Abstract
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. 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 immunofluorescent detection of DNA and alpha-tubulin, respectively. In addition, 18F-3'-fluoro-3'-deoxy-l-thymidine positron emission tomography (FLT-PET) was used to noninvasively measure effects of alisertib on in vivo tumor cell proliferation. 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. 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.
Highlights
Mitotic kinases, kinesins, and other mitotic enzymes are being pursued as targets for the generation of antimitotic therapies in oncology
In the cell-based assays, Aurora A activity was determined by measuring autophosphorylation of Aurora A on threonine 288, whereas Aurora B activity was determined by measuring phosphorylation of histone H3 on serine 10, in both cases, using high content imaging assays and as previously described [32]
In vitro studies show that alisertib inhibits Aurora A kinase (AAK) and is selective over family member Aurora B kinase (ABK) and other kinases
Summary
Kinesins, and other mitotic enzymes are being pursued as targets for the generation of antimitotic therapies in oncology. 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 incorporate 2 alternative outcomes following mitotic delays in metaphase [2, 3]. 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 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
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