CD301 and LSECtin glycan-binding receptors of innate immune cells serve as prognostic markers and potential predictors of immune response in breast cancer subtypes.

Glycosylation is a prominent posttranslational modification, and alterations in glycosylation are a hallmark of cancer. Glycan-binding receptors, primarily expressed on immune cells, play a central role in glycan recognition and immune response. Here, we used the recombinant C-type glycan-binding receptors CD301, Langerin, SRCL, LSECtin, and DC-SIGNR to recognize their ligands on tissue microarrays (TMA) of a large cohort (n = 1859) of invasive breast cancer of different histopathological types to systematically determine the relevance of altered glycosylation in breast cancer. Staining frequencies of cancer cells were quantified in an unbiased manner by a computer-based algorithm. CD301 showed the highest overall staining frequency (40%), followed by LSECtin (16%), Langerin (4%) and DC-SIGNR (0.5%). By Kaplan-Meier analyses, we identified LSECtin and CD301 as prognostic markers in different breast cancer subtypes. Positivity for LSECtin was associated with inferior disease-free survival in all cases, particularly in estrogen receptor positive (ER+) breast cancer of higher histological grade. In triple negative breast cancer, positivity for CD301 correlated with a worse prognosis. Based on public RNA single-cell sequencing data of human breast cancer infiltrating immune cells, we found CLEC10A (CD301) and CLEC4G (LSECtin) exclusively expressed in distinct subpopulations, particularly in dendritic cells and macrophages, indicating that specific changes in glycosylation may play a significant role in breast cancer immune response and progression.

Analyzing tyrosine kinase activity in head and neck cancer by functional kinomics: Identification of hyperactivated Src family kinases as prognostic markers and potential targets.

Signal transduction via protein kinases is of central importance in cancer biology and treatment. However, the clinical success of kinase inhibitors is often hampered by a lack of robust predictive biomarkers, which is also caused by the discrepancy between kinase expression and activity. Therefore, there is a need for functional tests to identify aberrantly activated kinases in individual patients. Here we present a systematic analysis of the tyrosine kinases in head and neck cancer using such a test-functional kinome profiling. We detected increased tyrosine kinase activity in tumors compared with their corresponding normal tissue. Moreover, we identified members of the family of Src kinases (Src family kinases [SFK]) to be aberrantly activated in the majority of the tumors, which was confirmed by additional methods. We could also show that SFK hyperphosphorylation is associated with poor prognosis, while inhibition of SFK impaired cell proliferation, especially in cells with hyperactive SFK. In summary, functional kinome profiling identified SFK to be frequently hyperactivated in head and neck squamous cell carcinoma. SFK may therefore be potential therapeutic targets. These results furthermore demonstrate how functional tests help to increase our understanding of cancer biology and support the expansion of precision oncology.

Resistance to anti-CD19/CD3 BiTE in acute lymphoblastic leukemia may be mediated by disrupted CD19 membrane trafficking.

The CD19 antigen is a promising target for immunotherapy of acute lymphoblastic leukemia (ALL), but CD19- relapses remain a major challenge in about 10% to 20% of patients. Here, we analyzed 4 CD19- ALL relapses after treatment with the CD19/CD3 bispecific T-cell engager (BiTE) blinatumomab. Three were on-drug relapses, with the CD19- escape variant first detected after only 2 treatment courses. In 1 patient, the CD19- clone appeared as a late relapse 19 months after completion of blinatumomab treatment. All 4 cases showed a cellular phenotype identical to the primary diagnosis except for CD19 negativity. This argued strongly in favor of an isolated molecular event and against a common lymphoid CD19- progenitor cell or myeloid lineage shift driving resistance. A thorough molecular workup of 1 of the cases with early relapse confirmed this hypothesis by revealing a disrupted CD19 membrane export in the post-endoplasmic reticulum compartment as molecular basis for blinatumomab resistance.

Immature O-glycans recognized by the macrophage glycoreceptor CLEC10A (MGL) are induced by 4-hydroxy-tamoxifen, oxidative stress and DNA-damage in breast cancer cells.

