells by overactivating B cell receptor signaling. DecryptM profiling of 31 cancer drugs in 13 cell lines demonstrates the broad applicability of the approach. The resulting 1.8 million dose-response curves are provided as an interactive molecular resource in ProteomicsDB.

Chemoproteomic profiling to identify activity changes and functional inhibitors of DNA-binding proteins.

DNA-binding proteins are promising therapeutic targets but are notoriously difficult to drug. Here, we evaluate a chemoproteomic DNA interaction platform as a complementary strategy for parallelized compound profiling. To enable this approach, we determined the proteomic binding landscape of 92 immobilized DNA sequences. Perturbation-induced activity changes of captured transcription factors in disease-relevant settings demonstrated functional relevance of the enriched subproteome. Chemoproteomic profiling of >300 cysteine-directed compounds against a coverage optimized bead mixture, which specifically captures >150 DNA binders, revealed competition of several DNA-binding proteins, including the transcription factors ELF1 and ELF2. We also discovered the first compound that displaces the DNA-repair complex MSH2-MSH3 from DNA. Compound binding to cysteine 252 on MSH3 was confirmed using chemoproteomic reactive cysteine profiling. Overall, these results suggested that chemoproteomic DNA bead pull-downs enable the specific readout of transcription factor activity and can identify functional "hotspots" on DNA binders toward expanding the druggable proteome.

Predicting the structural basis of targeted protein degradation by integrating molecular dynamics simulations with structural mass spectrometry.

Targeted protein degradation (TPD) is a promising approach in drug discovery for degrading proteins implicated in diseases. A key step in this process is the formation of a ternary complex where a heterobifunctional molecule induces proximity of an E3 ligase to a protein of interest (POI), thus facilitating ubiquitin transfer to the POI. In this work, we characterize 3 steps in the TPD process. (1) We simulate the ternary complex formation of SMARCA2 bromodomain and VHL E3 ligase by combining hydrogen-deuterium exchange mass spectrometry with weighted ensemble molecular dynamics (MD). (2) We characterize the conformational heterogeneity of the ternary complex using Hamiltonian replica exchange simulations and small-angle X-ray scattering. (3) We assess the ubiquitination of the POI in the context of the full Cullin-RING Ligase, confirming experimental ubiquitinomics results. Differences in degradation efficiency can be explained by the proximity of lysine residues on the POI relative to ubiquitin.

Chemical Phosphoproteomics Sheds New Light on the Targets and Modes of Action of AKT Inhibitors.

Due to its important roles in oncogenic signaling, AKT has been subjected to extensive drug discovery efforts leading to small molecule inhibitors investigated in advanced clinical trials. To better understand how these drugs exert their therapeutic effects at the molecular level, we combined chemoproteomic target affinity profiling using kinobeads and phosphoproteomics to analyze the five clinical AKT inhibitors AZD5363 (Capivasertib), GSK2110183 (Afuresertib), GSK690693, Ipatasertib, and MK-2206 in BT-474 breast cancer cells. Kinobead profiling identified between four and 29 nM targets for these compounds and showed that AKT1 and AKT2 were the only common targets. Similarly, measuring the response of the phosphoproteome to the same inhibitors identified ∼1700 regulated phosphorylation sites, 276 of which were perturbed by all five compounds. This analysis expanded the known AKT signaling network by 119 phosphoproteins that may represent direct or indirect targets of AKT. Within this new network, 41 regulated phosphorylation sites harbor the AKT substrate motif, and recombinant kinase assays validated 16 as novel AKT substrates. These included CEP170 and FAM83H, suggesting a regulatory function of AKT in mitosis and cytoskeleton organization. In addition, a specific phosphorylation pattern on the ULK1-FIP200-ATG13-VAPB complex was found to determine the active state of ULK1, leading to elevated autophagy in response to AKT inhibition.

Publisher Correction: A mass spectrometry-based proteome map of drug action in lung cancer cell lines.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Proteome activity landscapes of tumor cell lines determine drug responses.

