<scp>INKA</scp>
            , an integrative data analysis pipeline for phosphoproteomic inference of active kinases

Beekhof, Robin; Alphen, Carolien van; Henneman, Alex A.; Knol, Jaco C.; Pham, Thang V.; Rolfs, Frank; Labots, Mariëtte; Henneberry, Evan; Large, Tessa Y.S. Le; Haas, Richard R. de; Piersma, Sander R.; Vurchio, Valentina; Bertotti, Andrea; Trusolino, Livio; Verheul, Henk M.W.; Jiménez, Connie R.

doi:10.15252/msb.20198981

Cited by 54 publications

(52 citation statements)

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“…For standard proteomics analysis of the content of the different cell fractions, the samples were processed according to established protocols 82 , and deposited in the PRIDE repository under accession number PXD024426 . Briefly, following SDS-PAGE, sections were cut from the gel, and slices were digested with trypsin prior to LC-MS/MS.…”

Section: Methodsmentioning

confidence: 99%

“…Peptide counts were aggregated to proteins and normalized to the total count in each sample. Enrichment for a particular fraction was determined using a modified binomial test 82 . Peptide coverage for vimentin in each fraction was retrieved from the raw data and plotted as a count profile which reflects both the propensity to be analyzed (presence and frequency of lysines that are targeted by trypsin and determine inclusion in the analysis) as well as the distribution of the protein fragments of vimentin present in each fraction to determine any differences.…”

Section: Methodsmentioning

confidence: 99%

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Extracellular vimentin mimics VEGF and is a target for anti-angiogenic immunotherapy

et al. 2022

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Anti-angiogenic cancer therapies possess immune-stimulatory properties by counteracting pro-angiogenic molecular mechanisms. We report that tumor endothelial cells ubiquitously overexpress and secrete the intermediate filament protein vimentin through type III unconventional secretion mechanisms. Extracellular vimentin is pro-angiogenic and functionally mimics VEGF action, while concomitantly acting as inhibitor of leukocyte-endothelial interactions. Antibody targeting of extracellular vimentin shows inhibition of angiogenesis in vitro and in vivo. Effective and safe inhibition of angiogenesis and tumor growth in several preclinical and clinical studies is demonstrated using a vaccination strategy against extracellular vimentin. Targeting vimentin induces a pro-inflammatory condition in the tumor, exemplified by induction of the endothelial adhesion molecule ICAM1, suppression of PD-L1, and altered immune cell profiles. Our findings show that extracellular vimentin contributes to immune suppression and functions as a vascular immune checkpoint molecule. Targeting of extracellular vimentin presents therefore an anti-angiogenic immunotherapy strategy against cancer.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Extracellular vimentin mimics VEGF and is a target for anti-angiogenic immunotherapy

et al. 2022

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“…To further unveil the role of the observed phosphorylation dynamics in these cells, an R pipeline “InKA”, which integrates kinome, activation loop, phosphoSitePlus, and NetworKIN evidences, was applied to infer kinase activity. 23 Based on this substrate-centric kinase activity prediction model, a total of 131 kinases were predicted with a relative activity score ( Table S2B ). We observed that ERK1, ERK2, and CDK1 are the most-activated kinases, which is more obvious over time upon drug withdrawal in control, sgERK1, and sgJUNB, indicating their dominant roles in drug-resistant melanoma.…”

Section: Resultsmentioning

confidence: 99%

Proteomics and Phosphoproteomics Profiling of Drug-Addicted BRAFi-Resistant Melanoma Cells

Kong

Post

et al. 2021

J. Proteome Res.

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Acquired resistance to MAPK inhibitors limits the clinical efficacy in melanoma treatment. We and others have recently shown that BRAF inhibitor (BRAFi)-resistant melanoma cells can develop a dependency on the therapeutic drugs to which they have acquired resistance, creating a vulnerability for these cells that can potentially be exploited in cancer treatment. In drug-addicted melanoma cells, it was shown that this induction of cell death was preceded by a specific ERK2-dependent phenotype switch; however, the underlying molecular mechanisms are largely lacking. To increase the molecular understanding of this drug dependency, we applied a mass spectrometry-based proteomic approach on BRAFi-resistant BRAF MUT 451Lu cells, in which ERK1, ERK2, and JUNB were silenced separately using CRISPR-Cas9. Inactivation of ERK2 and, to a lesser extent, JUNB prevents drug addiction in these melanoma cells, while, conversely, knockout of ERK1 fails to reverse this phenotype, showing a response similar to that of control cells. Our analysis reveals that ERK2 and JUNB share comparable proteome responses dominated by reactivation of cell division. Importantly, we find that EMT activation in drug-addicted melanoma cells upon drug withdrawal is affected by silencing ERK2 but not ERK1. Moreover, transcription factor (regulator) enrichment shows that PIR acts as an effector of ERK2 and phosphoproteome analysis reveals that silencing of ERK2 but not ERK1 leads to amplification of GSK3 kinase activity. Our results depict possible mechanisms of drug addiction in melanoma, which may provide a guide for therapeutic strategies in drug-resistant melanoma.

