Richard R. de Haas

Proteomics of the TRAP-induced platelet releasate.

Upon stimulation, platelets release the soluble content of their cytoplasmic granules along with microparticles. This sub-proteome is of interest since many of its constituents are associated with coagulation, (tumor) angiogenesis, cell growth and adhesion. Previously, differential - antibody-based - serum analysis has yielded information on the proteins released from platelets upon stimulation. A promising alternative strategy is formed by identifying the proteins released by freshly isolated platelets from blood using proteomics. Here we report on the analysis of the thrombin receptor activating peptide (TRAP)-induced releasate from 3 different volunteers using high resolution, high mass accuracy hybrid LTQ-FT mass spectrometry in a GeLC-MS/MS workflow. We obtained an activated platelet releasate proteome comprising a total of 716 identified proteins with 225 proteins present in the releasate of 3/3 volunteers. This core dataset is characterized by gene ontology mining and signal peptide analysis. Meta-analysis of our dataset and two published datasets of platelet a-granules and microparticles reveals that 55% of our platelet releasate proteins can be annotated using these previous platelet subproteome data, of the remaining releasate proteome 5% overlaps with a published platelet secretome while 40% of our data consists of novel releasate proteins. This high-accuracy activated platelet releasate proteome represents the largest and most comprehensive analysis to date. This approach offers unique possibilities to analyse the role of platelet-secreted proteins in physiology and in diseases such as atherosclerosis and cancer.

Increased numbers of small circulating endothelial cells in renal cell cancer patients treated with sunitinib

Mature circulating endothelial cell (CEC) as well as endothelial progenitor populations may reflect the activity of anti-angiogenic agents on tumor neovasculature or even constitute a target for anti-angiogenic therapy. We investigated the behavior of CECs in parallel with hematopoietic progenitor cells (HPCs) in the blood of renal cell cancer patients during sunitinib treatment. We analyzed the kinetics of a specific population of small VEGFR2-expressing CECs (CD45neg/CD34bright), HPCs (CD45dim/CD34bright), and monocytes in the blood of 24 renal cell cancer (RCC) patients receiving 50xa0mg/day of the multitargeted VEGF inhibitor sunitinib, on a 4-week-on/2-week-off schedule. Blood was taken before treatment (C1D1), on C1D14, C1D28, and on C2D1 before the start of cycle 2. Also plasma VEGF and erythropoietin (EPO) were determined. Remarkably, while CD34bright HPCs and monocytes decreased during treatment, CD34bright CECs increased from 69 cells/ml (C1D1) to 180 cells/ml (C1D14; Pxa0=xa00.001) and remained high on C1D28. All cell populations recovered to near pre-treatment levels on C2D1. Plasma VEGF and EPO levels were increased on C1D14 and partly normalized to pre-treatment levels on C2D1. In conclusion, opposite kinetics of two circulating CD34bright cell populations, HPCs and small CECs, were observed in sunitinib-treated RCC patients. The increase in CECs is likely caused by sunitinib targeting of immature tumor vessels.

Cross-resistance to clinically used tyrosine kinase inhibitors sunitinib, sorafenib and pazopanib

PurposeWhen during cancer treatment resistance to a tyrosine kinase inhibitor (TKI) occurs, switching to another TKI is often considered as a reasonable option. Previously, we reported that resistance to sunitinib may be caused by increased lysosomal sequestration, leading to increased intracellular lysosomal storage and, thereby, inactivity. Here, we studied the effect of several other TKIs on the development of (cross-) resistance.MethodsTKI resistance was induced by continuous exposure of cancer cell lines to increasing TKI concentrations for 3–4xa0months. (Cross-) resistance was evaluated using MTT cell proliferation assays. Intracellular TKI concentrations were measured using LC-MS/MS. Western blotting was used to detect lysosome-associated membrane protein-1 and −2 (LAMP1/2) expression.ResultsThe previously generated sunitinib-resistant (SUN) renal cancer cells (786-O) and colorectal cancer cells (HT-29) were found to be cross-resistant to pazopanib, erlotinib and lapatinib, but not sorafenib. Exposure of 786-O and HT-29 cells to sorafenib, pazopanib or erlotinib for 3–4xa0months induced drug resistance to pazopanib and erlotinib, but not sorafenib. Intracellular drug accumulation was found to be increased in pazopanib- and erlotinib-, but not in sorafenib-exposed cells. Lysosomal capacity, reflected by LAMP1/2 expression, was found to be increased in resistant cells and, in addition, to be transient. No cross-resistance to the mTOR inhibitor everolimus was detected.ConclusionsOur data indicate that tumor cells can develop (cross-) resistance to TKIs, and that such resistance includes increased intracellular drug accumulation accompanied by increased lysosomal storage. Transient (cross-) resistance was found to occur for several of the TKIs tested, but not for everolimus, indicating that switching from a TKI to a mTOR inhibitor may be an attractive therapeutic option.

