Abstract YAP overactivation is an essential molecular event for cancer initiation and growth of most solid tumors, but pharmacologic targeting of the YAP or Hippo pathway has been proven to be challenging. In this regard, YAP activity is also required for stem and progenitor cell maintenance and function in multiple tissues, and as such, YAP is necessary for normal tissue homeostasis and regeneration. Thus, targeting YAP directly may result in normal cell growth arrest and tissue damage. The elucidation of the signaling networks sustaining YAP pervasive activation in each cancer type may afford the opportunity to identify novel suitable precision therapeutic targets for cancer treatment. GNAQ and GNA11 activate mutations (encoding GTPase deficient and constitutively active Gαq proteins) occurs in ~90% of uveal melanoma (UM) and 4% of skin cutaneous melanoma (SKCM), respectively, firmly established UM, and a subset of SKCM as Gαq-driven human malignancies. Approximately 50% of UM patients develop liver metastasis within 5-10 years after diagnosis, independently of the successful treatment of the primary lesions. To date, there are no effective treatment options for metastatic UM disease. Through the bioinformatics analysis of the TCGA UM dataset (n=80) using skin melanoma samples that do not harbor Gαq genomic alteration (n=209) as control, (1) we first identified the genes that are highly overexpressed in UM. (2) We then filtered the genes for those whose reduced expression leads to better patient survival in UM. (3) We finally used large datasets of gene essentiality and drug response screenings in cancer cells to identify genes that are predicted to reduce cell viability when targeted in Gαq-gain cells. (4) Finally, 7 top predicted drug targets were found from a set of genes that can be targeted by approved and experimental drugs (n=756). The top predicted gene target was PTK2, which encodes the nonreceptor tyrosine kinase FAK (focal adhesion kinase). We found that Gαq activates pY379-FAK (reflecting FAK activation) though a Trio-RhoA signaling pathway, independent of PLC-β regulated second messenger activation. Interestingly, genome-wide transcriptional analysis (RNA-seq) of UM cells treated with a FAK inhibitor (VS-4718) revealed that YAP signatures were highly inhibited by FAK blockade. This suggested that FAK signaling actives YAP downstream of Gαq in UM, in addition to our previously found Trio/Rho GTPase/F-actin polymerization regulation of YAP activation mechanism. Further signaling analysis uncovered that FAK stimulates YAP through direct YAP tyrosine phosphorylation (enhancing YAP protein stability) and, unexpectedly, MOB1 tyrosine phosphorylation on Y26. The latter disrupts MOB1/LATS interaction, causing Hippo signaling inhibition and YAP activation. Interestingly, FAK inhibitors are already under clinical evaluation, and showed strong inhibition of YAP activity and UM growth in vitro and in vivo, thus representing a potential precision therapeutic option for UM treatment. Citation Format: Xiaodong Feng, Nadia Arang, Damiano Cosimo Rigiracciolo, Joo Sang Lee, Huwate Yeerna, Zhiyong Wang, Simone Lubrano, Ayush Kishore, Jonathan A. Pachter, Gabriele M. König, Marcello Maggiolini, Evi Kostenis, David D. Schlaepfer, Pablo Tamayo, Qianming Chen, Eytan Ruppin, J. Silvio Gutkind. Gαq controls the Hippo pathway through MOB1 tyrosine phosphorylation by FAK [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A20.