Taxanes trigger cancer cell killing in vivo by inducing non-canonical T cell cytotoxicity.

Although treatment with taxanes does not always lead to clinical benefit, all patients are at risk of their detrimental side effects such as peripheral neuropathy. Understanding the in vivo mode of action of taxanes can help design improved treatment regimens. Here, we demonstrate that in vivo, taxanes directly trigger T cells to selectively kill cancer cells in a non-canonical, T cell receptor-independent manner. Mechanistically, taxanes induce T cells to release cytotoxic extracellular vesicles, which lead to apoptosis specifically in tumor cells while leaving healthy epithelial cells intact. We exploit these findings to develop an effective therapeutic approach, based on transfer of T cells pre-treated with taxanes ex vivo, thereby avoiding toxicity of systemic treatment. Our study reveals a different in vivo mode of action of one of the most commonly used chemotherapies, and opens avenues to harness T cell-dependent anti-tumor effects of taxanes while avoiding systemic toxicity.

Histidine phosphorylation in human cells; a needle or phantom in the haystack?

It has been suggested that in mammalian cells histidine residues in proteins may become as frequently phosphorylated as serine, threonine and tyrosine, and may play a key role in mammalian signaling. Here we applied a robust workflow that earlier allowed us to detect histidine phosphorylation in bacteria unambiguously, to probe for histidine phosphorylation in four human cell lines. Initially, seemingly hundreds of protein histidine phosphorylations were picked up in all studied human cell lines. However, careful examination of the data, and several control experiments, led us to the conclusion that >99% of these initially assigned pHis sites were not genuine, and should be site localized to neighboring Ser/Thr residues. Nevertheless, our methods are selective enough to detect just a handful of genuine pHis sites in mammalian cells, representing well-known enzymatic intermediates. Consequently, we do not find any evidence in our data supporting that protein histidine phosphorylation plays a role in mammalian signaling.

Widespread Arginine Phosphorylation in Staphylococcus aureus.

Arginine phosphorylation was only recently discovered to play a significant and relevant role in the Gram-positive bacterium Bacillus subtilis. In addition, arginine phosphorylation was also detected in Staphylococcus aureus, suggesting a widespread role in bacteria. However, the large-scale analysis of protein phosphorylation, and especially those that involve a phosphoramidate bond, comes along with several challenges. The substoichiometric nature of protein phosphorylation requires proper enrichment strategies prior to LC-MS/MS analysis, and the acid instability of phosphoramidates was long thought to impede those enrichments. Furthermore, good spectral quality is required, which can be impeded by the presence of neutral losses of phosphoric acid upon higher energy collision-induced dissociation. Here we show that pArg is stable enough for commonly used Fe3+-IMAC enrichment followed by LC-MS/MS and that HCD is still the gold standard for the analysis of phosphopeptides. By profiling a serine/threonine kinase (Stk1) and phosphatase (Stp1) mutant from a methicillin-resistant S. aureus mutant library, we identified 1062 pArg sites and thus the most comprehensive arginine phosphoproteome to date. Using synthetic arginine phosphorylated peptides, we validated the presence and localization of arginine phosphorylation in S. aureus. Finally, we could show that the knockdown of Stp1 significantly increases the overall amount of arginine phosphorylation in S. aureus. However, our analysis also shows that Stp1 is not a direct protein-arginine phosphatase but only indirectly influences the arginine phosphoproteome.

RANK signaling increases after anti-HER2 therapy contributing to the emergence of resistance in HER2-positive breast cancer.

