Arend H. Sikkema

Kinome Profiling in Pediatric Brain Tumors as a New Approach for Target Discovery

Progression in pediatric brain tumor growth is thought to be the net result of signaling through various protein kinase-mediated networks driving cell proliferation. Defining new targets for treatment of human malignancies, without a priori knowledge on aberrant cell signaling activity, remains exceedingly complicated. Here, we introduce kinome profiling using flow-through peptide microarrays as a new concept for target discovery. Comprehensive tyrosine kinase activity profiles were identified in 29 pediatric brain tumors using the PamChip kinome profiling system. Previously reported activity of epidermal growth factor receptor, c-Met, and vascular endothelial growth factor receptor in pediatric brain tumors could be appreciated in our array results. Peptides corresponding with phosphorylation consensus sequences for Src family kinases showed remarkably high levels of phosphorylation compared with normal tissue types. Src activity was confirmed applying Phos-Tag SDS-PAGE. Furthermore, the Src family kinase inhibitors PP1 and dasatinib induced substantial tumor cell death in nine pediatric brain tumor cell lines but not in control cell lines. Thus, this study describes a new high-throughput technique to generate clinically relevant tyrosine kinase activity profiles as has been shown here for pediatric brain tumors. In the era of a rapidly increasing number of small-molecule inhibitors, this approach will enable us to rapidly identify new potential targets in a broad range of human malignancies.

Galectin-4, a novel neuronal regulator of myelination

Myelination of axons by oligodendrocytes (OLGs) is essential for proper saltatory nerve conduction, i.e., rapid transmission of nerve impulses. Among others, extracellular matrix (ECM) molecules, neuronal signaling, and axonal adhesion regulate the biogenesis and maintenance of myelin membranes, driven by polarized transport of myelin‐specific proteins and lipids. Galectin‐4, a tandem‐repeat‐type lectin with affinity to sulfatide and nonsialylated termini of N‐glycans, has the ability to regulate adhesion of cells to ECM components and is also involved in polarized membrane trafficking. We, therefore, anticipated that galectin‐4 might play a role in myelination. Here, we show that in developing postnatal rat brains galectin‐4 expression is downregulated just before the onset of myelination. Intriguingly, when immature OLGs were treated with galectin‐4, OLG maturation was retarded, while a subset of the immature OLGs reverted to a morphologically less complex progenitor stage, displaying concomitantly an increase in proliferation. Similarly, myelination was inhibited when galectin‐4 or anti‐galectin‐4 antibodies were added to co‐cultures of dorsal root ganglion neurons and OLGs. Neurons and OLGs were identified as a possible source of galectin‐4, both in vitro and in vivo. In culture, neurons but not OLGs released galectin‐4. Interestingly, in co‐cultures, a reduced release of endogenous galectin‐4 correlated with the onset of myelination. Moreover, galectin‐4‐reactive sites are transiently expressed on processes of premyelinating primary OLGs, but not on neurons. Taken together, these results identify neuronal galectin‐4 as a candidate for a soluble regulator of OLG differentiation and, hence, myelination.

EphB2 activity plays a pivotal role in pediatric medulloblastoma cell adhesion and invasion

Eph/ephrin signaling has been implicated in various types of key cancer-enhancing processes, like migration, proliferation, and angiogenesis. In medulloblastoma, invading tumor cells characteristically lead to early recurrence and a decreased prognosis. Based on kinase-activity profiling data published recently, we hypothesized a key role for the Eph/ephrin signaling system in medulloblastoma invasion. In primary medulloblastoma samples, a significantly higher expression of EphB2 and the ligand ephrin-B1 was observed compared with normal cerebellum. Furthermore, medulloblastoma cell lines showed high expression of EphA2, EphB2, and EphB4. Stimulation of medulloblastoma cells with ephrin-B1 resulted in a marked decrease in in vitro cell adhesion and an increase in the invasion capacity of cells expressing high levels of EphB2. The cell lines that showed an ephrin-B1-induced phenotype possessed increased levels of phosphorylated EphB2 and, to a lesser extent, EphB4 after stimulation. Knockdown of EphB2 expression by short hairpin RNA completely abolished ephrin ligand-induced effects on adhesion and migration. Analysis of signal transduction identified p38, Erk, and mTOR as downstream signaling mediators potentially inducing the ephrin-B1 phenotype. In conclusion, the observed deregulation of Eph/ephrin expression in medulloblastoma enhances the invasive phenotype, suggesting a potential role in local tumor cell invasion and the formation of metastases.

