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Purpose: In precision oncology, tumor molecular profiles guide selection of therapy. Standardized snap freezing of tissue biospecimens is necessary to ensure reproducible, high-quality samples that preserve tumor biology for adequate molecular profiling. Quenching in liquid nitrogen (LN2) is the golden standard method, but LN2 has several limitations. We developed a LN2-independent snap freezer with adjustable cold sink temperature. To benchmark this device against the golden standard, we compared molecular profiles of biospecimens. Methods: Cancer cell lines and core needle normal tissue biopsies from five patients' liver resection specimens were used to compare mass spectrometry (MS)-based global phosphoproteomic and RNA sequencing profiles and RNA integrity obtained by both freezing methods. Results: Unsupervised cluster analysis of phosphoproteomic and transcriptomic profiles of snap freezer versus LN2-frozen K562 samples and liver biopsies showed no separation based on freezing method (with Pearson's r 0.96 (range 0.92–0.98) and >0.99 for K562 profiles, respectively), while samples with +2 h bench-time formed a separate cluster. RNA integrity was also similar for both snap freezing methods. Molecular profiles of liver biopsies were clearly identified per individual patient regardless of the applied freezing method. Two to 25 s freezing time variations did not induce profiling differences in HCT116 samples. Conclusion: The novel snap freezer preserves high-quality biospecimen and allows identification of individual patients' molecular profiles, while overcoming important limitations of the use of LN2. This snap freezer may provide a useful tool in clinical cancer research and practice, enabling a wider implementation of (multi-)omics analyses for precision oncology.

Supplementary Data from Tumor Drug Concentration and Phosphoproteomic Profiles After Two Weeks of Treatment With Sunitinib in Patients with Newly Diagnosed Glioblastoma

Purpose:Tyrosine kinase inhibitors (TKI) have poor efficacy in patients with glioblastoma (GBM). Here, we studied whether this is predominantly due to restricted blood–brain barrier penetration or more to biological characteristics of GBM.Patients and Methods:Tumor drug concentrations of the TKI sunitinib after 2 weeks of preoperative treatment was determined in 5 patients with GBM and compared with its in vitro inhibitory concentration (IC50) in GBM cell lines. In addition, phosphotyrosine (pTyr)-directed mass spectrometry (MS)-based proteomics was performed to evaluate sunitinib-treated versus control GBM tumors.Results:The median tumor sunitinib concentration of 1.9 μmol/L (range 1.0–3.4) was 10-fold higher than in concurrent plasma, but three times lower than sunitinib IC50s in GBM cell lines (median 5.4 μmol/L, 3.0–8.5; P = 0.01). pTyr-phosphoproteomic profiles of tumor samples from 4 sunitinib-treated versus 7 control patients revealed 108 significantly up- and 23 downregulated (P < 0.05) phosphopeptides for sunitinib treatment, resulting in an EGFR-centered signaling network. Outlier analysis of kinase activities as a potential strategy to identify drug targets in individual tumors identified nine kinases, including MAPK10 and INSR/IGF1R.Conclusions:Achieved tumor sunitinib concentrations in patients with GBM are higher than in plasma, but lower than reported for other tumor types and insufficient to significantly inhibit tumor cell growth in vitro. Therefore, alternative TKI dosing to increase intratumoral sunitinib concentrations might improve clinical benefit for patients with GBM. In parallel, a complex profile of kinase activity in GBM was found, supporting the potential of (phospho)proteomic analysis for the identification of targets for (combination) treatment.

While various GPCRs, including US28, display constitutive, ligand-independent activity, it remains to be established whether ligand-dependent and -independent active conformations differ and can be selectively modulated. Previously, the agonist-bound conformation of US28 was stabilized and its structure was solved using the anti-US28 nanobody Nb7. Here we report the recognition of the constitutively active, apo-conformation of US28 by another nanobody VUN103. While the Nb7 intrabody selectively inhibits ligand-induced signaling, the VUN103 intrabody blocks constitutive signaling, indicating the existence of distinct US28 conformational states. By displacing Gαq protein, VUN103 prevents US28 signaling and reduces tumor spheroids growth. Overall, nanobodies specific for distinct GPCR conformational states, i.e. apo- and agonist-bound, can selectively target and discern functional consequences of ligand-dependent versus independent signaling., Various GPCRs display constitutive ligand-independent activity, but it remains unclear whether ligand-dependent and -independent conformations differ. Here the authors demonstrate the recognition and blocking of G protein recruitment of either the ligand-bound active, or the constitutively active apo-conformation of the viral GPCR US28 by different nanobodies that target similar intracellular loops of the receptor.