BACKGROUND: Around 15-20% of primary breast cancers are characterized by HER2 protein overexpression and/or HER2 gene amplification. Despite the successful development of anti-HER2 drugs, intrinsic and acquired resistance represents a major hurdle. This study was performed to analyze the RANK pathway contribution in HER2-positive breast cancer and anti-HER2 therapy resistance. METHODS: RANK and RANKL protein expression was assessed in samples from HER2-positive breast cancer patients resistant to anti-HER2 therapy and treatment-naive patients. RANK and RANKL gene expression was analyzed in paired samples from patients treated with neoadjuvant dual HER2-blockade (lapatinib and trastuzumab) from the SOLTI-1114 PAMELA trial. Additionally, HER2-positive breast cancer cell lines were used to modulate RANK expression and analyze in vitro the contribution of RANK signaling to anti-HER2 resistance and downstream signaling. RESULTS: RANK and RANKL proteins are more frequently detected in HER2-positive tumors that have acquired resistance to anti-HER2 therapies than in treatment-naive ones. RANK (but not RANKL) gene expression increased after dual anti-HER2 neoadjuvant therapy in the cohort from the SOLTI-1114 PAMELA trial. Results in HER2-positive breast cancer cell lines recapitulate the clinical observations, with increased RANK expression observed after short-term treatment with the HER2 inhibitor lapatinib or dual anti-HER2 therapy and in lapatinib-resistant cells. After RANKL stimulation, lapatinib-resistant cells show increased NF-κB activation compared to their sensitive counterparts, confirming the enhanced functionality of the RANK pathway in anti-HER2-resistant breast cancer. Overactivation of the RANK signaling pathway enhances ERK and NF-κB signaling and increases lapatinib resistance in different HER2-positive breast cancer cell lines, whereas RANK loss sensitizes lapatinib-resistant cells to the drug. Our results indicate that ErbB signaling is required for RANK/RANKL-driven activation of ERK in several HER2-positive cell lines. In contrast, lapatinib is not able to counteract the NF-κB activation elicited after RANKL treatment in RANK-overexpressing cells. Finally, we show that RANK binds to HER2 in breast cancer cells and that enhanced RANK pathway activation alters HER2 phosphorylation status. CONCLUSIONS: Our data support a physical and functional link between RANK and HER2 signaling in breast cancer and demonstrate that increased RANK signaling may contribute to the development of lapatinib resistance through NF-κB activation. Whether HER2-positive breast cancer patients with tumoral RANK expression might benefit from dual HER2 and RANK inhibition therapy remains to be elucidated.

Thermal Proteome Profiling in Zebrafish Reveals Effects of Napabucasin on Retinoic Acid Metabolism.

Thermal proteome profiling (TPP) allows for the unbiased detection of drug-target protein engagements in vivo. Traditionally, 1 cell type is used for TPP studies, with the risk of missing important differentially expressed target proteins. The use of whole organisms would circumvent this problem. Zebrafish embryos are amenable to such an approach. Here, we used TPP on whole zebrafish embryo lysate to identify protein targets of napabucasin, a compound that may affect signal transducer and activator of transcription 3 (Stat3) signaling through an ill-understood mechanism. In zebrafish embryos, napabucasin induced developmental defects consistent with inhibition of Stat3 signaling. TPP profiling showed no distinct shift in Stat3 upon napabucasin treatment, but effects were detected on the oxidoreductase, Pora, which might explain effects on Stat3 signaling. Interestingly, thermal stability of several aldehyde dehydrogenases was affected. Moreover, napabucasin activated aldehyde dehydrogenase enzymatic activity in vitro. Aldehyde dehydrogenases have crucial roles in retinoic acid metabolism, and functionally, we validated napabucasin-mediated activation of the retinoic acid pathway in zebrafish in vivo. We conclude that TPP profiling in whole zebrafish embryo lysate is feasible and facilitates direct correlation of in vivo effects of small molecule drugs with their protein targets.

The IMiD target CRBN determines HSP90 activity toward transmembrane proteins essential in multiple myeloma.

The complex architecture of transmembrane proteins requires quality control (QC) of folding, membrane positioning, and trafficking as prerequisites for cellular homeostasis and intercellular communication. However, it has remained unclear whether transmembrane protein-specific QC hubs exist. Here we identify cereblon (CRBN), the target of immunomodulatory drugs (IMiDs), as a co-chaperone that specifically determines chaperone activity of HSP90 toward transmembrane proteins by means of counteracting AHA1. This function is abrogated by IMiDs, which disrupt the interaction of CRBN with HSP90. Among the multiple transmembrane protein clients of CRBN-AHA1-HSP90 revealed by cell surface proteomics, we identify the amino acid transporter LAT1/CD98hc as a determinant of IMiD activity in multiple myeloma (MM) and present an Anticalin-based CD98hc radiopharmaceutical for MM radio-theranostics. These data establish the CRBN-AHA1-HSP90 axis in the biogenesis of transmembrane proteins, link IMiD activity to tumor metabolism, and nominate CD98hc and LAT1 as attractive diagnostic and therapeutic targets in MM.

