Predicting treatment outcome using kinome activity profiling in HER2+ breast cancer biopsies.

In this study, we measured the kinase activity profiles of 32 pre-treatment tumor biopsies of HER2-positive breast cancer patients. The aim of this study was to assess the prognostic potential of kinase activity levels, to identify potential mechanisms of resistance and to predict treatment success of HER2-targeted therapy combined with chemotherapy. Indeed, our system-wide kinase activity analysis allowed us to link kinase activity to treatment response. Overall, high kinase activity in the HER2-pathway was associated with good treatment outcome. We found eleven kinases differentially regulated between treatment outcome groups, including well-known players in therapy resistance, such as p38a, ERK, and FAK, and an unreported one, namely MARK1. Lastly, we defined an optimal signature of four kinases in a multiple logistic regression diagnostic test for prediction of treatment outcome (AUC = 0.926). This kinase signature showed high sensitivity and specificity, indicating its potential as predictive biomarker for treatment success of HER2-targeted therapy.

Innovative strategies for measuring kinase activity to accelerate the next wave of novel kinase inhibitors.

The development of protein kinase inhibitors (PKIs) has gained significance owing to their therapeutic potential for diseases like cancer. In addition, there has been a rise in refining kinase activity assays, each possessing unique biological and analytical characteristics crucial for PKI development. However, the PKI development pipeline experiences high attrition rates and approved PKIs exhibit unexploited potential because of variable patient responses. Enhancing PKI development efficiency involves addressing challenges related to understanding the PKI mechanism of action and employing biomarkers for precision medicine. Selecting appropriate kinase activity assays for these challenges can overcome these attrition rate issues. This review delves into the current obstacles in kinase inhibitor development and elucidates kinase activity assays that can provide solutions.

Novel kinome profiling technology reveals drug treatment is patient and 2D/3D model dependent in glioblastoma.

Glioblastoma is the deadliest brain cancer. One of the main reasons for poor outcome resides in therapy resistance, which adds additional challenges in finding an effective treatment. Small protein kinase inhibitors are molecules that have become widely studied for cancer treatments, including glioblastoma. However, none of these drugs have demonstrated a therapeutic activity or brought more benefit compared to the current standard procedure in clinical trials. Hence, understanding the reasons of the limited efficacy and drug resistance is valuable to develop more effective strategies toward the future. To gain novel insights into the method of action and drug resistance in glioblastoma, we established in parallel two patient-derived glioblastoma 2D and 3D organotypic multicellular spheroids models, and exposed them to a prolonged treatment of three weeks with temozolomide or either the two small protein kinase inhibitors enzastaurin and imatinib. We coupled the phenotypic evidence of cytotoxicity, proliferation, and migration to a novel kinase activity profiling platform (QuantaKinome™) that measured the activities of the intracellular network of kinases affected by the drug treatments. The results revealed a heterogeneous inter-patient phenotypic and molecular response to the different drugs. In general, small differences in kinase activation were observed, suggesting an intrinsic low influence of the drugs to the fundamental cellular processes like proliferation and migration. The pathway analysis indicated that many of the endogenously detected kinases were associated with the ErbB signaling pathway. We showed the intertumoral variability in drug responses, both in terms of efficacy and resistance, indicating the importance of pursuing a more personalized approach. In addition, we observed the influence derived from the application of 2D or 3D models in in vitro studies of kinases involved in the ErbB signaling pathway. We identified in one 3D sample a new resistance mechanism derived from imatinib treatment that results in a more invasive behavior. The present study applied a new approach to detect unique and specific drug effects associated with pathways in in vitro screening of compounds, to foster future drug development strategies for clinical research in glioblastoma.

Afucosylated IgG characterizes enveloped viral responses and correlates with COVID-19 severity.

Immunoglobulin G (IgG) antibodies are crucial for protection against invading pathogens. A highly conserved N-linked glycan within the IgG-Fc tail, which is essential for IgG function, shows variable composition in humans. Afucosylated IgG variants are already used in anticancer therapeutic antibodies for their increased activity through Fc receptors (FcγRIIIa). Here, we report that afucosylated IgG (approximately 6% of total IgG in humans) are specifically formed against enveloped viruses but generally not against other antigens. This mediates stronger FcγRIIIa responses but also amplifies brewing cytokine storms and immune-mediated pathologies. Critically ill COVID-19 patients, but not those with mild symptoms, had high concentrations of afucosylated IgG antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), amplifying proinflammatory cytokine release and acute phase responses. Thus, antibody glycosylation plays a critical role in immune responses to enveloped viruses, including COVID-19.

Functional Attributes of Antibodies, Effector Cells, and Target Cells Affecting NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity.