Integrated analysis of genomes, transcriptomes, proteomes and drug responses of cancer cell lines (CCLs) is an emerging approach to uncover molecular mechanisms of drug action. We extend this paradigm to measuring proteome activity landscapes by acquiring and integrating quantitative data for 10,000 proteins and 55,000 phosphorylation sites (p-sites) from 125 CCLs. These data are used to contextualize proteins and p-sites and predict drug sensitivity. For example, we find that Progesterone Receptor (PGR) phosphorylation is associated with sensitivity to drugs modulating estrogen signaling such as Raloxifene. We also demonstrate that Adenylate kinase isoenzyme 1 (AK1) inactivates antimetabolites like Cytarabine. Consequently, high AK1 levels correlate with poor survival of Cytarabine-treated acute myeloid leukemia patients, qualifying AK1 as a patient stratification marker and possibly as a drug target. We provide an interactive web application termed ATLANTiC (http://atlantic.proteomics.wzw.tum.de), which enables the community to explore the thousands of novel functional associations generated by this work.

A mass spectrometry-based proteome map of drug action in lung cancer cell lines.

Mass spectrometry-based discovery proteomics is an essential tool for the proximal readout of cellular drug action. Here, we apply a robust proteomic workflow to rapidly profile the proteomes of five lung cancer cell lines in response to more than 50 drugs. Integration of millions of quantitative protein-drug associations substantially improved the mechanism of action (MoA) deconvolution of single compounds. For example, MoA specificity increased after removal of proteins that frequently responded to drugs and the aggregation of proteome changes across cell lines resolved compound effects on proteostasis. We leveraged these findings to demonstrate efficient target identification of chemical protein degraders. Aggregating drug response across cell lines also revealed that one-quarter of compounds modulated the abundance of one of their known protein targets. Finally, the proteomic data led us to discover that inhibition of mitochondrial function is an off-target mechanism of the MAP2K1/2 inhibitor PD184352 and that the ALK inhibitor ceritinib modulates autophagy.

Omomyc Reveals New Mechanisms To Inhibit the MYC Oncogene.

The MYC oncogene is upregulated in human cancers by translocation, amplification, and mutation of cellular pathways that regulate Myc. Myc/Max heterodimers bind to E box sequences in the promoter regions of genes and activate transcription. The MYC inhibitor Omomyc can reduce the ability of MYC to bind specific box sequences in promoters of MYC target genes by binding directly to E box sequences as demonstrated by chromatin immunoprecipitation (CHIP). Here, we demonstrate by both a proximity ligation assay (PLA) and double chromatin immunoprecipitation (ReCHIP) that Omomyc preferentially binds to Max, not Myc, to mediate inhibition of MYC-mediated transcription by replacing MYC/MAX heterodimers with Omomyc/MAX heterodimers. The formation of Myc/Max and Omomyc/Max heterodimers occurs cotranslationally; Myc, Max, and Omomyc can interact with ribosomes and Max RNA under conditions in which ribosomes are intact. Taken together, our data suggest that the mechanism of action of Omomyc is to bind DNA as either a homodimer or a heterodimer with Max that is formed cotranslationally, revealing a novel mechanism to inhibit the MYC oncogene. We find that in vivo, Omomyc distributes quickly to kidneys and liver and has a short effective half-life in plasma, which could limit its use in vivo.

Chemoproteomic Selectivity Profiling of PIKK and PI3K Kinase Inhibitors.

Chemical proteomic approaches utilizing immobilized, broad-selective kinase inhibitors (Kinobeads) have proven valuable for the elucidation of a compound's target profile under close-to-physiological conditions and often revealed potentially synergistic or toxic off-targets. Current Kinobeads enrich more than 300 native protein kinases from cell line or tissue lysates but do not systematically cover phosphatidylinositol 3-kinases (PI3Ks) and phosphatidylinositol 3-kinase-related kinases (PIKKs). Some PIKKs and PI3Ks show aberrant activation in many human diseases and are indeed validated drug targets. Here, we report the development of a novel version of Kinobeads that extends kinome coverage to these proteins. This is achieved by inclusion of two affinity probes derived from the clinical PI3K/MTOR inhibitors Omipalisib and BGT226. We demonstrate the utility of the new affinity matrix by the profiling of 13 clinical and preclinical PIKK/PI3K inhibitors. The large discrepancies between the PI3K affinity values obtained and reported results from recombinant assays led us to perform a phosphoproteomic experiment showing that the chemoproteomic assay is the better approximation of PI3K inhibitor action in cellulo. The results further show that NVP-BEZ235 is not a PI3K inhibitor. Surprisingly, the designated ATM inhibitor CP466722 was found to bind strongly to ALK2, identifying a new chemotype for drug discovery to treat fibrodysplasia ossificans progressiva.