Identification of new possible targets for leukemia treatment by kinase activity profiling

To date, the biology of acute leukemia has been unclear, and defining new therapeutic targets without prior knowledge remains complicated. The use of high-throughput techniques would enable us to learn more about the biology of the disease, and make it possible to directly assess a broader range of therapeutic targets. In this study we have identified comprehensive tyrosine kinase activity profiles in leukemia samples using the PamChip® kinase activity profiling system. Strikingly, 31% (44/120) of the detected peptides were active in all three groups of leukemia samples. The recently reported activity of platelet-derived growth factor receptor (PDGFR) and neurotrophic tyrosine kinase receptors (NTRK1 and NTRK2) in leukemia could be appreciated in our array results. In addition, high levels of peptide phosphorylation were demonstrated for peptides related to macrophage stimulating 1 receptor (MST1R). A provisional signal transduction scheme of the common active peptides was constructed and used to specifically select an inhibitor for leukemic blast cell survival assays. As expected, a dose-dependent decrease in leukemic blast cell survival was achieved for all leukemia samples. Our data demonstrate that kinase activity profiling in leukemic samples is feasible and provides novel insights into the pathogenesis of leukemia. This approach can be used for the rapid discovery of potential drug targets.

Optimizing targeted cancer therapy: Towards clinical application of systems biology approaches

In cancer, genetic and epigenetic alterations ultimately culminate in discordant activation of signal transduction pathways driving the malignant process. Pharmacological or biological inhibition of such pathways holds significant promise with respect to devising rational therapy for cancer. Thus, technical concepts pursuing robust characterization of kinase activity in tissue samples from cancer patients have been subject of investigation. In the present review we provide a comprehensive overview of these techniques and discuss their advantages and disadvantages for systems biology approaches to identify kinase targets in oncological disease. Recent advances in the development and application of array-based peptide-substrate kinase activity screens show great promise in overcoming the discrepancy between the evaluation of aberrant cell signaling in specific malignancies or even individual patients and the currently available ensemble of highly specific targeted treatment strategies. These developments have the potential to result in a more effective selection of kinase inhibitors and thus optimize mechanism-based patient-specific therapeutic strategies. Given the results from current research on the tumor kinome, generating network views on aberrant tumor cell signaling is critical to meet this challenge.

GD1a Overcomes Inhibition of Myelination by Fibronectin via Activation of Protein Kinase A: Implications for Multiple Sclerosis

Remyelination failure by oligodendrocytes contributes to the functional impairment that characterizes the demyelinating disease multiple sclerosis (MS). Since incomplete remyelination will irreversibly damage axonal connections, treatments effectively promoting remyelination are pivotal in halting disease progression. Our previous findings suggest that fibronectin aggregates, as an environmental factor, contribute to remyelination failure by perturbing oligodendrocyte progenitor cell (OPC) maturation. Here, we aim at elucidating whether exogenously added gangliosides (i.e., cell surface lipids with a potential to modulate signaling pathways) could counteract fibronectin-mediated inhibition of OPC maturation. Exclusive exposure of rat oligodendrocytes to GD1a, but not other gangliosides, overcomes aggregated fibronectin-induced inhibition of myelin membrane formation, in vitro, and OPC differentiation in fibronectin aggregate containing cuprizone-induced demyelinated lesions in male mice. GD1a exerts its effect on OPCs by inducing their proliferation and, at a late stage, by modulating OPC maturation. Kinase activity profiling revealed that GD1a activated a protein kinase A (PKA)-dependent signaling pathway and increased phosphorylation of the transcription factor cAMP response element-binding protein. Consistently, the effect of GD1a in restoring myelin membrane formation in the presence of fibronectin aggregates was abolished by the PKA inhibitor H89, whereas the effect of GD1a was mimicked by the PKA activator dibutyryl-cAMP. Together, GD1a overcomes the inhibiting effect of aggregated fibronectin on OPC maturation by activating a PKA-dependent signaling pathway. Given the persistent presence of fibronectin aggregates in MS lesions, ganglioside GD1a might act as a potential novel therapeutic tool to selectively modulate the detrimental signaling environment that precludes remyelination. SIGNIFICANCE STATEMENT As an environmental factor, aggregates of the extracellular matrix protein fibronectin perturb the maturation of oligodendrocyte progenitor cells (OPCs), thereby impeding remyelination, in the demyelinating disease multiple sclerosis (MS). Here we demonstrate that exogenous addition of ganglioside GD1a overcomes the inhibiting effect of aggregated fibronectin on OPC maturation, both in vitro and in vivo, by activating a PKA-dependent signaling pathway. We propose that targeted delivery of GD1a to MS lesions may act as a potential novel molecular tool to boost maturation of resident OPCs to overcome remyelination failure and halt disease progression.