Sunitinib activates Axl signaling in renal cell cancer

Mass spectrometry‐based phosphoproteomics provides a unique unbiased approach to evaluate signaling network in cancer cells. The tyrosine kinase inhibitor sunitinib is registered as treatment for patients with renal cell cancer (RCC). We investigated the effect of sunitinib on tyrosine phosphorylation in RCC tumor cells to get more insight in its mechanism of action and thereby to find potential leads for combination treatment strategies. Sunitinib inhibitory concentrations of proliferation (IC50) of 786‐O, 769‐p and A498 RCC cells were determined by MTT‐assays. Global tyrosine phosphorylation was measured by LC‐MS/MS after immunoprecipitation with the antiphosphotyrosine antibody p‐TYR‐100. Phosphoproteomic profiling of 786‐O cells yielded 1519 phosphopeptides, corresponding to 675 unique proteins including 57 different phosphorylated protein kinases. Compared to control, incubation with sunitinib at its IC50 of 2 µM resulted in downregulation of 86 phosphopeptides including CDK5, DYRK3, DYRK4, G6PD, PKM and LDH‐A, while 94 phosphopeptides including Axl, FAK, EPHA2 and p38α were upregulated. Axl‐ (y702), FAK‐ (y576) and p38α (y182) upregulation was confirmed by Western Blot in 786‐O and A498 cells. Subsequent proliferation assays revealed that inhibition of Axl with a small molecule inhibitor (R428) sensitized 786‐O RCC cells and immortalized endothelial cells to sunitinib up to 3 fold. In conclusion, incubation with sunitinib of RCC cells causes significant upregulation of multiple phosphopeptides including Axl. Simultaneous inhibition of Axl improves the antitumor activity of sunitinib. We envision that evaluation of phosphoproteomic changes by TKI treatment enables identification of new targets for combination treatment strategies.

Sunitinib‐induced changes in circulating endothelial cell‐related proteins in patients with metastatic renal cell cancer

Vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors are effective agents in the treatment of metastatic renal cell cancer (mRCC). We here investigated whether inhibition of VEGFR signalin by sunitinib causes changes in plasma proteins associated with tumor endothelium. Forty‐three patients with mRCC received sunitinib 50 mg/day in a 4‐weeks on 2‐weeks off schedule. Sequential plasma samples were obtained before treatment (C1D1), on C1D14, on C1D28, and on C2D1 before start of cycle 2. Plasma levels were assessed for VEGF, soluble vascular cell adhesion molecule‐1 (sVCAM‐1), soluble intercellular cell adhesion molecule‐1 (sICAM‐1), von Willebrand factor (vWF), circulating angiopoietin‐2 (Ang‐2) and soluble Tie‐2 (sTie‐2). Total tumor burden was calculated at baseline and at first evaluation. Progression‐free survival (PFS) and overall survival (OS) were determined. Tumor burden was positively associated with baseline circulating Ang‐2 [Spearmans rho (ρ) = 0.378, p = 0.028] and vWF (ρ = 0.417, p = 0.008). During sunitinib treatment, circulating Ang‐2 and sTie‐2 significantly decreased (p < 0.001 for both), plasma levels of sVCAM‐1 and VEGF significantly increased (p = 0.022 and p < 0.001), whereas those of sICAM‐1 and vWF remained stable. These protein changes had recovered on C2D1. The reduction in circulating Ang‐2 levels on C1D28 was positively correlated with the percentage decrease in tumor burden (ρ = 0.605; p = 0.002). Baseline protein levels and subsequent changes were not associated with PFS or OS. In conclusion, sunitinib‐induced changes in Ang‐2, sTie‐2, sVCAM‐1 and VEGF are related to the administration schedule, while reduction in Ang‐2 is also associated with decrease in tumor burden.