Introduction Intensive multi-agent treatment has boosted survival up to 80% of pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients. Nevertheless, relapsed patients have a poor prognosis due to acquired therapy resistance while most survivors have detrimental chemotherapy-induced side effects. Therefore, novel targeted therapies are urgently needed since further intensification of the current standard treatment regimen is not feasible for refractory/relapsed cases. Protein kinase inhibitors (PKIs) are amongst the most successful cancer treatments. Targetable kinases activated by gene fusions are rare in T-ALL and include subclonal NUP214-ABL1 fusion in 6% of cases or other rare clonal ABL1 fusions. Nevertheless, leukemic blasts rely on enhanced kinase signaling to sustain their dysregulated proliferation. Protein kinases can be hyper-activated even in the absence of defects in their genes. Thus, together with the identification of genomic aberrations, phospho-proteomics can provide information on pathway activation, signaling networks and aberrant kinase activities that offer important opportunities for targeted therapies. Aim Here, we aimed to identify and quantify kinase activation in T-ALL cell lines that may yield differential sensitivity to PKIs in vitro. This approach could pinpoint targetable leukemia vulnerabilities and provide effective (combination) treatment strategies. Methods Protein extracts from 11 T-ALL cell lines were enriched for phospho-peptides by titanium dioxide enrichment and anti-phospho tyrosine immunoprecipitation followed by liquid chromatography - tandem mass spectrometry (MS). Subsequently, the Integrative Inferred Kinase Activity (INKA) pipeline was used to rank activated kinases in our panel (Beekhof et al., 2019). Based on these data, selected kinase inhibitors were tested in vitro as single treatment or in combinations. Eventually, drugs of interest were further tested ex vivo in a cohort of T-ALL patient-derived xenografts (PDXs). Results MS-based phospho-proteome profiling of 11 T-ALL cell lines identified about 3700 tyrosine phospho-sites and more than 13300 serine/threonine phosphorylation sites. We found SRC-family kinases including LCK, SRC, FYN, and YES1 as most activated kinases in many T-ALL cell lines while ABL1, ZAP70, LYN, and FGR were detected only in specific lines. Additionally, other kinases including CDK1/2 and PAK1/2 were found to be activated in all the cell lines while activation of the INSR/IGF-1R axis was detected only in a subset of lines. We then tested cellular response to multiple clinically relevant PKIs based on predicted kinase activities. The in vitro drug screening showed an effective response and G1-arrest following treatment with the CDK1/2 inhibitor milciclib in all the cell lines tested, with IC50 values between 10nM and 1uM. Despite a general SRC-family kinases activation profile, dual SRC/ABL inhibitors like dasatinib reduced cellular viability only in cell lines with ABL1 fusions or LCK translocations (IC50 < 10nM) while other lines lacking ABL or LCK rearrangements were affected at much higher drug concentration (IC50 > 3uM, which is beyond the clinical achievable plasma concentration). Thus, PKIs were tested in combination with other relevant inhibitors based on additional kinase activities detected. Interestingly, the concomitant inhibition of the SRC-family kinases by dasatinib and the INSR/IGF-1R axis by BMS-754807 led to a drastic reduction of cell viability at nanomolar concentrations even in cell lines that did not respond to dasatinib, identifying a novel possible effective combination strategy for T-ALL. Eventually, we tested clinically relevant PKIs in 50 PDXs ex vivo and identified various T-ALL samples with a high sensitivity to dasatinib single treatment (IC50 < 100nM) as previously reported by others (Frismantas et al., 2017). Moreover, 70% of our PDXs efficiently responded to the broad spectrum kinase inhibitor midostaurin (IC50 < 1uM), highlighting the importance of targeting multiple signaling nodes simultaneously to tackle T-ALL vulnerabilities. Conclusions Ranking kinase activities and signaling networks from phospho-proteomic data can guide the use of PKIs as treatment option for T-ALL patients. Moreover, kinase activity profiling can provide insights for efficient treatment combination strategies to develop personalized medicine. Disclosures No relevant conflicts of interest to declare.