BACKGROUND: Ligands of the C-type lectin CLEC10A such as Tn and sialyl-Tn representing early intermediates of O-glycosylation are hallmarks of many human malignancies. A variety of regulatory mechanisms underlying their expression are being discussed. METHODS: CLEC10A ligands were detected in various tissues and cells using the recombinant glycan-binding domain of CLEC10A. In normal breast and endometrium, presence of ligands was correlated to the female cycle. Estrogen- and stress dependent induction of CLEC10A ligands was analyzed in MCF7 and T47D cells exposed to 4-hydroxy-tamoxifen (Tam), zeocin and hydrogen peroxide. The expression and localization of CLEC10A ligands was analyzed by Western blot and immunofluorescence. In breast cancer patients CLEC10A ligand expression and survival was correlated by Kaplan-Meyer analysis. RESULT: We observed binding of CLEC10A in normal endometrial and breast tissues during the late phase of the female hormonal cycle suggesting a suppressive effect of female sex hormones on CLEC10A ligand expression. Accordingly, CLEC10A ligands were induced in MCF7- and T47D breast cancer cells after Tam treatment and accumulated on the cell surface and in the endosomal/lysosomal compartment. Phagocytosis experiments indicate that macrophages preferentially internalize CLEC10A ligands coated beads and Tam treated MCF7 cells. CLEC10A ligands were also expressed after the addition of zeocin and hydrogen-peroxide. Each substance induced the production of ROS indicating reactive oxygen species as a unifying mechanism of CLEC10A ligand induction. Mechanistically, increased expression of GalNAc-transferase 6 (GalNT6) and translocation of GalNT2 and GalNT6 from cis- towards trans-Golgi compartment was observed, while protein levels of COSMC and T-synthase remained unaffected. In breast cancer patients, positivity for CLEC10A staining in tumor tissues was associated with improved outcome and survival. CONCLUSION: CLEC10A ligands are inducible by hormone depletion, 4-hydroxy-tamoxifen and agents inducing DNA damage and oxidative stress. Our results indicate that CLEC10A acts as a receptor for damaged and dead cells and may play an important role in the uptake of cell debris by macrophages and dendritic cells.

An in-depth multi-omics analysis in RLE-6TN rat alveolar epithelial cells allows for nanomaterial categorization.

BACKGROUND: Nanomaterials (NMs) can be fine-tuned in their properties resulting in a high number of variants, each requiring a thorough safety assessment. Grouping and categorization approaches that would reduce the amount of testing are in principle existing for NMs but are still mostly conceptual. One drawback is the limited mechanistic understanding of NM toxicity. Thus, we conducted a multi-omics in vitro study in RLE-6TN rat alveolar epithelial cells involving 12 NMs covering different materials and including a systematic variation of particle size, surface charge and hydrophobicity for SiO2 NMs. Cellular responses were analyzed by global proteomics, targeted metabolomics and SH2 profiling. Results were integrated using Weighted Gene Correlation Network Analysis (WGCNA). RESULTS: Cluster analyses involving all data sets separated Graphene Oxide, TiO2_NM105, SiO2_40 and Phthalocyanine Blue from the other NMs as their cellular responses showed a high degree of similarities, although apical in vivo results may differ. SiO2_7 behaved differently but still induced significant changes. In contrast, the remaining NMs were more similar to untreated controls. WGCNA revealed correlations of specific physico-chemical properties such as agglomerate size and redox potential to cellular responses. A key driver analysis could identify biomolecules being highly correlated to the observed effects, which might be representative biomarker candidates. Key drivers in our study were mainly related to oxidative stress responses and apoptosis. CONCLUSIONS: Our multi-omics approach involving proteomics, metabolomics and SH2 profiling proved useful to obtain insights into NMs Mode of Actions. Integrating results allowed for a more robust NM categorization. Moreover, key physico-chemical properties strongly correlating with NM toxicity were identified. Finally, we suggest several key drivers of toxicity that bear the potential to improve future testing and assessment approaches.

Reverse phase protein arrays enable glioblastoma molecular subtyping.