Alternative scheduling of pulsatile, high dose sunitinib efficiently suppresses tumor growth

BackgroundIncreased exposure to multitargeted kinase inhibitor sunitinib is associated with improved outcome, emphasizing the importance of maintaining adequate dosing and drug levels. The currently approved schedule (50xa0mg daily, four weeks on, two weeks off) precludes further dose-intensification. Recent data suggest that sunitinib, although initially developed as an antiangiogenic agent, has direct antitumor activity.MethodsIn this study, we tested whether a chemotherapy-like schedule of pulsatile high dose sunitinib would result in improved antitumor activity.ResultsIn vitro, a single exposure to 20xa0μM sunitinib for 6-9 h resulted in complete inhibition of tumor cell growth and cell death conveyed through activation of caspases and autophagy upregulation. Notably, repeated exposure of tumor cells to pulses of high concentrations of sunitinib did not induce resistance. In vivo, once-weekly treatment with high dose sunitinib of tumors growing on the chorioallantoic membrane (CAM) of the chicken embryo significantly impaired tumor growth by 57xa0% compared to vehicle, outperforming the daily, standard scheduling.ConclusionsThese results prompted the initiation of a phase I clinical trial, where intermittent, high dose sunitinib is being investigated in patients with advanced solid tumors (registration number and date: NCT02058901, 30 September 2013, respectively). The trial is actively recruiting patients and promising preliminary indications of antitumor activity have been observed.

Differential effects of inhibitors of the PI3K/mTOR pathway on the expansion and functionality of regulatory T cells.

The PI3K/mTOR pathway is commonly deregulated in cancer. mTOR inhibitors are registered for the treatment of several solid tumors and novel inhibitors are explored clinically. Notably, this pathway also plays an important role in immunoregulation. While mTOR inhibitors block cell cycle progression of conventional T cells (Tconv), they also result in the expansion of CD4(+)CD25(hi)FOXP3(+) regulatory T cells (Tregs), and this likely limits their clinical antitumor efficacy. Here, we compared the effects of dual mTOR/PI3K inhibition (using BEZ235) to single PI3K (using BKM120) or mTOR inhibition (using rapamycin and everolimus) on Treg expansion and functionality. Whereas rapamycin, everolimus and BEZ235 effected a relative expansion benefit for Tregs and increased their overall suppressive activity, BKM120 allowed for similar expansion rates of Tregs and Tconv without altering their overall suppressive activity. Therefore, PI3K inhibition alone might offer antitumor efficacy without the detrimental selective expansion of Tregs associated with mTOR inhibition.

Phosphotyrosine-based-phosphoproteomics scaled-down to biopsy level for analysis of individual tumor biology and treatment selection

Mass spectrometry-based phosphoproteomics of cancer cell and tissue lysates provides insight in aberrantly activated signaling pathways and potential drug targets. For improved understanding of individual patients tumor biology and to allow selection of tyrosine kinase inhibitors in individual patients, phosphoproteomics of small clinical samples should be feasible and reproducible. We aimed to scale down a pTyr-phosphopeptide enrichment protocol to biopsy-level protein input and assess reproducibility and applicability to tumor needle biopsies. To this end, phosphopeptide immunoprecipitation using anti-phosphotyrosine beads was performed using 10, 5 and 1mg protein input from lysates of colorectal cancer (CRC) cell line HCT116. Multiple needle biopsies from 7 human CRC resection specimens were analyzed at the 1mg-level. The total number of phosphopeptides captured and detected by LC-MS/MS ranged from 681 at 10mg input to 471 at 1mg HCT116 protein. ID-reproducibility ranged from 60.5% at 10mg to 43.9% at 1mg. Per 1mg-level biopsy sample, >200 phosphopeptides were identified with 57% ID-reproducibility between paired tumor biopsies. Unsupervised analysis clustered biopsies from individual patients together and revealed known and potential therapeutic targets.nnnSIGNIFICANCEnThis study demonstrates the feasibility of label-free pTyr-phosphoproteomics at the tumor biopsy level based on reproducible analyses using 1mg of protein input. The considerable number of identified phosphopeptides at this level is attributed to an effective down-scaled immuno-affinity protocol as well as to the application of ID propagation in the data processing and analysis steps. Unsupervised cluster analysis reveals patient-specific profiles. Together, these findings pave the way for clinical trials in which pTyr-phosphoproteomics will be performed on pre- and on-treatment biopsies. Such studies will improve our understanding of individual tumor biology and may enable future pTyr-phosphoproteomics-based personalized medicine.

