Activity-based Proteomic Profiling Research Articles

Increasing the Throughput and Reproducibility of Activity-Based Proteome Profiling Studies with Hyperplexing and Intelligent Data Acquisition.

Intelligent data acquisition (IDA) strategies, such as a real-time database search (RTS), have improved the depth of proteome coverage for experiments that utilize isobaric labels and gas phase purification techniques (i.e., SPS-MS3). In this work, we introduce inSeqAPI, an instrument application programing interface (iAPI) program that enables construction of novel data acquisition algorithms. First, we analyze biotinylated cysteine peptides from ABPP experiments to demonstrate that a real-time search method within inSeqAPI performs similarly to an equivalent vendor method. Then, we describe PairQuant, a method within inSeqAPI designed for the hyperplexing approach that utilizes protein-level isotopic labeling and peptide-level TMT labeling. PairQuant allows for TMT analysis of 36 conditions in a single sample and achieves ∼98% coverage of both peptide pair partners in a hyperplexed experiment as well as a 40% improvement in the number of quantified cysteine sites compared with non-RTS acquisition. We applied this method in the ABPP study of ligandable cysteine sites in the nucleus leading to an identification of additional druggable sites on protein- and DNA-interaction domains of transcription regulators and on nuclear ubiquitin ligases.

ABPP-CoDEL: Activity-Based Proteome Profiling-Guided Discovery of Tyrosine-Targeting Covalent Inhibitors from DNA-Encoded Libraries.

DNA-encoded chemical library (DEL) has been extensively used for lead compound discovery for decades in academia and industry. Incorporating an electrophile warhead into DNA-encoded compounds recently permitted the discovery of covalent ligands that selectively react with a particular cysteine residue. However, noncysteine residues remain underexplored as modification sites of covalent DELs. Herein, we report the design and utility of tyrosine-targeting DELs of 67 million compounds. Proteome-wide reactivity analysis of tyrosine-reactive sulfonyl fluoride (SF) covalent probes suggested three enzymes (phosphoglycerate mutase 1, glutathione s-transferase 1, and dipeptidyl peptidase 3) as models of tyrosine-targetable proteins. Enrichment with SF-functionalized DELs led to the identification of a series of tyrosine-targeting covalent inhibitors of the model enzymes. In-depth mechanistic investigation revealed their novel modes of action and reactive ligand-accessible hotspots of the enzymes. Our strategy of combining activity-based proteome profiling and covalent DEL enrichment (ABPP-CoDEL), which generated selective covalent binders against a variety of target proteins, illustrates the potential use of this methodology in further covalent drug discovery.

A Simplified and Ultrafast Pipeline for Site-Specific Quantitative Chemical Proteomics.

Activity-based proteome profiling (ABPP) is a powerful chemoproteomic technology for global profiling of protein activity and modifications. The tandem orthogonal proteolysis-ABPP (TOP-ABPP) strategy utilizes a clickable enrichment tag with cleavable linkers to enable direct identification of probe-labeled residue sites within the target proteins. However, such a site-specific chemoproteomic workflow requires a long operation time and complex sample preparation procedures, limiting its wide applications. In the current study, we developed a simplified and ultrafast peptide enrichment and release TOP-ABPP ("superTOP-ABPP") pipeline for site-specific quantitative chemoproteomic analysis with special agarose resins that are functionalized with azide groups and acid-cleavable linkers. The azide groups allow enrichment of peptides that are labeled by the alkynyl probe through a one-step click reaction, which can be conveniently released by acid cleavage for subsequent LC-MS/MS analysis. In comparison with the traditional TOP-ABPP method, superTOP-ABPP cuts down the averaged sample preparation time from 25 to 9 h, and significantly improves the sensitivity and coverage of site-specific cysteinome profiling. The method can also be seamlessly integrated with reductive dimethylation to enable quantitative chemoproteomic analysis with a high accuracy. The simplified and ultrafast superTOP-ABPP will become a valuable tool for site-specific quantitative chemoproteomic studies.