In the present study we investigated the phosphorylation status of the 12 most important signaling cascades in glioblastomas. More than 60 tumor and control biopsies from tumor center and periphery (based on neuronavigation) were subjected to selective protein expression analysis using reverse-phase protein arrays (RPPA) incubated with antibodies against posttranslationally modified cancer pathway proteins. The ratio between phosphorylated (or modified) and non-phosphorylated protein was assessed. All samples were histopathologically validated and proteomic profiles correlated with clinical and survival data. By RPPA, we identified three distinct activation patterns within glioblastoma defined by the ratios of pCREB1/CREB1, NOTCH-ICD/NOTCH1, and pGSK3ß/GSK3ß, respectively. These subclasses demonstrated distinct overall survival patterns in a cohort of patients from a single-institution and in an analysis of publicly available data. In particular, a high pGSK3ß/GSK3ß-ratio was associated with a poor survival. Wnt-activation/GSK3ß-inhibition in U373 and U251 cell lines halted glioma cell proliferation and migration. Gene expression analysis was used as an internal quality control of baseline proteomic data. The protein expression and phosphorylation had a higher resolution, resulting in a better class-subdivision than mRNA based stratification data. Patients with different proteomic profiles from multiple biopsies showed a worse overall survival. The CREB1-, NOTCH1-, GSK3ß-phosphorylation status correlated with glioma grades. RPPA represent a fast and reliable tool to supplement morphological diagnosis with pathway-specific information in individual tumors. These data can be exploited for molecular stratification and possible combinatorial treatment planning. Further, our results may optimize current glioma grading algorithms.

Time-resolved characterization of cAMP/PKA-dependent signaling reveals that platelet inhibition is a concerted process involving multiple signaling pathways.

One of the most important physiological platelet inhibitors is endothelium-derived prostacyclin which stimulates the platelet cyclic adenosine monophosphate/protein kinase A (cAMP/PKA)-signaling cascade and inhibits virtually all platelet-activating key mechanisms. Using quantitative mass spectrometry, we analyzed time-resolved phosphorylation patterns in human platelets after treatment with iloprost, a stable prostacyclin analog, for 0, 10, 30, and 60 seconds to characterize key mediators of platelet inhibition and activation in 3 independent biological replicates. We quantified over 2700 different phosphorylated peptides of which 360 were significantly regulated upon stimulation. This comprehensive and time-resolved analysis indicates that platelet inhibition is a multipronged process involving different kinases and phosphatases as well as many previously unanticipated proteins and pathways.

Deciphering of ADP-induced, phosphotyrosine-dependent signaling networks in human platelets by Src-homology 2 region (SH2)-profiling.

Tyrosine phosphorylation plays a central role in signal transduction controlling many important biological processes. In platelets, the activity of several signaling proteins is controlled by tyrosine phosphorylation ensuring proper platelet activation and aggregation essential for regulation of the delicate balance between bleeding and hemostasis. Here, we applied Src-homology 2 region (SH2)-profiling for deciphering of the phosphotyrosine state of human platelets activated by adenosine diphosphate (ADP). Applying a panel of 31 SH2-domains, rapid and complex regulation of the phosphotyrosine state of platelets was observed after ADP stimulation. Specific inhibition of platelet P2Y receptors by synthetic drugs revealed a major role for the P2Y1 receptor in tyrosine phosphorylation. Concomitant activation of protein kinase A (PKA) abolished ADP-induced tyrosine phosphorylation in a time and concentration-dependent manner. Given the fact that PKA activity is negatively regulated by the P2Y12 receptor, our data provide evidence for a novel link of synergistic control of the state of tyrosine phosphorylation by both P2Y receptors. By SH2 domain pull down and MS/MS analysis, we identified distinct tyrosine phosphorylation sites in cell adhesion molecules, intracellular adapter proteins and phosphatases suggesting a major, functional role of tyrosine phosphorylation of theses candidate proteins in ADP-dependent signaling in human platelets.

Low-grade and high-grade mammary carcinomas in WAP-T transgenic mice are independent entities distinguished by Met expression.

