Differentiating Benign from Malignant Thyroid Tumors by Kinase Activity Profiling and Dabrafenib BRAF V600E Targeting.

Differentiated non-medullary thyroid cancer (NMTC) can be effectively treated by surgery followed by radioactive iodide therapy. However, a small subset of patients shows recurrence due to a loss of iodide transport, a phenotype frequently associated with BRAF V600E mutations. In theory, this should enable the use of existing targeted therapies specifically designed for BRAF V600E mutations. However, in practice, generic or specific drugs aimed at molecular targets identified by next generation sequencing (NGS) are not always beneficial. Detailed kinase profiling may provide additional information to help improve therapy success rates. In this study, we therefore investigated whether serine/threonine kinase (STK) activity profiling can accurately classify benign thyroid lesions and NMTC. We also determined whether dabrafenib (BRAF V600E-specific inhibitor), as well as sorafenib and regorafenib (RAF inhibitors), can differentiate BRAF V600E from non-BRAF V600E thyroid tumors. Using 21 benign and 34 malignant frozen thyroid tumor samples, we analyzed serine/threonine kinase activity using PamChip®peptide microarrays. An STK kinase activity classifier successfully differentiated malignant (26/34; 76%) from benign tumors (16/21; 76%). Of the kinases analyzed, PKC (theta) and PKD1 in particular, showed differential activity in benign and malignant tumors, while oncocytic neoplasia or Graves' disease contributed to erroneous classifications. Ex vivo BRAF V600E-specific dabrafenib kinase inhibition identified 6/92 analyzed peptides, capable of differentiating BRAF V600E-mutant from non-BRAF V600E papillary thyroid cancers (PTCs), an effect not seen with the generic inhibitors sorafenib and regorafenib. In conclusion, STK activity profiling differentiates benign from malignant thyroid tumors and generates unbiased hypotheses regarding differentially active kinases. This approach can serve as a model to select novel kinase inhibitors based on tissue analysis of recurrent thyroid and other cancers.

Focal adhesion kinase (FAK) binds RET kinase via its FERM domain, priming a direct and reciprocal RET-FAK transactivation mechanism.

Whether RET is able to directly phosphorylate and activate downstream targets independently of the binding of proteins that contain Src homology 2 or phosphotyrosine binding domains and whether mechanisms in trans by cytoplasmic kinases can modulate RET function and signaling remain largely unexplored. In this study, oligopeptide arrays were used to screen substrates directly phosphorylated by purified recombinant wild-type and oncogenic RET kinase domain in the presence or absence of small molecule inhibitors. The results of the peptide array were validated by enzyme kinetics, in vitro kinase, and cell-based experiments. The identification of focal adhesion kinase (FAK) as a direct substrate for RET kinase revealed (i) a RET-FAK transactivation mechanism consisting of direct phosphorylation of FAK Tyr-576/577 by RET and a reciprocal phosphorylation of RET by FAK, which crucially is able to rescue the kinase-impaired RET K758M mutant and (ii) that FAK binds RET via its FERM domain. Interestingly, this interaction is abolished upon RET phosphorylation, indicating that RET binding to the FERM domain of FAK is a priming step for RET-FAK transactivation. Finally, our data indicate that FAK inhibitors could be used as potential therapeutic agents for patients with multiple endocrine neoplasia type 2 tumors because both, treatment with the FAK kinase inhibitor NVP-TAE226 and FAK down-regulation by siRNA reduced RET phosphorylation and signaling as well as the proliferation and survival of tumor and transfected cell lines expressing oncogenic RET.

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.

Prediction of response to preoperative chemoradiotherapy in rectal cancer by multiplex kinase activity profiling.

PURPOSE: Tumor response of rectal cancer to preoperative chemoradiotherapy (CRT) varies considerably. In experimental tumor models and clinical radiotherapy, activity of particular subsets of kinase signaling pathways seems to predict radiation response. This study aimed to determine whether tumor kinase activity profiles might predict tumor response to preoperative CRT in locally advanced rectal cancer (LARC). METHODS AND MATERIALS: Sixty-seven LARC patients were treated with a CRT regimen consisting of radiotherapy, fluorouracil, and, where possible, oxaliplatin. Pretreatment tumor biopsy specimens were analyzed using microarrays with kinase substrates, and the resulting substrate phosphorylation patterns were correlated with tumor response to preoperative treatment as assessed by histomorphologic tumor regression grade (TRG). A predictive model for TRG scores from phosphosubstrate signatures was obtained by partial-least-squares discriminant analysis. Prediction performance was evaluated by leave-one-out cross-validation and use of an independent test set. RESULTS: In the patient population, 73% and 15% were scored as good responders (TRG 1-2) or intermediate responders (TRG 3), whereas 12% were assessed as poor responders (TRG 4-5). In a subset of 7 poor responders and 12 good responders, treatment outcome was correctly predicted for 95%. Application of the prediction model on the remaining patient samples resulted in correct prediction for 85%. Phosphosubstrate signatures generated by poor-responding tumors indicated high kinase activity, which was inhibited by the kinase inhibitor sunitinib, and several discriminating phosphosubstrates represented proteins derived from signaling pathways implicated in radioresistance. CONCLUSIONS: Multiplex kinase activity profiling may identify functional biomarkers predictive of tumor response to preoperative CRT in LARC.

