Taisei Kinoshita

Preclinical characterization of Aurora kinase inhibitor R763/AS703569 identified through an image-based phenotypic screen

PurposeAurora kinases play a key role in mitotic progression. Over-expression of Aurora kinases is found in several human cancers and correlated with histological malignancy and clinical outcomes. Therefore, Aurora kinase inhibitors should be useful in the treatment of cancers.MethodsCell-based screening methods have an advantage over biochemical approaches because hits can be optimized to inhibit targets in the proper intracellular context. We developed a novel Aurora kinase inhibitor R763/AS703569 using an image-based phenotypic screen. The anti-proliferative effect was examined in a panel of tumor cell lines and primary cells. The efficacy was determined in a broad panel of xenograft models.ResultsR763/AS703569 inhibits Aurora kinases, along with a limited number of other kinases including FMS-related tyrosine kinase 3 (FLT3), and has potent anti-proliferative activity against many cell types accompanying unique phenotypic changes such as enlarged cell size, endoreduplication and apoptosis. The endoreduplication cycle induced by R763/AS703569 was irreversible even after the compound was withdrawn from the culture. Oral administration of R763/AS703569 demonstrated marked inhibition of tumor growth in xenograft models of pancreatic, breast, colon, ovarian, and lung tumors and leukemia. An acute myeloid leukemia cell line MV4-11, which carries a FLT3 internal tandem duplication mutation, is particularly sensitive to R763/AS703569 in vivo.ConclusionsR763/AS703569 is a potent inhibitor of Aurora kinases and exhibited significant anti-proliferative activity against a wide range of tumor cells both in vitro and in vivo. Inhibition of Aurora kinases has the potential to be a new addition to the treatment of cancers.

Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction

Controlled mechanical ventilation (CMV) is associated with the development of diaphragm atrophy and contractile dysfunction, and respiratory muscle weakness is thought to contribute significantly to delayed weaning of patients. Therefore, therapeutic strategies for preventing these processes may have clinical benefit. The aim of the current study was to investigate the role of the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in CMV‐mediated diaphragm wasting and weakness in rats. CMV‐induced diaphragm atrophy and contractile dysfunction coincided with marked increases in STAT3 phosphorylation on both tyrosine 705 (Tyr705) and serine 727 (Ser727). STAT3 activation was accompanied by its translocation into mitochondria within diaphragm muscle and mitochondrial dysfunction. Inhibition of JAK signaling during CMV prevented phosphorylation of both target sites on STAT3, eliminated the accumulation of phosphorylated STAT3 within the mitochondria, and reversed the pathologic alterations in mitochondrial function, reduced oxidative stress in the diaphragm, and maintained normal diaphragm contractility. In addition, JAK inhibition during CMV blunted the activation of key proteolytic pathways in the diaphragm, as well as diaphragm atrophy. These findings implicate JAK/STAT3 signaling in the development of diaphragm muscle atrophy and dysfunction during CMV and suggest that the delayed extubation times associated with CMV can be prevented by inhibition of Janus kinase signaling.—Smith, I. J., Godinez, G. L., Singh, B. K., McCaughey, K. M., Alcantara, R. R., Gururaja, T., Ho, M. S., Nguyen, H. N., Friera, A. M., White, K. A., McLaughlin, J. R., Hansen, D., Romero, J. M., Baltgalvis, K. A., Claypool, M. D., Li, W., Lang, W., Yam, G. C., Gelman, M. S., Ding, R., Yung, S. L., Creger, D. P., Chen, Y., Singh, R., Smuder, A. J., Wiggs, M. P., Kwon, O.‐S., Sollanek, K. J., Powers, S. K., Masuda, E. S., Taylor, V. C., Payan, D. G., Kinoshita, T., Kinsella, T. M. Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation‐induced diaphragm dysfunction. FASEB J. 28, 2790–2803 (2014). www.fasebj.org

