Abstract 703: A novel ATP-competitive MEK/Aurora kinase inhibitor BI-847325 reverses acquired BRAF inhibitor resistance through suppression of Mcl-1 and inhibition of MEK expression
Authors: Phadke Manali S. , Sini Patrizia , Smalley Keiran 
Published: August 2015

Abstract
BACKGROUND: BRAF/MEK inhibitors have shown promising levels of response in melanomas harboring the BRAF V600E mutation, however responses tend to be short-lived and resistance is a major clinical problem. In the current study, we evaluated the pharmacological activity of BI847325, a dual inhibitor of MEK and Aurora kinases against multiple melanoma cell line models.
METHODS: The cytotoxic effect of BI847325 was evaluated in vitro in a panel of selected BRAF-mutant vemurafenib resistant cell lines by Alamar blue and Annexin V binding assay. 3D spheroid model systems and colony formation assays demonstrated the long-term growth inhibitory effect of BI847325. Western blot analysis and qRT-PCR studies were carried out to evaluate the mechanism underlying BI5-mediated cytotoxicity. In vivo studies in Balb SCID mice were performed to assess the suppression of BRAF-mutant xenografts on treatment with BI847325.
RESULTS: BI847325 potently reduced the growth and survival of BRAF-mutant melanoma cell lines with acquired and intrinsic BRAF inhibitor resistance (NRAS mutations, BRAF splice forms, Cyclin D1 amplification, RTK upregulation, PTEN loss, COT amplification). We confirmed that BI847325 induced apoptosis through decrease in Mcl-1 mRNA and protein expression and increase in BIM expression. The effects of BI847325 upon BIM and Mcl-1 expression could not be mimicked by the combination of other MEK and aurora kinase inhibitors. For the first time we demonstrated that BI847325 reverses the acquired vemurafenib resistance by inhibiting MEK expression at mRNA and protein level. A strong suppression of MEK expression was observed without recovery following 72 h of washout. In vivo studies revealed complete tumor suppression with no recurrence over a period of 65 days of treatment with 70mg/kg/week dose of BI847325. In contrast, treatment with vemurafenib analog PLX4720 in the same mouse model was associated with tumor relapse after 30 days of treatment. BI847325 also successfully suppressed the long-term growth of xenografts with acquired vemurafenib resistance. Analysis of tumor samples complied with in vitro results demonstrating inhibition of phospho-ERK, phospho-Histone3, Mcl-1 and total MEK.
CONCLUSION: In conclusion we report for the first time that BI847325, a novel ATP-competitive MEK/ Aurora kinase inhibitor effectively inhibits BRAF-mutant melanoma and overcomes vemurafenib-resistance by decreasing expression of MEK and Mcl-1; in vitro and in vivo. Further preclinical and clinical investigations towards this would open new avenues in treatment of melanoma.
Citation Format: Manali S. Phadke, Patrizia Sini, Keiran Smalley. A novel ATP-competitive MEK/Aurora kinase inhibitor BI-847325 reverses acquired BRAF inhibitor resistance through suppression of Mcl-1 and inhibition of MEK expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 703. doi:10.1158/1538-7445.AM2015-703