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.

Osteoblast-induced EGFR/ERBB2 signaling in androgen-sensitive prostate carcinoma cells characterized by multiplex kinase activity profiling.

Bone metastases in prostate cancer are predominantly osteoblastic. To study regulatory mechanisms underlying the establishment of prostate cancer within an osteoblastic microenvironment, human androgen-sensitive prostate carcinoma cells (LNCaP) were treated with culture medium conditioned by human osteoblast-derived sarcoma cells (OHS), and activated signalling pathways in the carcinoma cells were analyzed using microarrays with tyrosine kinase substrates. Network interaction analysis of substrates with significantly increased phosphorylation levels revealed that signalling pathways mediated by EGFR and ERBB2 were activated in LNCaP cells under OHS influence but also by androgen treatment. Activation of EGFR/ERBB2 signalling was also found in LNCaP cells in cocultures with OHS cells or osteoblastic cells that had been differentiated from human mesenchymal stem cells. Our experimental data suggests osteoblast-directed induction of signalling activity via EGFR and ERBB2 in prostate carcinoma cells and may provide a rationale for the use of EGFR or ERBB2 inhibition in systemic prevention or treatment of metastatic prostate cancer in the androgen-sensitive stage of the disease.

Use of hybridization melting kinetics for detecting Phytophthora species using three-dimensional microarrays: demonstration of a novel concept for the differentiation of detection targets.

Microarray-based detection is limited by variable and inconsistent hybridization intensities across the diversity of probes used in each array. In this paper, we introduce a novel concept for the differentiation of detection targets using duplex melting kinetics. A microarray assay was developed on a PamChip microarray enabling the differentiation of target Phytophthora species using the melting kinetics of probe-target duplexes. In the majority of cases the hybridization kinetics of target and non-target duplexes differed significantly. Analysis of the melting kinetics of duplexes formed by probes with target and non-target DNA was found to be an effective method for determining specific hybridization and was independent of fluctuations in hybridization signal intensity. This form of analysis was more robust than the traditional approach based on hybridization intensity, and enabled the detection of individual Phytophthora species and mixtures thereof.

Llama antibodies against a lactococcal protein located at the tip of the phage tail prevent phage infection.

Bacteriophage p2 belongs to the most prevalent lactococcal phage group (936) responsible for considerable losses in industrial production of cheese. Immunization of a llama with bacteriophage p2 led to higher titers of neutralizing heavy-chain antibodies (i.e., devoid of light chains) than of the classical type of immunoglobulins. A panel of p2-specific single-domain antibody fragments was obtained using phage display technology, from which a group of potent neutralizing antibodies were identified. The antigen bound by these antibodies was identified as a protein with a molecular mass of 30 kDa, homologous to open reading frame 18 (ORF18) of phage sk1, another 936-like phage for which the complete genomic sequence is available. By the use of immunoelectron microscopy, the protein is located at the tip of the tail of the phage particle. The addition of purified ORF18 protein to a bacterial culture suppressed phage infection. This result and the inhibition of cell lysis by anti-ORF18 protein antibodies support the conclusion that the ORF18 protein plays a crucial role in the interaction of bacteriophage p2 with the surface receptors of Lactococcus lactis.

Quantitative assessment of a novel flow-through porous microarray for the rapid analysis of gene expression profiles.

A novel microarray system that utilizes a porous aluminum-oxide substrate and flow-through incubation has been developed for rapid molecular biological testing. To assess its utility in gene expression analysis, we determined hybridization kinetics, variability, sensitivity and dynamic range of the system using amplified RNA. To show the feasibility with complex biological RNA, we subjected Jurkat cells to heat-shock treatment and analyzed the transcriptional regulation of 23 genes. We found that trends (regulation or no change) acquired on this platform are in good agreement with data obtained from real-time quantitative PCR and Affymetrix GeneChips. Additionally, the system demonstrates a linear dynamic range of 3 orders of magnitude and at least 10-fold decreased hybridization time compared to conventional microarrays. The minimum amount of transcript that could be detected in 20 microl volume is 2-5 amol, which enables the detection of 1 in 300,000 copies of a transcript in 1 microg of amplified RNA. Hybridization and subsequent analysis are completed within 2 h. Replicate hybridizations on 24 identical arrays with two complex biological samples revealed a mean coefficient of variation of 11.6%. This study shows the potential of flow-through porous microarrays for the rapid analysis of gene expression profiles in clinical applications.