A Multiomic Approach to Reversing Therapy Resistance in Multiple Myeloma Using Paired Ex Vivo Drug Sensitivity Measures and RNA Sequencing Data

A key challenge in multiple myeloma (MM) is evolution of resistance to standard-of-care (SOC) therapies such as proteasome inhibitors (PI), monoclonal antibodies, and novel targeted therapies. We developed a computational framework that identifies gene programs associated with SOC therapy resistance by pairing patient RNAseq data with clinical response and ex vivo drug response to find patient-specific targets to overcome therapy resistance. We hypothesize that there are two types of mechanisms of resistance in MM; dynamic (involving cell cycle) and cytostatic (cell-intrinsic, EMDR, complement, etc.). We inferred the dynamic mechanism of resistance by comparing transcriptomic profiles of therapy-naïve vs refractory MM patients, therapy-resistant vs wildtype cell lines, drug perturbation transcriptomics from LINCS-L1000 and activity-based proteomic profiling of MM cell lines. The cytostatic mechanism of resistance was inferred by associating RNAseq data with ex vivo drug response, where CD138+ cells from MM patient samples were co-cultured with human bone marrow stromal cells and patient plasma in an ex vivo reconstruction of the tumor microenvironment and tested with SOC drugs in 399 MM patient samples. The computational framework begins with the construction of a topological map of MM transcriptome using t-SNE and fuzzy c-means clustering on z-normalized expression of 16,738 genes across 844 MM patient samples, which yielded MM-specific gene programs (clusters of co-expressing genes). Using publicly available ChIP-seq databases like ENCODE and ChEA, we identified regulatory proteins (RPs) upstream of these gene programs. Kinases phosphorylating these RPs were identified from proteomic databases like PhosphoSitePlus and PhosphoPoint; generating a cascading GRN of MM gene programs, their RPs, and kinases, described by a biophysical model representing transcription, translation, and post-translational effects. The model estimates expression of each gene as a function of gene expression of its upstream RPs and kinases. The model for transcriptional regulation for each gene is trained using RNAseq data of 422 patients and validated against the rest of the cohort (422). Figure 1 is a graphical representation of gene-wise Pearson's correlation of model-predicted and actual gene expression represented by the color of each point (gene) on the tSNE. The set of genes that have poor correlation in Figure 1 (blue) statistically significantly (hypergeometric test; p-value <1e-48 and representation factor = 7.27) coincide with genes that are epigenetically regulated (identified by their high variability of chromatin accessibility across MM patients). We implement this computational framework to identify gene programs associated with a dynamic mechanism of PI (frontline therapy in MM) resistance, which yielded a GRN comprising of 218 co-expressing genes, 13 upstream RPs, and 45 kinases (17 druggable by kinase inhibitors). Network analysis of this GRN revealed that FOXM1 as a key therapeutic vulnerability (characterized by the highest betweenness centrality - a hub for flow of information) that can be targeted by inhibiting PLK1; a key regulator of cell cycle that phosphorylates FOXM1 that, in turn regulates PLK1 expression. A statistically significant (unpaired t-test) increase in z-normalized expression of PLK1 was observed in MM patients (n = 233) clinically refractory to CFZ (p-value < 0.0001) and BTZ (p-value = 0.0004) compared to PI-naïve. Ex vivo functional validation in a cohort of 61 MM patient samples shows a statistically significant (unpaired t-test) lowering of Volasertib (PLK1 inhibitor) AUC in MM patients that were clinically refractory to CFZ (p-value = 0.03) and BTZ (p-value = 0.02) compared to PI-naïve (Figure 2). Similarly, we infer cytostatic mechanism of resistance to PIs by pairing ex vivo drug response from 399 MM patients with RNAseq data to identify gene programs that are enriched for PI-resistance and PI-sensitivity using gene set enrichment analysis; which identified PRCK, ROCK, and PLK1 inhibitors as therapeutic targets that can either synergize with PIs or exhibit synthetic lethality. Ongoing studies are focused on identifying novel targets to overcome resistance to DARA. The proposed computational approach can identify novel therapeutic strategies to overcome SOC therapy resistance using patient-specific targeted therapies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Increasing the Throughput of Activity-Based Proteome Profiling Studies with Real-Time Search and Sample Hyperplexing

Increasing the Throughput of Activity-Based Proteome Profiling Studies with Real-Time Search and Sample Hyperplexing

Activity-based proteomic profiling reveals reduction of lipid hydrolase activity levels in non-small cell lung cancer tumors

Activity-based proteomic profiling reveals reduction of lipid hydrolase activity levels in non-small cell lung cancer tumors

Abstract 1887: Discrimination of aggressive tumor profiles in lung adenocarcinomas using activity based protein profiling by SWATH/DIA-MS mode