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“…Phosphoproteomic alterations were analyzed and visualized using Integrative Inferred Kinase Activity (InKA) analysis v1.2.2 (https:// inkascore.org/) and PTMsigDB analysis v2.0 (https://github. com/broadinstitute/ssGSEA2.0) (23,24).…”

Section: Nanolc-ms/ms Acquisition and Data Processingmentioning

confidence: 99%

“…Integrative Inferred Kinase Activity (INKA) analysis was applied on the phosphoproteomic data to assess the kinase activity after moDC binding to a2-3sia in presence of LPS. This method integrates four phosphoproteomic analyses of one sample to a scoring system, allowing ranking of the kinase activity and visualization of the kinase-substrate networks (23). Multiple kinases were affected by a2-3sia and LPS stimulation (Figure 4A and Sl.…”

Section: Alterations In the Modc Phosphoproteome After A2-3 Sialic Acid Bindingmentioning

confidence: 99%

Quantitative Phosphoproteomic Analysis Reveals Dendritic Cell- Specific STAT Signaling After α2-3–Linked Sialic Acid Ligand Binding

Haas

Rodríguez

et al. 2021

Front. Immunol.

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Dendritic cells (DCs) are key initiators of the adaptive immunity, and upon recognition of pathogens are able to skew T cell differentiation to elicit appropriate responses. DCs possess this extraordinary capacity to discern external signals using receptors that recognize pathogen-associated molecular patterns. These can be glycan-binding receptors that recognize carbohydrate structures on pathogens or pathogen-associated patterns that additionally bind receptors, such as Toll-like receptors (TLRs). This study explores the early signaling events in DCs upon binding of α2-3 sialic acid (α2-3sia) that are recognized by Immune inhibitory Sialic acid binding immunoglobulin type lectins. α2-3sias are commonly found on bacteria, e.g. Group B Streptococcus, but can also be expressed by tumor cells. We investigated whether α2-3sia conjugated to a dendrimeric core alters DC signaling properties. Through phosphoproteomic analysis, we found differential signaling profiles in DCs after α2-3sia binding alone or in combination with LPS/TLR4 co-stimulation. α2-3sia was able to modulate the TLR4 signaling cascade, resulting in 109 altered phosphoproteins. These phosphoproteins were annotated to seven biological processes, including the regulation of the IL-12 cytokine pathway. Secretion of IL-10, the inhibitory regulator of IL-12 production, by DCs was found upregulated after overnight stimulation with the α2-3sia dendrimer. Analysis of kinase activity revealed altered signatures in the JAK-STAT signaling pathway. PhosphoSTAT3 (Ser727) and phosphoSTAT5A (Ser780), involved in the regulation of the IL-12 pathway, were both downregulated. Flow cytometric quantification indeed revealed de- phosphorylation over time upon stimulation with α2-3sia, but no α2-6sia. Inhibition of both STAT3 and -5A in moDCs resulted in a similar cytokine secretion profile as α-3sia triggered DCs. Conclusively, this study revealed a specific alteration of the JAK-STAT pathway in DCs upon simultaneous α2-3sia and LPS stimulation, altering the IL10:IL-12 cytokine secretion profile associated with reduction of inflammation. Targeted control of the STAT phosphorylation status is therefore an interesting lead for the abrogation of immune escape that bacteria or tumors impose on the host.

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

Cited by 54 publications

References 1 publication

Extracellular vimentin mimics VEGF and is a target for anti-angiogenic immunotherapy

Extracellular vimentin mimics VEGF and is a target for anti-angiogenic immunotherapy

Proteomics and Phosphoproteomics Profiling of Drug-Addicted BRAFi-Resistant Melanoma Cells

Quantitative Phosphoproteomic Analysis Reveals Dendritic Cell- Specific STAT Signaling After α2-3–Linked Sialic Acid Ligand Binding

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