P3-01-08: In Vitro and In Vivo Antitumor Activity of the Aurora and Angiogenic Kinase Inhibitor ENMD-2076 in Triple-Negative Breast Cancer Models.
Authors: Diamond JR , Eckhardt SG , Tan AC , Selby HM , Newton TP , Pitts TM , Bray MR , Fletcher GC , Tentler JJ 
Published: June 2012

Abstract
Background
Triple-negative breast cancer (TNBC) is an aggressive biologic subtype which lacks effective targeted anti-cancer agents and is characterized by a high mitotic index and dependence on angiogenesis. ENMD-2076 is a novel orally bioavailable Aurora and angiogenic kinase inhibitor currently in clinical development with favorable pharmacokinetics and a manageable toxicity profile. The purpose of this study was to use TNBC cell line-based in vitro and in vivo models to demonstrate the antitumor activity of ENMD-2076 towards this breast cancer subtype compared to the luminal and HER2−amplified subtypes. Additionally, we used baseline gene expression profiling and pathway analysis to explore molecular predictors of responsiveness to ENMD-2076 in TNBC.
Methods: Twenty-five breast cancer cell lines were exposed to ENMD-2076 and the effects on proliferation, apoptosis, and cell cycle distribution were evaluated. Proliferation was assessed using an SRB assay, apoptosis was analyzed using a caspase 3/7 assay and cell cycle was measured using flow cytometry. In vitro activity of ENMD-2076 was confirmed in 3-D cell culture and in MDA-MB-231 and MDA-MB-468 triple-negative breast cancer xenograft models and immunohistochemical analysis was performed for phosphor-histone H3 (pHH3). Gene array and gene set enrichment analysis (GSEA) was used to identify pathways differentially regulated in the sensitive and resistance cell lines, including within the triple-negative breast cancer subset.
Results: In vitro exposure to ENMD-2076 resulted in robust inhibition of proliferation in TNBC cell lines which was associated with a G2 cell cycle arrest and induction of caspase-dependent apoptosis. Of the TNBC cell lines screened, 1 of 10 had a mean IC50 value &gt; 5 μmol/L and 7 of 10 had a mean IC50 ≤ 1 μmol/L (Fisher's exact test, p-value = 0.009). In comparison, only 1 luminal (ER+) and one UER2-amplified breast cancer cell line had an IC50 value &lt; 1, whereas 7 and 5 resistant lines were luminal and HER2−amplified, respectively (Fisher's exact test, p-values = 0.02 and 0.11 for ER and HER2 status, respectively). ENMD-2076 exhibited antitumor activity towards MDA-MD-231 and MDA-MB-468 xenograft models of TNBC with statistically significant tumor growth inhibition compared to vehicle control (p&lt; 0.05 and p&lt; 0.01, respectively). A trend towards an increase in pHH3 staining cells in the MDA-MB-231 ENMD-2076 treated group compared to the vehicle control group was observed; however, this was not statistically significant (104.7 ± 36.2 positive cells/mm2 in treated vs. 79.9 ± 34.5 in control). Using significance of analysis of microarrays (SAM) analysis and GSEA, we identified Ran, a member of the mitotic spindle regulation pathway as upregulated in sensitive TNBC cell lines (p = 0.017). Interestingly, AURKA, the main target of ENMD-2076, is a core gene in this pathway.
Conclusions: ENMD-2076 exhibited robust anticancer activity towards preclinical models of TNBC, supporting future clinical investigations of this agent in TNBC with an emphasis on the continued development of biomarkers predictive of response in this breast cancer subset.
Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-08.