Abstract (a) Activity-based proteome profiling (ABPP) probes combined with mass spectrometry (MS) is the tool of functional proteomics for the quantification of catalytically active enzymes. Here, we applied a depletion-dependent ABPP for serine hydrolase (SH) profiling in 30-μm thickness cryosections of optimal-cutting-temperature (OCT)-embedded tissue of advanced lung adenocarcinomas (LUAD). (b) For the proof-of-concept, we profiled SH activities in advanced LUAD disease. We retrospectively analyzed hydrolase activity differences with prognostic values related to aggressive phenotype based on clinical data collected over the course of disease progression. Interestingly, detection of proteins correlating by active-enzyme capture uncover their functional links with the metabolic SHs related to the enhanced palmitoylation-depalmitoylation cycle of lipoproteins in aggressive LUAD. In contrast, the standard DDA-MS analysis of on-bead-depleted enzymes detected 95 SH’s of which less than 60 enzymes were quantified based on spectral counting. (c) We found enhanced activities of dozen SHs in all LUAD tumors. Remarkably, the activity of 11 hydrolases emphasized by IAH1, ABHD12, LYPLA2 and MST1 discriminate aggressive tumors at the time of diagnosis. Functional analysis leveraging enzymes and associated co-depleted proteins revealed catabolic processing of lipoproteins and enrichment of enzymes displaying increased de-palmitoylase activities. This is interesting because multiple isoforms of RAS protein (K-RAS, H-RAS and N-RAS), the most frequently mutated oncogene in human cancers including LUAD (i.e. 30% of LUADs contain somatic K-Ras mutation), attain oncogenic properties only in depalmitoylated state which favor protein intracellular trafficking by detachment from hydrophobic plasma membrane. In line, mutant K-Ras protein co-localize on outer mitochondrion membrane and interact with glycolytic enzymes (hexokinase-1 and -2) only in depalmitoylated state. This interaction induce K-Ras oncogenic properties and increased glucose uptake and glycolysis even in the presence of abundant oxygen (Warburg effect). Curiously, we observed co-depletion of mitochondrial proteins including glycolytic hexokinase 2 with several lipases displaying increase depalmitoylase activity in short LUAD survivals. (d) Surprisingly, an evident disagreement in the activity of enzymes involved in lipolysis and protein depalmitoylation (i.e. IAH1, ABHD12, LYPLA2) was discovered between LUAD survival subtypes. These essential metabolic differences were uncovered by our standard proteomic analysis and to our knowledge have not been reported. Since the inhibitors of serine lipases are promising targets in other RAS-mutant carcinomas, this metabolic vulnerabilities discovered in aggressive LUAD can be explored as promising therapeutic strategies in lung cancer. Citation Format: Tatjana Sajic, Stephan Arni, Rudolf Aebersold, Sven Hillinger. Discrimination of aggressive tumor profiles in lung adenocarcinomas using activity based protein profiling by SWATH/DIA-MS mode [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1887.

Azido Groups Hamper Glycan Acceptance by Carbohydrate Processing Enzymes.

Azido sugars have found frequent use as probes of biological systems in approaches ranging from cell surface metabolic labeling to activity-based proteomic profiling of glycosidases. However, little attention is typically paid to how well azide-substituted sugars represent the parent molecule, despite the substantial difference in size and structure of an azide compared to a hydroxyl. To quantitatively assess how well azides are accommodated, we have used glycosidases as tractable model enzyme systems reflecting what would also be expected for glycosyltransferases and other sugar binding/modifying proteins. In this vein, specificity constants have been measured for the hydrolysis of a series of azidodeoxy glucosides and N-acetylhexosaminides by a large number of glycosidases produced from expressed synthetic gene and metagenomic libraries. Azides at secondary carbons are not significantly accommodated, and thus, associated substrates are not processed, while those at primary carbons are productively recognized by only a small subset of the enzymes and often then only very poorly. Accordingly, in the absence of careful controls, results obtained with azide-modified sugars may not be representative of the situation with the natural sugar and should be interpreted with considerable caution. Azide incorporation can indeed provide a useful tool to monitor and detect glycosylation, but careful consideration should go into the selection of sites of azide substitution; such studies should not be used to quantitate glycosylation or to infer the absence of glycosylation activity.

