Abstract Background The acquired resistance to BRAF inhibitor poses a critical hurdle for the treatment of BRAF V600E mutant melanoma patients. Recent evidences suggest that the Hippo pathway effectors, YAP and its paralog TAZ, promote resistance to anti-cancer therapies. YAP/TAZ activity is tightly coupled to actin cytoskeleton dynamics and architecture. However, the influence of actin cytoskeletal remodeling on cancer drug resistance remains largely uncovered. Here, we report that actin remodeling in response to BRAF inhibitor treatment plays a pivotal role in YAP/TAZ-dependent resistance development in melanoma cells. Materials and Method Two vemurafenib resistant melanoma cell lines (SKMEL28 and WM3248) were established by two months of vemurafenib treatment. We inspected actin cytoskeletal structure changes in resistant cells. YAP/TAZ localization and target gene transcription were compared between parental and resistant melanoma cells. In addition, changes in the sensitivity to vemurafenib upon YAP/TAZ knockdown or overexpression were measured, and transcriptome-wide signatures supported by YAP/TAZ were investigated by microarray analysis. Lastly, a kinome siRNA library screening was performed in resistant cells to identify synthetic lethal targets to suppress resistant cell growth combined with BRAF inhibition. Results Vemurafenib resistant melanoma cells exhibit a substantial increase in both actin stress fiber formation and cell spreading force compared with the parental cells, which appears to promote the nuclear accumulation of YAP/TAZ and the upregulation of their transcriptional activity. Depletion of YAP/TAZ significantly restores vemurafenib sensitivity in resistant cells, whereas overexpression of constitutively active YAP induces vemurafenib resistance in parental cells, suggesting BRAF inhibitor resistance in melanoma cells is highly dependent on YAP/TAZ activity. The microarray analysis comparing control siRNA and YAP/TAZ siRNA knockdown in resistance cells suggests that E2F-related cell cycle progression accompanied by EGFR and c-MYC pathway activation is the major driver of YAP/TAZ-dependent cell proliferation and vemurafenib resistance. Remarkably, pharmacologic inhibition of actin polymerization or actomyosin contraction significantly promotes YAP/TAZ cytoplasmic retention and transcriptional downregulation. Moreover, resistant cell viability is suppressed by pharmacologic inhibition of actin polymerization and cytoskeletal tension. Consistent with the importance of the actin cytoskeleton, our kinome-wide RNAi screening indentifies TESK1, a kinase inhibiting cofilin activity, as a promising synthetic lethal target for BRAF inhibitor resistance. TESK1 knockdown suppresses both enhanced actin stress fiber formation and YAP/TAZ nuclear translocation, provoking growth arrest of vemurafenib-resistant melanoma cells. Conclusion Our results implicate the actin cytoskeleton in the induction of YAP/TAZ-dependent resistance to vemurafenib, and inhibition of actin remodeling might be a promising synthetic lethal strategy to suppress resistance in BRAF inhibitor therapies. Citation Format: Min Hwan Kim, Joon Kim. Actin remodeling confers BRAF inhibitor resistance to melanoma cells through YAP/TAZ activation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B50.