The target landscape of clinical kinase drugs.

Kinase inhibitors are important cancer therapeutics. Polypharmacology is commonly observed, requiring thorough target deconvolution to understand drug mechanism of action. Using chemical proteomics, we analyzed the target spectrum of 243 clinically evaluated kinase drugs. The data revealed previously unknown targets for established drugs, offered a perspective on the "druggable" kinome, highlighted (non)kinase off-targets, and suggested potential therapeutic applications. Integration of phosphoproteomic data refined drug-affected pathways, identified response markers, and strengthened rationale for combination treatments. We exemplify translational value by discovering SIK2 (salt-inducible kinase 2) inhibitors that modulate cytokine production in primary cells, by identifying drugs against the lung cancer survival marker MELK (maternal embryonic leucine zipper kinase), and by repurposing cabozantinib to treat FLT3-ITD-positive acute myeloid leukemia. This resource, available via the ProteomicsDB database, should facilitate basic, clinical, and drug discovery research and aid clinical decision-making.

Pharmacoproteomic characterisation of human colon and rectal cancer.

Most molecular cancer therapies act on protein targets but data on the proteome status of patients and cellular models for proteome-guided pre-clinical drug sensitivity studies are only beginning to emerge. Here, we profiled the proteomes of 65 colorectal cancer (CRC) cell lines to a depth of > 10,000 proteins using mass spectrometry. Integration with proteomes of 90 CRC patients and matched transcriptomics data defined integrated CRC subtypes, highlighting cell lines representative of each tumour subtype. Modelling the responses of 52 CRC cell lines to 577 drugs as a function of proteome profiles enabled predicting drug sensitivity for cell lines and patients. Among many novel associations, MERTK was identified as a predictive marker for resistance towards MEK1/2 inhibitors and immunohistochemistry of 1,074 CRC tumours confirmed MERTK as a prognostic survival marker. We provide the proteomic and pharmacological data as a resource to the community to, for example, facilitate the design of innovative prospective clinical trials.

Preferential microRNA targeting revealed by in vivo competitive binding and differential Argonaute immunoprecipitation.

MicroRNAs (miRNAs) have been described to simultaneously inhibit hundreds of targets, albeit to a modest extent. It was recently proposed that there could exist more specific, exceptionally strong binding to a subgroup of targets. However, it is unknown, whether this is the case and how such targets can be identified. Using Argonaute2-ribonucleoprotein immunoprecipitation and in vivo competitive binding assays, we demonstrate for miRNAs-21, -199-3p and let-7 exceptional regulation of a subset of targets, which are characterized by preferential miRNA binding. We confirm this finding by analysis of independent quantitative proteome and transcriptome datasets obtained after miRNA silencing. Our data suggest that mammalian miRNA activity is guided by preferential binding of a small set of 3'-untranslated regions, thereby shaping a steep gradient of regulation between potential targets. Our approach can be applied for transcriptome-wide identification of such targets independently of the presence of seed complementary sequences or other predictors.

Two serines in the distal C-terminus of the human ß1-adrenoceptor determine ß-arrestin2 recruitment.

G protein-coupled receptors (GPCRs) undergo phosphorylation at several intracellular residues by G protein-coupled receptor kinases. The resulting phosphorylation pattern triggers arrestin recruitment and receptor desensitization. The exact sites of phosphorylation and their function remained largely unknown for the human ß1-adrenoceptor (ADRB1), a key GPCR in adrenergic signal transduction and the target of widely used drugs such as ß-blockers. The present study aimed to identify the intracellular phosphorylation sites in the ADRB1 and to delineate their function. The human ADRB1 was expressed in HEK293 cells and its phosphorylation pattern was determined by mass spectrometric analysis before and after stimulation with a receptor agonist. We identified a total of eight phosphorylation sites in the receptor's third intracellular loop and C-terminus. Analyzing the functional relevance of individual sites using phosphosite-deficient receptor mutants we found phosphorylation of the ADRB1 at Ser461/Ser462 in the distal part of the C-terminus to determine ß-arrestin2 recruitment and receptor internalization. Our data reveal the phosphorylation pattern of the human ADRB1 and the site that mediates recruitment of ß-arrestin2.

