Abstract
Abstract Introduction: Targeted therapy against BRAF-mutated melanoma has shown clinical efficacy. However, resistance to therapy often occurs. CDK4 (INK4)-retinoblastoma (RB) pathway controls cell cycle progression by regulating the G1-S checkpoint. Defect in the p16INK4A: cycling D-CDK4/6: RB pathway is also detected in melanoma. We study the effect of combining CDK4-RB pathway inhibition with BRAF/MEK inhibition as a possible strategy to overcome resistance to therapy. Methods: Three BRAF wild-type and three BRAF-mutated melanoma cell lines were selected. CDK4/6 inhibitor (PD0332991; PD), BRAF inhibitor (PLX4032; PLX), MEK inhibitor (AZD6244; AZD), and PI3K/AKT inhibitor (XL765; XL) were used alone or in combination. Cell proliferation assay was performed using Cell Titer Blue assay. Cell migration assay was performed by artificial wounding of melanoma cell monolayer and observing migration of cells into the wound up to 48 hours. Western blotting was performed for expression of pERK/ERK, pRB/RB, Cyclin D1, CDK4, and GAPDH. Data were presented as means ± SD for triplicate experiments. For comparison between groups, the student’s t test was used and p<0.05 was considered to be statically significant. Results: Variable inhibition of cell proliferation and migration was observed in a dose- and time-dependent fashion. Enhanced inhibition of melanoma proliferation and migration was observed with PD+AZD and PD+PLX combination compared with single agent AZD or PLX. Differential enhancement was observed in BRAF wild-type cell lines with PD+AZD and in BRAF mutated cell lines with PD+PLX combination. Minimal change in tumor proliferation and migration inhibition was noted with PD+XL combination compared with PD or PLX alone. No correlation was observed between expression of CDK4 and cyclin D1 and sensitivity to singe agent or the combination. Inhibition of phosphorylated RB1 and ERK expression was observed with exposure to single agent PD, PLX, XL, AZD. Enhancement of pRB1 and pERK inhibition was noted with PD+PLX and PD+AZD combination but not PD+XL. Conclusion: CDK4 inhibition can enhance anti-proliferation and migration effects of BRAF/ MEK inhibition in melanoma cells. The mechanism is via augmented down-regulation of pRB expression. Citation Format: Yanping Zhang, Eddy C. Hsueh. CDK4 inhibition enhances anti-melanoma effects of BRAF/MEK inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2345. doi:10.1158/1538-7445.AM2017-2345
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