A CK1δ/CK1ε-to-β-Catenin Circuit Is a Therapeutic Vulnerability for Primary and Drug Resistant Multiple Myeloma

Multiple Myeloma (MM) remains an incurable malignancy, despite the advent of several new therapeutic agents, including immunomodulatory drugs (IMiDs, e.g., Lenalidomide (Len)) and proteasome inhibitors (PIs, e.g ., Bortezomib (Btz)). Accordingly, there is an urgent need to identify new targetable vulnerabilities for MM patients. We developed an ex vivo 384-well assay that allows one to define drug sensitivities of primary patient CD138+ MM cells in the context of a reconstructed tumor microenvironment (TME), including allogeneic BMSC, extracellular matrix and MM patient serum. Using this platform and activity-based proteomic profiling (ABPP), we identified shared signaling pathways induced by the interactions of MM with the TME and integrated these data with screens performed using a bank of protein kinase inhibitors (PKI) and current anti-MM therapeutics. These analyses revealed that the serine/threonine kinases casein kinase-1δ (CK1δ) and CK1ε are high priority targets for MM. In support of this data, a highly selective and potent dual inhibitor of CK1δ/CK1ε coined SR-3029 is the most potent PKI versus MM. Further, our studies revealed SR-3029 has potent activity in 75/86 primary patient MM specimens tested thus far, including quad and penta-refractory MM.Previous studies have shown that SR-3029 targets CK1δ in triple negative breast cancer (TNBC) and that the therapeutic response relied on disabling the Wnt/β-catenin signaling. Aberrant activation of the Wnt/β-catenin pathway has well established roles in the development and progression of cancer. Indeed, gain-of-function somatic mutations in β-catenin and upstream receptors such as LRP5, and loss-of-function mutations in components of the β-catenin destruction complex, including the scaffold proteins APC and Axin, are hallmarks of several tumor types.Our analyses of whole exome sequencing (WES) of MM patients revealed that ~40% of tumors have functional protein-altering mutations in the Wnt/β-catenin pathway, and that this is manifest in both treatment-naïve and relapsed MM. Additionally, RNA-seq and gene expression profiling analysis of over 1,300 MCC MM patients specimens have show that components of the Wnt/β-catenin signaling pathway are elevated in some subsets of MM. Further, we have shown that SR-3029 treatment or inducible knockdown of CK1δ or CK1ε rapidly compromises the growth and survival of MM cell lines. Importantly, the anti-MM activity of SR-3029 is augmented in MM cell lines with selected resistance to Btz and Len relative to parental myeloma cells. Further, treatment of MM cells with this kinase inhibitor leads to marked reductions in total β-catenin protein, decreases in β-catenin target genes (e.g., MYC, CCND1 and WNT3), and to marked changes in the expression of components of the Wnt/β-catenin signaling pathway. Finally, using the established 5TGM1/Kal-Ridge (C57B6/KaLwRijHsd) syngeneic mouse model of multiple myeloma, we show that tumors derived from 5TGM1 MM cells, which rapidly die following exposure to SR-3029 ex vivo, are also sensitive to CK1δ/CK1ε inhibition in vivo, where the SR-3029 treated cohort of animals demonstrated decreased tumor burden as assessed by IgG2b levels and imaging, and by significantly improved survival relative tovehicle treated recipients. Collectively, these findings demonstrate that SR-3029 has potent activity in both naïve and therapy resistant multiple myeloma and establish CK1δ and/or CK1ε as attractive targets for anti-myeloma therapy. DisclosuresSiqueira Silva:AbbVie: Research Funding. Teer:Interpares Biomedicine: Consultancy. Shain:Celgene Corporation: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Janssen: Consultancy; Takeda Pharmaceuticals: Consultancy.

Proteoform of Arabidopsis seed storage protein identified by functional proteomics approach exhibits acyl hydrolase activity during germination

Lipases play a crucial role in the life cycle of seed plants and the oil content of the seed is highly regulated by the lipase activity. Hence, understanding the role of lipases during germination and post-germination will provide insights into lipid mobilization. However, to date, no lipase gene has been identified in seeds except, Sugar-dependent-1 in Arabidopsis. Hence, in the present study, we employed a functional proteomic approach for the identification of seed-specific lipase. Activity-Based Proteome Profiling (ABPP) of Arabidopsis mature and germinating seeds revealed the expression of a functional serine hydrolase exclusively during germination. The mass-spectrometry analysis reveals the identity and amino acid sequence of the protein correspond to AT4G28520 gene, a canonical 12S Seed Storage Protein (SSP). Interestingly, the identified SSP was a proteoform of AT4G28520 (SL-AT4G28520) and exhibited >90% identity with the canonical AT4G28520 (FL-AT4G28520). Heterologous expression and enzyme assays indicated that SL-AT4G28520 protein indeed possesses monoacylglycerol lipase activity, while the FL-AT4G28520 protein didn't exhibit any detectable activity. Functional proteomics and lipidomics analysis demonstrated a catalytic function of this SSP. Collectively, this is the first report, which suggests that SL-AT4G28520 encodes a lipase, and the activity is depending on the physiological condition.