Lapatinib Resistance in Breast Cancer Cells Is Accompanied by Phosphorylation-Mediated Reprogramming of Glycolysis.

HER2/ERBB2-overexpressing breast cancers targeted effectively by the small-molecule kinase inhibitor lapatinib frequently acquire resistance to this drug. In this study, we employed explorative mass spectrometry to profile proteome, kinome, and phosphoproteome changes in an established model of lapatinib resistance to systematically investigate initial inhibitor response and subsequent reprogramming in resistance. The resulting dataset, which collectively contains quantitative data for >7,800 proteins, >300 protein kinases, and >15,000 phosphopeptides, enabled deep insight into signaling recovery and molecular reprogramming upon resistance. Our data-driven approach confirmed previously described mechanisms of resistance (e.g., AXL overexpression and PIK3 reactivation), revealed novel pharmacologically actionable targets, and confirmed the expectation of significant heterogeneity in molecular resistance drivers inducing distinct phenotypic changes. Furthermore, our approach identified an extensive and exclusively phosphorylation-mediated reprogramming of glycolytic activity, supported additionally by widespread changes of corresponding metabolites and an increased sensitivity towards glycolysis inhibition. Collectively, our multi-omic analysis offers deeper perspectives on cancer drug resistance and suggests new biomarkers and treatment options for lapatinib-resistant cancers. Cancer Res; 77(8); 1842-53. ©2017 AACR.

Optimized Enrichment of Phosphoproteomes by Fe-IMAC Column Chromatography.

Phosphorylation is among the most important post-translational modifications of proteins and has numerous regulatory functions across all domains of life. However, phosphorylation is often substoichiometric, requiring selective and sensitive methods to enrich phosphorylated peptides from complex cellular digests. Various methods have been devised for this purpose and we have recently described a Fe-IMAC HPLC column chromatography setup which is capable of comprehensive, reproducible, and selective enrichment of phosphopeptides out of complex peptide mixtures. In contrast to other formats such as StageTips or batch incubations using TiO2 or Ti-IMAC beads, Fe-IMAC HPLC columns do not suffer from issues regarding incomplete phosphopeptide binding or elution and enrichment efficiency scales linearly with the amount of starting material. Here, we provide a step-by-step protocol for the entire phosphopeptide enrichment procedure including sample preparation (lysis, digestion, desalting), Fe-IMAC column chromatography (column setup, operation, charging), measurement by LC-MS/MS (nHPLC gradient, MS parameters) and data analysis (MaxQuant). To increase throughput, we have optimized several key steps such as the gradient time of the Fe-IMAC separation (15 min per enrichment), the number of consecutive enrichments possible between two chargings (>20) and the column recharging itself (<1 h). We show that the application of this protocol enables the selective (>90 %) identification of more than 10,000 unique phosphopeptides from 1 mg of HeLa digest within 2 h of measurement time (Q Exactive Plus).

Hydrophilic Strong Anion Exchange (hSAX) Chromatography Enables Deep Fractionation of Tissue Proteomes.

The bottom-up proteomic analysis of cell line and tissue samples to a depth > 10,000 proteins still represents a considerable challenge because of the sheer number of peptides generated by proteolytic digestions and the high dynamic range of protein expression. As a result, comprehensive protein coverage requires multidimensional peptide separation. Recently, off-line hydrophilic strong cation exchange (hSAX) chromatography has proven its merits for high resolution separation of peptides due to its high degree of orthogonality to reversed-phase liquid chromatography. Here we describe the use of hSAX for the deep analysis of tissue proteomes. The protocol includes optimized sample preparation steps (lysis with the aid of mechanical disruption, one-step disulfide bridge reduction and alkylation), setup and operation of hSAX columns and gradients, desalting of hSAX fractions prior to LC-MS/MS analysis, and suggestions for the choice of data acquisition parameters and data analysis using MaxQuant. Application of the protocol to the fractionation of 300 µg human brain tissue digest led to the identification of more than 100,000 unique peptide sequences representing over 10,195 proteins and 9,500 genes in 3 days of measurement time on a Q Exactive Plus mass spectrometer.

