Abstract
Abstract INTRODUCTION: BRAF and NRAS oncogenic mutations occur in over half of all human melanomas. BRAF-mutant tumors are highly sensitive to small molecule inhibitors directed against the oncogenic BRAF protein. However, in tumors that lack the specific mutation, patients have few effective therapeutic options. To address the need for new therapies for patients with BRAF-wild-type (wt) melanoma, we hypothesized that the inhibition of the major nuclear export receptor CRM1 would result in nuclear accumulation of nuclear proteins, increase apoptosis and induce cell cycle arrest independent of BRAF mutational status. To test our hypothesis, we examined the effects of a novel CRM1 inhibitor on melanoma cell lines with various BRAF and NRAS mutational backgrounds. METHODS: We used human malignant melanoma cell lines with BRAF mutation (Sk-Mel-28, A375, Mel-624, UACC903, MalMe-3M), BRAF-wt (MeWo) and BRAF-wt and NRAS mutation (Hs940T) to assess the effects of CRM1 inhibition, BRAF inhibition (PLX4032) or MEK inhibition (U0126) using both mono- or combination therapy. MTT assays were used to evaluate the effects of treatment on cell proliferation. FACS analysis with annexin/PI staining was performed to assess effects on cell cycle and cell death. The Chou-Talalay method was used to assess synergistic drug combination effects. Treatment effects on cleaved caspase-3, PARP cleavage, P53, and FOXO3 were analyzed by western blot analysis. RESULTS: As mono therapy, CRM1 inhibition led to a significant decrease in cell proliferation, cell cycle arrest and an increase in apoptosis in both BRAF wt and mutant cell lines. The degree of inhibition and cell death achieved by CRM1 inhibition across all melanoma cell lines is comparable to the results achieved by BRAF inhibitor PLX4032 in BRAF mutant cell lines. Furthermore, the combination of CRM1 inhibition and BRAF inhibition results in a strongly synergistic combination against BRAF mutant cell lines with a statistically significant decrease in cell proliferation and increased apoptotic cell death. CONCLUSION: We found that relative to other cancer cell types, both BRAF-mutant and BRAF-wt melanoma cells are exquisitely sensitive to CRM1 inhibition. In addition, CRM1 inhibition significantly enhanced the effect of PLX4032 in BRAF-mutant cell lines, suggesting that the combination of these two drugs may address the emergence of acquired resistance to BRAF monotherapy. Based on these results, CRM1 inhibition warrants further investigation to determine its potential benefit in treating melanoma patients independent of BRAF mutation status. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1914. doi:1538-7445.AM2012-1914
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