Mammary carcinomas developing in SV40 transgenic WAP-T mice arise in two distinct histological phenotypes: as differentiated low-grade and undifferentiated high-grade tumors. We integrated different types of information such as histological grading, analysis of aCGH-based gene copy number and gene expression profiling to provide a comprehensive molecular description of mammary tumors in WAP-T mice. Applying a novel procedure for the correlation of gene copy number with gene expression on a global scale, we observed in tumor samples a global coherence between genotype and transcription. This coherence can be interpreted as a matched transcriptional regulation inherited from the cells of tumor origin and determined by the activity of cancer driver genes. Despite common recurrent genomic aberrations, e.g. gain of chr. 15 in most WAP-T tumors, loss of chr. 19 frequently occurs only in low-grade tumors. These tumors show features of "basal-like" epithelial differentiation, particularly expression of keratin 14. The high-grade tumors are clearly separated from the low-grade tumors by strong expression of the Met gene and by coexpression of epithelial (e.g. keratin 18) and mesenchymal (e.g. vimentin) markers. In high-grade tumors, the expression of the nonmutated Met protein is associated with Met-locus amplification and Met activity. The role of Met as a cancer driver gene is supported by the contribution of active Met signaling to motility and growth of mammary tumor-derived cells. Finally, we discuss the independent origin of low- and high-grade tumors from distinct cells of tumor origin, possibly luminal progenitors, distinguished by Met gene expression and Met signaling.

SLAMF receptors negatively regulate B cell receptor signaling in chronic lymphocytic leukemia via recruitment of prohibitin-2.

We identified a subset of Chronic Lymphocytic Leukemia (CLL) patients with high Signaling Lymphocytic Activation Molecule Family (SLAMF) receptor-related signaling that showed an indolent clinical course. Since SLAMF receptors play a role in NK cell biology, we reasoned that these receptors may impact NK cell-mediated CLL immunity. Indeed, our experiments showed significantly decreased degranulation capacity of primary NK cells from CLL patients expressing low levels of SLAMF1 and SLAMF7. Since the SLAMFlow signature was strongly associated with an unmutated CLL immunoglobulin heavy chain (IGHV) status in large datasets, we investigated the impact of SLAMF1 and SLAMF7 on the B cell receptor (BCR) signaling axis. Overexpression of SLAMF1 or SLAMF7 in IGHV mutated CLL cell models resulted in reduced proliferation and impaired responses to BCR ligation, whereas the knockout of both receptors showed opposing effects and increased sensitivity toward inhibition of components of the BCR pathway. Detailed molecular analyzes showed that SLAMF1 and SLAMF7 receptors mediate their BCR pathway antagonistic effects via recruitment of prohibitin-2 (PHB2) thereby impairing its role in signal transduction downstream the IGHV-mutant IgM-BCR. Together, our data indicate that SLAMF receptors are important modulators of the BCR signaling axis and may improve immune control in CLL by interference with NK cells.

A multi-omics approach reveals mechanisms of nanomaterial toxicity and structure-activity relationships in alveolar macrophages.

In respect to the high number of released nanomaterials and their highly variable properties, novel grouping approaches are required based on the effects of nanomaterials. Proper grouping calls for a combination of an experimental setup with a higher number of structurally similar nanomaterials and for employing integrated omics approaches to identify the mode of action. Here, we analyzed the effects of seven well-characterized NMs comprising different chemical compositions, sizes and chemical surface modifications on the rat alveolar macrophage cell line NR8383. The NMs were investigated at three doses ranging from 2.5 to 10 µg/cm2 after 24 h incubation using an integrated multi-omics approach involving untargeted proteomics, targeted metabolomics, and src homology 2 (SH2) profiling. By using Weighted Gene Correlation Network Analysis (WGCNA) for the integrative data, we identified correlations of molecular pathways with physico-chemical properties and toxicological endpoints. The three investigated SiO2 variants induced strong alterations in all three omics approaches and were, therefore, be classified as "active." Two organic phthalocyanines showed minor responses and Mn2O3 induced a different molecular response pattern than the other NMs. WGCNA revealed that agglomerate size and surface area as well as LDH release are among the most important parameters correlating with nanotoxicology. Moreover, we identified key drivers that can serve as representative biomarker candidates, supporting the value of multi-omics approaches to establish integrated approaches to testing and assessment (IATAs).

Approaching clinical proteomics: current state and future fields of application in cellular proteomics.