High pH Reversed-Phase Micro-Columns for Simple, Sensitive, and Efficient Fractionation of Proteome and (TMT labeled) Phosphoproteome Digests.

Despite recent advances in mass spectrometric sequencing speed and improved sensitivity, the in-depth analysis of proteomes still widely relies on off-line peptide separation and fractionation to deal with the enormous molecular complexity of shotgun digested proteomes. While a multitude of methods has been established for off-line peptide separation using HPLC columns, their use can be limited particularly when sample quantities are scarce. In this protocol, we describe an approach which combines high pH reversed-phase peptide separation into few fractions in StageTip micro-columns. This miniaturized sample preparation method enhances peptide recovery and hence improves sensitivity. This is particularly useful when working with limited sample amounts obtained from e.g., phosphopeptide enrichments or tissue biopsies. Essentially the same approach can also be applied for multiplexed analysis using tandem mass tags (TMT) and can be parallelized in order to deliver the required throughput. Here, we provide a step-by-step protocol for TMT6plex labeling of peptides, the construction of StageTips, sample fractionation and pooling schemes adjusted to different types of analytes, mass spectrometric sample measurement, and downstream data processing using MaxQuant. To illustrate the expected results using this protocol, we provide results from an unlabeled and a TMT6plex labeled phosphopeptide sample leading to the identification of >17,000 phosphopeptides in 8 h (Q Exactive HF) and >23,000 TMT6plex labeled phosphopeptides (Q Exactive Plus) in 12 h of measurement time. Importantly, this protocol is equally applicable to the fractionation of full proteome digests.

Phosphoproteome Profiling Reveals Molecular Mechanisms of Growth-Factor-Mediated Kinase Inhibitor Resistance in EGFR-Overexpressing Cancer Cells.

Although substantial progress has been made regarding the use of molecularly targeted cancer therapies, resistance almost invariably develops and presents a major clinical challenge. The tumor microenvironment can rescue cancer cells from kinase inhibitors by growth-factor-mediated induction of pro-survival pathways. Here we show that epidermal growth factor receptor (EGFR) inhibition by Gefitinib is counteracted by growth factors, notably FGF2, and we assessed the global molecular consequences of this resistance at the proteome and phosphoproteome level in A431 cells. Tandem mass tag peptide labeling and quantitative mass spectrometry allowed the identification and quantification of 22 000 phosphopeptides and 8800 proteins in biological triplicates without missing values. The data show that FGF2 protects the cells from the antiproliferative effect of Gefitinib and largely prevents reprogramming of the proteome and phosphoproteome. Simultaneous EGFR/FGFR or EGFR/GSG2 (Haspin) inhibition overcomes this resistance, and the phosphoproteomic experiments further prioritized the RAS/MEK/ERK as well as the PI3K/mTOR axis for combination treatment. Consequently, the MEK inhibitor Trametinib prevented FGF2-mediated survival of EGFR inhibitor-resistant cells when used in combination with Gefitinib. Surprisingly, the PI3K/mTOR inhibitor Omipalisib reversed resistance mediated by all four growth factors tested, making it an interesting candidate for mitigating the effects of the tumor microenvironment.

Chemical Proteomics and Structural Biology Define EPHA2 Inhibition by Clinical Kinase Drugs.

The receptor tyrosine kinase EPHA2 (Ephrin type-A receptor 2) plays important roles in oncogenesis, metastasis, and treatment resistance, yet therapeutic targeting, drug discovery, or investigation of EPHA2 biology is hampered by the lack of appropriate inhibitors and structural information. Here, we used chemical proteomics to survey 235 clinical kinase inhibitors for their kinase selectivity and identified 24 drugs with submicromolar affinities for EPHA2. NMR-based conformational dynamics together with nine new cocrystal structures delineated drug-EPHA2 interactions in full detail. The combination of selectivity profiling, structure determination, and kinome wide sequence alignment allowed the development of a classification system in which amino acids in the drug binding site of EPHA2 are categorized into key, scaffold, potency, and selectivity residues. This scheme should be generally applicable in kinase drug discovery, and we anticipate that the provided information will greatly facilitate the development of selective EPHA2 inhibitors in particular and the repurposing of clinical kinase inhibitors in general.