Abstract 375: Combination of MEK and PI3K inhibition in BRAF wild-type and mutant melanoma
Authors: Zhang Yanping , Peng Guangyong , Hsueh Eddy C. 
Published: July 2016

Introduction: Targeted therapy against BRAF-mutated melanoma with BRAF and MEK inhibitors has shown clinical efficacy. However, resistance to therapy occurred and lead to therapy failure. We evaluated the effect of combining downstream inhibition of RAS/RAF pathways with MEK (AZD6244) and PI3K (XL765) inhibition on BRAF wild-type and mutant melanoma with variable expression of NRAS as possible therapeutic option.

Methods: Fifteen melanoma cell lines were screened for BRAF mutation status and NRAS expression. BRAF wildtype (Mel628, Mel1098) and BRAF-mutated (Mel1861, Mel1890) cell lines were selected. Drug concentration study with AZD6244 (A, 2.5-40 uM) and XL765 (X, 2.5-40 uM) were performed on Mel628 cells. Subsequently, cells were treated with A (5 uM), X (5 uM), or combination. Cell proliferation assay was performed using Cell Titer Blue assay. Western blotting was performed to for expression of PARP, caspase-9, LC3A/B, Beclin1, pMEK/MEK, pERK/ERK, p-P70S6/P70S6, and p4EBP1/4EBP1. GAPDH was used as internal control. Apoptosis was analyzed using FITC Annexin V Apoptosis Detection Kit (BD Biosciences). Cell migration assay was performed by artificial wounding of melanoma cell monolayer and observing migration of cells into the wound up to 72 hours. Data were presented as means ± SD for the three separate experiments. For comparison between groups, the student's t test was used and p&lt; 0.05 was considered to be statically significant.

Results: There was dose-dependent inhibition of melanoma cell proliferation with both A and X. Combination A and X had synergistic effect on the anti-melanoma proliferative effect of BRAF mutated cells regardless of expression level of NRAS. Significantly increased apoptosis by Annnexin V assay was detected in BRAF mutated cells (Mel 1860 and Mel1890). Significantly increased expression (p&lt;0.05) of apoptosis markers (PARP and caspases-9) were observed in cells treated with A+X compared with A or X alone. Enhanced phosphorylation of ERK and P70S6 were observed in BRAF mutated cells. No significant difference in autophagy markers (LC3A/B and Beclin1) were observed between A, X, or combination. Variable to minimal additive effect of the combination treatment was observed in BRAF wild-type cells in terms of proliferation, apoptosis, and autophagy. The combination of A and X significantly inhibited melanoma migration compared with either drug alone regardless of BRAF mutation status in the wounding assay model.

Conclusion: We have observed a synergistic effect on suppressing BRAF-mutated melanoma proliferation with combined inhibition of MEK and PI3K. The effect is not dependent on NRAS expression levels of melanoma.

Citation Format: Yanping Zhang, Guangyong Peng, Eddy C. Hsueh. Combination of MEK and PI3K inhibition in BRAF wild-type and mutant melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 375.