Abstract 6482: Activity based protein profiling by SWATH/DIA-MS mode (ABPP-SWATH/DIA-MS) from OCT-embedded tissues biopsies reveals lung tumor-specific protease profiles

Abstract (a) Proteomics relies on protein digestion into smaller fragments/peptides and converge to proteome sequence information, classification, and expression levels without a screen of the cellular protein activities. Activity-based proteome profiling (ABPP) is emerging as a tool for functional proteomics based on mass spectrometry. We established an ABPP-SWATH/DIA MS platform compatible with OCT-embedded biopsies for differential global serine hydrolase (SHs) activity profiling and determine the Relative Depletion-Detected Activity (RDDA) index that reflects the relative activity of SHs in type IIIA lung adenocarcinoma biopsies. (b) To prevent digestion on streptavidin beads and MS spectra contamination, we used SWATH/DIA-MS2 signal intensities for the assessment of the ratio between the biopsy-extract solution either treated with DMSO or which had earlier been depleted with chemical probe for active enzyme molecules. We processed 24 lung biopsies (having a solution of depleted and non-depleted proteome) of long and short survivors with type IIIA lung adenocarcinoma tumor and their normal tissue counterparts and included peptides of 65 known endogenous inhibitors of SH. In parallel experiments, we analyzed the catalytically depleted part of the molecule by conventional ABPP-MS approach integrated on bead sample digestion. (c) We quantified 117 proteins annotated to the serine hydrolase family, of which as much as 60% was depleted with a CI of 90% in the biopsy-extract solution incubated with chemical probe. Among 133 enzymes monitored over the entire clinical cohort, we found as many as 89 enzymes with significant depletion that we further used to generate the activity SH profiles of each biopsy. We found 36 enzymes exclusively depleted in tumor tissue, both in short and long survivals, and with depletion absent from normal counterparts. By quantification of the catalytically active part of the molecule on the streptavidin bead sample pull-down digest, we found only 9 proteins that contained quality data for spectral counting comparisons. Partial Least Squares Discriminant Analysis (PLS-DA) analysis revealed the activity profiles of 9 enzymes perfectly discriminating the tumor tissues of short survivals, and these enzyme signatures were enhanced by the elevated activity of 2 enzymes - ADH1C and IAH1- in short-term survivors. (d) We have demonstrated that this approach can substantially increase the differential detection of protease activity between oncogenic lung tissue and their normal tissue counterparts. Citation Format: Tatjana Sajic, Stephan Arni, Rudolf Aebersold, Sven Hillinger. Activity based protein profiling by SWATH/DIA-MS mode (ABPP-SWATH/DIA-MS) from OCT-embedded tissues biopsies reveals lung tumor-specific protease profiles [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6482.

Systems level profiling of arginine starvation reveals MYC and ERK adaptive metabolic reprogramming

Arginine auxotrophy due to the silencing of argininosuccinate synthetase 1 (ASS1) occurs in many carcinomas and in the majority of sarcomas. Arginine deiminase (ADI-PEG20) therapy exploits this metabolic vulnerability by depleting extracellular arginine, causing arginine starvation. ASS1-negative cells develop resistance to ADI-PEG20 through a metabolic adaptation that includes re-expressing ASS1. As arginine-based multiagent therapies are being developed, further characterization of the changes induced by arginine starvation is needed. In order to develop a systems-level understanding of these changes, activity-based proteomic profiling (ABPP) and phosphoproteomic profiling were performed before and after ADI-PEG20 treatment in ADI-PEG20-sensitive and resistant sarcoma cells. When integrated with metabolomic profiling, this multi-omic analysis reveals that cellular response to arginine starvation is mediated by adaptive ERK signaling and activation of the Myc–Max transcriptional network. Concomitantly, these data elucidate proteomic changes that facilitate oxaloacetate production by enhancing glutamine and pyruvate anaplerosis and altering lipid metabolism to recycle citrate for oxidative glutaminolysis. Based on the complexity of metabolic and cellular signaling interactions, these multi-omic approaches could provide valuable tools for evaluating response to metabolically targeted therapies.

Re-Evaluating the Mechanism of Action of α,β-Unsaturated Carbonyl DUB Inhibitors b-AP15 and VLX1570: A Paradigmatic Example of Unspecific Protein Cross-linking with Michael Acceptor Motif-Containing Drugs.