Less tumor engraftment after anti-VEGF therapy in pediatric low grade astrocytoma

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction. Low grade astrocytomas are the most frequent brain tumors in children. Although children can benefit from treatments including neurosurgery, chemotherapy and radiotherapy, still children die due to tumor progression. Furthermore, morbidity can be serious. So new therapeutic strategies are warranted for these patients. Previously, we showed that angiogenesis is a characteristic of pediatric low grade astrocytoma (Sie et al, NAN 2010). Therefore, vascular endothelial growth factor (VEGF), the most critical angiogenic factor, may be a potential therapeutic target. The present study aimed to analyze the effect of VEGF inhibition in vitro and in vivo in pediatric low grade astrocytoma. Materials and methods. Three different pediatric low grade astrocytoma cell lines were used: Res-186 (WHO grade I astrocytoma), Res-259 and UW-467 (WHO grade II astrocytoma). Effects in vitro of the anti-VEGF monoclonal antibodies, bevacizumab (0 - 50 ug/ml, Avastin®, anti human VEGF) and B20-4.1.1 (0 - 200 ug/ml, anti human and mouse VEGF) were studied using a WST-1 cell viability assay and a cell proliferation assay with BRDU. Res-259 cells were orthotopically implanted in NOD-scid IL2Rgnull mice. After 6 weeks of treatment intraperitoneal twice weekly with bevacizumab (15 mg/kg), B20-4.1.1 (5 mg/kg) or phosphate buffered saline as control group, mice were euthanized and tumor engraftment was studied in brain slides using Aperios imagescope viewer. Results. In vitro cell growth was not changed as measured by WST-1 and BRDU incorporation. In vivo results showed less tumor engraftment in bevacizumab (n = 10) and B20-4.1.1 (n = 9) treated mice compared with the control group (n = 10) (resp. 70%, 44%, 100%). In the bevacizumab and B20-4.1.1 treated mice tumor mass was lower as compared with controls (resp. P = 0.062, P = 0.001). Conclusion(s). This study showed less tumor engraftment after anti-VEGF therapy in a newly developed pediatric low grade astrocytoma mouse model. In these tumors anti-VEGF seems to work especially on tumor microenvironment. Suggesting that anti-VEGF could have a potential role in the therapeutic strategy for children with low grade astrocytoma, thoughtfulness on possible tumor escape mechanisms that may arise will be crucial. [W.F.A. den Dunnen and E.S.J.M. de Bont shared senior authorship.] Citation Format: Mariska Sie, Arend H. Sikkema, Frank J.G. Scherpen, Arja ter Elst, Kim R. Kampen, Eelco W. Hoving, Wilfred F.a. den Dunnen, Eveline S.j.m. de Bont. Less tumor engraftment after anti-VEGF therapy in pediatric low grade astrocytoma. [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 5085. doi:10.1158/1538-7445.AM2013-5085

Abstract 1974: EphB2 signaling activity plays a vital role in medulloblastoma cell adhesion and invasion