In-Depth Characterization of the Staphylococcus aureus Phosphoproteome Reveals New Targets of Stk1.

Staphylococcus aureus is a major cause of infections worldwide, and infection results in a variety of diseases. As of no surprise, protein phosphorylation is an important game player in signaling cascades and has been shown to be involved in S. aureus virulence. Albeit long neglected, eukaryotic-type serine/threonine kinases in S. aureus have been implicated in this complex signaling cascades. Due to the substoichiometric nature of protein phosphorylation and a lack of suitable analysis tools, the knowledge of these cascades is, however, to date, still limited. Here, were apply an optimized protocol for efficient phosphopeptide enrichment via Fe3+-IMAC followed by LC-MS/MS to get a better understanding of the impact of protein phosphorylation on the complex signaling networks involved in pathogenicity. By profiling a serine/threonine kinase and phosphatase mutant from a methicillin-resistant S. aureus mutant library, we generated the most comprehensive phosphoproteome data set of S. aureus to date, aiding a better understanding of signaling in bacteria. With the identification of 3800 class I p-sites, we were able to increase the number of identifications by more than 21 times compared with recent literature. In addition, we were able to identify 74 downstream targets of the only reported eukaryotic-type Ser/Thr kinase of the S. aureus strain USA300, Stk1. This work allowed an extensive analysis of the bacterial phosphoproteome and indicates that Ser/Thr kinase signaling is far more abundant than previously anticipated in S. aureus.

Meltome atlas-thermal proteome stability across the tree of life.

We have used a mass spectrometry-based proteomic approach to compile an atlas of the thermal stability of 48,000 proteins across 13 species ranging from archaea to humans and covering melting temperatures of 30-90 °C. Protein sequence, composition and size affect thermal stability in prokaryotes and eukaryotic proteins show a nonlinear relationship between the degree of disordered protein structure and thermal stability. The data indicate that evolutionary conservation of protein complexes is reflected by similar thermal stability of their proteins, and we show examples in which genomic alterations can affect thermal stability. Proteins of the respiratory chain were found to be very stable in many organisms, and human mitochondria showed close to normal respiration at 46 °C. We also noted cell-type-specific effects that can affect protein stability or the efficacy of drugs. This meltome atlas broadly defines the proteome amenable to thermal profiling in biology and drug discovery and can be explored online at http://meltomeatlas.proteomics.wzw.tum.de:5003/ and http://www.proteomicsdb.org.

Loss of the Fanconi anemia-associated protein NIPA causes bone marrow failure.

Inherited bone marrow failure syndromes (IBMFSs) are a heterogeneous group of disorders characterized by defective hematopoiesis, impaired stem cell function, and cancer susceptibility. Diagnosis of IBMFS presents a major challenge due to the large variety of associated phenotypes, and novel, clinically relevant biomarkers are urgently needed. Our study identified nuclear interaction partner of ALK (NIPA) as an IBMFS gene, as it is significantly downregulated in a distinct subset of myelodysplastic syndrome-type (MDS-type) refractory cytopenia in children. Mechanistically, we showed that NIPA is major player in the Fanconi anemia (FA) pathway, which binds FANCD2 and regulates its nuclear abundance, making it essential for a functional DNA repair/FA/BRCA pathway. In a knockout mouse model, Nipa deficiency led to major cell-intrinsic defects, including a premature aging phenotype, with accumulation of DNA damage in hematopoietic stem cells (HSCs). Induction of replication stress triggered a reduction in and functional decline of murine HSCs, resulting in complete bone marrow failure and death of the knockout mice with 100% penetrance. Taken together, the results of our study add NIPA to the short list of FA-associated proteins, thereby highlighting its potential as a diagnostic marker and/or possible target in diseases characterized by hematopoietic failure.

Gaining Confidence in the Elusive Histidine Phosphoproteome.