Deubiquitinating enzymes (DUBs) are a growing target class across multiple disease states, with several inhibitors now reported. b-AP15 and VLX1570 are two structurally related USP14/UCH-37 inhibitors. Through a proteomic approach, we demonstrate that these compounds target a diverse range of proteins, resulting in the formation of higher molecular weight (MW) complexes. Activity-based proteome profiling identified CIAPIN1 as a submicromolar covalent target of VLX1570, and further analysis demonstrated that high MW complex formation leads to aggregation of CIAPIN1 in intact cells. Our results suggest that in addition to DUB inhibition, these compounds induce nonspecific protein aggregation, providing molecular explanation for general cellular toxicity.

A CK1δ/CK1ε Regulated Metabolic Circuit Is a Therapeutic Vulnerability for Multiple Myeloma

Multiple Myeloma (MM) remains an incurable malignancy, despite the advent of several new therapeutic agents, including immunomodulatory drugs (IMiDs, e.g., Lenalidomide (Len)) and proteasome inhibitors (PIs, e.g., Bortezomib (Btz)). Accordingly, there is an urgent need to identify new targetable vulnerabilities for MM patients. We developed an ex vivo 384-well platform that allows one to define drug sensitivities of primary patient CD138+ MM cells in the context of a reconstructed tumor microenvironment (TME), including allogeneic bone marrow stromal cells, extracellular matrix and MM patient serum. Using this platform and activity-based proteomic profiling (ABPP), we identified shared signaling pathways induced by the interactions of MM with stromal cells and integrated these data with screens performed using a bank of protein kinase inhibitors (PKI) and current anti-MM therapeutics. These analyses revealed that the serine/threonine kinases casein kinase-1δ (CK1δ) and CK1ε as high priority targets for MM. Indeed, a highly selective and potent dual inhibitor of CK1δ/CK1ε coined SR-3029 is the most potent PKI versus MM. Further, our studies revealed SR-3029 has potent activity in 138/153 primary patient MM specimens tested thus far, including quad and penta-refractory MM. Analysis of RNAseq data of over 600 Moffitt Cancer Center (MCC) MM patients revealed that patients with high expression of CK1ε had worse survival outcomes while no survival difference was seen with CK1δ expression. Importantly, using the established 5TGM1/Kal-Ridge (C57B6/KaLwRijHsd) syngeneic mouse model of multiple myeloma, we show that tumors derived from 5TGM1 MM cells, which rapidly die following exposure to SR-3029 ex vivo, are also sensitive to CK1δ/CK1ε inhibition in vivo, where SR-3029 treatment reduced tumor burden and significantly improved survival. Similar results were observed using NSG immune compromised animals inoculated with human MM1.S multiple myeloma cells (both flank and tail vein models), where SR-3029 treated animals had reduced tumor burden and extended survival. Analysis of RNAseq on patients' samples (on stroma) treated ex vivo with SR-3029 revealed CK1δ/CK1ε inhibition suppressed multiple metabolic pathways (oxidative phosphorylation, glycolysis, xenobiotic metabolism). Interestingly, analyses of MCC MM patient RNAseq data revealed upregulation of the genes identified in these metabolic pathways as patients progress from pre-treatment to relapse, and that patient MM samples that were resistant to CK1δ/CK1ε inhibition had an upregulation of some of these metabolic genes. Functional studies are being performed to define the mechanism(s) by which CK1δ/CK1ε inhibition disables MM metabolism. Collectively, these findings establish CK1ε and/or CK1δ as attractive targets for anti-myeloma therapy that are required to sustain MM metabolism. Disclosures Dai: M2Gen: Employment. Shain:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Sanofi Genzyme: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Prospective Application of Activity-Based Proteomic Profiling in Vision Research-Potential Unique Insights into Ocular Protease Biology and Pathology.

Activity-based proteomic profiling (ABPP) is a powerful tool to specifically target and measure the activity of a family of enzymes with the same function and reactivity, which provides a significant advantage over conventional proteomic strategies that simply provide abundance information. A number of inherited and age-related eye diseases are caused by polymorphisms/mutations or abnormal expression of proteases including serine proteases, cysteine proteases, and matrix metalloproteinases, amongst others. However, neither conventional genomic, transcriptomic, nor traditional proteomic profiling directly interrogate protease activities. Thus, leveraging ABPP to probe the activity of these enzyme classes as they relate to normal function and pathophysiology of the eye represents a unique potential opportunity for disease interrogation and possibly intervention.

