Abstract Introduction: We have previously reported that CDK4/6 inhibition enhanced the anti-proliferation and anti-migration effects of BRAF, MEK and PI3K inhibitors in melanoma cells. (Zhang Y et al, AACR 2017) In this study, we investigated the effect of combination CDK4/6 inhibition and BRAF, MEK and PI3K inhibition on induction of cell cycle arrest and apoptosis in melanoma. Methods: Five human melanoma cell lines were used. 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 apoptosis analysis was performed using the FITC Annexin V Apoptosis Detection Kit. Autophagy was evaluated by immunofluorescence using anti-LC3A/B and NECN-1 antibodies. Lysosomal morphology was evaluated using lysosomal associated membrane protein 1-green fluorescent protein fusion construct. Western blotting was performed for apoptosis markers (PARP and caspase-9) and autophagy markers (LC3A/B and Beclin1) using GAPDH as internal control. 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: Apoptosis was evaluated by Annexin V assay. Various levels of apoptosis were observed with single agent PD, PLX, XL and AZD in all 5 melanoma cell lines. Combination of PD enhanced the apoptosis effects of PLX, XL, and AZD in all 5 melanoma cell lines. Increased LC3 and BECN1 detection and increased density of autophagosomes on immunofluorescence was observed after combining CDK4 inhibition with BRAF, MEK, and PI3K/AKT inhibition. Inhibition of PARP and Beclin-1 expression and increase in caspase-9 expression were observed with combination of PD compared with single agent PLX, XL, and AZD. Conclusion: CDK4 inhibition enhanced anti-melanoma activity of BRAF , PI3K/AKT and MEK inhibitions through increased induction of early autophagy and caspase-mediated apoptosis. This suggests that inhibition of CDK4/6 plus BRAF , MEK and PI3K/AKT inhibition may help understanding the interplay between autophagy and apoptosis and inform the development of future targeted agents. Citation Format: Yanping Zhang, Guangyong Peng, Eddy C. Hsueh. Enhanced cytotoxic activity of BRAF, MEK and PI3K inhibition with combination CDK4/6 inhibition in melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2942.