Cancer Therapy: Preclinical Characterization of Alisertib (MLN8237), an Investigational Small-Molecule Inhibitor of Aurora A Kinase Using Novel In Vivo Pharmacodynamic Assays Mark G. Manfredi, Jeffrey A. Ecsedy, Arijit Chakravarty, Lee Silverman, Mengkun Zhang, Kara M. Hoar, Stephen G. Stroud, Wei Chen, Vaishali Shinde, Jessica J. Huck, Deborah R. Wysong, David A. Janowick, Marc L. Hyer, Patrick J. LeRoy, Rachel E. Gershman, Matthew D. Silva, Melissa S. Germanos, Joseph B. Bolen, Christopher F. Claiborne, and Todd B. Sells Abstract Purpose: Small-molecule inhibitors of Aurora A (AAK) and B (ABK) kinases, which play important roles in mitosis, are currently being pursued in oncology clinical trials. We developed three novel assays to quantitatively measure biomarkers of AAK inhibition in vivo. Here, we describe preclinical characterization of alisertib (MLN8237), a selective AAK inhibitor, incorporating these novel pharmacodynamic assays. Experimental Design: We investigated the selectivity of alisertib for AAK and ABK and studied the antitumor and antiproliferative activity of alisertib in vitro and in vivo. Novel assays were used to assess chromosome alignment and mitotic spindle bipolarity in human tumor xenografts using immunofluo- rescent detection of DNA and alpha-tubulin, respectively. In addition, 18F-3 0 -fluoro-3 0 -deoxy-L-thymidine positron emission tomography (FLT-PET) was used to noninvasively measure effects of alisertib on in vivo tumor cell proliferation. Results: Alisertib inhibited AAK over ABK with a selectivity of more than 200-fold in cells and produced a dose-dependent decrease in bipolar and aligned chromosomes in the HCT-116 xenograft model, a phenotype consistent with AAK inhibition. Alisertib inhibited proliferation of human tumor cell lines in vitro and produced tumor growth inhibition in solid tumor xenograft models and regressions in in vivo lymphoma models. In addition, a dose of alisertib that caused tumor stasis, as measured by volume, resulted in a decrease in FLT uptake, suggesting that noninvasive imaging could provide value over traditional measurements of response. Conclusions: Alisertib is a selective and potent inhibitor of AAK. The novel methods of measuring Aurora A pathway inhibition and application of tumor imaging described here may be valuable for clinical evaluation of small-molecule inhibitors. Clin Cancer Res; 17(24); 7614–24. Ó2011 AACR. Introduction Mitotic kinases, kinesins, and other mitotic enzymes are being pursued as targets for the next generation of antimi- totic therapies in oncology. Although several molecules have shown clinical efficacy, it is too early to know whether they will add benefit beyond classic microtubule antago- nists such as the taxanes and vinca alkaloids. So far, how- ever, it is clear that the newer agents are unlikely to cause the peripheral neuropathy, often observed in patients treated with microtubule-targeting drugs (1). The conventional view of antimitotic agents is that they cause prolonged mitotic arrest leading to cell death. In recent years, this perspective has been modified to incor- porate 2 alternative outcomes following mitotic delays in metaphase (2, 3). Studies using live-cell microscopy with a variety of antimitotic agents in a range of cell lines have reported a striking diversity of responses (4–6). In some sensitive cell lines, mitotic arrest is sustained until cells die directly from prometaphase. In other sensitive cell lines, the mitotic delay is transient, and it is followed by an inappropriate segregation of unaligned chromosomes (4, 5, 7). This mitotic slippage is followed by a variety of terminal outcomes that seem to include postmitotic death as well as terminal growth arrest (cellular senescence; refs. 8, 9). Evidence exists to support the view that cytostasis (10, 11) as well as postmitotic cell death (5) are significant drivers of the antiproliferative effects of taxanes. Authors' Affiliation: Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Author: Mark G. Manfredi, Millennium Pharmaceuticals, Inc., Cambridge, 40 Landsdowne Street, Cambridge, MA 02139. Phone: 617-679-7382; Fax: 617-551-8906; E-mail: mark.manfredi@mpi.com doi: 10.1158/1078-0432.CCR-11-1536 Ó2011 American Association for Cancer Research. Clinical Cancer Research Clin Cancer Res; 17(24) December 15, 2011 7614 on June 18, 2016. © 2011 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst October 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1536