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

Identifying (hyper)active kinases in cancer patient tumors is crucial to enable individualized treatment with specific inhibitors. Conceptually, kinase activity can be gleaned from global protein phosphorylation profiles obtained with mass spectrometry-based phosphoproteomics. A major challenge is to relate such profiles to specific kinases to identify (hyper)active kinases that may fuel growth/progression of individual tumors. Approaches have hitherto focused on phosphorylation of either kinases or their substrates. Here, we combine kinase-centric and substrate-centric information in an Integrative Inferred Kinase Activity (INKA) analysis. INKA utilizes label-free quantification of phosphopeptides derived from kinases, kinase activation loops, kinase substrates deduced from prior experimental knowledge, and kinase substrates predicted from sequence motifs, yielding a single score. This multipronged, stringent analysis enables ranking of kinase activity and visualization of kinase-substrate relation networks in a biological sample. As a proof of concept, INKA scoring of phosphoproteomic data for different oncogene-driven cancer cell lines inferred top activity of implicated driver kinases, and relevant quantitative changes upon perturbation. These analyses show the ability of INKA scoring to identify (hyper)active kinases, with potential clinical significance.

Abstract 5641: Sunitinib inhibits AXL phosphorylation in tumor cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnBackground Sunitinib is a multi-targeted tyrosine kinase inhibitor (TKI), that is used for treatment of patients with advanced renal cell cancer (RCC) Drug resistance to sunitinib is a major clinical problem. We recently reported that continuous exposure to sunitinib causes acquired resistance of tumor cells. The receptor tyrosine kinase Axl has been implicated in acquired resistance to multiple TKIs, including erlotinib and lapatinib. In addition, sunitinib was previously reported to be a potential inhibitor of Axl catalytic activity. We therefore investigated the potential role of Axl in sunitinib resistance. Methods 786-O cells resistant to sunitinib were obtained by continuous exposure to the drug as previously published (Gotink et al. Clin Cancer Res 2011). AXL expression was studied in 786-O cell lines and EC-RF24 endothelial cells by RT-PCR and Western blot. Sunitinib and AXL inhibitor R428 sensitivity of cells transfected with siRNAs against AXL or scrambled siRNAs was determined by MTT cell viability assays. For kinase activity experiments recombinant AXL and tyrosine peptide arrays (PamGene, ‘s Hertogenbosch, the Netherlands) were used. Phospho-AXL and total AXL in cell lysates were measured by ELISA. Results Axl phosphorylation in vitro in both 786-O and EC-RF24 cells was dose-dependently inhibited by sunitinib, with a 50% inhibitory concentration of 5 μM. Activity of recombinant Axl kinase was measured on tyrosine containing peptide arrays in presence of 0, 1 or 4 μM sunitinib or 2 μM R428. Sunitinib induced dose-dependent inhibition of Axl activity, where 2 μM R428 blocked all peptide phosphorylations. In addition, 786-O sunitinib resistant cells (786-O SUN) showed a 4-fold overexpression of Axl protein compared to 786-O parental cells. However, gene silencing of AXL in 786-O SUN cells by RNA interference did not sensitize the cells to sunitinib. Sunitinib resistant cells were shown to be cross-resistant to the Axl inhibitor R428 with respective IC50 values of 0.9 μM (±0.1 μM) and 1.8 μM (±0.1 μM). Conclusion Phosphorylation of Axl is inhibited by sunitinib in both 786-O RCC and EC-RF24 endothelial cells. Despite its overexpression in sunitinib resistant cells, gene silencing of AXL did not sensitize 786-O resistant cells to sunitinib, indicating that sunitinib resistance is not mediated by AXL.nnCitation Format: Johannes C. Van der Mijn, Kristy J. Gotink, Richard R. de Haas, Henk Dekker, Connie R. Jimenez, Henk J. Broxterman, Henk M.w. Verheul. Sunitinib inhibits AXL phosphorylation in tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5641. doi:10.1158/1538-7445.AM2013-5641