Identification of predictive biomarkers for targeted therapies requires information on drug exposure at the target site as well as its effect on the signaling context of a tumor. To obtain more insight in the clinical mechanism of action of protein kinase inhibitors (PKIs), we studied tumor drug concentrations of protein kinase inhibitors (PKIs) and their effect on the tyrosine-(pTyr)-phosphoproteome in patients with advanced cancer. Tumor biopsies were obtained from 31 patients with advanced cancer before and after 2 weeks of treatment with sorafenib (SOR), erlotinib (ERL), dasatinib (DAS), vemurafenib (VEM), sunitinib (SUN) or everolimus (EVE). Tumor concentrations were determined by LC-MS/MS. pTyr-phosphoproteomics was performed by pTyr-immunoprecipitation followed by LC-MS/MS. Median tumor concentrations were 2&ndash, 10 &micro, M for SOR, ERL, DAS, SUN, EVE and &gt, 1 mM for VEM. These were 2&ndash, 178 &times, higher than median plasma concentrations. Unsupervised hierarchical clustering of pTyr-phosphopeptide intensities revealed patient-specific clustering of pre- and on-treatment profiles. Drug-specific alterations of peptide phosphorylation was demonstrated by marginal overlap of robustly up- and downregulated phosphopeptides. These findings demonstrate that tumor drug concentrations are higher than anticipated and result in drug specific alterations of the phosphoproteome. Further development of phosphoproteomics-based personalized medicine is warranted.

e16556 Background: Sunitinib, a multi-targeted antiangiogenic tyrosine kinase inhibitor has improved the outcome of patients with advanced RCC considerably. Unfortunately, ± 30% of patients are intrinsically resistant, which underscores the urgent need to develop a clinically applicable method to select sunitinib treatment for patients. Since RCC is not single oncogene driven, but rather by multiple aberrantly activated kinase signaling pathways, we hypothesized that a functional read-out by large scale (phospho)proteomics can identify predictive biomarkers for sunitinib. Methods: Frozen tumor tissue of 26 patients (pts) with RCC, treated with sunitinib upon recurrence or progression, was obtained. Pts were classified as primary resistant (progression-free survival (PFS) < 3 months, n = 8) or sensitive (PFS ≥ 3 months, n = 18). Mass spectrometry-based tyrosine (pTyr) phosphoproteomics was performed by pTyr-immunoprecipitation followed by LC-MS/MS. Discriminatory phosphosites (p-sites) were identified (p < 0.05, fold-change (FC) > 2). Expression proteomics was performed by LC-MS/MS and differentially expressed proteins identified (p < 0.05, FC > 2, ≥ 50% data presence in group with highest abundance). Tumor biology was further analyzed by INferred Kinase Activity analysis, posttranslational modifications signature enrichment analysis (PTM-SEA, Krug et al 2019) and gene ontology mining. Results: pTyr-phosphoproteomics was successfully performed in 23 of 26 samples. Seventy-eight differential p-sites were identified, of which 22 (4 unique; BCAR3, NOP58, EIF4A2, GDI1) were upregulated in resistant and 56 in sensitive pts (35 unique). Supervised cluster analysis of these p-sites resulted in near-complete separation of the groups. EIF4A1 and its homolog EIF4A2 were differentially expressed in resistant pts both at the (phospho-)proteome and, in an independent cohort, at transcriptome level. Significantly higher inferred kinase activity of MAPK3 (p = 0.026) and EGFR (p = 0.045) was found in sensitive pts. PTM-SEA showed 3 p-site-centric signatures that were significantly enriched (p < 0.05) in resistant pts, including FGF1 and prolactin pathways. Fifteen signatures were significantly enriched (p < 0.05) in sensitive pts, including insulin, VEGF and FGF2 treatment and KIT receptor pathway. Expression proteomics revealed significantly higher expression (p < 0.05) of proteins related to vesicle mediated transport in resistant pts, while this was not the case in sensitive pts. Conclusions: This MS-based (phospho)proteomics analysis of RCC tissues revealed discriminatory phosphosite and protein signatures and differential kinase and pathway activities that are associated with sensitive and resistant tumors. These findings warrant validation in an independent cohort and the clinical utility for treatment selection remains to be demonstrated.