Eph/ephrin signaling has been implicated in various types of cancer, enhancing key processes like migration, proliferation and angiogenesis. In medulloblastoma, invading tumor cells characteristically lead to early recurrence and a decreased prognosis. Based on kinase activity profiling data published recently we hypothesized on a key function of the Eph/ephrin signaling system in medulloblastoma invasion. In a panel of medulloblastoma cell lines a limited subset of Eph receptors was found to be expressed of which EphB2 was the most predominant. Furthermore, a significantly higher expression of EphB2 and its ligands ephrin-B1 and ephrin-B3 was observed in medulloblastoma patient tissue compared to normal cerebellum. To determine the relative importance of the EphB and EphA receptor families, 3 medulloblastoma cell lines (Daoy, Uw-402, Res-256) were stimulated with either ephrin-A1 or ephrin-B1 followed by assessment of the migration and adhesion capacity of the cells. Upon stimulation with ephrin-A1 no phenotype could be observed. However, stimulation with ephrin-B1 resulted in a marked increase in migration capacity of Daoy and, to a lesser extent, Uw-402 in concentrations as low as 0.2 ug/mL. The adhesion capacity of these cell lines upon stimulation with ephrin-B1 was determined employing collagen and laminin coated culture dishes. Again, no effects could be observed on Res-256 whereas the adhesive capacity of Daoy and Uw-402 decreased significantly. In addition, stable transfection of Daoy medulloblastoma cells with shRNA against EphB2 significantly reduced the ephrin-B1 induced effects on tumor cell adhesion. The observed differences in the effects of EphB receptor stimulation correspond with the expression levels of EphB2 in the cell lines. Furthermore, a substantial increase of EphB2 and EphB4 phosphorylation could be observed for Daoy and to a lesser extent Uw-402. Res-256 had a low mRNA expression of EphB2 and B4 and also showed no increase in phosphorylation of these receptors upon stimulation. Interestingly, the increase in phosphorylated EphB2 and EphB4 is accompanied by a sharp decrease in EGFR phosphorylation. Possibly this plays a role in the ephrin-B1 induced effects, as has been reported recently for ephrin-A5 in glioblastoma. Recent studies report an epigenetic regulation by hypermethylation of Eph/ephrin family gene expression in acute lymphoblastic leukemia. To assess the presence of epigenetic regulation of EphB receptor expression in medulloblastoma we treated the medulloblastoma cell lines with the demethylating agent 5-aza-2′-deoxycytidine. This resulted in a substantial increase in expression of all EphB type receptors in Daoy. In conclusion, our results indicate a selective Eph expression profile in favor of medulloblastoma invasion. Furthermore, the expression of Eph receptors is regulated, at least in part, through DNA methylation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1974. doi:10.1158/1538-7445.AM2011-1974

Abstract 2377: Aminopeptidase A plays a role in VEGF-induced migration and proliferation through direct activation by VEGFR2 kinase activity

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Tyrosine kinases pose attractive targets for cancer therapy, as their abnormal signaling activity results in enhanced proliferation, migration and angiogenesis. Previously we have been able to generate a comprehensive description of the biological kinase activity present in pediatric brain tumor tissue lysate by the application of a peptide microarray containing 144 different tyrosine kinase peptide substrates. To establish which peptide substrates present on the array are most sensitive and discriminative for VEGFR activity we measured the peptide phosphorylation applying recombinant VEGFR1, 2 and 3. Furthermore, a panel of recombinant kinases with highly diverse VEGFR sequence homology was included to study substrate specificity. An aminopeptidase A (APA) derived peptide proved to be highly phosphorylated by VEGFR 1 and 2. No phosphorylation of this peptide could be observed applying any of the other kinases, arguing that this peptide is discriminative for VEGFR1 and 2 activity. In the peptide array data of pediatric brain tumors, phosphorylation of the APA peptide was observed, suggestive for VEGFR activation. Based on these results we hypothesize that APA can be activated through direct phosphorylation by VEGFR2 and exert functional effects on endothelial cell proliferation and migration. To test this hypothesis we stimulated human umbilical vein endothelial cells (HUVECs) with VEGF and observed phosphorylation of Aminopeptidase A. Literature states that inhibition of APA suppresses VEGF-induced migration and proliferation of endothelial cells and inhibits angiogenesis. Based on our data we hypothesize that these APA-mediated effects of VEGF stimulation are induced through direct activation by VEGFR2 kinase activity. Concluding, peptide microarray technology is able to provide a selective view on tyrosine kinase activation, as has been shown here for VEGFR1 and 2. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2377.