Recent developments in proteomics technology offer new opportunities for clinical applications in hospital or specialized laboratories including the identification of novel biomarkers, monitoring of disease, detecting adverse effects of drugs, and environmental hazards. Advanced spectrometry technologies and the development of new protein array formats have brought these analyses to a standard, which now has the potential to be used in clinical diagnostics. Besides standardization of methodologies and distribution of proteomic data into public databases, the nature of the human body fluid proteome with its high dynamic range in protein concentrations, its quantitation problems, and its extreme complexity present enormous challenges. Molecular cell biology (cytomics) with its link to proteomics is a new fast moving scientific field, which addresses functional cell analysis and bioinformatic approaches to search for novel cellular proteomic biomarkers or their release products into body fluids that provide better insight into the enormous biocomplexity of disease processes and are suitable for patient stratification, therapeutic monitoring, and prediction of prognosis. Experience from studies of in vitro diagnostics and especially in clinical chemistry showed that the majority of errors occurs in the preanalytical phase and the setup of the diagnostic strategy. This is also true for clinical proteomics where similar preanalytical variables such as inter- and intra-assay variability due to biological variations or proteolytical activities in the sample will most likely also influence the results of proteomics studies. However, before complex proteomic analysis can be introduced at a broader level into the clinic, standardization of the preanalytical phase including patient preparation, sample collection, sample preparation, sample storage, measurement, and data analysis is another issue which has to be improved. In this report, we discuss the recent advances and applications that fulfill the criteria for clinical proteomics with the focus on cellular proteomics (cytoproteomics) as related to preanalytical and analytical standardization and to quality control measures required for effective implementation of these technologies and analytes into routine laboratory testing to generate novel actionable health information. It will then be crucial to design and carry out clinical studies that can eventually identify novel clinical diagnostic strategies based on these techniques and validate their impact on clinical decision making.

Profiling the tyrosine phosphorylation state using SH2 domains.

Global monitoring of cellular signaling activity is of great importance for the understanding of the regulation of complex signaling networks and the characterization of signaling pathways deregulated in diseases. Tyrosine phosphorylation of intracellular signaling proteins followed by the recognition and binding of Src homology 2 (SH2) domains are key mechanisms in the downstream transmission of many important biological signals. SH2 domains, comprising 120 members in humans, are small modular protein binding domains that recognize tyrosine phosphorylated signaling proteins with high specificity. Based on these binding properties, the large number of naturally occurring and currently available SH2 domains serve as excellent probes for the comprehensive profiling of the cellular state of signaling activity. Here we have described different experimental strategies for global SH2 profiling: high-resolution phosphoproteomic scanning by far-Western Blot analysis and high-throughput profiling by our recently developed oligonucleotide-tagged multiplex assay (OTM) and Rosette assay.

Analyzing expression and phosphorylation of the EGF receptor in HNSCC.

Overexpression of the epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinomas (HNSCC) is considered to cause increased EGFR activity, which adds to tumorigenicity and therapy resistance. Since it is still unclear, whether EGFR expression is indeed associated with increased activity in HNSCC, we analyzed the relationship between EGFR expression and auto-phosphorylation as a surrogate marker for activity. We used a tissue micro array, fresh frozen HNSCC tumor and corresponding normal tissue samples and a large panel of HNSCC cell lines. While we observed substantial overexpression only in approximately 20% of HNSCC, we also observed strong discrepancies between EGFR protein expression and auto-phosphorylation in HNSCC cell lines as well as in tumor specimens using Western blot and SH2-profiling; for the majority of HNSCC EGFR expression therefore seems not to be correlated with EGFR auto-phosphorylation. Blocking of EGFR activity by cetuximab and erlotinib points to increased EGFR activity in samples with increased basal auto-phosphorylation. However, we could also identify cells with low basal phosphorylation but relevant EGFR activity. In summary, our data demonstrate that EGFR expression and activity are not well correlated. Therefore EGFR positivity is no reliable surrogate marker for EGFR activity, arguing the need for alternative biomarkers or functional predictive tests.

Lineage-specific control of TFIIH by MITF determines transcriptional homeostasis and DNA repair.