R-253 Disrupts Microtubule Networks in Multiple Tumor Cell Lines

Purpose: The design and development of synthetic small molecules to disrupt microtubule dynamics is an attractive therapeutic strategy for anticancer drug discovery research. Loss of clinical efficacy of many useful drugs due to drug resistance in tumor cells seems to be a major hurdle in this endeavor. Thus, a search for new chemical entities that bind tubulin, but neither are a substrate of efflux pump, P-glycoprotein 170/MDR1, nor cause undesired side effects, would potentially increase the therapeutic index in certain cancer treatments. Experimental Design: A high-content cell-based screen of a compound library led to the identification of a new class of compounds belonging to a thienopyrimidine series, which exhibited significant antitumor activities. On structure-activity relationship analysis, R-253 [N-cyclopropyl-2-(6-(3,5-dimethylphenyl)thieno[3,2-d]pyrimidin-4-yl)hydrazine carbothioamide] emerged as a potent antiproliferative agent (average EC50, 20 nmol/L) when examined in a spectrum of tumor cell lines. Results: R-253 is structurally unique and destabilizes microtubules both in vivo and in vitro. Standard fluorescence-activated cell sorting and Western analyses revealed that the effect of R-253 on cell growth was associated with cell cycle arrest in mitosis, increased select G2-M checkpoint proteins, and apoptosis. On-target activity of R-253 on microtubules was further substantiated by immunofluorescence studies and selected counter assays. R-253 competed with fluorescent-labeled colchicine for binding to tubulin, indicating that its binding site on tubulin could be similar to that of colchicine. R-253 neither is a substrate of P-glycoprotein 170/MDR1 nor is cytotoxic to nondividing human hepatocytes. Conclusion: Both biochemical and cellular mechanistic studies indicate that R-253 could become a promising new tubulin-binding drug candidate for treating various malignancies.

Ibrutinib Inhibits ERBB Receptor Tyrosine Kinases and HER2-Amplified Breast Cancer Cell Growth

Ibrutinib is a potent, small-molecule Bruton tyrosine kinase (BTK) inhibitor developed for the treatment of B-cell malignancies. Ibrutinib covalently binds to Cys481 in the ATP-binding domain of BTK. This cysteine residue is conserved among 9 other tyrosine kinases, including HER2 and EGFR, which can be targeted. Screening large panels of cell lines demonstrated that ibrutinib was growth inhibitory against some solid tumor cells, including those inhibited by other HER2/EGFR inhibitors. Among sensitive cell lines, breast cancer lines with HER2 overexpression were most potently inhibited by ibrutinib (<100 nmol/L); in addition, the IC50s were lower than that of lapatinib and dacomitinib. Inhibition of cell growth by ibrutinib coincided with downregulation of phosphorylation on HER2 and EGFR and their downstream targets, AKT and ERK. Irreversible inhibition of HER2 and EGFR in breast cancer cells was established after 30-minute incubation above 100 nmol/L or following 2-hour incubation at lower concentrations. Furthermore, ibrutinib inhibited recombinant HER2 and EGFR activity that was resistant to dialysis and rapid dilution, suggesting an irreversible interaction. The dual activity toward TEC family (BTK and ITK) and ERBB family kinases was unique to ibrutinib, as ERBB inhibitors do not inhibit or covalently bind BTK or ITK. Xenograft studies with HER2+ MDA-MB-453 and BT-474 cells in mice in conjunction with determination of pharmacokinetics demonstrated significant exposure-dependent inhibition of growth and key signaling molecules at levels that are clinically achievable. Ibrutinibs unique dual spectrum of activity against both TEC family and ERBB kinases suggests broader applications of ibrutinib in oncology. Mol Cancer Ther; 15(12); 2835–44. ©2016 AACR.

Noninvasive imaging of in vivo MuRF1 expression during muscle atrophy.

Numerous human diseases can lead to atrophy of skeletal muscle, and loss of this tissue has been correlated with increased mortality and morbidity rates. Clinically addressing muscle atrophy remains an unmet medical need, and the development of preclinical tools to assist drug discovery and basic research in this effort is important for advancing this goal. In this report, we describe the development of a bioluminescent gene reporter rat, based on the zinc finger nuclease-targeted insertion of a bicistronic luciferase reporter into the 3′ untranslated region of a muscle specific E3 ubiquitin ligase gene, MuRF1 (Trim63). In longitudinal studies, we noninvasively assess atrophy-related expression of this reporter in three distinct models of muscle loss (sciatic denervation, hindlimb unloading and dexamethasone-treatment) and show that these animals are capable of generating refined detail on in vivo MuRF1 expression with high temporal and anatomical resolution.