Ab-dependent cellular cytotoxicity (ADCC) is one of the most important effector mechanisms of tumor-targeting Abs in current immunotherapies. In ADCC and other Ab-dependent activation of myeloid effector cells, close cell-cell contact (between effector and target cell) and formation of immunological synapses are required. However, we still lack basic knowledge on the principal factors influencing ADCC potential by therapeutic Abs. In this study we investigated the combined roles of five factors affecting human NK cell-mediated ADCC, namely: 1) Ag density, 2) target cell membrane composition, 3) IgG FcγR polymorphism, 4) FcγR-blocking cytophilic Abs, and 5) Ab fucosylation. We demonstrate that the magnitude of NK cell-mediated ADCC responses is predominantly influenced by Ag density and Ab fucosylation. Afucosylation consistently induced efficient ADCC, even at very low Ag density, where fucosylated target Abs did not elicit ADCC. On the side of the effector cell, the FcγRIIIa-Val/Phe158 polymorphism influenced ADCC potency, with NK cells expressing the Val158 variant showing more potent ADCC. In addition, we identified the sialic acid content of the target cell membrane as an important inhibitory factor for ADCC. Furthermore, we found that the presence and glycosylation status of aspecific endogenous Abs bound to NK cell FcγRIIIa (cytophilic Abs) determine the blocking effect on ADCC. These five parameters affect the potency of Abs in vitro and should be further tested as predictors of in vivo capacity.

In-Source Decay and Pseudo-MS3 of Peptide and Protein Ions Using Liquid AP-MALDI.

Atmospheric pressure MALDI on a Q-Exactive instrument was optimized for in-source decay and pseudo-MS3. The dependence of AP-MALDI ISD on the MALDI liquid matrix was investigated for peptides and proteins. The liquid matrices enabled long-life ISD signal, and exhibited high fragment ion yield and signal stability. Extensive a-, b-, c-, y-, and z-type fragment series were observed depending on the matrix used but were most extensive with 2,5-DHB. Complete sequence coverage of small peptide and intact protein-terminus sequence tags were obtained and confirmed using HCD as a pseudo-MS3 method. Graphical Abstract ᅟ.

Electrophysiology of glioma: a Rho GTPase-activating protein reduces tumor growth and spares neuron structure and function.

BACKGROUND: Glioblastomas are the most aggressive type of brain tumor. A successful treatment should aim at halting tumor growth and protecting neuronal cells to prevent functional deficits and cognitive deterioration. Here, we exploited a Rho GTPase-activating bacterial protein toxin, cytotoxic necrotizing factor 1 (CNF1), to interfere with glioma cell growth in vitro and vivo. We also investigated whether this toxin spares neuron structure and function in peritumoral areas. METHODS: We performed a microarray transcriptomic and in-depth proteomic analysis to characterize the molecular changes triggered by CNF1 in glioma cells. We also examined tumor cell senescence and growth in vehicle- and CNF1-treated glioma-bearing mice. Electrophysiological and morphological techniques were used to investigate neuronal alterations in peritumoral cortical areas. RESULTS: Administration of CNF1 triggered molecular and morphological hallmarks of senescence in mouse and human glioma cells in vitro. CNF1 treatment in vivo induced glioma cell senescence and potently reduced tumor volumes. In peritumoral areas of glioma-bearing mice, neurons showed a shrunken dendritic arbor and severe functional alterations such as increased spontaneous activity and reduced visual responsiveness. CNF1 treatment enhanced dendritic length and improved several physiological properties of pyramidal neurons, demonstrating functional preservation of the cortical network. CONCLUSIONS: Our findings demonstrate that CNF1 reduces glioma volume while at the same time maintaining the physiological and structural properties of peritumoral neurons. These data indicate a promising strategy for the development of more effective antiglioma therapies.

Assessment of SRM, MRM(3) , and DIA for the targeted analysis of phosphorylation dynamics in non-small cell lung cancer.

Hypothesis-driven MS-based targeted proteomics has gained great popularity in a relatively short timespan. Next to the widely established selected reaction monitoring (SRM) workflow, data-independent acquisition (DIA), also referred to as sequential window acquisition of all theoretical spectra (SWATH) was introduced as a high-throughput targeted proteomics method. DIA facilitates increased proteome coverage, however, does not yet reach the sensitivity obtained with SRM. Therefore, a well-informed method selection is crucial for designing a successful targeted proteomics experiment. This is especially the case when targeting less conventional peptides such as those that contain PTMs, as these peptides do not always adhere to the optimal fragmentation considerations for targeted assays. Here, we provide insight into the performance of DIA, SRM, and MRM cubed (MRM(3) ) in the analysis of phosphorylation dynamics throughout the phosphoinositide 3-kinase mechanistic target of rapamycin (PI3K-mTOR) and mitogen-activated protein kinase (MAPK) signaling network. We observe indeed that DIA is less sensitive when compared to SRM, however demonstrates increased flexibility, by postanalysis selection of alternative phosphopeptide precursors. Additionally, we demonstrate the added benefit of MRM(3) , allowing the quantification of two poorly accessible phosphosites. In total, targeted proteomics enabled the quantification of 42 PI3K-mTOR and MAPK phosphosites, gaining a so far unachieved in-depth view mTOR signaling events linked to tyrosine kinase inhibitor resistance in non-small cell lung cancer.