The melanocytic lineage, which is prominently exposed to ultraviolet radiation (UVR) and radiation-independent oxidative damage, requires specific DNA-damage response mechanisms to maintain genomic and transcriptional homeostasis. The coordinate lineage-specific regulation of intricately intertwined DNA repair and transcription is incompletely understood. Here we demonstrate that the Microphthalmia-associated transcription factor (MITF) directly controls general transcription and UVR-induced nucleotide excision repair by transactivation of GTF2H1 as a core element of TFIIH. Thus, MITF ensures the rapid resumption of transcription after completion of strand repair and maintains transcriptional output, which is indispensable for survival of the melanocytic lineage including melanoma in vitro and in vivo. Moreover, MITF controls c-MYC implicated in general transcription by transactivation of far upstream binding protein 2 (FUBP2/KSHRP), which induces c-MYC pulse regulation through TFIIH, and experimental depletion of MITF results in consecutive loss of CDK7 in the TFIIH-CAK subcomplex. Targeted for proteasomal degradation, CDK7 is dependent on transactivation by MITF or c-MYC to maintain a steady state. The dependence of TFIIH-CAK on sequence-specific MITF and c-MYC constitutes a previously unrecognized mechanism feeding into super-enhancer-driven or other oncogenic transcriptional circuitries, which supports the concept of a transcription-directed therapeutic intervention in melanoma.

Combined inhibition of receptor tyrosine and p21-activated kinases as a therapeutic strategy in childhood ALL.

Receptor tyrosine kinase (RTK)-dependent signaling has been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL) of childhood. However, the RTK-dependent signaling state and its interpretation with regard to biological behavior are often elusive. To decipher signaling circuits that link RTK activity with biological output in vivo, we established patient-derived xenograft ALL (PDX-ALL) models with dependencies on fms-like tyrosine kinase 3 (FLT3) and platelet-derived growth factor receptor ß (PDGFRB), which were interrogated by phosphoproteomics using iTRAQ mass spectrometry. Signaling circuits were determined by receptor type and cellular context with few generic features, among which we identified group I p21-activated kinases (PAKs) as potential therapeutic targets. Growth factor stimulation markedly increased catalytic activities of PAK1 and PAK2. RNA interference (RNAi)-mediated or pharmacological inhibition of PAKs using allosteric or adenosine triphosphate (ATP)-competitive compounds attenuated cell growth and increased apoptosis in vitro. Notably, PAK1- or PAK2-directed RNAi enhanced the antiproliferative effects of the type III RTK and protein kinase C inhibitor midostaurin. Treatment of FLT3- or PDGFRB-dependent ALLs with ATP-competitive PAK inhibitors markedly decreased catalytic activities of both PAK isoforms. In FLT3-driven ALL, this effect was augmented by coadministration of midostaurin resulting in synergistic effects on growth inhibition and apoptosis. Finally, combined treatment of FLT3 D835H PDX-ALL with the ATP-competitive group I PAK inhibitor FRAX486 and midostaurin in vivo significantly prolonged leukemia progression-free survival compared with midostaurin monotherapy or control. Our study establishes PAKs as potential downstream targets in RTK-dependent ALL of childhood, the inhibition of which might help prevent the selection or acquisition of resistance mutations toward tyrosine kinase inhibitors.

SH2 Domain Histochemistry.

Among posttranslational modifications, the phosphorylation of tyrosine residues is a key modification in cell signaling. Because of its biological importance, characterization of the cellular state of tyrosine phosphorylation is of great interest. Based on the unique properties of endogenously expressed SH2 domains recognizing tyrosine phosphorylated signaling proteins with high specificity we have developed an alternative approach, coined SH2 profiling, enabling us to decipher complex patterns of tyrosine phosphorylation in various normal and cancerous tissues. So far, SH2 profiling has largely been applied for the analysis of protein extracts with the limitation that information on spatial distribution and intensity of tyrosine phosphorylation within a tissue is lost. Here, we describe a novel SH2 domain based strategy for differential characterization of the state of tyrosine phosphorylation in formaldehyde-fixed and paraffin-embedded tissues. This approach demonstrates that SH2 domains may serve as very valuable tools for the analysis of the differential state of tyrosine phosphorylation in primary tissues fixed and processed under conditions frequently applied by routine pathology laboratories.