The effect of Bruton's tyrosine kinase (BTK) inhibitors on collagen-induced platelet aggregation, BTK, and tyrosine kinase expressed in hepatocellular carcinoma (TEC)

Brutons tyrosine kinase (BTK) and tyrosine kinase expressed in hepatocellular carcinoma (TEC) are expressed by human platelets. These kinases participate in platelet activation through the collagen receptor glycoprotein VI and may perform overlapping functions. In clinical studies, BTK inhibitors (ibrutinib, acalabrutinib, tirabrutinib, zanubrutinib) have been associated with increased bleeding risk, which may result from inhibition of BTK alone or of both BTK and TEC, although the role of TEC in bleeding risk remains unclear.

Abstract B193: Ibrutinib potentiates the effects of mTOR inhibitors and pazopanib in renal cell carcinoma in vitro and in vivo

Introduction: Ibrutinib is a first-in-class Bruton9s tyrosine kinase (BTK) inhibitor approved for treatment of multiple B-cell malignancies that also inhibits EGFR and HER2 activity (Honigberg, 2010; Gao, 2014). Ibrutinib suppressed growth of EGFR-driven NSCLC or HER2-amplified breast cancer cell lines and reduced tumor growth in murine xenograft models demonstrating the potential of anti-tumor activity in solid tumors (Gao, 2014; Elias, 2013). Ibrutinib also modulated host tumor immunity and enhanced PD-L1 Mab activity in solid tumor models from tumor cells insensitive to BTK or HER kinase inhibition (Sagiv-Barfi, 2015). These results suggest the potential of ibrutinib to be clinically active across a variety of tumors via multiple mechanisms of action (MoA). Immunotherapeutic interventions have had varying success in renal cell carcinoma (RCC), the most common type of kidney cancer. In this study, we determined the therapeutic impact of ibrutinib alone and in combination with approved therapies in RCC in vitro and in vivo and further investigated its MoA. Methods: The effect of drugs on cell proliferation was determined with CellTiter-Glo (Promega), and apoptosis assayed with annexin-V/PI staining. Signaling pathways were evaluated with Western blotting. Single agent and combinations were studied in vivo by subcutaneous implantation of syngeneic Renca or human 786-0 RCC cell lines into mice. Results: Treating RCC cells with everolimus alone potently inhibited p-S6, a downstream mTOR target, and significantly inhibited proliferation. Pazopanib, a multi-kinase inhibitor, similarly inhibited pAkt. As reported, these inhibitors up-regulated pAkt and/or pErk, two key pro-survival molecules, after 1h and/or 24h treatment. Such compensatory changes to signaling pathways have been suggested to attenuate the anti-tumor activity of these inhibitors (Wang, 2008; Breuleux, 2009; Grabinski, 2012; Soares, 2013). Interestingly, addition of ibrutinib antagonized up-regulation of pAkt and pErk by everolimus or pazopanib, and enhanced their anti-proliferative activities. In contrast, ibrutinib did not enhance everolimus activity in Caki-1 cells where pAkt and pErk were unaffected by the combination. Ibrutinib alone did not or only weakly inhibited RCC proliferation in cell culture despite inhibition of EGF-induced pEGFR. In contrast, pAkt and pErk, were much less affected, suggesting that EGFR is not a primary driver of proliferation in RCC cells. In vivo, ibrutinib alone showed marginal or no anti-tumor activity in Renca or 786-0 mice models. However, ibrutinib significantly reduced tumor burden in combination with sirolimus (p Conclusion: These results suggest that ibrutinib, a kinase inhibitor with immune modulatory properties, has anti-tumor activity against RCC when combined with mTOR inhibitors or pazopanib with different target spectrums. Although EGFR is not critical for proliferation in these untreated RCC lines, ibrutinib may prevent ErbB kinases from contributing to feedback up-regulation of Akt/Erk pathways by established drugs. These results provide a preclinical rationale for investigation of ibrutinib as a novel agent for RCC through positive interaction with mTOR inhibitors and/or pazopanib. Citation Format: Jun Chen, Jeff Hsu, Yujun Huang, Danelle F. James, Taisei Kinoshita, Betty Y. Chang. Ibrutinib potentiates the effects of mTOR inhibitors and pazopanib in renal cell carcinoma in vitro and in vivo. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B193.