Abstract A286: The role of p53 family tumor suppressors in mediating response to the Aurora and angiogenic kinase inhibitor ENMD-2076 in triple-negative breast cancer.
Authors: Ionkina Anastasia A. , Tentler John J. , Newton Timothy P. , Brunkow Kelsey L. , Johnson Jared S. , Tan Aik Choon , Pitts Todd M. , Eckhardt S. Gail , Diamond Jennifer R. 
Published: August 2014

Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype defined by the lack of expression of ER, PR and HER2 over-expression. TNBC is heterogeneous in its biology, however, mutations in p53 are found in approximately 80% of tumors. The purpose of this study was to utilize TNBC cell line models to investigate the role of p53 and p73 in mediating sensitivity to ENMD-2076, a selective Aurora kinase A inhibitor (AurKi). p53 and p73 knockdown (KD) models were developed to determine their role in mediating induction of apoptosis and senescence in response to AurK inhibition. Additionally, the effects of combining ENMD-2076 with the p53 modulator nutlin-3 were assessed.

Methods: TNBC cell lines, MDA-MB-468 (p53 mut) and CAL51 (p53 WT) were transduced with shRNA constructs targeting p53 and p73 with &gt; 80% knockdown as determined by RT-PCR. These clones were exposed to escalating doses of ENMD-2076 and the effect on proliferation was determined using an SRB assay. Proliferation was assessed using an SRB assay, and confirmed using a direct cell counting to control for increase in cell size following ENMD-2076 exposure. In the CAL51 KD lines, the effects of ENMD-2076 treatment on cellular senescence were determined using a senescence associated beta-galactosidase (SA β-gal) assay. Combination studies with nutlin-3 in MDA-MB-468 and HCC1937 were assessed by SRB and synergy measured using Calcusyn software.

Results: KD of p53 and p73 in the MDA-MB-468p5310, MDA-MB-468p7355 cell lines resulted in an increase in the IC50 from 0.625 μM to 1.5 μM compared to scrambled. KD of CAL51 p53 and p73 increased from 0.16 µM to 0.41 µM, and 1.3 µM, in the CAL51scr, CAL51p5310, CAL51p7326, respectively. The sensitive MDA-MB-468 cell line (IC50 40 nM) and the sensitive CAL51 cell line (IC50 45 nM) were selected for further experimentation. Exposure to ENMD-2076 at concentrations of 0.5 μM and 1 μM for varying time points resulted in induction of senescence in the CAL51 cell line, but not in the MDA-MB-468 cell line, where induction of apoptosis at 48 hours was observed. In CAL51 p53 and p73 KD clones, induction of senescence was observed following exposure to ENMD-2076. Combination treatment with nutlin-3 showed synergistic activity.

Conclusions: ENMD-2076 exhibited robust anticancer activity towards preclinical models of TNBC, in both p53 mutated and p53 WT cell lines, supporting future clinical investigation of AurKi in TNBC. In p53 WT TNBC, both p53 and p73 mediate sensitivity to ENMD-2076 and p73 is essential for induction of senescence following exposure to ENMD-2076. Synergistic anticancer activity of ENMD-2076 and nutlin reinforces the role of p53 in Aurora kinase inhibition of TNBC. Biomarkers predictive of response to ENMD-2076 in p53 mutated TNBC are being developed. These results with p53 KD cell lines are currently being confirmed in vivo.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A286.

Citation Format: Anastasia A. Ionkina, John J. Tentler, Timothy P. Newton, Kelsey L. Brunkow, Jared S. Johnson, Aik Choon Tan, Todd M. Pitts, S. Gail Eckhardt, Jennifer R. Diamond. The role of p53 family tumor suppressors in mediating response to the Aurora and angiogenic kinase inhibitor ENMD-2076 in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A286.