Recent technological advances have made it possible to investigate the hitherto rather elusive protein histidine phosphorylation. However, confident site-specific localization of protein histidine phosphorylation remains challenging. Here, we address this problem, presenting a mass-spectrometry-based approach that outperforms classical HCD fragmentation without compromising sensitivity. We use the phosphohistidine immonium ion as a diagnostic tool as well as ETD-based fragmentation techniques to achieve unambiguous identification and localization of histidine-phosphorylation sites. The work presented here will allow more confident investigation of the phosphohistidine proteome to reveal the roles of histidine phosphorylation in cellular signaling events.

A New Tool to Reveal Bacterial Signaling Mechanisms in Antibiotic Treatment and Resistance.

The rapid emergence of antimicrobial resistance is a major threat to human health. Antibiotics modulate a wide range of biological processes in bacteria and as such, the study of bacterial cellular signaling could aid the development of urgently needed new antibiotic agents. Due to the advances in bacterial phosphoproteomics, such a systemwide analysis of bacterial signaling in response to antibiotics has recently become feasible. Here we present a dynamic view of differential protein phosphorylation upon antibiotic treatment and antibiotic resistance. Most strikingly, differential phosphorylation was observed on highly conserved residues of resistance regulating transcription factors, implying a previously unanticipated role of phosphorylation mediated regulation. Using the comprehensive phosphoproteomics data presented here as a resource, future research can now focus on deciphering the precise signaling mechanisms contributing to resistance, eventually leading to alternative strategies to combat antimicrobial resistance.

Adaptive Resistance to EGFR-Targeted Therapy by Calcium Signaling in NSCLC Cells.

Targeted therapies against oncogenic receptor tyrosine kinases (RTK) show promising results in the clinic. Unfortunately, despite the initial positive response, most patients develop therapeutic resistance. Most research has focused on acquired resistance occurring after an extensive time of treatment; however, the question remains as to how cells can survive an initial treatment, as early resistance to apoptosis will enable cells to develop any growth-stimulating mechanism. Here, the non-small cell lung cancer (NSCLC) PC9 cell line was used to systematically profile, by mass spectrometry, changes in the proteome, kinome, and phosphoproteome during early treatment with the EGFR inhibitor afatinib. Regardless of the response, initial drug-sensitive cells rapidly adapt to targeted therapy, and within days, cells regained the capacity to proliferate, despite persisting target inhibition. These data reveal a rapid reactivation of mTOR and MAPK signaling pathways after initial inhibition and an increase in abundance and activity of cytoskeleton and calcium signaling-related proteins. Pharmacologic inhibition of reactivated pathways resulted in increased afatinib efficacy. However more strikingly, cells that were restricted from accessing extracellular calcium were extremely sensitive to afatinib treatment. These findings were validated using three additional inhibitors tested in four different NSCLC cell lines, and the data clearly indicated a role for Ca2+ signaling during the development of adaptive resistance. From a therapeutic point of view, the increased inhibitor efficacy could limit or even prevent further resistance development.Implications: Combined targeting of calcium signaling and RTKs may limit drug resistance and improve treatment efficacy. Mol Cancer Res; 16(11); 1773-84. ©2018 AACR.

CTGF/VEGFA-activated Fibroblasts Promote Tumor Migration Through Micro-environmental Modulation.

Fibroblast activation is associated with tumor progression and implicated in metastasis, but the initial triggering signals required to kick-start this process remain largely unknown. Because small cancerous lesions share limited physical contact with neighboring fibroblasts, we reasoned the first tumor-derived signal for fibroblast activation should be secreted and diffusible. By pulsed metabolic labeling and click-chemistry based affinity enrichment, we sieved through the ductal carcinoma secretome for potential fibroblast activators. Using immuno-depletion/supplementation assays on various secreted factors, we pinpointed that tumor-secreted CTGF/VEGFA alone is sufficient to activate paired mammary fibroblasts from the same patient via ROCK1 and JunB signaling. Fibroblasts activated in this manner are distinct in morphology, growth, and adopt a highly tumor-like secretion profile, which in turn promotes tumor migration by counteracting oxidative and lactate stress. These findings reveal a profound division-of-labor between normal and cancer cells under the directive of the latter, and allude to potential metastatic prevention through inhibiting local fibroblast activation.

Defeating Major Contaminants in Fe3+- Immobilized Metal Ion Affinity Chromatography (IMAC) Phosphopeptide Enrichment.