Pharmacoproteomics Identifies PLK1 As Vulnerability for Aggressive B-Cell Lymphomas

Background: c-MYC is a transcription factor that promotes oncogenesis by activating and repressing its target genes that control cell growth, metabolism, and proliferation. MYC is deregulated in a large proportion of aggressive B-cell lymphomas. A typical example is the Double-Hit Lymphoma (DHL) and Double-Expression Lymphoma (DEL) which present with a rapidly progressing clinical course, refractory to treatment, poor clinical outcome, and currently considered incurable. Nevertheless, MYC is considered as an “undruggable” target since it has no “active site” amenable to binding by conventional small molecule inhibitors. Moreover, MYC has a broad spectrum of functions in cell proliferation, survival, metabolism, and others, so direct inhibition would likely cause severe side effects. Besides direct inhibition, another practical strategy is to target druggable proteins that are essential for the viability of MYC-driven tumors, inducing MYC-dependent “synthetic lethality”. The advantage of such approach is a capability of killing tumor cells discriminately, while leaving non-tumor cells intact or less influenced. This study is designed to identify such targets and explore practical novel strategies to treat MYC-driven lymphomas, especially DHL/DEL.Methods and Results: By integrating activity-based proteomic profiling and drug screens in isogenic MYC on/off lymphoma cells, we identified polo-like kinase-1 (PLK1) as an essential regulator of the MYC-dependent kinome in DHL/DEL. Notably, PLK1 was expressed at high levels in DHL, correlated with MYC expression and connoted poor outcome. Further, PLK1 is directly activated by MYC on transcriptional level and in turn, PLK1 signaling augmented MYC protein stability by promoting its phosphorylation and suppressing its degradation. Thus, MYC and PLK1 form a feed-forward circuit in lymphoma cells. Finally, both in vitro and in vivo studies demonstrated that inhibition of PLK1 triggered degradation of MYC and of the anti-apoptotic protein MCL1, and PLK1 inhibitors showed synergy with BCL-2 antagonists in blocking DHL/DEL cell growth, survival, and tumorigenicity. These data support that PLK1 is a promising therapeutic target in MYC-driven lymphomas.Brief summary: Functional pharmacoproteomics identified PLK1 as a therapeutic vulnerability for MYC-driven lymphoma, which was a synthetic lethal for DHL/DEL when targeted with BCL-2 inhibitors. [Display omitted] DisclosuresVose:Roche: Honoraria; Merck Sharp & Dohme Corp.: Research Funding; Acerta Pharma: Research Funding; Seattle Genetics, Inc.: Research Funding; Novartis: Honoraria, Research Funding; Kite Pharma: Research Funding; Bristol Myers Squibb: Research Funding; Epizyme: Honoraria; Legend Pharmaceuticals: Honoraria; Abbvie: Honoraria; Celgene: Research Funding; Incyte Corp.: Research Funding.

PLK1 stabilizes a MYC-dependent kinase network in aggressive B cell lymphomas.

Concordant activation of MYC and BCL-2 oncoproteins in double-hit lymphoma (DHL) results in aggressive disease that is refractory to treatment. By integrating activity-based proteomic profiling and drug screens, polo-like kinase-1 (PLK1) was identified as an essential regulator of the MYC-dependent kinome in DHL. Notably, PLK1 was expressed at high levels in DHL, correlated with MYC expression, and connoted poor outcome. Further, PLK1 signaling augmented MYC protein stability, and in turn, MYC directly induced PLK1 transcription, establishing a feed-forward MYC-PLK1 circuit in DHL. Finally, inhibition of PLK1 triggered degradation of MYC and of the antiapoptotic protein MCL-1, and PLK1 inhibitors showed synergy with BCL-2 antagonists in blocking DHL cell growth, survival, and tumorigenicity, supporting clinical targeting of PLK1 in DHL.

Impact of Mass Spectrometry-Based Technologies and Strategies on Chemoproteomics as a Tool for Drug Discovery.

Chemoproteomics is an invaluable tool to discover protein targets from phenotypic assays and to understand on- and off-target engagement of potential therapeutic compounds. Highlighted in this technology perspective is our view on how improvements in mass spectrometry (MS)-based proteomics technology have dramatically impacted chemoproteomics. Improvements in sample preparation, MS instrumentation, data acquisition, and quantification strategies have enabled medicinal chemists, chemical biologists, and mass spectrometrists to develop new chemoproteomic experiments and improve existing methods. As a result of improvements in MS, we will detail how bead-based affinity capture and activity-based proteome profiling methods have been reduced from multiple LC-MS runs for samples and controls down to a single LC-MS run each for sample and control. With improvements in scan duty cycle and sensitivity, sufficient depth of proteome coverage can be obtained for capture-free methods, which do not utilize an enrichment step.