Signal Transduction Reaction Monitoring Deciphers Site-Specific PI3K-mTOR/MAPK Pathway Dynamics in Oncogene-Induced Senescence.

We report a straightforward strategy to comprehensively monitor signal transduction pathway dynamics in mammalian systems. Combining targeted quantitative proteomics with highly selective phosphopeptide enrichment, we monitor, with great sensitivity, phosphorylation dynamics of the PI3K-mTOR and MAPK signaling networks. Our approach consists of a single enrichment step followed by a single targeted proteomics experiment, circumventing the need for labeling and immune purification while enabling analysis of selected phosphorylation nodes throughout signaling pathways. The need for such a comprehensive pathway analysis is illustrated by highlighting previously uncharacterized phosphorylation changes in oncogene-induced senescence, associated with diverse biological phenotypes and pharmacological intervention of the PI3K-mTOR pathway.

Flexibility in crosstalk between H2B ubiquitination and H3 methylation in vivo.

Histone H2B ubiquitination is a dynamic modification that promotes methylation of histone H3K79 and H3K4. This crosstalk is important for the DNA damage response and has been implicated in cancer. Here, we show that in engineered yeast strains, ubiquitins tethered to every nucleosome promote H3K79 and H3K4 methylation from a proximal as well as a more distal site, but only if in a correct orientation. This plasticity indicates that the exact location of the attachment site, the native ubiquitin-lysine linkage and ubiquitination cycles are not critical for trans-histone crosstalk in vivo. The flexibility in crosstalk also indicates that other ubiquitination events may promote H3 methylation.

Phosphoproteome dynamics in onset and maintenance of oncogene-induced senescence.

Expression of the BRAF(V600E) oncoprotein is known to cause benign lesions, such as melanocytic nevi (moles). Despite the oncogenic function of mutant BRAF, these lesions are arrested by a cell-autonomous mechanism called oncogene-induced senescence. Infrequently, nevi can progress to malignant melanoma, through mechanisms that are incompletely understood. To gain more insight into this vital tumor-suppression mechanism, we performed a mass-spectrometry-based screening of the proteome and phosphoproteome in cycling and senescent cells and in cells with abrogated senescence. Proteome analysis of senescent cells revealed the up-regulation of established senescence biomarkers, including specific cytokines, but also several proteins not previously associated with senescence, including extracellular matrix-interacting. Using both general and targeted phosphopeptide enrichment by Ti(4+)-IMAC and phosphotyrosine antibody enrichment, we identified over 15,000 phosphorylation sites. Among the regulated phosphorylation sites we encountered components of the interleukin, BRAF/MAPK, and CDK-retinoblastoma pathways and several other factors. The extensive proteome and phosphoproteome dataset of BRAF(V600E)-expressing senescent cells provides molecular clues as to how oncogene-induced senescence is initiated, maintained, or evaded, serving as a comprehensive proteomic basis for functional validation.

Improving SRM assay development: a global comparison between triple quadrupole, ion trap, and higher energy CID peptide fragmentation spectra.

In proteomics, selected reaction monitoring (SRM) is rapidly gaining importance for targeted protein quantification. The triple quadrupole mass analyzers used in SRM assays allow for levels of specificity and sensitivity hard to accomplish by more standard shotgun proteomics experiments. Often, an SRM assay is built by in silico prediction of transitions and/or extraction of peptide precursor and fragment ions from a spectral library. Spectral libraries are typically generated from nonideal ion trap based shotgun proteomics experiments or synthetic peptide libraries, consuming considerable time and effort. Here, we investigate the usability of beam type CID (or "higher energy CID" (HCD)) peptide fragmentation spectra, as acquired using an Orbitrap Velos, to facilitate SRM assay development. Therefore, peptide fragmentation spectra, obtained by ion-trap CID, triple-quadrupole CID (QqQ-CID) and Orbitrap HCD, originating from digested cellular lysates, were compared. Spectral comparison and a dedicated correlation algorithm indicated significantly higher similarity between QqQ-CID and HCD fragmentation spectra than between QqQ-CID and ion trap-CID spectra. SRM transitions generated using a constructed HCD spectral library increased SRM assay sensitivity up to 2-fold, when compared to the use of a library created from more conventionally used ion trap-CID spectra, showing that HCD spectra can assist SRM assay development.