Yusuke Kawase

Cutting Edge: IL-18-Transgenic Mice: In Vivo Evidence of a Broad Role for IL-18 in Modulating Immune Function

IL-18 has been shown to be a strong cofactor for Th1 T cell development. However, we previously demonstrated that when IL-18 was combined with IL-2, there was a synergistic induction of a Th2 cytokine, IL-13, in both T and NK cells. More recently, we and other groups have reported that IL-18 can potentially induce IgE, IgG1, and Th2 cytokine production in murine experimental models. Here, we report on the generation of IL-18-transgenic (Tg) mice in which mature mouse IL-18 cDNA was expressed. CD8+CD44high T cells and macrophages were increased, but B cells were decreased in these mice while serum IgE, IgG1, IL-4, and IFN-γ levels were significantly increased. Splenic T cells in IL-18 Tg mice produced higher levels of IFN-γ, IL-4, IL-5, and IL-13 than control wild-type mice. Thus, aberrant expression of IL-18 in vivo results in the increased production of both Th1 and Th2 cytokines.

Contribution of IL-18 to Th1 Response and Host Defense Against Infection by Mycobacterium tuberculosis: A Comparative Study with IL-12p40

The present study was conducted to critically determine the protective role of IL-18 in host response to Mycobacterium tuberculosis infection. IL-18-deficient (knockout (KO)) mice were slightly more prone to this infection than wild-type (WT) mice. Sensitivity of IL-12p40KO mice was lower than that of IL-12p40/IL-18 double KO mice. IFN-γ production caused by the infection was significantly attenuated in IL-18KO mice compared with WT mice, as indicated by reduction in the levels of this cytokine in sera, spleen, lung, and liver, and its synthesis by spleen cells restimulated with purified protein derivatives. Serum IL-12p40 level postinfection and its production by peritoneal exudate cells stimulated with live bacilli were also significantly lower in IL-18KO mice than WT mice, suggesting that attenuated production of IFN-γ was secondary to reduction of IL-12 synthesis. However, this was not likely the case, because administration of excess IL-12 did not restore the reduced IFN-γ production in IL-18KO mice. In further studies, IL-18 transgenic mice were more resistant to the infection than control littermate mice, and serum IFN-γ level and its production by restimulated spleen cells were increased in the former mice. Taken together, our results indicate that IL-18 plays an important role in Th1 response and host defense against M. tuberculosis infection although the contribution was not as profound as that of IL-12p40.

Comparison of the Cancer Gene Targeting and Biochemical Selectivities of All Targeted Kinase Inhibitors Approved for Clinical Use

The anti-proliferative activities of all twenty-five targeted kinase inhibitor drugs that are in clinical use were measured in two large assay panels: (1) a panel of proliferation assays of forty-four human cancer cell lines from diverse tumour tissue origins; and (2) a panel of more than 300 kinase enzyme activity assays. This study provides a head-on comparison of all kinase inhibitor drugs in use (status Nov. 2013), and for six of these drugs, the first kinome profiling data in the public domain. Correlation of drug activities with cancer gene mutations revealed novel drug sensitivity markers, suggesting that cancers dependent on mutant CTNNB1 will respond to trametinib and other MEK inhibitors, and cancers dependent on SMAD4 to small molecule EGFR inhibitor drugs. Comparison of cellular targeting efficacies reveals the most targeted inhibitors for EGFR, ABL1 and BRAF(V600E)-driven cell growth, and demonstrates that the best targeted agents combine high biochemical potency with good selectivity. For ABL1 inhibitors, we computationally deduce optimized kinase profiles for use in a next generation of drugs. Our study shows the power of combining biochemical and cellular profiling data in the evaluation of kinase inhibitor drug action.

Bone Malformations in Interleukin-18 Transgenic Mice†

The in vivo effects of IL‐18 on bone metabolism were investigated by histopathology in IL‐18 transgenic mice. Deformed cortical bone and decreased turnover rate of lumbar trabecular bone are consistent with increased expression of IFN‐γ and IL‐18 in the bone marrow.

Compound Selectivity and Target Residence Time of Kinase Inhibitors Studied with Surface Plasmon Resonance

Target residence time (τ) has been suggested to be a better predictor of the biological activity of kinase inhibitors than inhibitory potency (IC50) in enzyme assays. Surface plasmon resonance binding assays for 46 human protein and lipid kinases were developed. The association and dissociation constants of 80 kinase inhibitor interactions were determined. τ and equilibrium affinity constants (KD) were calculated to determine kinetic selectivity. Comparison of τ and KD or IC50 values revealed a strikingly different view on the selectivity of several kinase inhibitors, including the multi-kinase inhibitor ponatinib, which was tested on 10 different kinases. In addition, known pan-Aurora inhibitors resided much longer on Aurora B than on Aurora A, despite having comparable affinity for Aurora A and B. Furthermore, the γ/δ-selective PI3K inhibitor duvelisib and the δ-selective drug idelalisib had similar 20-fold selectivity for δ- over γ-isoform but duvelisib resided much longer on both targets.