Here we demonstrate that biomolecular contaminants, such as nucleic acid molecules, can seriously interfere with immobilized metal ion affinity chromatography (IMAC)-based phosphopeptide enrichments. We address and largely solve this issue, developing a robust protocol implementing methanol/chloroform protein precipitation and enzymatic digestion using benzonase, which degrades all forms of DNA and RNA, before IMAC-column loading. This simple procedure resulted in a drastic increase of enrichment sensitivity, enabling the identification of around 17,000 unique phosphopeptides and 12,500 unambiguously localized phosphosites in human cell-lines from a single LC-MS/MS run, constituting a 50% increase when compared with the standard protocol. The improved protocol was also applied to bacterial samples, increasing the number of identified bacterial phosphopeptides even more strikingly, by a factor 10, when compared with the standard protocol. For E. coli we detected around 1300 unambiguously localized phosphosites per LC-MS/MS run. The preparation of these ultra-pure phosphopeptide samples only requires marginal extra costs and sample preparation time and should thus be adoptable by every laboratory active in the field of phosphoproteomics.

Widespread bacterial protein histidine phosphorylation revealed by mass spectrometry-based proteomics.

For decades, major difficulties in analyzing histidine phosphorylation have limited the study of phosphohistidine signaling. Here we report a method revealing widespread and abundant protein histidine phosphorylation in Escherichia coli. We generated an extensive E. coli phosphoproteome data set, in which a remarkably high percentage (∼10%) of phosphorylation sites are phosphohistidine sites. This resource should help enable a better understanding of the biological function of histidine phosphorylation.

Differential oxidation of protein-tyrosine phosphatases during zebrafish caudal fin regeneration.

Zebrafish have the capacity to regenerate lost tissues and organs. Amputation of the caudal fin results in a rapid, transient increase in H2O2 levels emanating from the wound margin, which is essential for regeneration, because quenching of reactive oxygen species blocks regeneration. Protein-tyrosine phosphatases (PTPs) have a central role in cell signalling and are susceptible to oxidation, which results in transient inactivation of their catalytic activity. We hypothesized that PTPs may become oxidized in response to amputation of the caudal fin. Using the oxidized PTP-specific (ox-PTP) antibody and liquid chromatography-mass spectrometry, we identified 33 PTPs in adult zebrafish fin clips of the total of 44 PTPs that can theoretically be detected based on sequence conservation. Of these 33 PTPs, 8 were significantly more oxidized 40 min after caudal fin amputation. Surprisingly, Shp2, one of the PTPs that were oxidized in response to caudal fin amputation, was required for caudal fin regeneration. In contrast, Rptpα, which was not oxidized upon amputation, was dispensable for caudal fin regeneration. Our results demonstrate that PTPs are differentially oxidized in response to caudal fin amputation and that there is a differential requirement for PTPs in regeneration.

Combining Deep Sequencing, Proteomics, Phosphoproteomics, and Functional Screens To Discover Novel Regulators of Sphingolipid Homeostasis.

Sphingolipids (SLs) are essential components of cell membranes and are broad-range bioactive signaling molecules. SL levels must be tightly regulated as imbalances affect cellular function and contribute to pathologies ranging from neurodegenerative and metabolic disorders to cancer and aging. Deciphering how SL homeostasis is maintained and uncovering new regulators is required for understanding lipid biology and for identifying new targets for therapeutic interventions. Here we combine omics technologies to identify the changes of the transcriptome, proteome, and phosphoproteome in the yeast Saccharomyces cerevisiae upon SL depletion induced by myriocin. Surprisingly, while SL depletion triggers important changes in the expression of regulatory proteins involved in SL homeostasis, the most dramatic regulation occurs at the level of the phosphoproteome, suggesting that maintaining SL homeostasis demands rapid responses. To discover which of the phosphoproteomic changes are required for the cell's first-line response to SL depletion, we overlaid our omics results with systematic growth screens for genes required during growth in myriocin. By following the rate of SL biosynthesis in those candidates that are both affecting growth and are phosphorylated in response to the drug, we uncovered Atg9, Stp4, and Gvp36 as putative new regulators of SL homeostasis.

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).