All mammalian cells release small endosome-derived exosomes that function in intercellular communication, but the secretion process is poorly understood. Verweij et al. developed a live-imaging approach and demonstrate that external cues can trigger exosome release from a subpopulation of multivesicular bodies by phosphorylating the target membrane SNARE SNAP23 at serine residue 110., Exosomes are small endosome-derived extracellular vesicles implicated in cell–cell communication and are secreted by living cells when multivesicular bodies (MVBs) fuse with the plasma membrane (PM). Current techniques to study exosome physiology are based on isolation procedures after secretion, precluding direct and dynamic insight into the mechanics of exosome biogenesis and the regulation of their release. In this study, we propose real-time visualization of MVB–PM fusion to overcome these limitations. We designed tetraspanin-based pH-sensitive optical reporters that detect MVB–PM fusion using live total internal reflection fluorescence and dynamic correlative light–electron microscopy. Quantitative analysis demonstrates that MVB–PM fusion frequency is reduced by depleting the target membrane SNAREs SNAP23 and syntaxin-4 but also can be induced in single cells by stimulation of the histamine H1 receptor (H1HR). Interestingly, activation of H1R1 in HeLa cells increases Ser110 phosphorylation of SNAP23, promoting MVB–PM fusion and the release of CD63-enriched exosomes. Using this single-cell resolution approach, we highlight the modulatory dynamics of MVB exocytosis that will help to increase our understanding of exosome physiology and identify druggable targets in exosome-associated pathologies.

Background Colorectal cancer (CRC) develops in a multi-step-process from normal epithelium, through a pre-malignant lesion (adenoma), into a malignant lesion (adenocarcinoma). A minority of about 5% of adenomas will ultimately progress into cancer. To improve clinical practice it is important to identify the subgroup of colorectal adenomas that is at risk of progressing to cancer. By extensive genomic analysis we have shown that specific copy number alterations are associated with risk of progression. An example of such an alteration is the gain of chromosome arm 20q and we identified AURKA and TPX2 as major drivers of this amplicon. Yet, the activation status of these and other genes during colorectal carcinogenesis and the downstream signaling pathways affected with gene (in)activation are not fully known. In order to better understand the biology of adenoma to carcinoma transition, we performed a comprehensive analysis of phosphoproteomes at different stages of colorectal carcinogenesis. Methods Phosphotyrosine containing peptides were immunoprecipitated from 5 mg of colorectal adenoma (n=81) and colorectal carcinoma tissues (n=50) using agarose bead-coupled phosphotyrosine antibody P-Tyr-1000. A Q Exactive HF mass spectrometer was used to perform NanoLC-MS/MS. Spectral counts of phosphoproteins and ion intensities of phosphopeptides were defined by MaxQuant for relative quantitation of protein phosphorylation. Results Phosphotyrosine-based phosphoproteomics of the 131 colorectal tissue samples yielded 6056 phosphopeptides, corresponding to 2745 unique phosphoproteins including 183 phosphokinases. Data analysis to identify regulated phosphorylation states and inference of kinase activity is ongoing. Conclusions and future plans This is the first large phosphoproteomics dataset of colorectal adenomas and carcinomas. Preliminary data analysis shows promising differences in kinase activities between adenomas and carcinomas. After further analysis, we will manipulate the activity of the driver kinases in adenoma-derived organoids and monitor tumor outgrowth in mice. Citation Format: Sanne Martens-de Kemp, Alex Henneman, Richard de Goeij-de Haas, Sander Piersma, Thang Pham, Gerrit Meijer, Beatriz Carvalho, Connie Jimenez. Identification of signaling pathways involved in colorectal adenoma-to-carcinoma progression using phosphoproteomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4010.

Exosomes are small endosome-derived extracellular vesicles implicated in cell-cell communication and are secreted by living cells when multivesicular bodies (MVBs) fuse with the plasma membrane (PM). Current techniques to study exosome physiology are based on isolation procedures after secretion, precluding direct and dynamic insight into the mechanics of exosome biogenesis and the regulation of their release. In this study, we propose real-time visualization of MVB-PM fusion to overcome these limitations. We designed tetraspanin-based pH-sensitive optical reporters that detect MVB-PM fusion using live total internal reflection fluorescence and dynamic correlative light-electron microscopy. Quantitative analysis demonstrates that MVB-PM fusion frequency is reduced by depleting the target membrane SNAREs SNAP23 and syntaxin-4 but also can be induced in single cells by stimulation of the histamine H1 receptor (H1HR). Interestingly, activation of H1R1 in HeLa cells increases Ser110 phosphorylation of SNAP23, promoting MVB-PM fusion and the release of CD63-enriched exosomes. Using this single-cell resolution approach, we highlight the modulatory dynamics of MVB exocytosis that will help to increase our understanding of exosome physiology and identify druggable targets in exosome-associated pathologies.