Affinity-Driven Covalent Modulator of the Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Cascade.

Traditional medicines provide a fertile ground to explore potent lead compounds, yet their transformation into modern drugs is fraught with challenges in deciphering the target that is mechanistically valid for its biological activity. Herein we reveal that (Z)-(+)-isochaihulactone (1) exhibited significant inhibition against multiple-drug-resistant (MDR) cancer cell lines and mice xenografts. NMR spectroscopy showed that 1 resisted an off-target thiolate, thus indicating that 1 was a target covalent inhibitor (TCI). By identifying the pharmacophore of 1 (α,β-unsaturated moiety), a probe derived from 1 was designed and synthesized for TCI-oriented activity-based proteome profiling. By MS/MS and computer-guided molecular biology approaches, an affinity-driven Michael addition of the noncatalytic C247 residue of GAPDH was found to control the "ON/OFF" switch of apoptosis through non-canonically nuclear GAPDH translocation, which bypasses the common apoptosis-resistant route of MDR cancers.

Abstract TMEM-032: MULTIDISCIPLINARY CHARACTERIZATION OF OVARIAN CANCER SPHERES

Abstract Sphere-forming cultures have been widely used in stem cell biology. Ovarian cancer sphere culture is also a good model to study ovarian cancer ascites spheroids, which propagate without attachment to a substratum. We studied non-adherent ovarian cancer sphere cultures derived from ovarian cancer cell lines and epithelial cancer cells isolated from clinical samples. Western blot analysis and immunofluorescence showed that ovarian cancer spheres expressed elevated levels of stem cell markers CD133, c-Myc, Nanog, and Oct4. Metabolically, ovarian cancer sphere cells showed decreased ATP levels and were under constant oxidative stress, with significant depletion of the endogenous antioxidant glutathione. Accordingly, supplementation with antioxidant supplement N-acetylcysteine (NAC) in cancer sphere cultures relieved oxidative stress and improved growth of the sphere cultures in a dose-dependent manner. In contrast, adherent cancer cell cultures did not show any significant growth enhancement. Furthermore, microarray gene expression profiling and activity-based proteomic profiling using a sulfonate ester chemical probe revealed that the ovarian cancer spheres demonstrated increased level of FOS/JUN expression, which activated the expression and activity of the omega class of glutathione-S transferase GSTO1, an enzyme involved in the metabolism of xenobiotics and cisplatin resistance. Knockdown of FOS expression using siRNA reduced the levels of GSTO1 and abrogated chemoresistance of the sphere cultures. Separately, gene set enrichment analysis of the cancer sphere expression profiles revealed elevated expression of endosomal pathway genes for exosome secretion in the sphere cultures. Knockdown of one elevated gene, Rab27B, significantly reduced the exosome number in the spent medium of ovarian cancer sphere cultures. Clinically, measurement of the exosome number in patient body fluids using the NanoSight instrument and a flow cytometry method indicated that plasma and urine samples from a panel of ovarian cancer patients had increased number of exosomes compared with samples derived from benign patients. CONCLUSIONS: Our multidisciplinary studies have discovered a plethora of properties of ovarian cancer spheres that may promote ovarian cancer growth. Elevated expression and activity of GSTO1 in ovarian cancer spheres may represent a novel mechanism by which ovarian cancer spheroids respond to heightened oxidative stress and chemotherapeutic agents. Antioxidant supplements may not have the intended benefits to cancer survivors. Instead, they may promote cancer growth by relieving the oxidative stress of ovarian cancer spheroids. In contrast, inhibitors that target GSTO1 may have better clinical value in prolonging survivorship of ovarian cancer patients with malignant ascites. Ovarian cancer spheres also have increased expression of endosomal pathway genes for exosome secretion, which may promote the establishment of tumor microenvironment for cancer propagation, and is consistent with the increased exosomes in the plasma and urine samples of ovarian cancer patients. Citation Format: Shubai Liu, Junzheng Yang, Pui-Wah Choi, Jamie Sui-Lam Kwok, Kathleen Hasselblatt, Daniel Nomura, Wing Ping Fong, Stephen KW Tsui, Allison Vitonis, Daniel Cramer, William R. Welch, Benjamin Cravatt, Ross Berkowitz, and Shu-Wing Ng. MULTIDISCIPLINARY CHARACTERIZATION OF OVARIAN CANCER SPHERES [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-032.