Abstract The Ras family of small GTP binding proteins are frequently activated by mutations in melanoma, as shown for NRAS (20%), KRAS (2%) and HRAS (1%). Ras isoforms can also be activated by inactivation of Ras GTPase activating proteins (RasGAPs), such as NF1, RASA1, and RASA2. In our recent study, we observed that inactivation of RASA1 (RAS p21 protein activator 1, also called p120RasGAP) suppressed melanoma via its RasGAP activity toward the R-Ras (related RAS viral (r-ras) oncogene homolog) isoform and that R-Ras was required to promote anchorage-independent growth driven by RASA1 inactivation. Moreover, a low level of RASA1 mRNA expression is associated with decreased overall survival in melanoma patients with BRAF mutations. Based on these observations, we hypothesized that, although not mutated, R-Ras is activated in melanoma by inactivation of RasGAPs and that BRAF activation cooperates with this RasGAP/R-Ras pathway activation in melanoma tumorigenesis. In this study, we addressed the importance of R-Ras, a previously less appreciated member of the Ras small GTPases family, in melanoma tumorigenesis. We observed frequent activation of R-Ras in BRAF mutant human melanoma cell lines. In addition, RNAi-mediated reduced expression of R-Ras suppressed anchorage-independent colony growth and tumor growth. Moreover, among the 3 major RAS effector pathways, reduced R-Ras expression suppressed Ral-A activation, which may explain the mechanism of Ral-A activation in BRAF mutant melanoma. Interestingly, anchorage-independent growth driven by R-Ras activation downstream of RASA1 inactivation was suppressed by both genetic (siRNA targeting Ral-A) and pharmacological (Ral inhibitor BQU57) inhibition of Ral-A. To further investigate the impact of RASA1 loss, and thus R-Ras activation, on BRAF mutant melanoma development in vivo, we generated a RASA1L/L; BRAFCA/CA; Tyr-CreERT2 mouse model in which treatment with 4OHT results in expression of constitutively activated mutant BRAF and deletion of RASA1 in melanocytic lineage cells. Preliminary analysis shows hyperpigmentation of the ear, tail, and foot pad in RASA1L/L BRAFCA/CA mice compared to RASA1+/+ BRAFCA/CA littermates. Tumors generated in this animal model will be analyzed to determine the extent of R-Ras and Ral-A activity in vivo. This study demonstrates the importance of the RASA1/R-Ras/Ral-A signaling pathway in BRAF mutant melanoma and supports the possible combinatorial treatment strategy targeting both the BRAF/MAPK and Ral signaling pathways. Citation Format: Kristen Suzanne Hill, Xue Wang, Youngchul Kim, Minjung Kim. The importance of the RASA1/R-Ras/Ral-A signaling axis in melanoma tumorigenesis [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 1352. doi:10.1158/1538-7445.AM2017-1352