Limited Role for Interleukin-18 in the Host Protection Response to Pulmonary Infection with Pseudomonas aeruginosa in Mice

ABSTRACT We report that clearance of Pseudomonas aeruginosa, accumulation of neutrophils, and synthesis of tumor necrosis factor alpha and macrophage inflammatory protein 2 in the infected lung were not largely different in interleukin-18 (IL-18) knockout or transgenic mice compared with control mice. Our results suggest a limited role for IL-18 in the host defense against P. aeruginosa.

Abstract 2849: Analysis of the cancer gene targeting of clinical kinase inhibitor drugs by combining cellular and biochemical profiling

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA [Purpose] Kinase inhibitors are a prime example of the success of targeted therapy. Crucial to the development of targeted therapies is the ability to couple drug response to genetic markers, such as mutations in oncogenes and/or tumor suppressors, chromosomal rearrangements, and/or gene amplifications. The efficacy of response to such markers is linked with the biochemical characteristics of kinase inhibitors. However, it is poorly understood what profiles are required to achieve the best targeting. We have systematically compared the biochemical potency and selectivity profiles of all kinase inhibitor drugs in clinical use with their ability to inhibit cell proliferation in a panel of forty-four human cancer cell lines from diverse tumor tissue origins.[Experimental procedures] Proliferation assays were established for a panel of forty-four human cancer cell lines licensed from the American Type Culture Collection. The panel represents twenty-three of the most frequently identified genetic changes in the COSMIC Cell Lines (CCL) project in at least two cell lines. We profiled all twenty-five kinase inhibitors in clinical use on the cancer cell line panel, together with a number of classic cytotoxic agents for comparison. The proliferation assay data were linked to genetic data from the CCL database by Anova and volcano plot analysis. In parallel the biochemical profiles of all twenty-five kinase inhibitor drugs were determined on a panel of 313 protein kinase enzyme activity assays. The biochemical profiling data were used to interpret the cellular targeting compounds in terms of biochemical potency, selectivity and kinase poly-pharmacology.[Results] The suitability of the cell line panel was validated by confirming the known sensitivity of mutant B-RAF to vemurafenib, of BCR-ABL to imatinib, and EGFR overexpression to erlotinib. Mutation in SMAD4 was identified as a novel drug sensitivity marker for EGFR inhibitors, while mutation in the gene for β-catenin was identified as a sensitivity marker for MEK inhibitors, such as trametinib. When comparing compounds with similar mechanisms, dabrafenib is more efficient at selectively inhibiting BRAF-mutant transformed cells than vemurafenib. Of all ABL inhibitors, imatinib has the best targeting efficacy. The compound that most selectively inhibits growth of EGFR-overexpressing cell lines, is gefitinib.[Conclusions] Cancer cell line profiling can be used to identify novel sensitivity markers of drug response. Cancer cell line profiling in combination with biochemical kinase profiling revealed biochemical selectivity and potencies as prime determinants of on target efficacy of kinase inhibitor drugs, the extent of which is target-dependent. The combination of cell panel profiling and kinome profiling is an important tool for the development of optimal targeted therapies for kinases. Citation Format: Guido J.R. Zaman, Joost C.M. Uitdehaag, Rogier C. Buijsman, Jeroen A.D.M. de Roos, Antoon M. van Doornmalen, Martine B.W. Prinsen, Jos de Man, Yusuke Kawase, Yoshinori Tanizawa, Kohichiro Yoshino. Analysis of the cancer gene targeting of clinical kinase inhibitor drugs by combining cellular and biochemical profiling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2849. doi:10.1158/1538-7445.AM2014-2849

Abstract 4541: Highly sensitive kinase assays combining Carna Biosciences QSS Assist ELISA reagents with the Meso Scale Discovery MULTI-ARRAY platform.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC We developed a sensitive kinase asay using a QSS Assist ELISA kinase assay kit from Carna Biosciences, Inc. and the detection method from Meso Scale Discovery. The QSS Assist ELISA kit is optimized for screening of compounds. Comparing to most other kinase assay kits, QSS Assist ELISA kits are available for both peptide and protein substrates and several ELISA kits developed by Carna Biosciences employ a naturally occurring protein substrate, e.g. MAP kinases for MAPK kinases. Detection of kinase activity is achieved with a primary anti-phosphoserine/threonine or tyrosine antibody and a secondary anti-Ig-antibody. Commonly, such secondary antibodies are coupled to horseradish peroxidase (HRP), which allows for signal detection by adding a color reagent, i.e. an HRP substrate which can be detected upon reaction by measuring optical density. Here we applied an MSD SULFO-TAG labeled secondary antibody. These antibodies emit light upon electrochemical stimulation initiated at the electrode surfaces of MULTI-ARRAY microplates, and thus detect the primary antibody with ultimate sensitivity without direct labeling it. We have sucessfully miniaturized the highly sensitive assay and this new assay technology has been validated with published kinase inhibitors from AstraZeneca and Tocris Bioscience. Citation Format: Carsten Jacobi, Alain Schilb, Yusuke Kawase, Yasuyuki Kirii, Brigitte Boldyreff. Highly sensitive kinase assays combining Carna Biosciences QSS Assist ELISA reagents with the Meso Scale Discovery MULTI-ARRAY platform. [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 4541. doi:10.1158/1538-7445.AM2013-4541