Abstract P6-13-16: Ropidoxuridine (IPdR) potentiates alisertib (MLN8237) activity in triple-negative breast cancer

Abstract Introduction and Background: An estimated 234,190 new cases of invasive breast cancer will be diagnosed in 2015 with 40,730 deaths (American Cancer Society, 2015). Triple-negative breast cancer (TNBC) has an overall poor prognosis due to aggressive and early pattern of metastasis and a relative lack of therapeutic targets. Chemotherapy remains the cornerstone of treatment albeit with a dismal response rate (∼25%) to monotherapy. Synergism between two drugs could provide a key advance to enhance effectiveness, decrease resistance and reduce toxicities. Here we focused on discovery of synergism of antiproliferative agents given TNBC is highly proliferative. Experimental Methods: We assembled a library of antiproliferative agents with unique mechanisms and targets that have subtly different effects on cancer cell proliferation. We performed a novel synergy screen testing 105 unique two-drug combinations in MDA-MB-231 TNBC cells. Our screen was designed to distinguish between synergism and additivity through presence of an internal control and performed in sufficient replicates to identify changes in viability exceeding 5%. We validated hits through Chou Talalay Combination Index (CI), xenograft and mechanistic analyses. Results: We discovered strong synergy between Ropidoxuridine (IPdR) and Alisertib (MLN8237). Ropidoxuridine is an orally bioavailable pro-drug of IUdR (5-iodo-2'-deoxyuridine), with an improved therapeutic index and with promising activity as a radiosensitizer, although it lacks single agent activity. Alisertib is an inhibitor of Aurora A kinase and acts by impairing assembly of a bipolar mitotic spindle, thereby activating the spindle assembly checkpoint leading to cancer cell death. Alisertib has demonstrated activity in breast cancer with a response rate of 18% in heavily pretreated patients (Melichar B et.al. Lancet Oncol 16:395-405, 2015). First, Ropidoxuridine and Alisertib combination was validated in cell culture using CI analysis with a mean CI of 0.14; range 0.01-0.67 suggesting strong synergism. A separately performed Live Dead Cell Assay also confirmed synergism (mean CI of 0.79; range: 0.53-1.76) suggesting enhanced anti-proliferative activity of Alisertib in presence of Ropidoxuridine. To assess generalizability, this combination was validated in two other TNBC cell lines, MDA-MB-468 and CAL-51. To confirm our hypothesis that in vitro activity of Ropidoxuridine is from its metabolism to IUdR in cancer cells, we demonstrated strong synergy of IUdR and Alisertib (mean CI of 0.12; range 0.01-0.74) at clinically relevant concentrations. To validate synergy in an orthotopic tumor model, Ropidoxuridine and Alisertib single agents and in combination were tested in mice treated with Ropidoxuridine (750 mg/kg/day) and Alisertib (30 mg/kg/day) by gavage. This in vivo model also demonstrated strong synergism. Mechanistically, Ropidoxuridine and IUdR enhance G2/M arrest in response to Alisertib, allowing low dose exposures of Alisertib to be effective. Conclusions and Future Directions: This study identifies and validates a novel synergy between Ropidoxuridine and Alisertib with a potential for significant clinical implications. We propose evaluating this synergistic drug combination in an early phase clinical trial. Citation Format: Rampurwala MM, Choudhary A, Burkard ME. Ropidoxuridine (IPdR) potentiates alisertib (MLN8237) activity in triple-negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-13-16.