Abstract Uveal melanoma (UM) is characterized by gain-of-function mutations in GNAQ or GNA11, encoding Gα proteins from the Gq/11 family. UM is the most common eye malignancy in adults. Approximately 50% of UM patients develop liver metastasis (mUM) within 5-10 years after diagnosis, independently of the successful treatment of the primary lesions. mUM is refractory to cytotoxic, targeted, and immunotherapies, with most mUM patients dying within a year. Recent information suggests that GNAQ-oncogenic signaling involves a non-canonical pathway distinct from the activation of PLCβ and PKC-MEK-ERK, which may explain the failure of MEK inhibitors (MEKi) in increasing mUM patient survival. Instead, we found that GNAQ promotes the activation of YAP1, a key oncogenic driver, by a mechanism involving the activation of RhoA by the direct association of Gαq to TRIO, a Rho-GEF (Cancer Cell, 2014). In turn, YAP1 is essential for uveal melanoma cell growth, however no effective and safe YAP1 inhibitors are currently available. Using a novel bioinformatics pipeline, we recently found that PTK2, encoding Focal Adhesion Kinase (FAK), is a synthetic lethal gene with GNAQ activation, and uncovered that GNAQ controls YAP1 through FAK (Cancer Cell, 2019). This study identified FAK as a druggable signaling hub downstream from GNAQ in UM. However, activation of compensatory pathways often results in resistance to targeted agents. Here, we combined the use of CRISPR-Cas9 sgRNA screens with a recently described Cancer Signaling Toolkit approach to identify synthetic lethal interactions enhancing the response to FAKi and signaling networks mediating drug resistance, respectively. Remarkably, both approaches converged to reveal that co-targeting FAK and the MEK-ERK pathway would be a promising combination for treatment of UM. Indeed, MEK-ERK pathway inhibition by multiple approved MEKis (e.g., trametinib), combined with FAK inhibition (VS-4718 or defactinib), showed remarkable synergistic growth inhibitory effects in UM cells. Additionally, the novel RAF/MEK inhibitor RO5126766 also showed synergistic anti-proliferative effects with defactinib. Accordingly, FAKi combination with MEKi exerted cytotoxic effects (apoptotic death) leading to rapid tumor shrinkage in UM xenografts, whereas single drugs were primarily cytostatic. Furthermore, the FAKi/MEKi combination was successful at reducing tumor burden in recently developed liver metastasis UM models. By coupling the unique genetic landscape of UM with the power of unbiased computational pipelines and systems biology genetic screens, our studies revealed that FAK and MEK-ERK co-targeting may provide a new network-based precision therapeutic strategy for mUM treatment. Indeed, the combination of defactinib and RO5126766 is currently being evaluated in patients with various solid tumors (NCT03875820), and could be explored in mUM based on these preclinical findings. Citation Format: Justine S. Paradis, Monica Acosta, Nadia Arang, Robert Saddawi-Konefka, Ayush Kishore, Takahito Sugase, Xiaodong Feng, Kris C. Wood, Silvia Coma, Mizue Terai, Takami Sato, Jonathan A. Pachter, J. Silvio Gutkind. FAK and MEK co-targeting: A new multimodal precision therapy for GNAQ-driven uveal melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6406.